Multispecific polypeptide conjugates targeting GPRC5D

Abstract

The present invention is a multispecific polypeptide conjugate, an isolated polynucleotide encoding the same, a pharmaceutical composition comprising the same and uses thereof.

Description

[Technical Field]

[0001] The present application relates generally to multispecific polypeptide conjugates that target GPRC5D and uses thereof. [Background technology]

[0002] In the field of antibody therapy, bispecific or multispecific antibodies, which are currently being actively researched, are superior to monospecific antibodies in many respects because they can simultaneously recognize two or more different antigens, neutralize different pathogenic mediators, recruit different types of effector cells, and regulate signal pathways. Therefore, developing bispecific or multispecific antibodies as therapeutic agents for human diseases has important clinical significance, and in recent years, bispecific antibodies have become a widely used form in diagnostic and therapeutic applications.

[0003] In the development of bispecific or multispecific antibodies, G-protein-coupled receptor family C group 5 member D (GPRC5D), a seven-transmembrane protein and orphan receptor, has become one of the most sought-after targets. Overexpression of GPRC5D has been reported in patients with multiple myeloma. In particular, high expression significantly correlates with disease and poor treatment outcomes. Given the specific high expression of GPRC5D in tumor cells, GPRC5D may be a promising next target for treating multiple myeloma. If it is necessary to specifically target or eliminate cells overexpressing GPRC5D, multispecific antibodies that target GPRC5D as one of the targets (e.g., bispecific or multispecific antibodies that simultaneously target the tumor antigen GPRC5D and immune stimulatory antigens (e.g., CD3) or other tumor antigens (e.g., Her2)) are of great interest.

[0004] Therefore, there is an urgent need in the art for the development of novel multispecific antibodies that target GPRC5D as one of their targets. Summary of the Invention

[0005] Throughout this application, the articles "a" and "an" and "said" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or multiple antibodies.

[0006] The present application provides multispecific polypeptide conjugates, isolated polynucleotides encoding same, pharmaceutical compositions containing same and uses thereof.

[0007] In one aspect, the present application provides a multispecific polypeptide conjugate comprising a first antigen-binding domain and a second antigen-binding domain, wherein at least one of the first antigen-binding domain and the second antigen-binding domain binds to GPRC5D and comprises a GPRC5D-binding domain, wherein the GPRC5D-binding domain comprises three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and wherein the heavy chain complementarity determining regions are the heavy chain variable region (V) as set forth in SEQ ID NO: 13. H ) and the light chain complementarity determining region is the same as the light chain variable region (V L ) is the same as the three light chain complementarity determining regions contained within.

[0008] In some embodiments, the GPRC5D-binding domain comprises three heavy chain complementarity determining regions, HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions, LCDR1, LCDR2, and LCDR3, wherein a) the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 or a variant thereof having 3, 2, or no more than 1 amino acid substitutions, or SEQ ID NO: 9 or a variant thereof having 3, 2, or no more than 1 amino acid substitutions, b) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 or a variant thereof having 3, 2, or no more than 1 amino acid substitutions, or SEQ ID NO: 10 or a variant thereof having 3, 2, or no more than 1 amino acid substitutions, and c) the HCDR3 comprises SEQ ID NO: 6 or a variant thereof having 3, 2, or no more than 1 amino acid substitutions. or a variant thereof having 3, 2 or 1 or less amino acid substitutions; d) the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or a variant thereof having 3, 2 or 1 or less amino acid substitutions; or SEQ ID NO: 7 or a variant thereof having 3, 2 or 1 or less amino acid substitutions; e) the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or a variant thereof having 3, 2 or 1 or less amino acid substitutions; or SEQ ID NO: 8 or a variant thereof having 3, 2 or 1 or less amino acid substitutions; and f) the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3 or a variant thereof having 3, 2 or 1 or less amino acid substitutions.

[0009] In some embodiments, in the GPRC5D-binding domain, a) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof having three, two, or one or less amino acid substitutions; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof having three, two, or one or less amino acid substitutions; the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof having three, two, or one or less amino acid substitutions; the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof having three, two, or one or less amino acid substitutions; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof having three, two, or one or less amino acid substitutions; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof having three, two, or one or less amino acid substitutions. or the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof having 3, 2 or 1 or less amino acid substitutions.

[0010] In some embodiments, in the GPRC5D binding domain, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof having 3, 2 or 1 or less amino acid substitutions.

[0011] In some embodiments, the GPRC5D binding domain is humanized.

[0012] In some embodiments, the GPRC5D-binding domain comprises a heavy chain variable region (V H ) and / or light chain variable region (V L ), wherein a) the heavy chain variable region comprises an amino acid sequence selected from the group of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, or a variant thereof with no more than 3, 2, or 1 amino acid substitutions; and b) the light chain variable region is selected from the amino acid sequence of the group of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or a variant thereof with no more than 3, 2, or 1 amino acid substitutions.

[0013] In some embodiments, the GPRC5D-binding domain is a heavy chain variable region (V H ) and / or light chain variable region (V L), wherein a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof having three, two, or one or less amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14 or a variant thereof having three, two, or one or less amino acid substitutions, b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17 or a variant thereof having three, two, or one or less amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof having three, two, or one or less amino acid substitutions, and c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof having three, two, or one or less amino acid substitutions. the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof with no more than 3, 2 or 1 amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof with no more than 3, 2 or 1 amino acid substitutions, or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof with no more than 3, 2 or 1 amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof with no more than 3, 2 or 1 amino acid substitutions, wherein the amino acid substitutions are not within the CDR regions.

[0014] In some embodiments, the polypeptide conjugate further comprises an immunoglobulin constant region, optionally comprising a constant region of a human immunoglobulin, or optionally comprising a constant region of a human IgG.

[0015] In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain binds to GPRC5D and comprises a GPRC5D-binding domain described herein, and the other binds to an antigen different from GPRC5D. In some embodiments, the antigen different from GPRC5D is an immunostimulatory antigen, and optionally, the immunostimulatory antigen is CD3. In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain comprises a GPRC5D-binding domain described herein, and the other comprises a CD3-binding domain.

[0016] In some embodiments, the CD3-binding domain comprises three heavy chain complementarity determining regions, HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 49 or a variant thereof having three, two, or one or less amino acid substitutions; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 50 or a variant thereof having three, two, or one or less amino acid substitutions; the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 51 or a variant thereof having three, two, or one or less amino acid substitutions; the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof having three, two, or one or less amino acid substitutions; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 53 or a variant thereof having three, two, or one or less amino acid substitutions; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 54 or a variant thereof having three, two, or one or less amino acid substitutions. In some embodiments, in the CD3-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 92 or a variant thereof having three, two, or one or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 93 or a variant thereof having three, two, or one or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 94 or a variant thereof having three, two, or one or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 95 or a variant thereof having three, two, or one or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 96 or a variant thereof having three, two, or one or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 54 or a variant thereof having three, two, or one or less amino acid substitutions.

[0017] In some embodiments, the CD3 binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 55 or a variant thereof having three, two, or no more than one amino acid substitution, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 56 or a variant thereof having three, two, or no more than one amino acid substitution.

[0018] In some embodiments, the first antigen-binding domain and the second antigen-binding domain constitute a single DICAD domain, and the DICAD domain comprises: (i) a first polypeptide comprising, in an N-terminal to C-terminal direction, a first light chain variable domain VL1 that binds a first antigen and a second heavy chain variable domain VH2 that binds a second antigen, wherein VL1 and VH2 are linked directly or via a first linker; and (ii) a second polypeptide comprising, in an N-terminal to C-terminal direction, a second light chain variable domain VL2 that binds a second antigen and a first heavy chain variable domain VH1 that binds the first antigen, wherein VL2 and VH1 are linked directly or via a second linker, wherein VL1 and VH1 bind to the first antigen-binding domain, VL2 and VH2 bind to the second antigen-binding domain, and VL1 and VH1 are covalently linked via a disulfide bond.

[0019] In some embodiments, the first linker and / or the second linker each independently comprises 5 to 9 amino acid residues.

[0020] In some embodiments, the VL1 has a first cysteine ​​substitution in FR and the VH1 has a second cysteine ​​substitution in FR, and the first and second cysteines form a disulfide bond.

[0021] In some embodiments, the first and second cysteines are selected from the group consisting of 100C in VL1 and 44C in VH1, 43C in VL1 and 105C in VH1, 49C in VL1 and 100bC in VH1, 50C in VL1 and 100C in VH1, 46C in VL1 and 101C in VH1, wherein the numbering is according to Kabat numbering. In some embodiments, the disulfide bond is formed between 100C in VL1 and 44C in VH1.

[0022] In some embodiments, the VL1 and VH1 further have an electrostatic interaction between two oppositely charged residues, in some embodiments, the two oppositely charged residues are introduced into the VL1 and VH1 and replace residues at positions selected from the group consisting of a) Q38 in VL1 and Q39 in VH1, b) Q40 in VL1 and Q39 in VH1, or c) Q37 in VL1 and Q39 in VH1, wherein the numbering is according to Kabat numbering.

[0023] In some embodiments, there is an additional electrostatic interaction between two oppositely charged residues between the VL2 and VH2. In some embodiments, the two oppositely charged residues between the VL2 and VH2 are introduced to replace residues at positions selected from the group consisting of a) Q38 in VL2 and Q39 in VH2, b) Q40 in VL2 and Q39 in VH2, or c) Q37 in VL2 and Q39 in VH2, where the numbering is according to Kabat numbering. In some embodiments, the two oppositely charged residues comprise one negatively charged amino acid residue selected from the group of aspartic acid (D) or glutamic acid (E), and one positively charged amino acid residue selected from the group of lysine (K) or arginine (R).

[0024] In some embodiments, at least one residue in the FR of the VL1 is substituted with a negatively charged amino acid and at least one residue in the FR of the VH1 is substituted with a positively charged amino acid, or at least one residue in the FR of the VL1 is substituted with a positively charged amino acid and at least one residue in the FR of the VH1 is substituted with a negatively charged amino acid.

[0025] In some embodiments, the first antigen-binding domain comprises an antigen-binding fragment of an antibody that binds to GPRC5D described herein, and the second antigen-binding domain comprises a CD3-binding domain as defined herein. In some embodiments, the amino acid sequence of the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence of the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the second polypeptide further comprises, in N-terminal to C-terminal direction, a first Fc polypeptide. In some embodiments, the polypeptide complex further comprises a third polypeptide comprising, in N-terminal to C-terminal direction, a second Fc polypeptide.

[0026] In some embodiments, the polypeptide complex further comprises a third antigen-binding domain, optionally comprising a Fab domain. In some embodiments, the Fab domain comprises (i) a third polypeptide comprising, in an N-terminal to C-terminal direction, a third heavy chain variable domain VH3 that binds to a third antigen and a CH1 Domain, and (ii) a fourth polypeptide comprising, in an N-terminal to C-terminal direction, a third light chain variable domain VL3 that binds to the third antigen and a CL Domain, wherein VL3 and VH3 combine to form the third antigen-binding domain.

[0027] In some embodiments, the Fab domain binds to GPRC5D and comprises a GPRC5D-binding domain described herein. In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:30, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO:31, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:32.

[0028] In some embodiments, the polypeptide complex further comprises a third antigen-binding domain, optionally wherein the third antigen-binding domain comprises a Fab domain, wherein the Fab domain comprises (i) a third polypeptide comprising, in an N-terminal to C-terminal direction, a third heavy chain variable domain VH3 that binds to the third antigen and a CH1 Domain, and (ii) a fourth polypeptide comprising, in an N-terminal to C-terminal direction, a third light chain variable domain VL3 that binds to the third antigen and a CL Domain, wherein VL3 and VH3 combine to form the third antigen-binding domain.

[0029] In some embodiments, the first antigen, second antigen, and third antigen are each independently selected from GPRC5D, an immunostimulatory antigen, and a tumor antigen, and optionally, the immunostimulatory antigen is CD3 and the tumor antigen is Her2. In some embodiments, the first antigen is Her2, the second antigen is CD3, and the third antigen is GPRC5D.

[0030] In some embodiments, the Her2 binding domain comprises a heavy chain variable region (V H ) and the three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region (V L), wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 57 or a variant thereof having three, two or one or less amino acid substitutions; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 58 or a variant thereof having three, two or one or less amino acid substitutions; the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 59 or a variant thereof having three, two or one or less amino acid substitutions; the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 60 or a variant thereof having three, two or one or less amino acid substitutions; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 61 or a variant thereof having three, two or one or less amino acid substitutions; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 62 or a variant thereof having three, two or one or less amino acid substitutions.

[0031] In some embodiments, the Her2 binding domain comprises a light chain variable domain of the amino acid sequence set forth in SEQ ID NO:64, and VH3 comprises a heavy chain variable domain of the amino acid sequence set forth in SEQ ID NO:63.

[0032] In some embodiments, the first antigen-binding domain comprises a Her2-binding domain as defined herein, the second antigen-binding domain comprises a CD3-binding domain as defined herein, and the third antigen-binding domain comprises an antigen-binding fragment of an antibody that binds to GPRC5D as described herein. In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:25, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:26, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:28.

[0033] In some embodiments, the first antigen is GPRC5D, the second antigen is GPRC5D, the third antigen is CD3, and the first antigen-binding domain and the second antigen-binding domain comprise an antigen-binding fragment of an antibody that binds to GPRC5D described herein, and the third antigen-binding domain comprises the CD3-binding domain defined herein. In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 33, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 34, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 36.

[0034] In some embodiments, the second polypeptide further comprises, in an N-terminal to C-terminal direction, a first Fc polypeptide, and / or the third polypeptide further comprises, in an N-terminal to C-terminal direction, a second Fc polypeptide, and the first Fc polypeptide and the second Fc polypeptide can combine to form a dimer.

[0035] In some embodiments, the polypeptide conjugate comprises the first antigen-binding domain and the second antigen-binding domain, and the first antigen-binding domain comprises a first Fab domain comprising: (i) a first polypeptide comprising a first heavy chain variable domain VH1 and a first CH1 domain CH1a that bind to a first antigen in an N-terminal to C-terminal direction; and (ii) a second polypeptide comprising a first light chain variable domain VL1 and a first CL domain CLa that bind to the first antigen in an N-terminal to C-terminal direction; and the second antigen-binding domain comprises (iii) a second heavy chain variable domain VH1 that binds to a second antigen in an N-terminal to C-terminal direction. and (iv) a fourth polypeptide comprising, in an N-terminal to C-terminal direction, a second light chain variable domain VH2 and a second CH1 domain CH1b that binds to a second antigen, wherein VL1 and VH1 combine to form the first antigen-binding domain, VL2 and VH2 combine to form the second antigen-binding domain, CH1a and CLa can pair, and CH1b and CLb can pair, and the binding pairing of CH1a and CLa and the binding pairing of CH1b and CLb are configured to avoid mispairing between CH1a and CLb and / or CH1b and CLa.

[0036] In some embodiments, the first antigen-binding domain comprises an antigen-binding fragment of an antibody that binds to GPRC5D as described herein. In some embodiments, the second antigen-binding domain comprises a CD3-binding domain as defined herein.

[0037] In some embodiments, the CH1b and CLb binding pair in the polypeptide conjugate has at least one non-native disulfide bond that prevents mispairing between CH1b and CLa and / or CH1a and CLb.

[0038] In some embodiments, the first CH1 / CL binding pair and the second CH1 / CL binding pair in the polypeptide conjugate are selected from CH1b / CLb and CH1a / CLa, respectively, and the first CH1 / CL binding pair is bonded via a first pair of disulfide bonds, which are non-naturally occurring, and optionally, a naturally occurring disulfide bond in the first CH1 / CL binding pair is deleted or destroyed. In some embodiments, the second CH1 / CL binding pair is formed from a second pair of disulfide bonds, which are located at different positions from the first pair of disulfide bonds, and optionally, are naturally occurring disulfide bonds.

[0039] In some embodiments, the first pair of disulfide bonds is formed by two cysteines introduced at positions selected from the group consisting of: a) heavy chain EU numbering position 126 and light chain EU numbering position 121 in the first CH1 / CL binding pair; b) heavy chain EU numbering position 173 and light chain EU numbering position 160 in the first CH1 / CL binding pair; and c) heavy chain EU numbering position 128 and light chain EU numbering position 118 in the first CH1 / CL binding pair.

[0040] In some embodiments, the naturally occurring disulfide bond is formed between heavy chain EU-numbered position 220 and light chain EU-numbered position 214. In some embodiments, the first CH1 / CL pair comprises a CH1 mutated to a cysteine ​​residue at EU-numbered position 126 and a non-cysteine ​​residue at position 220, and a CL mutated to a cysteine ​​residue at EU-numbered position 121 and a non-cysteine ​​residue at position 214.

[0041] In some embodiments, the first CH1 / CL binding pair comprises a mutation of at least one uncharged amino acid residue to a charged amino acid residue and / or a mutation of at least one charged amino acid residue to an oppositely charged amino acid residue, such that the first CH1 / CL binding pair comprises a first pair of oppositely charged residues, the first pair of oppositely charged residues promoting pairing of the first CH1 / CL binding pair. In some embodiments, the second CH1 / CL pair comprises a mutation of at least one uncharged amino acid residue to a charged amino acid residue and / or a mutation of at least one charged amino acid residue to an oppositely charged amino acid residue, such that the second CH1 / CL binding pair comprises a second pair of oppositely charged residues, the second pair of oppositely charged residues promoting pairing of the second CH1 / CL binding pair, and optionally, the first pair of oppositely charged residues and the second pair of oppositely charged residues prevent pairing of CH1a and CLb or CH1b and CLa.

[0042] In some embodiments, the first pair of oppositely charged residues and the second pair of oppositely charged residues are designed so that both CH1a and CLb are positively charged or negatively charged, and / or so that both CH1b and CLa are positively charged or negatively charged.

[0043] In some embodiments, the first pair of oppositely charged residues and / or the second pair of oppositely charged residues are introduced at heavy chain-light chain EU numbering positions selected from the group consisting of: a) heavy chain EU numbering position 183 and light chain EU numbering position 176 in the first CH1 / CL binding pair; b) heavy chain EU numbering position 183 and light chain EU numbering position 133 in the first CH1 / CL binding pair; c) heavy chain EU numbering position 147 and light chain EU numbering position 176 in the first CH1 / CL binding pair; d) heavy chain EU numbering position 141 and light chain EU numbering position 116 in the first CH1 / CL binding pair; e) heavy chain EU numbering position 126 and light chain EU numbering position 121 in the first CH1 / CL binding pair; and f) heavy chain EU numbering position 218 and light chain EU numbering position 122 in the first CH1 / CL binding pair.

[0044] In some embodiments, the pair of oppositely charged amino acid residues comprises one positively charged amino acid residue and one negatively charged amino acid residue, wherein the positively charged amino acid residue is selected from the group of lysine (K), histidine (H) and arginine (R), and / or the negatively charged amino acid residue is selected from the group of aspartic acid (D) and glutamic acid (E).

[0045] In some embodiments, the CH1b and CLb binding pair in the polypeptide conjugate comprises a first pair of non-natural disulfide bonds and a first pair of oppositely charged residues that prevent mispairing between CH1b and CLa and / or between CH1a and CLb.

[0046] In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:39, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:40, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO:38, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:37.

[0047] In some embodiments, the third polypeptide of the polypeptide complex comprises, in an N-terminal to C-terminal direction, a first Fc polypeptide, and the first polypeptide further comprises, in an N-terminal to C-terminal direction, a second Fc polypeptide.

[0048] In some embodiments, the polypeptide complex further comprises a third antigen-binding domain, optionally a Fab domain. In some embodiments, the C-terminus of one of the third antigen-binding domains is linked to the N-terminus of one of the second antigen-binding domains. In some embodiments, the third antigen-binding domain is the same as the first antigen-binding domain and comprises (i) a first fragment comprising, in an N-terminal to C-terminal direction, a first heavy chain variable domain VH1 and a first CH1 domain CH1a that binds to a first antigen, and (ii) a second fragment comprising, in an N-terminal to C-terminal direction, a first light chain variable domain VL1 and a first CL domain CLa that binds to the first antigen, wherein the C-terminus of the first fragment is linked to the N-terminus of the fourth polypeptide.

[0049] In some embodiments, the polypeptide complex comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and a fifth polypeptide, wherein, from N-terminus to C-terminus, (i) the first polypeptide comprises VH1-CH1a, (ii) the third polypeptide comprises VH2-CH1b, (iii) the fourth polypeptide comprises VH1-CH1a-linker-VL2-CLb, and (iv) the second polypeptide and the fifth polypeptide are the same and both comprise VL1-CLa.

[0050] In some embodiments, the CH1b and CLb binding pair in the polypeptide conjugate has at least one non-natural disulfide bond that prevents mispairing between CH1b and CLa and / or between CH1a and CLb. In some embodiments, the CH1b and CLb binding pair in the polypeptide conjugate has one or more introduced amino acid mutations to form at least one introduced charged amino acid residue that prevents mispairing between CH1b and CLa and / or between CH1a and CLb.

[0051] In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 43, the second polypeptide or the fifth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 44, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 42, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 41.

[0052] In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:47, the second polypeptide or the fifth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:48, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO:46, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:45.

[0053] In some embodiments, the third polypeptide of the polypeptide complex further comprises, in N-terminal to C-terminal direction, a first Fc polypeptide, and the first polypeptide comprises, in N-terminal to C-terminal direction, a second Fc polypeptide.

[0054] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide in the polypeptide conjugate is derived from IgG1, IgG2, IgG3, or IgG4.

[0055] In some embodiments, the first Fc polypeptide and the second Fc polypeptide of the polypeptide complex have different amino acid sequences and are at least designed to promote heterodimerization of the first Fc polypeptide and the second Fc polypeptide.

[0056] In some embodiments, one of the first Fc polypeptide and the second Fc polypeptide comprises a first Fc mutation and the other comprises a second Fc mutation, wherein the first Fc mutation and the second Fc mutation are a) a combination of T366W or S354C with Y349C, T366S, L368A or Y407V, b) a combination of D399K or E356K with K392D or K409D, c) a combination of E356K, E357K or D399K with K370E, K370F, K370G, K370H ... d) a combination of S364H or F405A and Y349T or T394F; e) a combination of S364H or T394F and Y394T or F405A; f) a combination of K370D or K409D and E357K or D399K; or g) a combination of L351D or L368E and L351K or T366K, wherein the amino acid positions are numbered according to the EU numbering system.

[0057] In some embodiments, the first Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO:65 or SEQ ID NO:67, and the second Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO:66.

[0058] In another aspect, the present application provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide complex described herein.

[0059] In another aspect, the present application provides a vector comprising a nucleic acid described herein.

[0060] In another aspect, the present application provides a host cell comprising a nucleic acid described herein or a vector described herein.

[0061] In another aspect, the present application provides a pharmaceutical composition comprising a polypeptide conjugate described herein or a nucleic acid described herein and a pharmaceutically acceptable vector.

[0062] In another aspect, the present application provides a conjugate comprising a polypeptide conjugate as described herein and a payload conjugated thereto, wherein the payload is selected from the group consisting of a radioactive label, a fluorescent label, an enzyme substrate label, an affinity purification tag, a tracking molecule, an anti-cancer drug, and a cytotoxic molecule.

[0063] In another aspect, the present application provides a composition comprising a polypeptide complex described herein or a conjugate described herein and a pharmaceutically acceptable carrier.

[0064] In another aspect, the present application provides a method for treating or preventing a disease, condition, or symptom, comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide conjugate described herein, a pharmaceutical composition described herein, a conjugate described herein, or a composition described herein.

[0065] In one embodiment, the disease, condition or symptom is selected from the group of cancer, immune disorders and inflammation. [Brief explanation of the drawings]

[0066] [Figure 1] 1 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to HEK293 cells expressing hGPRC5D in an antigen-binding FACS experiment. [Figure 2] 1 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to CHOS cells expressing hGPRC5D in an antigen-binding FACS experiment. [Figure 3] 1 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to MM.1R cells that naturally express GPRC5D in an antigen-binding FACS experiment. [Figure 4] 1 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to NCI-H929 cells in an antigen binding FACS experiment. [Figure 5]1 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to RPMI-8226 cells in an antigen binding FACS experiment. [Figure 6] 1 shows the cytotoxic effects of GPRC5D antibodies (ch-72C7 and GC5B596) on NCI-H929 cells that naturally express GPRC5D in an ADCC effect evaluation experiment. [Figure 7] 1 shows the cytotoxic effects of GPRC5D antibodies (ch-72C7 and GC5B596) on MM.1R cells that naturally express GPRC5D in an ADCC effect evaluation experiment. [Figure 8] 1 shows CD3×GPRC5D bispecific antibody structure A. [Figure 9] 1 shows CD3×GPRC5D bispecific antibody structure B. [Figure 10] 1 shows CD3×GPRC5D bispecific antibody structure C. [Figure 11] 1 shows CD3×GPRC5D bispecific antibody structure D. [Figure 12] 1 shows the binding of the bispecific antibody to HEK293T-hGPRC5D (human GPRC5D). [Figure 13] Binding of the bispecific antibody to HEK293T-cynoGPRC5D (monkey GPRC5D) is shown. [Figure 14] 1 shows the inhibitory effect of bispecific antibodies (22A1-5 and 22B1) on the proliferation of PBMCs against NCI-H929 cells. [Figure 15] Figure 1 shows the inhibitory effect of bispecific antibodies (22A6 and 10B1) on the proliferation of PBMCs on NCI-H929 cells. [Figure 16] 1 shows the inhibitory effect of bispecific antibodies (22A8, 10B1 and 22B1) on the proliferation of PBMCs against NCI-H929 cells. [Figure 17] 1 shows the inhibitory effect of bispecific antibodies (22A8 and 22B1) on PBMC proliferation against MM1S cells. [Figure 18] Figure 1 shows the inhibitory effect of bispecific antibodies (22A8 and 22B1) on the proliferation of PBMCs on RPMI-8226 cells. [Figure 19] 1 shows the inhibitory effect of bispecific antibodies (22A8 and 22B1) on PBMC proliferation against KMS-12-BM cells. [Figure 20] 1 shows the inhibitory effect of bispecific antibodies (22A2 and 22A8) on human myeloma NCI-H929 tumors subcutaneously transplanted into human PBMC-immune-reconstituted mice. [Figure 21] 1 shows the inhibitory effect of bispecific antibodies (22B1 and 22A8) on human myeloma NCI-H929 tumors subcutaneously transplanted into human PBMC-immune-reconstituted mice. [Figure 22(A)] The full-length sequences of each specific antibody (22A1, 22A2, 22A3, 22A4, 22A5, 22A6, 22A8, and 22B1) of the present application are shown. Figures 22(A) to 22(H) show the amino acid sequences of the polypeptide conjugates provided in the present application, as well as the VH and VL variants in the antigen-binding domains targeting GPRC5D, CD3, and Her2, as well as the CH1 and CL variants. [Figure 22(B)] Same as above. [Figure 22(C)] Same as above. [Figure 22(D)] Same as above. [Figure 22(E)] Same as above. [Figure 22(F)] Same as above. [Figure 22(G)] Same as above. [Figure 22(H)] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0067] The following description of the present application is intended to merely illustrate various embodiments of the present application. Therefore, the specific modifications discussed should not be construed as limiting the scope of the present application. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present application, and it is understood that such equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0068] definition As used herein, the term "antibody" includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A natural, intact antibody contains two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and each heavy chain contains a variable region (V H ) and the first, second, third, and optionally fourth constant regions (C H1 , C H2 , C H3 , C H4 Mammalian light chains are classified as lambda or kappa, and each light chain consists of a variable region (V L) and constant regions. Antibodies are "Y" shaped, with the tail of the Y consisting of the second and third constant regions of two heavy chains joined via disulfide bonds. Each arm of the Y contains the variable region and first constant region of a single heavy chain bound to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2, and LCDR3, and heavy chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the definitions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., December 5, 186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature, December 21-28, 342(6252):877-83 (1989); Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 1999). Interest), 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), Chapter 26, 481-514 (2015).The three CDRs are interposed between flanking segments called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of the constant region of their heavy chains. The five major antibody classes or isotypes are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0069] In this application, the numbering designating amino acid residue positions in antibody constant regions is according to EU numbering, see e.g., Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969), and the numbering designating amino acid residue positions in antibody variable regions is according to Kabat numbering, see e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). These numbering systems and the correspondence between them are taken from the IMGT scientific chart, available from the website of the international ImMunoGeneTics information system.

[0070] As used herein, the term "bivalent" refers to an antibody or antigen-binding fragment that has two antigen-binding sites. The term "monovalent" refers to an antibody or antigen-binding fragment that has only a single antigen-binding site. The term "multivalent" refers to an antibody or antigen-binding fragment that has multiple antigen-binding sites. In some embodiments, an antibody or antigen-binding fragment thereof is monovalent, bivalent, or multivalent.

[0071] As used herein, a "bispecific" antibody is an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes, which may be on the same antigen or on two different antigens.

[0072] As used herein, a "multispecific" antibody is an artificial antibody that has fragments derived from two or more different monoclonal antibodies and is capable of binding to two or more different epitopes, which may be on the same antigen or on different antigens.

[0073] As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody containing one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain the intact native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), diabodies, multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments are capable of binding to the same antigen as the parent antibody.

[0074] "Fab," with respect to an antibody, refers to the portion of an antibody consisting of a single light chain (variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0075] "Fab'" refers to a Fab fragment that includes part of the hinge region.

[0076] "F(ab')2" refers to a dimer of Fab'. With respect to antibodies, "Fv" refers to the minimum antibody fragment containing a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0077] "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a "(dsFv)2" or "(dsFv-dsFv')" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. H The moieties are linked by a peptide linker (e.g., a long flexible linker) and each of the two V L In some embodiments, the dsFv-dsFv' is bispecific, with each disulfide paired heavy and light chain having a different antigen specificity.

[0078] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other either directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0079] "Fc," with respect to an antibody (e.g., an antibody of the IgG, IgA, or IgD isotype), refers to the portion of the antibody consisting of the second and third constant domains of a first heavy chain linked via disulfide bonds to the second and third constant domains of a second heavy chain. For antibodies of the IgM and IgE isotypes, Fc further comprises a fourth constant domain. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0080] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv connected to the Fc region of an antibody.

[0081] "Camelized single domain antibody", "heavy chain antibody", or "HCAb" refers to a camelized single domain antibody consisting of two V HIt refers to antibodies that contain heavy chains and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods, December 10, 231(1-2):25-38 (1999); Muyldermans S., J Biotechnol, June, 74(4):277-302 (2001); WO94 / 04678, WO94 / 25591, U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from the Camelidae family (camels, dromedaries, and llamas). Although lacking light chains, camelized antibodies possess a robust antigen-binding repertoire (Hamers-Casterman C. et al., Nature, June 3, 363(6428):446-8 (1993); Nguyen VK. et al., Immunogenetics, April, 54(1):39-47 (2002); Nguyen VK. et al., Immunology, May, 109(1):93-101 (2003)). The variable domain of heavy-chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J, November, 21(13):3490-8, published online June 15, 2007 (2007)).

[0082] "Nanobody" refers to an antibody fragment consisting of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.

[0083] A "diabody" or "dAb" comprises a small antibody fragment with two antigen-binding sites, wherein these fragments are V or V+ on the same polypeptide chain. L V connected to the domain H Domain (V H -V L or V L -V H) (see, e.g., Holliger P. et al., Proceedings of the National Academy of Sciences, July 15, 90(14):6444-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing of the two domains on the same chain, these domains are forced to pair with complementary domains on another chain, thus generating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes). In certain embodiments, an "scFv dimer" is a diabody that targets two different V H -V L V dimerized with the moiety H -V L (linked by a peptide linker), whereby the V of one part is a bivalent diabody or bispecific scFv (BsFv). H is another part of V L to form two binding sites that may target the same antigen (or epitope) or different antigens (or epitopes). In another embodiment, an "scFv dimer" is a dimer of V L1 -V H2 (linked by a peptide linker) and associated V H1 -V L2 (linked by a peptide linker), whereby V H1 and V L1 , and V H2 and V L2 are coordinated, and each coordinated pair has a different antigen specificity.

[0084] A "domain antibody" refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. H The domains are covalently linked with peptide linkers to generate bivalent or multivalent domain antibodies. H The domains may target the same or different antigens.

[0085] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment in which a portion of the heavy and / or light chain is derived from one species and the remaining portion of the heavy and / or light chain is derived from another species. In an illustrative example, a chimeric antibody can contain a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0086] As used herein, the term "humanized" means that the antibody or antigen-binding fragment contains CDRs derived from a non-human animal, FR regions derived from a human, and, if applicable, constant regions derived from a human.

[0087] As used herein, "GPRC5D" refers to G protein-coupled receptor family C group 5 member D derived from primates (e.g., humans, monkeys), etc. In certain embodiments, GPRC5D is human GPRC5D. Exemplary sequences of human GPRC5D include the human GPRC5D protein (UniProt number Q9NZD1). Exemplary sequences of monkey GPRC5D include, for example, the rhesus monkey GPRC5D protein (UniProt number F6Y5U7) or the cynomolgus monkey GPRC5D protein (UniProt number A0A2K5W6I7). GPRC5D, a relatively new target for multiple myeloma immunotherapy, is an orphan G protein-coupled receptor of unknown function that is highly expressed in malignant bone marrow plasma cells and in hard keratinous structures, including hair shafts, nails, and the central region of the tongue (see Smith EL et al., Sci Transl Med 2019;11:eaau7746; Pillarisetti K et al., Blood 2020;135:1232-43; and Inoue S et al., J Invest Dermatol 2004;122:565-73). High expression of GPRC5D is associated with poor prognosis in multiple myeloma (see Atamaniuk J et al., Eur J Clin Invest 2012;42:953-60). GPRC5D has been used as a target for CAR-T therapy for multiple myeloma with promising preclinical results (see de Larrea CF et al., Blood Cancer Discov 2020, 1:146.) and is currently the target of the bispecific antibody JNJ-64407564 (talquetamab) in four phase I clinical trials.

[0088] As used herein, the term "CD3" refers to cluster of differentiation 3, a protein complex and T cell coreceptor involved in the activation of cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). CD3 is a complex composed of four distinct chains. In mammals, the complex includes one CD3γ chain, one CD3δ chain, and two CD3ε chains. These chains bind to the T cell receptor (TCR) and the CD3ζ chain (ζ-chain), generating activation signals within T lymphocytes. The TCR, CD3ζ, and other CD3 molecules together comprise the TCR complex. Because CD3 is required for T cell activation, drugs targeting CD3 (usually monoclonal antibodies) are being investigated as immunosuppressant therapies for cancer and other autoimmune diseases. Based on the CD3 T cell coreceptor, new anticancer therapeutic approaches are being developed, in which molecules are designed to alter costimulatory signals to support cancer cell recognition and sufficient activation by T cells.

[0089] As used herein, the term "Her2" refers to human epidermal growth factor receptor 2, also known as receptor tyrosine protein kinase erbB-2, cluster of differentiation 340, or proto-oncogene neu, and is encoded by the gene ErbB2. ErbB is an abbreviation for erythroblastic oncogene B. It belongs to the epidermal growth factor receptor family and consists of an extracellular domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. In humans, the ErbB family includes four members: ErbB1 (Her1), ErbB2 (Her2), ErbB3 (Her3), and ErbB4 (Her4). Unlike other members of the ErbB family, Her2 does not directly bind to ligands. When Her2 levels are high (e.g., in a cancer environment), its homodimerization or heterodimerization with another ErbB member can lead to Her2 activation. Amplification or overexpression of the Her2 gene plays an important role in the development and progression of some aggressive breast cancers. In recent years, Her2 protein has become an important biomarker and therapeutic target for approximately 30% of breast cancer patients.

[0090] The term "anti-GPRC5D antibody" refers to an antibody capable of specifically binding to GPRC5D (eg, human GPRC5D). The term "anti-human GPRC5D antibody" refers to an antibody capable of specifically binding to human GPRC5D.

[0091] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, for example, between an antibody and an antigen. Specific binding can be characterized by binding affinity, e.g., K D value, i.e., the ratio of the dissociation rate to the association rate (k off / k on ) is expressed as K Dcan be determined by any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). -6 M (e.g., ≦5×10 -7 M, ≤ 2 × 10 -7 M, ≤10 -7 M, ≤ 5 × 10 -8 M, ≤ 2 × 10 -8 M, ≤10 -8 M, ≤ 5 × 10 -9 M, ≤ 4 × 10 -9 M, ≤ 3 × 10 -9 M, ≤ 3 × 1 -9 M or ≦10 -9 M)'s K D The value can indicate specific binding between the antibody or antigen-binding fragment thereof and GPRC5D (eg, human GPRC5D).

[0092] With respect to amino acid sequences, a "conservative substitution" refers to the replacement of an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues having hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), acidic side chains (e.g., Asp, Glu), basic side chains (e.g., His, Lys, and Arg), or aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions generally do not significantly alter the conformational structure of a protein, thereby preserving the biological activity of the protein.

[0093] As used herein, the term "homologous" refers to a nucleic acid sequence (or its complementary strand) or amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence when optimally aligned.

[0094] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in the candidate sequence that are identical with the amino acid (or nucleic acid) residues in the reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignments for purposes of determining percent amino acid (or nucleic acid) sequence identity can be performed using, for example, BLASTN, BLASTp (available at the website of the National Center for Biotechnology Information (NCBI) in the United States; see also Altschul SF et al., Journal of Molecular Biology 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res. 25:3389-3402 (1997)), ClustalW2 (available at the website of the European Bioinformatics Institute in the United States; see also Higgins DG et al., Methods in Enzymology 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, UK ... Altschul SF et al., Journal of Molecular Biology 215:403-410 (1990); This can be achieved by publicly available tools such as the ALIGN or Megalign (DNASTAR) software, see, for example, "The Journal of Molecular Biology and Biosciences, Vol. 1, No. 1, pp. 23(21):2947-8 (2007)," and by those skilled in the art who use the default parameters provided by these tools or customize the parameters for the alignment, e.g., by selecting a suitable algorithm.

[0095] As used herein, "effector function" refers to a biological activity resulting from the binding of the Fc region of an antibody to an effector such as the C1 complex and an Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC), which is mediated by the interaction of an antibody with C1q on the C1 complex, antibody-dependent cellular cytotoxicity (ADCC), which is mediated by the binding of the Fc region of an antibody to an Fc receptor on an effector cell, and phagocytosis. Effector function can be assessed by various assays (such as Fc receptor binding assays, C1q binding assays, and cytolytic assays).

[0096] An "isolated" material has been altered by artifical means from its natural state. When an "isolated" composition or material occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not "isolated," but the polynucleotide or polypeptide would be "isolated" if it is sufficiently free from the coexisting materials of its natural state so that it exists in a substantially pure state. An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that is at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure as measured by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, or capillary electrophoresis) or chromatographic methods (such as ion exchange chromatography or reverse-phase HPLC).

[0097] As used herein, the term "vector" refers to a vehicle into which a genetic element can be operably inserted and expressed to produce the protein, RNA, or DNA encoded by the genetic element or to copy the genetic element. A vector can be used to transform, transduce, or transfect a host cell to express the genetic element carried by the vector within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as λ phage or M13 phage, and animal viruses. A vector can contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, a vector can contain an origin of replication. A vector can also contain materials to aid in cell entry, including, but not limited to, viral particles, liposomes, or protein coatings. The vector may be an expression vector or a cloning vector. The present application provides vectors (e.g., expression vectors) containing a nucleic acid sequence encoding a polypeptide complex provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0098] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide and / or vector can be introduced or has been introduced.

[0099] As used herein, "treating" a condition includes alleviating the condition, delaying the onset or rate of progression of the condition, reducing the risk of developing the condition, delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0100] As used herein, a "GPRC5D-associated" disease or condition refers to any disease or condition caused, exacerbated, or otherwise associated with increased or decreased expression or activity of GPRC5D. In some embodiments, the GPRC5D-associated disease or condition is cancer, such as myeloma. In certain embodiments, the GPRC5D-associated disease or condition is characterized by overexpression of the GPRC5D gene. In one embodiment, the GPRC5D-associated disease or condition includes, but is not limited to, GPRC5D-positive breast cancer, multiple myeloma, Waldenstrom's macroglobulinemia, endometrial cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, blood cancer, lymphoma, or malignant melanoma.

[0101] The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation and physiologically compatible with the recipient thereof.

[0102] Multispecific Polypeptide Conjugates In one aspect, the present application provides a multispecific polypeptide complex comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises a first antigen-binding domain that binds to a first antigen, and the second antigen-binding domain comprises a second antigen-binding domain that binds to a second antigen. In some embodiments, the polypeptide complex further comprises a third antigen-binding domain comprising a third antigen-binding domain that binds to a third antigen.

[0103] i. Target of the Polypeptide Complex In one embodiment, at least one of the first antigen-binding domain and the second antigen-binding domain in the polypeptide complex described herein binds to GPRC5D and comprises a GPRC5D-binding domain. In some embodiments, at least one of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain in the polypeptide complex described herein binds to GPRC5D and comprises the GPRC5D-binding domain.

[0104] GPRC5D-binding domain The GPRC5D-binding domain in the polypeptide conjugate of the present application is capable of specifically binding to GPRC5D.

[0105] In certain embodiments, the GPRC5D binding domain has a Biacore analysis of 8×10 -8 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 8×10 -9 M or less, 5×10 -9 M or less, 2×10 -9 M or less, 10 -9 M or less, 8×10 -10 M or less, 7×10 -10 M or less or 6 x 10 -10 K below M D Biacore analysis is based on surface plasmon resonance technology, see for example Murphy, M. et al., Current protocols in protein science, Chapter 19, Unit 19.14, 2006.

[0106] The binding of the GPRC5D-binding domain to human GPRC5D was measured using the "half-maximal effective concentration" (EC 50 ) value. 50The EC value can be measured by binding assays known in the art, e.g., sandwich assays such as enzyme-linked immunosorbent assays (ELISAs), flow cytometry assays, and other binding assays. In certain embodiments, the antibodies and fragments thereof provided herein have an EC value of 1 μg / ml or less, 2 μg / ml or less, 3 μg / ml or less, 4 μg / ml or less, 5 μg / ml or less, or 10 μg / ml or less, as measured by, for example, a flow cytometry assay. 50 At this value (i.e., 50% binding concentration), the antibody specifically binds to cells expressing human GPRC5D.

[0107] As used herein, "binding ability" refers to the ability of a molecule (e.g., an antibody) to bind to another molecule (e.g., an antigen). This ability can be measured using any suitable binding assay known in the art, e.g., based on binding activity to an antigen of interest. For example, the antibody of interest may be labeled so that binding activity to the antigen can be directly quantified. As another example, the binding activity of an antibody of interest (i.e., a primary antibody) to its antigen may be detected using a labeled secondary antibody (e.g., an anti-species antibody), which detects the complex of the primary antibody and its antigen by binding to the primary antibody in the complex, thereby indirectly quantifying binding activity. Labeled antibodies can be detected by, for example, enzyme-linked immunosorbent assay (ELISA, e.g., the label is an enzyme), flow cytometry (e.g., the label is fluorescent), Western blotting (e.g., the label is a fluorescent or radioligand), colorimetric analysis, chemiluminescence-based methods, etc.

[0108] In certain embodiments, the GPRC5D-binding domain in the polypeptide conjugate of the present application is derived from the antigen-binding domain of the anti-GPRC5D monoclonal antibody ch-72C7.

[0109] As used herein, "ch-72C7" refers to a monoclonal antibody having a light chain variable region containing the sequence of SEQ ID NO: 12 and a heavy chain variable region containing the sequence of SEQ ID NO: 13. ch-72C7 is a mouse-derived antibody, and its CDR sequences can be classified by methods known in the art, including, but not limited to, CDR classification according to the IMGT system or CDR classification based on the Kabat numbering system.

[0110] Table 1 below shows the CDR sequences of antibody ch-72C7 classified according to the IMGT numbering system. Table 2 below shows the CDR sequences of antibody ch-72C7 classified according to the Kabat numbering system. Table 3 below shows the amino acid sequences of the heavy and light chain variable regions of GPRC5D. [Table 1] [Table 2] [Table 3]

[0111] In certain embodiments, the GPRC5D binding domain in the polypeptide composite of the present application comprises one or more (eg, 1, 2, 3, 4, 5, or 6) CDR sequences of antibody ch-72C7.

[0112] In a specific embodiment, the GPRC5D-binding domain in the polypeptide conjugate of the present application comprises three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the three heavy chain complementarity determining regions are the heavy chain variable region (V) shown in SEQ ID NO: 13. H ) and the light chain complementarity determining region is the same as the light chain variable region (V L ) are the same as the three light chain complementarity determining regions contained within

[0113] In certain embodiments, the GPRC5D binding domain in the polypeptide complex of the present application comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs, wherein the one or more CDRs comprise an sequence selected from the group consisting of SSVSF (SEQ ID NO: 1), DTT (SEQ ID NO: 2), QQWNSHPLT (SEQ ID NO: 3), GYPFTNYW (SEQ ID NO: 4), INPSNGRT (SEQ ID NO: 5), and ARGFAY (SEQ ID NO: 6).

[0114] In certain embodiments, the GPRC5D binding domain in the polypeptide complex of the present application comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs, wherein the one or more CDRs comprise an sequence selected from the group consisting of SASSSVSFMH (SEQ ID NO: 7), DTTKLAS (SEQ ID NO: 8), QQWNSHPLT (SEQ ID NO: 3), NYWMH (SEQ ID NO: 9), EINPSNGRTNYNEKFKS (SEQ ID NO: 10), and GFAY (SEQ ID NO: 11).

[0115] In certain embodiments, the GPRC5D-binding domain in the polypeptide composite of the present application comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 1, an LCDR2 having the sequence set forth in SEQ ID NO: 2, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 3, and / or an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 4, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the GPRC5D-binding domain in the polypeptide composite of the present application comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 7, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 8, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 3, and / or an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 9, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 10, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 11.

[0116] In a specific embodiment, in the GPRC5D-binding domain of the polypeptide complex of the present application, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof having 3, 2 or 1 or less amino acid substitutions.

[0117] In a specific embodiment, in the GPRC5D-binding domain of the polypeptide complex of the present application, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof having 3, 2 or 1 or less amino acid substitutions.

[0118] CDRs are known to be responsible for antigen binding. However, it has been found that not all six CDRs are essential or inalterable. In other words, one or more CDRs in the anti-GPRC5D antibody ch-72C7 may be replaced, altered, or modified while substantially maintaining the specific binding affinity to GPRC5D.

[0119] In certain embodiments, the GPRC5D-binding domain of the polypeptide conjugate provided herein comprises the heavy chain CDR3 sequence of antibody ch-72C7. In certain embodiments, the GPRC5D-binding domain of the polypeptide conjugate provided herein comprises the heavy chain CDR3 sequence of SEQ ID NO: 6, wherein the CDR3 is numbered according to the IMGT numbering system. In certain embodiments, the GPRC5D-binding domain of the polypeptide conjugate provided herein comprises the heavy chain CDR3 sequence of SEQ ID NO: 11, wherein the CDR3 is numbered according to the Kabat numbering system.

[0120] In certain embodiments, the GPRC5D-binding domain of the polypeptide complex provided herein comprises a suitable framework region (FR) sequence, as long as the GPRC5D-binding domain is capable of specifically binding to GPRC5D. The CDR sequences provided in Table 1 or Table 2 above are obtained from mouse antibodies, but can be grafted to suitable FR sequences of any suitable species, such as mouse, human, rat, or rabbit, by suitable methods known in the art, such as recombinant techniques.

[0121] In certain embodiments, the GPRC5D-binding domain in the polypeptide composites provided herein is humanized. Humanized antibodies or antigen-binding fragments thereof are desirable because they have reduced immunogenicity in humans. Humanized antibodies have chimeric variable regions in which non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can essentially be achieved by replacing the corresponding human CDR genes in a human immunoglobulin gene with non-human (e.g., murine) CDR genes (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0122] Suitable human heavy and light chain variable domains may be selected to achieve this goal by methods known in the art. In an illustrative example, a "best-fit" approach may be used, in which a non-human (e.g., rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as a human scaffold for grafting the non-human CDR sequences (see, e.g., Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Molecular Biology 196:901). Alternatively, a framework derived from the consensus sequence of all human antibodies may be used for grafting the non-human CDRs (see, e.g., Carter et al. (1992) Proceedings of the National Academy of Sciences of the United States of America 89:4285; Presta et al. (1993) J. Immunol. 151:2623).

[0123] In certain embodiments, the humanized GPRC5D-binding domains provided herein are composed substantially entirely of human sequences, except for the CDR sequences, which are non-human. In some embodiments, the variable region FR and constant region, if present, are derived completely or substantially from human immunoglobulin sequences. The human FR sequences and human constant region sequences may be derived from different human immunoglobulin genes, e.g., the FR sequences are derived from one human antibody and the constant region is derived from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment comprises human heavy chain HFR1-4 and / or light chain LFR1-4.

[0124] In some embodiments, a human-derived FR region may contain the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues in the human FR are substituted with the corresponding residue from the parent non-human antibody. In certain embodiments, it may be necessary to make the humanized antibody or fragment thereof closely resemble the non-human parent antibody structure. In certain embodiments, the humanized GPRC5D-binding domain provided herein contains no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each of the human FR sequences, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all FRs of the heavy or light chain variable domain. In some embodiments, such amino acid residue changes may occur only in the heavy chain FR region, only in the light chain FR region, or in both chains.

[0125] This application is 1) "22Mono5JO4" comprising a heavy chain variable region (22Mono5JO4-VH) represented by the amino acid sequence of SEQ ID NO: 15 and a light chain variable region (22Mono5JO4-VL) represented by the amino acid sequence of SEQ ID NO: 14; 2) "22Mono3L7F" including a heavy chain variable region (22Mono3L7F-VH) shown in the amino acid sequence of SEQ ID NO: 17 and a light chain variable region (22Mono3L7F-VL) shown in the amino acid sequence of SEQ ID NO: 16; 3) "22Mono4DN4" comprising a heavy chain variable region (22Mono4DN4-VH) shown in the amino acid sequence of SEQ ID NO: 19 and a light chain variable region (22Mono4DN4-VL) shown in the amino acid sequence of SEQ ID NO: 18; 4) "22Mono5UQY" comprising a heavy chain variable region (22Mono5UQY-VH) represented by the amino acid sequence of SEQ ID NO: 21 and a light chain variable region (22Mono5UQY-VL) represented by the amino acid sequence of SEQ ID NO: 20; Further provided are exemplary humanized GPRC5D binding domains derived from ch-72C7, including: [Table 4]

[0126] In certain embodiments, the application further provides a humanized GPRC5D-binding domain comprising an HFR1, HFR2, HFR3, and / or HFR4 sequence contained in a heavy chain variable region selected from the group consisting of 22Mono5JO4-VH (SEQ ID NO: 15), 22Mono3L7F-VH (SEQ ID NO: 17), 22Mono4DN4-VH (SEQ ID NO: 19), and 22Mono5UQY-VH (SEQ ID NO: 21).

[0127] In certain embodiments, the present application further provides a humanized GPRC5D binding domain comprising an LFR1, LFR2, LFR3 and / or LFR4 sequence contained in a light chain variable region, wherein the light chain variable region is selected from the group consisting of 22Mono5JO4-VL (SEQ ID NO: 14), 22Mono3L7F-VL (SEQ ID NO: 16), 22Mono4DN4-VL (SEQ ID NO: 18) and 22Mono5UQY-VL (SEQ ID NO: 20).

[0128] In certain embodiments, the humanized GPRC5D binding domain provided herein comprises a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 21, and / or a light chain variable domain sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20.

[0129] These exemplary humanized GPRC5D-binding domains retain specific binding ability or affinity to GPRC5D and are at least equivalent to, and even superior to, the parent murine antibody ch-72C7 in these respects. For example, data are provided in Examples 4 and 5.

[0130] In some embodiments, the GPRC5D-binding domain in the polypeptide complexes provided herein comprises all or a portion of a heavy chain variable domain and / or all or a portion of a light chain variable domain. In one embodiment, the GPRC5D-binding domain in the polypeptide complexes provided herein is a single-domain antibody consisting of all or a portion of a heavy chain variable domain provided herein. Details of such single-domain antibodies are available in the art (see, e.g., U.S. Patent No. 6,248,516).

[0131] Binding domains that target immunostimulatory antigens In some embodiments, the other of the first antigen-binding domain and the second antigen-binding domain in the polypeptide conjugate described herein binds to an antigen different from GPRC5D, hi some embodiments, the antigen different from GPRC5D is an immunostimulatory antigen.

[0132] Examples of immune stimulatory antigens include CD2, CD3, CD7, CD16, CD27, CD30, CD70, CD83, CD28, CD80 (B7-1), CD86 (B7-2), CD40, CD40L (CD154), CD47, CD122, CD137, CD137L, OX40 (CD134), OX40L (CD252), NKG2C, 4-1BB, LIGHT, PVRIG, SLAMF7, HVEM, BAFFR, ICAM-1, 2B4, LFA-1, GITR, ICOS (CD278), and ICOSLG. (CD275), LAG3 (CD223), A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), BTLA, CD160, CTLA-4 (CD152), IDO1, IDO2, TDO, KIR, LAIR-1, NOX2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, SIGLEC-7 (CD328), TIGIT, PVR (CD155), TGFβ, SIGLEC9 (CD329), or any combination thereof.

[0133] In some embodiments, the immunostimulatory antigen is CD3. In some embodiments, one of the first and second antigen-binding domains binds to GPRC5D and the other binds to CD3.

[0134] In some embodiments, the antigen-binding domain of the GPRC5D comprises any anti-GPRC5D antibody or antigen-binding fragment of anti-GPRC5D provided in the present application.

[0135] In some embodiments, the polypeptide conjugate comprises a CD3 binding domain. In some embodiments, the CD3 binding domain comprises a heavy chain variable region (V) as set forth in SEQ ID NO: 55. H ) and the light chain variable region (V L ) derived from an antibody having

[0136] In some specific embodiments, the CD3 binding domain has the CDR sequences or VH / VL sequences shown in Table 5 below. [Table 5]

[0137] In a specific embodiment, the CD3 binding domain comprises three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the three heavy chain complementarity determining regions are the heavy chain variable region (V) shown in SEQ ID NO: 55. H ) and the light chain complementarity determining region is the same as the light chain variable region (V) shown in SEQ ID NO: 56. L ) is the same as the light chain complementarity determining region contained within.

[0138] In certain embodiments, the CD3 binding domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs, wherein the one or more CDRs comprise a sequence selected from the group consisting of an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 49, an HCDR2 containing the sequence set forth in SEQ ID NO: 50, and an HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 51, and / or an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 52, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 53, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 54.

[0139] In certain embodiments, the CD3 binding domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs, wherein the one or more CDRs comprise a sequence selected from the group consisting of an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 92, an HCDR2 containing the sequence set forth in SEQ ID NO: 93, and an HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 94, and / or an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 95, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 96, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 54.

[0140] In a specific embodiment, the CD3 binding domain comprises an HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 92, an HCDR2 containing the sequence set forth in SEQ ID NO: 50 or SEQ ID NO: 93, and an HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 51 or SEQ ID NO: 94, and / or an LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 52 or SEQ ID NO: 95, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 96, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 54.

[0141] Binding domains targeting other tumor-associated antigens In some embodiments, the other of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain in the polypeptide conjugate described herein binds to a disease-associated antigen, which in some embodiments is a tumor-associated antigen, an immune disease-associated antigen, or an inflammatory disease-associated antigen.

[0142] Tumor-associated antigens include CD19, CD20, CD38, CD30, Her2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high-molecular-weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, 4-1BB, 5T4, adenocarcinoma antigen, fetoprotein, BAFF, B lymphoma cell, C242 antigen, carbonic anhydrase 9 (CA-IX), c-MET, CCR4, CD152, CD19, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, and CD44. v6, CD51, CD52, CD56, CD74, CD80, CNTO888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, These include, but are not limited to, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucoprotein CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin-C, TGF β2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, and the like.

[0143] In some embodiments, the tumor-associated antigen is Her2. In some embodiments, the polypeptide conjugate includes a Her2 binding domain.

[0144] In some embodiments, the Her2 binding domain comprises the heavy chain variable region (V) set forth in SEQ ID NO: 63. H) and the light chain variable region (V L ) derived from an antibody having

[0145] In some specific embodiments, the Her2 binding domain has the CDR sequences or VH / VL sequences shown in Table 4 below. [Table 6]

[0146] In a specific embodiment, the Her2 binding domain comprises three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the three heavy chain complementarity determining regions are the heavy chain variable region (V) shown in SEQ ID NO: 63. H ) and the light chain complementarity determining region is the same as the light chain variable region (V) shown in SEQ ID NO: 64. L ) is the same as the light chain complementarity determining region contained within.

[0147] In certain embodiments, the Her2 binding domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs, wherein the one or more CDRs comprise a sequence selected from the group consisting of an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 57, an HCDR2 containing the sequence set forth in SEQ ID NO: 58, and an HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 59, and / or an LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 60, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 61, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 62.

[0148] ii. Polypeptide complex structure The polypeptide complexes provided herein may be in a variety of suitable forms, hi some embodiments, the polypeptide complexes provided herein contain a DICAD domain.

[0149] Structure of a DICAD-containing polypeptide complex. In some embodiments, the first antigen domain and the second antigen domain in the polypeptide conjugate of the present application constitute a single DICAD domain. As used herein, the term "DICAD" refers collectively to a "disulfide and charge adjusted diabody," which incorporates a diabody structure at the VH-VL interface through covalent bonds and electrostatic charges, as disclosed, for example, in PCT application WO2019 / 120245, which is incorporated herein by reference in its entirety. Such a DICAD structure (1) preserves the affinity, avidity, potency, and other properties of each individual targeting domain; (2) provides greater stability and less aggregation than other antibodies; and (3) is easier to express and purify.

[0150] Specifically, the DICAD domain comprises: (i) a first polypeptide comprising, in an N-terminal to C-terminal direction, a first light chain variable domain VL1 that binds to a first antigen and a second heavy chain variable domain VH2 that binds to a second antigen, wherein VL1 and VH2 are linked directly or via a first linker; and (ii) a second polypeptide comprising, in an N-terminal to C-terminal direction, a second light chain variable domain VL2 that binds to a second antigen and a first heavy chain variable domain VH1 that binds to the first antigen, wherein VL2 and VH1 are linked directly or via a second linker, wherein VL1 and VH1 bind to the first antigen-binding domain, and VL2 and VH2 bind to the second antigen-binding domain.

[0151] In some embodiments, the C-terminus of the VL1 is linked to the N-terminus of the VH2, and the C-terminus of the VL2 is linked to the N-terminus of the VH1. In some embodiments, the C-terminus of the VH1 is linked to the N-terminus of the VL2, and the C-terminus of the VH2 is linked to the N-terminus of the VL1.

[0152] In some embodiments, VH2 and VL1 and / or VH1 and VL2 are covalently linked directly or indirectly (e.g., via a linker, such as a peptide linker). In some embodiments, the linker comprises a peptide linker. As used herein, the term "peptide linker" refers to any suitable polypeptide that connects two entities to form a single molecule or maintains a sufficiently close association of the two entities without affecting the biological activity of each of the two entities. The linker may consist of amino acid residues linked via peptide bonds and may optionally further comprise one or more unnatural amino acids. Any suitable polypeptide can be used as a linker. In some embodiments, the linker may be composed of a majority of sterically unhindered amino acids, such as glycine and alanine. In some embodiments, the linker is polyglycine, polyalanine, a combination of glycine and alanine (e.g., poly(Gly-Ala)), or a combination of glycine and serine (e.g., poly(Gly-Ser)). In some embodiments, the peptide linker comprises the amino acid sequence of RTVAA (SEQ ID NO: 74).

[0153] In some embodiments, VH1 is linked to VL2 via a first peptide linker, and VL1 is linked to VH2 via a second peptide linker. In some embodiments, the first peptide linker and the second peptide linker each independently comprise 5 to 9 amino acid residues. In some embodiments, the first peptide linker and the second peptide linker each independently comprise the amino acid sequence RTVAA (SEQ ID NO: 74).

[0154] In some embodiments, the DICADs may further comprise one or more modifications useful for promoting binding between corresponding heavy and light chains, e.g., between VH1 and VL1 or between VH2 and VL2. In some embodiments, non-natural covalent bonds (e.g., non-natural disulfide bonds) and / or electrostatic interactions may be introduced at the VH1-VL1 and / or VH2-VL2 interaction interfaces.

[0155] (a) Disulfide bond introduced into VH / VL In some embodiments, one of the first and second antigen-binding domains in the polypeptide conjugate comprises a first non-natural covalent bond, or optionally, the other does not comprise a non-natural covalent bond or comprises a second non-natural covalent bond that is different from the first non-natural covalent bond, e.g., the second non-natural covalent bond is formed between two amino acid residues that are different from the two amino acid residues that form the first non-natural covalent bond.

[0156] In some embodiments, the first non-natural covalent bond can be a non-natural disulfide bond. In some embodiments, the first non-natural disulfide bond is formed between two introduced cysteine ​​residues. In such embodiments, at least one of the first antigen-binding domain and the second antigen-binding domain is a disulfide-bond-stabilized Fv. Analysis of antibody crystal structures revealed that cysteine ​​mutations can be introduced into a relatively conserved sequence at the VL-VH interface to form a disulfide bond between the VL and VH, thereby covalently connecting them. The covalent bond between the VL and VH significantly improved the stability of the antibody. The first dsFv (disulfide Fv) was constructed by introducing disulfide bonds at the VH-VL interface through covalent interactions between cysteine ​​residues in the CDRs of each fragment (see Glockshuber, R. et al., Comparison of Strategies for Stabilizing Immunoglobulin Fv Fragments (1990) Biochemistry, 291362-1367). Although this method did not affect the antibody activity, it is difficult to make this method a universal solution for constructing various antibodies because detailed structural information of the CDRs of the original antibody is required for "customized" design to avoid interference with the antigen recognition / binding ability of the CDRs. To ensure the wide application of this method, amino acids at selected sites in the conserved FRs must be involved in the construction of dsFvs.

[0157] Since 1993, several VH-VL pairing sites suitable for covalent bond formation have been discovered, such as VH44-VL100, VH105-VL43, VH100b-VL49, VH100-VL150, and VH101-VL46, where the numbering is according to Kabat numbering (Reiter, Y. et al., Stabilization of Fv fragments of recombinant immunotoxins by disulfide bonds incorporated into conserved framework regions (1994) Biochemistry, 335451-5459, Jun. 2004). (See, for example, G, SH et al., Design of Interchain Disulfide Bonds in the Framework Region of the Fv Fragment of Monoclonal Antibody B3 (1994) Proteins, Structure, Function, Genes, 19, 35-47; Glockshuber, R. et al., Comparison of Strategies for Stabilizing Immunoglobulin Fv Fragments (1990) Biochemistry, 291362-1367; and Zhu, Z. et al., Remodeling of Domain Interfaces to Promote Heterodimer Formation (1997) Protein Science, 6, 781-788.) Among these, VH44-VL100 and VH105-VL43 are more widely used because they are superior to the others in many aspects (protein expression level, monolayer rate, Tm, affinity, etc.), although to different degrees.

[0158] In some embodiments, one of the first and second antigen-binding domains in a DICAD described herein comprises a first non-native disulfide bond, and in such embodiments, the other of the first and second antigen-binding domains does not comprise a disulfide bond or comprises a second non-native disulfide bond that is different from the first non-native disulfide bond.

[0159] In some embodiments, the first antigen-binding domain in the DICAD comprises a first non-native disulfide bond, and the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 has a first cysteine ​​substitution in a FR, and the VH1 has a second cysteine ​​substitution in a FR, and the first and second cysteines form a disulfide bond. In some embodiments, the first and second cysteines are located in the FR2 region of VL1 and the FR4 region of VH1, respectively.

[0160] In some embodiments, the first and second cysteines are at position 100 in VL1 and position 44 in VH1, respectively, or at position 43 in VL1 and position 105 in VH1, respectively, or at position 49 in VL1 and position 100 in VH1, respectively, or at position 50 in VL1 and position 100 in VH1, respectively, or at position 46 in VL1 and position 101 in VH1, respectively, wherein the numbering system is according to Kabat numbering. In some embodiments, the first and second cysteines are selected from the group: 100C in VL1 and 44C in VH1, 43C in VL1 and 105C in VH1, 49C in VL1 and 100bC in VH1, 50C in VL1 and 100C in VH1, or 46C in VL1 and 101C in VH1, wherein the numbering is according to Kabat numbering.

[0161] In some embodiments, the second antigen-binding domain of the DICAD does not contain any introduced non-native disulfide bond. In some embodiments, the native disulfide bond in the second antigen-binding domain of the DICAD is cleaved. In some other embodiments, the second antigen-binding domain of the DICAD contains a second non-native disulfide bond different from the first non-native disulfide bond, and the VL2 and VH2 are covalently linked via the second non-native disulfide bond. In some embodiments, the second non-native disulfide bond is formed between two non-native cysteine ​​residues in VL2 and VH2, respectively. In some embodiments, the VL2 has a third cysteine ​​substitution in a FR, and the VH2 has a fourth cysteine ​​substitution in a FR, and the third and fourth cysteines form a disulfide bond. In some embodiments, the third and fourth cysteines are located in the FR2 region of VL2 and the FR4 region of VH2, respectively. In some embodiments, the third and fourth cysteines are at position 100 in VL2 and position 44 in VH2, respectively, or at position 43 in VL2 and position 105 in VH2, respectively, or at position 49 in VL2 and position 100 in VH2, respectively, or at position 50 in VL2 and position 100 in VH2, respectively, or at position 46 in VL2 and position 101 in VH2, respectively, wherein the numbering system is according to Kabat numbering. In some embodiments, the third and fourth cysteines are selected from the group of 100C in VL2 and 44C in VH2, 43C in VL2 and 105C in VH2, 49C in VL2 and 100bC in VH2, 50C in VL2 and 100C in VH2, or 46C in VL2 and 101C in VH2, wherein the numbering is according to Kabat numbering.

[0162] In some specific embodiments, the first cysteine ​​in the VL1 is located at 100C in VL1 and the second cysteine ​​in the VH1 is located at 44C in VH1. In some embodiments, the disulfide bond is formed between 100C in VL1 and 44C in VH1. In some specific embodiments, the second antigen-binding domain in the DICAD does not contain a non-native disulfide bond.

[0163] (b) Charged amino acid replacement in VH / VL Strategies for enhancing the stable binding of corresponding VH and VL in bispecific antibodies by introducing charged amino acids are known in the art. Tan et al. successfully influenced the stability of scFv (single-chain FV variant) by adjusting amino acids at the VH-VL interface based on electrostatic properties (see Philip H. Tan et al., "Contribution of highly conserved VH-VL hydrogen-bonding interactions to scFv folding stability and refolding efficiency," Journal of Biophysics, September 1998, 75(3):1473-1482). Subsequently, Igawa et al. modified the method to modify scDb. To improve the uniformity of the product, two pairs of Q39-Q38 in the 4V fragment were replaced with amino acids bearing appropriate electrostatic charges to promote or inhibit specific isoforms (see Igawa T et al., Engineering the VH / VL Interface to Promote Selective Expression and Inhibit Conformational Isomerization of Thrombopoietin Receptor Agonist Single-Chain Diabodies, Protein Engineering Design and Selection, August 2010, 23(8):667-77, and WO2006106905A1). Gunasekaran et al. at Amgen further investigated this method and incorporated it into the modification of antibody Fab arms. The combination of electrostatic steering at the CH1-CL interface and modification of VH-VL at positions 38-39 promoted specific interactions between CH1-VH and CL-VL (see Gunasekaran K et al., "Promotion of antibody Fc heterodimer formation by electrostatic steering: application to bispecific molecules and monovalent IgG," Journal of Biochemistry, June 18, 2010, 285(25):19637-46; Liu Z et al., "A novel antibody engineering strategy for generating monovalent bispecific heterodimeric IgG antibodies via electrostatic steering," Journal of Biochemistry, March 20, 2015, 290(12):7535-62). These methods enabled each VH of the bispecific antibody to interact with the corresponding VL, resulting in a bispecific antibody capable of simultaneously binding to two antigens.

[0164] Therefore, in some embodiments, in addition to introducing non-native disulfide bonds, the DICAD in the polypeptide conjugates provided herein also alters the electrostatic steering of specific regions, thereby minimizing unwanted non-specific interactions. Modifications that introduce electrostatic interactions can improve the pharmacokinetic properties of the polypeptide conjugates, help remove bottlenecks in the downstream development process, and increase the probability of successful development of the polypeptide conjugates of the present application.

[0165] In some embodiments, the DICAD in the polypeptide conjugates provided herein promotes VH1 / VL1 pairing and / or VH2 / VL2 pairing by introducing electrostatic interactions into the first antigen-binding domain and / or the second antigen-binding domain.

[0166] In some embodiments, two oppositely charged amino acid residues are introduced into the VL1 and VH1, thereby promoting electrostatic interactions between VL1 and VH1, and in certain such embodiments, no such oppositely charged amino acid residues are introduced between VL2 and VH2.

[0167] In some other embodiments, two oppositely charged amino acid residues are introduced into the VL2 and VH2, thereby promoting electrostatic interactions between VL2 and VH2, and in certain such embodiments, no oppositely charged amino acid residues are introduced between VL1 and VH1.

[0168] In some other embodiments, the DICADs described herein are modified to introduce a first pair of oppositely charged amino acid residues that promote electrostatic interactions between VL1 and VH1, and a second pair of oppositely charged amino acid residues that promote electrostatic interactions between VL2 and VH2, thereby reducing mispairing between VH1 and VL2 and between VH2 and VL1 (e.g., due to electrostatic repulsion). For example, the charged residues introduced into VL2 and VH1 are similarly charged amino acid residues, and / or the charged residues introduced into VH2 and VL1 are similarly charged amino acid residues, thereby reducing mispairing between VL2 and VH1 and between VL1 and VH2.

[0169] In some embodiments, the oppositely charged residues are made up of positively charged and negatively charged amino acid residues.

[0170] In some embodiments, the first pair of oppositely charged amino acid residues comprises a negatively charged residue in VL1 and a positively charged residue in VH1, hi some embodiments, the second pair of oppositely charged amino acid residues comprises a positively charged residue in VL1 and a negatively charged residue in VH1.

[0171] In some other embodiments, the first pair of oppositely charged amino acid residues comprises a positively charged residue in VL1 and a negatively charged residue in VH1.

[0172] In some embodiments, the negatively charged amino acid may be selected from aspartic acid (D) or glutamic acid (E). In some embodiments, the positively charged amino acid may be selected from lysine (K), histidine (H), or arginine (R).

[0173] In some embodiments, the introduced charged residues in VL1 or VL2 are in a FR (e.g., FR2). In some embodiments, the introduced charged residues in VH1 or VH2 are in a FR (e.g., FR2).

[0174] In some embodiments, a first pair of oppositely charged amino acid residues is introduced into the VL1 and VH1 to replace Q38 in VL1 and Q39 in VH1, or Q40 in VL1 and Q39 in VH1, or Q37 in VL1 and Q39 in VH1, respectively, where numbering is according to Kabat numbering.

[0175] In some embodiments, a second pair of oppositely charged amino acid residues is further introduced into said VL2 and VH2 to replace Q40 in VL2 and Q39 in VH2, or Q40 in VL2 and Q39 in VH2, or Q37 in VL2 and Q39 in VH2, respectively, where numbering is according to Kabat numbering. In such embodiments, the introduced charged residues in VL2 and VH1 are similarly charged amino acid residues and / or the introduced charged residues in VH2 and VL1 are similarly charged amino acid residues, thereby reducing mispairing between VL2 and VH1 or VL1 and VH2.

[0176] In some specific embodiments, the first pair of oppositely charged amino acid residues comprises Q37K in VL1 and Q39D in VH1, respectively, where numbering is according to Kabat numbering, hi some specific embodiments, the second pair of oppositely charged amino acid residues comprises Q39D in VH2 and Q40K in VL2, respectively, where numbering is according to Kabat numbering.

[0177] The introduction of positively or negatively charged amino acids into antibodies is known in the art.

[0178] In some embodiments, a disulfide bond is introduced into VH1 and VL1, and charged amino acids are also introduced. In some embodiments, in the first antigen-binding domain, the first cysteine ​​introduced into VL1 and the second cysteine ​​introduced into VH1 are selected from the group consisting of 100C in VL1 and 44C in VH1, 43C in VL1 and 105C in VH1, 49C in VL1 and 100bC in VH1, 50C in VL1 and 100C in VH1, or 46C in VL1 and 101C in VH1, and the first pair of introduced oppositely charged amino acid residues replaces Q38 in VL1 and Q39 in VH1, or Q40 in VL1 and Q39 in VH1, or Q37 in VL1 and Q39 in VH1, respectively, wherein the numbering is according to Kabat numbering.

[0179] Polypeptide complex of structure A In some embodiments, the polypeptide complex of the present application comprises a first antigen-binding domain and a second antigen-binding domain, wherein at least one of the first antigen-binding domain and the second antigen-binding domain binds to GPRC5D and comprises a GPRC5D-binding domain provided in the present application.

[0180] In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain described herein binds to GPRC5D and comprises a GPRC5D-binding domain provided herein, and the other binds to an antigen different from GPRC5D. In some embodiments, the antigen different from GPRC5D is an immunostimulatory antigen, and optionally, the immunostimulatory antigen is CD3.

[0181] In some embodiments, one of the first and second antigen-binding domains comprises a GPRC5D-binding domain provided herein, and the other comprises a CD3-binding domain.

[0182] In some specific embodiments, the first antigen targeted by the first antigen-binding domain in the DICAD is GPRC5D. In certain embodiments, the VL1 in the first antigen-binding domain contains the mutation P100C and / or Q37K, and the VH1 contains the mutation G44C and / or Q39D, where the numbering is according to Kabat numbering. In certain embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 103. In certain embodiments, the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 104.

[0183] In certain such embodiments, the second antigen targeted by the second antigen-binding domain in the DICAD is CD3. In certain embodiments, VL2 and VH2 in the second antigen-binding domain contain no mutations. In certain embodiments, the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 56. In certain embodiments, the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 108. In certain specific embodiments, the amino acid sequence of the first polypeptide in the DICAD comprises the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence of the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, VL2 in the second antigen-binding domain contains the mutation Q40K and VH2 contains the mutation Q39D, the numbering being according to Kabat numbering. In certain embodiments, the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 106, and the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 107.

[0184] In some embodiments, the polypeptide complex of the present application further comprises an Fc polypeptide comprising a first Fc polypeptide and a second Fc polypeptide. In some embodiments, the first Fc polypeptide and the Fc polypeptide are the same or different. In some embodiments, the first Fc polypeptide and the second Fc polypeptide can combine to form a dimer. In some embodiments, the second polypeptide of the polypeptide complex further comprises the first Fc polypeptide at its N-terminus. In some embodiments, the polypeptide complex further comprises a third polypeptide comprising the second Fc polypeptide at its N-terminus.

[0185] In some specific embodiments, the polypeptide conjugate has the structure shown in FIG.

[0186] In some embodiments, the first antigen targeted by the polypeptide conjugate is GPRC5D and the second antigen is CD3. In some specific embodiments, the polypeptide conjugate comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 22 and a second polypeptide having the amino acid sequence set forth in SEQ ID NO: 23. In some specific embodiments, the polypeptide conjugate further comprises a first Fc polypeptide and a second Fc polypeptide, and comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 22, a second polypeptide having the amino acid sequence set forth in SEQ ID NO: 75, and a third polypeptide having the amino acid sequence set forth in SEQ ID NO: 24.

[0187] Polypeptide complex of structure B In some embodiments, the polypeptide complex comprises a first antigen-binding domain and a second antigen-binding domain that constitute a DICAD domain provided in the present application, and further comprises a third antigen-binding domain, optionally wherein the third antigen-binding domain comprises a Fab domain.

[0188] In some embodiments, the third antigen-binding domain in the polypeptide complex is a Fab domain, and the Fab domain comprises, in an N-terminal to C-terminal direction, a third polypeptide comprising a third heavy chain variable domain VH3 and a CH1 Domain that binds to a third antigen, and a fourth polypeptide comprising a third light chain variable domain VL3 and a CL Domain that binds to the third antigen, where VL3 and VH3 combine to form the third antigen-binding domain.

[0189] In some embodiments, the polypeptide complex of the present application further comprises an Fc polypeptide comprising a first Fc polypeptide and a second Fc polypeptide. In some embodiments, the first Fc polypeptide and the Fc polypeptide are the same or different. In some embodiments, the first Fc polypeptide and the second Fc polypeptide can combine to form a dimer. In some embodiments, the second polypeptide of the polypeptide complex further comprises the first Fc polypeptide at its N-terminus. In some embodiments, the polypeptide complex further comprises a third polypeptide comprising the second Fc polypeptide at its N-terminus.

[0190] In some specific embodiments, the polypeptide conjugate has the structure shown in FIG.

[0191] In some embodiments, the first antigen targeted by the polypeptide conjugate is GPRC5D, the second antigen is GPRC5D, and the third antigen is CD3. In certain embodiments, the first antigen targeted by the first antigen-binding domain in the DICAD is GPRC5D. In certain embodiments, the VL1 in the first antigen-binding domain contains mutations P100C and Q37K, and the VH1 contains mutations G44C and Q39D, where the numbering is according to Kabat numbering. In certain embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 103. In certain embodiments, the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 104.

[0192] In some specific embodiments, the second antigen targeted by the second antigen-binding domain in the DICAD is also GPRC5D, wherein VH2 does not contain disulfide bonds or charge mutations, and the numbering is according to Kabat numbering. In certain embodiments, the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 105.

[0193] In certain such embodiments, the third antigen targeted by the third antigen-binding domain is CD3, and the third antigen-binding domain comprises a Fab domain. In certain such embodiments, the VL3 in the third antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 56. In certain embodiments, the VH3 comprises the amino acid sequence set forth in SEQ ID NO: 108.

[0194] In certain such embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 33, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 34, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 36. In some specific embodiments, the polypeptide complex further comprises a first Fc polypeptide and a second Fc polypeptide, and comprises the amino acid sequences set forth in SEQ ID NO: 33, SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 36.

[0195] In some embodiments, the first antigen targeted by the polypeptide conjugate is Her2, the second antigen is CD3, and the third antigen is GPRC5D. In some embodiments, one of the first and second antigen-binding domains comprises a Her2-binding domain provided herein, and the other comprises a CD3-binding domain.

[0196] In certain embodiments, the first antigen targeted by the first antigen-binding domain in the DICAD is Her2. In certain embodiments, the VL1 in the first antigen-binding domain contains mutations Q38D and Q100C, and the VH1 contains mutations Q39K and G44C. In certain embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 109. In certain embodiments, the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 110.

[0197] In certain such embodiments, the second antigen targeted by the second antigen-binding domain in the DICAD is CD3, wherein VL2 contains the mutation Q40K and VH2 contains the mutation Q39D, and the numbering is according to Kabat numbering. In certain embodiments, the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 106. In certain embodiments, the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 107.

[0198] In certain such embodiments, the third antigen targeted by the third antigen-binding domain is GPRC5D and the third antigen-binding domain comprises a Fab domain. In certain such embodiments, the VL3 in the third antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the VH3 comprises the amino acid sequence set forth in SEQ ID NO: 105.

[0199] In certain such embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 25, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 26, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 27, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 28. In some specific embodiments, the polypeptide complex further comprises a first Fc polypeptide and a second Fc polypeptide, and comprises a first polypeptide of the amino acid sequence set forth in SEQ ID NO: 25, a second polypeptide of the amino acid sequence set forth in SEQ ID NO: 76, a third polypeptide of the amino acid sequence set forth in SEQ ID NO: 77, and a fourth polypeptide of the amino acid sequence set forth in SEQ ID NO: 28.

[0200] In some embodiments, the first antigen targeted by the polypeptide conjugate is GPRC5D, the second antigen is CD3, and the third antigen is GPRC5D. In certain such embodiments, the first antigen targeted by the first antigen-binding domain in DICAD is GPRC5D, and its VL1 contains mutations P100C and Q37K, and its VH1 contains mutations G44C and Q39D. In certain embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 103. In certain embodiments, the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 104.

[0201] In certain such embodiments, the second antigen targeted by the second antigen-binding domain in the DICAD is CD3, wherein VL2 contains the mutation Q40K and VH2 contains the mutation Q39D, and the numbering is according to Kabat numbering. In certain embodiments, the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 106. In certain embodiments, the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 107.

[0202] In certain such embodiments, the third antigen targeted by the third antigen-binding domain is GPRC5D, and the third antigen-binding domain comprises a Fab domain. In certain such embodiments, the VL3 in the third antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the VH3 comprises the amino acid sequence set forth in SEQ ID NO: 105. In certain such embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 30, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 31, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 32. In some specific embodiments, the polypeptide complex further comprises a first Fc polypeptide and a second Fc polypeptide, and comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 29, a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 78, a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 79, and a fourth polypeptide having the amino acid sequence set forth in SEQ ID NO: 32.

[0203] Polypeptide complexes containing at least two Fab domains: Structure C In some embodiments, the polypeptide complex comprises a first antigen-binding domain and the second antigen-binding domain, wherein the first antigen-binding domain comprises a first Fab domain and the second antigen-binding domain comprises a second Fab domain.

[0204] In some embodiments, the first antigen-binding domain comprises a first Fab domain comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, in the N-terminal to C-terminal direction, a first heavy chain variable domain VH1 and a first CH1 domain CH1a that bind to a first antigen, and the second polypeptide comprises, in the N-terminal to C-terminal direction, a first light chain variable domain VL1 and a first CL domain CLa that bind to the first antigen. In some embodiments, the second antigen-binding domain comprises a second Fab domain comprising a third polypeptide and a fourth polypeptide, wherein the third polypeptide comprises, in the N-terminal to C-terminal direction, a second heavy chain variable domain VH2 and a second CH1 domain CH1b that bind to a second antigen, and the fourth polypeptide comprises, in the N-terminal to C-terminal direction, a second light chain variable domain VL2 and a second CL domain CLb that bind to the second antigen.

[0205] In some embodiments, the polypeptide complex of the present application further comprises an Fc polypeptide comprising a first Fc polypeptide and a second Fc polypeptide. In some embodiments, the first Fc polypeptide and the Fc polypeptide are the same or different. In some embodiments, the first Fc polypeptide and the second Fc polypeptide can combine to form a dimer. In some embodiments, the second polypeptide of the polypeptide complex further comprises the first Fc polypeptide at its N-terminus. In some embodiments, the polypeptide complex further comprises a third polypeptide comprising the second Fc polypeptide at its N-terminus.

[0206] In some specific embodiments, the polypeptide conjugate has the structure shown in FIG.

[0207] In some embodiments, the VL1 and VH1 combine to form the first antigen-binding domain, and the VL2 and VH2 combine to form the second antigen-binding domain.

[0208] In some embodiments, CH1a and CLa can pair, CH1b and CLb can pair, and the binding pairs of CH1a and CLa and CH1b and CLb are configured to avoid mispairing between CH1a and CLb and / or CH1b and CLa.

[0209] (a) Introduction of a disulfide bond into the CH1 / CL region In some embodiments, at least one of the CH1b and CLb binding pair and the CH1a and CLa binding pair has at least one non-native disulfide bond that prevents mispairing between CH1b and CLa and / or CH1a and CLb.

[0210] In some embodiments, the first CH1 / CL binding pair and the second CH1 / CL binding pair are selected from a CH1b and CLb binding pair and a CH1a and CLa binding pair, wherein the first CH1 / CL binding pair is linked via a first pair of disulfide bonds. In some embodiments, the first pair of disulfide bonds is non-naturally occurring. In some embodiments, the original, naturally occurring disulfide bond in the first CH1 / CL binding pair is deleted or disrupted (e.g., disrupted by mutating the cysteine ​​residues that form the natural disulfide bond).

[0211] In some embodiments, the second CH1 / CL binding pair is formed by a second pair of disulfide bonds, and the second pair of disulfide bonds are in different positions than the first pair of disulfide bonds. In some embodiments, the second pair of disulfide bonds formed in the second CH1 / CL binding pair are native disulfide bonds or non-native disulfide bonds.

[0212] In some embodiments, the first pair of disulfide bonds is formed by two cysteine ​​residues introduced into heavy-light chain positions (numbered according to the EU numbering system) selected from the group consisting of: a) heavy chain position 134 and light chain position 116; b) heavy chain position 141 and light chain position 116; c) heavy chain position 128 and light chain position 118; d) heavy chain position 126 and light chain position 121; e) heavy chain position 127 and light chain position 121; f) heavy chain position 126 and light chain position 124; g) heavy chain position 170 and light chain position 162; h) heavy chain position 171 and light chain position 162; i) heavy chain position 173 and light chain position 162.

[0213] In some embodiments, the first pair of disulfide bonds is formed by two cysteine ​​residues introduced at heavy chain-light chain positions (numbered according to the EU numbering system) selected from the group consisting of j) heavy chain position 133 and light chain position 209, k) heavy chain position 131 and light chain position 119, l) heavy chain position 133 and light chain position 207, m) heavy chain position 170 and light chain position 176, n) heavy chain position 173 and light chain position 160, o) heavy chain position 133 and light chain position 117, and p) heavy chain position 129 and light chain position 121.

[0214] In some embodiments, the first pair of disulfide bonds is formed by two cysteine ​​residues introduced at heavy-light chain positions (numbered according to the EU numbering system) selected from the group consisting of a) heavy chain position 126 and light chain position 121, b) heavy chain position 173 and light chain position 160, and c) heavy chain position 128 and light chain position 118.

[0215] In some embodiments, the native disulfide bond is formed between heavy chain position 214 and a position selected from heavy chain position 131, position 219, and position 220, where numbering is according to the EU numbering system. In some embodiments, the native disulfide bond is formed between heavy chain position 220 and light chain position 214, where numbering is according to the EU numbering system.

[0216] In some embodiments, the first CH1 / CL pair comprises an altered CH1 and CL, where position 126 of the CH1 is substituted with a cysteine ​​residue and position 220 is substituted with a non-cysteine ​​residue, position 121 of the CL is substituted with a cysteine ​​residue and position 214 is substituted with a non-cysteine ​​residue, and numbering is according to the EU numbering system.

[0217] (b) Replacement of charged amino acids in the CH1 / CL region In some embodiments, the binding pair of CH1b and CLb has one or more introduced amino acid mutations, and at least one introduced charged amino acid residue is formed to prevent mispairing between CH1b and CLa and / or between CH1a and CLb.

[0218] In some embodiments, the first CH1 / CL binding pair and the second CH1 / CL binding pair are selected from a CH1b and CLb binding pair and a CH1a and CLa binding pair, wherein the first CH1 / CL binding pair comprises a mutation of at least one uncharged amino acid residue to a charged amino acid residue and / or a mutation of at least one charged amino acid residue to an uncharged amino acid residue, such that the first CH1 / CL binding pair comprises a first pair of oppositely charged residues that facilitates pairing of the first CH1 / CL binding pair. In some embodiments, the first CH1 / CL binding pair may comprise a combination of mutations that collectively provide a first pair of oppositely charged amino acid residues that facilitates pairing of the first CH1 / CL binding pair.

[0219] In other words, a pair of oppositely charged residues may be introduced into the first CH1 / CL binding pair to promote homologous pairing between the CH1 domain and the CL domain in the first CH1 / CL binding pair. For example, a charged amino acid residue may be introduced to replace an uncharged amino acid residue at a specific position in CH1 (or CL), allowing the introduced charged amino acid residue to form an electrostatic interaction with another oppositely charged amino acid residue already present in or to be introduced into CL (or CH1), thereby contributing to pairing of the first CH1 / CL binding pair. In another example, an existing charged amino acid residue at a specific position in CH1 (or CL) may be replaced with an oppositely charged amino acid residue, allowing the substituted charged amino acid residue to form an electrostatic interaction with another oppositely charged amino acid residue already present in or to be introduced into CL (or CH1), thereby contributing to pairing of the first CH1 / CL binding pair. In certain embodiments, an existing charged amino acid residue in CH1 or CL may be replaced with an uncharged amino acid residue, thereby reducing potential interference due to electrostatic interactions between the first CH1 / CL binding pair.

[0220] In some embodiments, the second CH1 / CL binding pair contains a mutation of at least one uncharged amino acid residue to a charged amino acid residue, and / or a mutation of at least one charged amino acid residue to an oppositely charged amino acid residue, such that the second CH1 / CL binding pair contains a second pair of oppositely charged amino acid residues, which aids in pairing of the second CH1 / CL binding pair, and optionally, the first pair of oppositely charged amino acid residues and the second pair of oppositely charged amino acid residues prevent pairing of CH1a and CLb or CH1b and CLa.

[0221] In some embodiments, the first pair of oppositely charged amino acid residues and the second pair of oppositely charged amino acid residues are configured such that both CH1a and CLb are positively charged or negatively charged, and / or both CH1b and CLa are positively charged or negatively charged.

[0222] In some embodiments, the first pair of oppositely charged residues and / or the second pair of oppositely charged residues are introduced at heavy-light chain EU numbered positions (numbered according to the EU numbering system) selected from the group consisting of a) heavy chain position 183 and light chain position 176, b) heavy chain position 183 and light chain position 133, c) heavy chain position 147 and light chain position 176, d) heavy chain position 141 and light chain position 116, e) heavy chain position 126 and light chain position 121, and f) heavy chain position 218 and light chain position 122.

[0223] In some embodiments, the first pair of oppositely charged amino acid residues and / or the second pair of oppositely charged amino acid residues are introduced into heavy-light chain positions (numbered according to the EU numbering system) selected from the group: g) heavy chain position 147 and light chain position 131; h) heavy chain position 168 and light chain position 174; i) heavy chain position 147 or 168 and light chain position 131 or 174.

[0224] In some embodiments, the pair of oppositely charged amino acid residues comprises one positively charged amino acid residue and one negatively charged amino acid residue, wherein the positively charged amino acid residue is selected from lysine (K), histidine (H) and arginine (R), and / or the negatively charged amino acid residue is selected from aspartic acid (D) and glutamic acid (E).

[0225] In some embodiments, the lysine at position 147 in CH1b is substituted with a negatively charged amino acid residue, the serine at position 176 in CLb is substituted with a positively charged amino acid residue, the serine at position 183 in CH1a is substituted with a positively charged amino acid residue, and the serine at position 176 in CLa is substituted with a negatively charged amino acid residue, where the numbering is according to the EU numbering system. In some embodiments, CH1b comprises the mutation K147D, CLb comprises the mutation S176K, CH1a comprises the mutation S183K, and CLa comprises the mutation S176D.

[0226] In some embodiments, the lysine at position 147 in CH1a is substituted with a negatively charged amino acid residue, the serine at position 176 in CLa is substituted with a positively charged amino acid residue, the serine at position 183 in CH1b is substituted with a positively charged amino acid residue, and the serine at position 176 in CLb is substituted with a negatively charged amino acid residue, where the numbering is according to the EU numbering system. In some embodiments, CH1a comprises the mutation K147D, CLa comprises the mutation S176K, CH1b comprises the mutation S183K, and CLb comprises the mutation S176D.

[0227] In some embodiments, the first CH1 / CL pair comprises at least one non-native disulfide bond in combination with a non-native electrostatic interaction.

[0228] In some embodiments, the first CH1 / CL pair is a binding pair of CH1b and CLb. In some embodiments, the amino acid residue at position 173 in CH1b is substituted with a cysteine, the amino acid residue at position 183 is substituted with a positively charged amino acid residue, and the amino acid residue at position 220 is replaced with a non-cysteine ​​amino acid residue; and in CLb, the amino acid residue at position 160 is substituted with a cysteine, the amino acid residue at position 176 is substituted with a negatively charged amino acid residue, and the amino acid residue at position 214 is substituted with a non-cysteine ​​amino acid residue, where the numbering is according to the EU numbering system. In some embodiments, CH1b comprises mutations V173C, S183K, and C220S, and CLb comprises mutations Q160C, S176D, and C214S.

[0229] In some embodiments, the first CH1 / CL pair is a binding pair of CH1b and CLb, in which the amino acid residue at position 173 in CH1b is substituted with a cysteine, the amino acid residue at position 183 is substituted with a negatively charged amino acid residue, and the amino acid residue at position 220 is substituted with a non-cysteine ​​amino acid residue, and the amino acid residue at position 160 in CLb is substituted with a cysteine, the amino acid residue at position 176 is substituted with a positively charged amino acid residue, and the amino acid residue at position 214 is substituted with a non-cysteine ​​amino acid residue, where the numbering is according to the EU numbering system.

[0230] In some embodiments, the first CH1 / CL pair comprises CH1a and CLa. In some embodiments, the amino acid residue at position 173 in CH1a is substituted with a cysteine, the amino acid residue at position 183 is substituted with a positively charged amino acid residue, and the amino acid residue at position 220 is substituted with a non-cysteine ​​amino acid residue; and in CLa, the amino acid residue at position 160 is substituted with a cysteine, the amino acid residue at position 176 is substituted with a negatively charged amino acid residue, and the amino acid residue at position 214 is substituted with a non-cysteine ​​amino acid residue, where the numbering is according to the EU numbering system. In some embodiments, CH1a comprises the mutations V173C, S183K, and C220S, and CLa comprises the mutations Q160C, S176D, and C214S.

[0231] In some embodiments, the amino acid residue at position 173 in CH1a is substituted with cysteine, the amino acid residue at position 183 is substituted with a negatively charged amino acid residue, and the amino acid residue at position 220 is replaced with a non-cysteine ​​amino acid residue; and the amino acid residue at position 160 in CLa is substituted with cysteine, the amino acid residue at position 176 is substituted with a positively charged amino acid residue, and the amino acid residue at position 214 is substituted with a non-cysteine ​​amino acid residue, where the numbering is according to the EU numbering system.

[0232] In some embodiments, CH1 in the first CH1 / CL pair includes the mutations A141K, V173C, and C220S, and CL includes the mutations F116D, Q160C, and C214S (numbering according to the EU numbering system).

[0233] In some embodiments, the first antigen targeted by the polypeptide conjugate is GPRC5D and the second antigen is CD3. In some embodiments, the second CH1 domain CH1b of the third polypeptide contains the mutations A141K, V173C, and C220S, and the second CL domain CLb of the fourth polypeptide contains the mutations F116D, Q160C, and C214S, where numbering is according to the EU numbering system. In some embodiments, the second CH1 domain CH1b of the third polypeptide contains the amino acid sequence set forth in SEQ ID NO:112, and the second CL domain CLb of the fourth polypeptide contains the amino acid sequence set forth in SEQ ID NO:111.

[0234] In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:39, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:40, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO:38, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:37.

[0235] In some specific embodiments, the polypeptide complex further comprises a first Fc polypeptide and a second Fc polypeptide, and comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 83, a second polypeptide having the amino acid sequence set forth in SEQ ID NO: 40, a third polypeptide having the amino acid sequence set forth in SEQ ID NO: 82, and a fourth polypeptide having the amino acid sequence set forth in SEQ ID NO: 37.

[0236] (c) Introduction of an orthogonal interaction interface into the CH1 / CL interaction (formation of orthogonal interactions) In certain embodiments, at least one of the CH1b and CLb binding pair and the CH1a and CLa binding pair has one or more introduced amino acid mutations to form a perpendicular CH1-CL interface and prevent mispairing between CH1b and CLa or between CH1a and CLb.

[0237] In certain embodiments, the first CH1 / CL pair and the second CH1 / CL pair are selected from a CH1b and CLb binding pair and a CH1a and CLa binding pair, wherein the first CH1 / CL pair comprises one or more introduced amino acid mutations to form a perpendicular CH1-CL interface.

[0238] In some embodiments, the amino acid mutations form a perpendicular CH1-CL interface that favors pairing between CH1b and CLb, and optionally prevents pairing between CH1a and CLb or CH1b and CLa. In some embodiments, the amino acid mutations form a perpendicular CH1-CL interface that favors pairing between CH1a and CLa, and optionally prevents pairing between CH1a and CLb or CH1b and CLa.

[0239] In some embodiments, the vertical CH1-CL interface comprises mutations H168A, F170G in the heavy chain and mutations L135Y, S176W in the light chain, hi some embodiments, the vertical CH1-CL interface comprises mutations H168A and F170G in the heavy chain and mutations L135Y and S176W in the light chain.

[0240] In some embodiments, CH1a comprises the mutation S183E, CLa comprises the mutation V133K, CH1b comprises the mutations A141I, F170S, S181M, S183A and V185A, and C1b comprises the mutations F116A, L235V, S174A, S176F and T178V.

[0241] In some embodiments, CH1b comprises the mutation S183E, CLb comprises the mutation V133K, CH1a comprises the mutations A141I, F170S, S181M, S183A and V185A, and CLa comprises the mutations F116A, L235V, S174A, S176F and T178V.

[0242] In some embodiments, the first CH1 / CL pair comprises one or more introduced amino acid mutations at heavy chain-light chain positions (numbered according to the EU numbering system) selected from the group consisting of A141I, F170S, S181M, S183A, and V185A in the heavy chain, and F116A, A235V, S174A, S176F, and T178V in the light chain.

[0243] In some embodiments, the first CH1 / CL pair comprises one or more introduced amino acid mutations at heavy-light chain positions (numbered according to the EU numbering system) selected from the group consisting of A141I, F170S, S181M, S183A, and V185A in the heavy chain and F116A, A235V, S174A, S176F, and T178V in the light chain, and the second CH1 / CL pair comprises introduced amino acid mutations that form a pair of oppositely charged residues, including S183E in CH1 and V133K in CL.

[0244] In some embodiments, the polypeptide complex of the present application further comprises an Fc polypeptide comprising a first Fc polypeptide and a second Fc polypeptide. In some embodiments, the first Fc polypeptide and the Fc polypeptide are the same or different. In some embodiments, the first Fc polypeptide and the second Fc polypeptide can combine to form a dimer. In some embodiments, the second polypeptide of the polypeptide complex further comprises the first Fc polypeptide at its N-terminus. In some embodiments, the polypeptide complex further comprises a third polypeptide comprising the second Fc polypeptide at its N-terminus.

[0245] Polypeptide complex containing at least two Fab domains: Structure D In some embodiments, the polypeptide conjugate further comprises a third antigen-binding domain under structure C, optionally wherein the third antigen-binding domain is a Fab domain. In some embodiments, the C-terminus of one of the third antigen-binding domains is linked to the N-terminus of one of the second antigen-binding domains.

[0246] In some embodiments, the third antigen-binding domain is the same as the first antigen-binding domain and comprises, in an N-terminal to C-terminal direction, a first fragment comprising a first heavy chain variable domain VH1 and a first CH1 domain CH1a that bind to the first antigen, and a second fragment comprising, in an N-terminal to C-terminal direction, a first light chain variable domain VL1 and a first CL domain CLa that bind to the first antigen, wherein the C-terminus of the first fragment is linked to the N-terminus of the fourth polypeptide.

[0247] In some embodiments, the polypeptide complex comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and a fifth polypeptide, wherein, from N-terminal to C-terminal, the first polypeptide comprises VH1-CH1a, the third polypeptide comprises VH2-CH1b, the fourth polypeptide comprises VH1-CH1a-linker-VL2-CLb, and the second and fifth polypeptides are the same and both comprise VL1-CLa.

[0248] In some embodiments, the CH1b and CLb binding pair has at least one non-native disulfide bond that prevents mispairing between CH1b and CLa and / or CH1a and CLb.

[0249] In some embodiments, the CH1b and CLb binding pair has one or more introduced amino acid mutations, and at least one introduced charged amino acid residue is formed to prevent mispairing between CH1b and CLa and / or between CH1a and CLb.

[0250] In some specific embodiments, CH1 in the CH1 / CL pair of the second Fab domain comprises the mutations A141K, V173C, and C220S, and CL comprises the mutations F116D, Q160C, and C214S, wherein the numbering is according to EU numbering. In some specific embodiments, CH1 in the second Fab domain comprises the amino acid sequence set forth in SEQ ID NO:112, and CL1 comprises the amino acid sequence set forth in SEQ ID NO:111.

[0251] In some embodiments, the polypeptide complex of the present application further comprises an Fc polypeptide comprising a first Fc polypeptide and a second Fc polypeptide. In some embodiments, the first Fc polypeptide and the Fc polypeptide are the same or different. In some embodiments, the first Fc polypeptide and the second Fc polypeptide can combine to form a dimer. In some embodiments, the second polypeptide of the polypeptide complex further comprises the first Fc polypeptide at its N-terminus. In some embodiments, the polypeptide complex further comprises a third polypeptide comprising the second Fc polypeptide at its N-terminus.

[0252] In some specific embodiments, the polypeptide conjugate has the structure shown in FIG.

[0253] In some embodiments, the first antigen targeted by the polypeptide conjugate is GPRC5D, the second antigen is CD3, and the third antigen is GPRC5D. In some embodiments, in the CH1 / CL pair of the second Fab domain, CH1 comprises the mutations A141K, V173C, and C220S, and CL comprises the mutations F116D, Q160C, and C214S, wherein the numbering is according to EU numbering. In some embodiments, CH1 in the second Fab domain comprises the amino acid sequence set forth in SEQ ID NO: 112, and CL1 comprises the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 43, the second polypeptide or the fifth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 44, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 42, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 41. In some specific embodiments, the polypeptide complex further comprises a first Fc polypeptide and a second Fc polypeptide, and comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 85, a second or fifth polypeptide having the amino acid sequence set forth in SEQ ID NO: 44, a third peptide having the amino acid sequence set forth in SEQ ID NO: 84, and a fourth polypeptide having the amino acid sequence set forth in SEQ ID NO: 41.

[0254] In some embodiments, the first antigen targeted by the polypeptide conjugate is GPRC5D, the second antigen is CD3, and the third antigen is GPRC5D. In some embodiments, the first antigen-binding domain and / or the third antigen-binding domain targeting GPRC5D are humanized. In some embodiments, the CH1 of the CH1 / CL pair of the second Fab domain comprises mutations A141K, V173C, and C220S, and the CL comprises mutations F116D, Q160C, and C214S, wherein the numbering is according to EU numbering. In some embodiments, the CH1 of the second Fab domain comprises the amino acid sequence set forth in SEQ ID NO: 112, and the CL1 comprises the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 47, the second polypeptide or the fifth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 48, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 46, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 45. In some specific embodiments, the polypeptide complex further comprises a first Fc polypeptide and a second Fc polypeptide, and comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 87, a second or fifth polypeptide having the amino acid sequence set forth in SEQ ID NO: 48, a third polypeptide having the amino acid sequence set forth in SEQ ID NO: 86, and a fourth polypeptide having the amino acid sequence set forth in SEQ ID NO: 45.

[0255] In some specific embodiments, the polypeptide conjugates have the specific sequences shown in Table 7. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8]

[0256] Fc variants In specific embodiments, said first Fc polypeptide and / or said second Fc polypeptide is derived from IgG1, IgG2, IgG3 or IgG4.

[0257] In certain embodiments, the polypeptide complex comprises one or more amino acid substitutions at the interface of the Fc region to support and / or promote heterodimerization. In certain embodiments, the first Fc polypeptide and the second Fc polypeptide have different amino acid sequences, at least designed to promote heterodimerization of the first Fc polypeptide and the second Fc polypeptide. For example, a protrusion may be introduced into the first Fc polypeptide and a cavity may be introduced into the second Fc polypeptide, where the protrusion may be positioned in the cavity to promote interaction between the first Fc polypeptide and the second Fc polypeptide to form a heterodimer or complex. Methods for producing antibodies with these modifications are known in the art, e.g., as described in U.S. Patent No. 5,731,168.

[0258] In some embodiments, in the polypeptide conjugates provided herein, one of the first Fc polypeptide and the second Fc polypeptide comprises a first Fc mutation and the other comprises a second Fc mutation, wherein the first Fc mutation and the second Fc mutation are selected from the group consisting of: a) T366W or S354C in combination with Y349C, T366S, L368A or Y407V; b) D399K or E356K in combination with K392D or K409D; c) E356K, E357K or D356K in combination with K392D or K409D; a) a combination of S364H or F405A with Y349T or T394F; b) a combination of S364H or T394F with Y394T or F405A; c) a combination of K370D or K409D with E357K or D399K; or g) a combination of L351D or L368E with L351K or T366K, wherein amino acid positions are numbered according to the EU numbering system.

[0259] In some embodiments, the first Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO:65 or SEQ ID NO:67, and the second Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO:66.

[0260] The polypeptide conjugates provided herein can be monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, labeled antibodies, bivalent antibodies, or anti-idiotypic antibodies. Recombinant antibodies are antibodies prepared in vitro by recombinant methods and not in an animal.

[0261] Antibody variants The polypeptide conjugates provided herein further encompass various antibody variants thereof.

[0262] In certain embodiments, antibody variants comprise one or more modifications or substitutions in one or more CDR sequences provided in Tables 1-2, 5-6 above, one or more variable region sequences provided in Tables 3-6 above (but not in any CDR sequences), and / or in the constant region (e.g., Fc region). Such variants retain the binding specificity of the parent antibody to a corresponding antigen (e.g., GPRC5D, CD3, or Her2) while possessing one or more desired properties conferred by one or more modifications or one or more substitutions. By way of example, antibody variants may have improved antigen-binding affinity, improved glycosylation patterns, reduced glycosylation risk, reduced deamination, reduced or eliminated one or more effector functions, improved FcRn receptor binding, and increased pharmacokinetic half-life, pH sensitivity, and / or conjugation suitability (e.g., one or more introduced cysteine ​​residues).

[0263] To identify suitable or preferred residues to modify or replace, parent antibody sequences may be screened by methods known in the art, such as "alanine scanning mutagenesis" (see, e.g., Cunningham and Wells (1989) Science 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) may be identified, and the target residues may be replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and the modified antibodies may be produced and screened for properties of interest. If substitutions at particular amino acid positions exhibit functional changes of interest, the positions may be identified as potential residues for modification or replacement. Potential residues may be further evaluated by substituting them with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0264] Affinity variants Affinity variants may contain modifications or substitutions in one or more of the CDR sequences provided in Tables 1-2, 5-6, one or more of the FR sequences provided herein, or the heavy or light chain variable region sequences provided in Tables 3-6. Because it is known in the art that a CDR region is flanked by two FR regions in the variable region, FR sequences can be easily identified by those skilled in the art based on the CDR sequences in Tables 1-2, 5-6, and the variable region sequences in Tables 3-6. Affinity variants retain the specific binding affinity of the parent antibody to the corresponding antigen (e.g., GPRC5D, CD3, or Her2) and thus have improved specific binding affinity to the corresponding antigen (e.g., GPRC5D, CD3, or Her2) over that of the parent antibody. In certain embodiments, substitutions in at least one (or all) of the CDR sequences, FR sequences, or variable region sequences comprise conservative substitutions.

[0265] Those skilled in the art will understand that one or more amino acid residues in the CDR and variable region sequences provided in Tables 1-6 above can be substituted while the resulting polypeptide complex still retains or even has improved binding affinity or capacity to the corresponding antigen (e.g., GPRC5D, CD3, or Her2). Various methods known in the art can be used to achieve this goal. For example, phage display technology can be used to generate and express a library of antibody variants (e.g., Fab or scFv variants), which can then be screened for binding affinity to the corresponding antigen (e.g., GPRC5D, CD3, or Her2). As another example, computer software can be used to virtually simulate the binding of an antibody to a corresponding antigen (e.g., GPRC5D, CD3, or Her2) to identify amino acid residues on the antibody that form the binding interface. Such residues may be avoided for substitution to prevent a decrease in binding affinity, or may be targeted for substitution to provide stronger binding.

[0266] In certain embodiments, the humanized polypeptide conjugates provided herein comprise one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences, hi certain embodiments, the affinity variants comprise a total of no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in the CDR and / or FR sequences.

[0267] In certain embodiments, the GPRC5D binding domain in the polypeptide composite comprises one, two or three CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with a sequence listed in Table 1 or Table 2 above (or their sequences), and retains binding affinity to GPRC5D at a level similar to, or even higher than, that of the parent antibody. In certain embodiments, the Her2-binding domain in the polypeptide conjugate comprises one, two, or three CDR sequences that share at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with a sequence listed in Table 5 above (or a sequence thereof), thereby maintaining a similar, and even higher, level of binding affinity to Her2 than the parent antibody. In certain embodiments, the CD3-binding domain in the polypeptide conjugate comprises one, two, or three CDR sequences that share at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with a sequence listed in Table 6 above (or a sequence thereof), thereby maintaining a similar, and even higher, level of binding affinity to CD3 than the parent antibody.

[0268] In certain embodiments, the GPRC5D-binding domain in the polypeptide composite comprises one or more variable region sequences that share at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with a sequence listed in Tables 3-4 above (or with those sequences), and retains a similar, or even higher, level of binding affinity to GPRC5D than the parent antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted in the variable region sequences listed in Table 3 above. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., within the FRs). In certain embodiments, the Her2-binding domain in the polypeptide composite comprises one or more variable region sequences that share at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with a sequence listed in Table 5 above (or with those sequences), and retains a similar, or even higher, level of binding affinity to Her2 than the parent antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted in the variable region sequences listed in Table 5 above. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., within the FRs). In certain embodiments, the CD3-binding domain in the polypeptide composite comprises one or more variable region sequences that share at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with a sequence listed in Table 6 above (or with those sequences), and retains a similar, or even higher, level of binding affinity to CD3 than the parent antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted in the variable region sequences listed in Table 6 above. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., within the FRs).

[0269] Glycosylation variants The polypeptide conjugates provided herein also encompass glycosylation variants that can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment or polypeptide conjugate.

[0270] Polypeptide conjugates may contain one or more modifications that introduce or remove glycosylation sites. Glycosylation sites are amino acid residues whose side chains can be attached to a carbohydrate moiety (e.g., an oligosaccharide structure). Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue (e.g., an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline). O-linked glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine. Removal of native glycosylation sites can be conveniently achieved, for example, by altering the amino acid sequence so that one of the above-mentioned tripeptide sequences (for N-linked glycosylation sites) or a serine or threonine residue (for O-linked glycosylation sites) present in the sequence is substituted. Similarly, new glycosylation sites can be created by introducing such tripeptide sequences or serine or threonine residues.

[0271] In certain embodiments, the polypeptide conjugates provided herein comprise a mutation at N297 (eg, N297A, N297Q, or N297G) to remove a glycosylation site.

[0272] Cysteine ​​engineered variants The polypeptide conjugates provided herein also include cysteine ​​engineered variants that contain one or more introduced free cysteine ​​amino acid residues.

[0273] A free cysteine ​​residue is one that is not part of a disulfide bridge. Cysteine ​​engineered variants are useful for conjugation at the engineered cysteine ​​site, e.g., via maleimide or haloacetyl, particularly with, for example, cytotoxic and / or imaging compounds, labels, or radioisotopes. Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine ​​residues are known in the art; see, e.g., WO2006 / 034488.

[0274] Fc variants The polypeptide conjugates provided herein also encompass Fc variants that comprise one or more amino acid residue modifications or substitutions in the Fc region and / or hinge region to provide altered effector functions, e.g., ADCC and CDC. Methods for altering ADCC activity by antibody engineering have been described in the art, e.g., Shields R L et al., J Biol Chem. 2001, 276(9):6591-604; Idusogie E E et al., J Immunol. 2000, 164(8):4178-84; Steurer W et al., J Immunol. 1995, 155(3):1165-74; Idusogie E E et al., J Immunol. 2001, 166(4):2571-5; Lazar G A et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS) 2006, 103(11):4005-4010; Ryan M C et al., Mol. Cancer Ther. 2007, 6:3009-3018; Richards See JO, et al., Molecular Cancer Therapeutics 2008, 7(8):2517-27; Shields RL et al., J. Biol. Chem 2002, 277:26733-26740; Shinkawa T. et al., J. Biol. Chem 2003, 278:3466-3473.

[0275] The CDC activity of the antibodies provided herein may also be altered, for example, by increasing or decreasing Clq binding and / or CDC (see, e.g., WO 99 / 51642; Duncan and Winter, Nature, 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants). One or more amino acids selected from amino acid residues 329, 331, and 322 of the Fc region may be replaced with another amino acid residue to alter Clq binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC) (see U.S. Pat. No. 6,194,551 by Idusogie et al.). One or more amino acid substitutions may be introduced to alter the ability of the antibody to fix complement (see PCT Publication WO 94 / 29351 by Bodmer et al.).

[0276] In certain embodiments, the polypeptide conjugates provided herein have reduced effector function and comprise one or more amino acid substitutions in IgG1 at positions selected from the group consisting of 234, 235, 237, 238, 268, 297, 309, 330, and 331. In certain embodiments, the polypeptide conjugates provided herein have an IgG1 isotype and comprise one or more amino acid substitutions selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A, L234F, P331S, and any combination thereof. In certain embodiments, the polypeptide conjugates provided herein have an IgG2 isotype and contain one or more amino acid substitutions selected from the group consisting of H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A, and any combination thereof (e.g., H268Q / V309L / A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S). In certain embodiments, the polypeptide conjugates provided herein have an IgG4 isotype and contain one or more amino acid substitutions selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A, and any combination thereof. In certain embodiments, the polypeptide conjugates provided herein have an IgG2 / IgG4 cross-isotype. An example of IgG2 / IgG4 cross-isotyping is described in Rother RP et al., Nat Biotechnol 25:1256-1264 (2007).

[0277] In certain embodiments, the polypeptide conjugates provided herein have an IgG1 isotype and contain one or more amino acid substitutions at one or more of positions 234, 235, and 331. In certain embodiments, the polypeptide conjugates provided herein have an IgG1 isotype and contain the triple mutation L234F / L235E / P331S in the Fc region.

[0278] In certain embodiments, the polypeptide conjugates provided herein have increased ADCC and / or increased affinity for Fcγ receptors and are capable of binding to 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 27 and one or more amino acid substitutions at one or more of positions 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439 (see WO 00 / 42072 by Presta). Particular mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. Furthermore, it is shown that combination mutants such as T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A can improve FcγRIII binding.

[0279] In certain embodiments, the polypeptide conjugates contain one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). By binding to FcRn at acidic pH, such variants avoid lysosomal degradation and are subsequently translocated and released extracellularly, which may result in an extended pharmacokinetic half-life. Methods for engineering polypeptide conjugates to improve their binding affinity to FcRn are well known in the art, see, e.g., Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Vol. 1, Chapter 27: Engineering of the Fc region for improved PK, Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., Journal of Immunology, 176:346-356 (2006).

[0280] Conjugates In some embodiments, the polypeptide conjugate further comprises a conjugate moiety. The conjugate moiety is linkable to the polypeptide conjugate. The conjugate moiety is a moiety linkable to the polypeptide conjugate. With consideration given, a variety of conjugate moieties can be linked to the polypeptide conjugates provided herein (see, e.g., "Conjugate Vaccines," in Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate moieties can be linked to the polypeptide conjugate by covalent bonding, affinity bonding, intercalation, coordinate bonding, complexation, association, blending, or addition, as well as other methods.

[0281] In certain embodiments, the polypeptide conjugates disclosed herein can be engineered to contain specific sites on the exterior of the epitope conjugate moiety that can be utilized for attachment to one or more conjugate moieties. By way of example, such sites can include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent attachment to the conjugate moiety.

[0282] In certain embodiments, the antibody may be linked to the conjugate moiety indirectly or through another conjugate moiety. For example, a polypeptide conjugate may be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin. The conjugate may be a clearance modifier, a toxin (e.g., a chemotherapeutic agent), a detectable label (e.g., a radioisotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label), or a purification moiety.

[0283] A "toxin" can be any agent that is harmful to or capable of damaging or killing cells. Examples of toxins include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, and the like. D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin CC) and cis-dichlorodiamineplatinum(II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), antimitotic agents (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin-binding agents.

[0284] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, glucose oxidase, or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi and 32 P, other lanthanides), luminescent labels, plastid moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0285] In certain embodiments, the conjugate moiety may be a clearance modifier, which serves to extend the half-life of the antibody. Illustrative examples include water-soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and ethylene glycol / propylene glycol copolymers. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when two or more polymers are attached, they may be the same or different molecules.

[0286] In certain embodiments, the conjugated moiety may be a purification moiety such as a magnetic bead.

[0287] In certain embodiments, the polypeptide complexes provided herein are used as substrates for conjugates.

[0288] Polynucleotides and Recombinant Methods The present application provides isolated polynucleotides encoding polypeptide complexes. The terms "nucleic acid" or "polynucleotide," as used herein, refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. In certain embodiments, the isolated polynucleotides comprise one or more nucleotide sequences set forth in SEQ ID NOs: 11, 12, 13, and 14, and / or homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and / or variants thereof having only degenerate substitutions, and encode the variable regions of the exemplary antibodies provided herein. Unless otherwise indicated, a particular polynucleotide sequence implicitly encompasses not only the sequence explicitly set forth, but also conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more (or all) selected codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acids Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0289] DNA encoding a monoclonal antibody is readily isolated and sequenced by conventional procedures (e.g., oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains). The encoding DNA may also be obtained by synthetic methods.

[0290] An isolated polynucleotide encoding a polypeptide complex (e.g., including a sequence shown in Table 3) may be inserted into a vector for further cloning (amplification of the DNA) or for expression by recombinant techniques known in the art. Many vectors are available. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0291] The present application provides an expression vector comprising an isolated polynucleotide provided herein. In certain embodiments, the polynucleotide provided herein encodes a polypeptide complex, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker. Exemplary vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage, and M13 phage, as well as the plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, and pCI. , pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

[0292] Vectors containing polynucleotide sequences encoding polypeptide complexes may be introduced into host cells for cloning or gene expression. Suitable host cells for DNA cloning or expression in the vectors herein are the prokaryotes, yeast, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillus subtilis. These include Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

[0293] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, numerous other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe, e.g., Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 56,500), Kluyveromyces spp. ... 36,906), Kluyveromyces hosts such as Kluyveromyces thermotolerans and Kluyveromyces marxianus, Yarrowia (EP 402,226), Pichia pastoris (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa, e.g., Schwanniomyces occidentalis, Schwanniomyces hosts include Schwanniomyces, such as A. occidentalis, and filamentous fungi, such as Neurospora, Penicillium, and Tolypocladium, as well as Aspergillus hosts, such as A. nidulans and A. niger.

[0294] Suitable host cells for expressing the glycosylated polypeptide conjugates provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 virus strain of Bombyx mori NPV, are publicly available, and such viruses can be used in accordance with the present invention as viruses herein, particularly for transfection of Spodoptera cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0295] However, vertebrate cells have received the most attention, and propagation of vertebrate cells in culture (tissue culture) has become common practice. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7, ATCC CRL 1651), human embryonic kidney (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proceedings of the National Academy of Sciences of the United States of America 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals of the New York Academy of Sciences, 383:44-68 (1982)), MRC5 cells, FS4 cells, and a human hepatocellular carcinoma line (Hep G2). In some preferred embodiments, the host cells are mammalian cultured cell lines such as CHO, BHK, NS0, 293, and their derivatives.

[0296] Host cells are transformed with expression or cloning vectors for producing the polypeptide conjugates described above and cultured in conventional nutrient media modified as needed for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In another embodiment, antibodies may be produced by homologous recombination, as known in the art. In certain embodiments, the host cells are capable of producing the polypeptide conjugates provided herein.

[0297] Host cells for producing the polypeptide conjugates provided herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma are suitable for culturing host cells. Additionally, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Pat. Reissue No. 30,985 can be used as a culture medium for the host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions, such as temperature and pH, will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0298] When using recombinant techniques, antibodies may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate cell debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992), describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) within approximately 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0299] The polypeptide conjugate prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being preferred.

[0300] In certain embodiments, solid-phase-immobilized protein A is used for immunoaffinity purification of polypeptide complexes. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Depending on the antibody recovered, other protein purification techniques can also be used, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0301] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer with a pH of about 2.5 to 4.5, preferably at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0302] Pharmaceutical Composition The present application further provides pharmaceutical compositions comprising a polypeptide conjugate of the present application or a polynucleotide of the present application and one or more pharmaceutically acceptable carriers.

[0303] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering / chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, or other ingredients known in the art, or various combinations thereof.

[0304] Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisol, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in compositions comprising the polypeptide conjugates and conjugates provided herein reduces oxidation of the polypeptide conjugates. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Accordingly, in certain embodiments, compositions are provided that include one or more polypeptide conjugates disclosed herein and one or more antioxidants, such as methionine. Further provided are methods for preventing oxidation of the polypeptide conjugates provided herein, extending their shelf life, and / or improving their efficacy by combining the polypeptide conjugates with one or more antioxidants, such as methionine.

[0305] As further described, pharmaceutically acceptable carriers include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; procaine hydrochloride; hydrochloride), suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN®-80), chelating agents such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antibacterial agents utilized as carriers may be added to pharmaceutical compositions in multidose containers and include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0306] The pharmaceutical compositions may be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0307] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition can be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or solid form suitable for preparing a liquid solution, suspension, or emulsion. Preparations for injection can include sterile and / or non-pyrogenic solutions prepared for injection, sterile dry soluble preparations such as lyophilized powders prepared to be combined with a solvent immediately before use, including hypodermic tablets, sterile suspensions prepared for injection, sterile dry insoluble preparations prepared to be combined with a vehicle immediately before use, and sterile and / or non-pyrogenic emulsions. The solution can be aqueous or non-aqueous.

[0308] In certain embodiments, unit dose parenteral preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration shall be sterile and non-pyrogenic, as known and practiced in the art.

[0309] In certain embodiments, a sterile, lyophilized powder is prepared by dissolving a polypeptide conjugate disclosed herein in a suitable solvent. The solvent may contain an excipient that improves the stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer such as citric acid, sodium phosphate, or potassium phosphate, or other such buffers known to those of skill in the art; in one embodiment, the buffer has a pH of about neutral. The desired formulation is then obtained by sterile filtration of the solution under standard conditions known to those of skill in the art, followed by lyophilization. In one embodiment, the resulting solution is apportioned into vials for lyophilization. Each vial may contain a single dose or multiple doses of the polypeptide conjugate or composition thereof. Overfilling the vial with a small amount (e.g., about 10%) beyond that required for a dose or set of doses may be acceptable to facilitate accurate sample draws and accurate dosing. The lyophilized powder may be stored under appropriate conditions, such as at about 4°C to room temperature.

[0310] Reconstitution of lyophilized powder with water for injection provides a formulation for use in parenteral administration.In one embodiment, sterile and / or non-pyrogenic water or other suitable liquid carrier is added to lyophilized powder for reconstitution.The exact amount depends on the given selected therapy and can be empirically determined.

[0311] How to use The present application further provides a method for treating a GPRC5D-related disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a polypeptide conjugate provided herein or a pharmaceutical composition provided herein.

[0312] In some embodiments, the GPRC5D-associated disease or condition is characterized by expression or overexpression of GPRC5D. Overexpression of GPRC5D has been documented in several autoimmune diseases, including myeloma.

[0313] In certain embodiments, GPRC5D-associated diseases or conditions include, but are not limited to, cancer and other hyperproliferative diseases, immune disorders, and inflammation.

[0314] The present application further provides a method of treating a Her2-associated disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide conjugate provided herein or a pharmaceutical composition provided herein.

[0315] In some embodiments, the Her2-associated disease or condition is characterized by expression or overexpression of Her2. Overexpression of Her2 has been established in several types of cancer, including breast and lung cancer.

[0316] In some embodiments, the cancer and other hyperproliferative disorders include benign or malignant tumors, leukemias, and lymphoid malignancies. Depending on the type of cell comprising the cancer or hyperproliferative disorder, examples include neurons, glial cells, astrocytes, hypothalamus, gland cells, macrophages, epithelial cells, endothelial cells, stromal malignancies, etc. Depending on the organ / site comprising the cancer or hyperproliferative disorder, examples include the head, neck, eye, oral cavity, larynx, esophagus, breast, skin, bone, lung, colon, rectum, stomach, spleen, kidney, skeletal muscle, subcutaneous tissue, metastatic melanoma, endometrium, prostate, breast, ovary, testis, thyroid, blood, lymph nodes, kidney, liver, pancreas, brain, or central nervous system.

[0317] In some embodiments, the immune disease and / or inflammation is selected from the group consisting of alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, adrenal autoimmune disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, Sjogren's syndrome, psoriasis, atherosclerosis, diabetic and other retinopathies, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasia (including Graves' disease), corneal and other tissue transplants, and chronic inflammation, sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion injury, septicemia, endotoxic shock, Cystic fibrosis, endocarditis, psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock, organ ischemia, reperfusion injury, spinal cord injury and allograft rejection, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue and immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Guillain-Barré syndrome Barre), Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, hypertrichosis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary non-cancer Inflammatory diseases include inflammatory bowel disease, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteoarthritis, and chronic inflammation resulting from chronic viral or bacterial infections.

[0318] In one embodiment, the GPRC5D-associated disease or condition is cancer, particularly multiple myeloma, hi one embodiment, the GPRC5D-associated disease or condition is an autoimmune disease such as systemic lupus erythematosus and / or rheumatoid arthritis.

[0319] In certain embodiments, the GPRC5D-related disease or condition is a GPRC5D-expressing cancer. As used herein, "GPRC5D-expressing cancer" refers to any cancer or tumor in which GPRC5D is expressed on the surface of cancer cells. In certain embodiments, the expression level of GPRC5D on GPRC5D-expressing cancer cells is significantly higher than the expression level of GPRC5D on normal cells.

[0320] In certain embodiments, the subject is identified as having cancer cells that express GPRC5D. The presence and / or expression level of GPRC5D on cancer cells can be determined by various methods known in the art. A biological sample containing or suspected to contain cancer cells can be obtained from the subject. In some embodiments, the biological sample may be derived from cancer cells or cancer tissue. In certain embodiments, the biological sample may be further processed to isolate analytes, such as nucleic acids or proteins. The presence and / or expression level of GPRC5D can be determined by, for example, quantitative fluorescence cytometry, immunohistochemistry (IHC), or nucleic acid-based methods. For example, a biological sample from a subject may be exposed to a polypeptide complex that binds to and detects expressed GPRC5D protein. Alternatively, GPRC5D may be detected at the nucleic acid expression level by methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, and FISH.

[0321] In one embodiment, the GPRC5D-related disease or condition includes, but is not limited to, GPRC5D-expressing breast cancer, multiple myeloma, Waldenstrom's macroglobulinemia, endometrial cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, blood cancer, lymphoma, or malignant melanoma.

[0322] The therapeutically effective amount of a polypeptide conjugate or pharmaceutical composition provided herein will depend on various factors known in the art, such as the subject's weight, age, past medical history, current drug treatment, health status and potential cross-reactions, allergies, hypersensitivity and side effects, as well as the route of administration and the severity of the disease. Doses can be proportionally increased or decreased by one skilled in the art (e.g., a physician or veterinarian) depending on these and other circumstances or requirements.

[0323] In certain embodiments, the polypeptide conjugates provided herein may be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the dosage may vary over the course of treatment. For example, in certain embodiments, an initial dosage may be higher than subsequent dosages. In certain embodiments, the dosage may vary over the course of treatment depending on the subject's response.

[0324] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered or several divided doses may be administered over time.

[0325] The polypeptide conjugates disclosed herein can be administered by any route known in the art, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous (including intravenous infusion), intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0326] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may be administered alone or in combination with one or more additional therapeutic procedures or agents. For example, the antibodies or antigen-binding fragments disclosed herein can be administered in combination with one or more treatments for a second therapeutic agent (e.g., a chemotherapeutic agent, an anti-cancer drug, a radiation therapy agent, an immunotherapy agent, an anti-angiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, or a palliative treatment), surgery for the treatment of cancer (e.g., tumor resection), or a chemotherapy-related complication.

[0327] The term "immunotherapy," as used herein, refers to a type of therapy that stimulates or generally strengthens the immune system to fight diseases such as cancer. Immunotherapy includes passive immunotherapy (e.g., antibody therapy or CAR-T cell therapy) by delivering drugs (e.g., effector cells) with confirmed tumor immune reactivity, which can directly or indirectly mediate anti-tumor effects and do not necessarily rely on the complete host immune system. Immunotherapy may also include active immunotherapy, in which treatment relies on in vivo stimulation of the endogenous host immune system to combat diseased cells by administration of immune response modifiers.

[0328] In certain of these embodiments, a polypeptide conjugate disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the polypeptide conjugate and the additional therapeutic agents may be administered as part of the same pharmaceutical composition. However, a polypeptide conjugate that is administered "in combination with" another therapeutic agent is not necessarily administered simultaneously with or in the same composition as the agent. A polypeptide conjugate that is administered before or after another agent is considered to be administered "in combination with" the agent, as that term is used herein, even if the polypeptide conjugate and the second agent are administered via different routes. When possible, additional therapeutic agents administered in combination with the polypeptide conjugates disclosed herein are administered according to the schedule listed on the product information sheet of the additional therapeutic agent or according to Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition, Medical Economics Company, ISBN: 1563634457, 57th Edition (November 2002)), or protocols known in the art.

[0329] In some embodiments, the present application provides a method for detecting the presence or level of GPRC5D in a sample, the method comprising contacting the sample with a polypeptide complex provided herein.

[0330] In some embodiments, the present application provides a detection or treatment kit comprising a polypeptide conjugate provided herein and, optionally, instructions for use in combination with a detectable moiety, which kit can be used for detecting GPRC5D or for treating a GPRC5D-related disease or condition.

[0331] In some embodiments, the present application further provides the use of the polypeptide conjugates provided herein for the manufacture of a medicament for treating a GPRC5D-related disease or condition in a subject, or for the manufacture of a diagnostic reagent for diagnosing a GPRC5D-related disease or condition.

[0332] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, are within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but are merely intended to illustrate specific embodiments within the scope of the invention. Those skilled in the art will be able to develop equivalent compositions, materials, and methods without inventive effort and without departing from the scope of the invention. It will be understood that many variations can be made to the procedures described herein within the scope of the invention. It is the inventor's intention that such variations be included within the scope of the invention.

[0333] Example Example 1 Preparation of GPRC5D monoclonal antibody In this example, monoclonal antibodies were prepared using mice immunized with a tumor cell line expressing GPRC5D.

[0334] 1.1 Construction of 293T-GPRC5D and CHOS-GPRC5D cell lines Human GPRC5D (hGPRC5D) was overexpressed in HEK293 cells (ATCC) and CHOS cells (Invitrogen) by lentiviral infection (MOI = 3-10, 5 μg / ml polybrene). Seventy-two hours after cell infection, the corresponding antibiotic was added and the cells were cultured for 2-4 weeks. The cells were then expanded and cryopreserved to obtain two overexpressing cell lines, HEK293-hGPRC5D and CHOS-hGPRC5D, which were used in subsequent immunoassays.

[0335] 1.2 Construction of control antibody GC5B596 The HC and LC plasmids for the GC5B596 antibody were constructed using the following sequence constructions, and then transiently transfected into CHO cells and cultured. The supernatant was then purified by affinity chromatography to obtain the control antibody GC5B596. [Table 8]

[0336] 1.3 Mouse immunization / hybridoma fusion To generate anti-human GPRC5D antibodies, Balb / c mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., Category 216) were immunized with HEK293-GPRC5D cells overexpressing human GPRC5D. Complete Freund's adjuvant CFA (InvivoGen, product number vac-cfa-60) was used as the primary adjuvant, followed by IFA (InvivoGen, product number vac-ifa-60). Multiple immunizations were performed subcutaneously. After multiple immunizations, spleen cells from the immunized mice were fused with mouse myeloma cells SP2 / 0 using polyethylene glycol fusion. The fusion cells were cultured in HAT selection medium to obtain hybridoma cells capable of expressing antibodies and capable of indefinite in vitro proliferation. The hybridoma cells were then plated and cultured in 96-well cell culture plates.

[0337] 1.4 Hybridoma cloning and screening The binding ability of antibodies secreted by hybridoma cells to GPRC5D was detected at the cellular level in a 96-well cell culture plate. GPRC5D-highly expressing cells were cultured in DMEM medium containing 10% FBS, digested with TrypLE trypsin, centrifuged, and resuspended in DPBS solution containing 2% BSA (FACS buffer, 4°C). 5 × 10 5 Cells were added to a U-bottom 96-well plate at 50 μl per well and placed in a round-bottom low-binding 96-well plate. 50 μl of mouse hybridoma supernatant was added and incubated at 4°C for 1 hour. The supernatant was removed by centrifugation and washed twice with FACS buffer. A secondary antibody (DyLight488 goat anti-human IgG, Abcam, ab97003) was added to each well and incubated at 4°C for 0.5 hours. The supernatant was removed by centrifugation and washed twice with FACS buffer. The cells were then resuspended in FACS buffer and fluorescence measurements were performed on the cells in the experimental plate using a flow cytometer (BD, model number Canto II) to determine the binding status of the hybridoma supernatant to the cells. At the same time, a similar binding assay was performed using the background cell line HEK293, which was used to construct GPRC5D-overexpressing cells. Positive HEK293-hGPRC5D binding and negative HEK293 cell binding were used as the standard. Clones that were positive for GPRC5D binding were selected and subjected to two to three rounds of subcloning.

[0338] 1.5 Hybridoma sequencing / recombinant expression vector construction The Ch-72C7 clone was obtained by screening. Selected hybridoma clones were subjected to hybridoma sequencing according to standard hybridoma sequencing methods to obtain the heavy and light chain variable regions (VH and VL) of the selected clones. VH and VL were synthesized using total gene synthesis and linked to human IgG1 and kappa chain constant regions. The heavy and light chain sequences were ligated into the pcDNA3.4 vector and transiently expressed in the 293 system followed by protein A / G purification. The resulting chimeric recombinant antibody was ultrafiltered and the buffer was replaced with PBS solution. The sequencing results for the Ch-72C7 clone are shown in Table 9. [Table 9]

[0339] Example 2 Antigen Binding FACS Experiment HEK293-hCD22GPRC5D and CHOS-hGPRC5D cells expressing hGPRC5D were centrifuged and resuspended in DPBS solution containing 2% BSA (FACS buffer, 4°C), and 5 × 10 5 Cells were added to a U-bottom 96-well plate at 100 μl per well, and gradient-diluted antibodies were added. The cells were incubated at 4°C for 1 hour, centrifuged, and the supernatant was discarded. 100 μl of anti-human IgG Fc-APC secondary antibody was added per well and incubated at 4°C for 1 hour. The cells were then washed once with FACS buffer, resuspended in 200 μl of FACS buffer, and the fluorescent signal was read using a BD Canto II. Results showed that the ch-72C7 antibody bound to HEK293-hGPRC5D (Figure 1) and CHOS-hGPRC5D (Figure 2).

[0340] MM.1R cells, NCI-H929 cells, and RPMI-8226 cells (GPRC5D high expression, ATCC, CL-188) were cultured in RPMI1640 medium containing 10% FBS. The cells were digested with TrypLE trypsin, centrifuged, and resuspended in DPBS solution containing 2% BSA (FACS buffer, 4°C). 5 × 10 5 Cells were added to a U-bottom 96-well plate at 100 μl per well, and gradient-diluted antibodies were added. The mixture was incubated at 4°C for 1 hour, centrifuged, and the supernatant was discarded. 100 μl of anti-human IgG Fc-APC secondary antibody was added per well and incubated at 4°C for 1 hour. The cells were then washed once with FACS buffer, resuspended in 200 μl of FACS buffer, and the fluorescent signal was read using a BD Canto II. Results showed that the ch-72C7 antibody bound to MM.1R cells (Figure 3), NCI-H929 cells (Figure 4), and RPMI-8226 cells (Figure 5). [Table 10]

[0341] Example 3 Evaluation of ADCC effect of antibodies Target cells were tumor cells naturally expressing GPRC5D (NCI-H929, Nanjing Kebai Biosciences; MM.1R, Nanjing Kebai Biosciences), and effector cells were stably transfected with an in-house constructed Jurkat-NFAT-Luc-CD16 cell line expressing the CD16 receptor and NFAT response element. Experiments were performed in 96-well flat-bottom cell plates (Corning 3903). Gradient-diluted antibodies were added to the target cells and incubated at 37°C for 30 minutes. 60,000 effector cells were added per 10,000 target cells and incubated at 37°C for 6 hours. After the incubation, One-Glo™ reagent (Promega, E6110) was added for fluorescence development, and the cell plates were read using a Tecan Spark10 microplate reader. Data analysis was performed using GraphPad. The horizontal axis represents the logarithm of antibody concentration, and the vertical axis represents the luminescence readings from the corresponding wells. The EC50 values ​​for antibody-dependent cellular cytotoxicity of anti-GPRC5D antibodies were fitted to a curve. The results showed that the blank control isotype (ISO) had no cytotoxic effect on NCI-H929 and MM.1R cells. Both the ch-72C7 and CG5B596 antibodies had cytotoxic effects on tumor cells NCI-H929 (Figure 6) and RPMI-8226 (Figure 7), which naturally express GPRC5D. Compared to the CG5B596 antibody, the ch-72C7 antibody exhibited stronger ADCC activity.

[0342] Example 4 Humanized antibody design and expression The ch-72C7 antibody was compared with the IMGT database, and the human Framework sequence with the highest homology to its VH / VL was selected. CDR grafting was performed, and computational chemistry simulations were performed to maintain antigen binding. The design of the humanized antibody is shown in Table 11. [Table 11-1] [Table 11-2]

[0343] The VH and VL regions of the above antibodies were linked to the human IgG1 Fc region and kappa constant region, and the antibody heavy and light chain sequences were inserted into the pcDNA3.4 vector. The antibodies were transiently expressed in HEK293 cells and purified using protein A or G.

[0344] At the same time, the VH and VL of the ch-72C7 antibody were replaced with the VH and VL of a human IgG1 antibody, respectively, to form a chimeric antibody, 22mono, which was used as a control to evaluate the humanization results of each antibody. [Table 12]

[0345] Example 5 Affinity testing of humanized GPRC5D antibodies Recombinant GPRC5D antigen (Human GPRC5D protein-Flag-His tag (51.8 KD) ACRO Cat: GPD-H52D3) was immobilized on the chip and detected using the Octet® R8 Biomolecular Interaction Analysis System, and the results are shown in Table 13. [Table 13]

[0346] To summarize the above data, the VH and VL of 22Mono5JO4 were used to construct a humanized antibody, followed by the construction of a diabody.

[0347] Example 6 Construction of multispecific antibodies Using the above-mentioned CPRC5D antibody, a series of CD3xGPRC5D bispecific antibodies or Her2xCD3xGPRC5D trispecific antibodies were constructed employing four different antibody structures (Figures 8 to 11), as shown in Table 14. [Table 14-1] [Table 14-2] [Table 14-3]

[0348] Example 7 Binding of bispecific antibodies to monkey or human GPRC5D, respectively The purpose of this experiment was to compare the binding ability of the constructed diabodies to monkey or human GPRC5D by inoculating a fixed amount of HEK293T cells overexpressing human GPRC5D or monkey GPRC5D (HEK293T-hGPRC5D or HEK293T-cynoGPRC5D) and adding gradient dilutions of the test antibody.

[0349] Experimental Method Logarithmic growth phase HEK293T-hGPRC5D or HEK293T-cynoGPRC5D cells were seeded into 96-well culture plates and treated with antibodies at different concentrations (200, 66.7, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09, 0.03, and 0.01 nM). Each concentration was treated with two replicate wells, along with a negative control (PBS) well. The cells were incubated at 25°C for approximately 60 minutes before color development and detection. The raw data was the difference between the detection wavelength and the reference wavelength. Using statistical software, dose-response curves were plotted based on the OD values ​​and the logarithmic concentrations, and the EC50 values ​​for the curves were calculated. [Table 15]

[0350] Experimental results The results of this experiment are shown in Figures 12 and 13. The results showed that 22A8 had similar binding activity to monkey and human GPRC5D, with EC50s of 10.07 nM and 3.8862 nM, respectively. The positive control antibody 22B1 had significantly higher binding activity to human GPRC5D (EC50 of 23.1 nM) than to monkey (EC50 > 200 nM). The negative antibody 10B1 had no binding activity to either monkey or human GPRC5D protein.

[0351] Example 8 In vitro killing assay of bispecific antibodies 8.1 Detection of cell growth inhibitory effect of bispecific antibodies on GPRC5D highly expressing cells NCI-H929 The purpose of this experiment was to compare the growth inhibitory effects of the constructed bispecific antibodies mediated by PBMC on NCI-H929 cells.

[0352] Experimental Method Logarithmic growth phase H929 cells were prepared. A portion of the cells was reserved as a CFSE negative control, and the remaining cells were stained with 1 μM CFSE. E cells:T cells (PBMC:H929) were seeded at a 20:1 ratio into 96-well culture plates and treated with antibodies at different concentrations (4500, 1500, 500, 166.67, 55.56, 18.52, 6.17, 2.06, 0.69, and 0.23 pM). Each concentration was replicated in duplicate, along with a negative control (PBS) well. Cells were incubated at 37°C for 24 h, then stained with PI and subjected to flow cytometry (FACS). FACS data files were analyzed using Thermo Attune NxT software, and EC50 values ​​were analyzed using Graphpad Prism 6.0.

[0353] Experimental results The 22 test antibodies (22A1-22A8) and the positive antibody 22B1 were added at different concentrations to co-cultures of H929 cells and PBMCs (pre-stained with CFSE). The proportion of CFSE- and PI-double positive cells among CFSE-positive cells was measured using a flow cytometer to assess the antibody-mediated killing of H929 cells by PBMCs. As shown in Figures 14-16, both 22A1-22A5 and 22B1 mediated the killing of H929 cells by PBMCs in a dose-dependent manner.

[0354] 8.2 Detection of the growth inhibitory effect of 22A8 and 22B1 antibodies on MM1S cells highly expressing GPRC5D The purpose of this experiment was to compare the growth inhibitory effects of 22A8 and 22B1 mediated PBMC on MM1S cells.

[0355] Experimental Method MM1S cells in logarithmic growth phase were prepared. A portion of the cells was reserved as a CFSE negative control, and the remaining cells were stained with 1 μM CFSE. E cells:T cells (PBMC:MM1S) were seeded at a 20:1 ratio into 96-well culture plates and treated with different concentrations of antibody (4500, 1500, 500, 166.67, 55.56, 18.52, 6.17, 2.06, 0.69, 0.23 pM). Each concentration was replicated in two wells, along with a negative control (PBS). Cells were incubated at 37°C for 24 h, then stained with PI and analyzed by flow cytometry (FACS).

[0356] Experimental results We added different concentrations of 22A8, 22A16, and the positive antibody 22B1 to a co-culture system of MM1S and PBMC (pre-stained with CFSE), and used a flow cytometer to measure the proportion of CFSE- and PI-double-positive cells among CFSE-positive cells to evaluate the antibody-mediated killing of PBMC MM1S cells. As shown in Figure 17, both 22A8 and 22B1 mediated MM1S cell killing in PBMC in a dose-dependent manner, with EC50 values ​​of 13.34 and 83.79 pM, respectively.

[0357] 8.3 Detection of cell growth inhibitory effect of 22A8 and 22B1 antibodies on RPMI8226 or KMS-12-BM cells with low GPRC5D expression The purpose of this experiment was to compare the 22A8- and 22B1-mediated inhibitory effects of PBMC proliferation on RPMI8226 or KMS-12-BM cells.

[0358] Experimental Method Logarithmic growth phase RPMI8226 or KMS-12-BM cells were prepared. A portion of the cells was reserved as a CFSE negative control, and the remaining cells were stained with 1 μM CFSE. E cells:T cells (PBMC:MM1S) were seeded at a 20:1 ratio into 96-well culture plates and treated with different concentrations of antibody (4500, 1500, 500, 166.67, 55.56, 18.52, 6.17, 2.06, 0.69, 0.23 pM). Each concentration was replicated in duplicate, along with a negative control (PBS) well. Cells were incubated at 37°C for 24 h, then stained with PI and subjected to flow cytometry (FACS). FACS data files were analyzed using Thermo Attune NxT software, and EC50 values ​​were analyzed using Graphpad Prism 6.0.

[0359] Experimental results The 22A8 and 22B1 antibodies were added at different concentrations to RPMI8226 or KMS-12-BM and PBMC (pre-stained with CFSE) cocultures. The proportion of CFSE- and PI-double-positive cells among CFSE-positive cells was measured using a flow cytometer to evaluate the antibody-mediated killing of MM1S cells in PBMC. The data showed that 22A8 and 22B1 had EC50 values ​​of 19.91 pM and NA against RPMI8226 (Figure 18), and 132.4 and 303.4 pM against KMS-12-BM (Figure 19).

[0360] Example 9 Affinity testing of humanized CD3×GPRC5D bispecific antibody (22A8) Recombinant CD3e antigen (Human CD3 epsilon Protein, His Tag, ACRO Cat: CDE-H5223) and recombinant GPRC5D antigen (Human GPRC5D protein-Flag-His tag (51.8KD) ACRO Cat: GPD-H52D3) were immobilized on chips, respectively, and detected using the Octet® R8 Biomolecular Interaction Analysis System. The results are shown in Table 16. [Table 16]

[0361] The results showed that the humanized CD3xGPRC5D bispecific antibody (22A8) had strong affinity for the CPRG5D and CD3 antigens, respectively.

[0362] Example 9 In vivo killing assay of bispecific antibodies 9.1 Human Myeloma NCI-H929 Animal Model Experimental Method Six-week-old female NPG mice were used, with 5 × 10 per mouse. 6 NCI-H929 cells were subcutaneously inoculated, and 5 × 10 PBMCs were added on the day of NCI-H929 cell inoculation (day 0). 6 When the tumor volume reached approximately 113 mm, the mice were divided into groups according to tumor volume and intravenously injected with 1 mg / kg of drug 22A2 or 1 mg / kg of drug 22A8 twice weekly (BIW) at a volume of 0.1 mL / 10 g body weight.

[0363] The effect of the drug on tumor growth is considered, and the specific indicators are the tumor growth rate (T / C%) or tumor inhibition rate (TGI%). Tumor diameters were measured with a caliper three times a week.

[0364] Tumor volume calculation formula: Tumor volume TV (mm3) = 1 / 2 × (a × b2) In the formula, a represents the major axis and b represents the minor axis.

[0365] Tumor growth rate formula: T / C% = TmTV / CmTV × 100% where TmTV: mean of treatment group, CmTV: mean of control group.

[0366] Tumor inhibition rate formula: TGI%=(1-T / C)×100% where T / C% is the tumor growth rate, which is the percentage of relative tumor volume (or body weight) between the treatment and control groups at a given time point.

[0367] Partial regression (PR) of the tumor was defined as a reduction in tumor volume below the initial volume, and complete disappearance of the tumor was defined as complete regression (CR).

[0368] Experimental results The results are shown in Figure 20 and Table 17. The results showed that 22A2 and 22A8 (1 mg / kg, iv, twice weekly, a total of 5 doses) had significant inhibitory effects on the growth of subcutaneously transplanted tumors in human myeloma NCI-H929 human PBMC-immune reconstituted mice. The TGI at the end of the study, D17, was 84.84% and 97.71%, respectively. Of these, 5 / 6 mice in the 22A8 group experienced complete tumor regression. The tumor-bearing mice tolerated all of these drugs well, and no significant weight loss or other symptoms occurred during the treatment period. [Table 17]

[0369] 9.2 Human Myeloma NCI-H929 Animal Model Experimental Method Six-week-old female NPG mice were used, with 5 × 10 per mouse. 6 NCI-H929 cells were subcutaneously inoculated, and 5 × 10 PBMCs were added on the day of NCI-H929 cell inoculation (day 0). 6 The average tumor volume was approximately 97 mm. 3 Once tumors reached the target size, the mice were divided into groups according to tumor volume and administered 0.1 mg / kg and 1 mg / kg of drug 22A8 intravenously (iv) twice weekly (BIW), with an injection volume of 0.1 mL / 10 g body weight.

[0370] The effect of the drug on tumor growth is considered, and the specific indicators are the tumor growth rate (T / C%) or tumor inhibition rate (TGI%). Tumor diameters were measured with a caliper three times a week.

[0371] Tumor volume calculation formula: Tumor volume TV (mm3) = 1 / 2 × (a × b2) In the formula, a represents the major axis and b represents the minor axis.

[0372] Tumor growth rate formula: T / C% = TmTV / CmTV × 100% where TmTV: mean of treatment group, CmTV: mean of control group.

[0373] Tumor inhibition rate formula: TGI%=(1-T / C)×100% where T / C% is the tumor growth rate, which is the percentage of relative tumor volume (or body weight) between the treatment and control groups at a given time point.

[0374] Partial regression (PR) of the tumor was defined as a reduction in tumor volume below the initial volume, and complete disappearance of the tumor was defined as complete regression (CR).

[0375] Experimental results The results are shown in Figure 21 and Table 18. The results showed that 22A8 (0.1 mg / kg or 1 mg / kg, iv, twice weekly, a total of 5 doses) had a significant inhibitory effect on the growth of subcutaneously transplanted human myeloma NCI-H929 tumors in human PBMC-immune reconstituted mice, with 22A8 TGIs of 85.61% and 95.65%, respectively. Partial tumor regression occurred in 1 / 5 mice in the 0.1 mg / kg 22A8 group, and in 2 / 5 mice in the 1 mg / kg 22A8 group. Aside from slight weight loss in tumor-bearing mice in the 0.1 mg / kg 22A8 group (3.6% weight loss on Day 16 compared to pre-treatment), the remaining tumor-bearing mice tolerated all drugs well and showed no significant weight loss or other symptoms during the treatment period. [Table 18]

[0376] 9.3 Subcutaneous or intravenous ascending dose PK study in cynomolgus monkeys Experimental Method Four female cynomolgus monkeys were numbered 1#, 2#, 5#, and 6#. Animals 1# and 2# were administered test article 22A8 subcutaneously, and animals 5# and 6# were administered test article 22A8 intravenously, with administration completed within 90 min. Animal 1# received 0.3 mg / kg, 1 mg / kg, and 3 mg / kg doses at 1 mL / kg, administered three times on days 0, 3, and 6. Animal 2# received 1 mg / kg, 3 mg / kg, and 10 mg / kg doses at 1 mL / kg or 1.65 mL / kg, administered three times on days 0, 3, and 6. The doses for animal 5 were 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg, with a dosing volume of 5 mL / kg. Administration was sequentially given on days 0, 3, and 6, for a total of three doses. The doses for animal 6 were 0.3 mg / kg, 1 mg / kg, and 3 mg / kg, with a dosing volume of 5 mL / kg. Administration was sequentially given on days 0, 3, and 6, for a total of three doses. Of the four animals, 1# and 5# were administered simultaneously, and 2# and 6# were administered simultaneously. The specific design is shown in Table 19. During the study, blood samples were taken at different time points, and clinical observations, clinical pathology, serum cytokines, immunophenotyping, and blood drug concentration were also performed to calculate PK parameters. [Table 19]

[0377] Experimental results During the experimental period, all animals showed no obvious abnormalities in clinical observation and did not lose weight.

[0378] Penalty kick The results showed that 22A8 was well absorbed in the animals. When administered subcutaneously, Cmax and AUC increased essentially dose-proportionally. Animal #1 (0.3 / 1 / 3 mg / kg, Q3D x 3, sc) had a HL half-life of 26-64 hours. Animal #2 (1 / 3 / 10 mg / kg, Q3D x 3, sc) had a HL half-life of 36-74 hours. Animal #5 (0.1 / 0.3 / 1 mg / kg, Q3D x 3, iv) had a HL half-life of 23-38 hours. Animal #6 (0.3 / 1 / 3 mg / kg, Q3D x 3, iv) had a HL half-life of 30-47 hours.

[0379] While the present application has been particularly shown and described with reference to specific embodiments, some of which are preferred embodiments, it should be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the present application as disclosed herein.

Claims

1. A multispecific polypeptide conjugate comprising a first antigen-binding domain and a second antigen-binding domain, wherein at least one of the first antigen-binding domain and the second antigen-binding domain binds to GPRC5D and comprises a GPRC5D-binding domain, wherein the GPRC5D-binding domain comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), and wherein the heavy chain complementarity determining regions are the heavy chain variable region (V) as set forth in SEQ ID NO:

13. Ｈ ) and the light chain complementarity determining region is the same as the light chain variable region (V Ｌ ) a polypeptide complex identical to the three light chain complementarity determining regions contained within the

2. In the GPRC5D-binding domain, a) said HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, or SEQ ID NO: 9 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; b) said HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions, or SEQ ID NO: 10 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions; c) the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions, or SEQ ID NO: 11 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions; d) said LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, or SEQ ID NO: 7 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; e) the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, or SEQ ID NO: 8 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; and f) The polypeptide conjugate of claim 1, wherein the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions.

3. In the GPRC5D-binding domain, a) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, or b) The polypeptide conjugate of any one of the preceding claims, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof having 3, 2 or 1 or less amino acid substitutions.

4. 10. The polypeptide conjugate of claim 1, wherein in the GPRC5D binding domain, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof having 3, 2 or 1 or less amino acid substitutions.

5. 10. The polypeptide conjugate of claim 1, wherein the GPRC5D binding domain is humanized.

6. The GPRC5D binding domain is a heavy chain variable region (V Ｈ ) and / or light chain variable region (V Ｌ ), and a) the heavy chain variable region comprises an amino acid sequence selected from the group of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, or a variant thereof having no more than 3, 2, or 1 amino acid substitution; b) The polypeptide conjugate of any one of the preceding claims, wherein the light chain variable region is selected from the amino acid sequences of the group SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or variants thereof having no more than 3, 2, or 1 amino acid substitutions.

7. The GPRC5D binding domain is a heavy chain variable region (V Ｈ ) and / or light chain variable region (V Ｌ ), and a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof having three, two, or one or fewer amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, or a variant thereof having three, two, or one or fewer amino acid substitutions; b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17 or a variant thereof having three, two, or no more than one amino acid substitution; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof having three, two, or no more than one amino acid substitution; c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof with no more than 3, 2, or 1 amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof with no more than 3, 2, or 1 amino acid substitutions; or d) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof with no more than 3, 2, or 1 amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof with no more than 3, 2, or 1 amino acid substitutions; 10. The polypeptide conjugate of claim 1, wherein the amino acid substitution is not within a CDR region.

8. 10. The polypeptide conjugate of any one of the preceding claims, further comprising an immunoglobulin constant region, optionally comprising the constant region of a human immunoglobulin, or optionally comprising the constant region of a human IgG.

9. 10. The polypeptide conjugate of claim 1, wherein the other of the first antigen-binding domain and the second antigen-binding domain binds to an antigen different from GPRC5D.

10. 10. The polypeptide conjugate of claim 9, wherein the antigen different from GPRC5D is an immunostimulatory antigen, and optionally, the immunostimulatory antigen is CD3.

11. The polypeptide conjugate of claim 9 , wherein the other of the first antigen-binding domain and the second antigen-binding domain comprises a CD3-binding domain.

12. the CD3-binding domain comprises three heavy chain complementarity determining regions, HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:49 or a variant thereof having three, two, or one or less amino acid substitutions; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:50 or a variant thereof having three, two, or one or less amino acid substitutions; the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:51 or a variant thereof having three, two, or one or less amino acid substitutions; the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:52 or a variant thereof having three, two, or one or less amino acid substitutions; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:53 or a variant thereof having three, two, or one or less amino acid substitutions; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:54 or a variant thereof having three, two, or one or less amino acid substitutions; or 12. The polypeptide conjugate of claim 11, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 92 or a variant thereof having three, two, or one or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 93 or a variant thereof having three, two, or one or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 94 or a variant thereof having three, two, or one or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 95 or a variant thereof having three, two, or one or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 96 or a variant thereof having three, two, or one or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 54 or a variant thereof having three, two, or one or less amino acid substitutions.

13. 13. The polypeptide conjugate of claim 12, wherein the CD3 binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 55 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 56 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions.

14. The first antigen-binding domain and the second antigen-binding domain constitute one DICAD domain, and the DICAD domain comprises: (i) a first polypeptide comprising, in an N-terminal to C-terminal direction, a first light chain variable domain VL1 that binds to a first antigen and a second heavy chain variable domain VH2 that binds to a second antigen, wherein VL1 and VH2 are linked directly or via a first linker; (ii) a second polypeptide comprising, in an N-terminal to C-terminal direction, a second light chain variable domain VL2 that binds to a second antigen and a first heavy chain variable domain VH1 that binds to a first antigen, wherein VL2 and VH1 are linked directly or via a second linker; VL1 and VH1 combine to form the first antigen-binding domain; VL2 and VH2 combine to form the second antigen-binding domain; The polypeptide conjugate of any one of claims 1 to 13, wherein VL1 and VH1 are covalently linked via a disulfide bond.

15. The polypeptide conjugate of claim 14, wherein the first linker and / or the second linker each independently comprises 5 to 9 amino acid residues.

16. 16. The polypeptide conjugate of claim 14 or 15, wherein the VL1 has a first cysteine ​​substitution in the FR and the VH1 has a second cysteine ​​substitution in the FR, and the first and second cysteines form a disulfide bond.

17. The first and second cysteines are 100C in VL1 and 44C in VH1, 43C in VL1 and 105C in VH1, 49C in VL1 and 100bC in VH1, 50C in VL1 and 100C in VH1, 46C in VL1 and 101C in VH1, is selected from the group 17. The polypeptide conjugate of claim 16, wherein the numbering is according to Kabat numbering.

18. The polypeptide conjugate of claim 17, wherein the disulfide bond is formed between 100C in VL1 and 44C in VH1.

19. The polypeptide conjugate of any one of claims 14 to 18, wherein said VL1 and VH1 further have an electrostatic interaction between two oppositely charged residues.

20. the two oppositely charged residues are introduced into the VL1 and VH1; and a) Q38 in VL1 and Q39 in VH1; b) Q40 in VL1 and Q39 in VH1, or c) replacing a residue at a position selected from the group consisting of Q37 in VL1 and Q39 in VH1, wherein said numbering is according to Kabat numbering.

21. The polypeptide conjugate of any one of claims 14 to 20, wherein the VL2 and VH2 further have electrostatic interactions between two oppositely charged residues.

22. The two oppositely charged residues between the VL2 and VH2 are a) Q38 in VL2 and Q39 in VH2; b) Q40 in VL2 and Q39 in VH2, or c) Q37 in VL2 and Q39 in VH2, wherein said numbering is according to Kabat numbering.

23. 23. The polypeptide conjugate of any one of claims 19 to 22, wherein the two oppositely charged residues comprise one negatively charged amino acid residue selected from the group of aspartic acid (D) or glutamic acid (E) and one positively charged amino acid residue selected from the group of lysine (K) or arginine (R).

24. 24. The polypeptide conjugate of claim 23, wherein at least one of the residues in the FR of the VL1 is substituted with a negatively charged amino acid and at least one of the residues in the FR of the VH1 is substituted with a positively charged amino acid, or at least one of the residues in the FR of the VL1 is substituted with a positively charged amino acid and at least one of the residues in the FR of the VH1 is substituted with a negatively charged amino acid.

25. 25. The polypeptide conjugate of any one of claims 17 to 24, wherein the first antigen-binding domain comprises a GPRC5D-binding domain as defined in any one of claims 1 to 7, and the second antigen-binding domain comprises a CD3-binding domain as defined in claim 11 or 12.

26. The polypeptide conjugate of any one of claims 16 to 25, wherein the amino acid sequence of the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence of the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

23.

27. The polypeptide conjugate of any one of claims 14 to 26, wherein the second polypeptide further comprises a first Fc polypeptide at the C-terminus.

28. 28. The polypeptide conjugate of any one of claims 14 to 27, further comprising a third polypeptide further comprising a second Fc polypeptide at its C-terminus.

29. The polypeptide conjugate of any one of claims 14 to 26, further comprising a third antigen-binding domain, optionally wherein the third antigen-binding domain comprises a Fab domain.

30. The Fab domain is (i) a third polypeptide comprising, in an N-terminal to C-terminal direction, a third heavy chain variable domain VH3 and a CH1 domain that binds a third antigen; and (ii) a fourth polypeptide comprising, in an N-terminal to C-terminal direction, a third light chain variable domain VL3 and a CL domain that binds a third antigen; 30. The polypeptide conjugate of claim 29, wherein VL3 and VH3 combine to form the third antigen-binding domain.

31. 31. The polypeptide conjugate of claim 30, wherein the Fab domain binds to GPRC5D and comprises a GPRC5D binding domain as defined in any one of claims 1 to 7.

32. The polypeptide complex of claim 31 , wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 30, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 31, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

32.

33. The polypeptide complex further comprises a third antigen-binding domain, and optionally, the third antigen-binding domain comprises a Fab domain, wherein the Fab domain comprises: (iii) a third polypeptide comprising, in an N-terminal to C-terminal direction, a third heavy chain variable domain VH3 and a CH1 domain that binds a third antigen; and (iv) a fourth polypeptide comprising, in an N-terminal to C-terminal direction, a third light chain variable domain VL3 and a CL domain that binds a third antigen; The polypeptide conjugate of any one of claims 14 to 24, wherein VL3 and VH3 combine to form the third antigen-binding domain.

34. 34. The polypeptide conjugate of claim 33, wherein the first antigen, second antigen, and third antigen are each independently selected from GPRC5D, an immunostimulatory antigen, and a tumor antigen, and optionally, the immunostimulatory antigen is CD3 and the tumor antigen is Her2.

35. 35. The polypeptide conjugate of claim 34, wherein the first antigen is Her2, the second antigen is CD3, and the third antigen is GPRC5D.

36. The Her2 binding domain is a heavy chain variable region (V Ｈ ) and three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region (V Ｌ 36. The polypeptide conjugate of claim 35, comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within the sequence set forth in SEQ ID NO: 57, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 57 or a variant thereof having three, two, or one or less amino acid substitutions; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 58 or a variant thereof having three, two, or one or less amino acid substitutions; the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 59 or a variant thereof having three, two, or one or less amino acid substitutions; the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 60 or a variant thereof having three, two, or one or less amino acid substitutions; the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 61 or a variant thereof having three, two, or one or less amino acid substitutions; and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 62 or a variant thereof having three, two, or one or less amino acid substitutions.

37. 37. The polypeptide conjugate of claim 36, wherein the Her2 binding domain comprises a light chain variable domain of the amino acid sequence set forth in SEQ ID NO: 64, and VH3 comprises a heavy chain variable domain of the amino acid sequence set forth in SEQ ID NO:

63.

38. The polypeptide conjugate of any one of claims 35 to 37, wherein the first antigen-binding domain comprises a Her2-binding domain as defined in any one of claims 39 to 40, the second antigen-binding domain comprises a CD3-binding domain as defined in any one of claims 15 to 16, and the third antigen-binding domain comprises an antigen-binding fragment of an antibody that binds to GPRC5D as defined in any one of claims 1 to 10.

39. The polypeptide complex of claim 38, wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:25, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:26, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

28.

40. The polypeptide conjugate of claim 33, wherein the first antigen is GPRC5D, the second antigen is GPRC5D, the third antigen is CD3, and the first antigen-binding domain and the second antigen-binding domain comprise a GPRC5D-binding domain defined in any one of claims 1 to 7, and the third antigen-binding domain comprises a CD3-binding domain defined in claim 12 or 13.

41. The polypeptide complex of claim 40, wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 33, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 34, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

36.

42. The polypeptide conjugate of any one of claims 30 to 41, wherein the second polypeptide further comprises, in an N-terminal to C-terminal direction, a first Fc polypeptide, and / or the third polypeptide further comprises, in an N-terminal to C-terminal direction, a second Fc polypeptide, and the first Fc polypeptide and the second Fc polypeptide are capable of combining to form a dimer.

43. the polypeptide complex comprises the first antigen-binding domain and the second antigen-binding domain; and The first antigen-binding domain (i) a first polypeptide comprising, in an N-terminal to C-terminal direction, a first heavy chain variable domain VH1 and a first CH1 domain CH1a that binds a first antigen; and (ii) a second polypeptide comprising, in an N-terminal to C-terminal direction, a first light chain variable domain VL1 and a first CL domain CLa that binds to a first antigen; and a first Fab domain comprising: The second antigen-binding domain (iii) a third polypeptide comprising, in an N-terminal to C-terminal direction, a second heavy chain variable domain VH2 and a second CH1 domain CH1b that binds a second antigen; and (iv) a fourth polypeptide comprising, in N-terminal to C-terminal direction, a second light chain variable domain VL2 and a second CL domain CLb that binds a second antigen; and a second Fab domain comprising: wherein VL1 and VH1 bind to form the first antigen-binding domain, and VL2 and VH2 bind to form the second antigen-binding domain; The polypeptide conjugate of any one of claims 1 to 8, wherein CH1a and CLa can pair, CH1b and CLb can pair, and the binding pairs of CH1a and CLa and CH1b and CLb are configured to avoid mispairing between CH1a and CLb and / or between CH1b and CLa.

44. 44. The polypeptide conjugate of claim 43, wherein the first antigen-binding domain comprises a GPRC5D-binding domain as defined in any one of claims 1 to 7.

45. 45. The polypeptide conjugate of claim 43 or 44, wherein the second antigen-binding domain comprises a CD3-binding domain as defined in claim 12 or 13.

46. 46. ​​The polypeptide conjugate of any one of claims 43 to 45, wherein the binding pair of CH1b and CLb has at least one non-natural disulfide bond that prevents mispairing between CH1b and CLa and / or between CH1a and CLb.

47. The polypeptide conjugate of claim 46, wherein the first CH1 / CL binding pair and the second CH1 / CL binding pair are selected from CH1b / CLb and CH1a / CLa, respectively, and the first CH1 / CL binding pair is bound via a first pair of disulfide bonds, the first pair of disulfide bonds being non-natural, and optionally, the originally naturally occurring disulfide bond in the first CH1 / CL binding pair is deleted or destroyed.

48. The polypeptide conjugate of claim 47, wherein the second CH1 / CL binding pair is formed from a second pair of disulfide bonds, the second pair of disulfide bonds being in a different position than the first pair of disulfide bonds and optionally being a naturally occurring disulfide bond.

49. The first pair of disulfide bonds is a) heavy chain EU numbering position 126 and light chain EU numbering position 121 in the first CH1 / CL binding pair; b) heavy chain EU numbering position 173 and light chain EU numbering position 160 in the first CH1 / CL binding pair; and c) heavy chain EU numbering position 128 and light chain EU numbering position 118 in the first CH1 / CL binding pair; 49. The polypeptide conjugate of claim 47 or 48, formed by two cysteines introduced at positions selected from the group:

50. 50. The polypeptide conjugate of any one of claims 47 to 49, wherein the naturally occurring disulfide bond is formed between heavy chain EU numbering position 220 and light chain EU numbering position 214.

51. 51. The polypeptide conjugate of any one of claims 47 to 50, wherein the first CH1 / CL pair comprises CH1 mutated to a cysteine ​​residue at EU numbering position 126 and to a non-cysteine ​​residue at position 220, and CL mutated to a cysteine ​​residue at EU numbering position 121 and to a non-cysteine ​​residue at position 214.

52. 52. The polypeptide conjugate of any one of claims 47 to 51, wherein the first CH1 / CL binding pair comprises a mutation of at least one uncharged amino acid residue to a charged amino acid residue and / or a mutation of at least one charged amino acid residue to an oppositely charged amino acid residue, such that the first CH1 / CL binding pair comprises a first pair of oppositely charged residues, and the first pair of oppositely charged residues promotes pairing of the first CH1 / CL binding pair.

53. The polypeptide conjugate of claim 52, wherein the second CH1 / CL pair comprises a mutation of at least one uncharged amino acid residue to a charged amino acid residue and / or a mutation of at least one charged amino acid residue to an oppositely charged amino acid residue, such that the second CH1 / CL binding pair comprises a second pair of oppositely charged residues, the second pair of oppositely charged residues promoting pairing of the second CH1 / CL binding pair, and optionally, the first pair of oppositely charged residues and the second pair of oppositely charged residues preventing pairing of CH1a and CLb or pairing of CH1b and CLa.

54. 54. The polypeptide conjugate of claim 53, wherein the first pair of oppositely charged residues and the second pair of oppositely charged residues are designed so that both CH1a and CLb are positively or negatively charged, and / or so that both CH1b and CLa are positively or negatively charged.

55. The first pair of oppositely charged residues and / or the second pair of oppositely charged residues are a) heavy chain EU numbering position 183 and light chain EU numbering position 176 in the first CH1 / CL binding pair; b) heavy chain EU numbering position 183 and light chain EU numbering position 133 in the first CH1 / CL binding pair; c) heavy chain EU numbering position 147 and light chain EU numbering position 176 in the first CH1 / CL binding pair; d) heavy chain EU numbering position 141 and light chain EU numbering position 116 in the first CH1 / CL binding pair; e) heavy chain EU numbering position 126 and light chain EU numbering position 121 in the first CH1 / CL binding pair, and f) heavy chain EU numbering position 218 and light chain EU numbering position 122 in the first CH1 / CL binding pair; 55. The polypeptide conjugate of any one of claims 52 to 54, wherein a pair of oppositely charged amino acid residues is introduced at a heavy chain-light chain EU number position selected from the group consisting of:

56. 56. The polypeptide conjugate of any one of claims 52 to 55, wherein the pair of oppositely charged amino acid residues comprises one positively charged amino acid residue and one negatively charged amino acid residue, the positively charged amino acid residue being selected from the group of lysine (K), histidine (H) and arginine (R), and / or the negatively charged amino acid residue being selected from the group of aspartic acid (D) and glutamic acid (E).

57. 57. The polypeptide conjugate of any one of claims 52 to 56, wherein the binding pair of CH1b and CLb comprises a first pair of non-natural disulfide bonds and a first pair of oppositely charged residues that prevent mispairing between CH1b and CLa and / or between CH1a and CLb.

58. 58. The polypeptide complex of claim 57, wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 39, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 40, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 38, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

37.

59. 59. The polypeptide conjugate of any one of claims 43 to 58, wherein the third polypeptide further comprises a first Fc polypeptide at its C-terminus, and the first polypeptide further comprises a second Fc polypeptide at its C-terminus.

60. 60. The polypeptide complex of any one of claims 43 to 59, wherein the polypeptide complex further comprises a third antigen-binding domain, and optionally, the third antigen-binding domain is a Fab domain.

61. 61. The polypeptide conjugate of claim 60, wherein the C-terminus of one of the third antigen-binding domains is linked to the N-terminus of one of the second antigen-binding domains.

62. the third antigen-binding domain is the same as the first antigen-binding domain; and (i) a first fragment comprising, in an N-terminal to C-terminal direction, a first heavy chain variable domain VH1 and a first CH1 domain CH1a that binds a first antigen; and (ii) a second fragment comprising, in an N-terminal to C-terminal direction, a first light chain variable domain VL1 and a first CL domain CLa that binds to the first antigen; and the C-terminus of the first fragment is linked to the N-terminus of the fourth polypeptide.

63. The polypeptide complex comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and a fifth polypeptide, in the N-terminal to C-terminal direction: (i) the first polypeptide comprises VH1-CH1a; (ii) the third polypeptide comprises VH2-CH1b; (iii) the fourth polypeptide comprises VH1-CH1a-linker-VL2-CLb; and (iv) the second polypeptide and the fifth polypeptide are the same and both comprise VL1-CLa.

64. 64. The polypeptide conjugate of claim 63, wherein the binding pair of CH1b and CLb has at least one non-natural disulfide bond that prevents mispairing between CH1b and CLa and / or between CH1a and CLb.

65. The polypeptide conjugate of claim 64, wherein the binding pair of CH1b and CLb has one or more introduced amino acid mutations to form at least one introduced charged amino acid residue that prevents mispairing between CH1b and CLa and / or between CH1a and CLb.

66. The polypeptide complex of claim 65, wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 43, the second polypeptide or the fifth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 44, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 42, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

41.

67. The polypeptide complex of claim 65, wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 47, the second polypeptide or the fifth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 48, the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 46, and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

45.

68. 68. The polypeptide conjugate of any one of claims 43 to 67, wherein the third polypeptide further comprises, in an N-terminal to C-terminal direction, a first Fc polypeptide, and the first polypeptide comprises, in an N-terminal to C-terminal direction, a second Fc polypeptide.

69. 69. The polypeptide conjugate of claim 28, 42, 59 or 68, wherein the first Fc polypeptide and / or the second Fc polypeptide is derived from IgG1, IgG2, IgG3 or IgG4.

70. 70. The polypeptide conjugate of claim 69, wherein the first Fc polypeptide and the second Fc polypeptide have different amino acid sequences and are at least designed to promote heterodimerization of the first Fc polypeptide and the second Fc polypeptide.

71. one of the first Fc polypeptide and the second Fc polypeptide comprises a first Fc mutation and the other comprises a second Fc mutation, and the first Fc mutation and the second Fc mutation comprise: a) combinations of T366W or S354C with Y349C, T366S, L368A or Y407V; b) D399K or E356K in combination with K392D or K409D; c) E356K, E357K or D399K in combination with K370E, K409D or K439E; d) S364H or F405A in combination with Y349T or T394F; e) S364H or T394F in combination with Y394T or F405A; f) a combination of K370D or K409D with E357K or D399K, or g) L351D or L368E in combination with L351K or T366K; 71. The polypeptide conjugate of claim 70, comprising:

72. 72. The polypeptide conjugate of claim 71, wherein the first Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 65 or SEQ ID NO: 67, and the second Fc polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

66.

73. A nucleic acid comprising a nucleotide sequence encoding a polypeptide conjugate according to any one of claims 1 to 72.

74. A vector comprising the nucleic acid of claim 73.

75. 75. A host cell comprising the nucleic acid of claim 73 or the vector of claim 74.

76. A pharmaceutical composition comprising the polypeptide complex of any one of claims 1 to 72 or the nucleic acid of claim 73, and a pharmaceutically acceptable vector.

77. 73. A conjugate comprising the polypeptide conjugate of any one of claims 1 to 72 and a payload conjugated thereto, wherein the payload is selected from the group consisting of a radioactive label, a fluorescent label, an enzyme substrate label, an affinity purification tag, a tracking molecule, an anti-cancer drug and a cytotoxic molecule.

78. A composition comprising a polypeptide conjugate according to any one of claims 1 to 72 or a conjugate according to claim 77, and a pharmaceutically acceptable carrier.

79. 78. A method for treating or preventing a disease, condition or symptom, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide conjugate of any one of claims 1 to 72, a pharmaceutical composition of claim 76, a conjugate of claim 77 or a composition of claim 78.

80. 80. The method of claim 79, wherein the disease, condition or symptom is selected from the group of cancer, immune disease, inflammation.

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