Multi-specific polypeptide complexes targeting GPRC5D

Abstract

A multispecific polypeptide complex, an isolated polynucleotide encoding the same, a pharmaceutical composition comprising the same, and uses thereof.

Description

Multispecific peptide complex targeting GPRC5D Field of the Invention

[0001] The present application generally relates to multispecific polypeptide complexes targeting GPRC5D and their uses. Background Art

[0002] In the field of antibody therapy, bispecific or multispecific antibodies, which are currently being actively studied, can simultaneously recognize two or more different antigens, neutralize different pathogenic mediators, recruit different types of effector cells, and regulate signaling pathways, making them superior to monospecific antibodies in many aspects. Therefore, the development of bispecific or multispecific antibodies as therapeutic agents for human diseases has important clinical significance. In recent years, bispecific antibodies have become a widely used modality for 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 an orphan receptor, has become a popular target. Overexpression of GPRC5D has been reported in patients with multiple myeloma. In particular, high expression is significantly correlated with poor disease and treatment outcomes. Given the specific high expression of GPRC5D on tumor cells, GPRC5D is likely to become the next hot candidate for the treatment of multiple myeloma. When specific targeting or killing of cells overexpressing GPRC5D is desired, multispecific antibodies with GPRC5D as one of the targets (e.g., bispecific or multispecific antibodies that simultaneously target the tumor antigen GPRC5D and an immunostimulatory antigen (such as CD3) or other tumor antigens (such as Her2)) are of great research interest.

[0004] Therefore, there is an urgent need in this field to develop new multispecific antibodies with GPRC5D as one of the targets.

[0005] Summary of the Invention

[0006] Throughout this application, the articles "a", "an" and "the" are used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, "an antibody" means one antibody or more than one antibody.

[0007] The present application provides a multispecific polypeptide complex, an isolated polynucleotide encoding the same, a pharmaceutical composition comprising the same, and uses thereof.

[0008] In one aspect, the present application provides a multispecific polypeptide complex 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 complementary determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein the heavy chain complementary determining regions are bound to the heavy chain variable region (V L L R ... H ) are identical to the three heavy chain complementary determining regions contained in the light chain variable region (V L ) are identical in the three light chain complementarity determining regions.

[0009] In some embodiments, the GPRC5D binding domain comprises three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3), and three light chain complementary 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 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) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 or a variant thereof having no more than 3, 2, or 1 amino acid substitutions, or SEQ ID NO: 10 or a variant thereof having no more than 3, 2, or 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 no more than 3, 2, or 1 amino acid substitutions, or SEQ ID NO: 11 or a variant thereof having no more than 3, 2, or 1 amino acid substitutions; d) the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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 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.

[0010] In some embodiments, in the GPRC5D binding domain, a) the HCDR1 comprises the amino acid sequence of SEQ ID NO:4 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR2 comprises the amino acid sequence of SEQ ID NO:5 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR3 comprises the amino acid sequence of SEQ ID NO:6 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR1 comprises the amino acid sequence of SEQ ID NO:1 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR2 comprises the amino acid sequence of SEQ ID NO:2 or a variant having no more than 3, 2 or 1 amino acid substitutions, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:3 or a variant having no more than 3, 2 or 1 amino acid substitutions; or the HCDR1 comprises the amino acid sequence of SEQ ID NO:9 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR2 comprises the amino acid sequence of SEQ ID NO:10 or a variant having no more than 3, 2 or 1 amino acid substitutions. NO: 10 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 11 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 7 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 8 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 3 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions.

[0011] In some embodiments, in the GPRC5D binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13 or a variant having no more than 3, 2, or 1 amino acid substitutions, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12 or a variant having no more than 3, 2, or 1 amino acid substitutions.

[0012] In some embodiments, the GPRC5D binding domain is humanized.

[0013] In some embodiments, the GPRC5D binding domain includes 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 consisting of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, or a variant having no more than 3, 2, or 1 amino acid substitutions; b) the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or a variant having no more than 3, 2, or 1 amino acid substitutions.

[0014] In some embodiments, the GPRC5D binding domain includes 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 of SEQ ID NO: 15 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 of SEQ ID NO: 14 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17 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 of SEQ ID NO: 16 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 19 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 of SEQ ID NO: 18 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21 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 of SEQ ID NO: The amino acid sequence shown in NO:20 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; wherein the amino acid substitutions are not in the CDR regions.

[0015] In some embodiments, the polypeptide complex 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.

[0016] In some embodiments, one of the first and second antigen-binding domains binds to GPRC5D and comprises a GPRC5D binding domain as described herein, and the other binds to an antigen different from GPRC5D. In some embodiments, the antigen different from GPRC5D is an immunostimulatory antigen, optionally CD3. In some embodiments, one of the first and second antigen-binding domains comprises a GPRC5D binding domain as described herein, and the other comprises a CD3 binding domain.

[0017] In some embodiments, the CD3 binding domain comprises three heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO:49, or a variant thereof having no more than 3, 2, or 1 amino acid substitutions, the HCDR2 comprises the amino acid sequence of SEQ ID NO:50, or a variant thereof having no more than 3, 2, or 1 amino acid substitutions, the HCDR3 comprises the amino acid sequence of SEQ ID NO:51, or a variant thereof having no more than 3, 2, or 1 amino acid substitutions, the LCDR1 comprises the amino acid sequence of SEQ ID NO:52, or a variant thereof having no more than 3, 2, or 1 amino acid substitutions, the LCDR2 comprises the amino acid sequence of SEQ ID NO:53, or a variant thereof having no more than 3, 2, or 1 amino acid substitutions, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:54, or a variant thereof having no more than 3, 2, or 1 amino acid substitutions. In some embodiments, in the CD3 binding domain, the HCDR1 comprises the amino acid sequence of SEQ ID NO:92 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR2 comprises the amino acid sequence of SEQ ID NO:93 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR3 comprises the amino acid sequence of SEQ ID NO:94 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR1 comprises the amino acid sequence of SEQ ID NO:95 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR2 comprises the amino acid sequence of SEQ ID NO:96 or a variant having no more than 3, 2 or 1 amino acid substitutions, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:54 or a variant having no more than 3, 2 or 1 amino acid substitutions.

[0018] In some embodiments, the CD3 binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 55 or a variant having no more than 3, 2 or 1 amino acid substitutions, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 56 or a variant having no more than 3, 2 or 1 amino acid substitutions.

[0019] In some embodiments, the first antigen-binding domain and the second antigen-binding domain constitute a DICAD domain, which comprises: (i) a first polypeptide comprising, in the 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 directly connected or connected through a first linker; and (ii) a second polypeptide comprising, in the 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 directly connected or connected through a second linker; wherein VL1 and VH1 combine to form the first antigen-binding domain, VL2 and VH2 combine to form the second antigen-binding domain, and VL1 and VH1 are covalently linked through a disulfide bond.

[0020] In some embodiments, the first linker and / or the second linker each independently comprises 5 to 9 amino acid residues.

[0021] 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 cysteine ​​and the second cysteine ​​form a disulfide bond.

[0022] In some embodiments, the first and second cysteine ​​residues 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.

[0023] 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.

[0024] In some embodiments, the VL2 and VH2 further have an electrostatic interaction between two oppositely charged residues. In some embodiments, the two oppositely charged residues between the VL2 and VH2 are introduced and 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, wherein the numbering is according to Kabat numbering. In some embodiments, the two oppositely charged residues comprise a negatively charged amino acid residue selected from the group consisting of aspartic acid (D) or glutamic acid (E), and a positively charged amino acid residue selected from the group consisting of lysine (K) or arginine (R).

[0025] In some embodiments, at least one of the residues in the FR of VL1 is substituted with a negatively charged amino acid and at least one of the residues in the FR of VH1 is substituted with a positively charged amino acid, or at least one of the residues in the FR of VL1 is substituted with a positively charged amino acid and at least one of the residues in the FR of VH1 is substituted with a negatively charged amino acid.

[0026] In some embodiments, the first antigen-binding domain comprises an antigen-binding fragment of an antibody that binds GPRC5D as 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 a first Fc polypeptide in the N-terminal to C-terminal direction. In some embodiments, the polypeptide complex further comprises a third polypeptide that comprises a second Fc polypeptide in the N-terminal to C-terminal direction.

[0027] 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, from the N-terminus to the C-terminus, a third heavy chain variable domain VH3 and a CH1 domain that bind to a third antigen; and (ii) a fourth polypeptide comprising, from the N-terminus to the C-terminus, a third light chain variable domain VL3 and a CL domain that bind to a third antigen; wherein VL3 and VH3 combine to form the third antigen-binding domain.

[0028] In some embodiments, the Fab domain binds to GPRC5D and comprises a GPRC5D binding domain as described herein. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO:29, the second polypeptide comprises the amino acid sequence of SEQ ID NO:30, the third polypeptide comprises the amino acid sequence of SEQ ID NO:31, and the fourth polypeptide comprises the amino acid sequence of SEQ ID NO:32.

[0029] In some embodiments, the polypeptide complex further comprises a third antigen-binding domain, optionally, the third antigen-binding domain comprises a Fab domain, wherein the Fab domain comprises: (i) a third polypeptide comprising, in the N-terminal to C-terminal direction, a third heavy chain variable domain VH3 and a CH1 domain that binds to a third antigen; and (ii) a fourth polypeptide comprising, in the N-terminal to C-terminal direction, a third light chain variable domain VL3 and a CL domain that binds to a third antigen; wherein VL3 and VH3 combine to form the third antigen-binding domain.

[0030] In some embodiments, the first antigen, the second antigen, and the third antigen are independently selected from GPRC5D, an immunostimulatory antigen, and a tumor antigen. 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.

[0031] In some embodiments, the Her2 binding domain is contained in the heavy chain variable region (V H ) within the three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and within the light chain variable region (V L), the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 57 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 58 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 59 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 60 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 61 or a variant having no more than 3, 2 or 1 amino acid substitutions, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 62 or a variant having no more than 3, 2 or 1 amino acid substitutions.

[0032] In some embodiments, the Her2 binding domain comprises a light chain variable domain having an amino acid sequence as shown in SEQ ID NO:64, and VH3 comprises a heavy chain variable domain having an amino acid sequence as shown in SEQ ID NO:63.

[0033] 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 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.

[0034] In some embodiments, the first antigen is GPRC5D, the second antigen is GPRC5D, and the third antigen is CD3, and the first and second antigen-binding domains comprise an antigen-binding fragment of an antibody that binds GPRC5D as described herein, and the third antigen-binding domain comprises a CD3-binding domain as defined herein. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO:33, the second polypeptide comprises the amino acid sequence of SEQ ID NO:34, the third polypeptide comprises the amino acid sequence of SEQ ID NO:35, and the fourth polypeptide comprises the amino acid sequence of SEQ ID NO:36.

[0035] In some embodiments, the second polypeptide further comprises a first Fc polypeptide in the N-terminal to C-terminal direction, and / or the third polypeptide further comprises a second Fc polypeptide in the N-terminal to C-terminal direction, and the first Fc polypeptide and the second Fc polypeptide are capable of binding to form a dimer.

[0036] In some embodiments, the polypeptide complex 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, from N-terminus to C-terminus, a first heavy chain variable domain VH1 and a first CH1 domain CH1a that bind to a first antigen; and (ii) a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain VL1 and a first CL domain CLa that bind to a first antigen; the second antigen-binding domain comprises a second Fab domain comprising: (iii) a third polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain VH1 and a first CH1 domain CH1a that bind to a first antigen. In the N-terminal to C-terminal direction, it comprises a second heavy chain variable domain VH2 and a second CH1 domain CH1b that binds to a second antigen; and (iv) a fourth polypeptide, which comprises a second light chain variable domain VL2 and a second CL domain CLb that binds to a second antigen in the N-terminal to C-terminal direction; 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 bind, CH1b and CLb can pair and bind, and the CH1a and CLa binding pair and the CH1b and CLb binding pair are configured to avoid mispairing between CH1a and CLb and / or between CH1b and CLa.

[0037] 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.

[0038] In some embodiments, the CH1b and CLb binding pair in the polypeptide complex has at least one non-native disulfide bond that hinders mispairing between CH1b and CLa and / or between CH1a and CLb.

[0039] In some embodiments, the first CH1 / CL binding pair and the second CH1 / CL binding pair in the polypeptide complex are selected from CH1b / CLb and CH1a / CLa, respectively, and the first CH1 / CL binding pair is bound by a first pair of disulfide bonds, the first pair of disulfide bonds is non-natural, and optionally, the naturally occurring disulfide bonds in the first CH1 / CL binding pair are deleted or disrupted. In some embodiments, the second CH1 / CL binding pair is formed by a second pair of disulfide bonds, the second pair of disulfide bonds is located at a different position than the first pair of disulfide bonds, and is optionally a naturally occurring disulfide bond.

[0040] 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.

[0041] In some embodiments, the naturally occurring disulfide bond is formed between EU numbering position 220 of the heavy chain and EU numbering position 214 of the light chain. In some embodiments, the first CH1 / CL pair comprises a CH1 in which EU numbering position 126 is mutated to a cysteine ​​residue and position 220 is mutated to a non-cysteine ​​residue; and a CL in which EU numbering position 121 is mutated to a cysteine ​​residue and position 214 is mutated to a non-cysteine ​​residue.

[0042] In some embodiments, the first CH1 / CL binding pair comprises at least one mutation from an uncharged amino acid residue to a charged amino acid residue, and / or at least one mutation from a 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. In some embodiments, the second CH1 / CL pair comprises at least one mutation from an uncharged amino acid residue to a charged amino acid residue, and / or at least one mutation from a 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, and the second pair of oppositely charged residues promotes 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 hinder pairing of CH1a and CLb or pairing of CH1b and CLa.

[0043] In some embodiments, the first pair of oppositely charged residues and the second pair of oppositely charged residues are designed such that CH1a and CLb are both positively charged or both negatively charged, and / or CH1b and CLa are both positively charged or both negatively charged.

[0044] In some embodiments, the first pair of oppositely charged residues and / or the second pair of oppositely charged residues introduce an oppositely charged pair of amino acid residues at a heavy chain-light chain EU numbering position 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.

[0045] In some embodiments, the pair of oppositely charged amino acid residues includes a positively charged amino acid residue and a negatively charged amino acid residue, wherein the positively charged amino acid residue is selected from the group consisting of lysine (K), histidine (H) and arginine (R), and / or the negatively charged amino acid residue is selected from the group consisting of aspartic acid (D) and glutamic acid (E).

[0046] In some embodiments, the CH1b and CLb binding pair in the polypeptide complex has a first pair of non-native disulfide bonds and a first pair of oppositely charged residues that hinder mispairing between CH1b and CLa and / or between CH1a and CLb.

[0047] In some embodiments, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:39, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:40, the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:38, and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO:37.

[0048] In some embodiments, the third polypeptide of the polypeptide complex comprises a first Fc polypeptide in the N-terminal to C-terminal direction, and the first polypeptide further comprises a second Fc polypeptide in the N-terminal to C-terminal direction.

[0049] In some embodiments, the polypeptide complex further comprises a third antigen-binding domain, optionally, the third antigen-binding domain is a Fab domain. In some embodiments, a C-terminus of the third antigen-binding domain is connected to an N-terminus of the second antigen-binding domain. In some embodiments, the third antigen-binding domain is identical to the first antigen-binding domain and comprises: (i) a first fragment comprising a first heavy chain variable domain VH1 and a first CH1 domain CH1a that bind to a first antigen in the N-terminal to C-terminal direction; and (ii) a second fragment comprising a first light chain variable domain VL1 and a first CL domain CLa that bind to a first antigen in the N-terminal to C-terminal direction; and wherein the C-terminus of the first fragment is connected to the N-terminus of the fourth polypeptide.

[0050] In some embodiments, the polypeptide complex comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and a fifth polypeptide, wherein 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, each comprising VL1-CLa.

[0051] In some embodiments, the CH1b and CLb binding pair in the polypeptide complex has at least one non-native disulfide bond that hinders mispairing between CH1b and CLa and / or between CH1a and CLb. In some embodiments, the CH1b and CLb binding pair in the polypeptide complex has one or more introduced amino acid mutations and forms at least one introduced charged amino acid residue that hinders mispairing between CH1b and CLa and / or between CH1a and CLb.

[0052] In some embodiments, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:43, the second polypeptide or the fifth polypeptide comprises the amino acid sequence shown in SEQ ID NO:44, the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:42, and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO:41.

[0053] In some embodiments, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:47, the second polypeptide or the fifth polypeptide comprises the amino acid sequence shown in SEQ ID NO:48, the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:46, and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO:45.

[0054] In some embodiments, the third polypeptide of the polypeptide complex further comprises a first Fc polypeptide in the N-terminal to C-terminal direction, and the first polypeptide further comprises a second Fc polypeptide in the N-terminal to C-terminal direction.

[0055] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide in the polypeptide complex is derived from IgG1, IgG2, IgG3, or IgG4.

[0056] 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.

[0057] 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 comprise the following combinations: 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 D39 d) S364H or F405A, in combination with Y349T or T394F; e) S364H or T394F, in combination with Y394T or F405A; f) K370D or K409D, in combination with E357K or D399K; or g) L351D or L368E, in combination with L351K or T366K; wherein amino acid positions are numbered according to the EU numbering system.

[0058] 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.

[0059] In another aspect, the present application provides a nucleic acid comprising a nucleotide sequence encoding the polypeptide complex described herein.

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

[0061] In another aspect, the present application provides a host cell comprising the nucleic acid described herein or the vector described herein.

[0062] In another aspect, the present application provides a pharmaceutical composition comprising the polypeptide complex described herein or the nucleic acid described herein, and a pharmaceutically acceptable carrier.

[0063] On the other hand, the present application provides a conjugate comprising the polypeptide complex described herein and a cargo conjugated thereto, wherein the cargo is selected from the group consisting of a radioactive label, a fluorescent label, an enzyme substrate label, an affinity purification label, a tracer molecule, an anticancer drug, and a cytotoxic molecule.

[0064] In another aspect, the present application provides a composition comprising the polypeptide complex or the conjugate described herein, and a pharmaceutically acceptable carrier.

[0065] 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 the polypeptide complex described herein, the pharmaceutical composition described herein, the conjugate described herein or the composition described herein.

[0066] In one embodiment, the disease, condition or symptom is selected from the group consisting of cancer, immune disease, and inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG1 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to hGPRC5D-expressing HEK293 cells in an antigen binding FACS experiment.

[0068] FIG2 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to CHOS cells expressing hGPRC5D in an antigen binding FACS experiment.

[0069] FIG3 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to MM.1R cells naturally expressing GPRC5D in an antigen binding FACS experiment.

[0070] FIG4 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to NCI-H929 cells in an antigen binding FACS experiment.

[0071] FIG5 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to RPMI-8226 cells in an antigen binding FACS experiment.

[0072] FIG6 shows the cytotoxic effects of GPRC5D antibodies (ch-72C7 and GC5B596) on NCI-H929 cells naturally expressing GPRC5D in an ADCC effect evaluation experiment.

[0073] FIG7 shows the cytotoxic effects of GPRC5D antibodies (ch-72C7 and GC5B596) on MM.1R cells naturally expressing GPRC5D in an ADCC effect evaluation experiment.

[0074] FIG8 shows the CD3×GPRC5D bispecific antibody structure A.

[0075] FIG9 shows the CD3×GPRC5D bispecific antibody structure B.

[0076] FIG10 shows the CD3×GPRC5D bispecific antibody structure C.

[0077] FIG11 shows the CD3×GPRC5D bispecific antibody structure D.

[0078] FIG12 shows the binding of the bispecific antibody to HEK293T-hGPRC5D (human GPRC5D).

[0079] FIG13 shows the binding of the bispecific antibody to HEK293T-cynoGPRC5D (monkey GPRC5D).

[0080] FIG14 shows the bispecific antibody (22A1-5 and 22B1)-mediated PBMC-induced proliferation inhibition of NCI-H929 cells.

[0081] FIG15 shows the bispecific antibody (22A6 and 10B1)-mediated PBMC-induced proliferation inhibition of NCI-H929 cells.

[0082] FIG16 shows the bispecific antibody (22A8, 10B1 and 22B1)-mediated PBMC-induced proliferation inhibition of NCI-H929 cells.

[0083] FIG17 shows the bispecific antibody (22A8 and 22B1)-mediated PBMC-mediated inhibition of MM1S cell proliferation.

[0084] FIG18 shows the bispecific antibody (22A8 and 22B1)-mediated inhibitory effect of PBMC on the proliferation of RPMI-8226 cells.

[0085] FIG19 shows the bispecific antibody (22A8 and 22B1)-mediated PBMC-mediated inhibition of KMS-12-BM cell proliferation.

[0086] FIG20 shows the inhibitory effect of bispecific antibodies (22A2 and 22A8) on subcutaneous transplanted tumors in mice immunized with human PBMCs and reconstituted with human myeloma NCI-H929.

[0087] FIG21 shows the inhibitory effect of bispecific antibodies (22B1 and 22A8) on subcutaneous xenograft tumors in mice immunized with human PBMCs and reconstituted with human myeloma NCI-H929.

[0088] Figure 22 shows the full-length sequences of each of the specific antibodies of the present application (22A1, 22A2, 22A3, 22A4, 22A5, 22A6, 22A8 and 22B1).

[0089] Figures 22 (A) to (H) show the amino acid sequences in the polypeptide complex provided by the present application, as well as the mutant sequences of VH and VL in each antigen-binding domain targeting GPRC5D, CD3 and Her2, and the mutant sequences of CH1 and CL regions. DETAILED DESCRIPTION

[0090] The following description of the present application is intended only to illustrate various embodiments of the present application. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present application. It will be apparent to those skilled in the art that various equivalents, variations and modifications can be made without departing from the scope of the present application, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0091] definition

[0092] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that binds to a specific antigen. Natural intact antibodies include two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ and μ, each of which consists of a variable region (V H ) and the first, second, third and optionally fourth constant regions (respectively C H1 、C H2 、C H3 、C H4 ); mammalian light chains are classified as λ or κ, and each light chain consists of a variable region (V L) and constant regions. Antibodies are "Y" shaped, where the stem of the Y consists of the second and third constant regions of two heavy chains bound together by 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 of the two chains generally contain three highly variable loops called complementarity determining regions (CDRs) (the light chain CDRs comprise LCDR1, LCDR2, and LCDR3, and the heavy chain CDRs comprise HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol. 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., Sequences of Proteins of Immunological Interest. 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 inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold that supports 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 classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are 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).

[0093] In this application, the numbering of amino acid residue positions in the constant region of an antibody is based on the EU numbering system, which can be found in, for example, Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969), and the numbering of amino acid residue positions in the variable region of an antibody is based on the Kabat numbering system, which can be found in, for example, 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 can be found in the IMGT scientific table, which is available on the website of the international ImMunoGeneTics information system.

[0094] 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; and the term "multivalent" refers to an antibody or antigen-binding fragment that has multiple antigen-binding sites. In some embodiments, the antibody or antigen-binding fragment thereof is monovalent, bivalent, or multivalent.

[0095] As used herein, a "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes can be present on the same antigen, or they can be present on two different antigens.

[0096] As used herein, a "multispecific" antibody refers to 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. The two or more different epitopes may be present on the same antigen, or they may be present on different antigens.

[0097] As used herein, the term "antigen binding fragment" refers to an antibody fragment formed by a portion of an antibody including one or more CDRs, or any other antibody fragment that binds to an antigen but does not include a complete native antibody structure. Examples of antigen binding fragments include, but are not limited to, bifunctional antibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (divalent bifunctional antibodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen binding fragment is capable of binding to the same antigen to which the parent antibody binds.

[0098] "Fab" with respect to antibodies refers to the portion of an antibody consisting of a single light chain (variable and constant regions) bound by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0099] "Fab'" refers to the Fab fragment including a portion of the hinge region.

[0100] "F(ab')2" refers to a dimer of Fab'. "Fv" with respect to antibodies refers to the smallest antibody fragment with a complete antigen-binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0101] "dsFv" refers to a disulfide-stabilized Fv fragment in which the linkage 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, "(dsFv)2" or "(dsFv-dsFv')" comprises three peptide chains: one connected by a peptide linker (e.g., a longer flexible linker) and each linked to two V chains by a disulfide bridge. L Partially combined two V H In some embodiments, the dsFv-dsFv' is bispecific, wherein each disulfide-bonded paired heavy and light chain has a different antigen specificity.

[0102] 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 linked to each other directly or through a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0103] "Fc" in relation to antibodies (e.g., antibodies of the IgG, IgA, or IgD isotype) refers to the portion of the antibody consisting of the second and third constant domains of the first heavy chain bound by disulfide bonds to the second and third constant domains of the second heavy chain. The Fc of antibodies of the IgM and IgE isotypes further includes a fourth constant domain. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.

[0104] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc region of an antibody.

[0105] A "camelized single domain antibody," "heavy chain antibody," or "HCAb" is a H Heavy chain antibodies are antibodies that contain a heavy chain domain and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;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). Despite the lack of light chains, camelid antibodies have a bona fide antigen-binding repertoire (Hamers-Casterman C. et al. Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK. et al. Immunogenetics. Apr;54(1):39-47 (2002); Nguyen VK. et al. Immunology. May;109(1):93-101 (2003)). The variable domain of a heavy-chain antibody (VHH domain) represents the smallest known antigen-binding unit produced by an acquired immune response (Koch-Nolte F. et al. FASEB J Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0106] "Nanobody" refers to an antibody fragment consisting of the VHH domain from a heavy chain antibody and the two constant domains CH2 and CH3.

[0107] "Diabodies" or "dAbs" comprise small antibody fragments with two antigen-binding sites, wherein the fragments comprise both V and V domains in the same polypeptide chain. L Domain-linked V H Domain (V H -V L or V L -V H (See, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA Jul 15;90(14):6444-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen binding sites. The antigen binding sites can target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds bifunctional antibody" is a bifunctional antibody that targets two different antigens (or epitopes). In certain embodiments, a "scFv dimer" is a bivalent bifunctional antibody or bivalent scFv (BsFv) that includes V H -V L (connected by a peptide linker) to another V H -V L Partial dimerization makes a part of V H With another part of the V L Coordinate and form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific bifunctional antibody that includes V H1 -V L2 (connected by a peptide linker) and V L1 -V H2 (also linked by a peptide linker) are combined so that V H1 With V L1 Coordinated and V H2 With V L2 Each ligand pair has a different antigen specificity.

[0108] "Domain antibodies" are antibody fragments containing only the variable region of a heavy chain or the variable region of a light chain. H The domains are covalently joined using peptide linkers to produce bivalent or multivalent domain antibodies. H The domains may target the same or different antigens.

[0109] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment in which a portion of the heavy and / or light chains are derived from one species and the remainder of the heavy and / or light chains are derived from a different species. In an illustrative example, a chimeric antibody may include 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.

[0110] As used herein, the term "humanized" means that the antibody or antigen-binding fragment includes CDRs derived from non-human animals, FR regions derived from humans, and, when applicable, constant regions derived from humans.

[0111] As used herein, "GPRC5D" refers to a G protein-coupled receptor family C group 5 member D from primates (e.g., humans, monkeys). In certain embodiments, GPRC5D is human GPRC5D. Exemplary sequences of human GPRC5D include human GPRC5D protein (UniProt number Q9NZD1). Exemplary sequences of monkey GPRC5D include, for example, rhesus macaque GPRC5D protein (UniProt number F6Y5U7) or cynomolgus macaque GPRC5D protein (UniProt number A0A2K5W6I7). GPRC5D is a relatively new target for immunotherapy of multiple myeloma. It is an orphan G protein-coupled receptor of unknown function that is highly expressed on malignant bone marrow plasma cells and on hard keratinized 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 in multiple myeloma with promising results in preclinical studies (see de Larrea CF et al., Blood Cancer Discov 2020;1:146.) and is now the target of the bispecific antibody JNJ-64407564 (talquetamab) in four phase I trials.

[0112] As used herein, the term "CD3" refers to cluster of differentiation 3, which is a protein complex and T cell co-receptor that is 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 different chains. In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains bind to the T cell receptor (TCR) and the CD3ζ chain (ζ-chain) to generate activation signals in T lymphocytes. TCR, CD3ζ, and other CD3 molecules together constitute the TCR complex. Since CD3 is required for T cell activation, drugs targeting CD3 (usually monoclonal antibodies) are being studied as immunosuppressant therapies for cancer and other autoimmune diseases. Based on the CD3T cell co-receptor, new anticancer drug treatments are being developed, and molecules are designed to change co-stimulatory signals to help T cells recognize cancer cells and fully activate.

[0113] As used herein, the term "Her2" refers to human epidermal growth factor receptor 2 (human epidermal growth factor receptor 2), also known as receptor tyrosine protein kinase erbB-2, differentiation cluster 340 (cluster of differentiation 340) or proto-oncogene neu, which is encoded by gene ErbB2. ErbB is the abbreviation of erythroblastic oncogene B (erythroblastic oncogene B), belongs to the epidermal growth factor receptor family, and is composed 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). In contrast to other members of the ErbB family, Her2 does not directly bind to a ligand. When the concentration of Her2 is high (e.g., in a cancer environment), its homodimerization or its formation of a heterodimer with another ErbB member can cause the activation of Her2. Amplification or overexpression of the Her2 gene plays a significant role in the development and progression of certain aggressive breast cancers. In recent years, the Her2 protein has become an important biomarker and therapeutic target for approximately 30% of breast cancer patients.

[0114] The term "anti-GPRC5D antibody" refers to an antibody that can specifically bind to GPRC5D (eg, human GPRC5D). The term "anti-human GPRC5D antibody" refers to an antibody that can specifically bind to human GPRC5D.

[0115] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as, for example, an antibody and an antigen. Specific binding can be characterized by binding affinity, for example, as measured by K. D The value represents the ratio of the dissociation rate to the association rate (k) when the binding between the antigen and the antigen-binding molecule reaches equilibrium. off / k on ). K D It can be determined by using any conventional method known in the art, including but not limited to surface plasmon resonance, microthermophoresis, 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)K D The value can indicate specific binding between the antibody or antigen-binding fragment thereof and GPRC5D (eg, human GPRC5D).

[0116] "Conservative substitutions" with respect to amino acid sequences refer to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made between amino acid residues having hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues having neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues having acidic side chains (e.g., Asp, Glu), between amino acids having basic side chains (e.g., His, Lys, and Arg), or between residues having aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions generally do not cause significant changes in the conformational structure of the protein, and therefore can maintain the biological activity of the protein.

[0117] 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.

[0118] "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 to the amino acid (or nucleic acid) residues in the reference sequence, after aligning the candidate sequence with the reference sequence and, if necessary, introducing gaps to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for the purpose of determining percent amino acid (or nucleic acid) sequence identity can be performed, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI), see also Altschul SF et al., Journal of Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin et al., Methods in Enzymology, 267:383-402 (1997)). MA et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007) and ALIGN or Megalign (DNASTAR) software. One skilled in the art can use the default parameters provided by the tools, or can customize parameters suitable for alignment, such as by selecting an appropriate algorithm.

[0119] As used herein, "effector function" refers to the biological activity caused by the binding of the Fc region of an antibody to its effector, such as the C1 complex, and an Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC) mediated by the interaction of the antibody with C1q on the C1 complex; antibody-dependent cellular cytotoxicity (ADCC) mediated by the binding of the antibody Fc region to Fc receptors on effector cells; and phagocytosis. Effector function can be assessed using various assays, such as Fc receptor binding assays, C1q binding assays, and cell lysis assays.

[0120] An "isolated" material has been altered by the hand of man from its natural state. If an "isolated" composition or substance occurs in nature, it has been altered from its original environment, removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated," but if the polynucleotide or polypeptide is sufficiently separated from the coexisting materials of its natural state so that it exists in a substantially pure state, then the polynucleotide or polypeptide is "isolated." 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 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%, 99% pure as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatography (e.g., ion exchange chromatography or reverse phase HPLC).

[0121] As used herein, the term "vector" refers to a vehicle into which a genetic element can be operably inserted to achieve expression of the genetic element, thereby producing a protein, RNA or DNA encoded by the genetic element, or replicating the genetic element. A vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in 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 lambda phages or M13 phages; and animal viruses. A vector can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain an origin of replication. A vector can also contain substances that facilitate its entry into cells, including but not limited to viral particles, liposomes or protein envelopes. A vector can be an expression vector or a cloning vector. The present application provides a vector (eg, an expression vector) containing a nucleic acid sequence encoding a polypeptide complex provided herein, at least one promoter (eg, SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0122] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced.

[0123] As used herein, "treating" or "treatment" of a condition includes palliating the condition, slowing the rate of onset or development of the condition, reducing the risk of developing the condition, delaying the development of symptoms associated with the condition, reducing or eliminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0124] As used herein, a "GPRC5D-associated" disease or condition refers to any disease or condition caused by, exacerbated by, or otherwise associated with an increase or decrease in the expression or activity of GPRC5D. In some embodiments, the GPRC5D-associated disease or condition is cancer, such as, for example, 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, hematological cancer, lymphoma, or malignant melanoma.

[0125] 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 making up the formulation and physiologically compatible with the recipient thereof.

[0126] Multispecific peptide complexes

[0127] 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, wherein the third antigen-binding domain comprises a third antigen-binding domain that binds to a third antigen.

[0128] i. Target of the peptide complex

[0129] In one embodiment, at least one of the first and second antigen-binding domains in the polypeptide complex described herein binds to GPRC5D and comprises the GPRC5D binding domain. In some embodiments, at least one of the first, second, and third antigen-binding domains in the polypeptide complex described herein binds to GPRC5D and comprises the GPRC5D binding domain.

[0130] GPRC5D binding domain

[0131] The GPRC5D binding domain in the polypeptide complex of the present application can specifically bind to GPRC5D.

[0132] In certain embodiments, the GPRC5D binding domain is detected at a concentration of no more than 8×10 -8 M, not more than 5×10 -8 M, not more than 2×10 -8 M, not more than 8×10 -9 M, not more than 5×10 -9 M, not more than 2×10 -9 M, no more than 10 -9 M, not more than 8×10 -10 M, not more than 7×10 -10 M or no more than 6×10 -10 M's K D The value specifically binds to human GPRC5D. 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.

[0133] The binding of the GPRC5D binding domain to human GPRC5D can also be determined by the "half maximal effective concentration" (EC 50 Value) indicates that EC 50 EC refers to the antibody concentration at which 50% of its maximum binding is observed. 50 The EC values ​​can be measured by binding assays known in the art, such as sandwich assays, such as enzyme-linked immunosorbent assays (ELISA), flow cytometric assays, and other binding assays. In certain embodiments, the antibodies and fragments thereof provided herein are measured at an EC value of no more than 1 μg / ml, no more than 2 μg / ml, no more than 3 μg / ml, no more than 4 μg / ml, no more than 5 μg / ml, no more than 10 μg / ml, as measured by flow cytometry. 50 The value (i.e., 50% binding concentration) specifically binds to cells expressing human GPRC5D.

[0134] As used herein, "binding capacity" refers to the ability of a molecule (such as an antibody) to bind to another molecule (such as an antigen). The ability can be measured by, for example, binding activity with an antigen of interest using any suitable binding assay known in the art. For example, the antibody of interest can be labeled to allow direct quantitative binding activity with an antigen. For another example, the binding activity of an antibody of interest (i.e., a primary antibody) with its antigen can also be detected by using a labeled secondary antibody (e.g., an anti-species antibody), which detects the primary antibody bound to its antigen by the primary antibody in the complex, and thus indirectly quantifies the binding activity. The labeled antibody can be detected by, for example, enzyme-linked immunosorbent assay (ELISA, for example, wherein the label is an enzyme), flow cytometry (e.g., wherein the label is fluorescent), western blotting (e.g., wherein the label is fluorescent or a radioligand), colorimetry, chemiluminescence-based methods, etc.

[0135] In certain embodiments, the GPRC5D binding domain in the polypeptide complex of the present application is derived from the antigen binding domain of the anti-GPRC5D monoclonal antibody ch-72C7.

[0136] As used herein, "ch-72C7" refers to a monoclonal antibody having a light chain variable region comprising the sequence of SEQ ID NO: 12 and a heavy chain variable region comprising the sequence of SEQ ID NO: 13. ch-72C7 is a mouse antibody, and the CDR sequences therein can be divided using methods known in the art, including but not limited to the IMGT method or the Kabat numbering method.

[0137] Table 1 below shows the CDR sequences of antibody ch-72C7 divided by IMGT numbering. Table 2 below shows the CDR sequences of antibody ch-72C7 divided by Kabat numbering. Table 3 below shows the amino acid sequences of the heavy and light chain variable regions of GPRC5D.

[0138] Table 1. CDR amino acid sequences of antibody ch-72C7 based on IMGT numbering

[0139] Table 2. CDR amino acid sequences of antibody ch-72C7 based on Kabat numbering

[0140] Table 3. Amino acid sequence of the variable region of ch-72C7

[0141] In certain embodiments, the GPRC5D binding domain in the polypeptide complex of the present application comprises one or more (eg, 1, 2, 3, 4, 5, or 6) CDR sequences of the antibody ch-72C7.

[0142] In certain embodiments, the GPRC5D binding domain in the polypeptide complex 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 bound to the heavy chain variable region (V H ) are identical to the three heavy chain complementary determining regions contained in the light chain variable region (V L ) are identical in the three light chain complementarity determining regions.

[0143] In certain embodiments, the GPRC5D binding domain in the polypeptide complex of the present application includes one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs, wherein the one or more CDRs include a 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).

[0144] In certain embodiments, the GPRC5D binding domain in the polypeptide complex of the present application includes one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs, wherein the one or more CDRs include a sequence selected from the group consisting of: SASSVSFMH (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).

[0145] In certain embodiments, the GPRC5D binding domain in the polypeptide complex of the present application comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a LCDR2 comprising the sequence of SEQ ID NO: 2, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the GPRC5D binding domain in the polypeptide complex of the present application comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 11.

[0146] In certain embodiments, in the GPRC5D binding domain in the polypeptide complex of the present application, the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:4 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:5 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:6 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:1 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:2 or a variant having no more than 3, 2 or 1 amino acid substitutions, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:3 or a variant having no more than 3, 2 or 1 amino acid substitutions.

[0147] In certain embodiments, in the GPRC5D binding domain of the polypeptide complex of the present application, the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:9 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:10 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:11 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:7 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:8 or a variant having no more than 3, 2 or 1 amino acid substitutions, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:3 or a variant having no more than 3, 2 or 1 amino acid substitutions.

[0148] It is known that CDRs are responsible for antigen binding. However, it has been found that not all six CDRs are essential or unchangeable. In other words, it is possible to replace, change, or modify one or more CDRs in the anti-GPRC5D antibody ch-72C7 while substantially maintaining specific binding affinity for GPRC5D.

[0149] In certain embodiments, the GPRC5D binding domain of the polypeptide complexes provided herein comprises the heavy chain CDR3 sequence of the antibody ch-72C7. In certain embodiments, the GPRC5D binding domain of the polypeptide complexes 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 complexes provided herein comprises the heavy chain CDR3 sequence of SEQ ID NO: 11, wherein the CDR3 is numbered according to the Kabat numbering system.

[0150] 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 can specifically bind to GPRC5D. The CDR sequences provided in Table 1 or Table 2 above are obtained from mouse antibodies, but can be transplanted into any suitable FR sequence of any suitable species, such as mouse, human, rat, rabbit, etc., using suitable methods known in the art, such as recombinant technology.

[0151] In certain embodiments, the GPRC5D binding domain in the polypeptide complexes provided herein is humanized. Humanized antibodies or antigen-binding fragments are desirable in that they reduce human immunogenicity. Humanized antibodies are chimeric in their variable regions because non-human CDR sequences are transplanted to human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can be performed essentially by replacing non-human (e.g., mouse) CDR genes with corresponding human CDR genes in human immunoglobulin 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).

[0152] Suitable human heavy chain and light chain variable domains can be selected using methods known in the art to achieve this purpose. In an illustrative example, a "best fit" method can be used, wherein the database screening or BLASTization of non-human (e.g., rodent) antibody variable domain sequences for known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified, and used as the human support for transplanting non-human CDR sequences (see, for example, Sims et al., (1993) "Journal of Immunology" 151: 2296; Chothia et al. (1987) "Journal of Molecular Biology" 196: 901). Alternatively, the framework derived from the consensus sequence of all human antibodies can be used for the transplantation of non-human CDR (see, for example, Carter et al. (1992) "Proceedings of the National Academy of Sciences of the United States of America", 89: 4285; Presta et al. (1993) "Journal of Immunology", 151: 2623).

[0153] In certain embodiments, the humanized GPRC5D binding domains provided herein are composed essentially entirely of human sequences, except for CDR sequences that are non-human sequences. In some embodiments, the variable region FRs and constant regions, if present, are derived entirely or essentially from human immunoglobulin sequences. The human FR sequences and human constant region sequences can be derived from different human immunoglobulin genes, for example, the FR sequences are derived from one human antibody, while the constant regions are derived from another human antibody. In some embodiments, the humanized antibodies or antigen-binding fragments comprise human heavy chain HFR1-4 and / or light chain LFR1-4.

[0154] In some embodiments, the FR region derived from a human can include an amino acid sequence identical to the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues in a human FR are replaced with corresponding residues from a parent non-human antibody. In certain embodiments, it may be necessary to closely approximate the structure of the non-human parent antibody to produce a humanized antibody or its fragment. In certain embodiments, the humanized GPRC5D binding domain provided herein includes no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue replacements in each human FR sequence, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue replacements in all FRs of the heavy or light chain variable domains. In some embodiments, such amino acid residue changes can be present only in the heavy chain FR region, only in the light chain FR region, or in both chains.

[0155] The present application also provides an exemplary humanized GPRC5D binding domain derived from ch-72C7, comprising:

[0156] 1) “22Mono5JO4”, which includes a heavy chain variable region (22Mono5JO4-VH) as shown in the amino acid sequence of SEQ ID NO: 15 and a light chain variable region (22Mono5JO4-VL) as shown in the amino acid sequence of SEQ ID NO: 14;

[0157] 2) “22Mono3L7F”, which includes a heavy chain variable region (22Mono3L7F-VH) as set forth in the amino acid sequence of SEQ ID NO: 17 and a light chain variable region (22Mono3L7F-VL) as set forth in the amino acid sequence of SEQ ID NO: 16;

[0158] 3) “22Mono4DN4”, which includes a heavy chain variable region (22Mono4DN4-VH) as set forth in the amino acid sequence of SEQ ID NO: 19 and a light chain variable region (22Mono4DN4-VL) as set forth in the amino acid sequence of SEQ ID NO: 18;

[0159] 4) “22Mono5UQY”, which includes a heavy chain variable region (22Mono5UQY-VH) as shown in the amino acid sequence of SEQ ID NO: 21 and a light chain variable region (22Mono5UQY-VL) as shown in the amino acid sequence of SEQ ID NO: 20.

[0160] Table 4. Amino acid sequences of the variable regions of the humanized GPRC5D binding domain

[0161] In certain embodiments, the present application also provides a humanized GPRC5D binding domain, which includes HFR1, HFR2, HFR3, and / or HFR4 sequences 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).

[0162] In certain embodiments, the present application also provides a humanized GPRC5D binding domain, which includes LFR1, LFR2, LFR3 and / or LFR4 sequences contained in the 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).

[0163] 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.

[0164] These exemplary humanized GPRC5D binding domains retain specific binding ability or affinity to GPRC5D and are at least comparable to or even superior to the parental mouse antibody ch-72C7 in this regard. For example, data are provided in Examples 4 and 5.

[0165] In some embodiments, the GPRC5D binding domain in the polypeptide complex provided herein includes all or a portion of the heavy chain variable domain and / or all or a portion of the light chain variable domain. In one embodiment, the GPRC5D binding domain of the polypeptide complex provided herein is a single domain antibody composed of all or a portion of the heavy chain variable domain provided herein. More information on such single domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0166] Binding domain targeting immunostimulatory antigens

[0167] In some embodiments, the other of the first antigen-binding domain and the second antigen-binding domain in the polypeptide complex described herein binds to an antigen different from GPRC5D. In some embodiments, the antigen different from GPRC5D is an immunostimulatory antigen.

[0168] Examples of immunostimulatory antigens include, but are not limited to, 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 (

[0015] The present invention relates to a novel phosphatase inhibitory protein α (ATPase inhibitory protein α) that is specifically targeted to a phosphatase inhibitory protein α (ATPase inhibitory protein α) that is ...

[0169] In some embodiments, the immunostimulatory antigen is CD3. In some embodiments, one of the first antigen binding domain and the second antigen binding domain binds GPRC5D and the other binds CD3.

[0170] In some embodiments, the antigen-binding domain that binds to GPRC5D comprises any anti-GPRC5D antibody or anti-GPRC5D antigen-binding fragment provided herein.

[0171] In some embodiments, the polypeptide complex comprises a CD3 binding domain. In some embodiments, the CD3 binding domain is derived from a heavy chain variable region (V H ) and the light chain variable region (V L ) antibodies.

[0172] In some specific embodiments, the CD3 binding domain has a CDR sequence or VH / VL sequence as shown in Table 5 below.

[0173] Table 6. Exemplary CD3 binding domain sequences

[0174] In certain 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 three heavy chain complementarity determining regions are bound to the heavy chain variable region (V H ) are identical to the heavy chain complementary determining regions within the variable region of the light chain (V L ) are identical to the light chain complementarity determining regions within the .

[0175] 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 a HCDR1 comprising the amino acid sequence of SEQ ID NO:49, a HCDR2 comprising the sequence of SEQ ID NO:50, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:51, and / or a LCDR1 comprising the amino acid sequence of SEQ ID NO:52, a LCDR2 comprising the amino acid sequence of SEQ ID NO:53, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:54.

[0176] 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 a HCDR1 comprising the amino acid sequence of SEQ ID NO: 92, a HCDR2 comprising the sequence of SEQ ID NO: 93, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 94, and / or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 95, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 96, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 54.

[0177] In certain embodiments, the CD3 binding domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO:49 or SEQ ID NO:92, a HCDR2 comprising the sequence of SEQ ID NO:50 or SEQ ID NO:93, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:51 or SEQ ID NO:94, and / or a LCDR1 comprising the amino acid sequence of SEQ ID NO:52 or SEQ ID NO:95, a LCDR2 comprising the amino acid sequence of SEQ ID NO:53 or SEQ ID NO:96, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:54.

[0178] Binding domains targeting other tumor-associated antigens

[0179] In some embodiments, another 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 a disease-associated antigen. In some embodiments, the disease-associated antigen is a tumor-associated antigen, an immune disease-associated antigen, or an inflammatory disease-associated antigen.

[0180] Tumor-associated antigens include but are not limited to: 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, alpha-fetoprotein, BAFF, B lymphoma cells, C242 antigen, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD51, CD52, CD56, CD74, CD80, CNTO8 88, 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, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, Mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, etc.

[0181] In some embodiments, the tumor-associated antigen is Her2. In some embodiments, the polypeptide complex comprises a Her2 binding domain.

[0182] In some embodiments, the Her2 binding domain is derived from a heavy chain variable region (V H ) and the light chain variable region (V L ) antibodies.

[0183] In some specific embodiments, the Her2 binding domain has a CDR sequence or VH / VL sequence as shown in Table 4 below.

[0184] Table 5. Exemplary Her2 binding domains

[0185] In certain embodiments, the Her2 binding domain comprises three heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein the three heavy chain complementary determining regions are bound to the heavy chain variable region (V H ) are identical to the heavy chain complementary determining regions within the variable region of the light chain (V L ) are identical to the light chain complementarity determining regions within the .

[0186] 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: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 57, a HCDR2 comprising the sequence of SEQ ID NO: 58, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 59, and / or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 60, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 62.

[0187] ii. Structure of the polypeptide complex

[0188] The polypeptide complex provided herein can be in various suitable forms. In some embodiments, the polypeptide complex provided herein contains a DICAD domain.

[0189] Structure of a peptide complex containing DICAD

[0190] In some embodiments, the first antigenic domain and the second antigenic domain in the polypeptide complex of the present application constitute a DICAD domain. As used herein, the full name of the term "DICAD" is "Disulfide and Charge Adjusted Diabody", which refers to a dibody structure that introduces covalent bonds and electrostatic charges at the VH-VL interface, as disclosed in, for example, PCT application WO2019 / 120245, which is incorporated herein by reference in its entirety. This DICAD structure has the following advantages: (1) retaining the affinity, avidity, potency and other properties of each individual targeting domain; (2) having high stability and less aggregation compared to other antibodies; and (3) being easy to express and purify.

[0191] Specifically, the DICAD domain comprises (i) a first polypeptide comprising, in the 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 directly connected or connected through a first linker, and (ii) a second polypeptide comprising, in the 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 directly connected or connected through a second linker; wherein VL1 and VH1 combine to form the first antigen-binding domain, and VL2 and VH2 combine to form the second antigen-binding domain.

[0192] In some embodiments, the C-terminus of the VL1 is connected to the N-terminus of the VH2, and the C-terminus of the VL2 is connected to the N-terminus of the VH1. In some embodiments, the C-terminus of the VH1 is connected to the N-terminus of the VL2, and the C-terminus of the VH2 is connected to the N-terminus of the VL1.

[0193] 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" can be any suitable polypeptide that is capable of binding two entities to form a molecule, or maintaining an association of two entities close enough, but substantially does not interfere with the biological activity of the two entities. The linker can be composed of amino acid residues linked together by a peptide bond, and can optionally further comprise one or more non-natural amino acids. Any suitable polypeptide can be used as a linker. In some embodiments, the linker can be composed of amino acids that are not sterically hindered, such as glycine and alanine. In some embodiments, the linker is a combination of polyglycine, polyalanine, 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 following amino acid sequence RTVAA (SEQ ID NO: 74).

[0194] In some embodiments, VH1 is connected to VL2 via a first peptide linker, and VL1 is connected 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).

[0195] In some embodiments, the DICAD may further comprise one or more modifications to promote binding between the corresponding heavy and light chains, for example, between VH1 and VL1 or between VH2 and VL2. In some embodiments, non-native covalent bonds (e.g., non-native disulfide bonds) and / or electrostatic interactions may be introduced into the VH1-VL1 interaction interface and / or the VH2-VL2 interaction interface.

[0196] (a) Introducing disulfide bonds in VH / VL

[0197] In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain in the polypeptide complex comprises a first non-natural covalent bond, or alternatively, 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. For example, 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.

[0198] 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-stabilized Fv. Analysis of antibody crystal structures has shown that cysteine ​​mutations can be introduced into some relatively conserved sequences at the VL-VH interface, thereby forming a disulfide bond between VL and VH, so that they are covalently linked. The covalent bond between VL and VH significantly improves the stability of the antibody. The original dsFv (disulfide bond Fv) was constructed by introducing a disulfide bond into the VH-VL interface through covalent interactions between the cysteine ​​residues in the CDRs of each fragment (see Glockshuber, R. et al., Comparison of Strategies for Stabilizing Immunoglobulin Fv Fragments, (1990) Biochemistry, 29, 1362-1367). Although this method does not affect the activity of the antibody, the "custom" design requires detailed structural information of the original antibody CDR to avoid interfering with the antigen recognition / binding ability of the CDR, which makes this method difficult to become a universal solution for constructing various antibodies. To ensure the broad application of this method, it is necessary to conserve the amino acids at selected positions in the FR for the construction of dsFv.

[0199] Since 1993, several pairs of sites suitable for forming covalent bonds in VH-VL have been discovered, including VH44-VL100, VH105-VL43, VH100b-VL49, VH100-VL150 and VH101-VL46, wherein the numbering is based on the Kabat numbering (see Reiter, Y. et al., Stabilization of Fv fragments in recombinant immunotoxins by designing disulfide bonds in conserved framework regions, (1994) Biochemistry, 335451-5459). ; Jung, 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, 29, 1362-1367; and Zhu, Z. et al., Reshaping domain interfaces to enhance heterodimer formation, (1997) Protein Science, 6, 781-788). Among them, VH44-VL100 and VH105-VL43 are superior to other sites in various aspects (such as protein expression level, purity, Tm and affinity), and therefore are more widely used.

[0200] In some embodiments, one of the first and second antigen-binding domains in the DICAD described herein comprises a first non-native disulfide bond. In such embodiments, the other of the first and second antigen-binding domains does not contain a disulfide bond, or comprises a second non-native disulfide bond that is different from the first non-native disulfide bond.

[0201] In some embodiments, the first antigen-binding domain of the DICAD comprises a first non-native disulfide bond, and the VL1 and VH1 are covalently linked by a disulfide bond. In some embodiments, the VL1 has a first cysteine ​​substitution in the FR and the VH1 has a second cysteine ​​substitution in the FR, and the first cysteine ​​and the second cysteine ​​form a disulfide bond. In some embodiments, the first cysteine ​​and the second cysteine ​​are located in the FR2 region of the VL1 and the FR4 region of the VH1, respectively.

[0202] In some embodiments, the first cysteine ​​and the second cysteine ​​are located at position 100 in VL1 and position 44 in VH1, or at position 43 in VL1 and position 105 in VH1, or at position 49 in VL1 and position 100 in VH1, or at position 50 in VL1 and position 100 in VH1, or at position 46 in VL1 and position 101 in VH1, respectively, and the numbering is according to Kabat numbering. In some embodiments, the first cysteine ​​and the second cysteine ​​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; wherein the numbering is according to Kabat numbering.

[0203] In some embodiments, the second antigen-binding domain of the DICAD does not contain any introduced non-native disulfide bonds. In some embodiments, the native disulfide bonds in the second antigen-binding domain of the DICAD are disrupted. In some other embodiments, the second antigen-binding domain of the DICAD contains a second non-native disulfide bond that is 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 the FR and the VH2 has a fourth cysteine ​​substitution in the FR, and the third and fourth cysteine ​​residues form a disulfide bond. In some embodiments, the third and fourth cysteine ​​residues are located in the FR2 region of VL2 and the FR4 region of VH2, respectively. In some embodiments, the third cysteine ​​and the fourth cysteine ​​are located at position 100 in VL2 and position 44 in VH2, or at position 43 in VL2 and position 105 in VH2, or at position 49 in VL2 and position 100 in VH2, or at position 50 in VL2 and position 100 in VH2, or at position 46 in VL2 and position 101 in VH2, respectively, and the numbering is according to Kabat numbering. In some embodiments, the third cysteine ​​and the fourth cysteine ​​are selected from the group consisting 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.

[0204] In some embodiments, the first cysteine ​​in VL1 is located at 100C in VL1, and the second cysteine ​​in 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 embodiments, the second antigen-binding domain in DICAD does not contain a non-native disulfide bond.

[0205] (b) Replacement of charged amino acids in VH / VL

[0206] The strategy of enhancing the stable association of the corresponding VH and VL in bispecific antibodies by introducing charged amino acids is well known in the art. Tan et al. tried to influence the stability of scFv (single-chain F variant) by adjusting the amino acids at the VH-VL interface based on their electrostatic properties (see Philip H. Tan et al., Contribution of highly conserved VH-VL hydrogen bonding interactions to scFv folding stability and refolding efficiency, Biophysical Journal 1998 Sep;75(3):1473–1482.). Later, Igawa et al. modified this approach to improve scDb. The two pairs of Q39-Q38 in the 4V segment were substituted with amino acids with appropriate charges to promote or inhibit the production of certain isoforms, thereby improving the homogeneity of the product (see Igawa T et al., VH / VL interface engineering promotes the selective expression of thrombopoietin receptor agonist single-chain diabodies and inhibits conformational isomerization, Protein Engineering Design Options, August 2010; 23(8): 667-77. and WO2006106905A1). Gunasekaran et al. of Amgen further studied this method and applied it to the improvement of antibody Fab arms. Modulating electrostatic interactions at the CH1-CL interface and at 38-39 of VH-VL promotes specific interactions between CH1-VH and CL-VL (see Gunasekaran K et al., Enhancement of antibody Fc heterodimer formation by electrostatic interactions: Application to bispecific molecules and monovalent IgG. Journal of Biol. Chem. 2010 Jun 18, 285(25):19637-46.; Liu Z et al., A novel antibody engineering strategy for preparing monovalent bispecific heterodimeric IgG antibodies via an electrostatic-directed mechanism. Journal of Biol. Chem. 2015 Mar 20;290(12):7535-62.). Through these methods, each VH of a bispecific antibody can interact with the corresponding VL, thereby generating a bispecific antibody capable of binding to two antigens simultaneously.

[0207] Therefore, in some embodiments, in addition to introducing non-native disulfide bonds, DICAD in the polypeptide complexes provided herein also modifies the electrostatic orientation of specific regions to minimize undesirable nonspecific interactions. Modifications that introduce electrostatic interactions can improve the pharmacokinetic properties of the polypeptide complexes, help eliminate obstacles in downstream development processes, and increase the probability of successful development of the polypeptide complexes of this application.

[0208] In some embodiments, DICAD in the polypeptide complex provided herein introduces electrostatic interactions in the first antigen-binding domain and / or the second antigen-binding domain, thereby promoting pairing between VH1 / VL1 and / or promoting pairing between VH2 / VL2.

[0209] In some embodiments, two amino acid residues with opposite charges are introduced into VL1 and VH1 to promote electrostatic interaction between VL1 and VH1. In certain such embodiments, no such amino acid residues with opposite charges are introduced between VL2 and VH2.

[0210] In other embodiments, two oppositely charged amino acid residues are introduced into VL2 and VH2 to promote electrostatic interaction between VL2 and VH2. In such certain embodiments, no such oppositely charged amino acid residues are introduced between VL1 and VH1.

[0211] In other embodiments, the DICAD described herein is 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 mismatches between VH1 and VL2 and mismatches between VH2 and VL1 (e.g., by electrostatic repulsion). For example, the charged residues introduced into VL2 and VH1 are amino acid residues with the same charge, and / or the charged residues introduced into VH2 and VL1 are amino acid residues with the same charge, thereby reducing mismatches between VL2 and VH1 and between VL1 and VH2.

[0212] In some embodiments, the oppositely charged residues consist of a positively charged amino acid residue and a negatively charged amino acid residue.

[0213] 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. In 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.

[0214] 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.

[0215] In some embodiments, the negatively charged amino acid can be selected from aspartic acid (D) or glutamic acid (E). In some embodiments, the positively charged amino acid can be selected from lysine (K), histidine (H) or arginine (R).

[0216] In some embodiments, the charged residues introduced in VL1 or VL2 are in FR (eg, FR2). In some embodiments, the charged residues introduced in VH1 or VH2 are in FR (eg, FR2).

[0217] In some embodiments, a first pair of oppositely charged amino acid residues are 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 the numbering is according to Kabat numbering.

[0218] In some embodiments, a second pair of oppositely charged amino acid residues is further introduced into the 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, wherein the numbering is according to Kabat numbering. In such embodiments, the charged residues introduced into VL2 and VH1 are amino acid residues with the same charge, and / or the charged residues introduced into VH2 and VL1 are amino acid residues with the same charge, such that mispairing between VL2 and VH1 or between VL1 and VH2 is reduced.

[0219] In some specific embodiments, the first pair of oppositely charged amino acid residues comprises Q37K in VL1 and Q39D in VH1, respectively, wherein the numbering is according to Kabat numbering. In some specific embodiments, the second pair of oppositely charged amino acid residues comprises Q39D in VH2 and Q40K in VL2, respectively, wherein the numbering is according to Kabat numbering.

[0220] The introduction of positively or negatively charged amino acids into antibodies is known in the art.

[0221] In some embodiments, both disulfide bonds and charged amino acids are introduced in VH1 and VL1. In some embodiments, in the first antigen-binding domain, the first cysteine ​​introduced in VL1 and the second cysteine ​​introduced in 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 oppositely charged amino acid residues introduced 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.

[0222] Peptide complex of structure A:

[0223] 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 the GPRC5D-binding domain provided herein.

[0224] In some embodiments, one of the first and second antigen-binding domains herein binds to GPRC5D and comprises a GPRC5D binding domain as provided herein, and the other binds to an antigen different from GPRC5D. In some embodiments, the antigen different from GPRC5D is an immunostimulatory antigen, optionally CD3.

[0225] In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain comprises the GPRC5D binding domain provided herein, and the other comprises a CD3 binding domain.

[0226] In some specific embodiments, the first antigen targeted by the first antigen-binding domain of DICAD is GPRC5D. In certain embodiments, the VL1 of the first antigen-binding domain comprises the P100C and / or Q37K mutations, and the VH1 comprises the G44C and / or Q39D mutations, wherein 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.

[0227] In certain such embodiments, the second antigen-binding domain in the DICAD targets CD3. In certain embodiments, the VL2 and VH2 in the second antigen-binding domain do not contain 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, the VL2 in the second antigen-binding domain comprises the mutation Q40K, and the VH2 comprises the mutation Q39D, wherein the numbering is based on 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.

[0228] 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 second Fc polypeptide are identical or different. In some embodiments, the first Fc polypeptide and the second Fc polypeptide are capable of binding 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, the third polypeptide comprising the second Fc polypeptide at its N-terminus.

[0229] In some specific embodiments, the polypeptide complex has a structure as shown in FIG8 .

[0230] In some embodiments, the first antigen targeted by the polypeptide complex is GPRC5D and the second antigen is CD3. In some specific embodiments, the polypeptide complex comprises a first polypeptide having an amino acid sequence as set forth in SEQ ID NO:22 and a second polypeptide having an amino acid sequence as set forth in SEQ ID NO:23. In some specific embodiments, the polypeptide complex further comprises a first Fc polypeptide and a second Fc polypeptide, and comprises a first polypeptide having an amino acid sequence as set forth in SEQ ID NO:22, a second polypeptide having an amino acid sequence as set forth in SEQ ID NO:75, and a third polypeptide having an amino acid sequence as set forth in SEQ ID NO:24.

[0231] Peptide complex of structure B:

[0232] In some embodiments, the polypeptide complex comprises a first antigen binding domain and a second antigen binding domain, which constitute a DICAD domain provided herein, and further comprises a third antigen binding domain, optionally, the third antigen binding domain comprises a Fab domain.

[0233] In some embodiments, the third antigen-binding domain in the polypeptide complex is a Fab domain, which comprises: a third polypeptide comprising, in the N-terminal to C-terminal direction, a third heavy chain variable domain VH3 and a CH1 domain that binds to a third antigen; and a fourth polypeptide comprising, in the N-terminal to C-terminal direction, a third light chain variable domain VL3 and a CL domain that binds to a third antigen, wherein VL3 and VH3 combine to form the third antigen-binding domain.

[0234] 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 second Fc polypeptide are identical or different. In some embodiments, the first Fc polypeptide and the second Fc polypeptide are capable of binding 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, the third polypeptide comprising the second Fc polypeptide at its N-terminus.

[0235] In some specific embodiments, the polypeptide complex has a structure as shown in FIG9 .

[0236] In some embodiments, the first antigen targeted by the polypeptide complex 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 of DICAD is GPRC5D. In certain embodiments, the VL1 of the first antigen-binding domain contains mutations P100C and Q37K, and the VH1 contains mutations G44C and Q39D, where numbering is based on 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.

[0237] In some specific embodiments, the second antigen-binding domain of DICAD also targets GPRC5D, wherein the VH2 does not contain disulfide bonds or charge mutations, and the numbering is based on 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.

[0238] 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.

[0239] 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.

[0240] In some embodiments, the first antigen targeted by the polypeptide complex is Her2, the second antigen is CD3, and the third antigen is GPRC5D. In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain comprises the Her2 binding domain provided herein, and the other comprises a CD3 binding domain.

[0241] In certain embodiments, the first antigen targeted by the first antigen-binding domain of DICAD is Her2. In certain embodiments, the VL1 of the first antigen-binding domain comprises the mutations Q38D and Q100C, and the VH1 comprises the 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.

[0242] In certain such embodiments, the second antigen-binding domain of the DICAD targets CD3, wherein VL2 comprises the Q40K mutation and VH2 comprises the Q39D mutation, 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.

[0243] 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.

[0244] 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 comprises a first polypeptide further comprising a first Fc polypeptide and a second Fc polypeptide and comprising the amino acid sequence set forth in SEQ ID NO: 25, a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 76, a third polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 77, and a fourth polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 28.

[0245] In some embodiments, the first antigen targeted by the polypeptide complex is GPRC5D, the second antigen is CD3, and the third antigen is GPRC5D. In certain such embodiments, the first antigen-binding domain of the DICAD targets GPRC5D, wherein the VL1 comprises the mutations P100C and Q37K, and the VH1 comprises the 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.

[0246] In certain such embodiments, the second antigen-binding domain of the DICAD targets CD3, wherein VL2 comprises the Q40K mutation and VH2 comprises the Q39D mutation, 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.

[0247] 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 of the third antigen-binding domain comprises the amino acid sequence of SEQ ID NO:12. In certain embodiments, the VH3 comprises the amino acid sequence of SEQ ID NO:105. In certain such embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO:29, the second polypeptide comprises the amino acid sequence of SEQ ID NO:30, the third polypeptide comprises the amino acid sequence of SEQ ID NO:31, and the fourth polypeptide comprises the amino acid sequence of 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 of SEQ ID NO:29, a first polypeptide having the amino acid sequence of SEQ ID NO:78, a first polypeptide having the amino acid sequence of SEQ ID NO:79, and a fourth polypeptide having the amino acid sequence of SEQ ID NO:32.

[0248] A polypeptide complex containing at least two Fab domains: Structure C

[0249] In some embodiments, the polypeptide complex comprises a first antigen-binding domain and a 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.

[0250] 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 CHIa 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 CHIb 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 a second antigen.

[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 second Fc polypeptide are identical or different. In some embodiments, the first Fc polypeptide and the second Fc polypeptide are capable of binding 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, the third polypeptide comprising the second Fc polypeptide at its N-terminus.

[0252] In some specific embodiments, the polypeptide complex has a structure as shown in FIG10 .

[0253] In some embodiments, the VL1 and VH1 combine to form the first antigen-binding domain; and VL2 and VH2 combine to form the second antigen-binding domain.

[0254] In some embodiments, CH1a and CLa can bind in pairs, CH1b and CLb can bind in pairs, and the CH1a and CLa binding pairs and the CH1b and CLb binding pairs are configured to avoid mispairing between CH1a and CLb and / or between CH1b and CLa.

[0255] (a) Introduction of disulfide bonds into the CH1 / CL region

[0256] 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 between CH1a and CLb.

[0257] 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 bound by a first pair of disulfide bonds. In some embodiments, the first pair of disulfide bonds is non-natural. In some embodiments, the naturally occurring disulfide bond in the first CH1 / CL binding pair is deleted or disrupted (e.g., by mutating a cysteine ​​residue that forms a native disulfide bond).

[0258] In some embodiments, the second CH1 / CL binding pair is formed by a second pair of disulfide bonds, wherein the second pair of disulfide bonds is located at a different position than the first pair of disulfide bonds. In some embodiments, the second pair of disulfide bonds formed in the second CH1 / CL binding pair is a native disulfide bond or a non-native disulfide bond.

[0259] In some embodiments, the first pair of disulfide bonds is formed by two cysteine ​​residues introduced at heavy chain-light chain positions selected from the group consisting of (numbered according to EU numbering): 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.

[0260] In some embodiments, the first pair of disulfide bonds is formed by two cysteine ​​residues introduced at heavy chain-light chain positions selected from the group consisting of (numbered according to EU numbering): 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.

[0261] In some embodiments, the first pair of disulfide bonds is formed by two cysteine ​​residues introduced at heavy chain-light chain positions selected from the group consisting of (numbered according to EU numbering): 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.

[0262] In some embodiments, the native disulfide bond is formed between a position selected from the group consisting of position 131, 219, and 220 of the heavy chain and position 214 of the light chain, numbered according to EU numbering. In some embodiments, the native disulfide bond is formed between position 220 of the heavy chain and position 214 of the light chain, numbered according to EU numbering.

[0263] In some embodiments, the first CH1 / CL pair comprises an engineered CH1 and CL; 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; numbered according to EU numbering.

[0264] (b) Replacement of charged amino acids in the CH1 / CL region

[0265] In some embodiments, the CH1b and CLb binding pair has one or more introduced amino acid mutations and forms at least one introduced charged amino acid residue that hinders mispairing between CH1b and CLa and / or between CH1a and CLb.

[0266] 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 at least one mutation from an uncharged amino acid residue to a charged amino acid residue, and / or at least one mutation from a 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 promote pairing of the first CH1 / CL binding pair. In some embodiments, the first CH1 / CL binding pair can comprise a combination of mutations that together provide a first pair of oppositely charged amino acid residues that facilitate pairing of the first CH1 / CL binding pair.

[0267] In other words, a pair of oppositely charged residues can 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 pair. For example, a charged amino acid residue can be introduced to replace an uncharged amino acid residue at a certain position in CH1 (or CL), such that the introduced charged amino acid residue will form an electrostatic interaction with another oppositely charged amino acid residue already present or to be introduced in CL (or CH1), thereby promoting pairing of the first CH1 / CL binding pair. In another example, an existing charged amino acid residue at a certain position in CH1 (or CL) can be replaced with an oppositely charged amino acid residue, such that the replaced charged amino acid residue will form an electrostatic interaction with another oppositely charged amino acid residue already present or to be introduced in CL (or CH1), thereby promoting pairing of the first CH1 / CL binding pair. In certain embodiments, an existing charged amino acid residue in CH1 or CL can be replaced with an uncharged amino acid residue to reduce potential interference with electrostatic interactions between the first CH1 / CL binding pair.

[0268] In some embodiments, the second CH1 / CL binding pair contains at least one mutation from an uncharged amino acid residue to a charged amino acid residue, and / or at least one mutation from a 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 that facilitates 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 hinder pairing of CH1a with CLb or CH1b with CLa.

[0269] 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 CH1a and CLb are both positively charged or both negatively charged, and / or CH1b and CLa are both positively charged or both negatively charged.

[0270] In some embodiments, the first pair of oppositely charged residues and / or the second pair of oppositely charged residues introduce an oppositely charged amino acid residue pair at a heavy chain-light chain EU numbering position 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.

[0271] 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 at a heavy chain-light chain position selected from the following group (numbered according to the EU numbering method): g) heavy chain position 147 and light chain position 131, h) heavy chain position 168 and light chain position 174, i) heavy chain positions 147 and 168 and light chain positions 131 and 174.

[0272] In some embodiments, the pair of oppositely charged amino acid residues includes a positively charged amino acid residue and a 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).

[0273] In some embodiments, the lysine at position 147 in CHIb 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 CHIa 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, numbered according to EU numbering. In some embodiments, CHIb includes mutation K147D; CLb includes mutation S176K; CHIa includes mutation S183K; and CLa includes mutation S176D.

[0274] In some embodiments, the lysine at position 147 in CHa 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 CHa 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, numbered according to EU numbering. In some embodiments, CHa comprises mutation K147D; CLa comprises mutation S176K; CHa comprises mutation S183K; and CLa comprises mutation S176D.

[0275] In some embodiments, the first CH1 / CL pair comprises a combination of at least one non-native disulfide bond and a non-native electrostatic interaction.

[0276] In some embodiments, the first CH1 / CL pair is a CH1b and CLb binding pair. In some embodiments, the amino acid residue at position 173 of CH1b is substituted with 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 the amino acid residue at position 160 of CLb is substituted with 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, numbered according to EU numbering. In some embodiments, CH1b includes mutations V173C, S183K, and C220S, and CLb includes mutations Q160C, S176D, and C214S.

[0277] In some embodiments, the first CH1 / CL pair is a CH1b and CLb binding pair. In some embodiments, the amino acid residue at position 173 of CH1b 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 of CLb 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, numbered according to EU numbering.

[0278] In some embodiments, the first CH1 / CL pair includes CH1a and CLa. In some embodiments, the amino acid residue at position 173 of CH1a is substituted with 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 the amino acid residue at position 160 of CLa is substituted with 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 ​​residue, numbered according to EU numbering. In some embodiments, CH1a includes mutations V173C, S183K, and C220S, and CLa includes mutations Q160C, S176D, and C214S.

[0279] In some embodiments, the amino acid residue at position 173 of 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 of 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, numbered according to the EU numbering method.

[0280] In some embodiments, the first CH1 / CL pair comprises mutations A141K, V173C, and C220S in CH1 and mutations F116D, Q160C, and C214S in CL (numbered according to EU numbering).

[0281] In some embodiments, the first antigen targeted by the polypeptide complex is GPRC5D, and the second antigen is CD3. In some embodiments, the second CH1 domain CH1b of the third polypeptide contains mutations A141K, V173C, and C220S, and the second CL domain CLb of the fourth polypeptide contains mutations F116D, Q160C, and C214S, numbered according to EU numbering. 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.

[0282] In some embodiments, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:39, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:40, the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:38, and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO:37.

[0283] In some specific embodiments, the polypeptide complex further comprises a first Fc polypeptide and a second Fc polypeptide, and comprises a first polypeptide having an amino acid sequence as shown in SEQ ID NO:83, a second polypeptide having an amino acid sequence as shown in SEQ ID NO:40, a third polypeptide having an amino acid sequence as shown in SEQ ID NO:82, and a fourth polypeptide having an amino acid sequence as shown in SEQ ID NO:37.

[0284] (c) Introducing an orthogonal interaction into the CH1 / CL interaction

[0285] 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.

[0286] 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.

[0287] In some embodiments, the amino acid mutations form a perpendicular CH1-CL interface that favors pairing of CH1b and CLb and optionally prevents pairing of CH1a with CLb or CH1b with CLa. In some embodiments, the amino acid mutations form a perpendicular CH1-CL interface that favors pairing of CH1a and CLa and optionally prevents pairing of CH1a with CLb or CH1b with CLa.

[0288] In some embodiments, the vertical CH1-CL interface comprises mutations H168A, F170G in the heavy chain and mutations L135Y, S176W in the light chain. In 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.

[0289] In some embodiments, CH1a comprises mutation S183E, CLa comprises mutation V133K, CH1b comprises mutations A141I, F170S, S181M, S183A, and V185A, and CLb comprises mutations F116A, L235V, S174A, S176F, and T178V.

[0290] In some embodiments, CH1b comprises mutation S183E, CLb comprises mutation V133K, CH1a comprises mutations A141I, F170S, S181M, S183A, and V185A, and CLa comprises mutations F116A, L235V, S174A, S176F, and T178V.

[0291] In some embodiments, the first CH1 / CL pair comprises one or more introduced amino acid mutations located at heavy chain-light chain positions 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 (numbered according to EU numbering).

[0292] In some embodiments, the first CH1 / CL pair comprises one or more introduced amino acid mutations located at heavy chain-light chain positions 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 (numbered according to EU numbering); 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.

[0293] 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 second Fc polypeptide are identical or different. In some embodiments, the first Fc polypeptide and the second Fc polypeptide are capable of binding 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, the third polypeptide comprising the second Fc polypeptide at its N-terminus.

[0294] Polypeptide complex containing at least two Fab domains: Structure D

[0295] In some embodiments, the polypeptide complex further comprises a third antigen-binding domain based on Structure C. Optionally, the third antigen-binding domain is a Fab domain. In some embodiments, a C-terminus of the third antigen-binding domain is linked to an N-terminus of the second antigen-binding domain.

[0296] In some embodiments, the third antigen-binding domain is the same as the first antigen-binding domain and comprises: a first fragment comprising, in the N-terminal to C-terminal direction, 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 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, and wherein the C-terminus of the first fragment is connected to the N-terminus of the fourth polypeptide.

[0297] In some embodiments, the polypeptide complex comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and a fifth polypeptide, wherein in the N-terminal to C-terminal direction: the first polypeptide comprises VH1-CH1a; the third polypeptide comprises VH2-CH1b; the fourth polypeptide comprises VH1-CH1a-linker-VL2-CLb; and the second polypeptide and the fifth polypeptide are the same, each comprising VL1-CLa.

[0298] In some embodiments, the CH1b and CLb binding pair has at least one non-native disulfide bond that discourages mispairing between CH1b and CLa and / or between CH1a and CLb.

[0299] In some embodiments, the CH1b and CLb binding pair has one or more introduced amino acid mutations and forms at least one introduced charged amino acid residue that hinders mispairing between CH1b and CLa and / or between CH1a and CLb.

[0300] In some specific 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, according to EU numbering. In some specific 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.

[0301] 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 second Fc polypeptide are identical or different. In some embodiments, the first Fc polypeptide and the second Fc polypeptide are capable of binding 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, the third polypeptide comprising the second Fc polypeptide at its N-terminus.

[0302] In some specific embodiments, the polypeptide complex has a structure as shown in FIG11 .

[0303] In some embodiments, the first antigen targeted by the polypeptide complex is GPRC5D, the second antigen is CD3, and the third antigen is GPRC5D. 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, 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:43, the second or 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 an amino acid sequence as shown in SEQ ID NO:85, a second polypeptide or a fifth polypeptide having an amino acid sequence as shown in SEQ ID NO:44, a third peptide having an amino acid sequence as shown in SEQ ID NO:84, and a fourth polypeptide having an amino acid sequence as shown in SEQ ID NO:41.

[0304] In some embodiments, the first antigen targeted by the polypeptide complex is GPRC5D, the second antigen is CD3, and the third antigen is GPRC5D. In some embodiments, the first and / or third antigen-binding domains 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, as numbered 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 or 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 the amino acid sequence shown as the first polypeptide of the amino acid sequence shown in SEQ ID NO:87, the second polypeptide or the fifth polypeptide of the amino acid sequence shown in SEQ ID NO:48, the third polypeptide of the amino acid sequence shown in SEQ ID NO:86, and the fourth polypeptide of the amino acid sequence shown in SEQ ID NO:45.

[0305] In some specific embodiments, the polypeptide complex has a specific sequence as shown in Table 7.

[0306] Table 7. Sequences of exemplary polypeptide complexes

[0307] Fc variants

[0308] In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide are derived from IgG1, IgG2, IgG3, or IgG4.

[0309] In certain embodiments, the polypeptide complex comprises one or more amino acid substitutions at the interface of the Fc region to facilitate and / or promote heterodimerization. In certain embodiments, 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. For example, a protrusion can be introduced into the first Fc polypeptide and a cavity can be introduced into the second Fc polypeptide, wherein the protrusion can 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, for example, as described in U.S. Patent No. 5,731,168.

[0310] In some embodiments, in the polypeptide complex 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 comprise the following combinations: a) T366W or S354C in combination with Y349C, T366S, L368A or Y407V; b) D399K or E356K in combination with K392D or K409D; c) E356K, E354C, and Y349C, T366S, L368A or Y407V; or g) L351D or L368E in combination with L351K or T366K; wherein amino acid positions are numbered according to the EU numbering system.

[0311] 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.

[0312] The polypeptide complexes 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 produced in vitro using recombinant methods rather than in animals.

[0313] Antibody variants

[0314] The polypeptide complexes provided herein also encompass various antibody variants thereof.

[0315] In certain embodiments, the antibody variants include one or more modifications or substitutions in one or more CDR sequences as provided in Tables 1-2, 5-6 above, one or more variable region sequences (but not in any CDR sequence) provided in Tables 3-6 above, and / or constant regions (e.g., Fc regions). Such variants retain the binding specificity of their parent antibodies to the corresponding antigens (e.g., GPRC5D, CD3, or Her2), but have one or more desired properties conferred by one or more modifications or one or more substitutions. For example, the antibody variants may have increased antigen binding affinity, improved glycosylation patterns, reduced glycosylation risk, reduced deamination, reduced or depleted one or more effector functions, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or compatibility with binding (e.g., one or more introduced cysteine ​​residues).

[0316] Parent antibody sequences can be screened using methods known in the art, such as "alanine scanning mutagenesis" (see, for example, Cunningham and Wells (1989) Science, 244: 1081-1085) to identify suitable or preferred residues for modification or substitution. In brief, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies are produced and screened for properties of interest. If the substitution at a particular amino acid position shows a functional change of interest, the position can be determined as a potential residue for modification or substitution. Potential residues can be further evaluated by replacing them with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0317] Affinity variants

[0318] Affinity variants may contain modifications or substitutions in one or more CDR sequences as provided in Tables 1-2, 5-6 above, one or more FR sequences as provided herein, or the heavy or light chain variable region sequences provided in Tables 3-6 above. FR sequences can be easily identified by those skilled in the art based on the CDR sequences in Tables 1-2, 5-6 above and the variable region sequences in Tables 3-6 above, because it is well known in the art that the CDR region is flanked by two FR regions in the variable region. Affinity variants retain the specific binding affinity of the parent antibody to the corresponding antigen (e.g., GPRC5D, CD3, or Her2), or even have an improved specific binding affinity for the corresponding antigen (e.g., GPRC5D, CD3, or Her2) that is superior to that of the parent antibody. In certain embodiments, at least one (or all) substitutions in the CDR sequence, FR sequence, or variable region sequence comprise conservative substitutions.

[0319] It will be understood by those skilled in the art that one or more amino acid residues may be substituted in the CDR sequences and variable region sequences provided in Tables 1-6 above, while the resulting polypeptide complex still maintains binding affinity or binding capacity for the corresponding antigen (e.g., GPRC5D, CD3 or Her2), or even has an improved binding affinity or capacity. Various methods known in the art can be used to achieve this goal. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed using phage display technology, and then screened for binding affinity to the corresponding antigen (e.g., GPRC5D, CD3 or Her2). For another example, computer software can be used to actually simulate the binding of an antibody to a corresponding antigen (e.g., GPRC5D, CD3 or Her2), and to identify the amino acid residues on the antibody that form the binding interface. Such residues can be avoided from being substituted in order to prevent a decrease in binding affinity, or can be targeted for substitution to achieve stronger binding.

[0320] In certain embodiments, the humanized polypeptide complexes provided herein include one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences. In certain embodiments, the affinity variants include no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in a total of CDR sequences and / or FR sequences.

[0321] In certain embodiments, the GPRC5D binding domain in the polypeptide complex comprises 1, 2 or 3 CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequences listed in Table 1 or Table 2 above, while maintaining a similar or even higher level of binding affinity for GPRC5D than its parent antibody. In certain embodiments, the Her2 binding domain in the polypeptide complex comprises 1, 2 or 3 CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequences listed in Table 5 above, while maintaining a similar or even higher level of binding affinity for Her2 than its parent antibody. In certain embodiments, the CD3 binding domain in the polypeptide complex 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 to the sequences listed in Table 6 above (or those), while maintaining a similar or even higher level of binding affinity to CD3 as its parent antibody.

[0322] In certain embodiments, the GPRC5D binding domain in the polypeptide complex comprises one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequences listed in Tables 3-4 above (or those), while maintaining a similar or even higher level of binding affinity for GPRC5D than its 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 of the CDRs (e.g., in the FRs). In certain embodiments, the Her2 binding domain in the polypeptide complex comprises one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequences listed in Table 5 above, and at the same time maintaining a binding affinity to Her2 similar to or even higher than that of its 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 substitution, insertion, or deletion occurs in a region outside the CDR (e.g., in the FR). In certain embodiments, the CD3 binding domain in the polypeptide complex comprises one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequences listed in Table 6 above (or those sequences), while maintaining a similar or even higher level of binding affinity for 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 of the CDRs (e.g., in the FRs).

[0323] Glycosylation variants

[0324] The polypeptide complexes provided herein also encompass glycosylation variants, which can be obtained to increase or decrease the extent of glycosylation of the antibody or antigen-binding fragment or polypeptide complex.

[0325] The polypeptide complex may include one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue whose side chain can be linked to a carbohydrate moiety (e.g., an oligosaccharide structure). Antibody glycosylation is typically either N-linked or O-linked. N-linkage refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue (e.g., the asparagine residue in tripeptide sequences 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 the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine. Removal of a native glycosylation site can be conveniently achieved, for example, by altering the amino acid sequence so that one of the tripeptide sequences described above (for N-linked glycosylation sites) or a serine or threonine residue present in the sequence (for O-linked glycosylation sites) is substituted. New glycosylation sites can be generated in a similar manner by introducing such tripeptide sequences or serine or threonine residues.

[0326] In certain embodiments, the polypeptide complexes provided herein include a mutation at N297 (eg, N297A, N297Q, or N297G) to remove the glycosylation site.

[0327] Cysteine ​​engineered variants

[0328] The polypeptide complexes provided herein also encompass cysteine ​​engineered variants comprising one or more introduced free cysteine ​​amino acid residues.

[0329] A free cysteine ​​residue is a cysteine ​​residue that is not part of a disulfide bridge. Cysteine ​​engineered variants can be used to bind to engineered cysteine ​​sites, for example, via maleimide or haloacetyl groups, 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, for example, WO2006 / 034488.

[0330] Fc variants

[0331] The polypeptide complexes provided herein also encompass Fc variants comprising one or more amino acid residue modifications or substitutions at the Fc region and / or hinge region thereof, eg, to provide altered effector functions, such as ADCC and CDC. Methods for altering ADCC activity by antibody engineering have been described in the art, see, for example, Shields RL. et al., J Biol Chem. 2001. 276(9):6591-604; Idusogie EE. et al., J Immunol. 2000. 164(8):4178-84; Steurer W. et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE. et al., J Immunol. 2001, 166(4):2571-5; Lazar GA. et al., PNAS, 2006, 103(11):4005-4010; Ryan MC. et al., Mol. Cancer Therapeutics. Ther., 2007, 6: 3009-3018; Richards JO, et al., Mol 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.

[0332] The CDC activity of the antibodies provided herein can also be altered, for example, by improving or reducing C1q binding and / or CDC (see, e.g., WO 99 / 51642; Duncan and Winter, Nature, 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821); and for other examples of Fc region variants, WO 94 / 29351. One or more amino acids selected from amino acid residues 329, 331, and 322 of the Fc region can be substituted with a different amino acid residue to alter C1q binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC) (see U.S. Patent No. 6,194,551 to Idusogie et al.). One or more amino acid substitutions can also be introduced to alter the ability of an antibody to fix complement (see PCT Publication No. WO 94 / 29351 to Bodmer et al.).

[0333] In certain embodiments, the polypeptide complexes 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 and 238, 268, 297, 309, 330, and 331. In certain embodiments, the polypeptide complexes 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 complexes disclosed herein have an IgG2 isotype and include 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 complexes disclosed herein have an IgG4 isotype and include 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 complexes disclosed herein have an IgG2 / IgG4 crossover isotype. Examples of IgG2 / IgG4 cross-isotypes are described in Rother RP et al., Nat Biotechnol 25: 1256-1264 (2007).

[0334] In certain embodiments, the polypeptide complexes provided herein have an IgG1 isotype and include one or more amino acid substitutions at one or more of 234, 235, and 331. In certain embodiments, the polypeptide complexes provided herein have an IgG1 isotype and include the triple mutation L234F / L235E / P331S in the Fc region.

[0335] In certain embodiments, the polypeptide complexes provided herein have increased ADCC and / or increased affinity for an Fcγ receptor and include one or more amino acid substitutions at one or more of the following positions: 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, 297, 298, 299, 300, 301, 302, 303, 304, 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 96, 298, 301, 303, 305, 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 to Presta). Specific mutations at positions 256, 290, 298, 333, 334 and 339 have been shown to improve binding to FcγRIII. Additionally, the following combination mutants were shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A.

[0336] In certain embodiments, the polypeptide complex comprises one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants may have a prolonged pharmacokinetic half-life because they bind to FcRn at acidic pH, allowing them to avoid degradation in lysosomes and subsequently translocate and be released from cells. Methods for engineering polypeptide complexes to increase binding affinity to FcRn are well known in the art, see, for example, 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 Publishing, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., J. Immunol., 176:346-356 (2006).

[0337] Conjugate

[0338] In some embodiments, the polypeptide complex further includes a binding moiety. The binding moiety can be connected to the polypeptide complex. The binding moiety is a portion that can be connected to the polypeptide complex. It is contemplated that a variety of binding moieties can be connected to the polypeptide complex provided herein (see, for example, "Conjugate Vaccines," in Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These binding moieties can be connected to the polypeptide complex by covalent binding, affinity binding, embedding, coordination binding, compounding, association, blending or addition, as well as other methods.

[0339] In certain embodiments, the polypeptide complexes disclosed herein can be engineered to contain specific sites outside of an epitope binding moiety that can be used for binding to one or more binding moieties. For example, such sites can comprise one or more reactive amino acid residues, such as, for example, cysteine ​​or histidine residues, to facilitate covalent attachment to a binding moiety.

[0340] In certain embodiments, the antibody can be linked to a binding moiety indirectly or through another binding moiety. For example, a polypeptide complex can be conjugated to biotin, which in turn can be indirectly bound to a second binding partner that is conjugated to avidin. The binding partner can be a clearance modulator, 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.

[0341] A "toxin" can be any agent that is harmful to cells or that can damage or kill cells. Examples of toxins include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, and cytochrome P60. 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), cyclothiophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C ( C) and cis-dichlorodiamine platinum (II) (DDP, cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), antimitotics (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin-binding agents.

[0342] Examples of detectable labels can include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide 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 Bihe 32 P, other lanthanides), luminescent labels, chromogenic moieties, digoxigenin, biotin / avidin, DNA molecules, or gold labels are used for detection.

[0343] In some embodiments, binding moiety can be a clearance regulator that helps to increase the antibody half-life. Illustrative examples include water-soluble polymers, such as copolymers of PEG, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol / propylene glycol, etc. Polymer can have any molecular weight and can be side chain or non-side chain. The number of the polymer connected with the antibody can change, and if connection exceeds a polymer, it can be identical or different molecules so.

[0344] In certain embodiments, the binding moiety can be a purification moiety, such as a magnetic bead.

[0345] In certain embodiments, the polypeptide complexes provided herein are used as substrates for conjugates.

[0346] Polynucleotides and recombinant methods

[0347] The present application provides isolated polynucleotides encoding polypeptide complexes. As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single or double-stranded form and polymers thereof. In certain embodiments, the isolated polynucleotide comprises one or more nucleotide sequences as shown in SEQ ID NOs: 11, 12, 13, and 14, and / or homologous sequences thereof having at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and / or variants thereof having only degenerate substitutions, and encoding the variable regions of the exemplary antibodies provided herein. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) 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)).

[0348] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Encoding DNA can also be obtained synthetically.

[0349] The isolated polynucleotide encoding the polypeptide complex (e.g., comprising a sequence as shown in Table 3) can be inserted into a vector for further cloning (DNA amplification) or expression using recombinant techniques known in the art. A variety of vectors are available. Vector components generally include, but are not limited to, one or more of the following: 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.

[0350] The application provides expression vectors, which include isolated polynucleotides provided herein. In certain embodiments, the polynucleotide encoding polypeptide complex provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to a nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phages and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0351] The vector comprising the polynucleotide sequence encoding the polypeptide complex can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast, or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, for example, Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescens. marcescans); and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces.

[0352] In addition to prokaryotes, eukaryotic microorganisms 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 lower eukaryotic host microorganism. However, a variety of other genera, species and strains are commonly used and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as, for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0353] Suitable host cells for expressing the glycosylated polypeptide complexes provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains and variants and corresponding permissive insect host cells from the following hosts have been identified: Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses herein according to the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0354] However, the greatest interest has been in vertebrate cells, whose propagation in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (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., Proc. Natl. Acad. Sci. USA 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 CCL2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL3A, 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 NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2). In some preferred embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293, and derivatives thereof.

[0355] Host cells are transformed with the expression or cloning vectors described above for producing polypeptide complexes and cultured in conventional nutrient media modified as needed to induce promoters, select transformants, or amplify genes encoding the desired sequences. In another embodiment, antibodies can be produced by homologous recombination as known in the art. In certain embodiments, host cells are capable of producing the polypeptide complexes provided herein.

[0356] Host cells for producing the polypeptide complexes disclosed herein can be cultured in a variety of culture media. Commercially available culture media, such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, 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. Reissue Patent No. 30,985 can be used as culture medium for the host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium salts, magnesium salts, and phosphates), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN TM The culture medium comprises at least one of the following: a) a 5-10 μg / mL lysine phosphate (LPP) phosphate buffer (PBS) , b) a 5-10 μg / mL lysine phosphate (LPP) phosphate buffer (PBS) , c) a 5-10 μg / mL lysine phosphate (LPP) phosphate buffer (PBS) , e ...

[0357] When using recombinant technology, antibodies can be produced in the intracellular, periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, then as a first step, the microparticle debris of the host cell or cleavage fragment is removed, for example, by centrifugation or ultrafiltration. Carter et al., " Biotechnology (Bio / Technology) " 10:163-167 (1992) describes a procedure for separating antibodies secreted into the periplasmic space of Escherichia coli. In brief, in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF), the cell paste is thawed in about 30 minutes. Cell debris can be removed by centrifugation. In the case where the antibody is secreted into the culture medium, commercially available protein concentration filters are generally used first, such as Amicon (Amicon) or Millipore (Millipore) Pellicon ultrafiltration units are concentrated from the supernatant of such expression systems. Protease inhibitors, such as PMSF, can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0358] The polypeptide complex produced by the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique.

[0359] In certain embodiments, protein A immobilized on a solid phase 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 commonly agarose, but other matrices are also available. Mechanically stable matrices, such as controlled pore glass or poly (styrene divinyl) benzene, allow faster flow rates and shorter processing times than can be achieved with agarose. When the antibody includes a CH3 domain, Bakerbond ABX TM Resins (JT Baker, Phillipsburg, NJ) can be used for purification. Other techniques for protein purification include fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, heparin SEPHAROSE TMChromatography on ELISA, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatographing, SDS-PAGE, and ammonium sulfate precipitation are also useful, depending on the antibody to be recovered.

[0360] Following any preliminary purification steps, the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably at low salt concentration (e.g., about 0-0.25 M salt).

[0361] Pharmaceutical composition

[0362] The present application further provides a pharmaceutical composition comprising the polypeptide complex or the polynucleotide of the present application, and one or more pharmaceutically acceptable carriers.

[0363] The pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering / chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0364] Suitable components 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 hydroxyanisole (butylated hydroxanisol), butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, one or more antioxidants, such as methionine, are included in the compositions comprising polypeptide complexes and conjugates as provided herein to reduce oxidation of the polypeptide complexes. This oxidation reduction prevents or reduces the loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Therefore, in certain embodiments, compositions comprising one or more polypeptide complexes as disclosed herein and one or more antioxidants, such as methionine, are provided. Also provided are methods of preventing oxidation, extending the shelf life, and / or improving the efficacy of polypeptide complexes as provided herein by mixing the polypeptide complexes with one or more antioxidants, such as methionine.

[0365] To further illustrate, a pharmaceutically acceptable carrier can include, for example, an aqueous vehicle, such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; a non-aqueous vehicle, such as a fixed oil of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; an antimicrobial agent at bacteriostatic or fungistatic concentrations; an isotonic agent, such as sodium chloride or dextrose; a buffer, such as a phosphate or citrate buffer; an antioxidant, such as sodium bisulfate; a local anesthetic, such as procaine hydrochloride; hydrochloride); suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinyl pyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); chelating agents such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multidose containers and include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0366] The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0367] In certain embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as, for example, liquid solutions, suspensions, emulsions, or solid forms suitable for producing liquid solutions, suspensions, or emulsions. Injectable formulations may include sterile and / or pyrogen-free solutions that can be used immediately for injection; sterile dry soluble products that are prepared for combination with solvents just before use, such as lyophilized powders, including subcutaneous tablets; sterile suspensions that can be used immediately for injection; sterile dry insoluble products that are prepared for combination with vehicles just before use; and sterile and / or pyrogen-free emulsions. Solutions can be aqueous or non-aqueous.

[0368] In certain embodiments, unit dose parenteral formulations are packaged in ampoules, vials, or syringes with needles.All preparations for parenteral administration should be sterile and pyrogen-free, as is known and practiced in the art.

[0369] In certain embodiments, a sterile lyophilized powder is prepared by dissolving a polypeptide complex as disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability of the powder or a reconstituted solution prepared from the powder, or other pharmacological components. Useful excipients include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may contain a buffer, such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art. In one embodiment, the buffer is at approximately neutral pH. The solution is then sterile filtered under standard conditions known to those skilled in the art, followed by lyophilization to provide the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single or multiple doses of the polypeptide complex or combination thereof. Overfilling the vial by a small amount (e.g., about 10%) beyond that required for a single dose or a series of doses is acceptable to facilitate accurate sampling and dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0370] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. In one embodiment, for reconstitution, sterile and / or pyrogen-free water or other liquid suitable carrier is added to the lyophilized powder. The exact amount depends on the selected therapy given and can be determined empirically.

[0371] How to use

[0372] The present application also provides a method for treating a GPRC5D-related disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide complex provided herein or the pharmaceutical composition provided herein.

[0373] In some embodiments, the GPRC5D-related disease or condition is characterized by expression or overexpression of GPRC5D. Overexpression of GPRC5D has been demonstrated in several autoimmune diseases, including myeloma.

[0374] In certain embodiments, GPRC5D-associated diseases or conditions include, but are not limited to, cancer and other hyperproliferative diseases, immune diseases, and inflammation.

[0375] The present application also provides a method for treating a Her2-related disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide complex provided herein or the pharmaceutical composition provided herein.

[0376] In some embodiments, the Her2-related disease or condition is characterized by expression or overexpression of Her2. Overexpression of Her2 has been demonstrated in several cancers, including breast and lung cancer.

[0377] In some embodiments, the cancer and other hyperproliferative diseases include benign or malignant tumors, leukemias, and lymphoid malignancies. Depending on the cell type harboring the cancer or hyperproliferative disease, examples include neurons, glial cells, astrocytes, hypothalamus, glandular cells, macrophages, epithelial cells, endothelial cells, and mesenchymal malignancies. Depending on the organ / site harboring the cancer or hyperproliferative disease, examples include: head, neck, eye, mouth, throat, esophagus, chest, skin, bone, lung, colon, rectum, colon, 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.

[0378] In some embodiments, the immune disease and / or inflammation includes: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, adrenal autoimmune disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, Sjögren's syndrome, psoriasis, atherosclerosis, diabetic and other retinopathy, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangioma, thyroid hyperplasia (including Graves' disease), corneal and other tissue transplants, as well as chronic inflammation, sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion injury, sepsis, 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 dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus erythematosus, 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, hirsutism, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-person syndrome, systemic lupus erythematosus, lupus erythematosus, Alpine arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis. Inflammatory diseases may further include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic diseases, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation caused by chronic viral or bacterial infection.

[0379] In one embodiment, the disease or condition associated with GPRC5D is cancer, in particular multiple myeloma. In one embodiment, the disease or condition associated with GPRC5D is an autoimmune disease, such as systemic lupus erythematosus and / or rheumatoid arthritis.

[0380] In certain embodiments, the disease or condition associated with GPRC5D 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.

[0381] In certain embodiments, the subject is identified as having cancer cells expressing 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 of containing cancer cells can be obtained from a subject. In some embodiments, the biological sample can be derived from cancer cells or cancerous tissue. In certain embodiments, the biological sample can be further processed to, for example, separate 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 can be exposed to a polypeptide complex that binds to and detects the expressed GPRC5D protein. Alternatively, methods such as qPCR, reverse transcriptase PCR, microarrays, SAGE, FISH, etc. can also be used to detect GPRC5D at the nucleic acid expression level.

[0382] In one embodiment, the disease or condition associated with GPRC5D includes, but is not limited to, 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 that expresses GPRC5D.

[0383] The therapeutically effective amount of a polypeptide complex or pharmaceutical composition as provided herein will depend on various factors known in the art, such as the subject's weight, age, past medical history, current medication, health status and the possibility of cross-reactions, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of disease progression. As indicated by these and other circumstances or requirements, one of ordinary skill in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dose.

[0384] In certain embodiments, the polypeptide complexes as provided herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the dosage can be varied during the course of treatment. For example, in certain embodiments, the initial dosage can be higher than subsequent dosages. In certain embodiments, the dosage can be varied during the course of treatment, depending on the subject's response.

[0385] Dosage regimens can be adjusted to provide the optimal desired response (eg, a therapeutic response). For example, a single dose can be administered, or several divided doses can be administered over time.

[0386] The polypeptide complexes disclosed herein can be administered by any route known in the art, such as, 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.

[0387] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein can be administered alone or in combination with one or more additional therapeutic means or agents. For example, the antibodies or antigen-binding fragments disclosed herein can be administered in combination with a second therapeutic agent (e.g., a chemotherapeutic agent, an anticancer drug, radiation therapy, immunotherapy, an anti-angiogenic agent, a targeted therapy, a cell therapy, a gene therapy, a hormone therapy, palliative care), a surgery for cancer treatment (e.g., tumor resection), or one or more treatments for complications caused by chemotherapy.

[0388] As used herein, the term "immunotherapy" refers to a type of therapy that stimulates the immune system to fight diseases such as cancer or enhances the immune system in a general manner. Immunotherapy includes passive immunotherapy, i.e., passive immunotherapy by delivering a drug (e.g., effector cells) with a determined tumor immune reactivity that can directly or indirectly mediate an anti-tumor effect without having to rely on a complete host immune system (e.g., antibody therapy or CAR-T cell therapy). Immunotherapy can further include active immunotherapy, in which treatment relies on in vivo stimulation of the endogenous host immune system to fight diseased cells by administering immune response regulators.

[0389] In certain of these embodiments, a polypeptide complex as disclosed herein administered in combination with one or more additional therapeutic agents can be administered concurrently with the one or more additional therapeutic agents, and in certain of these embodiments, the polypeptide complex and the additional therapeutic agent can be administered as part of the same pharmaceutical composition. However, a polypeptide complex administered "in combination" with another therapeutic agent need not be administered concurrently with the agent or in the same composition. A polypeptide complex administered before or after another agent is considered to be administered "in combination" with the agent, as the phrase is used herein, even if the polypeptide complex and the second agent are administered by different routes. When possible, additional therapeutic agents administered in combination with the polypeptide complexes disclosed herein are administered according to the schedule listed in the additional therapeutic agent's product information sheet or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)) or protocols well known in the art.

[0390] In some embodiments, the present application provides a method for detecting the presence or level of GPRC5D in a sample, comprising contacting the sample with a polypeptide complex provided herein.

[0391] In some embodiments, the present application provides a detection or therapeutic kit comprising a polypeptide complex as provided herein and instructions for use, optionally in combination with a detectable moiety. The kit can be suitable for detecting GPRC5D or for treating a GPRC5D-related disease or condition.

[0392] In some embodiments, the present application also provides use of the polypeptide complex provided herein for manufacturing a medicament for treating a GPRC5D-related disease or condition in a subject, or for manufacturing a diagnostic reagent for diagnosing a GPRC5D-related disease or condition.

[0393] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All of the following specific compositions, materials, and methods fall within the scope of the present invention in whole or in part. These specific compositions, materials, and methods are not intended to limit the present invention, but are merely for the purpose of illustrating specific embodiments falling within the scope of the present invention. Without departing from the scope of the present invention, those skilled in the art can develop equivalent compositions, materials, and methods without having to perform inventive abilities. It should be understood that many changes can be made within the procedures described herein while still remaining within the scope of the present invention. The inventors hope that such variations will be included within the scope of the present invention.

[0394] Example:

[0395] Example 1: Preparation of GPRC5D monoclonal antibody

[0396] In this example, a tumor cell line expressing GPRC5D was used to immunize mice to prepare monoclonal antibodies.

[0397] 1.1 Construction of 293T-GPRC5D and CHOS-GPRC5D cell lines

[0398] Human GPRC5D (hGPRC5D) was overexpressed in HEK293 cells (ATCC) and CHOS cells (Invitrogen) using lentiviral infection (MOI = 3-10, 5 μg / ml polybrene). 72 hours after infection, the cells were cultured with the appropriate antibiotics for 2-4 weeks, expanded, and cryopreserved to generate two overexpressing cell lines, HEK293-hGPRC5D and CHOS-hGPRC5D, for subsequent immunotherapy experiments.

[0399] 1.2 Construction of control antibody GC5B596

[0400] The HC and LC plasmids of the GC5B596 antibody were constructed using the following sequences and transiently transformed into CHO cells. The supernatant was harvested and purified by affinity chromatography to obtain the control antibody GC5B596.

[0401] Table 8.

[0402] 1.3 Mouse Immunization / Hybridoma Fusion

[0403] To obtain anti-human GPRC5D antibodies, Balb / c mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., strain code 216) were immunized with HEK293-GPRC5D cells overexpressing human GPRC5D. Complete Freund's adjuvant (CFA) (InvivoGen, catalog number vac-cfa-60) was used as the primary adjuvant, and subsequent immunizations were administered with IFA (InvivoGen, catalog number vac-ifa-60). Multiple immunizations were performed subcutaneously. After multiple immunizations, spleen cells from the immunized mice were fused with mouse myeloma SP2 / 0 cells using the polyethylene glycol method and cultured in HAT selective medium to obtain hybridoma cells that both expressed the antibody and could proliferate indefinitely in vitro. The hybridoma cells were plated and cultured in 96-well cell culture plates.

[0404] 1.4 Hybridoma cloning and screening

[0405] The ability of the antibodies secreted by hybridoma cells in 96-well cell culture plates to bind to GPRC5D at the cellular level was tested. GPRC5D-high-expressing cells were cultured in DMEM 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 50 μl of cells were added to a U-bottom 96-well plate placed in a round-bottom low-adhesion 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. Secondary antibody (DyLight 488 goat anti-human IgG, Abcam, ab97003) was added to each well and incubated at 4°C for 0.5 hour. The supernatant was removed by centrifugation and washed twice with FACS buffer. The cells were resuspended in FACS buffer in each well and the binding of the hybridoma supernatant to the cells was determined by fluorescence measurement using a flow cytometer (BD Biosciences, Canto II). The same binding analysis was performed using HEK293 cells, a background cell line used to construct GPRC5D overexpression cells. Using HEK293-hGPRC5D binding-positive and HEK293 cell-negative clones as the standard, GPRC5D-binding clones were selected and subcloned for two to three rounds.

[0406] 1.5 Hybridoma Sequencing / Recombinant Expression Vector Construction

[0407] The ch-72C7 clone was screened and obtained. The selected hybridoma clones were sequenced using standard hybridoma sequencing methods to obtain the heavy and light chain variable regions (VH and VL) of the selected clones. The VH and VL sequences were synthesized by whole-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 a 293 system and purified using protein A / G. The resulting chimeric recombinant antibody was then ultrafiltered and buffer exchanged into PBS. The sequencing results of the Ch-72C7 clone are shown in Table 9.

[0408] Table 9.

[0409] Example 2: Antigen binding FACS experiment

[0410] HEK293-hCD22GPRC5D and CHOS-hGPRC5D cells expressing hGPRC5D were centrifuged and resuspended in DPBS solution containing 2% BSA (FACS buffer, 4°C) and plated at a concentration of 5×10 5100 μl of cells / well were added to a U-bottom 96-well plate, and serially diluted antibodies were added. The cells were incubated at 4°C for 1 hour, centrifuged, and the supernatant discarded. 100 μl of anti-human IgG Fc-APC secondary antibody was added to each well and incubated at 4°C for 1 hour. The cells were washed once with FACS buffer, resuspended in 200 μl of FACS buffer, and fluorescence signals were read on a BD CantoII. Results showed that the ch-72C7 antibody bound to both HEK293-hGPRC5D (Figure 1) and CHOS-hGPRC5D (Figure 2).

[0411] 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 5 100 μl of cells / well were added to a U-bottom 96-well plate, and serially diluted antibodies were added. The cells were incubated at 4°C for 1 hour, centrifuged, and the supernatant discarded. 100 μl of anti-human IgG Fc-APC secondary antibody was added to each well and incubated at 4°C for 1 hour. The cells were washed once with FACS buffer, resuspended in 200 μl of FACS buffer, and fluorescence signals were read on a BD CantoII. 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).

[0412] Table 10.

[0413] Example 3: Evaluation of ADCC effect of antibodies

[0414] Target cells used GPRC5D naturally expressing tumor cells (NCI-H929, Nanjing Kebai Biotechnology; MM.1R, Nanjing Kebai Biotechnology), and effector cells used the in-house constructed Jurkat-NFAT-Luc-CD16 cell line stably transfected with CD16 receptor and NFAT reaction original. The experiment was performed in a 96-well flat-bottom cell plate (Corning 3903). Serially diluted antibodies were added to the target cells and incubated at 37°C for 30 minutes. 60,000 effector cells were added for every 10,000 target cells, and the reaction was carried out at 37°C for 6 hours. At the end of the reaction, One-Glo was added. TMFluorescence was developed using a reagent (Promega, E6110), and luminescence readings were measured on a Tecan Spark10 microplate reader. Data were analyzed using GraphPad, with the logarithm of the antibody concentration plotted on the horizontal axis and the luminescence readings of the corresponding wells plotted on the vertical axis. The EC50 values ​​for the antibody-dependent cytotoxicity of the anti-GPRC5D antibody were calculated based on curve fitting. The results showed that the blank isotype control (ISO) had no cytotoxic effect on NCI-H929 and MM.1R cells. Both the ch-72C7 antibody and the CG5B596 antibody were able to induce cytotoxicity against NCI-H929 (Figure 6) and RPMI-8226 (Figure 7), tumor cells naturally expressing GPRC5D. The ch-72C7 antibody demonstrated superior ADCC activity compared to the CG5B596 antibody.

[0415] Example 4: Humanized Antibody Design and Expression

[0416] 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 for CDR grafting. Computational chemical simulation was then performed to maintain its antigen binding. The humanized antibody design is shown in Table 11.

[0417] Table 11.

[0418] The VH and VL regions of the above antibodies were linked to the human IgG1 Fc region and kappa constant region, and the heavy and light chain sequences of the antibodies were inserted into the pcDNA3.4 vector and transiently expressed in HEK293 cells. The antibodies were purified by protein A or G.

[0419] At the same time, the VH and VL of the ch-72C7 antibody were replaced with the VH and VL of the human IgG1 antibody, respectively, to generate the chimeric antibody 22mono, which was used as a control to evaluate the results of humanization of each antibody.

[0420] Table 12.

[0421] Example 5: Humanized GPRC5D antibody affinity test

[0422] The recombinant GPRC5D antigen (Human GPRC5D protein-Flag-His tag (51.8KD) ACRO Cat: GPD-H52D3) was immobilized on the chip. The results are shown in Table 13.

[0423] Table 13.

[0424] Based on the above data, the VH and VL of 22Mono5JO4 were used as the humanized antibody results for the subsequent construction of bispecific antibodies.

[0425] Example 6: Construction of multispecific antibodies

[0426] Using the above-mentioned CPRC5D antibody and adopting four different antibody structures ( Figures 8 to 11 ), a series of CD3×GPRC5D bispecific antibodies or Her2×CD3×GPRC5D trispecific antibodies were constructed, as shown in Table 14.

[0427] Table 14.

[0428] Example 7: Detection of the binding of bispecific antibodies to monkey or human GPRC5D

[0429] The purpose of this experiment was to inoculate a certain amount of HEK293T cells overexpressing human GPRC5D or monkey GPRC5D (HEK293T-hGPRC5D or HEK293T-cynoGPRC5D), add serially diluted test antibodies, and compare the binding ability of the constructed bispecific antibodies to monkey or human GPRC5D.

[0430] Experimental methods:

[0431] HEK293T-hGPRC5D or HEK293T-cynoGPRC5D cells in the logarithmic growth phase were seeded in 96-well plates. Antibodies were added at varying concentrations (200, 66.7, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09, 0.03, and 0.01 nM) in duplicate wells for each concentration, along with a negative control (PBS). Cells were incubated at 25°C for approximately 60 minutes before color development and detection. Raw data were calculated as the difference between the detection wavelength and the reference wavelength. Statistical software was used to construct a dose-response curve based on the OD values ​​and the logarithmic concentrations, and the EC50 values ​​were calculated.

[0432] Table 15.

[0433] Experimental results:

[0434] The results of this experiment are shown in Figures 12 and 13. The results show 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 GPRC5D (EC50 > 200 nM). The negative antibody 10B1 had no binding activity to either monkey or human GPRC5D proteins.

[0435] Example 8: In vitro killing test of bispecific antibodies

[0436] 8.1 Detection of the inhibitory effect of bispecific antibodies on GPRC5D-overexpressing NCI-H929 cells

[0437] The purpose of this experiment was to compare the inhibitory effect of the constructed bispecific antibody on PBMC proliferation of NCI-H929 cells.

[0438] Experimental methods:

[0439] H929 cells in logarithmic growth phase were prepared. A portion of cells was reserved as a CFSE negative control, and the remaining cells were stained with 1 μM CFSE. Cells were seeded in 96-well plates at a 20:1 ratio of E:T cells (PBMC:H929). Antibodies were added at various concentrations (4500, 1500, 500, 166.67, 55.56, 18.52, 6.17, 2.06, 0.69, and 0.23 μM) in duplicate, with a negative control (PBS) well included. Cells were incubated at 37°C for 24 hours and then stained with PI for flow cytometry (FACS). FACS data were analyzed using Thermo Attune NxT software, and EC50 values ​​were calculated using Graphpad Prism 6.0.

[0440] Experimental results:

[0441] Different concentrations of the 22 test antibodies (22A1-22A8) and the positive antibody 22B1 were added to a co-culture system of H929 cells and PBMCs (pre-stained with CFSE). Flow cytometry was used to measure the proportion of CFSE- and PI-positive cells relative to CFSE-positive cells to evaluate the antibody-mediated PBMC-mediated killing of H929 cells. As shown in Figures 14-16, antibodies 22A1 to 22A5 and 22B1 all mediated the cytotoxicity of H929 cells by PBMCs in a dose-dependent manner.

[0442] 8.2 Detection of the inhibitory effect of 22A8 and 22B1 antibodies on the growth of GPRC5D-overexpressing MM1S cells

[0443] The purpose of this experiment was to compare the inhibitory effects of 22A8 and 22B1 on MM1S cell proliferation mediated by PBMC.

[0444] Experimental methods:

[0445] Prepare logarithmically growing MM1S cells, retain a portion as a CFSE negative control, and stain the remaining cells with 1 μM CFSE. Plate cells in a 96-well culture plate at a 20:1 ratio of E:T cells (PBMC:MM1S). Add different concentrations of antibodies (4500, 1500, 500, 166.67, 55.56, 18.52, 6.17, 2.06, 0.69, and 0.23 μM), with two replicate wells for each concentration. A negative control (PBS) well was also included. After incubation at 37°C for 24 hours, cells were stained with PI and analyzed by flow cytometry (FACS).

[0446] Experimental results:

[0447] Different concentrations of 22A8, 22A16, and the positive antibody 22B1 were added to a co-culture system of MM1S and PBMC (pre-stained with CFSE). The ratio of CFSE- and PI-positive cells to CFSE-positive cells was measured by flow cytometry to evaluate the ability of the test antibodies to mediate PBMC-mediated killing of MM1S cells. As shown in Figure 17, both 22A8 and 22B1 mediated the killing effect of PBMCs on MM1S cells in a dose-dependent manner, with EC50 values ​​of 13.34 and 83.79 pM, respectively.

[0448] 8.3 Detection of the inhibitory effect of 22A8 and 22B1 antibodies on the growth of GPRC5D low-expressing RPMI 8226 or KMS-12-BM cells

[0449] The purpose of this experiment was to compare the inhibitory effects of 22A8 and 22B1 on the proliferation of RPMI8226 or KMS-12-BM cells mediated by PBMC.

[0450] Experimental methods:

[0451] RPMI 8226 or KMS-12-BM cells were prepared in the logarithmic growth phase. A portion of cells was reserved as a CFSE negative control, and the remaining cells were stained with 1 μM CFSE. Cells were seeded in 96-well plates at a 20:1 ratio of E:T cells (PBMC:MM1S). Antibodies were added at various concentrations (4500, 1500, 500, 166.67, 55.56, 18.52, 6.17, 2.06, 0.69, and 0.23 μM) in duplicate for each concentration. A negative control (PBS) well was also included. Cells were incubated at 37°C for 24 hours and then stained with PI for flow cytometry (FACS). FACS data were analyzed using Thermo Attune NxT software, and EC50 values ​​were calculated using Graphpad Prism 6.0.

[0452] Experimental results:

[0453] Different concentrations of 22A8 and the positive antibody 22B1 were added to a co-culture system of RPMI 8226 or KMS-12-BM and PBMC (pre-stained with CFSE). Flow cytometry was used to measure the proportion of CFSE- and PI-positive cells relative to CFSE-positive cells to assess the antibody-mediated PBMC-mediated killing of MM1S cells. The data showed that the EC50 values ​​for 22A8 and 22B1 against RPMI 8226 were 19.91 pM and NA, respectively (Figure 18); and for KMS-12-BM, the EC50 values ​​were 132.4 and 303.4 pM, respectively (Figure 19).

[0454] Example 9: Affinity test of humanized CD3×GPRC5D bispecific antibody (22A8)

[0455] 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 the chip respectively. The results are shown in Table 16.

[0456] Table 16.

[0457] The results showed that the humanized CD3×GPRC5D bispecific antibody (22A8) had strong affinity for CPRG5D and CD3 antigens, respectively.

[0458] Example 9: In vivo killing test of bispecific antibodies

[0459] 9.1 Human Myeloma NCI-H929 Animal Model

[0460] Experimental methods:

[0461] Six-week-old female NPG mice were subcutaneously inoculated with 5×10 6 NCI-H929 cells were inoculated and 5×10 PBMC were injected intraperitoneally (ip) on the day of NCI-H929 cell inoculation (day 0). 6 When the average tumor volume grew to about 113 mm3, the mice were divided into groups according to tumor volume and intravenously injected (iv) with 1 mg / kg of drug 22A2 and 1 mg / kg of drug 22A8 twice a week (BIW) at an injection volume of 0.1 mL / 10 g body weight.

[0462] Investigate the effect of drugs on tumor growth, with specific indicators being tumor proliferation rate (T / C%) or tumor inhibition rate (TGI%)

[0463] Tumor diameters were measured with a vernier caliper three times a week.

[0464] Tumor volume calculation formula: tumor volume TV (mm3) = 1 / 2 × (a × b2);

[0465] Where a represents the major diameter and b represents the minor diameter.

[0466] Tumor proliferation rate formula: T / C%=TmTV / CmTV×100%;

[0467] Wherein, TmTV: mean value of treatment group; CmTV: mean value of control group.

[0468] Tumor inhibition rate formula: TGI% = (1-T / C) × 100%;

[0469] Wherein, T / C% is the tumor proliferation rate, which is the percentage value of the relative tumor volume (or body weight) of the treatment group and the control group at a certain time point.

[0470] If the tumor is smaller than its initial size, it is defined as partial tumor regression (PR); if the tumor disappears completely, it is defined as complete tumor regression (CR).

[0471] Experimental results:

[0472] The results are shown in Figure 20 and Table 17. They demonstrate that 22A2 and 22A8 (1 mg / kg, IV, twice weekly for five doses) significantly inhibited the growth of subcutaneous xenografts in mice reconstituted with human PBMCs for the human myeloma NCI-H929 tumor. The TGIs at the endpoint of D17 were 84.84% and 97.71%, respectively. Five of six mice in the 22A8 group experienced complete tumor regression. All of the drugs were well tolerated by the tumor-bearing mice, with no significant weight loss or other symptoms observed during dosing.

[0473] Table 17.

[0474] 9.2 Human myeloma NCI-H929 animal model.

[0475] Experimental methods:

[0476] Six-week-old female NPG mice were subcutaneously inoculated with 5×10 6 NCI-H929 cells were inoculated and 5×10 PBMC were injected intraperitoneally (ip) on the day of NCI-H929 cell inoculation (day 0). 6 cells / mouse. When the average tumor volume grows to about 97mm 3 The patients were divided into groups according to tumor volume and injected intravenously (iv) with 0.1 mg / kg and 1 mg / kg of 22A8 twice a week (BIW) with an injection volume of 0.1 mL / 10 g body weight.

[0477] Investigate the effect of drugs on tumor growth, with specific indicators being tumor proliferation rate (T / C%) or tumor inhibition rate (TGI%)

[0478] Tumor diameters were measured with a vernier caliper three times a week.

[0479] Tumor volume calculation formula: tumor volume TV (mm3) = 1 / 2 × (a × b2);

[0480] Where a represents the major diameter and b represents the minor diameter.

[0481] Tumor proliferation rate formula: T / C%=TmTV / CmTV×100%;

[0482] Wherein, TmTV: mean value of treatment group; CmTV: mean value of control group.

[0483] Tumor inhibition rate formula: TGI% = (1-T / C) × 100%;

[0484] Wherein, T / C% is the tumor proliferation rate, which is the percentage value of the relative tumor volume (or body weight) of the treatment group and the control group at a certain time point.

[0485] If the tumor is smaller than its initial size, it is defined as partial tumor regression (PR); if the tumor disappears completely, it is defined as complete tumor regression (CR).

[0486] Experimental results:

[0487] 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 for a total of 5 doses) significantly inhibited the growth of subcutaneous xenografts in mice reconstituted with human PBMCs for the human myeloma NCI-H929 tumor, with TGIs of 85.61% and 95.65%, respectively. Partial tumor regression was observed in 1 / 5 mice in the 0.1 mg / kg 22A8 group and in 2 / 5 mice in the 1 mg / kg 22A8 group. With the exception of the 0.1 mg / kg 22A8 group, which exhibited slight weight loss (a 3.6% decrease on D16 compared to pre-dose weight loss), the drug was well tolerated by the tumor-bearing mice in the remaining groups, with no significant weight loss or other symptoms observed during the dosing period.

[0488] Table 18.

[0489] 9.3 PK study of subcutaneous or intravenous dose escalation in cynomolgus monkeys.

[0490] Experimental methods:

[0491] Four female cynomolgus monkeys were used, numbered 1#, 2#, 5#, and 6#. Animals 1# and 2# were administered test article 22A8 subcutaneously, while animals 5# and 6# were administered intravenously. Dosing was completed within 90 minutes. Among them, the dosage of animal #1 was 0.3 mg / kg, 1 mg / kg and 3 mg / kg, with a dosage volume of 1 mL / kg, and the drugs were administered on day 0, day 3 and day 6, for a total of 3 times; the dosage of animal #2 was 1 mg / kg, 3 mg / kg and 10 mg / kg, with a dosage volume of 1 mL / kg or 1.65 mL / kg, and the drugs were administered on day 0, day 3 and day 6, for a total of 3 times; the dosage of animal #5 was 0.1 mg / kg, 0.3 mg / kg and 1 mg / kg, with a dosage volume of 5 mL / kg, and the drugs were administered on day 0, day 3 and day 6, for a total of 3 times; the dosage of animal #6 was 0.3 mg / kg, 1 mg / kg and 3 mg / kg, with a dosage volume of 5 mL / kg, and the drugs were administered on day 0, day 3 and day 6, for a total of 3 times. Among the above 4 animals, 1# and 5# were administered at the same time, and 2# and 6# were administered at the same time. The specific design is shown in Table 19. During the trial, blood was collected at different time points, and clinical observation, clinical pathology, serum cytokine, immune phenotype detection and blood drug concentration detection were carried out to calculate PK parameters.

[0492] Table 19.

[0493] Experimental results:

[0494] During the experiment, no obvious abnormalities were found in the clinical observations of all animals, and no weight loss was observed.

[0495] PK results showed that 22A8 was well absorbed in animals. Subcutaneous administration of 22A8 demonstrated a dose-dependent increase in Cmax and AUC. Animal #1 (0.3 / 1 / 3 mg / kg, Q3D x 3, sc) demonstrated a half-life of 26-64 hours. Animal #2 (1 / 3 / 10 mg / kg, Q3D x 3, sc) demonstrated a half-life of 36-74 hours. Animal #5 (0.1 / 0.3 / 1 mg / kg, Q3D x 3, iv) demonstrated a half-life of 23-38 hours. Animal #6 (0.3 / 1 / 3 mg / kg, Q3D x 3, iv) demonstrated a half-life of 30-47 hours.

[0496] While the present application has been particularly shown and described with reference to specific embodiments, some of which are preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the application disclosed herein.

Claims

1. A multispecific polypeptide complex 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 comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein the heavy chain complementarity determining regions bind to the heavy chain variable region (V H ) are identical to the three heavy chain complementary determining regions contained in the light chain variable region (V L ) are identical in the three light chain complementarity determining regions.

2. The polypeptide complex according to claim 1, wherein in the GPRC5D binding domain: a) the 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) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, or SEQ ID NO: 10 or a variant thereof having no more than 3, 2 or 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 no more than 3, 2, or 1 amino acid substitutions, or SEQ ID NO: 11 or a variant thereof having no more than 3, 2, or 1 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 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 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. The polypeptide complex according to any one of the preceding claims, wherein in the GPRC5D binding domain, a) the HCDR1 comprises the amino acid sequence of SEQ ID NO:4 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, the HCDR2 comprises the amino acid sequence of SEQ ID NO:5 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, the HCDR3 comprises the amino acid sequence of SEQ ID NO:6 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, the LCDR1 comprises the amino acid sequence of SEQ ID NO:1 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, the LCDR2 comprises the amino acid sequence of SEQ ID NO:2 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:3 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; or b) the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 9 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 10 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 11 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 7 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 8 or a variant having no more than 3, 2 or 1 amino acid substitutions, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 3 or a variant having no more than 3, 2 or 1 amino acid substitutions.

4. The polypeptide complex according to any one of the preceding claims, wherein in the GPRC5D binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13 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 shown in SEQ ID NO: 12 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions.

5. The polypeptide complex according to any one of the preceding claims, wherein the GPRC5D binding domain is humanized.

6. The polypeptide complex according to any one of the preceding claims, wherein the GPRC5D binding domain comprises 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 consisting of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, or a variant having no more than 3, 2, or 1 amino acid substitutions; b) the light chain variable region is selected from the amino acid sequence of the following group: SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or a variant having no more than 3, 2, or 1 amino acid substitutions.

7. The polypeptide complex according to any one of the preceding claims, wherein the GPRC5D binding domain comprises 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 of SEQ ID NO: 15 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 of SEQ ID NO: 14 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17 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 of SEQ ID NO: 16 or a variant thereof having no more than 3, 2, or 1 amino acid substitutions; c) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19 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 shown in SEQ ID NO: 18 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; or d) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21 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 of SEQ ID NO: 20 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; wherein the amino acid substitution is not within a CDR region.

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

9. The polypeptide complex of any one of the preceding claims, wherein the other of the first antigen binding domain and the second antigen binding domain binds to an antigen different from GPRC5D.

10. The polypeptide complex according to claim 9, wherein the antigen different from GPRC5D is an immunostimulatory antigen, optionally the immunostimulatory antigen is CD3. The polypeptide complex 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 polypeptide complex of claim 11 , wherein 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 of SEQ ID NO: 49 or a variant thereof having no more than 3, 2, or 1 amino acid substitutions, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 50 or a variant thereof having no more than 3, 2, or 1 amino acid substitutions, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 51 or a variant thereof having no more than 3, 2, or 1 amino acid substitutions, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 52 or a variant thereof having no more than 3, 2, or 1 amino acid substitutions, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 53 or a variant thereof having no more than 3, 2, or 1 amino acid substitutions, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 54 or a variant thereof having no more than 3, 2, or 1 amino acid substitutions; or The HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 92 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 93 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 94 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 95 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 96 or a variant having no more than 3, 2 or 1 amino acid substitutions, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 54 or a variant having no more than 3, 2 or 1 amino acid substitutions.

13. The polypeptide complex according to 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 shown in SEQ ID NO: 55 or a variant having no more than 3, 2 or 1 amino acid substitutions, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 56 or a variant having no more than 3, 2 or 1 amino acid substitutions.

14. The polypeptide complex according to any one of claims 1 to 13, wherein the first antigen-binding domain and the second antigen-binding domain constitute a DICAD domain, and the DICAD domain comprises: (i) a first polypeptide comprising, in the 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 directly connected or connected through a first linker; (ii) a second polypeptide comprising, in the 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 directly linked or linked via a second linker; in: VL1 and VH1 combine to form the first antigen-binding domain, VL2 and VH2 combine to form the second antigen-binding domain, VL1 and VH1 are covalently linked by a disulfide bond. The polypeptide complex according to claim 14 , wherein the first linker and / or the second linker independently comprise 5 to 9 amino acid residues. The polypeptide complex according to claim 14 or 15, wherein the VL1 has a first cysteine ​​substitution in FR and the VH1 has a second cysteine ​​substitution in FR, and the first cysteine ​​and the second cysteine ​​form a disulfide bond.

17. The polypeptide complex according to claim 16, wherein the first cysteine ​​and the second cysteine ​​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; The numbering therein is according to Kabat numbering. The polypeptide complex according to claim 17 , wherein the disulfide bond is formed between 100C in VL1 and 44C in VH1. The polypeptide complex according to any one of claims 14 to 18, wherein the VL1 and VH1 further have an electrostatic interaction between two oppositely charged residues.

20. The polypeptide complex according to any one of claims 14 to 19, wherein 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.

21. The polypeptide complex according to any one of claims 14 to 20, wherein VL2 and VH2 further have an electrostatic interaction between two oppositely charged residues.

22. The polypeptide complex according to any one of claims 14 to 21, wherein the two oppositely charged residues between VL2 and VH2 are introduced and 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, wherein the numbering is according to Kabat numbering.

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

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

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

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

23.

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

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

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

30. The polypeptide complex of claim 29, wherein the Fab domain comprises: (i) a third polypeptide comprising, in the 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 the N-terminal to C-terminal direction, a third light chain variable domain VL3 and a CL domain that binds a third antigen, Wherein VL3 and VH3 combine to form the third antigen-binding domain. The polypeptide complex according to 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 shown in SEQ ID NO: 29, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 30, the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 31, and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO:

32.

33. The polypeptide complex according to any one of claims 14 to 24, wherein the polypeptide complex further comprises a third antigen-binding domain, optionally, the third antigen-binding domain comprises a Fab domain, wherein the Fab domain comprises: (iii) a third polypeptide comprising, in the 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 the N-terminal to C-terminal direction, a third light chain variable domain VL3 and a CL domain that binds a third antigen, Wherein VL3 and VH3 combine to form the third antigen-binding domain. The polypeptide complex according to claim 33 , wherein the first antigen, the second antigen and the third antigen are independently selected from GPRC5D, an immunostimulatory antigen and a tumor antigen, respectively. Optionally, the immunostimulatory antigen is CD3 and the tumor antigen is Her2. The polypeptide complex according to claim 34 , wherein the first antigen is Her2, the second antigen is CD3, and the third antigen is GPRC5D.

36. The polypeptide complex according to claim 35, wherein the Her2 binding domain is contained in the heavy chain variable region (V H ) within the three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and within the light chain variable region (V L ), the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 57 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 58 or a variant having no more than 3, 2 or 1 amino acid substitutions, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 59 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 60 or a variant having no more than 3, 2 or 1 amino acid substitutions, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 61 or a variant having no more than 3, 2 or 1 amino acid substitutions, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 62 or a variant having no more than 3, 2 or 1 amino acid substitutions.

37. The polypeptide complex of claim 36, wherein the Her2 binding domain comprises a light chain variable domain having an amino acid sequence as shown in SEQ ID NO: 64, and VH3 comprises a heavy chain variable domain having an amino acid sequence as shown in SEQ ID NO:

63.

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

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

28.

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

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

36.

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

43. The polypeptide complex according to any one of claims 1 to 8, wherein the polypeptide complex 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, in the N-terminal to C-terminal direction, a first heavy chain variable domain VH1 and a first CH1 domain CHIa that binds to a first antigen; and (ii) a second polypeptide comprising, in the 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; The second antigen binding domain comprises a second Fab domain comprising: (iii) a third polypeptide comprising, in the 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 the N-terminal to C-terminal direction, a second light chain variable domain VL2 and a second CL domain CLb that binds 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 up and bind, CH1b and CLb can pair up and bind, and CH1a and CLa can pair up and bind. The CH1b and CLb binding pair is configured to avoid mismatches between CH1a and CLb and / or between CH1b and CLa. The polypeptide complex 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. The polypeptide complex of claim 43 or 44, wherein the second antigen binding domain comprises a CD3 binding domain as defined in claim 12 or 13.

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

47. The polypeptide complex 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 by a first pair of disulfide bonds, the first pair of disulfide bonds is non-natural, and optionally the naturally occurring disulfide bond in the first CH1 / CL binding pair is deleted or destroyed.

48. The polypeptide complex of claim 47, wherein the second CH1 / CL binding pair is formed by a second pair of disulfide bonds, the second pair of disulfide bonds being located at a different position than the first pair of disulfide bonds and optionally being naturally occurring disulfide bonds.

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

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

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

52. The polypeptide complex of any one of claims 47-51, wherein the first CH1 / CL binding pair comprises at least one mutation from an uncharged amino acid residue to a charged amino acid residue, and / or at least one mutation from a 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 complex of claim 52, wherein the second CH1 / CL pair comprises at least one mutation from an uncharged amino acid residue to a charged amino acid residue, and / or at least one mutation from a 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 hinder pairing of CH1a and CLb or pairing of CH1b and CLa. The polypeptide complex according to claim 53 , wherein the first pair of oppositely charged residues and the second pair of oppositely charged residues are designed such that CH1a and CLb are both positively charged or both negatively charged, and / or CH1b and CLa are both positively charged or both negatively charged.

55. The polypeptide complex according to any one of claims 52 to 54, wherein the first pair of oppositely charged residues and / or the second pair of oppositely charged residues introduce an oppositely charged amino acid residue pair at a heavy chain-light chain EU numbering position 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) position 126 by EU numbering for the heavy chain and position 121 by EU numbering for the light chain in the first CH1 / CL binding pair; and f) Heavy chain position 218 by EU numbering and light chain position 122 by EU numbering in the first CH1 / CL binding pair.

56. A polypeptide complex as described in any one of claims 52-55, wherein the pair of oppositely charged amino acid residues includes a positively charged amino acid residue and a negatively charged amino acid residue, wherein the positively charged amino acid residue is selected from the following group: lysine (K), histidine (H) and arginine (R), and / or the negatively charged amino acid residue is selected from the following group: aspartic acid (D) and glutamic acid (E).

57. The polypeptide complex of any one of claims 52-56, wherein the CH1b and CLb binding pair has a first pair of non-native disulfide bonds and a first pair of oppositely charged residues that hinder mispairing between CH1b and CLa and / or between CH1a and CLb.

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

37.

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

60. The polypeptide complex according to any one of claims 43 to 59, wherein the polypeptide complex further comprises a third antigen-binding domain, optionally, the third antigen-binding domain is a Fab domain. The polypeptide complex of claim 60 , wherein a C-terminus of the third antigen-binding domain is linked to an N-terminus of the second antigen-binding domain.

62. The polypeptide complex of claim 60 or 61, wherein the third antigen binding domain is identical to the first antigen binding domain and comprises: (i) a first fragment comprising, in the 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 the 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 wherein the C-terminus of the first fragment is connected to the N-terminus of the fourth polypeptide.

63. The polypeptide complex of claim 62, wherein the polypeptide complex comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and a fifth polypeptide, wherein 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 identical and each comprises VL1-CLa.

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

65. The polypeptide complex of claim 64, wherein the CH1b and CLb binding pair has one or more introduced amino acid mutations and forms at least one introduced charged amino acid residue that hinders 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 shown in SEQ ID NO: 43, the second polypeptide or the fifth polypeptide comprises the amino acid sequence shown in SEQ ID NO: 44, the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 42, and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO:

41.

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

45.

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

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

71. The polypeptide complex of claim 70, wherein 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 comprise a combination of: a) T366W or S354C in combination with Y349C, T366S, L368A or Y407V; b) a combination of D399K or E356K and K392D or K409D; c) The combination of E356K, E357K or D399K and 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; in, Amino acid positions are numbered according to the EU numbering system.

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

66.

73. A nucleic acid comprising a nucleotide sequence encoding the polypeptide complex of any one of claims 1-72.

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

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 1-72 or the nucleic acid of claim 73, and a pharmaceutically acceptable carrier.

77. A conjugate comprising the polypeptide complex of any one of claims 1-72 and a cargo conjugated thereto, wherein the cargo is selected from the group consisting of a radioactive label, a fluorescent label, an enzyme substrate label, an affinity purification label, a tracer molecule, an anticancer drug, and a cytotoxic molecule.

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

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

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