Gprc5d binding moieties, chimeric antigen receptors, and uses thereof

Abstract

The present invention relates to GPRC5D binding moieties, chimeric antigen receptors (CARs) having these GPRC5D binding moieties, and uses thereof. The present invention also relates to genetically modified immune cells having chimeric antigen receptors for use in adoptive cell therapy for treating a GPRC5D expressing cancer or tumor in a subject in need thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to International Application No. PCT / CN2023 / 131797, filed on November 15, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] sequence declaration

[0004] The following content, submitted as an XML file, is incorporated herein by reference in its entirety: Computer-readable Sequence List (filename: IEC240497PCT_SEQUENCE LISTING.xml, record date: 13 November 2024, size: 359,094 bytes). Technical Field

[0005] This disclosure relates to GPRC5D binding moieties, chimeric antigen receptors (CARs) having these GPRC5D binding moieties, and their uses. This disclosure also relates to genetically modified immune cells having chimeric antigen receptors for use in adoptive cell therapy for treating subjects with GPRC5D-expressing cancers or tumors. Background Technology

[0006] G protein-coupled receptor family C group 5 member D (GPRC5D) is a type C 7-transmembrane receptor protein. It is an orphan receptor whose ligand and signal transduction mechanisms are not yet determined. Located on chromosome 12p13.3, the GPRC5D gene contains three exons and spans approximately 9.6 kb. The large first exon encodes a 7-transmembrane domain. High messenger RNA (mRNA) expression of GPRC5D has been observed in patients with multiple myeloma, while only low expression has been detected in normal tissues. Furthermore, GPRC5D mRNA expression has shown a significant correlation with poor overall survival. Through immunohistochemical analysis, Eric L. Smith and colleagues demonstrated that GPRC5D is expressed on malignant bone marrow plasma cells, while expression in normal tissues is limited to hair follicles. Overexpression in high-risk myeloma, low expression in normal tissues, and cell surface expression suggest that GPRC5D is an attractive target for cancer immunotherapy. Currently, immunotherapies targeting GPRC5D have not yet become successful treatments.

[0007] Therefore, therapeutic options targeting GPRC5D represent an unmet need. There is a need for next-generation antibodies targeting GPRC5D to treat various cancers or other diseases. Summary of the Invention

[0008] Given the shortcomings of existing technologies, one of the main objectives of this disclosure is to provide a GPRC5D resistant component. Therefore, this disclosure provides a GPRC5D resistant component (e.g., V...). H H), chimeric antigen receptors (CARs) containing these portions, pharmaceutical compositions containing these portions, nucleic acids encoding these portions, vectors and host cells for preparing these portions, and methods for treating subjects using these portions, and these portions disclosed herein can be used for detecting and / or treating tumors.

[0009] On the one hand, this disclosure provides protection against GPRC5D V. H H, the anti-GPRC5D V H H contains: (1) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 4 respectively; (2) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 8 respectively; (3) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 12 respectively; (4) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 16 respectively; (5) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 20 respectively; (6) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 24 respectively; (7) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 28 respectively; (8) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 32, respectively; (9) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 36 respectively; (10) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 40, respectively; (11) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 44 respectively; (12) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 48 respectively; (13) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 52 respectively; (14) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 56 respectively; (15) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 60, respectively; (16) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 64 respectively; (17) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 68 respectively; (18) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 72, respectively; (19) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 76 respectively; (20) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 80, respectively; (21) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 84 respectively; (22) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 88 respectively; (23) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 92 respectively; (24) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 96 respectively; (25) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 100 respectively; (26) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 104 respectively; (27) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 108 respectively; (28) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 112 respectively; (29) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 115 respectively; (30) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 119 respectively; (31) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 123 respectively; (32) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 127 respectively; (33) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 131 respectively; (34) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 135 respectively; (35) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 139 respectively; (36) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 143 respectively; (37) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 147 respectively; (38) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 151, respectively; (39) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 155 respectively; (40) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 159 respectively; (41) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 163 respectively; (42) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 167 respectively; (43) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 171 respectively; (44) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 175 respectively; (45) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 179 respectively; (46) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 183 respectively; (47) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 187 respectively; (48) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 190 respectively; (49) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 193 respectively; (50) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 197 respectively; (51) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 201 respectively; (52) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 205 respectively; (53) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 209 respectively; (54) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 213 respectively; (55) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 216 respectively; (56) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 220 respectively; (57) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 224, respectively; (58) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 228, respectively; (59) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 232, respectively; (60) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 236, respectively; (61) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 240, respectively; (62) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 244 respectively; (63) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 248 respectively; (64) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 252, respectively; (65) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 256, respectively; (66) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 260, respectively; (67) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 264, respectively; (68) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 266 respectively; (69) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 270 respectively; (70) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 274, respectively; (71) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 278 respectively; (72) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 282, respectively; (73) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 286 respectively; (74) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 290, respectively; (75) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 294, respectively; (76) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 298, respectively; (77) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 302 respectively; (78) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 306 respectively; (79) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 310, respectively.

[0010] In some embodiments, the CDR1, CDR2, or CDR3 is determined according to the AbM numbering scheme, the IMGT numbering scheme, the Kabat numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or any combination thereof.

[0011] In some embodiments, the CDR1, CDR2, or CDR3 is determined according to the AbM numbering scheme.

[0012] On the other hand, this disclosure provides an anti-GPRC5D V H H, the anti-GPRC5D V H H contains CDR1, CDR2, and CDR3, which respectively contain the following amino acid sequences: (1) SEQ ID NO: 5, 6 and 7; or (2) SEQ ID NO: 9, 10 and 11; or (3) SEQ ID NO: 13, 14 and 15; or (4) SEQ ID NO: 17, 18 and 19; or (5) SEQ ID NO: 21, 22 and 23; or (6) SEQ ID NO: 25, 26 and 27; or (7) SEQ ID NO: 29, 30 and 31; or (8) SEQ ID NO: 33, 34 and 35; or (9) SEQ ID NO: 37, 38 and 39; or (10) SEQ ID NO: 41, 42 and 43; or (11) SEQ ID NO: 45, 46 and 47; or (12) SEQ ID NO: 49, 50 and 51; or (13) SEQ ID NO: 53, 54 and 55; or (14) SEQ ID NO: 57, 58 and 59; or (15) SEQ ID NO: 61, 62 and 63; or (16) SEQ ID NO: 65, 66 and 67; or (17) SEQ ID NO: 69, 70 and 71; or (18) SEQ ID NO: 73, 74 and 75; or (19) SEQ ID NO: 77, 78 and 79; or (20) SEQ ID NO: 81, 82 and 83; or (21) SEQ ID NO: 85, 86 and 87; or (22) SEQ ID NO: 89, 90 and 91; or (23) SEQ ID NO: 93, 94 and 95; or (24) SEQ ID NO: 97, 98 and 99; or (25) SEQ ID NO: 101, 102 and 103; or (26) SEQ ID NO: 105, 106 and 107; or (27) SEQ ID NO: 109, 110 and 111; or (28) SEQ ID NO: 81, 113 and 114; or (29) SEQ ID NO: 116, 117 and 118; or (30) SEQ ID NO: 120, 121 and 122; or (31) SEQ ID NO: 124, 125 and 126; or (32) SEQ ID NO: 128, 129 and 130; or (33) SEQ ID NO: 132, 133 and 134; or (34) SEQ ID NO: 136, 137 and 138; or (35) SEQ ID NO: 140, 141 and 142; or (36) SEQ ID NO: 144, 145 and 146; or (37) SEQ ID NO: 148, 149 and 150; or (38) SEQ ID NO: 152, 153 and 154; or (39) SEQ ID NO: 156, 157 and 158; or (40) SEQ ID NO: 160, 161 and 162; or (41) SEQ ID NO: 164, 165 and 166; or (42) SEQ ID NO: 168, 169 and 170; or (43) SEQ ID NO: 172, 173 and 174; or (44) SEQ ID NO: 176, 177 and 178; or (45) SEQ ID NO: 180, 181 and 182; or (46) SEQ ID NO: 184, 185 and 186; or (47) SEQ ID NO: 136, 188 and 189; or (48) SEQ ID NO: 191, 192 and 138; or (49) SEQ ID NO: 194, 195 and 196; or (50) SEQ ID NO: 198, 199 and 200; or (51) SEQ ID NO: 202, 203 and 204; or (52) SEQ ID NO: 206, 207 and 208; or (53) SEQ ID NO: 210, 211 and 212; or (54) SEQ ID NO: 120, 214 and 215; or (55) SEQ ID NO: 217, 218 and 219; or (56) SEQ ID NO: 221, 222 and 223; or (57) SEQ ID NO: 225, 226 and 227; or (58) SEQ ID NO: 229, 230 and 231; or (59) SEQ ID NO: 233, 234 and 235; or (60) SEQ ID NO: 237, 238 and 239; or (61) SEQ ID NO: 241, 242 and 243; or (62) SEQ ID NO: 245, 246 and 247; or (63) SEQ ID NO: 249, 250 and 251; or (64) SEQ ID NO: 253, 254 and 255; or (65) SEQ ID NO: 257, 258 and 259; or (66) SEQ ID NO: 261, 262 and 263; or (67) SEQ ID NO: 5, 6 and 265; or (68) SEQ ID NO: 267, 268 and 269; or (69) SEQ ID NO: 271, 272 and 273; or (70) SEQ ID NO: 275, 276 and 277; or (71) SEQ ID NO: 279, 280 and 281; or (72) SEQ ID NO: 283, 284 and 285; or (73) SEQ ID NO: 287, 288 and 289; or (74) SEQ ID NO: 291, 292 and 293; or (75) SEQ ID NO: 295, 296 and 297; or (76) SEQ ID NO: 299, 300 and 301; or (77) SEQ ID NO: 303, 304 and 305; or (78) SEQ ID NO: 307, 308 and 309; or (79) SEQ ID NO: 311, 312 and 313.

[0013] In some embodiments, the V H H contains SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 190, 193, 197, 201, 205, 209, 213, 216, 220 The amino acid sequence of 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 266, 270, 274, 278, 282, 286, 290, 294, 298, 302, 306, or 310, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0014] In some embodiments, the V H H represents camels (V) H H, interlocking V H H or humanized V H H.

[0015] In some embodiments, the VH H contains an amino acid sequence of SEQ ID NO: 16, 24, 48, 143, 179, 187, 193, 220, 244, 274, 298, 302, 306 or 310, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it.

[0016] In some embodiments, the V H H contains an amino acid sequence of any one of the following: (1) SEQ ID NO: 328, 329, 330, 331, 332 or 333; (2) SEQ ID NO: 334, 335, 336 or 337; (3) SEQ ID NO: 338, 339, 340, 341 or 342; (4) SEQ ID NO: 343, 344, 345 or 346; (5) SEQ ID NO: 347, 348 or 349; (6) SEQ ID NO: 350, 351 or 352; (7) SEQ ID NO: 353, 354, 355, 356, 357 or 358; (8) SEQ ID NO: 359, 360, 361, 362 or 363; (9) SEQ ID NO: 364, 365, 366 or 367; (10) SEQ ID NO: 368, 369, 370 or 371; (11) SEQ ID NO: 372, 373, 374, 375 or 376; (12) SEQ ID NO: 377, 378 or 379; (13) SEQ ID NO: 380, 381, 382, ​​383 or 384; (14) SEQ ID NO: 385, 386, 387, 388, 389 or 390.

[0017] On the other hand, this disclosure provides a polypeptide construct comprising the V disclosed herein. H H.

[0018] In some embodiments, the construct is a single-specific molecule, a bispecific molecule, a multispecific molecule, an immunoconjugate, or a fusion protein.

[0019] In some embodiments, the single-specific molecule is sdAb or its antigen-binding fragment.

[0020] In some embodiments, the construct contains additional polypeptides.

[0021] In some embodiments, the additional polypeptide is the Fc region of an immunoglobulin.

[0022] In some embodiments, the immunoglobulin Fc region is an IgG Fc region, such as an IgG1, IgG2, IgG3, or IgG4 Fc region.

[0023] In some embodiments, the additional polypeptide is an additional binding domain of a second target other than GPRC5D.

[0024] On the other hand, this disclosure provides a multispecific molecule comprising V containing the present disclosure. H H binds to the GPRC5D binding domain and to another binding domain that binds to a second target other than GPRC5D.

[0025] In some embodiments, the multispecific molecule is a bispecific antibody.

[0026] In some embodiments, the additional binding domain is a monovalent antibody fragment, such as Fab, Fv, scFv, or V. H H.

[0027] In some embodiments, the second target is selected from CD3, CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, mesothelin 6, mesothelin 18.2, GPC3, GPC2, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

[0028] On the other hand, this disclosure provides a nucleic acid molecule containing encoding the V disclosed herein. H H. The nucleotide sequence of the polypeptide construct or the multispecific molecule disclosed herein.

[0029] On the other hand, this disclosure provides a carrier that contains nucleic acid molecules as described above.

[0030] On the other hand, this disclosure provides a host cell that contains nucleic acid molecules or vectors as described above.

[0031] On the other hand, this disclosure provides a V for generating this disclosure. HH, or a polypeptide construct of the present disclosure, or a multispecific molecule of the present disclosure, the method comprising: culturing a host cell or cell containing a nucleic acid molecule of the present disclosure or a vector of the present disclosure under conditions allowing protein expression, and recovering the V from a culture of the cultured cells. H H, the polypeptide construct or the multispecific molecule.

[0032] On the other hand, this disclosure provides a GPRC5D bonding portion that includes the V disclosed herein. H H.

[0033] In some embodiments, the binding portion is sdAb or its antigen-binding fragment.

[0034] On the other hand, this disclosure provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain includes a first binding portion that specifically binds to a first antigen or epitope, wherein the first binding portion includes the V disclosed herein. H H. The polypeptide or multispecific molecule disclosed herein.

[0035] In some embodiments, the extracellular antigen-binding domain further includes a second binding portion that specifically binds to a second antigen or epitope.

[0036] In some embodiments, the first antigen or epitope is the same as the second antigen or epitope. In some embodiments, the first antigen or epitope is different from the second antigen or epitope. In some embodiments, the first binding portion and the second binding portion are connected in series.

[0037] In some embodiments, the second binding portion is an extracellular domain of sdAb, scFv, or the receptor.

[0038] In some embodiments, the second binding portion is an antitumor antigen sdAb or scFv, and a tumor antigen selected from CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL3, duracin 6, duracin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

[0039] In some embodiments, the second bonding portion is anti-GPRC5D sdAb and includes the V disclosed herein. H H. The polypeptide construct disclosed herein or the multispecific molecule disclosed herein.

[0040] In some embodiments, the CAR further comprising a spacer region domain is selected from the hinge domain and / or CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4). In some embodiments, the hinge domain comprises the hinge region of CD8α, IgG4, PD1, CD152, or CD154. In some embodiments, the spacer region domain comprises the hinge region of CD8α (e.g., the sequence shown in SEQ ID NO: 316).

[0041] In some embodiments, the transmembrane domain is selected from one or more transmembrane regions selected from the group consisting of: the α, β, or ζ chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD28, CD137, CD152, CD154, and PD1. In some embodiments, the transmembrane domain comprises a transmembrane region of CD8α (e.g., the sequence shown in SEQ ID NO: 317).

[0042] In some embodiments, the intracellular signal transduction domain includes a primary signal transduction domain and / or a co-stimulatory signal transduction domain.

[0043] In some embodiments, the intracellular signal transduction domain includes a primary signal transduction domain and at least one co-stimulatory signal transduction domain.

[0044] In some embodiments, the primary signal transduction domain comprises intracellular signal transduction domains of proteins selected from the group consisting of: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0045] In some embodiments, the co-stimulatory signal transduction domain comprises an intracellular signal transduction domain of a protein selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAGS), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, and DAP12.

[0046] In some embodiments, the intracellular signal transduction domain comprises a primary signal transduction domain and a co-stimulatory signal transduction domain, wherein the primary signal transduction domain comprises an intracellular signal transduction domain of CD3ζ (e.g., the sequence shown in SEQ ID NO: 319), and the co-stimulatory signal transduction domain comprises an intracellular signal transduction domain of CD137 (e.g., the sequence shown in SEQ ID NO: 318).

[0047] In some embodiments, the chimeric antigen receptor further comprises a signal peptide and / or a tag.

[0048] In some embodiments, the tag is selected from myc tags, His tags, short linear peptide sequences from yeast transcription factor GCN4, leucine zipper sequences, or short linear peptide sequences from human nuclear proteins.

[0049] In some embodiments, the tag is a myc tag, and the myc tag contains a sequence as shown in SEQ ID NO: 315.

[0050] In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain.

[0051] In some embodiments, the signal peptide comprises the sequence shown in SEQ ID NO: 314.

[0052] In some embodiments, the chimeric antigen receptor comprises, in sequence from the N-terminus to the C-terminus, the following domains: a signal peptide, a tag, an antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signal transduction domain.

[0053] On the other hand, this disclosure provides a chimeric antigen receptor (CAR) comprising: A first engineered receptor, comprising the chimeric antigen receptor disclosed herein; and The second engineered receptor comprises: a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular domain.

[0054] In some embodiments, the second extracellular antigen-binding domain is an antitumor antigen sdAb or scFv, and a tumor antigen selected from BCMA, CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, DLL3, duracin 6, duracin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

[0055] On the other hand, this disclosure provides a nucleic acid molecule containing a nucleotide sequence encoding the chimeric antigen receptor or bichimeric antigen receptor disclosed herein.

[0056] In some embodiments, the CAR is expressed by introducing the nucleic acid encoding it into cells either in vivo (in vivo cell therapy) or in vitro (including autologous cell therapy and allogeneic cell therapy). In some embodiments, the cells are immune cells.

[0057] On the other hand, this disclosure provides a carrier containing the nucleic acid molecules disclosed herein.

[0058] On the other hand, this disclosure provides a host cell containing the nucleic acid molecules or vectors disclosed herein.

[0059] In some embodiments, the host cell is selected from immune cells (e.g., human immune cells).

[0060] In some embodiments, the immune cell is selected from the group consisting of T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells and any combination thereof.

[0061] On the other hand, this disclosure provides an engineered immune cell that expresses the chimeric antigen receptor or bi-chimeric antigen receptor disclosed herein.

[0062] In some embodiments, the chimeric antigen receptor or the bichimeric antigen receptor is expressed on the surface of the engineered immune cell.

[0063] In some embodiments, the engineered immune cells also express chimeric antigen receptors that are not specific to GPRC5D.

[0064] In some embodiments, the immune cell is selected from the group consisting of T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells and any combination thereof.

[0065] In some embodiments, the immune cells are obtained from a patient or a healthy donor.

[0066] On the other hand, this disclosure provides a pharmaceutical composition comprising the V disclosed herein. H H. The polypeptide construct disclosed herein, the multispecific molecule disclosed herein, or the chimeric antigen receptor disclosed herein or the bichimeric antigen receptor disclosed herein; and pharmaceutically acceptable carriers and / or excipients.

[0067] In some embodiments, the pharmaceutical compositions disclosed herein further comprise additional therapeutic agents.

[0068] In some embodiments, the additional therapeutic agent is an antitumor agent.

[0069] In some embodiments, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a radionuclide.

[0070] On the other hand, this disclosure provides the V of this disclosure. H H, or the use of the polypeptide construct disclosed herein, or the multispecific molecule disclosed herein, or the nucleic acid molecule disclosed herein, or the vector disclosed herein, or the cell disclosed herein, or the chimeric antigen receptor disclosed herein, or the dual chimeric antigen receptor disclosed herein, or the engineered immune cell disclosed herein, or the pharmaceutical composition disclosed herein in the manufacture of a medicament for use in the prevention and / or treatment of a subject’s disease.

[0071] In some embodiments, the disease is a tumor or an autoimmune disease.

[0072] In some embodiments, the tumor expresses GPRC5D.

[0073] In some embodiments, the tumor is a solid tumor, such as ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcoma, or brain tumors (e.g., gliomas, such as glioblastoma).

[0074] In some embodiments, the tumor is a hematologic malignancy, such as leukemia, lymphoma, and myeloma, including acute myeloid leukemia, adult T-cell leukemia, T-cell large granular lymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute monocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma.

[0075] In some embodiments, the subject is a mammal, such as a human.

[0076] In some embodiments, the V H H, or the polypeptide construct, or the multispecific molecule, or the chimeric antigen receptor, or the bi-chimeric antigen receptor, or the engineered immune cell, or the pharmaceutical composition used in combination with other therapeutic agents or other therapies.

[0077] In some embodiments, the additional therapeutic agent is an antitumor agent.

[0078] In some embodiments, the additional therapeutic agent is another immune checkpoint inhibitor, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, or an anti-CTLA-4 antibody.

[0079] In some embodiments, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a radionuclide.

[0080] In some embodiments, the additional therapy is standard cancer treatment, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

[0081] On the other hand, this disclosure provides the V of this disclosure. H H, or the polypeptide construct disclosed herein, or the multispecific molecule disclosed herein, or the nucleic acid molecule disclosed herein, or the vector disclosed herein, or the cell disclosed herein, or the chimeric antigen receptor disclosed herein, or the dual chimeric antigen receptor disclosed herein, or the engineered immune cell disclosed herein, or the pharmaceutical composition disclosed herein, for use in the prevention and / or treatment of a subject's disease.

[0082] In some embodiments, the disease is a tumor or an autoimmune disease.

[0083] In some embodiments, the tumor expresses GPRC5D.

[0084] In some embodiments, the tumor is a solid tumor, such as ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcoma, or brain tumors (e.g., gliomas, such as glioblastoma).

[0085] In some embodiments, the tumor is a hematologic malignancy, such as leukemia, lymphoma, and myeloma, including acute myeloid leukemia, adult T-cell leukemia, T-cell large granular lymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute monocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma.

[0086] In some embodiments, the subject is a mammal, such as a human.

[0087] In some embodiments, the V H H, or the polypeptide construct, or the multispecific molecule, or the chimeric antigen receptor, or the bi-chimeric antigen receptor, or the engineered immune cell, or the pharmaceutical composition used in combination with other therapeutic agents or other therapies.

[0088] In some embodiments, the additional therapeutic agent is an antitumor agent.

[0089] In some embodiments, the additional therapeutic agent is another immune checkpoint inhibitor, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, or an anti-CTLA-4 antibody.

[0090] In some embodiments, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a radionuclide.

[0091] In some embodiments, the additional therapy is standard cancer treatment, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

[0092] On the other hand, this disclosure provides a method for preventing and / or treating a disease in a subject, the method comprising administering an effective amount of the V of this disclosure to the subject in need. H H, or the polypeptide construct disclosed herein, or the multispecific molecule disclosed herein, or the nucleic acid molecule disclosed herein, or the vector disclosed herein, or the cell disclosed herein, or the chimeric antigen receptor disclosed herein, or the dual chimeric antigen receptor disclosed herein, or the engineered immune cell disclosed herein, or the pharmaceutical composition disclosed herein.

[0093] In some embodiments, the disease is a tumor or an autoimmune disease.

[0094] In some embodiments, the tumor expresses GPRC5D.

[0095] In some embodiments, the tumor is a solid tumor, such as ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcoma, or brain tumors (e.g., gliomas, such as glioblastoma).

[0096] In some embodiments, the tumor is a hematologic malignancy, such as leukemia, lymphoma, and myeloma, including acute myeloid leukemia, adult T-cell leukemia, T-cell large granular lymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute monocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma.

[0097] In some embodiments, the subject is a mammal, such as a human.

[0098] In some embodiments, the V HH, or the polypeptide construct, or the multispecific molecule, or the chimeric antigen receptor, or the bi-chimeric antigen receptor, or the engineered immune cell, or the pharmaceutical composition used in combination with other therapeutic agents or other therapies.

[0099] In some embodiments, the additional therapeutic agent is an antitumor agent.

[0100] In some embodiments, the additional therapeutic agent is another immune checkpoint inhibitor, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, or an anti-CTLA-4 antibody.

[0101] In some embodiments, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a radionuclide.

[0102] In some embodiments, the additional therapy is standard cancer treatment, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care. Attached Figure Description

[0103] Figure 1 20 anti-GPRC5D antibodies AS300399 ( Figure 1 a) AS300413 ( Figure 1 b), AS300442 ( Figure 1 c), AS300452 ( Figure 1 d), AS300533 ( Figure 1 e), AS302376 ( Figure 1 f), AS302399 ( Figure 1 g), AS302509 ( Figure 1 h), AS302553 ( Figure 1 i), AS302558 ( Figure 1 j), AS302643 ( Figure 1 k), AS307587 ( Figure 1 l), AS307685 ( Figure 1 m), AS307858 ( Figure 1 n), AS307879 ( Figure 1 o), AS307946 ( Figure 1 p), AS308386 ( Figure 1 q), AS308506 ( Figure 1 r), AS310012 ( Figure 1 s) and AS310027 ( Figure 1(t) Binding to HEK293 / human GPRC5D cells. Comparisons were performed using 5E11 IgG, G109 scFv-Fc, and isotype control antibodies in each plot.

[0104] Figure 2 20 anti-GPRC5D antibodies AS300399 ( Figure 2 a) AS300413 ( Figure 2 b), AS300442 ( Figure 2 c), AS300452 ( Figure 2 d), AS300533 ( Figure 2 e), AS302376 ( Figure 2 f), AS302399 ( Figure 2 g), AS302509 ( Figure 2 h), AS302553 ( Figure 2 i), AS302558 ( Figure 2 j), AS302643 ( Figure 2 k), AS307587 ( Figure 2 l), AS307685 ( Figure 2 m), AS307858 ( Figure 2 n), AS307879 ( Figure 2 o), AS307946 ( Figure 2 p), AS308386 ( Figure 2 q), AS308506 ( Figure 2 r), AS310012 ( Figure 2 s) and AS310027 ( Figure 2 The binding of t) to H929 was measured. 5E11 IgG and an isotype control antibody were used for comparison in each figure.

[0105] Figure 3 A schematic diagram of the chimeric GPRC5D protein. Chimeric DB1 was constructed by replacing the extracellular region or loop with its corresponding portion from GPRC5A or GPRC5B. Figure 3 a) Chimeric DA2 ( Figure 3 b) Chimeric DA3 ( Figure 3 c) and chimeric DB4 ( Figure 3 d).

[0106] Figure 4 20 anti-GPRC5D antibodies AS300399, AS300413, AS300442, AS300452 ( Figure 4 a), AS300533, AS302376, AS302399, AS302509 ( Figure 4b), AS302553, AS302558, AS302643, AS307587 ( Figure 4 c), AS307685, AS307858, AS307879, AS307946 ( Figure 4 d), AS308386, AS308506, AS310012 and AS310027 ( Figure 4 e) Binding to chimeric GPRC5D expressed on FreeStyle™ 293-F cells. Comparisons were made using 5E11 IgG, G109 scFv-Fc, and an isotype control antibody in each plot.

[0107] Figure 5A Showing monospecific antibody GPRC5D V H Long-term in vitro cytotoxicity of H CAR-T cells against the GPRC5D-positive cell line MM.1S, with the baseline 5E11bbz as a control.

[0108] Figure 5B Showing monospecific antibody GPRC5D V H In vitro proliferation of H CAR-T cells against the GPRC5D positive cell line MM.1S with repeated stimulation, with the baseline 5E11bbz as a control.

[0109] Figure 6 Following stimulation of the GPRC5D-positive cell line MM.1S, a single-specific anti-GPRC5D V was detected. H In vitro IFN-γ production of H CAR-T cells, with baseline 5E11bbz as a control.

[0110] Figure 7A Showing monospecific antibody GPRC5D V H H CAR-T cells demonstrated short-term in vitro cytotoxicity against the GPRC5D-overexpressing cell line Raji-luc, with baseline 5E11bbz and UnT serving as controls.

[0111] Figure 7B Showing monospecific antibody GPRC5D V H H CAR-T cells demonstrated short-term in vitro cytotoxicity against the Raji-luc cell line expressed in GPRC5D, with baseline 5E11bbz and UnT serving as controls.

[0112] Figure 7C Showing monospecific antibody GPRC5D V H H CAR-T cells demonstrated short-term in vitro cytotoxicity against the GPRC5D-low-expressing cell line Raji-luc, with baseline 5E11bbz and UnT serving as controls.

[0113] Figure 8 Showing single-specific GPRC5D V H H CAR-T cells exhibit in vitro cytotoxicity against the GPRC5D-positive cell line MM.1S. All GPRC5D V H H CAR-T cells effectively demonstrated cytotoxicity against GPRC5D-positive cells. UnT refers to T cells not transduced with CAR, serving as a control.

[0114] Figure 9 Showing single-specific GPRC5D V H Results of in vitro long-term cytotoxicity assays of H CAR-T cells against the GPRC5D-low expression cell line (Hep3B2.1-7-5D). Results showed that the single-specific GPRC5D V H H CAR-T cells can rapidly eliminate tumor cells. Detailed Implementation

[0115] Anti-GPRC5D V H H

[0116] The term "V" H H" or "V" H The term "H domain" can be used interchangeably in this article to refer to the antigen-binding portion of a single-domain antibody, such as camel heavy chain antibodies or shark heavy chain antibodies. V H H typically contains three CDRs and four frame regions, designated as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some cases, V H H can be truncated at the N-terminus or C-terminus, so that it contains only a portion of FR1 and / or FR4, or lacks one or both of those frame regions, as long as V H H essentially maintains antigen binding and specificity.

[0117] On the one hand, this disclosure provides protection against GPRC5D V. H H, the anti-GPRC5D V H H contains: (1) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 4 respectively; (2) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 8 respectively; (3) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 12 respectively; (4) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 16 respectively; (5) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 20 respectively; (6) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 24 respectively; (7) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 28 respectively; (8) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 32, respectively; (9) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 36 respectively; (10) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 40, respectively; (11) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 44 respectively; (12) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 48 respectively; (13) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 52 respectively; (14) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 56 respectively; (15) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 60, respectively; (16) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 64 respectively; (17) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 68 respectively; (18) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 72, respectively; (19) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 76 respectively; (20) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 80, respectively; (21) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 84 respectively; (22) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 88 respectively; (23) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 92 respectively; (24) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 96 respectively; (25) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 100 respectively; (26) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 104 respectively; (27) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 108 respectively; (28) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 112 respectively; (29) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 115 respectively; (30) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 119 respectively; (31) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 123 respectively; (32) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 127 respectively; (33) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 131 respectively; (34) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 135 respectively; (35) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 139 respectively; (36) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 143 respectively; (37) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 147 respectively; (38) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 151, respectively; (39) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 155 respectively; (40) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 159 respectively; (41) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 163 respectively; (42) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 167 respectively; (43) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 171 respectively; (44) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 175 respectively; (45) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 179 respectively; (46) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 183 respectively; (47) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 187 respectively; (48) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 190 respectively; (49) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 193 respectively; (50) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 197 respectively; (51) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 201 respectively; (52) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 205 respectively; (53) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 209 respectively; (54) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 213 respectively; (55) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 216 respectively; (56) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 220 respectively; (57) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 224, respectively; (58) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 228, respectively; (59) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 232, respectively; (60) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 236, respectively; (61) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 240, respectively; (62) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 244 respectively; (63) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 248 respectively; (64) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 252, respectively; (65) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 256, respectively; (66) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 260, respectively; (67) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 264, respectively; (68) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 266 respectively; (69) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 270 respectively; (70) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 274, respectively; (71) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 278 respectively; (72) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 282, respectively; (73) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 286 respectively; (74) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 290, respectively; (75) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 294, respectively; (76) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 298, respectively; (77) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 302 respectively; (78) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 306 respectively; (79) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 310, respectively.

[0118] The CDR1, CDR2, or CDR3 mentioned above can be determined according to the AbM numbering scheme, IMGT numbering scheme, Kabat numbering scheme, Chothia numbering scheme, Contact numbering scheme, or any combination thereof.

[0119] V HH may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 5, 6, and 7. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 9, 10, and 11. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 13, 14, and 15. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 17, 18, and 19. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 21, 22, and 23. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 25, 26, and 27. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 29, 30, and 31. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 33, 34, and 35. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 37, 38, and 39. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 41, 42, and 43. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 45, 46, and 47. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 49, 50, and 51. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 53, 54, and 55. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 57, 58, and 59. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 61, 62, and 63. V HH may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences SEQ ID NO: 65, 66, and 67. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 69, 70, and 71. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 73, 74, and 75. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 77, 78, and 79. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 81, 82, and 83. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 85, 86, and 87. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 89, 90, and 91. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 93, 94, and 95. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 97, 98, and 99. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 101, 102, and 103. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences SEQ ID NO: 105, 106, and 107. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 109, 110, and 111. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 81, 113, and 114. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 116, 117, and 118. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 120, 121, and 122. V HH may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences SEQ ID NO: 124, 125, and 126. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 128, 129, and 130. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 132, 133, and 134. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 136, 137, and 138. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 140, 141, and 142. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 144, 145, and 146. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 148, 149, and 150. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 152, 153, and 154. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 156, 157, and 158. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 160, 161, and 162. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences SEQ ID NO: 164, 165, and 166. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 168, 169, and 170. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 172, 173, and 174. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 176, 177, and 178. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences SEQ ID NO: 180, 181, and 182. VH H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences SEQ ID NO: 184, 185, and 186. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 136, 188, and 189. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences SEQ ID NO: 191, 192, and 138. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 194, 195, and 196. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 198, 199, and 200. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 202, 203, and 204. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 206, 207, and 208. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 210, 211, and 212. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 120, 214, and 215. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 217, 218, and 219. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 221, 222, and 223. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 225, 226, and 227. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 229, 230, and 231. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 233, 234, and 235. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 237, 238, and 239. VH H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 241, 242, and 243. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 245, 246, and 247. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 249, 250, and 251. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences SEQ ID NO: 253, 254, and 255. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO:257, 258, and 259. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 261, 262, and 263. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 5, 6, and 265. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 267, 268, and 269. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 271, 272, and 273. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 275, 276, and 277. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 279, 280, and 281. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 283, 284, and 285. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 287, 288, and 289. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 291, 292, and 293. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 295, 296, and 297. VH H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 299, 300, and 301. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 303, 304, and 305. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 307, 308, and 309. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 311, 312, and 313.

[0120] The V disclosed in this article H H can further include a frame region (FR).

[0121] V H H may contain a framework region (FR) derived from camel heavy chain antibodies.

[0122] V H H can contain SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 190, 193, 197, 201, 205, 209, 213, 216, 220 The amino acid sequence of 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 266, 270, 274, 278, 282, 286, 290, 294, 298, 302, 306, or 310, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0123] V H H can be a camel (V) H H, interlocking V H H or humanized V H H.

[0124] V HH can be a humanized version of one or more of the frame regions that have been largely replaced by human frame regions. H H. V H H may contain a framework region (FR) derived from the variable region of the human immunoglobulin heavy chain, one or more of which can be mutated back to their camel counterparts (i.e., reversion mutation).

[0125] V H H may contain the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 328, 329, 330, 331, 332 or 333.

[0126] V H H may contain the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 334, 335, 336 or 337.

[0127] V H H may contain the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 338, 339, 340, 341 or 342.

[0128] V H H may contain the amino acid sequence of SEQ ID NO: 143, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 343, 344, 345 or 346.

[0129] V H H may contain the amino acid sequence of SEQ ID NO: 179, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 347, 348 or 349.

[0130] V H H may contain the amino acid sequence of SEQ ID NO: 187, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 350, 351 or 352.

[0131] V H H may contain the amino acid sequence of SEQ ID NO: 193, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 353, 354, 355, 356, 357 or 358.

[0132] V H H may contain the amino acid sequence of SEQ ID NO: 220, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 359, 360, 361, 362 or 363.

[0133] V HH may contain the amino acid sequence of SEQ ID NO: 244, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 364, 365, 366 or 367.

[0134] V H H may contain the amino acid sequence of SEQ ID NO: 274, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 368, 369, 370 or 371.

[0135] V H H may contain the amino acid sequence of SEQ ID NO: 298, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 372, 373, 374, 375 or 376.

[0136] V H H may contain the amino acid sequence of SEQ ID NO: 302, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 377, 378 or 379.

[0137] V H H may contain the amino acid sequence of SEQ ID NO: 306, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V HH may contain the amino acid sequence of SEQ ID NO: 380, 381, 382, ​​383 or 384.

[0138] V H H may contain the amino acid sequence of SEQ ID NO: 310, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. V H H may contain the amino acid sequence of SEQ ID NO: 385, 386, 387, 388, 389 or 390.

[0139] peptide constructs

[0140] On the other hand, this disclosure provides a polypeptide construct comprising the V disclosed herein. H H.

[0141] The construct can be a single-specific molecule, a bispecific molecule, a multispecific molecule, an immunoconjugate, or a fusion protein. A single-specific molecule can be an sdAb or its antigen-binding fragment. The construct may contain additional peptides. These additional peptides can be immunoglobulin Fc regions. The immunoglobulin Fc region can be an IgG Fc region, such as the Fc region of IgG1, IgG2, IgG3, or IgG4. The additional peptide can be an additional binding domain that binds to a second target besides GPRC5D.

[0142] On the other hand, this disclosure provides a polypeptide construct comprising the V disclosed herein. H H and the Fc region of immunoglobulins.

[0143] The immunoglobulin Fc region can be an IgG Fc region, such as the IgG1, IgG2, IgG3, or IgG4 Fc region. The immunoglobulin Fc region can further be a human IgG Fc region, such as the human IgG1, IgG2, IgG3, or IgG4 Fc region.

[0144] The Fc region of an immunoglobulin can be a natural Fc region containing the same amino acid sequence as that found in naturally occurring Fc regions.

[0145] Alternatively, the immunoglobulin Fc region can be a variant Fc region containing an amino acid sequence that differs from the native Fc region due to at least one amino acid mutation. One or more mutations (e.g., one or more amino acid substitutions) can be introduced into the constant region's Fc region to alter (increase, decrease, or eliminate) one or more effector functions of the same antibody, such as ADCC, CDC, or ADCP, compared to an antibody without such mutations. The immunoglobulin Fc region can have one or more effector functions that are reduced or eliminated.

[0146] V H H can fuse with the N-terminus of the Fc region of immunoglobulin (e.g., at its C-terminus) without a linker.

[0147] Alternatively, V H H can fuse with the N-terminus of the Fc region of an immunoglobulin (e.g., at its C-terminus) in the presence of a linker. The linker can be selected from cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, and non-helical linkers. The linker can be a peptide linker. The linker can contain a sequence of (GmS)n, where m is selected from an integer of 1-6 (e.g., 1, 2, 3, or 4), and n is selected from an integer of 1-6 (e.g., 1, 2, or 3).

[0148] Multispecific molecules

[0149] On the other hand, this disclosure provides a multispecific molecule comprising V containing the present disclosure. H H binds to the GPRC5D binding domain and to another binding domain that binds to a second target other than GPRC5D.

[0150] Multispecific molecules can be bispecific antibodies.

[0151] Other binding domains can be monovalent antibody fragments, such as Fab, Fv, scFv, or V. H H.

[0152] The second target can be selected from CD3, CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, mesothelin 6, mesothelin 18.2, GPC3, GPC2, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

[0153] The multispecific molecules described herein may further include an immunoglobulin Fc domain. Exemplary Fc domains may be selected from the heavy chain constant region of IgG (e.g., IgG1, IgG2, IgG3, or IgG4); more particularly, the heavy chain constant region of human IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0154] The Fc domain can be composed of a first subunit and a second subunit. The GPRC5D binding domain and other binding domains can each be fused with the first and second Fc subunits. The first and second Fc subunits can be engineered for heterodimerization.

[0155] V H H production

[0156] On the other hand, V is provided for generating the disclosure herein. H H, nucleic acid molecules, vectors, and host cells of polypeptide constructs or multispecific molecules.

[0157] On the other hand, this disclosure provides a nucleic acid molecule containing encoding the V disclosed herein. H H. The nucleotide sequence of the polypeptide construct or the multispecific molecule disclosed herein.

[0158] On the other hand, this disclosure provides a carrier that contains nucleic acid molecules as described above.

[0159] The vectors disclosed in this article may be, for example, plasmids, granules, or bacteriophages.

[0160] The carrier may contain encoding the V disclosed herein. H The nucleotide sequence of H.

[0161] The vector may contain a nucleotide sequence encoding the polypeptide construct disclosed herein.

[0162] Vectors can contain different nucleotide sequences of different polypeptide chains that encode multispecific molecules. Different nucleotide sequences can be located on the same or different vectors.

[0163] On the other hand, this disclosure provides a host cell that contains nucleic acid molecules or vectors as described above. Such host cells include, but are not limited to, prokaryotic cells (such as Escherichia coli cells) and eukaryotic cells (such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells and human cells)).

[0164] A V for generating this disclosure is also provided. HH, or a polypeptide construct of the present disclosure, or a multispecific molecule of the present disclosure, the method comprising: culturing a host cell or cell containing a nucleic acid molecule of the present disclosure or a vector of the present disclosure under conditions allowing protein expression, and recovering the V from a culture of the cultured cells. H H, the polypeptide construct or the multispecific molecule.

[0165] Chimeric antigen receptor

[0166] On the other hand, this disclosure provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain includes a first binding portion that specifically binds to a first antigen or epitope, wherein the first binding portion includes the V disclosed herein. H H. The polypeptide or multispecific molecule disclosed herein.

[0167] "Binding moiety" refers to a molecule that contains a portion of a complete antibody and binds to the antigen bound by that complete antibody. Examples of binding moieties include, but are not limited to, Fv, Fab, Fab', F(ab')2; biantibodies; linear antibodies; heavy chain variable (VH) regions; single-chain antibody molecules (such as scFv); and single-domain antibodies containing only VH regions.

[0168] The extracellular antigen-binding domain may further include a second binding portion that specifically binds to a second antigen or epitope.

[0169] The first antigen or epitope may be the same as the second antigen or epitope. The first antigen or epitope may be different from the second antigen or epitope. The first binding region and the second binding region may be connected in tandem.

[0170] The second binding portion can be sdAb, scFv, or the extracellular domain of the receptor.

[0171] The second binding part can be an antitumor antigen sdAb or scFv, and a tumor antigen selected from CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL3, micin 6, micin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

[0172] The second binding portion may be anti-GPRC5D sdAb and includes the V disclosed herein. H H. The polypeptide construct disclosed herein or the multispecific molecule disclosed herein.

[0173] The CAR may further include a spacer domain selected from the hinge domain and / or CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4). Typically, the spacer is an amino acid sequence located between the extracellular antigen-binding domain and the transmembrane domain of the CAR, connecting the extracellular antigen-binding domain and the transmembrane domain.

[0174] Such a spacer region contains portions of human immunoglobulins or their modified forms, including those longer than 125 amino acids, such as those longer than 150, 180, 200, or more amino acids. The hinge domain may contain hinge regions of CD8α, IgG4, PD1, CD152, or CD154. The spacer region domain may contain a hinge region of CD8α. The spacer region domain may contain a sequence as shown in SEQ ID NO: 316.

[0175] The transmembrane domain of a CAR may contain a hydrophobic α-helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, the receptor cluster and signal are transmitted to the cell. According to the subject matter disclosed in this invention, the transmembrane domain may be selected from one or more transmembrane regions of the group consisting of: the α, β, or ζ chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD28, CD137, CD152, CD154, and PD1.

[0176] The transmembrane domain may contain the transmembrane region of CD8α. The transmembrane domain may contain the sequence shown in SEQ ID NO: 317.

[0177] Intracellular signal transduction domains may contain primary signal transduction domains and / or co-stimulatory signal transduction domains. Intracellular signal transduction domains may contain primary signal transduction domains and at least one co-stimulatory signal transduction domain.

[0178] Primary signal transduction domains may contain intracellular signal transduction domains of proteins selected from the group consisting of: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0179] Co-stimulatory signal transduction domains may contain intracellular signal transduction domains of proteins selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAGS), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, and DAP12.

[0180] The intracellular signal transduction domain may include a primary signal transduction domain and a co-stimulatory signal transduction domain, wherein the primary signal transduction domain includes the intracellular signal transduction domain of CD3ζ, and the co-stimulatory signal transduction domain includes the intracellular signal transduction domain of CD137. The primary signal transduction domain may contain the sequence shown in SEQ ID NO: 319. The co-stimulatory signal transduction domain may contain the sequence shown in SEQ ID NO: 318.

[0181] Chimeric antigen receptors may further include signal peptides.

[0182] Chimeric antigen receptors can further include tags.

[0183] The tag can be selected from myc tags, His tags, short linear peptide sequences from yeast transcription factor GCN4, leucine zipper sequences, or short linear peptide sequences from human nuclear proteins. The tag can be a myc tag, and the myc tag can contain the sequence shown in SEQ ID NO: 315.

[0184] Signal peptides can be derived from molecules consisting of CD8α, GM-CSF receptor α, and the IgG1 heavy chain. Signal peptides can contain sequences as shown in SEQ ID NO: 314.

[0185] Chimeric antigen receptors can contain the following domains in sequence from the N-terminus to the C-terminus: signal peptide, tag, antigen-binding domain, spacer domain, transmembrane domain, and intracellular signal transduction domain.

[0186] In some cases, in addition to minimizing relapse due to antigen escape, simultaneous targeting of two antigens can improve the depth and durability of a patient's response. As demonstrated by data from CAR T-cell trials in B-cell malignancies, the mechanism of resistance to CAR T-cell therapy may be the loss or downregulation of the target antigen (“escape”). (Robbie G. Majzner and Crystal L. Mackall, Cancer Discov 2018 Aug 22; DOI 10.1158 / 2159-8290.CD-18-0442). Compared to monotherapy approaches involving only a single antigen, such combined or dual-targeting strategies can achieve synergistic or improved tumor responses based on targeting two antigens.

[0187] Therefore, this disclosure provides a chimeric antigen receptor (CAR) comprising: A first engineered receptor, comprising the chimeric antigen receptor disclosed herein; and The second engineered receptor comprises: a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular domain.

[0188] The second extracellular antigen-binding domain can be an antitumor antigen sdAb or scFv, or a tumor antigen selected from BCMA, CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, DLL3, mesothelin 6, mesothelin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

[0189] In some cases, CARs are expressed by introducing the nucleic acid encoding the CAR into cells either in vivo (in vivo cell therapy) or in vitro (including autologous cell therapy and allogeneic cell therapy). In some cases, the cells are immune cells.

[0190] Also provided are nucleic acid molecules, vectors, and host cells for expressing chimeric or bichimeric antigen receptors and for generating engineered immune cells expressing such chimeric or bichimeric antigen receptors. Genetic engineering typically involves introducing nucleic acids encoding chimeric or bichimeric antigen receptors into cells, such as through lentiviral transduction, retroviral transduction, transfection, or transformation.

[0191] Therefore, this disclosure provides a nucleic acid molecule containing a nucleotide sequence encoding the chimeric antigen receptor or bichimeric antigen receptor disclosed herein.

[0192] On the other hand, this disclosure provides a carrier containing the nucleic acid molecules disclosed herein.

[0193] On the other hand, this disclosure provides a host cell containing the nucleic acid molecules or vectors disclosed herein.

[0194] The host cell can be selected from immune cells (e.g., human immune cells).

[0195] Immune cells can be selected from T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells and any combination thereof.

[0196] On the other hand, this disclosure provides an engineered immune cell that expresses the chimeric antigen receptor or bi-chimeric antigen receptor disclosed herein.

[0197] Optionally, chimeric antigen receptors or dual chimeric antigen receptors are expressed on the surface of engineered immune cells.

[0198] Optionally, engineered immune cells also express chimeric antigen receptors or bi-chimeric antigen receptors that are not specific to GPRC5D.

[0199] Immune cells can be selected from T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells and any combination thereof.

[0200] Immune cells can be obtained from patients or healthy donors.

[0201] Pharmaceutical Composition

[0202] On the other hand, this disclosure provides a pharmaceutical composition comprising the V disclosed herein. H H. The polypeptide construct disclosed herein, the multispecific molecule disclosed herein, or the chimeric antigen receptor disclosed herein or the bichimeric antigen receptor disclosed herein; and pharmaceutically acceptable carriers and / or excipients.

[0203] The pharmaceutical composition may contain an effective amount of V H H. V H H may be the only active ingredient contained in a pharmaceutical composition.

[0204] A pharmaceutical composition may contain an effective amount of a peptide construct. The peptide construct may be the only active ingredient contained in the pharmaceutical composition.

[0205] A pharmaceutical composition may contain an effective amount of a multispecific molecule. A multispecific molecule may be the only active ingredient contained in the pharmaceutical composition.

[0206] A pharmaceutical composition may contain an effective amount of a chimeric antigen receptor. The chimeric antigen receptor may be the only active ingredient contained in the pharmaceutical composition.

[0207] A pharmaceutical composition may contain an effective amount of a bichirpedic antigen receptor. The bichirpedic antigen receptor may be the only active ingredient contained in the pharmaceutical composition.

[0208] The pharmaceutical compositions disclosed herein may further comprise additional therapeutic agents. These additional therapeutic agents may be antitumor agents. They may also be cytotoxic agents, such as alkylating agents, antimitotic agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, or radionuclides.

[0209] V H H, peptide constructs, multispecific molecules, chimeric antigen receptors, dual chimeric antigen receptors, and other therapeutic agents may be provided as separate components or as components of a single composition. V of this article H H. Peptide constructs, multispecific molecules, chimeric antigen receptors, and bichimeric antigen receptors can be used simultaneously, separately, or in combination with other drugs.

[0210] The pharmaceutical composition may be provided in unit dosage form (i.e., the dose for a single administration).

[0211] Pharmaceutical compositions can be formulated using one or more pharmaceutically acceptable carriers and / or excipients. The formulation depends on the chosen route of administration. For parenteral administration, the pharmaceutical composition is preferably sterile and substantially isotonic and manufactured under GMP conditions. For example, the V disclosed herein can be... H H, heavy chain antibodies, polypeptide constructs, multispecific molecules, chimeric antigen receptors, or dual chimeric antigen receptors are formulated in an aqueous solution for injection, preferably in a physiologically compatible buffer such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer), or Ringer's solution. The solution may contain formulations such as suspending agents, stabilizers, and / or dispersants. Alternatively, V disclosed herein... H H, peptide constructs, multispecific molecules, chimeric antigen receptors, or dual chimeric antigen receptors can be in lyophilized form so that they can be prepared with a suitable medium (e.g., sterile pyrogen-free water) before use.

[0212] The pharmaceutical compositions described herein can be used to treat diseases such as tumors.

[0213] Methods or uses for treating diseases / tumors

[0214] On the other hand, it provides the use of V as described in this article. HH, or a method or use of a polypeptide construct, or a multispecific molecule, or a nucleic acid molecule, or a carrier, or a cell, or a chimeric antigen receptor, or a dual chimeric antigen receptor, or an engineered immune cell, or a pharmaceutical composition for the treatment of a disease / tumor.

[0215] In one respect, this disclosure provides the V of this disclosure. H H, or the use of the polypeptide construct disclosed herein, or the multispecific molecule disclosed herein, or the nucleic acid molecule disclosed herein, or the vector disclosed herein, or the cell disclosed herein, or the chimeric antigen receptor disclosed herein, or the dual chimeric antigen receptor disclosed herein, or the engineered immune cell disclosed herein, or the pharmaceutical composition disclosed herein in the manufacture of a medicament for use in the prevention and / or treatment of tumors in a subject.

[0216] On the other hand, this disclosure provides the V of this disclosure. H H, or the polypeptide construct disclosed herein, or the multispecific molecule disclosed herein, or the nucleic acid molecule disclosed herein, or the vector disclosed herein, or the cell disclosed herein, or the chimeric antigen receptor disclosed herein, or the dual chimeric antigen receptor disclosed herein, or the engineered immune cell disclosed herein, or the pharmaceutical composition disclosed herein, for use in the prevention and / or treatment of tumors in a subject.

[0217] On the other hand, this disclosure provides a method for preventing and / or treating tumors in a subject, the method comprising administering an effective amount of the V of this disclosure to the subject in need. H H, or the polypeptide construct disclosed herein, or the multispecific molecule disclosed herein, or the nucleic acid molecule disclosed herein, or the vector disclosed herein, or the cell disclosed herein, or the chimeric antigen receptor disclosed herein, or the dual chimeric antigen receptor disclosed herein, or the engineered immune cell disclosed herein, or the pharmaceutical composition disclosed herein.

[0218] Tumors can express GPRC5D.

[0219] Tumors can be solid tumors, such as ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell lung cancer or non-small cell lung cancer), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, stomach cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcoma, or brain tumors (e.g., gliomas, such as glioblastoma).

[0220] Tumors can be blood malignancies, such as leukemia, lymphoma, and myeloma, including acute myeloid leukemia, adult T-cell leukemia, T-cell large granular lymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute monocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma.

[0221] Subjects can be mammals, such as humans.

[0222] V H H, or peptide constructs, or multispecific molecules, or chimeric antigen receptors, or engineered immune cells, or pharmaceutical compositions may be used in combination with other therapeutic agents or other therapies.

[0223] Other treatment options could be antitumor agents.

[0224] Other treatment options could be other immune checkpoint inhibitors, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, or anti-CTLA-4 antibodies.

[0225] Other therapeutic agents can be cytotoxic agents, such as alkylating agents, antimitotic agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, or radionuclides.

[0226] Other treatment options include standard cancer treatments such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

[0227] definition

[0228] In this disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions and explanations of the terms are provided below for a better understanding of this disclosure.

[0229] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Unless otherwise stated or clear from the context, the term "antibody" as used herein can include complete antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains. The term "antibody" can refer to a conventional antibody that typically comprises at least one heavy chain and at least one light chain. Antibody light chains can be classified as κ light chains and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes can be defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V... H ) and heavy chain constant region (C H It consists of three structural domains (C). The heavy chain constant region is composed of three structural domains (C). H 1. C H 2 and C H 3) Composition. Each light chain consists of a light chain variable region (V L) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. V H and V L The region can be further subdivided into highly variable regions (called complementary determinant regions (CDRs)), interspersed with relatively conservative regions called framing regions (FRs). V H and V L Each of these consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V L These form antibody binding sites. The term "antibody" is not limited to any particular method used to produce antibodies.

[0230] As used in this article, the term "V" H H" or "V" H The "H domain" refers to the antigen-binding portion of a single-domain antibody (such as a camel heavy chain antibody or a shark heavy chain antibody). H H can contain three CDRs and four frame regions, designated as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. V H H can be truncated at the N-terminus or C-terminus, so that it contains only a portion of FR1 and / or FR4, or lacks one or both of those frame regions, as long as V H H essentially maintains antigen binding and specificity. As used in this article, "humanized V" H "H" refers to a V where one or more frame regions have been substantially replaced by human frame regions. H H. In some cases, certain framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. Furthermore, humanized V... H H can contain residues that are not present in the original V. H These residues were found in H and not in human framework sequences, but were included to further refine and optimize V. H The performance of H. As will be understood, humanized sequences can be identified by their primary sequence and do not necessarily represent the process of antibody production.

[0231] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example, according to the following definitions: the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al., (1989) Nature 342: 878-883), the AbM numbering system (Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art can readily identify the CDR defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). The CDR of the antibodies disclosed herein can be defined according to the Kabat, AbM, IMGT, or Chothia numbering systems, or any combination thereof. Unless otherwise specified or clear from the context, the CDR of the antibodies disclosed herein is preferably defined according to the AbM numbering system.

[0232] As used herein, the term “frame region” or “FR” residues refer to those amino acid residues in the variable region of an antibody, other than the CDR residues as defined above.

[0233] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide containing a full-length antibody fragment that retains the ability to specifically bind the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific antigen binding, and this polypeptide is also referred to as an "antigen-binding moiety." Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Antigen-binding fragments can contain fragments of Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining regions (CDRs), single-chain antibodies (e.g., scFv), chimeric antibodies, biantibodies, and polypeptides containing at least a portion of an antibody (that portion is sufficient to confer specific antigen-binding ability upon the polypeptide).

[0234] As used herein, the term "heavy chain-only antibody" or "HCAb" refers to a functional antibody that contains a heavy chain but lacks the light chain typically found in 4-chain antibodies. HCAbs are known to be produced by camels (such as camels, llamas, or alpacas). HCAbs may contain sdAbs fused to the Fc region. HCAbs may also contain sdAbs fused to both the hinge and Fc region of human IgG1.

[0235] As used in this article, the term "Fd fragment" refers to a fragment composed of V H and C H 1. Antibody fragments composed of structural domains; the term "dAb fragment" refers to antibody fragments composed of V... H Antibody fragments composed of structural domains; the term "Fab fragment" refers to an antibody fragment composed of V... L V H C L and C H An antibody fragment consisting of a 1-domain structure; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments linked by disulfide bonds in the hinge region.

[0236] As used herein, the term "Fv fragment" refers to the V fragment formed by the antibody single arm. L and V H Antibody fragments are composed of structural domains. Fv fragments are generally considered to be the smallest antibody fragments capable of forming a complete antigen-binding site. It is believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (e.g., an Fd fragment containing only three CDRs specific to the antigen) can recognize and bind to the antigen, but with less affinity than the entire binding site.

[0237] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains, having a generic structure of NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art consist of repeating G4S amino acid sequences or variants thereof. In some cases, a disulfide bond may also exist between the VH and VL domains of scFv.

[0238] As used herein, the term "biantibody" refers to an scFv dimer consisting of VH and VL domains linked by short peptide linkers. Linkers that are too short to form intra-chain pairing of the VH and VL domains are not possible. Instead, two such scFv fragments are co-expressed to form a multimer through inter-chain pairing (cross-pairing) of the VH and VL domains.

[0239] Each antigen-binding fragment maintains the ability to specifically bind the same antigen bound by the full-length antibody and / or competes with the full-length antibody for the ability to specifically bind that antigen. Antigen-binding fragments can be obtained from a given antibody (e.g., the complete antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques, or enzymatic or chemical cleavage methods), and these fragments can be screened for specificity in the same manner as the complete antibody is screened.

[0240] As used herein, the term "Fc region" refers to a portion of the heavy chain constant region containing CH2 and CH3. The Fc region may contain the hinge, CH2, and CH3. The Fc region can be any antibody heavy chain constant region isotype discussed herein. The Fc region can be IgG1, IgG2, IgG3, or IgG4.

[0241] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody type or subtype), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody type or subtype), provided that the antibody retains its activity in binding to the antigen of interest. For example, the term "chimeric antibody" can include antibodies (e.g., human-cameloid chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a cameloid antibody), while the constant regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0242] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its sequence homology with that of human antibodies. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies typically retain the intended properties of the donor antibody, including but not limited to the ability to specifically bind GPRC5D.

[0243] As used herein, the term "specifically bind" refers to the non-random binding of two molecules, such as the reaction between an antibody and its target antigen. Antibodies that specifically bind to antigens (or antibodies that are specific to antigens) can refer to molecules that bind in a manner less than approximately 10-1. -5 M (for example, less than about 10) -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity (K) of M or smaller D Antibodies that bind to antigens.

[0244] As used in this article, the term "K" D "" refers to the dissociation constant of a specific antibody-antigen interaction, used to describe the binding affinity of an antibody to an antigen. The smaller the dissociation constant, the tighter the antibody binding and the higher the affinity between the antibody and the antigen. Typically, antibodies (e.g., the antibodies disclosed herein) have a dissociation constant of less than about 10. -5 M (for example, less than about 10) -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K (M or smaller) D The K binds to an antigen (e.g., GPRC5D). D Determined by, for example, surface plasmon resonance (SPR) in a BIACORE device.

[0245] As used herein, the term "vector" refers to a nucleic acid medium into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. A vector can express its carried genetic material elements in a host cell through transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, kinases, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC)); bacteriophages (such as λ phage or M13 phage); and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papova viruses (such as SV40). A vector may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector may contain an origin of replication.

[0246] As used herein, the term "host cell" refers to a cell that can be introduced into or transformed by a vector, including but not limited to prokaryotic cells (such as Escherichia coli or Bacillus subtilis). Bacillus subtilis Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, or MDCKII cells.

[0247] As used herein, the term "identity" refers to the degree of matching between two polypeptides or two nucleic acids. Two sequences used for comparison are identical at a site when they share the same base or amino acid monomer subunit (e.g., each of two DNA molecules has an adenine at a site, or each of two polypeptides has a lysine at a site). The percentage of identity between two sequences is calculated by dividing the number of shared identical sites by the total number of sites used for comparison and then multiplying by 100. For example, if six out of ten sites in two sequences match, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six sites match). Typically, comparisons of two sequences are performed in a manner that yields the greatest possible identity. Such comparisons can be performed using computer programs such as the Align program (DNAstar, Inc.) based on the method of Needleman et al. (J. Mol. Biol. 48:443-453, 1970). The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) in the ALIGN program (version 2.0), using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) in the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0248] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient. Such carriers and / or excipients include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate-buffered saline. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, etc. Absorption delaying agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols, and polyols (e.g., glycerol), etc. Stabilizers have the meaning commonly understood by those skilled in the art, meaning they stabilize the desired activity of an active ingredient in a pharmaceutical product (including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or their degradation products (e.g., lactalbumin hydrolysates)). Pharmaceutically acceptable carriers or excipients may include sterile injectable liquids (e.g., aqueous or non-aqueous suspensions or solutions). Such sterile injectable liquids may be selected from the group consisting of: water for injection (WFI), antimicrobial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0249] As used herein, the term "treatment" refers to a method undertaken to achieve a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease reduction, stabilization (i.e., non-aggravation) of the disease state, delay or slowing disease progression, and symptom relief (partial or complete), whether detectable or undetectable. Furthermore, "treatment" also refers to prolonged survival compared to expected survival (without treatment). Beneficial or desired clinical outcomes described herein may include, but are not limited to: slowing tumor progression, cancer regression, enhanced anti-tumor immune response, reduction of tumor growth or size, tumor necrosis, reduction of the severity of at least one disease symptom, increased frequency and duration of asymptomatic periods of disease, prevention of damage or disability due to disease suffering, or other ways of improving a patient's disease symptoms.

[0250] As used herein, the term “subject” refers to any human or non-human animal receiving treatment. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0251] As used herein, the term "effective amount" means an amount sufficient to achieve or at least partially achieve the intended effect. For example, an effective amount for treating a disease means an amount that effectively cures or at least partially blocks the disease and its complications in a patient suffering from the disease. The determination of such an effective amount is within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes depends on the severity of the disease to be treated, the general state of the patient's immune system, the patient's general condition (such as age, weight, and sex), the route of administration, and any concurrent therapies used.

[0252] As used herein, the term “immune cells” includes cells that have a hematopoietic origin and play a role in the immune response, such as lymphocytes, like B cells and T cells; natural killer (NK) cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0253] As used herein, the term "effective function" refers to those biological activities attributable to the Fc region (of the native sequence or an amino acid sequence variant) of an antibody, which vary with antibody isotype. Examples of antibody effector functions include, but are not limited to, Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, half-life / clearance of antibodies and antigen-antibody complexes, etc. Methods for altering antibody effector functions are known in the art, such as by introducing mutations into the Fc region.

[0254] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens onto immune effector cells, such as T cells. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." CARs may contain extracellular ligand-binding domains or extracellular antigen-binding domains, transmembrane domains, and intracellular signaling domains that are specific to one or more ligands or antigens, such as tumor antigens. "CAR-T cells" refers to T cells that express CARs.

[0255] As used herein, the terms “cancer” and “tumor” are used interchangeably and refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0256] Example

[0257] Unless otherwise stated, the examples provided below are for illustrative purposes only and are not intended to be limiting. Therefore, this disclosure should in no way be construed as limited to the examples below, but rather as covering any and all variations that become apparent as a result of the teachings provided herein.

[0258] Example 1: Production of anti-GPRC5D antibody

[0259] In accordance with all current animal welfare regulations, two camels and two llamas were immunized sequentially with different forms of human GPRC5D antigen. For protein immunization, human GPRC5D protein-VLP (Q9NZD1-1, SEQ ID No: 1, Kactus, catalog number GPR-HM05P) was formulated into an emulsion with 2% Alhydrogel® adjuvant (InvivoGen, catalog number vac-alu-250). The antigen emulsion was administered via a two-point subcutaneous injection in the neck. For cell immunization, HEK293 / GPRC5D cells (ACRO, catalog number CHEK-STP042) were administered intramuscularly via a two-point injection in the neck. Animals received four injections of the emulsion containing 100–400 μg of human GPRC5D protein-VLP at 2-week intervals, followed by 2 × 10⁶ doses per week. 7 Three injections of HEK293 / GPRC5D cells were administered. Three days after terminal immunization, 150 mL of blood samples were collected from the immunized animals. Approximately 3 × 10⁶ cells were isolated from the blood. 8 Peripheral blood lymphocytes (PBLs) serve as the genetic source of heavy chain antibodies.

[0260] Using TRIZOL ® The reagent (Invitrogen™, catalog number 15596026) was used to extract total RNA from lymphocytes of immunized camels or llamas. PrimeScrit was used based on the RNA template. ™ The first-strand cDNA synthesis kit uses oligo(dT)20 primers (Takara, catalog number 6110A) to synthesize cDNA. The cDNA encoding V is amplified from camel or llama cDNA. HDNA of H (the variable region of heavy chain antibodies only, also known as single-domain antibodies, sdAbs) was purified and ligated into an internal phage vector (see patent number US 20170089914 A1). Figure 1 Transform SS320 electrocompetent cells using the ligation product. Supplement the sdAb phage plasmid library with 20% glycerol and store at -80°C.

[0261] The sdAb phage particle library was rescued, and the resulting phage library was stored at 4°C for further use after filtration sterilization. Binding proteins were isolated from the phage library using both protein-based and cell-based panning methods. A single round of panning was performed using both protein-based and cell-based methods. At least 50% of the GPRC5D-positive clones were identified by ELISA, and high sequence diversity was observed for all output phages. These outputs were then used for subsequent high-throughput screening.

[0262] Thousands of colonies from the exported phages were individually picked and grown in 96-well plates containing 1.4 mL of 2YT medium. The ability of the sdAb to bind phages to human GPRC5D protein-VLP, mouse GPRC5D protein-VLP (Kactus, catalog number GPR-MM05P), and human enveloped VLP control (ctrl VLP, Kactus, catalog number VLP-HM00C) was demonstrated by ELISA analysis. Human GPRC5D binding proteins with unique sequences were selected for further characterization. To assess the cross-species reactivity and specificity of the selected clones, human MM cell line H929 (American Type Culture Collection (ATCC) was tested with or without transient expression of cynomolgus monkey GPRC5D (A0A2K5W6I7, SEQ ID NO: 2) and mouse GPRC5D (Q9JIL6-2, SEQ ID NO: 3). ® Flow cytometry was performed on selected clones (Catalog No. CRL-3580™), HEK293 / human GPRC5D stable cells, and FreeStyle™ 293-F cells (Thermo Fisher, Catalog No. R79007). The binding activity of the selected clones is summarized in Table 1. The IDs of the full-length sdAb and CDR sequences are listed in Table 2. CDRs are defined according to the AbM numbering system.

[0263] Table 1. Anti-GPRC5D antibodies that bind to target proteins and cells

[0264] Table 2. Anti-GPRC5D antibody sequences in camels

[0265] Example 2: In vitro efficacy of anti-GPRC5D camel CAR construct

[0266] Seventy-nine anti-GPRC5D sdAbs identified in Example 1 were used as the GPRC5D binding moiety to construct CARs. G109scFv (from patent no. WO 2016090312, VH SEQ ID NO: 37, VL SEQ ID NO: 38) was used as the GPRC5D binding moiety to construct a positive control CAR. The sequences of these CARs from N-terminus to C-terminus are: leader sequence (SEQ ID No: 314), myc tag (SEQ ID NO: 315), target binding moiety (e.g., anti-GPRC5D sdAb / scFv), CD8α hinge (SEQ ID NO: 316), CD8α transmembrane (TM) region (SEQ ID NO: 317), cytoplasmic portion of 4-1BB (CD137) molecule (SEQ ID NO: 318), and cytoplasmic portion of CD3ζ molecule (SEQ ID NO: 319). These constructs were designated as "[GPRC5D binding moiety]bbz". The CAR construct using G109 scFv as the target binding site is designated as G109bbz (SEQ ID NO: 320).

[0267] To achieve high-throughput retrovirus production, the HEK 293-based packaging cell line 293Vec-BaEV, which generates a pseudotyped retroviral vector derived from baboon envelope proteins, was seeded in 24-well plates. Using Lipofectamine 3000 transfection reagent (Thermo Fisher, catalog number L3000075), 293Vec-BaEV cells were transfected in 250 μL of OptiMEM (Gibco, catalog number 31985-070) with an in-house designed sinCMV CAR-encoding plasmid tagged with myc (at a 2:1 DNA:Lipofectamine 3000 ratio). The medium was changed after 6 hours, and the 293Vec-BaEV cells were incubated for 3 days, after which the retroviral supernatant was collected. On the day of T cell transduction, the retrovirus was collected by centrifuging the packaging cell line plate and collecting the supernatant. The retrovirus was then applied directly to the preactivated T cells.

[0268] Peripheral blood mononuclear cells (PBMCs, Hemacare) were thawed from frozen vials in TexMACS (Miltenyi, catalog number 130-097-196) supplemented with 200 IU / mL rhIL-2 (Miltenyi, catalog number 170-076-147) for one day. Human T cells were purified from PBMCs using human CD4 and human CD8 microbeads (Miltenyi, catalog numbers 130-045-101 and 130-045-201, respectively) on an LS magnetic column (Miltenyi, catalog number 130-042-401). The purified T cells were then pre-activated for 24 hours in the presence of 200 IU / mL rhIL-2 using human T cell TransAct (Miltenyi, catalog number 130-111-160). Then, 2.5 × 10⁶ cells were... 5 Pre-activated T cells (in 250 μL of TexMACS supplemented with 200 IU / mL rhIL-2) were transferred to 24-well plates coated with retroectin (20 μg / mL) (RetroNectin® GMP grade, Takara, catalog number T202) and transduced with 250 μL of retroviral supernatant harvested from 293Vec-BaEV packaging cells. The plates were centrifuged at 1200 xg for 1 hour at 32°C in the presence of 10 μL 1 M HEPES (Gibco, catalog number 15630-080). The T cells were then cultured at 37°C for 10 days, with the medium refreshed every 2 days with fresh TexMACS supplemented with 200 IU / mL rhIL-2 to maintain a T cell concentration of approximately 1–2 × 10⁶ cells / day. 6 Density of cells / mL.

[0269] Ten days after transduction, CAR expression on transduced T cells was assessed by flow cytometry after staining with antimyc antibody (Cell Signaling Technology, catalog number 2233S). Eleven days after transduction, CAR T cells were co-incubated with H929 cells (a multiple myeloma cell line with high BCMA expression and approximately 70% population expression of GPRC5D) in 96-well plates at an E:T ratio of 2:1 (i.e., 10,000 CAR T cells + 5,000 H929 cells) or an E:T ratio of 1:1 (i.e., 5,000 CAR T cells + 5,000 H929 cells) in 200 μL of 1:1 mixed medium (TexMACS + RPMI 1640 (Gibco, catalog 11875-093)) supplemented with 10% FBS (Gibco, catalog 16140-071). After twenty-four hours of co-incubation, the supernatant was collected for the LDH-releasing cytotoxicity assay (LDH-Glo™ cytotoxicity assay, Promega, catalog 11875-093).

[0270] Based on the results of target cell-specific lysis (i.e., cytotoxicity) in Table 3, most CAR constructs showed higher killing efficiency than the baseline G109bbz CAR T cells (positive control CAR T cells). The constructs are: AS300398bbz, AS300399bbz, AS300409bbz, AS300413bbz, AS300424bbz, AS300435bbz, AS300442bbz, AS300452bbz, AS300463bbz, AS300469bbz, AS300477bbz, AS300488bbz, AS300494bbz, AS300506bbz, AS300513bbz, AS300524bbz, AS300527bbz, AS300529bbz, AS300535bbz, AS300573bbz, AS300579bbz, AS300614bbz, AS300629bbz, and AS300637bbz.

[0271] Table 3. Cytotoxicity of CAR T cells after co-culturing with H929 tumor cells for 24 hours

[0272] As described above, the second group of anti-GPRC5D CAR constructs was tested. CAR T cells were co-cultured with H929 target cells at a 2:1 E:T ratio for 24 hours. Then, every two days, the CAR T cells were re-attacked with irradiated H929 target cells until most CAR constructs ceased killing target cells. Based on the results of target cell-specific lysis (i.e., cytotoxicity) in Table 4, all CAR constructs exhibited higher killing efficiency than the baseline G109bbz CAR T cells (positive control CAR T cells).

[0273] Table 4. Cytotoxicity of CAR T cells after co-culturing with H929 tumor cells at an E:T ratio of 2:1

[0274] As described above, the third group of anti-GPRC5D CAR constructs was tested. CAR T cells were co-cultured with H929 target cells at a 2:1 E:T ratio for 24 hours. Based on the data in Table 5, seven constructs (AS307858bbz, AS308386bbz, AS310027bbz, AS307587bbz, AS307685bbz, AS307879bbz, and AS307946bbz) showed comparable to or better cytotoxicity than the baseline G109bbz CAR T cells (positive control CAR T cells) in the first round of tumor cell killing. These CAR T cells were then re-challenged with 5,000 irradiated H929 cells, and their cytotoxicity was assessed after 24 hours. After one round of re-challenging, all seven constructs were comparable to or better than G109bbz.

[0275] Table 5. Cytotoxicity of CAR T cells after co-culturing with H929 tumor cells at an E:T ratio of 2:1

[0276] 5E11 scFv (VH-(GGGGS)3-VL, VH SEQ ID NO: 321, VL SEQ ID NO: 322 (from patent number WO 2019154890 A1), VH SEQ ID NO: 19, amino acids 1-117, VL SEQ ID NO: 17, amino acids 1-110) was used as the GPRC5D binding moiety to construct the positive control CAR as described above. CAR constructs with cytotoxic activity comparable to or better than G109bbz were further evaluated and compared with the baseline 5E11.

[0277] CAR-T cells were co-cultured with MM.1S (ATCC, catalog number CRL-2974) cells (a multiple myeloma cell line expressing endogenous GPRC5D) at a 1:1 effector-to-target cell (E:T) ratio. Cell counts were performed every 2-3 days, and the number of T cells and CAR-T positivity rate were detected using FACS. If the T cell frequency was greater than or equal to 80%, the T cells were re-challenged with MM.1S cells at a 1:1 E:T ratio until the end of the experiment. Results ( Figures 5A to 5B The results showed that the proliferation and efficacy of all test groups were superior to those of the baseline 5E11.

[0278] CAR-T cells were mixed with MM.1S tumor cells at an E:T ratio of 1:2, and the co-culture supernatant was collected 72 hours after initial co-culture. IFN-γ production was detected using the HTRF human IFN-γ detection kit (Revity, catalog number 62HIFNGPEH). Results ( Figure 6 The results showed that IFN-γ production levels in all test groups were significantly lower than the baseline 5E11, suggesting a potentially safer clinical profile.

[0279] High, medium, and low levels of GPRC5D mRNA were overexpressed in Raji-luc cells using the Cell Line Nucleofector™ Kit V (LONZA, catalog number VCA-1003) to construct Raji-luc GPRC5D（高） Raji-luc GPRC5D（中） and Raji-luc GPRC5D（低） In Raji-luc GPRC5D（高） In the middle, the logarithmic offset of GPRC5D is 0.63 ( Figure 7A ), in Raji-luc GPRC5D （中） In the middle, the logarithmic offset of GPRC5D is 0.38 ( Figure 7B ), and in Raji-luc GPRC5D（低） In the middle, the logarithmic offset of GPRC5D is 0.17 ( Figure 7C ) CAR-T cells with Raji-luc GPRC5D（高） Raji-luc GPRC5D（中） and Raji-luc GPRC5D（低） Mixed at E:T ratios of 3:1, 1.5:1, and 0.75:1 for 24 hours. Tumor cell viability was assessed using the ONE-Glo™ luciferase assay system (PROMEGA, catalog number B6110). Results ( Figures 7A to 7CThe results showed that, compared with the baseline 5E11, the CAR-T cells in the test group exhibited stronger efficacy against Raji-luc cells with different GPRC5D expression levels at different ratios, demonstrating excellent sensitivity profile.

[0280] Example 3: Characterization and epitope mapping of anti-GPRC5D antibody

[0281] In all anti-GPRC5D antibodies, 20 camel-derived sdAb sequences were fused with the human IgG1 hinge and Fc fragment to construct heavy-chain-only antibodies (HCAbs). The VH and VL sequences derived from 5E11 (VH SEQ ID NO: 321, VL SEQ ID NO: 322, disclosed herein, from patent number WO 2019154890 A1, VH SEQ ID NO: 19, amino acids 1-117, VL SEQ ID NO: 17, amino acids 1-110) were used to construct the conventional IgG heavy and light chains. The scFv sequence derived from G109 (SEQ ID NO: 323, disclosed herein, from patent number WO 2016090312, SEQ ID NO: 109) was fused with the human IgG1 hinge and Fc fragment.

[0282] Heavy chain, light chain, and scFv-Fc plasmids were prepared and used to produce HCAb, IgG, and scFv-Fc from FreeStyle™ 293-F cells. Antibodies were purified using a Protein A Agarose column (Genscript, catalog number L00464) followed by size exclusion chromatography (Citiva, catalog number GE28-9893-35). Two different forms of anti-GPRC5D antibody, 5E11 and G109, were used as positive controls in the following assays.

[0283] The binding affinity of the selected molecule to human GPRC5D protein (ACRO, catalog number GPD-H52D3) was determined by surface plasmon resonance (SPR) on a BIAcore T200 instrument (GE Healthcare). Experiments were performed as follows: the antibody was captured onto a sensor chip pre-coated with goat anti-human pAb (Jackson Immuno Research, catalog number 109-005-098) via the interaction between a polyclonal antibody and human Fc. Incremental concentrations (ranging from 10 nM to 640 nM) of human GPRC5D were injected onto the sensor chip surface, allowing binding of the captured antibody for 100 s, followed by injection of run buffer to allow dissociation of the complex for 300 s. After each cycle, the surface was regenerated by injection of 10 mM glycine-HCl buffer (pH 2.0). The experimental data were fitted using the Langmuir model (which describes a simple 1:1 interaction between a ligand molecule and an analyte) to obtain the association rate. k a ) and dissociation rate ( k d The kinetic values ​​of ) are used to calculate the equilibrium dissociation constant ( ). K D Based on the results summarized in Table 6, the antibodies exhibited an affinity ranging from 220 nM to 23 nM. K D Combined with human GPRC5D.

[0284] Table 6. Binding affinity of purified antibody to human GPRC5D protein

[0285] To determine the binding activity of the selected molecules with cells expressing human GPRC5D, HEK293 / GPRC5D and H929 cells were incubated with gradient concentrations of 20 HCAbs, followed by incubation at 37°C for 30 min with Alexa Fluor 647-labeled goat anti-human IgG secondary antibody (Jackson ImmunoResearch, catalog number 109-605-098). Samples were analyzed by flow cytometry. The binding activity of the anti-GPRC5D antibody is shown in [data missing]. Figure 1 , Figure 2 And in Table 7. Most of the selected antibodies showed better or comparable binding activity than the 5E11 and G109 antibodies, as evidenced by lower EC50 values ​​and higher maximum binding signal (mean fluorescence intensity, MFI), especially on H929 cells.

[0286] Table 7. Binding activity of purified antibody to cells expressing human GPRC5D

[0287] To identify the antigenic regions / loops crucial for antigen-antibody interactions, four plasmids encoding chimeric GPRC5D proteins were constructed by replacing each extracellular region / loop of human GPRC5D with its corresponding portion from GPRC5A or GPRC5B. Figure 3 These plasmids were used to transfect FreeStyle™ 293-F cells. Transfected cells transiently expressed the chimeric GPRC5D proteins (chimeric DB1, chimeric DA2, chimeric DA3, chimeric DB4) and were subsequently subjected to binding with the aforementioned 20 HCAbs and control antibodies. Flow cytometry was performed to determine whether these antibodies bound cells expressing wild-type and chimeric antigens. For example, the fact that AS300399 bound both wild-type human GPRC5D and chimeric DB4 cells with a high binding signal, but bound chimeric DA2 and DA3 cells with a low or no binding signal, indicates that the epitopes of AS300399 are located in the second and third extracellular loops. Figure 4 As shown in Table 8, the epitopes of the selected antibodies exhibit a high degree of diversity.

[0288] Table 8. Epitope grouping of selected antibodies

[0289] Example 4: Humanization of anti-GPRC5D antibody fragment

[0290] Fourteen camel sdAb sequences were humanized using CDR transplantation technology. Camel sdAb sequences were BLASTed in the NCBI Human V Gene Database to identify human VH germline sequences with the highest identity to the camel sdAb sequences. Table 9 lists the most suitable human frames (hereinafter referred to as human recipients) for establishing CDR transplantation of VH sequences.

[0291] Table 9. Human receptors selected for camel sdAb

[0292] In the CDR transplantation method, the CDR of the human recipient is replaced by the CDR of the parental sdAb, resulting in a straight-graft sequence. Straight-grafted antibodies typically lose binding activity, which can be restored by gradually replacing crucial framework residues with camel-derived animal residues (i.e., reversion mutations), thereby producing humanized sdAbs (SEQ ID NO: 328-390) with varying degrees of humanization.

[0293] Cameloid and humanized sdAb sequences were fused with the hinge and Fc fragment of human IgG1. DNA encoding these fusion proteins was synthesized and inserted into the pcDNA3.1 vector. HEK293 cells were transfected using the fusion protein expression plasmid. H929 cell binding of the crude fusion protein secreted into the culture medium was assessed by flow cytometry, and expression levels were assessed by SPR.

[0294] To evaluate the binding activity between the parental and humanized sdAb, 25-fold, 125-fold, and 625-fold dilutions of the aforementioned medium containing the crude fusion protein were incubated with H929 cells expressing GPRC5D at 4°C for 30 min. Cells were washed three times and then incubated for 30 min at 4°C with Alexa Fluor 647-labeled goat anti-human IgG secondary antibody (Jackson ImmunoResearch, catalog number 109-605-098). Cells were washed three times and resuspended in DPBS (Servicebio, catalog number G4200-500 mL). Samples were acquired using a BD FACSCelesta flow cytometer (BD Biosciences) to determine the geometric mean fluorescence intensity (MFI) at each antibody concentration. Raw experimental data were analyzed using FlowJo software.

[0295] Expression levels of the fusion protein in the supernatant were assessed using SPR. Briefly, the capture antibody anti-human Fc pAb (Jackson ImmunoResearch, catalog number 109-605-098) was immobilized on a Biacore™ CM5 chip to approximately 6,000 RU using EDC-activated amine coupling chemistry. The recombinant fusion protein was captured onto the sensor chip surface for 15 seconds via interaction between the polyclonal antibody and human Fc. Expression levels of the parental and humanized antibodies were reflected by the capture levels in the corresponding supernatant. The binding activity and expression levels of the humanized variant were compared to those of the camel sdAb (Table 10). Humanized clones retaining H929 binding ability were selected for CAR construction and further characterization.

[0296] Table 10. Expression levels of camel-derived and humanized sdAb and their binding activity on H929 cells.

[0297] Example 5: In vitro cytotoxicity of humanized anti-GPRC5D CAR construct

[0298] A CAR construct using a humanized variant as the target binding site was synthesized and inserted into an internal lentiviral transfer plasmid. The transfer plasmid encoding the CAR was mixed with lentiviral packaging plasmids (Addgene, catalog number 12259) including pMDLG / PRE, PRSV-REV, psPAX2, and pMD2.G at approximately equal molar ratios. The plasmid mixture was then mixed with polyethyleneimine (PEI) at a plasmid:PEI ratio of 1:3. HEK293 cells were transfected with this mixture and cultured for 48 hours. The culture supernatant was collected and centrifuged to remove cell debris. The supernatant was concentrated with polyethylene glycol (PEG)-6K. Viral particles were resuspended, aliquoted, and immediately stored at -80°C. Viral titers were determined by flow cytometry to measure the transduction efficiency of the CHO cell lines.

[0299] T lymphocytes were activated for 24 hours and then transduced using the aforementioned lentiviral vector. Seven days after transduction, CAR T cells were harvested and used for in vitro functional evaluation.

[0300] CAR T cells were mixed with H929-Luc cells (containing a luciferase marker) at four different E:T ratios of 10:1, 5:1, 2.5:1, and 1:1, and then in Corning ® Incubate overnight in 96-well solid white plates. Luciferase activity was measured using the ONE-Glo™ luciferase assay system (PROMEGA, catalog number B6110). Add 50 μL of ONE-Glo™ reagent to each well of the 96-well plate, incubate, and then place on a Spark™ 10M multimode microplate reader (TECAN) for fluorescence measurement, reflecting the cytotoxicity of different CAR constructs to target cells.

[0301] The cytotoxicity of CAR T cells using camel sdAb as the GPRC5D binding moiety (Table 11, bold) and its humanized counterpart was compared. All constructs appeared to have at least one humanized variant with similar or better functional activity (Table 11, italics), indicating that humanization was successfully implemented.

[0302] Table 11. Cytotoxicity of CAR T cells co-cultured with H929-Luc at different E:T ratios

[0303] The cytotoxicity of seven CAR constructs using the humanized GPRC5D binding motif against MM.1S-Luc cells (containing luciferase markers) was further tested. CAR-T cells were co-incubated with MM.1S-Luc cells at an E:T ratio of 1:2 for 20–24 hours. To determine the cytotoxicity of CAR-T cells against tumor cells, the ONE-Glo™ luciferase assay system (PROMEGA, catalog number B6120) was prepared according to the manufacturer's protocol and added to the co-cultured cells to detect residual luciferase activity in the wells. Residual luciferase activity was directly correlated with the number of surviving target cells in the wells. Specific cytotoxicity was calculated using the following formula: Specific cytotoxicity % = 100% × (1 - (RLU) 样品 -RLU min ) / (RLU UnT -RLU min RLU 样品 Represents luciferase activity measured in wells containing CAR-T cells transduced with the GPRC5D CAR disclosed herein. RLU min This refers to the luciferase activity determined at the start of a cytotoxicity assay in wells supplemented with a final concentration of 1% Triton X-100, while RLU... UnT This refers to the luciferase activity determined in pores containing untransduced T cells.

[0304] like Figure 8 The results showed that all single-specific GPRC5D CAR-T cells exhibited effective killing effects against MM.1S-Luc cells.

[0305] Example 6: Real-time Cell Analysis (RTCA) Assay

[0306] The real-time cytotoxicity of GPRC5D CAR-T cells co-cultured with tumor cells expressing low levels of GPRC5D (Hep3B2.1-7-5D, Hep3B2.1-7 (ATCC, catalog number HB-8064) cells transduced with a lentivirus containing GPRC5D) was measured using RTCA assay. Tumor cells expressing low levels of GPRC5D (6 × 10⁻⁶) were used in this assay. 3 Target cells (1 cell / well) were seeded in 96-well E plates and monitored overnight using an RTCA analyzer. Target cells were then incubated with GPRC5D CAR-T or UnT cells at a 1:1 E:T ratio, with T cell cytotoxicity assessed in real-time during this period. Cell indices indicating target cell activity, adhesion, and number were normalized (Normalized Cell Index (NCI) = Real-time Cell Index / Cell Index at the time point before effector cell addition). Specific cytotoxicity was calculated using the following formula: Specific cytotoxicity % = 100% × (1 - NCI)样品 / NCI UnT ).

[0307] like Figure 9 The results showed that the cell index of tumor cells with low GPRC5D expression co-cultured with UnT cells increased over time, with a normalized cell index of 1.32 after 90 hours of incubation and at the end of the experiment. With prolonged incubation, single-specific GPRC5D CAR-T cells exhibited varying degrees of cytotoxicity against tumor cells with low GPRC5D expression. At the end of the experiment, the specific cytotoxicity of single-specific GPRC5D CAR-T cells was greater than 90%. After 90 hours of co-culture, the normalized cell indexes of single-specific GPRC5D CAR-T cells were 0.12 (AS300399VH7bbz), 0.11 (AS302399VH4bbz), 0.06 (AS302553VH5bbz), and 0.05 (AS302558VH5bbz). The specific cytotoxicity of monospecific GPRC5DCAR-T cells was 90.74% (AS300399VH7bbz), 91.32% (AS302399VH4bbz), 95.40% (AS302553VH5bbz), and 96.55% (AS302558VH5bbz).

[0308] sequence .

Claims

1. A GPRC5D V resistant H H, the anti-GPRC5D V H H contains: (1) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 4 respectively; (2) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 8 respectively; (3) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 12 respectively; (4) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 16 respectively; (5) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 20 respectively; (6) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 24 respectively; (7) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 28 respectively; (8) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 32, respectively; (9) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 36 respectively; (10) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 40, respectively; (11) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 44 respectively; (12) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 48 respectively; (13) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 52 respectively; (14) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 56 respectively; (15) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 60, respectively; (16) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 64 respectively; (17) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 68 respectively; (18) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 72, respectively; (19) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 76 respectively; (20) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 80, respectively; (21) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 84 respectively; (22) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 88 respectively; (23) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 92 respectively; (24) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 96 respectively; (25) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 100 respectively; (26) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 104 respectively; (27) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 108 respectively; (28) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 112 respectively; (29) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 115 respectively; (30) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 119 respectively; (31) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 123 respectively; (32) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 127 respectively; (33) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 131 respectively; (34) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 135 respectively; (35) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 139 respectively; (36) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 143 respectively; (37) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 147 respectively; (38) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 151, respectively; (39) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 155 respectively; (40) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 159 respectively; (41) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 163 respectively; (42) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 167 respectively; (43) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 171 respectively; (44) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 175 respectively; (45) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 179 respectively; (46) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 183 respectively; (47) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 187 respectively; (48) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 190 respectively; (49) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 193 respectively; (50) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 197 respectively; (51) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 201 respectively; (52) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 205 respectively; (53) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 209 respectively; (54) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 213 respectively; (55) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 216 respectively; (56) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 220 respectively; (57) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 224, respectively; (58) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 228, respectively; (59) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 232, respectively; (60) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 236, respectively; (61) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 240, respectively; (62) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 244 respectively; (63) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 248 respectively; (64) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 252, respectively; (65) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 256, respectively; (66) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 260, respectively; (67) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 264, respectively; (68) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 266 respectively; (69) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 270 respectively; (70) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 274, respectively; (71) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 278 respectively; (72) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 282, respectively; (73) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 286 respectively; (74) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 290, respectively; (75) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 294, respectively; (76) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 298, respectively; (77) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 302 respectively; (78) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 306, respectively; or (79) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 310, respectively.

2. The V as described in claim 1 H H, wherein CDR1, CDR2 or CDR3 is determined according to the AbM numbering scheme, IMGT numbering scheme, Kabat numbering scheme, Chothia numbering scheme, Contact numbering scheme or any combination thereof.

3. A type of anti-GPRC5D V H H, the anti-GPRC5D V H H contains CDR1, CDR2, and CDR3, which respectively contain the following amino acid sequences: (1) SEQ ID NO: 5, 6 and 7; or (2) SEQ ID NO: 9, 10 and 11; or (3) SEQ ID NO: 13, 14 and 15; or (4) SEQ ID NO: 17, 18 and 19; or (5) SEQ ID NO: 21, 22 and 23; or (6) SEQ ID NO: 25, 26 and 27; or (7) SEQ ID NO: 29, 30 and 31; or (8) SEQ ID NO: 33, 34 and 35; or (9) SEQ ID NO: 37, 38 and 39; or (10) SEQ ID NO: 41, 42 and 43; or (11) SEQ ID NO: 45, 46 and 47; or (12) SEQ ID NO: 49, 50 and 51; or (13) SEQ ID NO: 53, 54 and 55; or (14) SEQ ID NO: 57, 58 and 59; or (15) SEQ ID NO: 61, 62 and 63; or (16) SEQ ID NO: 65, 66 and 67; or (17) SEQ ID NO: 69, 70 and 71; or (18) SEQ ID NO: 73, 74 and 75; or (19) SEQ ID NO: 77, 78 and 79; or (20) SEQ ID NO: 81, 82 and 83; or (21) SEQ ID NO: 85, 86 and 87; or (22) SEQ ID NO: 89, 90 and 91; or (23) SEQ ID NO: 93, 94 and 95; or (24) SEQ ID NO: 97, 98 and 99; or (25) SEQ ID NO: 101, 102 and 103; or (26) SEQ ID NO: 105, 106 and 107; or (27) SEQ ID NO: 109, 110 and 111; or (28) SEQ ID NO: 81, 113 and 114; or (29) SEQ ID NO: 116, 117 and 118; or (30) SEQ ID NO: 120, 121 and 122; or (31) SEQ ID NO: 124, 125 and 126; or (32) SEQ ID NO: 128, 129 and 130; or (33) SEQ ID NO: 132, 133 and 134; or (34) SEQ ID NO: 136, 137 and 138; or (35) SEQ ID NO: 140, 141 and 142; or (36) SEQ ID NO: 144, 145 and 146; or (37) SEQ ID NO: 148, 149 and 150; or (38) SEQ ID NO: 152, 153 and 154; or (39) SEQ ID NO: 156, 157 and 158; or (40) SEQ ID NO: 160, 161 and 162; or (41) SEQ ID NO: 164, 165 and 166; or (42) SEQ ID NO: 168, 169 and 170; or (43) SEQ ID NO: 172, 173 and 174; or (44) SEQ ID NO: 176, 177 and 178; or (45) SEQ ID NO: 180, 181 and 182; or (46) SEQ ID NO: 184, 185 and 186; or (47) SEQ ID NO: 136, 188 and 189; or (48) SEQ ID NO: 191, 192 and 138; or (49) SEQ ID NO: 194, 195 and 196; or (50) SEQ ID NO: 198, 199 and 200; or (51) SEQ ID NO: 202, 203 and 204; or (52) SEQ ID NO: 206, 207 and 208; or (53) SEQ ID NO: 210, 211 and 212; or (54) SEQ ID NO: 120, 214 and 215; or (55) SEQ ID NO: 217, 218 and 219; or (56) SEQ ID NO: 221, 222 and 223; or (57) SEQ ID NO: 225, 226 and 227; or (58) SEQ ID NO: 229, 230 and 231; or (59) SEQ ID NO: 233, 234 and 235; or (60) SEQ ID NO: 237, 238 and 239; or (61) SEQ ID NO: 241, 242 and 243; or (62) SEQ ID NO: 245, 246 and 247; or (63) SEQ ID NO: 249, 250 and 251; or (64) SEQ ID NO: 253, 254 and 255; or (65) SEQ ID NO: 257, 258 and 259; or (66) SEQ ID NO: 261, 262 and 263; or (67) SEQ ID NO: 5, 6 and 265; or (68) SEQ ID NO: 267, 268 and 269; or (69) SEQ ID NO: 271, 272 and 273; or (70) SEQ ID NO: 275, 276 and 277; or (71) SEQ ID NO: 279, 280 and 281; or (72) SEQ ID NO: 283, 284 and 285; or (73) SEQ ID NO: 287, 288 and 289; or (74) SEQ ID NO: 291, 292 and 293; or (75) SEQ ID NO: 295, 296 and 297; or (76) SEQ ID NO: 299, 300 and 301; or (77) SEQ ID NO: 303, 304 and 305; or (78) SEQ ID NO: 307, 308 and 309; or (79) SEQ ID NO: 311, 312 and 313.

4. The V as described in any one of claims 1-3 H H, the V H H contains SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 190, 193, 197, 201, 205, 209, 213, 216, 220 The amino acid sequence of 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 266, 270, 274, 278, 282, 286, 290, 294, 298, 302, 306, or 310, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

5. The V as described in any one of claims 1-4 H H, the V H H represents camels (V) H H, interlocking V H H or humanized V H H.

6. The V as described in claim 5 H H, the V H H contains an amino acid sequence of SEQ ID NO: 16, 24, 48, 143, 179, 187, 193, 220, 244, 274, 298, 302, 306 or 310, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it; Preferably, the V H H contains an amino acid sequence of any one of the following: (1) SEQ ID NO: 328, 329, 330, 331, 332 or 333; (2) SEQ ID NO: 334, 335, 336 or 337; (3) SEQ ID NO: 338, 339, 340, 341 or 342; (4) SEQ ID NO: 343, 344, 345 or 346; (5) SEQ ID NO: 347, 348 or 349; (6) SEQ ID NO: 350, 351 or 352; (7) SEQ ID NO: 353, 354, 355, 356, 357 or 358; (8) SEQ ID NO: 359, 360, 361, 362 or 363; (9) SEQ ID NO: 364, 365, 366 or 367; (10) SEQ ID NO: 368, 369, 370 or 371; (11) SEQ ID NO: 372, 373, 374, 375 or 376; (12) SEQ ID NO: 377, 378 or 379; (13) SEQ ID NO: 380, 381, 382, ​​383 or 384; (14) SEQ ID NO: 385, 386, 387, 388, 389 or 390.

7. A polypeptide construct comprising V as described in any one of claims 1 to 6. H H.

8. The polypeptide construct of claim 7, wherein the construct is a single-specific molecule, a bispecific molecule, a multispecific molecule, an immunoconjugate, or a fusion protein; Preferably, the single-specific molecule is sdAb or its antigen-binding fragment.

9. The polypeptide construct of claim 7 or 8, wherein the construct comprises an additional polypeptide.

10. The polypeptide construct of claim 9, wherein the additional polypeptide is an immunoglobulin Fc region; Preferably, the immunoglobulin Fc region is an IgG Fc region, such as an IgG1, IgG2, IgG3, or IgG4 Fc region.

11. The polypeptide construct of claim 9, wherein the additional polypeptide is a different binding domain of the second target besides GPRC5D; Preferably, the second target is selected from CD3, CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, mesothelin 6, mesothelin 18.2, GPC3, GPC2, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

12. The polypeptide construct of claim 11, wherein the additional binding domain is a monovalent antibody fragment, such as Fab, Fv, scFv, sdAb, or V. H H.

13. A nucleic acid molecule comprising encoding V as described in any one of claims 1 to 6. H The nucleotide sequence of H or the polypeptide construct as described in claims 7 to 12.

14. A vector comprising the nucleic acid molecule as described in claim 13.

15. A cell comprising a nucleic acid molecule as claimed in claim 13 or a carrier as claimed in claim 14.

16. A method for producing V as described in any one of claims 1 to 6 H H or the method for constructing a polypeptide as described in claims 7 to 12, the method comprising: Culture host cells containing the nucleic acid molecule as described in claim 13 or the vector as described in claim 14, or the cells as described in claim 15, under conditions that allow protein expression, and recover the V from the culture of the cultured cells. H H or the polypeptide construct thereof.

17. A chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises a first binding portion that specifically binds a first antigen or epitope, wherein the first binding portion comprises V as described in any one of claims 1 to 6. H H or the polypeptide construct as described in claims 7 to 12.

18. The chimeric antigen receptor of claim 17, wherein the extracellular antigen-binding domain further comprises a second binding portion that specifically binds a second antigen or epitope; Preferably, the first antigen or epitope is the same as the second antigen or epitope; Preferably, the first antigen or epitope is different from the second antigen or epitope; Preferably, the first connecting portion and the second connecting portion are connected in series.

19. The chimeric antigen receptor of claim 17 or 18, wherein the second binding portion is an sdAb, scFv, or an extracellular domain of the receptor. Preferably, the second binding portion is an antitumor antigen sdAb or scFv, and a tumor antigen selected from CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL3, micrin 6, micrin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA; Preferably, the second bonding portion is anti-GPRC5D sdAb and contains V as claimed in any one of claims 1 to 6. H H or the polypeptide construct as described in claims 7 to 12.

20. The chimeric antigen receptor of any one of claims 17 to 19, wherein the CAR further comprising a spacer region domain is selected from the hinge domain and / or CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4); Preferably, the hinge structure domain includes a hinge region of CD8α, IgG4, PD1, CD152, or CD154; Preferably, the spacer region structural domain includes the hinge region of CD8α (e.g., the sequence shown in SEQ ID NO: 316).

21. The chimeric antigen receptor of any one of claims 17 to 20, wherein the transmembrane domain is selected from one or more transmembrane regions selected from the group consisting of: the α, β or ζ chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD28, CD137, CD152, CD154 and PD1; Preferably, the transmembrane domain comprises a transmembrane region of CD8α (e.g., the sequence shown in SEQ ID NO: 317).

22. The chimeric antigen receptor of any one of claims 17 to 21, wherein the intracellular signal transduction domain comprises a primary signal transduction domain and / or a co-stimulatory signal transduction domain; Preferably, the intracellular signal transduction domain comprises a primary signal transduction domain and at least one co-stimulatory signal transduction domain; Preferably, the primary signal transduction domain comprises intracellular signal transduction domains of proteins selected from the group consisting of: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d; Preferably, the co-stimulatory signal transduction domain comprises intracellular signal transduction domains of proteins selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAGS), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, and DAP12; Preferably, the intracellular signal transduction domain comprises a primary signal transduction domain and a co-stimulatory signal transduction domain, wherein the primary signal transduction domain comprises an intracellular signal transduction domain of CD3ζ (e.g., the sequence shown in SEQ ID NO: 319), and the co-stimulatory signal transduction domain comprises an intracellular signal transduction domain of CD137 (e.g., the sequence shown in SEQ ID NO: 318).

23. The chimeric antigen receptor of any one of claims 17 to 22, wherein the chimeric antigen receptor further comprises a signal peptide and / or a tag; Preferably, the tag is selected from myc tag, His tag, short linear peptide sequence from yeast transcription factor GCN4, leucine zipper sequence, or short linear peptide sequence from human nuclear protein; Preferably, the tag is a myc tag, and the myc tag contains a sequence as shown in SEQ ID NO: 315; Preferably, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain; Preferably, the signal peptide comprises the sequence shown in SEQ ID NO: 314; Preferably, the chimeric antigen receptor comprises, in sequence from the N-terminus to the C-terminus, the following domains: the signal peptide, the tag, the antigen-binding domain, the spacer domain, the transmembrane domain, and the intracellular signal transduction domain.

24. A chimeric antigen receptor (CAR), said chimeric antigen receptor comprising: A first engineered receptor, comprising a chimeric antigen receptor as described in any one of claims 17-23; and The second engineered receptor comprises: a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular domain; Preferably, the second extracellular antigen-binding domain is an antitumor antigen sdAb or scFv, and a tumor antigen selected from BCMA, CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, DLL3, duracin 6, duracin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

25. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor as described in any one of claims 17 to 23 or a bichimeric antigen receptor as described in claim 24.

26. A vector comprising the nucleic acid molecule as described in claim 25.

27. A host cell comprising a nucleic acid molecule as described in claim 25 or a vector as described in claim 26; Preferably, the host cell is selected from immune cells (e.g., human immune cells). Preferably, the immune cells are selected from the group consisting of T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof.

28. An engineered immune cell expressing a chimeric antigen receptor as described in any one of claims 17 to 23 or a dual chimeric antigen receptor as described in claim 24; Optionally, the chimeric antigen receptor or the dual chimeric antigen receptor is expressed on the surface of the engineered immune cells; Optionally, the engineered immune cells also express a chimeric antigen receptor that is not specific to GPRC5D; Preferably, the immune cells are selected from the group consisting of T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof; Preferably, the immune cells are obtained from a patient or a healthy donor.

29. A pharmaceutical composition comprising V as claimed in any one of claims 1 to 6 H H. The polypeptide construct as described in claims 7 to 12, or the chimeric antigen receptor as described in any one of claims 17 to 23, or the bi-chimeric antigen receptor as described in claim 24; and pharmaceutically acceptable carriers and / or excipients.

30. The pharmaceutical composition of claim 29, wherein the pharmaceutical composition further comprises an additional therapeutic agent; Preferably, the additional therapeutic agent is an antitumor agent; Preferably, the additional therapeutic agent is a cytotoxic agent, such as an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a radionuclide.

31. A method for preventing and / or treating a disease in a subject, the method comprising administering to the subject in need an effective amount of V as described in any one of claims 1 to 6 H H, or the polypeptide construct as described in claims 7 to 12, or the nucleic acid molecule as described in claims 13 or 25, or the carrier as described in claims 14 or 26, or the cell as described in claims 15 or 27, or the chimeric antigen receptor as described in any one of claims 17 to 23, or the dual chimeric antigen receptor as described in claim 24, or the engineered immune cell as described in claim 28, or the pharmaceutical composition as described in claim 29 or 30. Preferably, the disease is a tumor or an autoimmune disease; Preferably, the tumor expresses GPRC5D; Preferably, the tumor is a solid tumor; Preferably, the tumor is a hematologic malignancy, such as multiple myeloma; Preferably, the subject is a mammal, such as a human.

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