Novel Anti-GPRC5d antibodies, bispecific antigen-binding molecules binding GPRC5d and CD3, and uses thereof

Abstract

The present disclosure provides anti-GPRC5D antibodies or antigen binding fragments thereof, bispecific antigen binding molecules that bind GPRC5D and CD3, isolated polynucleotides encoding the same, pharmaceutical compositions comprising the same, and uses thereof.

Description

Technical Field

[0001] The present disclosure generally relates to novel anti-GPRC5D antibodies and antibody fragments thereof, bispecific antigen binding molecules that bind GPRC5D and CD3, and uses thereof. Background Art

[0002] G protein-coupled receptor class C group 5 member D (GPRC5D) is an orphan atypical class C G protein-coupled receptor (GPCR) first identified in 2001 (Brauner-Osborne et al., Biochim Biophys Acta. 1518(3):237-248, 2001). GPRC5D is a specific surface protein expressed by plasma cells in multiple myeloma and may also be a relevant target on plasma cells in autoimmunity. It has been reported that GPRC5D is associated with prognosis and tumor burden in multiple myeloma patients (Atamaniuk, J. et al., Eur J Clin Invest, 42(9):953-960, 2012; and Cohen, Y. et al., Hematology, 18(6):348-351, 2013). Various anti-GPRC5D antibodies have been described in the art (e.g., WO 2018017786A2 and WO 2021018925A1).

[0003] CD3 (differentiation cluster 3) T cell co-receptor is a protein complex and consists of four different chains, namely CD3γ chain, CD3δ chain and two CD3ε chains. These chains associate with a molecule called T cell receptor (TCR) and ζ chain to produce activation signals in T lymphocytes. TCR, ζ chain and CD3 molecules together form TCR-CD3 complex, in which TCR recognizes and binds to antigen as a subunit, and CD3 transfers and transmits antigen stimulation to the signal transduction pathway as a subunit, and ultimately regulates T cell activity. CD3 protein is actually present in all T cells. The recent application of CD3 antibodies is in the form of bispecific antibodies, which bind CD3 on the one hand and tumor cell antigens on the other hand. The simultaneous binding of such antibodies to their two targets will force a temporary interaction between target cells and T cells, leading to the activation of any cytotoxic T cells and the subsequent lysis of target cells.

[0004] There remains a need for additional drugs for the treatment of cancer, particularly multiple myeloma. Summary of the invention

[0005] Throughout this disclosure, the articles "a", "an", and "the" as used herein refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an antibody" means one antibody or more than one antibody.

[0006] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to GPRC5D, the antibody or antigen-binding fragment thereof comprising:

[0007] one or two or three heavy chain complementarity determining regions (HCDR1, HCDR2 and / or HCDR3) contained in any one of the heavy chain variable (VH) region sequences selected from the group consisting of: SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183 and 191; and / or

[0008] One or two or three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3), wherein the light chain complementary determining regions are contained in any one of the light chain variable (VL) region sequences selected from the group consisting of: SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184 and 192.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises at least one heavy chain or light chain complementarity determining region (CDR) comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:1,2,3,4,5,6,9,10,11,12,13,14,17,18,19,20,21,22,25,26,27,28,29,30,33,34,35,36,37,38,41,42,43,44,45,46,49,50 ,51,52,53,54,57,58,59,60,61,62,65,66,67,68,69,70,73,74,75,76,77,78,97,98,99,100,101,102,105,106,107,108,109,1 10, 113, 114, 115, 116, 117, 118, 121, 122, 123, 124, 125, 126, 129, 130, 131, 132, 133, 134, 137, 138, 139, 140, 141, 142, 145, 146, 147, 148, 149, 150, 153, 154, 155, 156, 157, 158, 169, 170, 171, 172, 173, 174, 177, 178, 179, 180, 181, 182, 185, 186, 187, 188, 189 and 190.

[0010] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a VH region, wherein the VH region comprises one, two, or three of HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:1,2,3,9,10,11,17,18,19,25,26,27,33,34,35,41,42,43,49,50,51,57,58,59,65,66,67,73,74,75,97,98,99,105,106,107 ,113,114,115,121,122,123,129,130,131,137,138,139,145,146,147,153,154,155,169,170,171,177,178,179,185,186 and 187.

[0011] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a VL region comprising one, two, or three of LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:4,5,6,12,13,14,20,21,22,28,29,30,36,37,38,44,45,46,52,53,54,60,61,62,68,69,70,76,77,78,100,101,102,108,109,1 10, 116, 117, 118, 124, 125, 126, 132, 133, 134, 140, 141, 142, 148, 149, 150, 156, 157, 158, 172, 173, 174, 180, 181, 182, 188, 189 and 190.

[0012] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise:

[0013] i. HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 97, 105, 113, 121, 129, 137, 145, 153, 169, 177 and 185;

[0014] ii. a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 98, 106, 114, 122, 130, 138, 146, 154, 170, 178 and 186; and

[0015] iii. HCDR3, wherein the HCDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 99, 107, 115, 123, 131, 139, 147, 155, 171, 179 and 187.

[0016] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise:

[0017] i. LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 100, 108, 116, 124, 132, 140, 148, 156, 172, 180 and 188;

[0018] ii. LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 101, 109, 117, 125, 133, 141, 149, 157, 173, 181 and 189; and

[0019] iii. LCDR3, wherein the LCDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 102, 110, 118, 126, 134, 142, 150, 158, 174, 182 and 190.

[0020] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise:

[0021] i. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;

[0022] ii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;

[0023] iii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19;

[0024] iv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27;

[0025] v. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:33, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:34, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:35;

[0026] vi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:43;

[0027] vii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:49, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:50, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:51;

[0028] viii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 57, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 58, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 59;

[0029] ix. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 65, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 66, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 67;

[0030] x. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 73, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 74, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 75;

[0031] xi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 97, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 98, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 99;

[0032] xii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 105, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 106, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 107;

[0033] xiii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 114, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 115;

[0034] xiv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 121, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 122, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 123;

[0035] xv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 129, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 130, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 131;

[0036] xvi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 138, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 139;

[0037] xvii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 145, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 146, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 147;

[0038] xviii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 153, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 154, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 155;

[0039] xix. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 169, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 170, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 171;

[0040] xx. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 177, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 178, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 179; or

[0041] xxi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:185, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:186, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:187.

[0042] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise:

[0043] i. LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6;

[0044] ii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 12, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14;

[0045] iii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22;

[0046] iv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:28, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:29, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:30;

[0047] v. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:36, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:37, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:38;

[0048] vi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:44, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:45, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:46;

[0049] vii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:53, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:54;

[0050] viii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:60, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:61, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:62;

[0051] ix. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:68, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:69, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:70;

[0052] x. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:76, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:77, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:78;

[0053] xi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 100, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 101, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 102;

[0054] xii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 108, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 109, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 110;

[0055] xiii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 116, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 117, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 118;

[0056] xiv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 124, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 125, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126;

[0057] xv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 132, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 133, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 134;

[0058] xvi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 140, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 141, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 142;

[0059] xvii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 148, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 149, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 150;

[0060] xviii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 156, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 157, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 158;

[0061] xix. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 172, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 173, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 174;

[0062] xx. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 180, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 181, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 182; or

[0063] xxi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:188, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:189, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:190.

[0064] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise:

[0065] i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6;

[0066] ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:12, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:13, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:14;

[0067] iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22;

[0068] iv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:28, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:29, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:30;

[0069] v. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:33, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:34, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:35, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:36, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:37, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:38;

[0070] vi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:43, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:44, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:45, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:46;

[0071] vii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:49, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:50, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:51, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:53, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:54;

[0072] viii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:57, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:58, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:59, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:60, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:61, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:62;

[0073] ix. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:65, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:66, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:67, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:68, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:69, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:70;

[0074] x. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:73, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:74, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:75, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:76, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:77, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:78;

[0075] xi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:97, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:98, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:99, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:100, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:101, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:102;

[0076] xii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 105, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 106, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 107, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 108, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 109, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 110;

[0077] xiii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 114, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 115, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 116, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 117, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 118;

[0078] xiv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 121, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 122, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 123, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 124, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 125, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126;

[0079] xv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 129, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 130, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 131, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 132, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 133, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 134;

[0080] xvi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 138, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 139, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 140, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 141, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 142;

[0081] xvii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 145, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 146, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 147, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 148, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 149, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 150;

[0082] xviii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 153, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 154, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 155, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 156, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 157, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 158;

[0083] xix. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 169, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 170, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 171, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 172, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 173, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 174;

[0084] xx. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 177, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 178, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 179, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 180, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 181, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 182; or

[0085] xxi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:185, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:186, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:187, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:188, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:189, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:190.

[0086] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a VH region having an amino acid sequence as shown in SEQ ID NO:7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183 or 191, or a homologous sequence thereof having at least 80% sequence identity to SEQ ID NO:7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183 or 191.

[0087] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a VL region having an amino acid sequence as shown in SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184 or 192, or a homologous sequence thereof having at least 80% sequence identity to SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184 or 192.

[0088] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a VH / VL amino acid sequence pair selected from the group consisting of SEQ ID NO: 7 / 8, 15 / 16, 23 / 24, 31 / 32, 39 / 40, 47 / 48, 55 / 56, 63 / 64, 71 / 72, 79 / 80, 103 / 104, 111 / 112, 119 / 120, 127 / 128, 135 / 136, 143 / 144, 151 / 152, 159 / 160, 175 / 176, 183 / 184, or 191 / 192.

[0089] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention further comprises one or more amino acid residue substitutions or modifications, but still retains specific binding affinity for GPRC5D. In some embodiments, at least one of the substitutions or modifications is located in one or more CDR sequences in the VH region or VL region. In some embodiments, at least one of the substitutions or modifications is located in one or more non-CDR sequences in the VH region or VL region. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention further comprises one or more non-natural amino acids (NNAA) substitutions. In some embodiments, NNAA can be conjugated.

[0090] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure have one or more binding properties selected from the group consisting of:

[0091] i. Cross-reactive to human, cynomolgus monkey and mouse GPRC5D;

[0092] ii. capable of binding to human GPRC5D, cynomolgus monkey GPRC5D or mouse GPRC5D as measured by FACS assay;

[0093] iii. With an EC no greater than 10 nM as measured by FACS assay 50 Binds to human GPRC5D;

[0094] iv. exhibit T cell-dependent cytotoxic effects as measured by FACS assay.

[0095] In another aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof as described above for binding to GPRC5D.

[0096] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are chimeric antibodies, humanized antibodies, or human antibodies or antigen-binding fragments thereof.

[0097] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are labeled antibodies, bivalent antibodies, anti-idiotypic antibodies, or fusion proteins.

[0098] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are diabodies, Fab, Fab', F(ab') 2 、Fd、Fv fragment、Fv fragment stabilized by disulfide bonds (dsFv)、(dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), camelized single domain antibodies, nanobodies, domain antibodies or bivalent domain antibodies.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure further comprises an Fc region. In some embodiments, the Fc region is the Fc region of human immunoglobulin (Ig). In some embodiments, the Fc region is the Fc region of human IgG. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 161-163.

[0100] In some embodiments, the light chain of the antibodies or antigen-binding fragments thereof of the present disclosure is a lambda light chain or a kappa light chain.

[0101] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are bispecific antibodies or multispecific antibodies or antigen-binding fragments thereof. In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are capable of specifically binding to one or more additional antigens other than GPRC5D, or a second epitope on GPRC5D. In some embodiments, the one or more additional antigens other than GPRC5D are selected from the group consisting of KRAS, ERK, XPO1, mTORC1 / 2, PAK4, NAMPT, ATR, EGFR, FGFR, VEGF, LILRB, c-MET, Her2, Her3, CTLA4, GITA, CD112R, CD2, CD3, CD7, CD16, CD19, CD20, CD24, CD27, CD30, CD34, CD37, CD39, CD70, CD73, CD83, CD28, CD80 (B7-1), CD86 (B7-2), CD40, CD40L (CD154), CD47, SIRPα, CD122, CD137, CD137L, OX40 (CD134), OX40L (CD252), BCMA (e.g., BCMA02), PSMA , CLDN18 (e.g., CLDN18.2), NKG2C, 4-1BB, LIGHT, PVRIG, SLAMF7, HVEM, BAFFR, ICAM-1, 2B4, LFA-1, G ITR, ICOS(CD278), ICOSLG(CD275), LAG3(CD223), A2AR, B7-H3(CD276), B7-H4(VTCN1), B7-H5, BT LA(CD272), BTLA, CD160, CTLA-4(CD152), IDO1, IDO2, ILT3, TDO, KIR, LAIR-1, NOX2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, SIGLEC-7(CD328), SIGLEC-9(CD329), SIGLEC-15, TIGIT, PVR(CD155) and TGFβ.

[0102] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are bispecific antibodies or antigen-binding fragments thereof that bind to GPRC5D and CD3. In some embodiments, the bispecific antibodies or antigen-binding fragments thereof comprise a GPRC5D binding portion and a CD3 binding portion. In some embodiments, the CD3 binding portion comprises:

[0103] One or two or three heavy chain complementarity determining regions (HCDR1, HCDR2 and / or HCDR3) contained in the heavy chain variable (VH) region sequence of SEQ ID NO: 87 or 95; and / or

[0104] One or two or three light chain complementarity determining regions (LCDR1, LCDR2 and / or LCDR3), which are contained in the light chain variable (VL) region sequence of SEQ ID NO: 88 or 96.

[0105] In some embodiments, the CD3 binding moiety comprises:

[0106] HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 81 or 89, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 82 or 90, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 83 or 91; and / or

[0107] LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:84 or 92, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:85 or 93, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86 or 94.

[0108] In some embodiments, the CD3 binding moiety comprises:

[0109] i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:81, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:82, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:83, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:84, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:85, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86; or

[0110] ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:89, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:90, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:91, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:92, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:93, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:94.

[0111] In some embodiments, the CD3 binding portion comprises the VH / VL amino acid sequence pair of SEQ ID NO: 87 / 88 or 95 / 96.

[0112] In some embodiments, the CD3 binding portion further comprises a constant domain CL and a constant domain CH1. In some embodiments, the constant domains CL and CH1 are replaced with each other.

[0113] In some embodiments, the bispecific antibody or multispecific antibody of the present disclosure or its antigen-binding fragment further comprises an Fc region. In some embodiments, the bispecific antibody or multispecific antibody of the present disclosure or its antigen-binding fragment further comprises an Fc region of human immunoglobulin (Ig). In some embodiments, the bispecific antibody or multispecific antibody of the present disclosure or its antigen-binding fragment further comprises an Fc region of human IgG. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region comprises one or more amino acid substitutions selected from the group consisting of: L234A, L235A, S354C, T366W, Y349C, T366S, L368A and Y407V (according to EU numbering). In some embodiments, the Fc region comprises L234A and L235A (according to EU numbering) amino acid substitutions. In some embodiments, the Fc region of one heavy chain comprises S354C and T366W (by EU numbering) amino acid substitutions, and the Fc region of the other heavy chain comprises Y349C, T366S, L368A, and Y407V (by EU numbering) amino acid substitutions.

[0114] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are linked to one or more conjugate moieties. In some embodiments, the conjugate moieties comprise clearance modulators, chemotherapeutic agents, toxins, radioisotopes, lanthanides, detectable labels, DNA alkylating agents, topoisomerase inhibitors, tubulin binding agents, purification moieties or other anticancer drugs. In some embodiments, the conjugate moieties are covalently linked directly or via a linker.

[0115] On the other hand, the present disclosure provides a chimeric antigen receptor, the chimeric antigen receptor comprises an antibody or its antigen binding fragment provided herein, a transmembrane region and an intracellular signal region. In some embodiments, the transmembrane region comprises the transmembrane region of CD3, CD4, CD8 or CD28. In some embodiments, the intracellular signal region is selected from the group consisting of: CD3 (e.g., CD3ζ), FcγRI, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLR or a combination thereof The intracellular signal region sequence. In some embodiments, the antigen binding fragment of the chimeric antigen receptor is scFv. In some embodiments, the chimeric antigen receptor is transplanted into allogeneic cells, autologous cells or xenogeneic cells. In some embodiments, the chimeric antigen receptor is transplanted into immune effector cells. In some embodiments, the chimeric antigen receptor is transplanted into T cells, natural killer cells, macrophages or tumor infiltrating lymphocytes.

[0116] In another aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof and / or chimeric antigen receptor of the present disclosure, and one or more pharmaceutically acceptable carriers.

[0117] In another aspect, the present disclosure provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof and / or chimeric antigen receptor of the present disclosure.

[0118] In another aspect, the present disclosure provides a vector comprising the isolated polynucleotide of the present disclosure.

[0119] On the other hand, the present disclosure provides a host expression system comprising a vector of the present disclosure, or having a polynucleotide of the present disclosure integrated into its genome. In some embodiments, the host expression system of the present disclosure is a microorganism, a yeast, or a mammalian cell. In some embodiments, the microorganism is selected from the group consisting of Escherichia coli and Bacillus subtilis. In some embodiments, the yeast is Saccharomyces. In some embodiments, the mammalian cell is selected from the group consisting of: COS, CHO-S, CHO-K1, HEK-293, and 3T3 cells.

[0120] In another aspect, the present disclosure provides a virus comprising the vector of the present disclosure.

[0121] In another aspect, the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof of the present disclosure and / or the chimeric antigen receptor of the present disclosure and / or the pharmaceutical composition of the present disclosure, and a second therapeutic agent.

[0122] In another aspect, the present disclosure provides a method of expressing the antibody or antigen-binding fragment thereof and / or the chimeric antigen receptor of the present disclosure, comprising culturing the host expression system of the present disclosure under conditions for expressing the antibody or antigen-binding fragment of the present disclosure.

[0123] In another aspect, the present disclosure provides a method for treating, preventing or alleviating a disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure and / or a chimeric antigen receptor of the present disclosure and / or a pharmaceutical composition of the present disclosure.

[0124] In another aspect, the present disclosure provides use of an antibody or antigen-binding fragment thereof of the present disclosure and / or a chimeric antigen receptor of the present disclosure and / or a pharmaceutical composition of the present disclosure in the preparation of a medicament for treating a GPRC5D-related disease, disorder or condition in a subject.

[0125] In another aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof and / or chimeric antigen receptor and / or pharmaceutical composition of the present disclosure in the preparation of a diagnostic reagent for diagnosing a GPRC5D-related disease, disorder or condition.

[0126] In some embodiments, the disease, disorder or condition is cancer. In some embodiments, the cancer is a solid tumor or a blood tumor. In some embodiments, the disease, disorder or condition is a B cell cancer expressing GPRC5D. In some embodiments, the disease, disorder or condition is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lung adenocarcinoma or lung squamous cell carcinoma), peritoneal cancer, carcinoid, bone cancer, pancreatic cancer, primitive neuroectodermal tumors, skin cancer, gallbladder cancer, cancer of the head or neck, squamous cell carcinoma, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial carcinoma), anal cancer (e.g., anal squamous cell carcinoma), stomach cancer or stomach cancer (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatocarcinoma or liver cancer), bile duct cancer. carcinoma, sarcoma, colorectal cancer, fallopian tube cancer, salivary gland cancer, cervical cancer, endometrial cancer or uterine cancer, osteosarcoma, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, nasopharyngeal cancer, soft tissue sarcoma, polycythemia vera, urethral cancer, penile cancer, kidney or ureter cancer (e.g., rhabdoid tumor of the kidney), cutaneous T-cell lymphoma, medulloblastoma, Wilms tumor, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, choroid plexus papilloma, renal cell carcinoma, renal pelvic cancer, central nervous system (CNS) tumors, soft tissue sarcoma (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi's sarcoma) , spinal axis tumors, gliomas (e.g., ependymomas, astrocytomas, anaplastic astrocytomas, oligodendrogliomas, eye cancers (e.g., retinoblastomas), brain stem gliomas, or mixed gliomas such as oligoastrocytomas), brain tumors (e.g., glioblastoma / glioblastoma multiforme (GBM), non-glioblastoma brain tumors, or meningiomas), skin or intraocular melanoma, thrombocythaemia, mesothelioma, mycosis fungoides, Sezary syndrome, idiopathic myelofibrosis, solitary plasmacytoma, vestibular schwannoma, Ewing sarcoma, chondrosarcoma, MYH-associated polyposis, pituitary adenomas, pediatric cancers such as pediatric sarcomas (e.g., neurofibromatosis), blastoma, rhabdomyosarcoma, and osteosarcoma), blood cancers, Hodgkin lymphoma, non-Hodgkin lymphoma, leukemias (e.g., lymphocytic / lymphoblastic leukemias), chronic or acute leukemias, mast cell leukemia, lymphocytic lymphoma, primary CNS lymphomas, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia (HCL), Burkitt lymphoma (BL), multiple myeloma (e.g.,In some embodiments, the disease, disorder or condition is multiple myeloma. ,

[0127] In some embodiments, the subject is a human.

[0128] In some embodiments, administration is by parenteral routes, including subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection; or non-parenteral routes, including transdermal, oral, intranasal, intraocular, sublingual, rectal, or topical.

[0129] In some embodiments, the method for treating, preventing or alleviating a disease, disorder or condition in a subject further comprises administering an additional therapeutic agent to a subject in need thereof. In some embodiments, the additional therapeutic agent is selected from the group consisting of: an active agent, an imaging agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a co-stimulatory molecule agonist, a co-inhibitory molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or its functional fragment, a detectable marker or reporter molecule, an antimicrobial agent, a gene editor, a beta agonist, a viral RNA inhibitor, a polymerase inhibitor, an interferon and a microRNA. In some embodiments, the additional therapeutic agent is administered to a subject in need thereof before, after or simultaneously with the antibody or its antigen-binding fragment and / or pharmaceutical composition disclosed herein.

[0130] In another aspect, the present disclosure provides a method of activating cells expressing GPRC5D in vivo or in vitro, comprising contacting cells expressing GPRC5D with the antibody or antigen-binding fragment thereof and / or the chimeric antigen receptor and / or the pharmaceutical composition of the present disclosure.

[0131] In another aspect, the present disclosure provides a method of modulating GPRC5D activity in a cell expressing GPRC5D, comprising exposing the cell expressing GPRC5D to an antibody or antigen-binding fragment thereof and / or a chimeric antigen receptor and / or a pharmaceutical composition of the present disclosure.

[0132] In another aspect, the present disclosure provides a method for detecting the presence or amount of GPRC5D in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof of the present disclosure and / or the chimeric antigen receptor of the present disclosure and / or the pharmaceutical composition of the present disclosure, and determining the presence or amount of GPRC5D in the sample.

[0133] In another aspect, the present disclosure provides a method for diagnosing a GPRC5D-related disease, disorder or condition in a subject, comprising: a) obtaining a sample from the subject, b) contacting the sample obtained from the subject with an antibody or antigen-binding fragment thereof and / or chimeric antigen receptor and / or pharmaceutical composition of the present disclosure; c) determining the presence or amount of GPRC5D in the sample; and d) establishing a correlation between the presence or amount of GPRC5D in the subject and the presence or status of a GPRC5D-related disease, disorder or condition.

[0134] In another aspect, the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof and / or chimeric antigen receptor and / or pharmaceutical composition of the present disclosure for detecting GPRC5D, optionally recombinant GPRC5D, GPRC5D expressed on the surface of a cell, or a cell expressing GPRC5D. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1 The selected chimeric antibodies are shown to be resistant to human GPRC5D ( Figure 1 A), cynomolgus monkey GPRC5D ( Figure 1 B) and mouse GPRC5D ( Figure 1 C) binding affinity.

[0136] Figure 2 The T cell-dependent cytotoxic effects of selected bispecific hIgG-scFv antibodies on CHOS-hGPRC5D cells are shown.

[0137] Figure 3 Shown are the T cell-dependent cytotoxic effects of selected bispecific Crossmab antibodies on MM.1S cells.

[0138] Figure 4 Tumor growth curves are shown, illustrating the in vivo efficacy of selected bispecific Crossmab antibodies.

[0139] Figure 5 CAR-T cell cytotoxicity of selected clones is shown. DETAILED DESCRIPTION

[0140] The following description of the present disclosure is only intended to illustrate each embodiment of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that such equivalent embodiments will be included herein. All documents cited in this article, including publications, patents and patent applications are incorporated herein by reference in their entirety.

[0141] definition

[0142] The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that binds to a specific antigen. A natural complete antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are divided into α, δ, ε, γ and μ, each heavy chain comprising a variable region (VH) and a first constant region, a second constant region, a third constant region and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are divided into λ or κ, and each light chain comprises a variable region (VL) and a constant region. The antibody is "Y" shaped, wherein the stem of the Y-shaped structure comprises the second constant region and the third constant region of two heavy chains bound together by disulfide bonds. Each arm of the Y comprises the variable region and the first constant region of a single heavy chain bound to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region of each chain usually contains three hypervariable loops, called complementary determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2, LCDR3, and heavy chain CDRs include HCDR1, HCDR2, HCDR3). The three CDRs are separated by flanking segments called framework regions (FRs) (light chain FRs include LFR1, LFR2, LFR3, and LFR4, and heavy chain FRs include HFR1, HFR2, HFR3, and HFR4), which are more highly conserved than CDRs and form a scaffold to support the highly variable loops. The constant regions of the heavy and light chains are not related to antigen binding, but exhibit a variety of effector functions. Antibodies can be divided into several categories based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0143] In certain embodiments, the antibodies provided herein encompass any antigen-binding fragments thereof. As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed by a portion of an antibody comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs, or any other antibody fragment that binds to an antigen but does not contain a complete native antibody structure. Examples of antigen-binding fragments include, but are not limited to, bifunctional antibodies, Fab, Fab', F(ab'), 2 、Fd、Fv fragment、Fv fragment stabilized by disulfide bonds (dsFv)、(dsFv) 2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies, single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies or bivalent domain antibodies. The antigen-binding fragment can bind to the same antigen or epitope as the parent antibody.

[0144] The "Fab" of an antibody refers to a portion of an antibody composed of a single light chain (including a variable region and a constant region) and the variable region and first constant region of a single heavy chain bound together by disulfide bonds.

[0145] "Fab'" refers to the Fab fragment including a portion of the hinge region.

[0146] “F(ab') 2 ” refers to a dimer of Fab′.

[0147] The "Fc" of an antibody (e.g., IgG, IgA or IgD isotype) refers to a portion of an antibody consisting of the second constant domain and the third constant domain of the first heavy chain combined with the second constant domain and the third constant domain of the second heavy chain via a disulfide bond. The Fc of IgM and IgE isotype antibodies further comprises a fourth constant domain. The Fc portion of an antibody is responsible for a variety of different effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.

[0148] The "Fv" of an antibody refers to the smallest antibody fragment that contains a complete antigen binding site. The Fv fragment consists of the variable region of a single light chain combined with the variable region of a single heavy chain.

[0149] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are directly connected to each other or connected to each other by a linker (e.g., a peptide sequence) (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0150] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc region of an antibody.

[0151] "Camelized single domain antibody", "heavy chain antibody" or "HCAb" refers to an antibody comprising two VH domains but no light chain (Riechmann L. and Muyldermans S., J Immunol Methods Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Pat. No. 6,005,079). Heavy chain antibodies were originally derived from the Camelidae (camels, dromedaries and llamas). Although lacking light chains, camelid antibodies have a well-established full complement of antigen binding functions (Hamers-Casterman C. et al., Nature Jun 3;363(6428):446-8 (1993); Nguyen VK. et al., Immunogenetics Apr;54(1):39-47 (2002); Nguyen VK. et al., Immunology May;109(1):93-101 (2003)). The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen binding unit produced by an adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8 Epub 2007 Jun 15 (2007)).

[0152] "Nanobody" refers to an antibody fragment consisting of the VHH domain from a heavy chain antibody and the two constant domains CH2 and CH3.

[0153] "Diabodies", "diabodies" or "dAbs" include small antibody fragments with two antigen binding sites, wherein the fragments comprise a VH domain and a VL domain connected on the same polypeptide chain (VH-VL or VL-VH) (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA Jul. 15;90(14):6444-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby generating two antigen binding sites. The antigen binding sites can target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds bifunctional antibody" is a bifunctional antibody that targets two different antigens (or epitopes).

[0154] "Domain antibodies" refer to antibody fragments that contain only the variable region of a heavy chain or a variable region of a light chain. In some cases, two or more VH domains are covalently joined by a peptide linker to produce a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody can target the same or different antigens (or epitopes).

[0155] As used herein, the term "valence" refers to the presence of a specified number of antigen binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen binding fragment having only one antigen binding site; and the term "multivalent" refers to an antibody or antigen binding fragment having multiple antigen binding sites. Thus, the terms "bivalent", "tetravalent" and "hexavalent" refer to the presence of two antigen binding sites, four antigen binding sites and six antigen binding sites in an antigen binding molecule, respectively. In some embodiments, the antibody or its antigen binding fragment is bivalent.

[0156] As used herein, a "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes may be present on the same antigen, or they may be present on two different antigens.

[0157] As used herein, a "multispecific" antibody refers to an antibody that specifically binds to at least two different antigens or at least two different epitopes of the same antigen. A multispecific antibody can bind to, for example, two, three, four, five or more different antigens, or can bind to two, three, four, five or more different epitopes of the same antigen.

[0158] In certain embodiments, "scFv dimer" is a bivalent bifunctional antibody (diabody) or a bispecific scFv (BsFv), which comprises two VH-VL (connected by a peptide linker) parts of dimerization, so that the VH of one part cooperates with the VL of the other part to form two binding sites, and the two binding sites can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, "scFv dimer" is a bispecific bifunctional antibody, which comprises mutually associated VH1-VL2 (connected by a peptide linker) and VL1-VH2 (also connected by a peptide linker), so that VH1 and VL1 cooperate, VH2 and VL2 cooperate, and each cooperative pairing has a different antigen specificity.

[0159] "dsFv" refers to a disulfide-stabilized Fv fragment, in which the connection between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, "(dsFv)2" or "(dsFv-dsFv')" comprises three peptide chains: two VH parts are connected by a peptide linker (e.g., a long flexible linker) and are respectively bound to two VL parts by disulfide bonds. In some embodiments, dsFv-dsFv' has dual specificity, in which each pair of heavy and light chains paired by disulfide bonds has different antigenic specificity.

[0160] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of a heavy chain and / or light chain derived from one species and the remainder of the heavy chain and / or light chain derived from another different species. In illustrative embodiments, a chimeric antibody may comprise a constant region derived from a human and a variable region derived from a non-human animal (e.g., a mouse). In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0161] As used herein, the term "humanized" means that the antibody or antigen-binding fragment comprises a CDR derived from a non-human animal, a FR region derived from a human, and a constant region derived from a human (when applicable). The CDR of the humanized antibody provided by the present disclosure may comprise a mutation compared to the CDR of its parent antibody.

[0162] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (ie, antibody) or antigen-binding fragment thereof and an antigen.

[0163] An antibody or antigen binding fragment thereof that "specifically binds" (specifically binds / specific binding) to a target (e.g., an epitope) is a term well known in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with greater affinity than it does with alternative cells or substances. An antibody "specifically binds" to a target if it binds with greater affinity, higher affinity, more readily, and / or longer duration than it binds to other substances. For example, an antibody that specifically binds to a GPRC5D epitope is an antibody that binds to this GPRC5D epitope with greater affinity, higher affinity, more readily, and / or longer duration than it binds to other GPRC5D epitopes or non-GPRC5D epitopes. It should also be understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" (specifically bind) does not necessarily require (although it can include) exclusive binding. Typically, but not necessarily, references to binding refer to specific binding.

[0164] As used herein, the ability to "compete for binding to GPRC5D" refers to the ability of a first antibody or antigen-binding fragment to inhibit the binding interaction between GPRC5D and a second anti-GPRC5D antibody to any detectable extent. In certain embodiments, the antibody or antigen-binding fragment that competes for binding to GPRC5D inhibits the binding interaction between GPRC5D and a second anti-GPRC5D antibody by at least 85%, or at least 90%. In certain embodiments, the inhibition may be greater than 95% or greater than 99%.

[0165] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitopes within the antigen. An epitope can be linear or spatially conformational (i.e., comprising spaced-apart amino acid residues). For example, if an antibody or antigen-binding fragment blocks the binding of a reference antibody to an antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or antigen-binding fragment can be considered to bind to the same or closely related epitope as the reference antibody.

[0166] As used herein, the term "amino acid" refers to a 2) and carboxyl (-COOH) functional groups and side chains unique to each amino acid. Amino acid names are also represented in this disclosure by standard single-letter or three-letter codes, which are summarized as follows:

[0167]

[0168]

[0169] "Conservative substitution" with respect to an amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between amino acid residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between amino acid residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between amino acid residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As known in the art, conservative substitutions generally do not cause significant changes in protein conformational structure, and therefore the biological activity of the protein can be retained.

[0170] As used herein, the term "homologous" refers to a nucleic acid sequence (or its complementary strand) or an amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence when optimally aligned.

[0171] "Percentage (%) of sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the percentage (%) of sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence to which it is compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence (whichever is shorter). Conservative substitutions of amino acid residues may or may not be considered identical residues. For example, publicly available tools such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods In Enzymology, 266:383-402 (1996); Larkin et al., Methods In Enzymology, 267:383-402 (1997); MA et al., " Bioinformatics (Bioinformatics) " (Oxford, England), 23 (21): 2947-8 (2007) and ALIGN or Megalign (DNASTAR) software to achieve comparison to determine the amino acid (or nucleic acid) sequence identity percentage. Those skilled in the art can use the default parameters provided by the tool or can suitably customize parameters according to the needs of the comparison, for example, by selecting a suitable algorithm.

[0172] As used herein, "effector function" refers to the biological activity caused by the binding of the Fc region of an antibody to its effector (e.g., C1 complex and Fc receptor). Exemplary effector functions include complement-dependent cytotoxicity (CDC) mediated by the interaction of the antibody and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by the binding of the Fc region of the antibody to Fc receptors on effector cells; and phagocytosis. Effector function can be assessed using various assays, such as Fc receptor binding assays, C1q binding assays, and cell lysis assays.

[0173] As used herein, "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which effector cells expressing Fc receptors (FcRs) recognize bound antibodies or antigen-binding fragments on target cells and subsequently cause lysis of the target cells. "ADCC activity" or "ADCC effector" refers to the ability of an antibody or antigen-binding fragment bound to a target cell to elicit an ADCC reaction as described above.

[0174] As used herein, "complement dependent cytotoxicity" or "CDC" refers to a mechanism by which antibodies can mediate specific target cell lysis by activating the complement system of an organism. In CDC, C1q binds to the antibody and this binding triggers the complement cascade, which results in the formation of a membrane attack complex (MAC) (C5b to C9) at the surface of the target cell as a result of complement activation of the classical pathway. "CDC activity" or "CDC effect" refers to the ability of an antibody or antigen-binding fragment bound to a target cell to elicit a CDC reaction as described above.

[0175] As used herein, "target cell" refers to a cell to which an antibody comprising an Fc region specifically binds (usually through a protein portion located at the C-terminus of the Fc region). "Effector cell" is a leukocyte that expresses one or more Fc receptors and performs effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; wherein PBMC and NK cells are preferred. Effector cells can be isolated from their natural sources, such as from blood or PBMC as known in the art.

[0176] An "isolated" substance has been artificially altered from its natural state. If an "isolated" composition or substance occurs in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated", but it may be considered "isolated" if the polynucleotide or polypeptide is sufficiently separated from materials with which it coexists in nature and is present in a substantially pure state. An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In certain embodiments, an “isolated antibody or antigen-binding fragment thereof” refers to an antibody or antigen-binding fragment thereof that is at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% pure as determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (e.g., ion exchange chromatography or reversed phase HPLC).

[0177] As used herein, the term "vector" refers to a medium into which a polynucleotide encoding a protein can be operably inserted so as to cause the expression of the protein. A vector can be used to transform, transduce or transfect a host cell so that the genetic elements it carries are expressed in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) from P1 sources, phages such as lambda phage or M13 phage, and animal viruses. The categories of animal viruses used as vectors include retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses and papovaviruses (e.g., SV40). A vector can include a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements and reporter genes. In addition, a vector can also contain a replication origin. A vector can also include materials that assist it in entering a cell, including but not limited to viral particles, liposomes or protein coatings. A vector can be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably connected to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phages and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0178] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced.

[0179] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians and reptiles. Except where noted, the terms "patient," "subject," or "individual" are used interchangeably herein.

[0180] The term "anti-tumor activity" means a decrease in tumor cell proliferation, viability or metastatic activity. For example, compared to a control not using therapy, the anti-tumor activity can be represented by a decrease in the growth rate of abnormal cells that appear during treatment or a stability or decrease in tumor size or a longer survival rate due to treatment. Such activity can be evaluated using recognized in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, mouse mammary tumor virus (MMTV) models, and other known models known in the art to study anti-tumor activity.

[0181] As used herein, "treating" or "treatment" of a disease, disorder or condition includes preventing or ameliorating the disease, disorder or condition, slowing the onset or rate of development of a disease, disorder or condition, reducing the risk of developing a disease, disorder or condition, preventing or delaying the development of symptoms associated with the disease, disorder or condition, reducing or ending symptoms associated with the disease, disorder or condition, causing complete or partial regression of the disease, disorder or condition, curing the disease, disorder or condition, or some combination thereof.

[0182] The term "diagnosis" refers to the identification of a pathological state, disease or condition, such as the identification of a GPRC5D-related disease, or the identification of a subject with a GPRC5D-related disease who may benefit from a particular treatment regimen. In some embodiments, the diagnosis comprises the identification of an abnormal amount or activity of GPRC5D. In some embodiments, the diagnosis refers to the identification of cancer in a subject.

[0183] As used herein, the term "biological sample" or "sample" refers to a biological composition obtained from or derived from a subject of interest, the biological composition containing, for example, cells and / or other molecular entities to be characterized and / or identified based on physical, biochemical, chemical and / or physiological properties. Biological samples include, but are not limited to, cells, tissues, organs and / or biological fluids of a subject obtained by any method known to those skilled in the art. In some embodiments, the biological sample is a body fluid sample. In some embodiments, the body fluid sample is whole blood, plasma, serum, mucus (including nasal drainage and sputum), peritoneal fluid, pleural fluid, pleural fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, peritoneal effusion, ascites or pericardial fluid. In some embodiments, the biological sample is a tissue or cell obtained from the stomach, heart, liver, spleen, lung, kidney, skin or blood vessel of the subject.

[0184] As used herein, "GPRC5D" refers to a G protein coupled receptor C family 5 group D member, and includes any variant, conformation, isoform, and species homolog of GPRC5D expressed naturally by cells or expressed by cells transfected with the GPRC5D gene. For example, GPRC5D described herein may refer to a G protein coupled receptor C family 5 group D member derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). For example, exemplary sequences of human GPRC5D proteins are described in GenBank accession number BC069341, NCBI reference sequence: NP_061124.1, and UniProtKB / Swiss-Prot accession number Q9NZD1 (see also Brauner-Osborne, H. et al., 2001, "Acta Biochimica et Biophysica Sinica" 1518, 237-248). As used herein, the term "GPRC5D" is intended to encompass any form of GPRC5D, e.g., 1) a naturally occurring unprocessed GPRC5D molecule, a "full-length" GPRC5D chain or a naturally occurring GPRC5D variant, including, e.g., a splice variant or an allelic variant; 2) any form of GPRC5D produced by processing in a cell; or 3) full-length, fragments (e.g., truncated forms, extracellular / transmembrane domains), or modified forms (e.g., mutant forms, glycosylated / pegylated, His-tag / immunofluorescence fusion forms) of a GPRC5D subunit produced by recombinant methods.

[0185] As used herein, "CD3" refers to a cluster of differentiation 3 protein, and includes any variant, conformation, isoform, and species homolog of CD3 expressed naturally by cells or expressed by cells transfected with the CD3 gene. For example, the CD3 described herein may refer to a cluster of differentiation 3 protein derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). In mammals, the CD3 molecule is a six-chain multiprotein complex, including: a homodimer of a CD3γ chain, a CD3δ chain, two CD3ε chains, and a CD3ζ chain, wherein the CD3ζ chain is the intracellular tail of the CD3 molecule, and the CD3γ, CD3δ, and CD3ε chains are all included in an extracellular domain (ECD) expressed on the surface of T cells. Exemplary sequences of human CD3 include human CD3 epsilon protein (NCBI Reference Sequence No. NP_000724), human CD3 delta protein (NCBI Reference Sequence No. NP_000723), and human CD3 gamma protein (NCBI Reference Sequence No. NP_000064). Exemplary sequences of non-human CD3 include cynomolgus macaque (monkey) CD3 epsilon protein (NCBI Reference Sequence No. NP_001270544), cynomolgus macaque (Macaca fascicularis) (monkey) CD3 delta protein (NCBI Reference Sequence No. NP_001274617), cynomolgus macaque (monkey) CD3 gamma protein (NCBI Reference Sequence No. NP_001270839); Mus musculus (mouse) CD3 epsilon protein (NCBI Reference Sequence No. NP_031674), Mus musculus (mouse) CD3 delta protein (NCBI Reference Sequence No. NP_038515), Mus musculus (mouse) CD3 gamma protein (NCBI Reference Sequence No. AAA37400); Rattus norvegicus (Rattus norvegicus) (rat) CD3ε protein (NCBI reference sequence number NP_001101610), Rattus norvegicus (rat) CD3δ protein (NCBI reference sequence number NP_037301), Rattus norvegicus (rat) CD3γ protein (NCBI reference sequence number NP_001071114). In certain embodiments, the CD3 used herein may also be recombinant CD3, for example, including recombinant CD3ε protein, recombinant CD3δ protein and recombinant CD3γ protein, and the recombinant CD3 may optionally be expressed in the form of a recombinant CD3 complex. The recombinant CD3 complex may be expressed on the cell surface, or may be expressed in a soluble form that is not bound to the cell surface. In certain embodiments, CD3 is human CD3. The terms "CD3", "CD-3", "CD 3", and "differentiation cluster 3" may be used interchangeably in the present disclosure.

[0186] The term "anti-GPRC5D antibody" refers to an antibody that binds to GPRC5D (eg, human GPRC5D). The term "anti-human GPRC5D antibody" or "anti-hGPRC5D antibody" refers to an antibody that specifically binds to human GPRC5D.

[0187] The term "anti-CD3 antibody" refers to an antibody that specifically binds to CD3 (e.g., human CD3). The term "anti-human CD3 antibody" refers to an antibody that specifically binds to human CD3. In some embodiments, the anti-CD3 antibodies provided herein specifically bind to CD3γ protein. In some embodiments, the anti-CD3 antibodies provided herein specifically bind to CD3δ protein. In some embodiments, the anti-CD3 antibodies provided herein specifically bind to CD3ε protein.

[0188] As used herein, the term "CD3γ" is intended to encompass any form of CD3γ, e.g., 1) a naturally occurring unprocessed CD3γ molecule, a "full-length" CD3γ chain, or a naturally occurring CD3γ variant, including, e.g., a splice variant or an allelic variant; 2) any form of CD3γ produced by processing in a cell; or 3) full-length, fragments (e.g., truncated forms, extracellular / transmembrane domains), or modified forms (e.g., mutant forms, glycosylated / pegylated, His-tag / immunofluorescence fusion forms) of a CD3γ subunit produced by recombinant methods.

[0189] As used herein, the term "CD3δ" is intended to encompass any form of CD3δ, e.g., 1) the native unprocessed CD3δ molecule, a "full-length" CD3δ chain or a naturally occurring CD3δ variant, including, e.g., a splice variant or an allelic variant; 2) any form of CD3δ produced by processing in a cell; or 3) full-length, fragments (e.g., truncated forms, extracellular / transmembrane domains), or modified forms (e.g., mutant forms, glycosylated / pegylated, His-tag / immunofluorescence fusion forms) of a CD3δ subunit produced by recombinant methods.

[0190] As used herein, the term "CD3ε" is intended to encompass any form of CD3ε, e.g., 1) a naturally occurring unprocessed CD3ε molecule, a "full-length" CD3ε chain or a naturally occurring CD3ε variant, including, e.g., a splice variant or an allelic variant; 2) any form of CD3ε produced by processing in a cell; or 3) full-length, fragments (e.g., truncated forms, extracellular / transmembrane domains) or modified forms (e.g., mutant forms, glycosylated / pegylated, His-tag / immunofluorescence fusion forms) of a CD3ε subunit produced by recombinant methods.

[0191] As used herein, a "GPRC5D related / GPRC5D-related" disease, disorder or condition refers to any disease, disorder or condition caused, aggravated or otherwise connected by an increase or decrease in the expression or activity of GPRC5D. In some embodiments, a GPRC5D related disease, disorder or condition is a disorder associated with excessive cell proliferation, such as cancer. In certain embodiments, a GPRC5D related disease or condition is characterized by expression or overexpression of GPRC5D and / or a GPRC5D related gene.

[0192] The term "pharmaceutically acceptable" means that the specified carrier, vehicle, diluent, excipient and / or salt are generally chemically and / or physically compatible with the other ingredients making up the formulation and physiologically compatible with the recipient.

[0193] As used herein, the term "cell expressing GPRC5D" refers to a cell expressing GPRC5D on the surface of the cell.

[0194] Anti-GPRC5D Antibody

[0195] The present disclosure provides anti-GPRC5D antibodies and antigen-binding fragments thereof.The anti-GPRC5D antibodies and antigen-binding fragments provided herein are capable of binding (eg, specifically binding) to GPRC5D (eg, human GPRC5D).

[0196] The binding affinity of the antibodies or antigen-binding fragments thereof provided herein can be determined by K D The value represents the ratio of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium (k off / k on Antigen binding affinity (e.g., K) can be suitably determined using appropriate methods known in the art, including, for example, flow cytometry assays. D In some embodiments, the binding of an antibody or antigen-binding fragment thereof to different concentrations of an antigen can be measured by flow cytometry, and the determined mean fluorescence intensity (MFI) can be first plotted against the antibody concentration, and then K can be calculated by fitting the dependence of specific binding fluorescence intensity (Y) and antibody concentration (X) to a sitesaturation equation using Prism version 5 (GraphPad Software, San Diego, CA). D Value: Y = B max *X / (K D +X), where B maxIt refers to the maximum specific binding of the tested antibody to the antigen.

[0197] The binding of the antibodies or antigen-binding fragments thereof provided herein to GPRC5D can also be measured by "half maximal effective concentration" (EC 50 ) value, which refers to the concentration of the antibody at which 50% of its maximal binding is observed. 50 The values ​​can be measured by binding assays known in the art, such as direct or indirect binding assays, such as enzyme-linked immunosorbent assay (ELISA), FACS assays, and other binding assays.

[0198] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to human GPRC5D, cynomolgus monkey GPRC5D or mouse GPRC5D (as measured by FACS assay). In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are cross-reactive to human, cynomolgus monkey and mouse GPRC5D.

[0199] As used herein, the term "cross-reactivity" refers to the ability of a binding protein to bind to a target other than the target for which it is produced. Typically, a binding protein will bind to its target tissue / antigen with a suitably high affinity, but will exhibit a suitably low affinity to non-target normal tissues / antigens. Individual binding proteins are typically selected to meet the following two criteria: (1) binding of the antibody to a known expressed tissue suitable for the antibody target, as visualized using staining methods known in the art; and (2) similar staining patterns between human and toxicological species (tox species) (e.g., mice and cynomolgus monkeys) tissues from the same organ. These and other methods for assessing cross-reactivity are known to those skilled in the art (e.g., US20090311253A1).

[0200] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have an EC of no more than 10 nM (e.g., no more than 9 nM, no more than 8 nM, no more than 7 nM, no more than 6 nM, no more than 5 nM, no more than 4 nM, no more than 3 nM, no more than 2 nM, no more than 1 nM) as measured by FACS assay. 50 In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to human GPRC5D with an EC of no more than 20 nM (e.g., no more than 15 nM, no more than 10 nM, no more than 9 nM, no more than 8 nM, no more than 7 nM, no more than 6 nM, no more than 5 nM, no more than 4 nM, no more than 3 nM, no more than 2 nM, no more than 1 nM) as measured by FACS assay. 50 Binds to cynomolgus monkey GPRC5D and / or mouse GPRC5D.

[0201] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein show a T cell-dependent cytotoxic effect (e.g., as measured by a FACS assay). In certain embodiments, the T cell-dependent cytotoxic effect is measured by a method as described in Example 3 of the present disclosure.

[0202] Exemplary anti-GPRC5D antibodies

[0203] In certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to GPRC5D, comprising:

[0204] one or two or three heavy chain complementarity determining regions (HCDR1, HCDR2 and / or HCDR3) contained in any one of the heavy chain variable (VH) region sequences selected from the group consisting of: SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183 and 191; and / or

[0205] One or two or three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3), wherein the light chain complementary determining regions are contained in any one of the light chain variable (VL) region sequences selected from the group consisting of: SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184 and 192.

[0206] A person skilled in the art can define or determine the CDR boundaries of a VH or VL region by methods known in the art, as long as the amino acid sequence of the VH or VL region is known. For example, the CDR boundaries of an antibody or antigen-binding fragment thereof may be defined or determined by the Kabat, IMGT, Chothia or Al-Lazikani rules (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th Public Health Edition). Service), National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (Second Edition), Chapter 26, 481-514, (2015). In some embodiments, the CDR boundaries of the antibodies or antigen-binding fragments thereof provided herein are determined according to the Kabat rule. In some embodiments, the CDR boundaries of the antibodies or antigen-binding fragments thereof provided herein are determined according to the IMGT rule. In some embodiments, the CDR boundaries of the antibodies or antigen-binding fragments thereof provided herein are determined according to the Chothia rule. In some embodiments, the CDR boundaries of the antibodies or antigen-binding fragments thereof provided herein are determined according to the Al-Lazikani rule.

[0207] In certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to GPRC5D, wherein the antibody or antigen-binding fragment thereof comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of anti-GPRC5D antibodies 36A3G8, 28H9C12, 26E2H6, 20C4E1, 15D6G2, 12G12C2, 71F8H2, 64G9C1, 98A3A8, 96C9E7, 90G6H3, 89F11E3, 87A3G8, 83A9B8, 82A1C10, 119E4B4, 116G1G4, 116F1G6, 110B4H5, 108C4C12, or 101B3G3.

[0208] As used herein, antibody "36A3G8" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:7 and a light chain variable region having the sequence of SEQ ID NO:8.

[0209] As used herein, antibody "28H9C12" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:15 and a light chain variable region having the sequence of SEQ ID NO:16.

[0210] As used herein, antibody "26E2H6" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:23 and a light chain variable region having the sequence of SEQ ID NO:24.

[0211] As used herein, antibody "20C4E1" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:31 and a light chain variable region having the sequence of SEQ ID NO:32.

[0212] As used herein, antibody "15D6G2" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:39 and a light chain variable region having the sequence of SEQ ID NO:40.

[0213] As used herein, antibody "12G12C2" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:47 and a light chain variable region having the sequence of SEQ ID NO:48.

[0214] As used herein, antibody "71F8H2" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:55 and a light chain variable region having the sequence of SEQ ID NO:56.

[0215] As used herein, antibody "64G9C1" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:63 and a light chain variable region having the sequence of SEQ ID NO:64.

[0216] As used herein, antibody "98A3A8" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:71 and a light chain variable region having the sequence of SEQ ID NO:72.

[0217] As used herein, antibody "96C9E7" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:79 and a light chain variable region having the sequence of SEQ ID NO:80.

[0218] As used herein, antibody "90G6H3" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:103 and a light chain variable region having the sequence of SEQ ID NO:104.

[0219] As used herein, antibody "89F11E3" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:111 and a light chain variable region having the sequence of SEQ ID NO:112.

[0220] As used herein, antibody "87A3G8" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:119 and a light chain variable region having the sequence of SEQ ID NO:120.

[0221] As used herein, antibody "83A9B8" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:127 and a light chain variable region having the sequence of SEQ ID NO:128.

[0222] As used herein, antibody "82A1C10" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:135 and a light chain variable region having the sequence of SEQ ID NO:136.

[0223] As used herein, antibody "119E4B4" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:143 and a light chain variable region having the sequence of SEQ ID NO:144.

[0224] As used herein, antibody "116G1G4" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:151 and a light chain variable region having the sequence of SEQ ID NO:152.

[0225] As used herein, antibody "116F1G6" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:159 and a light chain variable region having the sequence of SEQ ID NO:160.

[0226] As used herein, antibody "110B4H5" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:175 and a light chain variable region having the sequence of SEQ ID NO:176.

[0227] As used herein, antibody "108C4C12" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:183 and a light chain variable region having the sequence of SEQ ID NO:184.

[0228] As used herein, antibody "101B3G3" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:191 and a light chain variable region having the sequence of SEQ ID NO:192.

[0229] The specific amino acid sequences of the heavy chain variable region and the light chain variable region of each exemplary antibody as described above are shown in Table 3 below.

[0230] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO:7 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO:8.

[0231] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO: 15 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO: 16.

[0232] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO:23 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO:24.

[0233] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO:31 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO:32.

[0234] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO:39 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO:40.

[0235] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO:47 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO:48.

[0236] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO:55 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO:56.

[0237] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO:63 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO:64.

[0238] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO:71 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO:72.

[0239] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO:79 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO:80.

[0240] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO: 103 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO: 104.

[0241] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO:111 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO:112.

[0242] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO: 119 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO: 120.

[0243] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO: 127 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO: 128.

[0244] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO: 135 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO: 136.

[0245] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO: 143 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO: 144.

[0246] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO: 151 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO: 152.

[0247] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO: 159 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO: 160.

[0248] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO: 175 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO: 176.

[0249] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO: 183 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO: 184.

[0250] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein include three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence shown in SEQ ID NO: 191 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence shown in SEQ ID NO: 192.

[0251] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise at least one (e.g., 1, 2, or 3) heavy chain or light chain CDR comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:1,2,3,4,5,6,9,10,11,12,13,14,17,18,19,20,21,22,25,26,27,28,29,30,33,34,35,36,37,38,41,42,43,44,45,46,49,50 ,51,52,53,54,57,58,59,60,61,62,65,66,67,68,69,70,73,74,75,76,77,78,97,98,99,100,101,102,105,106,107,108,109,1 10, 113, 114, 115, 116, 117, 118, 121, 122, 123, 124, 125, 126, 129, 130, 131, 132, 133, 134, 137, 138, 139, 140, 141, 142, 145, 146, 147, 148, 149, 150, 153, 154, 155, 156, 157, 158, 169, 170, 171, 172, 173, 174, 177, 178, 179, 180, 181, 182, 185, 186, 187, 188, 189 and 190.

[0252] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a VH region, wherein the VH region comprises one, two, or three of HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:1,2,3,9,10,11,17,18,19,25,26,27,33,34,35,41,42,43,49,50,51,57,58,59,65,66,67,73,74,75,97,98,99,105,106,107 ,113,114,115,121,122,123,129,130,131,137,138,139,145,146,147,153,154,155,169,170,171,177,178,179,185,186 and 187.

[0253] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a VL region comprising one, two or three of LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:4,5,6,12,13,14,20,21,22,28,29,30,36,37,38,44,45,46,52,53,54,60,61,62,68,69,70,76,77,78,100,101,102,108,109,1 10, 116, 117, 118, 124, 125, 126, 132, 133, 134, 140, 141, 142, 148, 149, 150, 156, 157, 158, 172, 173, 174, 180, 181, 182, 188, 189 and 190.

[0254] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 97, 105, 113, 121, 129, 137, 145, 153, 169, 177 and 185; the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 98, 106, 114, 122, 130, 138, 146, 154, 170, 178 and 186; and the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 99, 107, 115, 123, 131, 139, 147, 155, 171, 179 and 187.

[0255] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 100, 108, 116, 124, 132, 140, 148, 156, 172, 180 and 188; the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 101, 109, 117, 125, 133, 141, 149, 157, 173, 181 and 189; the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 102, 110, 118, 126, 134, 142, 150, 158, 174, 182 and 190.

[0256] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise:

[0257] i. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;

[0258] ii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;

[0259] iii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19;

[0260] iv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27;

[0261] v. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:33, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:34, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:35;

[0262] vi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:43;

[0263] vii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:49, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:50, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:51;

[0264] viii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 57, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 58, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 59;

[0265] ix. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 65, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 66, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 67;

[0266] x. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 73, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 74, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 75;

[0267] xi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 97, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 98, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 99;

[0268] xii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 105, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 106, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 107;

[0269] xiii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 114, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 115;

[0270] xiv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 121, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 122, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 123;

[0271] xv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 129, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 130, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 131;

[0272] xvi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 138, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 139;

[0273] xvii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 145, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 146, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 147;

[0274] xviii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 153, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 154, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 155;

[0275] xix. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 169, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 170, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 171;

[0276] xx. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 177, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 178, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 179; or

[0277] xxi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:185, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:186, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:187.

[0278] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise:

[0279] i. LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6;

[0280] ii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 12, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14;

[0281] iii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22;

[0282] iv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:28, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:29, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:30;

[0283] v. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:36, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:37, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:38;

[0284] vi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:44, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:45, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:46;

[0285] vii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:53, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:54;

[0286] viii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:60, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:61, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:62;

[0287] ix. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:68, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:69, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:70;

[0288] x. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:76, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:77, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:78;

[0289] xi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 100, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 101, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 102;

[0290] xii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 108, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 109, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 110;

[0291] xiii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 116, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 117, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 118;

[0292] xiv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 124, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 125, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126;

[0293] xv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 132, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 133, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 134;

[0294] xvi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 140, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 141, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 142;

[0295] xvii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 148, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 149, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 150;

[0296] xviii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 156, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 157, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 158;

[0297] xix. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 172, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 173, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 174;

[0298] xx. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 180, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 181, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 182; or

[0299] xxi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:188, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:189, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:190.

[0300] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise:

[0301] i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6;

[0302] ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:12, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:13, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:14;

[0303] iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22;

[0304] iv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:28, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:29, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:30;

[0305] v. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:33, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:34, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:35, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:36, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:37, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:38;

[0306] vi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:43, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:44, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:45, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:46;

[0307] vii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:49, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:50, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:51, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:53, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:54;

[0308] viii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:57, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:58, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:59, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:60, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:61, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:62;

[0309] ix. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:65, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:66, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:67, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:68, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:69, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:70;

[0310] x. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:73, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:74, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:75, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:76, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:77, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:78;

[0311] xi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:97, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:98, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:99, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:100, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:101, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:102;

[0312] xii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 105, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 106, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 107, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 108, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 109, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 110;

[0313] xiii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 114, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 115, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 116, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 117, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 118;

[0314] xiv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 121, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 122, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 123, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 124, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 125, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126;

[0315] xv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 129, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 130, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 131, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 132, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 133, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 134;

[0316] xvi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 138, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 139, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 140, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 141, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 142;

[0317] xvii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 145, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 146, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 147, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 148, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 149, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 150;

[0318] xviii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 153, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 154, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 155, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 156, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 157, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 158;

[0319] xix. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 169, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 170, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 171, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 172, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 173, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 174;

[0320] xx. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 177, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 178, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 179, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 180, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 181, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 182; or

[0321] xxi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:185, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:186, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:187, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:188, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:189, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:190.

[0322] The SEQ ID NOs of the heavy chain (represented by "H") variable region, light chain (represented by "L") variable region, HCDR and LCDR of each of the above 21 monoclonal antibodies are shown in Table 1 below. The amino acid sequence of each CDR of 21 exemplary monoclonal antibodies is shown in Table 2 below. Unless otherwise stated, the CDR boundaries as described in Table 2 below are defined or determined by the Kabat rule. The amino acid sequences of each VH and VL of 21 exemplary monoclonal antibodies are shown in Table 3 below.

[0323] Table 1. SEQ ID NOs of VH, VL, HCDR and LCDR of 21 exemplary monoclonal antibodies

[0324]

[0325]

[0326]

[0327] Table 2. Amino acid sequences of each CDR of 21 exemplary monoclonal antibodies

[0328]

[0329]

[0330]

[0331]

[0332] Table 3. Amino acid sequences of each VH and VL of 21 exemplary monoclonal antibodies

[0333]

[0334]

[0335]

[0336]

[0337] Considering that each of the 21 exemplary monoclonal antibodies can bind to GPRC5D, and the antigen binding specificity is mainly provided by the CDR1 region, the CDR2 region and the CDR3 region, the HCDR1 sequence, HCDR2 sequence and HCDR3 sequence and the LCDR1 sequence, LCDR2 sequence and LCDR3 sequence of each of the 21 exemplary monoclonal antibodies can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched, but each antibody must contain HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3) to produce the anti-GPRC5D antibodies or antigen-binding fragments thereof of the present disclosure. The binding assays described above and in the Examples can be used to test the binding of such "mixed and matched" antibodies to GPRC5D. Preferably, when the VH CDR sequences are mixed and matched, the HCDR1 sequence, HCDR2 sequence and / or HCDR3 sequence from a specific VH sequence is replaced by a structurally similar CDR sequence. Likewise, when VL CDR sequences are mixed and matched, the LCDR1 sequence, LCDR2 sequence, and / or LCDR3 sequence from a particular VL sequence is preferably replaced with a structurally similar CDR sequence. For example, the HCDR1 of antibodies 26E2H6 and 71F8H2 share some structural similarities and are therefore easily mixed and matched. It will be apparent to one skilled in the art that novel VH and VL sequences can be generated by replacing one or more VH and / or VL CDR sequences with structurally similar sequences from the CDR sequences disclosed herein of the 21 exemplary monoclonal antibodies.

[0338] It is known that CDRs are responsible for antigen binding. However, it has been found that not all 6 CDRs are indispensable or unchangeable. In other words, one or more CDRs of each of the 21 exemplary monoclonal antibodies can be replaced or changed or modified, but the specific binding affinity for GPRC5D is substantially retained.

[0339] In certain embodiments, the anti-GPRC5D antibodies and antigen-binding fragments provided herein comprise the heavy chain CDR3 sequence of one of the anti-GPRC5D antibodies 36A3G8, 28H9C12, 26E2H6, 20C4E1, 15D6G2, 12G12C2, 71F8H2, 64G9C1, 98A3A8, 96C9E7, 90G6H3, 89F11E3, 87A3G8, 83A9B8, 82A1C10, 119E4B4, 116G1G4, 116F1G6, 110B4H5, 108C4C12, or 101B3G3. In certain embodiments, the anti-GPRC5D antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain CDR3 sequence selected from the group consisting of: SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 99, 107, 115, 123, 131, 139, 147, 155, 171, 179 and 187. The heavy chain CDR3 region is located at the center of the antigen binding site and is therefore believed to have the most contact with the antigen and provide the most free energy for the antibody's affinity for the antigen. It is also believed that, through various diversification mechanisms, the heavy chain CDR3 is by far the most diverse CDR in terms of length, amino acid composition and conformation in the antigen binding site (Tonegawa S. Nature 302: 575-81). The diversity of the heavy chain CDR3 is sufficient to generate most antibody specificities (Xu JL, Davis MM. Immunity. 13:37-45) and ideal antigen binding affinity (Schier R et al., Journal of Mol. Biol. 263:551-67).

[0340] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a VH region having an amino acid sequence as shown in SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183 or 191 or a sequence similar to SEQ ID NO: NO:7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183 or 191 has a homologous sequence with at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity.

[0341] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a VL region having an amino acid sequence as shown in SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184 or 192 or a sequence similar to SEQ ID NO: NO:8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184 or 192 has a homologous sequence with at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity.

[0342] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a VH / VL amino acid sequence pair selected from the group consisting of SEQ ID NO: 7 / 8, 15 / 16, 23 / 24, 31 / 32, 39 / 40, 47 / 48, 55 / 56, 63 / 64, 71 / 72, 79 / 80, 103 / 104, 111 / 112, 119 / 120, 127 / 128, 135 / 136, 143 / 144, 151 / 152, 159 / 160, 175 / 176, 183 / 184, or 191 / 192.

[0343] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, as long as the antibodies and antigen-binding fragments thereof can specifically bind to GPRC5D. The CDR sequences provided in Table 2 above are obtained from mouse antibodies, but the CDR sequences can be transplanted into any suitable FR sequence of any suitable species such as mouse, human, rat, rabbit, etc. using suitable methods known in the art such as recombinant technology.

[0344] In certain embodiments, the antibodies and Fabs thereof provided herein are humanized. The desired humanized antibodies or Fabs thereof have reduced immunogenicity in humans. Humanized antibodies are chimeric in their variable regions because non-human CDR sequences are transplanted to human or substantially human FR sequences. The humanization of antibodies or Fabs can be carried out by replacing the corresponding human CDR genes in human immunoglobulin genes with non-human (such as mouse) CDR genes (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0345] Suitable human heavy chain and light chain variable domains can be selected using methods known in the art to achieve this purpose. In an illustrative embodiment, a "best fit" method can be used, wherein the database screening or BLAST non-human (e.g., rodent) antibody variable domain sequences for known human variable domain sequences, the human sequence closest to the non-human query sequence is identified, and used as a human framework for transplanting non-human CDR sequences (see, e.g., Sims et al., (1993) "Journal of Immunology (J.Immunol.)" 151:2296; Chothia et al. (1987) "Journal of Molecular Biology" 196:901). Alternatively, the framework derived from the consensus sequence of all human antibodies can be used for transplanting non-human CDR (see, e.g., Carter et al. (1992) "Proceedings of the National Academy of Sciences of the United States of America", 89:4285; Presta et al. (1993) "Journal of Immunology", 151:2623).

[0346] In some embodiments, the anti-GPRC5D antibody or its Fab provided herein is humanized. In certain embodiments, except the inhuman CDR sequence, the humanized antibody or its Fab provided herein is composed of substantially all human sequences. In some embodiments, the variable region FR and constant region (if present) are completely or substantially from human immunoglobulin sequences. People's FR sequence and people's constant region sequence can be derived from different human immunoglobulin genes, for example, FR sequence is derived from a kind of human antibody and constant region is derived from another kind of human antibody. In some embodiments, humanized antibody or its Fab comprises people's heavy chain HFR1, HFR2, HFR3 and HFR4 and / or light chain LFR1, LFR2, LFR3 and LFR4.

[0347] In some embodiments, the FR region derived from people can include the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of people FR are replaced by corresponding residues from parent non-human antibodies. In some embodiments, the replacement may be desired, so that the structure of humanized antibodies or their fragments is very close to the non-human parent antibodies, thereby optimizing their binding properties (for example, increasing binding affinity). In some embodiments, the humanized antibodies or their antigen-binding fragments provided herein include no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue replacements in each FR sequence in the people FR sequence, or include no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue replacements in all FR sequences of heavy chain variable domains or light chain variable domains. In some embodiments, such changes of amino acid residues can be present only in the heavy chain FR region, only in the light chain FR region or in two chains. In some embodiments, one or more amino acids of the people FR sequence are randomly mutated to increase binding affinity. In certain embodiments, one or more amino acids of the human FR sequence are back mutated to the corresponding amino acids of the parent non-human antibody to increase binding affinity.

[0348] In some embodiments, the anti-GPRC5D antibodies and antigen-binding fragments provided herein comprise all or a portion of a heavy chain variable domain and / or all or a portion of a light chain variable domain. In one embodiment, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein are single domain antibodies consisting of all or a portion of a heavy chain variable domain provided herein. More information on such single domain antibodies can be obtained in the art (see, e.g., U.S. Patent No. 6,248,516).

[0349] In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein further comprises an Fc region. In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein further comprises an Fc region of human immunoglobulin (Ig). In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein further comprises a constant region, and the constant region optionally further comprises a heavy chain and / or light chain constant region. In certain embodiments, the heavy chain constant region comprises CH1, hinge and / or CH2-CH3 region (or optionally CH2-CH3-CH4 region). In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgM. In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein comprises a λ (lambda) light chain or a κ (kappa) light chain. The constant region of the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein may be identical to the wild-type constant region sequence or may differ in the presence of one or more mutations.

[0350] In certain embodiments, the heavy chain constant region comprises an Fc region. It is known that the Fc region mediates effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of antibodies. The Fc regions of different Ig isotypes have different abilities to induce effector functions. For example, the Fc regions of IgG1 and IgG3 have been recognized to be more effective than the Fc regions of IgG2 and IgG4 in inducing both ADCC and CDC. In certain embodiments, the anti-GPRC5D antibodies and antigen-binding fragments thereof provided herein comprise an Fc region of an IgG1 or IgG3 isotype, which can induce ADCC or CDC; or alternatively, a constant region of an IgG4 or IgG2 isotype, which has a reduced or missing effector function. In some embodiments, the Fc region is derived from human IgG1 with enhanced effector functions. In some embodiments, the Fc region comprises an amino acid sequence as shown in SEQ ID NO: 161.

[0351] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:161)

[0352] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have specific binding affinity for human GPRC5D sufficient to provide diagnostic and / or therapeutic uses.

[0353] The antibody or antigen-binding fragment thereof provided herein can be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a multispecific antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody or a fusion protein. Recombinant antibodies are antibodies prepared in vitro rather than in an animal using a recombinant method.

[0354] In certain embodiments, the disclosure provides an anti-GPRC5D antibody or an antigen-binding fragment thereof, which competes with an antibody or an antigen-binding fragment thereof provided herein for binding to GPRC5D. In certain embodiments, the disclosure provides an anti-GPRC5D antibody or an antigen-binding fragment thereof, which competes with any one of antibodies 36A3G8, 28H9C12, 26E2H6, 20C4E1, 15D6G2, 12G12C2, 71F8H2, 64G9C1, 98A3A8, 96C9E7, 90G6H3, 89F11E3, 87A3G8, 83A9B8, 82A1C10, 119E4B4, 116G1G4, 116F1G6, 110B4H5, 108C4C12 or 101B3G3 for binding to human GPRC5D. In some embodiments, the present disclosure provides an anti-GPRC5D antibody or antigen-binding fragment thereof that competes for the same epitope as an antibody or antigen-binding fragment provided herein.

[0355] As used herein, the ability to "block binding" or "compete for the same epitope" refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human GPRC5D and anti-GPRC5D antibodies) to any detectable extent. In certain embodiments, the antibody or antigen-binding fragment that blocks the binding between the two molecules inhibits the binding interaction between the two molecules by at least 85% or at least 90%. In certain embodiments, the inhibition may be greater than 85% or greater than 90%.

[0356] One skilled in the art will recognize that whether a human monoclonal antibody binds to the same epitope as an antibody of the disclosure (e.g., mouse monoclonal antibody 36A3G8, 28H9C12, 26E2H6, 20C4E1, 15D6G2, 12G12C2, 71F8H2, 64G9C1, 98A3A8, 96C9E7, 90G6H3, 89F11E3, 87A3G8, 83A9B8, 82A1C10, 119E4B4, 116G1G4, 116F1G6, 110B4H5, 108C4C12, or 101B3G3) can be determined without undue experimentation by determining whether the human monoclonal antibody prevents the antibody of the disclosure from binding to the GPRC5D antigen polypeptide. If the test antibody competes with the antibody of the present invention, as shown by reduced binding of the antibody of the present invention to the GPRC5D antigen polypeptide, the two antibodies bind to the same or closely related epitopes. Alternatively, if the binding of the test antibody to the GPRC5D antigen polypeptide is inhibited by the antibody of the present invention, the two antibodies bind to the same or closely related epitopes.

[0357] In certain embodiments, the disclosure provides anti-GPRC5D antibodies or antigen-binding fragments thereof that compete with ATG596 for binding to GPRC5D. In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment that competes with the antibodies or antigen-binding fragments thereof provided herein for binding to GPRC5D is not ATG596.

[0358] As used herein, "ATG596" refers to an antibody or antigen-binding fragment thereof comprising one or two or three heavy chain CDRs contained in a heavy chain variable region having an amino acid sequence of SEQ ID NO: 199 and one or two or three light chain CDRs contained in a light chain variable region having an amino acid sequence of SEQ ID NO: 200. In certain embodiments, ATG596 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises an amino acid sequence of SEQ ID NO: 193, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 194, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 195, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 196, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 197, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 198. The amino acid sequences of each CDR, VH region, and VL region of ATG596 are shown in Table 4 below. The CDR boundaries of ATG596 in Table 4 below are identified by Kabat convention.

[0359] Table 4. Amino acid sequences of HCDR, LCDR, VH region and VL region of ATG596

[0360]

[0361] Antibody variants

[0362] The antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.

[0363] In certain embodiments, the antibody variant comprises one or more amino acid residue substitutions or modifications, but still retains binding affinity to GPRC5D. In certain embodiments, at least one of the substitutions or modifications is located in one or more CDR sequences in the VH region or the VL region. In certain embodiments, at least one of the substitutions or modifications is located in one or more non-CDR sequences in the VH region or the VL region. In certain embodiments, the antibody or its antigen-binding fragment of the present disclosure further comprises one or more non-natural amino acids (NNAA) substitutions. In certain embodiments, the NNAA can be conjugated.

[0364] For example, the antibody variant comprises one or more of the CDR sequences provided in Table 2 above, one or more of the non-CDR sequences of the heavy chain variable region or light chain variable region provided in Table 3 above, and / or one or more amino acid residues in the constant region (e.g., Fc region) are substituted or modified. Such variants retain the binding specificity of their parent antibody to GPRC5D, but have one or more desired properties conferred by the modification or substitution. For example, the antibody variant may have improved antigen binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, enhanced effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity and / or compatibility with conjugation (e.g., one or more introduced cysteine ​​residues), etc.

[0365] Parent antibody sequences can be screened to identify suitable or preferred residues to be modified or replaced using methods known in the art, such as "alanine scanning mutagenesis" (see, for example, Cunningham and Wells (1989) Science, 244: 1081-1085). Briefly, target residues (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) can be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies are produced and screened for properties of interest. If the substitution at a particular amino acid position shows a functional change of interest, the position can be identified as a potential residue for modification or replacement. The potential residue can be further evaluated by replacing it with another residue (e.g., a cysteine ​​residue, a positively charged residue, etc.).

[0366] Affinity variants

[0367] The affinity variants of the antibody may comprise modifications or substitutions in one or more CDR sequences provided in Table 2 above, one or more FR sequences, or heavy chain variable regions or light chain variable region sequences provided in Table 3 above. Those skilled in the art can easily identify FR sequences based on the CDR sequences in Table 2 above and the variable region sequences in Table 3 above, because it is well known in the art that in the variable region, the CDR region is flanked by two FR regions. The affinity variants retain the specific binding affinity of the parent antibody to GPRC5D, or even have a higher GPRC5D binding affinity than the parent antibody. In certain embodiments, at least one (or all) substitutions in the CDR sequence, FR sequence, or variable region sequence comprise conservative substitutions.

[0368] Those skilled in the art will appreciate that in the CDR sequences provided in Table 2 above and the variable region sequences provided in Table 3 above, one or more amino acid residues may be substituted, but the resulting antibody or antigen-binding fragment still retains binding affinity or binding capacity to GPRC5D, or even has improved binding affinity or binding capacity. Various methods known in the art may be used to achieve this purpose. For example, a library of antibody variants (such as Fab or scFv variants) may be generated and expressed using phage display technology, and then screened for affinity for binding to human GPRC5D. For another example, computer software may be used to virtually simulate the binding of antibodies to human GPRC5D, and to identify the amino acid residues on the antibody that form the binding interface. Such residues may be avoided in the substitution to prevent a reduction in binding affinity, or may be used as a target for substitution to obtain a stronger binding.

[0369] In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein comprises one or more amino acid residues in one or more CDR sequences and / or one or more FR sequences in the CDR sequences and / or FR sequences. In certain embodiments, the affinity variant comprises no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 substitutions in total in the CDR sequences and / or FR sequences.

[0370] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise 1, 2 or 3 CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the sequences listed in Table 2 above, but still retain specific binding affinity for GPRC5D at a similar or even higher level relative to its parent antibody.

[0371] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the sequences listed in Table 3 above, but still retaining specific binding affinity to GPRC5D at a similar or even higher level relative to its parent antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted or deleted in the variable region sequences listed in Table 3 above. In some embodiments, the substitution, insertion or deletion occurs in a region outside of a CDR (e.g., in a FR).

[0372] Glycosylation variants

[0373] The anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein also encompass glycosylation variants, which can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment thereof.

[0374] The antibodies or antigen-binding fragments thereof provided herein may comprise one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) may be attached. Glycosylation of an antibody is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue (e.g., an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine), wherein X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly to serine or threonine. Natural glycosylation sites can be conveniently removed, for example, by altering the amino acid sequence so that one of the above tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence is replaced. New glycosylation sites can be generated in a similar manner by the introduction of such tripeptide sequences or serine or threonine residues.

[0375] Cysteine ​​engineered variants

[0376] The anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein also encompass cysteine ​​engineered variants comprising one or more introduced free cysteine ​​amino acid residues.

[0377] A free cysteine ​​residue is a cysteine ​​residue that is not part of a disulfide bond. Cysteine ​​engineered variants can be used to conjugate, for example, with a cytotoxic compound and / or imaging compound, a label or a radioisotope, etc., at the site of the engineered cysteine, for example, via a maleimide or haloacetyl group. Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine ​​residues are known in the art, see, for example, WO2006 / 034488.

[0378] Fc variants

[0379] The anti-GPRC5D antibodies and antigen-binding fragments provided herein also encompass Fc variants comprising one or more amino acid residue modifications or substitutions at the Fc region and / or hinge region thereof.

[0380] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise one or more amino acid substitutions that improve pH-dependent binding to neonatal Fc receptors (FcRn). Such variants may have a prolonged pharmacokinetic half-life because they bind to FcRn at acidic pH, thereby preventing them from being degraded in lysosomes and subsequently being transferred and released outside the cell. Methods for engineering antibodies or antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art, see, e.g., Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Vol. 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., Journal of Immunology, 176:346-356 (2006).

[0381] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise one or more amino acid substitutions that alter ADCC. Certain amino acid residues at the CH2 domain of the Fc region may be substituted to provide enhanced ADCC activity. Alternatively or additionally, the carbohydrate structure on the antibody may be changed to enhance ADCC activity. Methods for altering ADCC activity by antibody engineering have been described in the art, see, for example, Shields RL. et al., J Biol Chem. 2001 276(9):6591-604; Idusogie EE. et al., J Immunol. 2000. 164(8):4178-84; Steurer W. et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE. et al., J Immunol. 2001, 166(4):2571-5; Lazar GA. et al., Proc. Natl. Acad. Sci. USA, 2006, 103(11):4005-4010; Ryan MC. et al., Mol. Cancer Ther., 2007, 6:3009-3018; Richards J. et al., J Immunol. 2008, 6:3111-3113; JO et al., Mol Cancer Therapy, 2008, 7(8):2517-27; Shields RL et al., J Biol Chem, 2002, 277:26733-26740; Shinkawa T. et al., J Biol Chem, 2003, 278:3466-3473.

[0382] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise one or more amino acid substitutions that alter CDC, for example by improving or reducing C1q binding and / or CDC (see, e.g., WO 99 / 51642; Duncan and Winter, Nature, 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821); and WO 94 / 29351 for other examples of Fc region variants. One or more amino acids selected from amino acid residues 329, 331, and 322 of the Fc region may be replaced with different amino acid residues to alter C1q binding and / or enhance CDC (see U.S. Pat. No. 6,194,551 to Idusogie et al.). One or more amino acid substitutions may also be introduced to alter the ability of the antibody to fix complement (see PCT Publication WO 94 / 29351 to Bodmer et al.).

[0383] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise one or more amino acid substitutions at positions 234 and / or 235 (according to EP numbering) in a human immunoglobulin (e.g., IgG1). In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise two amino acid substitutions at positions 234 and 235 (according to EP numbering) in a human immunoglobulin (e.g., IgG1). In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise L234A and L235A (according to EU numbering) amino acid substitutions.

[0384] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise one or more amino acid substitutions in the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include introducing a protrusion into a first Fc polypeptide and introducing a cavity into a second Fc polypeptide, wherein the protrusion can be positioned in the cavity to promote the interaction of the first Fc polypeptide with the second Fc polypeptide to form a heterodimer or complex. Methods for producing antibodies with these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0385] In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein comprises an amino acid substitution at position 366 (according to EP numbering) of a first Fc polypeptide, and comprises one, two or three amino acid substitutions at one, two or three positions among positions 366, 368 and 407 (according to EP numbering) of a second Fc polypeptide. In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein comprises a T366W (according to EP numbering) substitution of a first Fc polypeptide, and comprises a T366S+L368A+Y407V (according to EP numbering) substitution of a second Fc polypeptide.

[0386] In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein further comprises one or more amino acid substitutions of the first Fc polypeptide, and comprises one or more amino acid substitutions of the second Fc polypeptide, thereby introducing a non-natural disulfide bond between the two Fc polypeptides. For example, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein comprises an amino acid substitution at position 354 (according to EU numbering) of the first Fc polypeptide, and comprises an amino acid substitution at position 349 (according to EU numbering) of the second Fc polypeptide. In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein comprises an S354C (according to EP numbering) substitution of the first Fc polypeptide, and comprises a Y349C (according to EP numbering) substitution of the second Fc polypeptide.

[0387] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) amino acid substitutions selected from the group consisting of L234A, L235A, S354C, T366W, Y349C, T366S, L368A, and Y407V (according to EU numbering). In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise S354C and T366W (according to EU numbering) amino acid substitutions of a first Fc polypeptide, and comprise Y349C, T366S, L368A, and Y407V (according to EU numbering) amino acid substitutions of a second Fc polypeptide.

[0388] In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein comprises a first Fc polypeptide comprising the amino acid sequence shown in SEQ ID NO: 162 and a second Fc polypeptide comprising the amino acid sequence shown in SEQ ID NO: 163.

[0389] KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0390] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0391] AKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:162)

[0392] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

[0393] DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0394] KAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:163)

[0395] Fab

[0396] Anti-GPRC5D antigen-binding fragments are also provided herein. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-GPRC5D antibodies provided herein, including, for example, exemplary antibodies and their different variants (such as affinity variants, glycosylation variants, Fc variants, cysteine ​​engineered variants, etc.), the CDRs of the exemplary antibodies are shown in Table 2 above and their variable sequences are shown in Table 3 above.

[0397] In certain embodiments, the anti-GPRC5D antigen-binding fragments provided herein are bifunctional antibodies, Fab, Fab', F(ab') 2 、Fd、Fv fragment、Fv fragment stabilized by disulfide bonds (dsFv)、(dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide-bonded stabilized bifunctional antibodies, single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), camelized single domain antibodies, nanobodies, domain antibodies or bivalent domain antibodies.

[0398] Such antigen-binding fragments can be produced using a variety of techniques. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science 229: 81 (1985)), recombinant expression by host cells such as Escherichia coli (e.g., for Fab, Fv and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., for ScFv) and chemical coupling of two Fab'-SH fragments to form F(ab')2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). Other techniques for producing antibody fragments are apparent to those skilled in the art.

[0399] In certain embodiments, the antigen binding fragment is a scFv. The production of scFv is described in, for example, WO 93 / 16185; U.S. Pat. No. 5,571,894; and No. 5,587,458. ScFv can be fused to an effector protein at the amino or carboxyl terminus to provide a fusion protein (see, for example, Antibody Engineering, Borrebaeck, ed.).

[0400] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent or multivalent. Any molecule with more than two valencies is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0401] If both binding sites specifically bind to the same antigen or the same epitope, the bivalent molecule can be monospecific. In some embodiments, this provides stronger binding to the antigen or epitope than the monovalent counterpart. Similarly, multivalent molecules can also be monospecific. In some embodiments, in a bivalent or multivalent antigen binding portion, the first valence of the binding site and the second valence of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences but have the same specificity).

[0402] A bivalent can also be bispecific if the two binding sites are specific for different antigens or epitopes. This also applies to multivalent molecules. For example, a trivalent molecule can be bispecific when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0403] Bispecific or multispecific antibodies

[0404] In certain embodiments, the anti-GPRC5D antibody or its antigen-binding fragment provided herein is bispecific or multispecific. In certain embodiments, the anti-GPRC5D antibody or its antigen-binding fragment provided herein is further connected to a second binding moiety having a binding specificity different from that of the anti-GPRC5D antibody or its antigen-binding fragment. In some embodiments, the bispecific antibody or multispecific antibody or its antigen-binding fragment provided herein has a first specificity to GPRC5D and has a second specificity. In some embodiments, the second specificity is for GPRC5D but for a different epitope. In some embodiments, the second specificity is for a second antigen different from GPRC5D.

[0405] In certain embodiments, the second specificity is for a tumor-associated antigen or an epitope thereof. The term "tumor-associated antigen" refers to an antigen that appears on or can be presented on the surface of a tumor cell, or can be located on or in a tumor cell. In some embodiments, tumor-associated antigens can only be presented by tumor cells, but not by normal cells (i.e., non-tumor cells). In some other embodiments, tumor-associated antigens can be expressed exclusively on tumor cells or can represent tumor-specific mutations compared to non-tumor cells. In some other embodiments, tumor-associated antigens can be found in both tumor cells and non-tumor cells, but they are overexpressed on tumor cells compared to non-tumor cells, or they can be used for antibody binding in tumor cells due to the less compact structure of tumor tissue compared to non-tumor tissue. In some embodiments, tumor-associated antigens are located on the vasculature of the tumor.

[0406] In certain embodiments, the bispecific antibodies or multispecific antibodies or antigen-binding fragments thereof provided herein are capable of binding to one or more (e.g., 1, 2, 3, 4, 5 or more) additional antigens in addition to GPRC5D. In certain embodiments, the one or more additional antigens other than GPRC5D are selected from the group consisting of KRAS, ERK, XPO1, mTORC1 / 2, PAK4, NAMPT, ATR, EGFR, FGFR, VEGF, LILRB, c-MET, Her2, Her3, CTLA4, GITA, CD112R, CD2, CD3, CD7, CD16, CD19, CD20, CD24, CD27, CD30, CD34, CD37, CD38, CD39, CD70, CD73, CD83, CD28, CD80 (B7-1), CD86 (B7-2), CD40, CD40L (CD154), CD47, SIRPα, CD122, CD137, CD137L, OX40 (CD134), OX40L (CD252), BCMA (e.g., BCMA02), FcRH 5. PSMA, CLDN18 (for example, CLDN18.2), NKG2C, 4-1BB, LIGHT, PVRIG, SLAMF7, HVEM, BAFFR, ICAM-1, 2B4, LF A-1, GITR, ICOS(CD278), ICOSLG(CD275), LAG3(CD223), A2AR, B7-H3(CD276), B7-H4(VTCN1), B7-H5 , BTLA(CD272), BTLA, CD160, CTLA-4(CD152), IDO1, IDO2, ILT3, TDO, KIR, LAIR-1, NOX2, PD-1, PD-L1 , PD-L2, TIM-3, VISTA, SIGLEC-7(CD328), SIGLEC-9(CD329), SIGLEC-15, TIGIT, PVR(CD155) and TGFβ.

[0407] In certain embodiments, the bispecific antibodies or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and CD3. In certain embodiments, the bispecific antibodies or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and BCMA. In certain embodiments, the bispecific antibodies or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and CD38. In certain embodiments, the bispecific antibodies or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and CD19. In certain embodiments, the bispecific antibodies or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and FcRH5. In certain embodiments, the bispecific antibodies or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and PD-L1. In certain embodiments, the bispecific antibodies or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and PD-1.

[0408] In certain embodiments, the bispecific antibodies or multispecific antibodies or antigen-binding fragments thereof provided herein comprise a GPRC5D binding portion and a CD3 binding portion.

[0409] As used herein, the term "GPRC5D binding portion" with respect to a bispecific antibody or a multispecific antibody or an antigen-binding fragment thereof refers to a portion that is capable of binding to GPRC5D (e.g., human GPRC5D, mouse GPRC5D, and cynomolgus monkey GPRC5D). The GPRC5D binding portion may take any form that allows specific recognition of the target GPRC5D. For example, the GPRC5D binding portion may be an antibody or an antigen-binding fragment thereof, such as an IgG (e.g., IgG1, IgG2, IgG3, and IgG4) antibody, an IgA antibody, or an IgM antibody.

[0410] The GPRC5D binding portion of the bispecific antibody or multispecific antibody provided herein can be derived from any of the anti-GPRC5D antibodies described above. In certain embodiments, the GPRC5D binding portion of the bispecific antibody or multispecific antibody provided herein includes HCDR1, HCDR2 and / or HCDR3 contained in any of the heavy chain variable region sequences shown in Table 3 above and LCDR1, LCDR2 and / or LCDR3 contained in any of the light chain variable region sequences shown in Table 3 above. In certain embodiments, the GPRC5D binding portion of the bispecific antibody or multispecific antibody provided herein includes HCDR1, HCDR2 and / or HCDR3 comprising the amino acid sequence shown in Table 2 above. In certain embodiments, the GPRC5D binding portion of the bispecific antibody or multispecific antibody provided herein includes LCDR1, LCDR2 and / or LCDR3 comprising the amino acid sequence shown in Table 2 above. In certain embodiments, the GPRC5D binding portion of the bispecific or multispecific antibodies provided herein comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences shown in Table 2 above. In certain embodiments, the GPRC5D binding portion of the bispecific or multispecific antibodies provided herein includes a HCDR1, HCDR2, and / or HCDR3 derived from the illustrative anti-GPRC5D antibodies 36A3G8, 28H9C12, 26E2H6, 20C4E1, 15D6G2, 12G12C2, 71F8H2, 64G9C1, 98A3A8, 96C9E7, 90G6H3, 89F11E3, 87A3G8, 83A9B8, 82A1C10, 119E4B4, 116G1G4, 116F1G6, 110B4H5, 108C4C12, or 101B3G3 as described above. In certain embodiments, the GPRC5D binding portion of the bispecific or multispecific antibodies provided herein includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 derived from the illustrative anti-GPRC5D antibodies 36A3G8, 28H9C12, 26E2H6, 20C4E1, 15D6G2, 12G12C2, 71F8H2, 64G9C1, 98A3A8, 96C9E7, 90G6H3, 89F11E3, 87A3G8, 83A9B8, 82A1C10, 119E4B4, 116G1G4, 116F1G6, 110B4H5, 108C4C12, or 101B3G3 as described above.

[0411] In certain embodiments, the GPRC5D binding portion of the bispecific antibody or multispecific antibody provided herein comprises a heavy chain variable region having an amino acid sequence as shown in Table 3 above, or a homologous sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%) sequence identity to the amino acid sequence as shown in Table 3 above. In certain embodiments, the GPRC5D binding portion of the bispecific antibody or multispecific antibody provided herein comprises a light chain variable region having an amino acid sequence as shown in Table 3 above, or a homologous sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%) sequence identity to the amino acid sequence as shown in Table 3 above. In certain embodiments, the GPRC5D binding portion of the bispecific or multispecific antibodies provided herein comprises a VH / VL amino acid sequence pair selected from the group consisting of SEQ ID NO: 7 / 8, 15 / 16, 23 / 24, 31 / 32, 39 / 40, 47 / 48, 55 / 56, 63 / 64, 71 / 72, 79 / 80, 103 / 104, 111 / 112, 119 / 120, 127 / 128, 135 / 136, 143 / 144, 151 / 152, 159 / 160, 175 / 176, 183 / 184, or 191 / 192.

[0412] As used herein, the term "CD3 binding portion" when referring to a bispecific antibody or a multispecific antibody or its antigen-binding fragment refers to a portion that can bind to CD3 (e.g., human CD3, mouse CD3, and cynomolgus CD3). The CD3 binding portion can take any form that allows specific recognition of the target CD3. For example, the CD3 binding portion can be an antibody or its antigen-binding fragment, such as an IgG (such as IgG1, IgG2, IgG3, and IgG4) antibody, an IgA antibody, or an IgM antibody. In some embodiments, the CD3 binding portion of the bispecific antibody or multispecific antibody provided herein is derived from an anti-CD3 antibody that binds and activates primary T cells. The CD3 binding portion of the bispecific antibody or multispecific antibody provided herein can be derived from any one of anti-CD3 antibodies known in the art, such as the anti-CD3 antibodies described in US20200048348A1, WO 2013186613A1, WO 2015001085A1, etc.

[0413] In some embodiments, the CD3 binding portion of the bispecific or multispecific antibodies provided herein comprises one, two or three heavy chain complementary determining regions (HCDR1, HCDR2 and / or HCDR3) contained within the heavy chain variable (VH) region sequence of SEQ ID NO: 87 or 95; and / or comprises one, two or three light chain complementary determining regions (LCDR1, LCDR2 and / or LCDR3) contained within the light chain variable (VL) region sequence of SEQ ID NO: 88 or 96. In some embodiments, the CD3 binding portion of the bispecific or multispecific antibodies provided herein comprises HCDR1, HCDR2 and / or HCDR3 contained within the VH region sequence of SEQ ID NO: 87, and LCDR1, LCDR2 and / or LCDR3 contained within the VL region sequence of SEQ ID NO: 88. In some embodiments, the CD3 binding portion of the bispecific antibody or multispecific antibody provided herein comprises HCDR1, HCDR2 and / or HCDR3 contained in the VH region sequence of SEQ ID NO: 95, and LCDR1, LCDR2 and / or LCDR3 contained in the VL region sequence of SEQ ID NO: 96. In some embodiments, the CDR boundaries of the CD3 binding portion are determined according to the Kabat rule. In some embodiments, the CDR boundaries of the CD3 binding portion are determined according to the IMGT rule. In some embodiments, the CDR boundaries of the CD3 binding portion are determined according to the Chothia rule. In some embodiments, the CDR boundaries of the CD3 binding portion are determined according to the Al-Lazikani rule.

[0414] In some embodiments, the CD3 binding portion of the bispecific antibody or multispecific antibody provided herein comprises a HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 81 or 89, a HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 82 or 90, and a HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 83 or 91; and / or a LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 84 or 92, a LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 85 or 93, and a LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 86 or 94.

[0415] In some embodiments, the CD3 binding portion of the bispecific antibody or multispecific antibody provided herein comprises:

[0416] i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:81, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:82, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:83, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:84, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:85, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86; or

[0417] ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:89, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:90, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:91, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:92, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:93, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:94.

[0418] In some embodiments, the CD3 binding portion of the bispecific antibody or multispecific antibody provided herein comprises the VH / VL amino acid sequence pair of SEQ ID NO: 87 / 88 or 95 / 96.

[0419] The amino acid sequences of the CDRs, VH regions and VL regions of illustrative CD3 binding portions are shown below in Table 5. The boundaries of the CDRs as described in Table 5 below are all defined or determined by the Kabat rules.

[0420] Table 5. Amino acid sequences of HCDR, LCDR, VH region and VL region of two exemplary CD3 binding portions

[0421]

[0422] Exemplary bispecific antibodies or antigen-binding fragments thereof provided herein comprise:

[0423] (1) a GPRC5D binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence as shown in SEQ ID NO: 127, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence as shown in SEQ ID NO: 128; and a CD3 binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence as shown in SEQ ID NO: 87, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence as shown in SEQ ID NO: 88;

[0424] (2) a GPRC5D binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence set forth in SEQ ID NO: 183, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence set forth in SEQ ID NO: 184; and a CD3 binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence set forth in SEQ ID NO: 87, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence set forth in SEQ ID NO: 88;

[0425] (3) a GPRC5D binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence set forth in SEQ ID NO: 151, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence set forth in SEQ ID NO: 152; and a CD3 binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence set forth in SEQ ID NO: 87, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence set forth in SEQ ID NO: 88;

[0426] (4) a GPRC5D binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence set forth in SEQ ID NO: 159, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence set forth in SEQ ID NO: 160; and a CD3 binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence set forth in SEQ ID NO: 87, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence set forth in SEQ ID NO: 88;

[0427] (5) a GPRC5D binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence as shown in SEQ ID NO:39, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence as shown in SEQ ID NO:40; and a CD3 binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence as shown in SEQ ID NO:87, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence as shown in SEQ ID NO:88;

[0428] (6) a GPRC5D binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence set forth in SEQ ID NO:71, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence set forth in SEQ ID NO:72; and a CD3 binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence set forth in SEQ ID NO:87, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence set forth in SEQ ID NO:88;

[0429] (7) a GPRC5D binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence as shown in SEQ ID NO:79, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence as shown in SEQ ID NO:80; and a CD3 binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence as shown in SEQ ID NO:87, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence as shown in SEQ ID NO:88;

[0430] (8) a GPRC5D binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence as shown in SEQ ID NO: 175, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence as shown in SEQ ID NO: 176; and a CD3 binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence as shown in SEQ ID NO: 87, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence as shown in SEQ ID NO: 88; or

[0431] (9) a GPRC5D binding portion, comprising HCDR1, HCDR2 and HCDR3 contained in the VH region sequence as shown in SEQ ID NO:23, and LCDR1, LCDR2 and LCDR3 contained in the VL region sequence as shown in SEQ ID NO:24; and a CD3 binding portion, comprising HCDR1, HCDR2 and HCDR3 contained in the VH region sequence as shown in SEQ ID NO:87, and LCDR1, LCDR2 and LCDR3 contained in the VL region sequence as shown in SEQ ID NO:88.

[0432] Another exemplary bispecific antibody or antigen-binding fragment thereof provided herein comprises:

[0433] (1) a GPRC5D binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 121, 122, 123, 124, 125 and 126, respectively; and a CD3 binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 81, 82, 83, 84, 85 and 86, respectively;

[0434] (2) a GPRC5D binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 177, 178, 179, 180, 181 and 182, respectively; and a CD3 binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 81, 82, 83, 84, 85 and 86, respectively;

[0435] (3) a GPRC5D binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 145, 146, 147, 148, 149 and 150, respectively; and a CD3 binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 81, 82, 83, 84, 85 and 86, respectively;

[0436] (4) a GPRC5D binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 153, 154, 155, 156, 157 and 158, respectively; and a CD3 binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 81, 82, 83, 84, 85 and 86, respectively;

[0437] (5) a GPRC5D binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 33, 34, 35, 36, 37 and 38, respectively; and a CD3 binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 81, 82, 83, 84, 85 and 86, respectively;

[0438] (6) a GPRC5D binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 65, 66, 67, 68, 69 and 70, respectively; and a CD3 binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 81, 82, 83, 84, 85 and 86, respectively;

[0439] (7) a GPRC5D binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 73, 74, 75, 76, 77 and 78, respectively; and a CD3 binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 81, 82, 83, 84, 85 and 86, respectively;

[0440] (8) a GPRC5D binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 169, 170, 171, 172, 173 and 174, respectively; and a CD3 binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 81, 82, 83, 84, 85 and 86, respectively; or

[0441] (9) a GPRC5D binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 17, 18, 19, 20, 21 and 22, respectively; and a CD3 binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 81, 82, 83, 84, 85 and 86, respectively.

[0442] Another exemplary bispecific antibody or antigen-binding fragment thereof provided herein comprises:

[0443] (1) a GPRC5D binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence as shown in SEQ ID NO: 183, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence as shown in SEQ ID NO: 184; and a CD3 binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence as shown in SEQ ID NO: 95, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence as shown in SEQ ID NO: 96;

[0444] (2) a GPRC5D binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence as shown in SEQ ID NO:71, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence as shown in SEQ ID NO:72; and a CD3 binding portion comprising HCDR1, HCDR2 and HCDR3 contained within the VH region sequence as shown in SEQ ID NO:95, and LCDR1, LCDR2 and LCDR3 contained within the VL region sequence as shown in SEQ ID NO:96; or

[0445] (3) a GPRC5D binding portion, comprising HCDR1, HCDR2 and HCDR3 contained in the VH region sequence as shown in SEQ ID NO:79, and LCDR1, LCDR2 and LCDR3 contained in the VL region sequence as shown in SEQ ID NO:80; and a CD3 binding portion, comprising HCDR1, HCDR2 and HCDR3 contained in the VH region sequence as shown in SEQ ID NO:95, and LCDR1, LCDR2 and LCDR3 contained in the VL region sequence as shown in SEQ ID NO:96.

[0446] Another exemplary bispecific antibody or antigen-binding fragment thereof provided herein comprises:

[0447] (1) a GPRC5D binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 177, 178, 179, 180, 181 and 182, respectively; and a CD3 binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 89, 90, 91, 92, 93 and 94, respectively;

[0448] (2) a GPRC5D binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 65, 66, 67, 68, 69 and 70, respectively; and a CD3 binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 89, 90, 91, 92, 93 and 94, respectively; or

[0449] (3) a GPRC5D binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 73, 74, 75, 76, 77 and 78, respectively; and a CD3 binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 89, 90, 91, 92, 93 and 94, respectively.

[0450] Different formats of bispecific antibodies or multispecific antibodies have been described in the art (e.g., in Chames and Baty (2009) Curr Opin Drug Disc Dev 12:276). The bispecific antibodies or multispecific antibodies or antigen-binding fragments thereof provided herein are not limited to any particular bispecific format, and all may have different formats known in the art.

[0451] For example, the bispecific antibody or multispecific antibody or its antigen-binding fragment provided herein can have a typical full-length antibody structure, i.e., an antibody with two different full-length antibody heavy chains and two different full-length antibody light chains. The full-length antibody heavy chain comprises a heavy chain variable region (VH) and constant domains CH1, CH2, CH3, and optionally CH4. The full-length antibody light chain comprises a light chain variable region (VL) and a constant domain CL. For example, the bispecific antibody or its antigen-binding fragment provided herein can comprise a first light chain, a first heavy chain, a second heavy chain, and a second light chain, wherein the first light chain and the first heavy chain are paired to form a first antigen-binding site that is bound to GPRC5D, and the second light chain and the second heavy chain are paired to form a second antigen-binding site that is bound to CD3. For another embodiment, the bispecific antibody or its antigen-binding fragment provided herein can comprise a first light chain, a first heavy chain, a second heavy chain, and a second light chain, wherein the first light chain and the first heavy chain are paired to form a first antigen-binding site that is bound to CD3, and the second light chain and the second heavy chain are paired to form a second antigen-binding site that is bound to GPRC5D.

[0452] For another example, the bispecific antibodies or multispecific antibodies or antigen-binding fragments thereof provided herein may include, but are not limited to, bispecific antibodies with complementary CH3 domains that force heterodimerization, knob-in-hole molecules (Genentech, WO9850431), CrossMAb (Roche, WO 2009080253), or electrostatic matching molecules (Amgen EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO 2010129304). For example, with respect to the CrossMab format, the CD3 binding portion of the bispecific antibody or multispecific antibody provided herein further comprises a constant domain CL and a constant domain CH1, and in some embodiments, the constant domains CL and CH1 are replaced with each other; or the GPRC5D binding portion of the bispecific antibody or multispecific antibody provided herein further comprises a constant domain CL and a constant domain CH1, and in some embodiments, the constant domains CL and CH1 are replaced with each other.

[0453] In some embodiments, the bispecific antibody or multispecific antibody provided herein further comprises an Fc region. In some embodiments, the bispecific antibody or multispecific antibody provided herein further comprises an Fc region of human immunoglobulin (Ig), or optionally an Fc region of human IgG. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3 or IgG4.

[0454] For another example, the bispecific antibody or antigen-binding fragment thereof provided herein can be a bifunctional fusion protein targeting GPRC5D and CD3, wherein the GPRC5D binding portion is a full-length antibody, and the CD3 binding portion is an antigen-binding fragment thereof (e.g., scFv), wherein the CD3 binding portion is directly or through a linker connected to the GPRC5D binding portion. The CD3 binding portion can be connected to the N-terminus of the variable region of the GPRC5D binding portion, can be connected to the C-terminus of the Fc region of the GPRC5D binding portion, or can be connected to the C-terminus of the CL constant domain of the GPRC5D binding portion. In some embodiments, the CD3 binding portion can be or include scFv, the scFv includes a VH region, the VH region is directly or through a linker connected to the VL region, and the VH region of the CD3 binding portion is directly or through a linker connected to the GPRC5D binding portion. In some embodiments, the CD3 binding moiety can be or comprise a scFv comprising a VH region, which is directly or through a linker connected to a VL region, and the VL region of the CD3 binding moiety is directly or through a linker connected to the GPRC5D binding moiety.

[0455] For another example, the bispecific antibody or antigen-binding fragment thereof provided herein can be a bifunctional fusion protein targeting GPRC5D and CD3, wherein the CD3 binding portion is a full-length antibody, and the GPRC5D binding portion is an antigen-binding fragment thereof (e.g., scFv), wherein the GPRC5D binding portion is directly or through a linker connected to the CD3 binding portion. The GPRC5D binding portion can be connected to the N-terminus of the variable region of the CD3 binding portion, can be connected to the C-terminus of the Fc region of the CD3 binding portion, or can be connected to the C-terminus of the CL constant domain of the CD3 binding portion. In some embodiments, the GPRC5D binding portion can be or include scFv, the scFv includes a VH region, the VH region is directly or through a linker connected to the VL region, and the VH region of the GPRC5D binding portion is directly or through a linker connected to the CD3 binding portion. In some embodiments, the GPRC5D binding moiety can be or comprise an scFv comprising a VH region connected directly or through a linker to a VL region, and the VL region of the GPRC5D binding moiety is connected directly or through a linker to the CD3 binding moiety.

[0456] As used herein, the term "linker" refers to an artificial amino acid sequence having 1, 2, 3, 4 or 5 amino acid residues or a length between 5 and 15, 20, 30, 50 or more amino acid residues, connected by peptide bonds and used to connect one or more polypeptides. The linker may or may not have a secondary structure. Linker sequences are known in the art, see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., Structure 2:1121-1123 (1994).

[0457] In certain embodiments, the linker is selected from the group consisting of: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a spiral linker, and a non-spiral linker. Any suitable linker known in the art can be used. In certain embodiments, the linker comprises a peptide linker. For example, the linker available in the present disclosure can be rich in glycine and serine residues. Examples include a linker having a single or repeated sequence (such as TGGGG (SEQ ID NO: 164), GGGGS (SEQ ID NO: 165) or SGGGG (SEQ ID NO: 166) or its tandem repeat sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeat sequences)) comprising threonine / serine and glycine. In certain embodiments, the linker used in the present disclosure comprises GGGGSGGGGSGGGGS (SEQ ID NO: 167). In certain embodiments, the GS linker comprises one or more repeat sequences of GGGS (SEQ ID NO: 168). In certain embodiments, the first linker comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs: 164-168.

[0458] Conjugate

[0459] In some embodiments, the anti-GPRC5D antibody or its antigen-binding fragment provided herein further comprises one or more conjugate parts. The conjugate part can be connected to the antibody or its antigen-binding fragment. The conjugate part is a part that can be connected to the antibody or its antigen-binding fragment. It is conceivable that a variety of conjugate parts can be connected to the antibody or its antigen-binding fragment provided herein (see, for example, "Conjugate Vaccines", "Contributions to Microbiology and Immunology", JM Cruse and RE Lewis, Jr. (ed.), Carger Press, New York, (1989)). These conjugate parts can be connected to the antibody or its antigen-binding fragment by covalent bonding (e.g., disulfide bonds), affinity bonding, embedding, coordination bonding, compounding, association, blending or adding methods. In some embodiments, the antibody or its antigen-binding fragment can be connected to one or more conjugates by a linker or a cross-linking agent. The linker or cross-linking agent contains a reactive chemical group that can react with the anti-GPRC5D antibody or its fragment. The reactive chemical group may be N-succinimidyl ester and N-sulfosuccinimidyl ester. In addition, the linker comprises a reactive chemical group, which may be a dithiopyridyl group that can react with the drug to form a disulfide bond. Linker molecules include, for example, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (see, e.g., Carlsson et al., J. Biol. Chem., 173:723-737 (1978)), N-succinimidyl 4-(2-pyridyldithio) butyrate (SPDB) (see, e.g., U.S. Pat. No. 4,563,304), N-succinimidyl 4-(2-pyridyldithio) 2-sulfobutyrate (sulfo-SPDB) (see U.S. Publication No. 20090274713), N-succinimidyl 4-(2-pyridyldithio) pentanoate (SPP) (see, e.g., CAS Reg. No. 341498-08-6), 2-iminothiolane, or acetyl succinic anhydride. For example, an antibody or cell binding agent can be modified with a cross-linking agent, and the resulting antibody or cell binding agent containing free or protected thiol groups is then reacted with a maytansinoid containing a disulfide or thiol to produce a conjugate. The conjugate can be purified by chromatography, including but not limited to HPLC, size exclusion, adsorption, ion exchange and affinity capture, dialysis or tangential flow filtration.

[0460] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein can be engineered to include specific sites outside of the epitope binding moiety that can be used to bind to one or more conjugate moieties. For example, such sites can include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent attachment to the conjugate moieties.

[0461] In certain embodiments, the antibody or antigen binding fragment thereof provided herein can be connected to the conjugate portion indirectly or through another conjugate portion. For example, the antibody or antigen binding fragment thereof provided herein can be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin. In some embodiments, the conjugate portion comprises a scavenging modifier (e.g., a polymer that extends the half-life, such as PEG), a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a detectable label (e.g., a luminescent label, a fluorescent label, an enzyme substrate label), a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, a purification portion or other anticancer drugs (e.g., toll-like receptor 7 (TLR-7), an agonist of TLR-8 and / or TLR-9, siRNA, an antibody or its antigen binding fragment, a peptide (e.g., a short peptide), etc.).

[0462] A "toxin" can be any agent that is harmful to cells or that can damage or kill cells. Examples of toxins include, but are not limited to, paclitaxel, taxanes, CC-1065 and CC-1065 analogs, duocarmycin and duocarmycin analogs, enediynes such as calicheamicin, dolastatin and dolastatin analogs (including auristatin), tomaymycin derivatives, leptomycin derivatives, and the like. derivatives), cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil and morpholino doxorubicin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, DM4, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., nitrogen mustard, thioepa chlorambucil, chlorambucil), melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and dichlorodiamine platinum (II) (DDP cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g.,dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), antimitotic agents (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin-binding agents.

[0463] Examples of detectable labels can include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase, or β-D-galactosidase), radioactive isotopes (e.g., 123 I. 124 I. 125 I. 131 I. 35 S. 3 H. 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y. 88 Y. 90 Y. 177 Lu, 211 At 186 Re, 188 Re, 153 Sm, 212 Bihe 32 P, other lanthanides), luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules or gold for detection.

[0464] In certain embodiments, the conjugate moiety may be a clearance modifier that helps increase the half-life of the antibody. Illustrative examples include water-soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol / propylene glycol, and the like. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules.

[0465] In certain embodiments, the conjugate moiety may be a purification moiety, such as a magnetic bead.

[0466] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are used as substrates for conjugates.

[0467] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are conjugated with a signal peptide. Signal peptides (sometimes referred to as signal sequences, leader sequences or leader peptides) can be used to promote the secretion and separation of antibodies or antigen-binding fragments thereof provided herein. Signal peptides are generally characterized as a core of hydrophobic amino acids that are typically cut from mature proteins during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow the signal sequence to be cut from the mature protein as the mature protein passes through the secretory pathway. Therefore, the present invention relates to the described polypeptides with signal sequences and polypeptides (i.e., cleavage products) whose signal sequences have been proteolytically cut. In one embodiment, a nucleic acid sequence encoding a signal sequence can be operably connected in an expression vector to a protein of interest, such as a protein that is not usually secreted or otherwise difficult to separate. The signal sequence directs the secretion of the protein, such as from an expression vector transformed into a eukaryotic host therein, and the signal sequence is subsequently or simultaneously cut. The protein can then be easily purified from the extracellular medium by methods recognized in the art. Alternatively, the signal sequence can be connected to the protein of interest using a sequence that promotes purification, such as using a GST domain.

[0468] Chimeric Antigen Receptor

[0469] In certain embodiments, the present disclosure provides a chimeric antigen receptor comprising an antibody or antigen-binding fragment thereof provided herein, a transmembrane region, and an intracellular signaling region.

[0470] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that is transplanted to a cell (e.g., a T cell, such as a primary T cell, a central memory T cell, an effector memory T cell, a regulatory T cell, or any combination thereof). CAR is also referred to as an artificial cell receptor, a chimeric cell receptor, or a chimeric immune receptor. In some embodiments, CAR comprises an antigen-specific targeting region (e.g., an antigen-binding fragment of an anti-GPRC5D antibody as provided herein), an extracellular region, a transmembrane region, one or more costimulatory regions, and an intracellular signaling region.

[0471] In some embodiments, the antigen-specific targeting region is scFv. In some embodiments, the transmembrane region comprises a transmembrane region of CD3, CD4, CD8 or CD28. In some embodiments, the costimulatory region comprises a costimulatory domain of CD28, ICOS, CD27, 4-1BB, OX40 and CD40L. In some embodiments, the intracellular signal region is selected from the group consisting of: CD3 (e.g., CD3ζ), FcγRI, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLR or a combination thereof. In some embodiments, the CD8 transmembrane region comprises an amino acid sequence as shown in SEQ ID NO: 203. In some embodiments, the 4-1BB intracellular signal region comprises an amino acid sequence as shown in SEQ ID NO: 204. In some embodiments, the CD3ζ intracellular signal region comprises an amino acid sequence as shown in SEQ ID NO: 205. In some embodiments, the intracellular signaling region includes a 4-1BB intracellular signaling region comprising an amino acid sequence as shown in SEQ ID NO: 204 and a CD3ζ intracellular signaling region comprising an amino acid sequence as shown in SEQ ID NO: 205.

[0472] CARs can be transplanted into a variety of cells, for example, allogeneic cells, autologous cells, or xenogeneic cells.

[0473] As used herein, the term "allogeneic cell" refers to any cell derived from a different subject of the same species.

[0474] As used herein, the term "autologous cells" refers to any cells derived from the same individual that are subsequently reintroduced into the subject.

[0475] As used herein, the term "xenogeneic cell" refers to any cell originating from a different subject of a different species.

[0476] In certain embodiments, the CAR is transplanted into immune effector cells, such as T cells, natural killer cells, macrophages, tumor infiltrating lymphocytes, etc.

[0477] In some embodiments, compared with known CARs targeting GPRC5D in the art (e.g., CARs disclosed in WO 2020092854A2), CARs provided herein exhibit comparable or better cytotoxicity. In some embodiments, compared with BMK-203, CARs disclosed herein exhibit comparable or better cytotoxicity. The term "BMK-203" includes scFvs, CD8 transmembrane regions, 4-1BB intracellular signal regions, and CD3 ζ intracellular signal regions targeting GPRC5D, wherein the scFv targeting GPRC5D includes a VH region comprising an amino acid sequence as shown in SEQ ID NO: 201 and a VL region comprising an amino acid sequence as shown in SEQ ID NO: 202; CD8 transmembrane regions include an amino acid sequence as shown in SEQ ID NO: 203; 4-1BB intracellular signal regions include an amino acid sequence as shown in SEQ ID NO: 204; and CD3 ζ intracellular signal regions include an amino acid sequence as shown in SEQ ID NO: 205. The amino acid sequences of SEQ ID NOs: 201, 202, 203, 204 and 205 are shown below.

[0478]

[0479] Polynucleotides and recombinant methods

[0480] The disclosure provides isolated polynucleotides, and the isolated polynucleotide encoding antibodies or their antigen-binding fragments and / or chimeric antigen receptors provided herein. As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise noted, specific polynucleotide sequences also implicitly encompass variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences of their conservative modifications and sequences explicitly noted. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acids Research 19:5081 (1991); Ohtsuka et al., Journal of Biol. Chem., 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0481] The DNA encoding the antibodies or antigen-binding fragments thereof provided herein is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of antibodies). Encoding DNA can also be obtained by synthetic methods.

[0482] Recombinant techniques known in the art can be used to encode the antibody or its antigen-binding fragment and / or chimeric antigen receptor provided herein. The isolated polynucleotides are inserted into a vector for further cloning (amplification of DNA) or expression. Many vectors are available. The vector composition generally includes but is not limited to one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α) and a transcription termination sequence.

[0483] The disclosure provides a vector comprising a polynucleotide of separation provided herein. In certain embodiments, the polynucleotide encoding antibody or its antigen binding fragment and / or chimeric antigen receptor provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably connected to the nucleic acid sequence and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), λ phages and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0484] The carrier comprising the polynucleotide sequence of the antibody or its Fab and / or chimeric antigen receptor provided by this paper can be introduced into the host expression system (for example, host cell) for cloning or gene expression.In some embodiments, the host expression system provided by this paper is a microorganism, yeast or mammalian cell.In some embodiments, the microorganism is selected from the group consisting of Escherichia coli and Bacillus subtilis.In some embodiments, the yeast is a genus Saccharomyces.In some embodiments, the mammalian cell is selected from the group consisting of: COS, CHO-S, CHO-K1, HEK-293 and 3T3 cells.

[0485] Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotic cells, yeast cells or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0486] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding anti-GPRC5D antibodies. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the lower eukaryotic host microorganisms. However, many other genera, species and strains are commonly used and suitable for use herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts (such as A. nidulans and A. niger).

[0487] Suitable host cells for expressing the glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. A variety of baculovirus strains and variants and corresponding permissive insect host cells have been identified, and the permissive insect host cells are derived from hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori. A variety of virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain variant of Bombyx mori NPV, and such viruses can be used as viruses herein according to the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.

[0488] However, of greatest interest are vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36 / 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; mouse forestomach carcinoma cells (MFC), SNU620 cells and human hepatoma line (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293, MFC, SNU620 and derivatives thereof.

[0489] The host cell is transformed with the above-mentioned expression or cloning vector for antibody production, and the host cell is cultivated in a conventional nutrient medium, and the conventional nutrient medium is modified into a gene that is suitable for inducing promoters, selecting transformants or amplifying the sequence of the desired coding. In another embodiment, the antibody can be produced by homologous recombination known in the art. In some embodiments, the host cell can produce the antibody or its Fab provided by this paper.

[0490] The disclosure also provides a method for expressing the antibody or its Fab and / or chimeric antigen receptor provided herein, the method comprising culturing the host expression system provided herein under the condition of expressing the antibody or its Fab and / or chimeric antigen receptor. The host expression system for producing the antibody or its Fab and / or chimeric antigen receptor provided herein can be cultivated in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Methods in Enzymology 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Rep. 30,985 can be used as culture medium for the host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM Drugs), trace elements (defined as inorganic compounds whose final concentrations are usually in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0491] When using recombinant technology, antibodies can be produced in the intracellular, periplasmic space, or directly secreted into the culture medium. If the antibody is produced in the cell, then as the first step, the particulate fragments of the fragments of the host cell or dissolution can be removed by, for example, centrifugation or ultrafiltration. Carter et al., "Biology / Technology" 10:163-167 (1992) describes a program for separating antibodies secreted into the periplasmic space of Escherichia coli. In short, the cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. When the antibody is secreted into the culture medium, usually first the supernatant from this type of expression system is concentrated using a commercially available protein concentration filter (such as Amicon or MilliporePellicon ultrafiltration unit). Protease inhibitors such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0492] Antibodies or antigen-binding fragments thereof and / or chimeric antigen receptors produced by the host expression system can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique.

[0493] In certain embodiments, protein A fixed on a solid phase is used for immunoaffinity purification of antibodies and their antigen-binding fragments and / or chimeric antigen receptors. Whether protein A is suitable as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used for purifying antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., Journal of Immunological Methods 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., Journal of the European Molecular Biology Association (EMBO J.) 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Compared with the flow rate and processing time that can be achieved with agarose, mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene can achieve faster flow rates and shorter processing times. Where the antibody comprises a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) can be used for purification. Other techniques for protein purification are also available, depending on the antibody to be recovered, such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0494] Following any preliminary purification steps, the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5-4.5, preferably at low salt concentrations (e.g., about 0-0.25 M salt).

[0495] Pharmaceutical composition

[0496] The present disclosure further provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof and / or a chimeric antigen receptor provided herein, and one or more pharmaceutically acceptable carriers.

[0497] Pharmaceutically acceptable carriers for the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / partitioning agents, sequestering agents or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0498] Suitable components can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole (butylated hydroxanisol), butylated hydroxytoluene and / or propyl gallate. As disclosed herein, in the composition comprising the antibody or its Fab provided herein and conjugate, including one or more antioxidants such as methionine reduces the oxidation of the antibody or its Fab. This oxidation reduction can prevent or reduce the loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Therefore, in certain embodiments, a pharmaceutical composition comprising one or more antibodies or its Fab as disclosed herein and one or more antioxidants such as methionine is provided. Further provided are methods for preventing oxidation, extending shelf life and / or improving efficacy of the antibodies or antigen-binding fragments provided herein by mixing the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0499] To further illustrate, pharmaceutically acceptable carriers may include, for example: aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid or lactic acid. Antimicrobial agents used as carriers can be added to the pharmaceutical compositions in the multidose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0500] The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0501] In some embodiments, pharmaceutical composition is formulated into injectable pharmaceutical composition.Injectable composition can be prepared in any conventional form, such as liquid solution, suspension, emulsion or solid form suitable for producing liquid solution, suspension or emulsion.Injection preparation can include sterile and / or pyrogen-free solution prepared for injection, sterile dry soluble product (such as lyophilized powder, including subcutaneous injection tablets) prepared for injection before use with solvent combination, sterile suspension prepared for injection, sterile dry insoluble product prepared for use with vehicle combination before use and sterile and / or pyrogen-free emulsion.Solution can be aqueous or non-aqueous.

[0502] In certain embodiments, the unit dose parenteral formulation is packaged in an ampoule, a vial, or a syringe with a needle. As is known and practiced in the art, all preparations for parenteral administration should be sterile and pyrogen-free.

[0503] In certain embodiments, a sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment thereof as disclosed herein in a suitable solvent. The solvent may include an excipient that can improve the stability of the powder or a reconstituted solution prepared from the powder or other pharmacological components. Excipients that can be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may include a buffer such as citrate, sodium phosphate or potassium phosphate, or other such buffers known to those skilled in the art, and in one embodiment, the buffer is about neutral pH. The solution is then sterile filtered and then freeze-dried under standard conditions known to those skilled in the art to provide the desired formulation. In one embodiment, the resulting solution is dispensed into vials for freeze-drying. Each vial may contain a single dose or multiple doses of an antibody or antigen-binding fragment thereof or a composition thereof. It is acceptable to overfill the vial with an amount slightly higher than that required for each dose or multiple doses (e.g., about 10%) to facilitate accurate sampling and accurate dosing. The freeze-dried powder may be stored under appropriate conditions (e.g., at about 4°C to room temperature).

[0504] The lyophilized powder is reconstituted with water for injection to provide a formulation for parenteral administration. In one embodiment, sterile and / or pyrogen-free water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the selected therapy to be administered and can be determined empirically.

[0505] Reagent test kit

[0506] In certain embodiments, the disclosure provides a kit comprising an antibody or antigen binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein. In certain embodiments, the disclosure provides a kit comprising an antibody or antigen binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein, and a second therapeutic agent. In certain embodiments, the second therapeutic agent is selected from the group consisting of: chemotherapeutic agents, anticancer drugs, radiotherapy agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormone therapy agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators and cytokines.

[0507] It is obvious to those skilled in the art that such pharmaceutical kits may further include one or more of the various conventional kit components, if desired, such as a container with one or more pharmaceutically acceptable carriers, additional containers, etc. Instructions indicating the quantities of the components to be administered (either as an insert or as a label), instructions for administration, and / or instructions for mixing the components may also be included in the kit.

[0508] How to use

[0509] The disclosure also provides a method for treating, preventing or alleviating a disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein. In certain embodiments, the disease, disorder or condition is a GPRC5D-related disease, disorder or condition. In certain embodiments, the subject is human.

[0510] In some embodiments, the GPRC5D-associated disease, disorder, or condition is characterized by expression or overexpression of GPRC5D.

[0511] In some embodiments, the disease, disorder or condition is cancer. In some embodiments, the cancer is a cancer expressing GPRC5D. As used herein, "cancer expressing GPRC5D" refers to a cancer characterized by expressing GPRC5D protein in cancer cells (i.e., tumor infiltrating immune cells) or expressing GPRC5D in cancer cells (i.e., tumor infiltrating immune cells), and its expression level is significantly higher than the expected level of normal cells. Various methods can be used to determine the presence and / or amount of GPRC5D in the test biological sample of the subject. For example, the test biological sample can be exposed to an anti-GPRC5D antibody or its antigen binding fragment, which binds and detects the expressed GPRC5D protein. Alternatively, methods such as qPCR, reverse transcriptase PCR, microarrays, SAGE, FISH, etc. can also be used to detect GPRC5D at the nucleic acid expression level. In some embodiments, the test sample is derived from cancer cells or tissues or tumor infiltrating immune cells. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from which the test sample is obtained. For example, the reference sample can be a non-diseased sample adjacent to or near the test sample (e.g., tumor). In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the cancer is a B-cell cancer expressing GPRC5D.

[0512] In certain embodiments, the disease, disorder or condition is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lung adenocarcinoma or lung squamous cell carcinoma), peritoneal cancer, carcinoid, bone cancer, pancreatic cancer, primitive neuroectodermal tumors, skin cancer, gallbladder cancer, cancer of the head or neck, squamous cell carcinoma, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial carcinoma), anal region cancer (e.g., anal squamous cell carcinoma), stomach cancer or gastric cancer (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatocarcinoma or hepatocellular carcinoma), bile duct cancer, sarcoma, colorectal cancer, fallopian tube cancer, salivary gland cancer, cervical cancer, endometrial cancer or uterine cancer, osteosarcoma, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, nasopharyngeal cancer, soft tissue sarcoma, polycythemia vera, urethral cancer, penile cancer, kidney or ureter cancer (e.g., rhabdoid tumor of the kidney), cutaneous T-cell lymphoma, medulloblastoma, Wilms tumor, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, choroid plexus papilloma, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, soft tissue sarcomas (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi's sarcoma), spinal axis tumors, gliomas (e.g., ependymomas, astrocytic tumors), astrocytoma, anaplastic astrocytoma, oligodendroglioma, eye cancer (e.g., retinoblastoma), brain stem glioma or mixed glioma such as oligoastrocytoma), brain tumor (e.g., glioblastoma / glioblastoma multiforme (GBM), non-glioblastoma brain tumor or meningioma), skin or intraocular melanoma, thrombocythemia, mesothelioma, mycosis fungoides, Sezary syndrome, idiopathic myelofibrosis, solitary plasmacytoma, vestibular schwannoma, Ewing sarcoma, chondrosarcoma, MYH-associated polyposis, pituitary adenoma, pediatric cancers such as pediatric sarcomas (e.g., neuroblastoma, rhabdomyosarcoma, and osteosarcoma), blood cancers, Hodgkin lymphoma, non-Hodgkin lymphoma lymphoma, leukemia (e.g., lymphocytic / lymphoblastic leukemia), chronic or acute leukemia, mast cell leukemia, lymphocytic lymphoma, primary CNS lymphoma, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia (HCL), Burkitt lymphoma (BL), multiple myeloma (e.g., relapsed or refractory multiple myeloma), T-cell lymphoma or B-cell lymphoma, mantle cell lymphoma (MCL) (e.g.,relapsed or refractory mantle cell lymphoma), malignant melanoma, diffuse large B-cell lymphoma (DLBCL), DLBCL arising from follicular lymphoma, high-grade B-cell lymphoma, primary mediastinal large B-cell lymphoma, follicular lymphoma (FL), and primary mediastinal B-cell lymphoma. In some embodiments, the disease, disorder, or condition is multiple myeloma. ,

[0513] In some embodiments, the subject has been identified as having cancer cells or tumor-infiltrating immune cells that express GPRC5D, optionally at levels significantly higher than those normally found on non-cancerous cells.

[0514] In another aspect, a method for treating, preventing or alleviating a disease, disorder or condition in a subject who would benefit from modulation of GPRC5D activity is provided, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein and / or a chimeric antigen receptor provided herein. In certain embodiments, the disease, disorder or condition is a GPRC5D-related disease, disorder or condition as defined above.

[0515] The therapeutically effective amount of the antibody or antigen-binding fragment provided herein will depend on various factors known in the art, for example, body weight, age, past medical history, current medication, the health status of the subject and the potential for cross-reactions, allergies, sensitivities and adverse side effects, and the route of administration and the extent of disease development. Those skilled in the art (e.g., physicians or veterinarians) can proportionally reduce or increase the dosage according to these and other circumstances or required instructions.

[0516] In certain embodiments, the antibody or Fab provided herein and / or the chimeric receptor provided herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the administered dose can be changed during the course of treatment. For example, in certain embodiments, the initial administered dose can be higher than the subsequent administered dose. In certain embodiments, the administered dose can change during the course of treatment according to the reaction of the subject.

[0517] Dosage regimens may be adjusted to provide the optimal desired response (eg, a therapeutic response). For example, a single dose may be administered, or multiple divided doses may be administered over time.

[0518] The antibodies or antigen-binding fragments thereof provided herein and / or the chimeric antigen receptors provided herein can be administered by any route known in the art, for example, by a parenteral route including subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection or intradermal injection; or by a non-parenteral route, including transdermal, oral, intranasal, intraocular, sublingual, rectal or topical.

[0519] In some embodiments, the antibody or its antigen binding fragment provided herein and / or the chimeric antigen receptor provided herein can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibody or its antigen binding fragment disclosed herein and / or the chimeric antigen receptor disclosed herein can be administered in combination with a second therapeutic agent, and the second therapeutic agent is, for example, a detection agent, a chemotherapeutic agent, an anticancer drug, a radiotherapeutic agent, an immunotherapy agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, a cytokine, an active agent, an imaging agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a costimulatory molecule agonist, a co-inhibitory molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or its functional fragment, a detectable marker or a reporter gene, an antimicrobial agent, a gene editor, a beta agonist, a viral RNA inhibitor, a polymerase inhibitor, an interferon or a microRNA.

[0520] As used herein, the term "immunotherapy" refers to a type of therapy that stimulates the immune system to fight diseases such as cancer or enhances the immune system in a general manner. Examples of immunotherapy include, but are not limited to, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

[0521] "Targeted therapy" is a type of therapy that acts on specific molecules associated with cancer, such as specific proteins that are present in cancer cells but not in normal cells or are more abundant in cancer cells, or target molecules in the cancer microenvironment that contribute to cancer growth and survival. Targeted therapy targets the therapeutic agent to the tumor, sparing normal tissue from the effects of the therapeutic agent.

[0522] In some of these embodiments, the antibody or its antigen binding fragment provided herein and / or the chimeric antigen receptor provided herein and / or the pharmaceutical composition provided herein administered in combination with one or more other therapeutic agents can be administered simultaneously with one or more other therapeutic agents, and in some of these embodiments, the antibody or its antigen binding fragment provided herein and / or the pharmaceutical composition and the other therapeutic agent can be administered as part of the same pharmaceutical composition. However, the antibody or its antigen binding fragment provided herein and / or the chimeric antigen receptor and / or the pharmaceutical composition provided herein administered in combination with another therapeutic agent need not be administered simultaneously with the agent or administered with the same composition as the agent. As the phrase used herein, administering an antibody or its antigen binding fragment, or a chimeric antigen receptor, or a pharmaceutical composition before or after another agent is considered to be administered in combination with the agent, even if the antibody or antigen binding fragment, or a pharmaceutical composition, or a chimeric antigen receptor and a second therapeutic agent are administered by different routes. Where possible, the additional therapeutic agent administered in combination with an antibody or antigen-binding fragment thereof, chimeric antigen receptor, or pharmaceutical composition disclosed herein is administered according to the schedule listed in the product information sheet of the additional therapeutic agent or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457 57th Edition (November 2002)) or regimens well known in the art.

[0523] The present disclosure further provides a method for activating cells expressing GPRC5D in vivo or in vitro, comprising contacting cells expressing GPRC5D with an antibody or antigen-binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein.

[0524] The present disclosure further provides methods of modulating GPRC5D activity in a cell expressing GPRC5D, comprising exposing the cell expressing GPRC5D to an antibody or antigen-binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein.

[0525] In another aspect, the present disclosure provides a method for detecting the presence or amount of GPRC5D in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof provided herein and / or the chimeric antigen receptor provided herein and / or the pharmaceutical composition provided herein, and determining the presence or amount of GPRC5D in the sample.

[0526] In another aspect, the present disclosure provides a method for diagnosing a GPRC5D-related disease, disorder or condition in a subject, comprising: a) obtaining a sample from the subject, b) contacting the sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein and / or a chimeric antigen receptor provided herein and / or a pharmaceutical composition provided herein; c) determining the presence or amount of GPRC5D in the sample; and d) establishing a correlation between the presence or amount of GPRC5D in the subject and the presence or status of a GPRC5D-related disease, disorder or condition.

[0527] In another aspect, the disclosure provides a kit comprising an antibody or antigen binding fragment thereof as provided herein and / or a chimeric antigen receptor as provided herein and / or a pharmaceutical composition as provided herein, optionally conjugated to a detectable moiety, the kit being useful for detecting GPRC5D, optionally recombinant GPRC5D, GPRC5D expressed on the cell surface, or a cell expressing GPRC5D. The kit may further comprise instructions for use.

[0528] On the other hand, the present disclosure also provides a use of the antibody or antigen-binding fragment thereof provided herein and / or the chimeric antigen receptor provided herein and / or the pharmaceutical composition provided herein in the preparation of a medicament for treating, preventing or alleviating a GPRC5D-related disease, disorder or condition in a subject, and in the preparation of a diagnostic reagent for diagnosing a GPRC5D-related disease, disorder or condition.

[0529] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials and methods described below fall within the scope of the invention in whole or in part. These specific compositions, materials and methods are not intended to limit the present invention, but are only used to illustrate specific embodiments falling within the scope of the present invention. Those skilled in the art can develop equivalent compositions, materials and methods without using inventive abilities and without departing from the scope of the present invention. It will be understood that many changes can be made in the procedures described herein while still remaining within the limits of the present invention. It is the intention of the inventor of the present invention that such changes are all included within the scope of the present invention.

[0530] Example

[0531] Example 1. Antibody Generation

[0532] 1.1 Immunity

[0533] To generate antibodies against GPRC5D, in each group, Balb / c and SJL mice were immunized with cells (i.e., human GPRC5D overexpressing HEK293 cells (HEK293-hGPRC5D) or OPM2 cells, referred to as "cellular immunization"), or genetic material (i.e., full-length human GPRC5D expression DNA construct pTT5-H5 (human GPRC5D), referred to as "genetic immunization")) or GPRC5D protein (i.e., recombinant human GPRC5D virus-like particles (hGPRC5D VLPs), referred to as "protein immunization") (as shown in Table 6 below). The primary immunization was followed by several booster immunizations until the animals produced satisfactory antiserum titers suitable for hybridoma development. All immunization strategies were performed in parallel to compare performance at serum levels with immune responses. The immunization schedule for each group of animals is shown in Table 6 below.

[0534] Table 6. Animal groups

[0535] Group Immunogen way Animal / breed Group size dose 1 HEK293-hGPRC5D Intraperitoneal injection SJL 5 <![CDATA[0.5~1x10 7 cells]]> 2 HEK293-hGPRC5D Intraperitoneal injection Balb / c 5 <![CDATA[0.5~1x10 7 cells]]> 3 pTT5-H5 (human GPRC5D) Gene Gun SJL 5 4 μg 4 pTT5-H5 (human GPRC5D) Gene Gun Balb / c 5 4 μg 5 hGPRC5D VLP Intraperitoneal injection SJL 5 25–50 μg 6 hGPRC5D VLP Intraperitoneal injection Balb / c 5 25–50 μg

[0536] Table 7. Immunization schedule for Group 1 and Group 2

[0537]

[0538] Table 8. Immunization schedule for groups 3 and 4

[0539]

[0540]

[0541] Table 9. Immunization schedule for Groups 5 and 6

[0542]

[0543] 1.2 Hybridoma production and screening

[0544] As determined by test blood sampling FACS, spleen cell fusion was performed on the mice with the best immune response. Lymphocytes from spleen and lymph nodes were fused with a mouse myeloma cell line (Sp2 / 0-Ag14) using an optimized electrofusion protocol. Multiple fusions were performed to ensure the success of the project.

[0545] The fused cells were seeded (1×10 4 to 10 5Cells) are added to a pile of 96-well plates. The growth of cells in the monitoring plate is fed once a week. The holes with cell growth are screened by the primary screening assay of Mirrorball assay and / or other feasible assays within 10-14 days. Every kind of targeted antigen is fused multiple times and screened. The positive parent clones that show the positive binding with the HEK293-hGPRC5D from the preliminary screening are expanded into 24-well plates for secondary screening. After the primary screening, the positive parent clones that are expanded into 24-well plates are screened again by the assay described in the hybridoma screening conditions below. Select interested hybridomas to carry out subcloning.

[0546] Parental hybridomas with the desired reactivity and isotype from the above screening conditions are then subcloned by multiple rounds of limiting dilution or single cell sorting until a single clone is obtained.

[0547] Subclone plates were screened by Mirrorball assay, and subclones with good binding capacity were expanded to 24 wells for confirmation testing. The specificity and cross-reactivity of these subclones were confirmed with FACS analysis. In short, parental HEK293T, HEK293T cells (i.e., HEK293T-hGPRC5D) that stably overexpressed human GPRC5D protein, HEK293T cells (i.e., HEK293T-cynoGPRC5D) that stably overexpressed cynomolgus monkey GPRC5D protein, HEK293T cells (i.e., HEK293T-mGPRC5D) that stably overexpressed mouse GPRC5D protein and OPM2 cells were incubated with antibodies produced by each subclone, respectively. The secondary antibody conjugated with fluorescent dye was used to detect the combination of primary antibodies with cells. Median fluorescence intensity (MFI) was measured by FACS analysis.

[0548] The desired subcloned cell lines were sequenced and further expanded into culture flasks for cryopreservation. Initially, 0.5-1.0 x 10 7 Cryopreserve 4 to 6 vials of each cell line per cell line. If necessary, a master cell bank and working cell bank can be established for the most valuable cell lines selected.

[0549] As a result, 21 antibodies with unique sequences were found, which showed positive binding to HEK293T-hGPRC5D and OPM2 cells. Some of these clones can also bind to HEK293T-cynoGPRC5D and / or HEK293T-mGPRC5D. The MFI and positive percentage of antibodies stained with OPM2 cells, parent HEK293T, HEK293T-hGPRC5D, HEK293T-cynoGPRC5D and HEK293T-mGPRC5D detected by FACS are summarized in Tables 10A, 10B and 10C below, which show that these antibodies are antibodies that recognize GPRC5D. ATG596 is a positive human IgG1 control antibody comprising a VH region having an amino acid sequence as shown in SEQ ID NO: 199 and a VL region having an amino acid sequence as shown in SEQ ID NO: 200.

[0550] Table 10A. FACS MFI and % positivity of antibodies (generated by protein immunization) binding to different cell lines expressing GPRC5D

[0551]

[0552]

[0553] Table 10B. FACS MFI and % positivity of antibodies binding to different cell lines expressing GPRC5D (antibodies generated by cell immunization)

[0554]

[0555] Table 10C. FACS MFI and % positivity of antibodies (generated by genetic immunization) binding to different cell lines expressing GPRC5D

[0556]

[0557] Example 2. Antibody characterization: Binding affinity to human, cynomolgus monkey and mouse GPRC5D.

[0558] 2.1 Antibodies

[0559] Hybridoma antibody clones 36A3G8, 28H9C12, 26E2H6, 20C4E1, 15D6G2, 12G12C2, 71F8H2, 64G9C1, 98A3A8, 96C9E7, 90G6H3, 89F11E3, 87A3G8, 83A9B8, 82A1C10, 119E4B4, 116G1G4, 116F1G6, 110B4H5, 108C4C12, and 101B3G3 were characterized.

[0560] 2.2 Hybridoma Sequencing

[0561] Total RNA was isolated from hybridoma cells according to the technical manual of RNAiso Plus (TAKARA catalog number 9109). Total RNA was then reverse transcribed into cDNA using isoform-specific antisense primers or universal primers according to the technical manual of PrimeScript II 1st Strand cDNA Synthesis Kit (TAKARA catalog number 6210A). TM (Catalog No. R001A) amplifies antibody fragments of VH and VL. The amplified antibody fragments are cloned individually into standard cloning vectors. Colony PCR is performed to screen clones with inserts of the correct size. No less than five colonies with inserts of the correct size for each fragment are sequenced. The sequences of different clones are compared, and the consensus sequences of these clones are provided. The variable region sequences of hybridoma antibodies are provided in Table 3 above.

[0562] 2.3 Production of chimeric antibodies

[0563] All sequences from hybridoma antibodies (i.e., 36A3G8, 28H9C12, 26E2H6, 20C4E1, 15D6G2, 12G12C2, 71F8H2, 64G9C1, 98A3A8, 96C9E7, 90G6H3, 89F11E3, 87A3G8, 83A9B8, 82A1C10, 119E4B4, 116G1G4, 116F1G6, 110B4H5, 108C4C12, and 101B3G3) and reference antibody ATG596 from Table 10A, Table 10B, and Table 10C were selected to generate and produce human IgG1 chimeric antibodies. DNA encoding the variable regions of 21 hybridoma antibodies and reference antibody ATG596 was synthesized and subcloned into an expression vector pre-included with a human IgG1 constant gene. The vector was transfected into mammalian cells for recombinant protein expression, and the expressed antibodies were purified using a protein A affinity chromatography column. The resulting chimeric antibodies are referred to herein as ch36A3G8, ch28H9C12, ch26E2H6, ch20C4E1, ch15D6G2, ch12G12C2, ch71F8H2, ch64G9C1, ch98A3A8, ch96C9E7, ch90G6H3, ch89F11E3, ch87A3G8, ch83A9B8, ch82A1C10, ch119E4B4, ch116G1G4, ch116F1G6, ch110B4H5, ch108C4C12, and ch101B3G3, where the prefix "ch" means "chimeric" and the following portion indicates a hybridoma antibody clone. For example, ch36A3G8 indicates that it is a chimeric antibody derived from hybridoma antibody clone 36A3G8.

[0564] 2.4 Chimeric Antibody Characterization (Cell-based Binding Affinity to GPRC5D)

[0565] The binding affinity of the chimeric antibody and the benchmark antibody ATG596 to CHOS-hGPRC5D, HEK293T-cynoGPRC5D and HEK293T-mGPRC5D was determined by FACS analysis. The protocol used for FACS analysis is described as follows:

[0566] (a) Wash the cells with staining buffer (PBS containing 1% BSA) and adjust the cell density to 2 x 10 5 cells / well.

[0567] (b) 50 μL of 2× serially diluted test antibodies and cells were added to each well and incubated at 4°C for 1 hour.

[0568] (c) Centrifuge at 300 g for 3 minutes and discard the supernatant.

[0569] (d) Wash the cells with 200 μL of staining buffer and repeat step (c).

[0570] (e) Cells were incubated with 100 μL of secondary antibody (APC rat anti-human IgG Fc antibody, diluted 1:100) in staining buffer for 0.5 h at 4°C.

[0571] (f) Centrifuge at 300 g for 3 minutes and discard the supernatant.

[0572] (g) Wash cells with 200 μL staining buffer and repeat step (f).

[0573] (h) Cells were resuspended in 120 μl staining buffer and analyzed by FACS.

[0574] like Figure 1 A. Figure 1 B. Figure 1 C and as shown in Table 11 below, 18, 13 and 15 of the chimeric antibodies exhibited positive binding affinities to human, cynomolgus monkey and mouse GPRC5D, respectively.

[0575] Table 11. Binding affinity of antibodies to human, cynomolgus monkey and mouse GPRC5D

[0576]

[0577]

[0578] Example 3. Antibody Characterization: T Cell-Dependent Cytotoxicity

[0579] 3.1 Generation of bispecific hIgG-scFv antibodies

[0580] Bispecific antibodies were generated to evaluate the T cell-dependent cytotoxic potential mediated by all candidates. hIgG-scFv was used as the format of the generated bispecific antibodies. Each bispecific antibody comprised the chimeric antibody generated in Example 2 and a scFv targeting CD3, wherein the scFv targeting CD3 (comprising a VH region comprising an amino acid sequence as shown in SEQ ID NO: 87 and a VL region comprising an amino acid sequence as shown in SEQ ID NO: 88) was expressed by (GGGGS) 3The linker is connected to the C-terminus of the Fc domain of the chimeric antibody. The LALA mutation (L234A, L235A, according to Eu numbering) is introduced into each bispecific antibody to eliminate Fc receptor binding ability. The resulting bispecific antibodies are referred to herein as Bis-36A3G8, Bis-28H9C12, Bis-26E2H6, Bis-20C4E1, Bis-15D6G2, Bis-12G12C2, Bis-71F8H2, Bis-64G9C1, Bis-98A3A8, Bis-96C9E7, Bis-90G6H3, Bis-89F11E3, Bis-87A3G8, Bis-83A9B8, Bis-82A1C10, Bis-119E4B4, Bis-116G1G4, Bis-116F1G6, Bis-110B4H5, Bis-108C4C12 and Bis-101B3G3, wherein the prefix "Bis-" indicates "bispecific", and the following portion indicates the hybridoma antibody clone from which the chimeric antibody is derived. For example, "Bis-83A9B8" indicates that it is a bispecific antibody comprising a chimeric antibody from hybridoma antibody clone 83A9B8 and a scFv targeting CD3 (which includes a VH region comprising an amino acid sequence as shown in SEQ ID NO: 87 and a VL region comprising an amino acid sequence as shown in SEQ ID NO: 88).

[0581] 3.2 T cell-dependent cytotoxicity of bispecific hIgG-scFv antibody against CHOS cells

[0582] The T cell-dependent cytotoxicity of the bispecific hIgG-scFv antibodies on CHOS cells was tested by FACS analysis. The protocol used for FACS analysis is described as follows:

[0583] (a) Isolation of CD3 from normal donors + T cells.

[0584] (b) 1x 10 4 target cells (CHOS-hGPRC5D-GFP) and 1x 10 5 T cells were added to the wells of a 96-well U-bottom plate (10:1 effector:target ratio).

[0585] (c) After target cells and T cells were mixed, a 1:3 serial dilution of the bispecific hIgG-scFv antibody was added to each well at a final concentration of 100 nM for 7 data points.

[0586] (d) Incubate the plate at 37°C with 5% CO 2 Incubate together for 48 hours.

[0587] (e) Two days (48 hours) later, the plates were centrifuged and analyzed by FACS.

[0588] As shown in Table 12 and Figure 2 As shown, several selected bispecific antibodies showed potent T cell-dependent cytotoxic effects on CHOS-hGPRC5D cells.

[0589] Table 12. T cell-dependent cytotoxic effects of selected antibodies on CHOS-hGPRC5D cells

[0590] Clone number Top Kill % <![CDATA[EC 50 (nM)]]> IgG1 12.07 Unstable Bis-83A9B8 66.9 1.897 Bis-108C4C12 67.93 1.439 Bis-116G1G4 69.25 1.876 Bis-116F1G6 71.81 1.658 Bis-15D6G2 45.86 6.693 Bis-98A3A8 64.44 2.95 Bis-96C9E7 66.19 2.337 Bis-110B4H5 61.52 3.615 Bis-26E2H6 79.12 30.54

[0591] 3.3 Generation of bispecific Crossmab antibodies

[0592] To further evaluate cytotoxicity mediated by the antibody candidates, another series of bispecific antibodies were constructed using the Crossmab format as described in WO 2009 / 080253. Typically, each of the produced bispecific Crossmab antibodies is an IgG-like bispecific antibody comprising an anti-GPRC5D Fab domain, an anti-CD3 Fab domain and a constant domain, wherein the anti-GPRC5D Fab domain comprises a GPRC5D binding portion comprising the VH region and the VL region of each of the hybridoma antibody clones screened in Example 1; the anti-CD3 Fab domain comprises a CD3 binding portion comprising a VH region comprising the amino acid sequence shown in SEQ ID NO: 95 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 96, and the CH1 region and the CL region of the anti-CD3 Fab domain are replaced with each other; the constant region is a human IgG1 constant region, and the LALA mutation (L234A, L235A, Eu numbering) is introduced into the IgG1 Fc region to eliminate Fc receptor binding. In addition, knob-and-hole mutations (S354C, T366W on CH3 of the first heavy chain, Y349C, T366S, L368A, Y407V on CH3 of the second heavy chain) were introduced to avoid heavy chain mispairing. ATG596 was used to construct a bispecific antibody under the condition that the Crossmab format was used as a positive control. The bispecific Crossmab ATG596 (i.e., Bis-ATG596-CM) differs only in the amino acid sequences of the VH and VL regions of the anti-GPRC5D Fab domain compared to the tested bispecific Crossmab antibody. JNJ-64407564 was also used as a positive control. The construct and amino acid sequence of JNJ-64407564 can be obtained from WO 2018017786A2. Specifically, JNJ-64407564 includes a GPRC5D binding portion comprising a VH region as shown in SEQ ID NO: 199 and a VL region as shown in SEQ ID NO: 200, and a CD3 binding portion comprising a VH region as shown in SEQ ID NO: 95 and a VL region as shown in SEQ ID NO: 96.

[0593] The resulting bispecific antibodies were designated Bis-36A3G8-CM, Bis-28H9C12-CM, Bis-26E2H6-CM, Bis-20C4E1-CM, Bis-15D6G2-CM, Bis-12G12C2-CM, Bis-71F8H2-CM, Bis-64G9C1-CM, Bis-98A3A8-CM, Bis-96C9E7-CM, Bis-90G6H3-CM, Bis-89F11E3-CM, Bis- s-87A3G8-CM, Bis-83A9B8-CM, Bis-82A1C10-CM, Bis-119E4B4-CM, Bis-116G1G4-CM, Bis-116F1G6-CM, Bis-110B4H5-CM, Bis-108C4C12-CM, and Bis-101B3G3-CM, wherein the prefix "Bis-" indicates "bispecific", the middle indicates a hybridoma antibody clone, and the suffix "-CM" indicates "Crossmab". For example, "Bis-108C4C12-CM" indicates that it is a bispecific Crossmab antibody comprising a GPRC5D binding portion of the VH region and the VL region of the hybridoma antibody clone 108C4C12.

[0594] 3.4 T cell-dependent cytotoxicity of bispecific Crossmab antibody against MM.1S cells

[0595] The cytotoxicity of the bispecific Crossmab antibody to MM.1S cells was tested by FACS analysis. The protocol used for FACS analysis is described as follows:

[0596] (a) Isolation of CD3 from normal donors + T cells.

[0597] (b) 1x 10 4 target cells (CFSE-labeled MM.1S) and 1x 10 5 T cells were added to the wells of a 96-well U-bottom plate (10:1 effector:target ratio).

[0598] (c) After target cells and T cells were mixed, a 1:4 serial dilution of the bispecific Crossmab antibody was added to each well at a final concentration of 5 nM for 9 data points.

[0599] (d) Incubate the plate at 37°C with 5% CO 2 Incubate together overnight.

[0600] (e) After incubation, the plates were centrifuged and analyzed by FACS.

[0601] As shown in Table 13 and Figure 3As shown, all selected antibodies showed effective T cell-dependent cytotoxicity against MM.1S cells. The selected antibodies showed comparable maximum cytotoxicity against MM.1S cells, and three clones (i.e., Bis-108C4C12-CM, Bis-98A3A8-CM, and Bis-96C9E7-CM) had lower EC compared to the benchmark antibodies, JNJ-64407564, and Bis-ATG596-CM. 50 .

[0602] Table 13. T cell-dependent cytotoxic effects of selected antibodies on MM.1S cells

[0603]

[0604]

[0605] Example 4. Antibody Characterization: In Vivo Efficacy

[0606] 4.1 Generation of humanized PBMC MM.1S mouse model

[0607] To evaluate the activity of the bispecific antibodies in vivo, a human PBMC humanized MM.1S model was used. Each NCG (NOD CRISPR Prkdc Il2rγ) mouse received 1 x 10 7 MM.1S human MM (multiple myeloma) cells. When the tumor volume reaches 60-80mm 3 At 1 2 hr, animals received an intravenous injection of 1 x 10 7 Human PBMC. When the tumor volume reaches 100-140mm 3 When, mice were randomly divided into groups according to similar starting tumor volumes, and on the same day, each mouse received intravenous administration of the test article. Treatment was administered at a dose every three days. JNJ-64407564 and Bis-ATG596-CM were used as benchmark antibodies. The tumor growth inhibition mean value was calculated by using the following equation, wherein the mean value (control) indicates the tumor volume mean value of mice treated with hIgG isotype control; the mean value (control D0) indicates the tumor volume mean value of mice treated with hIgG isotype control at day 0; the mean value (test) indicates the tumor volume mean value of mice treated with the tested bispecific antibody; the mean value (test D0) indicates the tumor volume mean value of mice treated with the tested bispecific antibody at day 0.

[0608]

[0609] As shown in Table 14 and Figure 4As shown, most candidates exhibited more potent anti-tumor efficacy in vivo than that exhibited by Bis-ATG596-CM in the same format, indicating that the candidates have great in vivo therapeutic potential. In addition, Bis-108C4C12-CM even showed better efficacy than the benchmark antibody JNJ-64407564.

[0610] Table 14. Mean values ​​of tumor growth inhibition showing the in vivo efficacy of selected antibodies.

[0611] Clone number D0 D2 D5 D9 D12 D16 hIgG Isotype Control Bis-108C4C12-CM 14.43％ -19.71％ 8.68％ 56.76％ 72.89％ Bis-98A3A8-CM 16.15％ -40.13％ -7.87％ 37.10％ 61.71％ Bis-96C9E7-CM 12.67％ -23.76％ -2.22％ 44.78％ 61.95％ Bis-ATG596-CM 11.86％ -25.31％ 12.99％ 31.97％ 37.94％ JNJ-64407564 19.60％ -2.57％ 12.09％ 33.01％ 66.75％

[0612] Example 5. Antibody Characterization: CAR-T

[0613] To evaluate the cytotoxic efficacy of chimeric antigen receptor (CAR) T cells generated by the selected antibodies, CARs were constructed and transfected into T cells, which were then incubated with NCI-H929 cells. BMK-203 was used as a benchmark control. The protocol is described below.

[0614] 5.1 Plasmid transfection and recombinant virus collection

[0615] Day 0: Collect 293T cells and count them when the cell confluence reaches 80%. Inoculate 8.2x 10^6 293T cell suspension into a T75 flask. Return the cell plate to 37°C, 5% CO 2 incubator.

[0616] Day 1: After 24 hours of incubation, the cells reached >90% confluence. The medium was removed and replaced with 11 mL of fresh Opti-MEM medium.

[0617] Transfection system preparation: Tube A: 2 ml Opti-MEM + 25 μg DNA in total (12.5 μg CAR vector + 7.5 μg pGP + 5 μg VSV-G). Tube B: 2 ml Opti-MEM + 75 μg PEI. Add tube A to tube B and mix well. Incubate the mixture at room temperature for 20 minutes. Slowly add the mixture to 293T cells and incubate at 37°C with 5% CO 2 Incubate together for 6 hours. Remove the culture medium and replace with 15 mL of fresh complete DMEM medium (10% FBS + 1% P / S).

[0618] Day 3: After 48 hours post-transfection, all viral supernatants were collected and filtered through a 0.45 μm filter and stored at 4°C. The medium was then removed and replaced with 15 mL of fresh complete DMEM medium and incubated at 37°C with 5% CO. 2Incubate together overnight.

[0619] Day 4: 72 hours after transfection, collect all viral supernatants and filter them with a 0.45 μm filter and combine with the previous viral supernatant. Centrifuge the supernatant at 20,000 × g for 4 hours at 4 °C. Discard the supernatant and resuspend in 400 μl X-VIVO +++ culture medium (5ng / ml IL-2+10ng / ml IL-7+5ng / ml IL-15).

[0620] 5.2 Rotary inoculation

[0621] Isolation of CD3 from 50M frozen human PBMC + T cells were stimulated with CD3 / CD28 Dynabeads (Gibco, #11131D) at a 1:1 ratio and cultured in X-VIVO +++ grown in culture medium.

[0622] Day 1: After three days of activation, T cells were inoculated with CAR lentivirus. The prepared CAR lentivirus expresses CAR, which comprises an extracellular antigen binding region having the amino acid sequences of the VH and VL regions of each selected antibody, a CD8 transmembrane region having an amino acid sequence of SEQ ID NO: 203, a 4-1BB intracellular signal region having an amino acid sequence of SEQ ID NO: 204, and a CD3ζ intracellular signal region having an amino acid sequence as shown in SEQ ID NO: 205.

[0623] Add concentrated virus and polybrene reagent (final concentration 8 μg / ml) to 5 x 10^5 activated T cells (in 2 ml X-VIVO +++ Culture medium) (MOI≈100:1), with a total volume of 700-800 μl per well.

[0624] The mixture was centrifuged at 1000 x g for 2 h at room temperature and then returned to 37°C, 5% CO 2 After 24 hours of incubation, fresh X-VIVO +++ Medium replacement.

[0625] Day 5: 4 days after infection, CD3 / CD28 Dynabeads were removed and transduction efficiency (GFP positive %) was measured.

[0626] CAR-T cell cytotoxicity assay was performed on target cells.

[0627] 5.3 Cytotoxicity assay

[0628] Cell Trace-Far red labeled 3x 10 4 NCI-H929 cells were added to 96-well U-bottom plates (100 μl / well).

[0629] 100 μl / well CAR-T or vehicle CD3 T cells were added to the wells at specific effector to tumor cell (E:T) ratios, E=GFP positive cells.

[0630] After 48 h of co-culture, the number of viable cells of Far Red signaling-positive tumor cells was detected by FACS.

[0631] The cytotoxicity was calculated as follows:

[0632] Cytotoxicity % / CD3 T = (Far Red + Cell number-Far Red in CAR-T group + Far Red cell number / CD3 T group + Cell number*100%

[0633] 5.4 Results

[0634] like Figure 5 As shown, all tested antibodies showed potent CAR-T cell cytotoxicity against NCI-H929 cells, with comparable or better cell killing efficacy compared to the benchmark control BMK-203.

Claims

1. An antibody or antigen-binding fragment thereof that binds to GPRC5D, comprising: one or two or three heavy chain complementarity determining regions (HCDR1, HCDR2 and / or HCDR3) contained in any one of the heavy chain variable (VH) region sequences selected from the group consisting of: SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183 and 191; and / or One or two or three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3), wherein the light chain complementary determining regions are contained in any one of the light chain variable (VL) region sequences selected from the group consisting of: SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184 and 192.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises at least one heavy chain or light chain complementarity determining region (CDR), wherein the heavy chain or light chain CDR comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:1,2,3,4,5,6,9,10,11,12,13,14,17,18,19,20,21,22,25,26,27,28,29,30,33,34,35,36,37,38,41,42,43,44,45,46,49,50 ,51,52,53,54,57,58,59,60,61,62,65,66,67,68,69,70,73,74,75,76,77,78,97,98,99,100,101,102,105,106,107,108,109,1 10, 113, 114, 115, 116, 117, 118, 121, 122, 123, 124, 125, 126, 129, 130, 131, 132, 133, 134, 137, 138, 139, 140, 141, 142, 145, 146, 147, 148, 149, 150, 153, 154, 155, 156, 157, 158, 169, 170, 171, 172, 173, 174, 177, 178, 179, 180, 181, 182, 185, 186, 187, 188, 189 and 190.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a VH region, wherein the VH region comprises one, two or three of HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:1,2,3,9,10,11,17,18,19,25,26,27,33,34,35,41,42,43,49,50,51,57,58,59,65,66,67,73,74,75,97,98,99,105,106,107 ,113,114,115,121,122,123,129,130,131,137,138,139,145,146,147,153,154,155,169,170,171,177,178,179,185,186 and 187.

4. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising a VL region, wherein the VL region comprises one or two or three of LCDR1, LCDR2 and LCDR3, wherein the LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:4,5,6,12,13,14,20,21,22,28,29,30,36,37,38,44,45,46,52,53,54,60,61,62,68,69,70,76,77,78,100,101,102,108,109,1 10, 116, 117, 118, 124, 125, 126, 132, 133, 134, 140, 141, 142, 148, 149, 150, 156, 157, 158, 172, 173, 174, 180, 181, 182, 188, 189 and 190.

5. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising: i. HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 97, 105, 113, 121, 129, 137, 145, 153, 169, 177 and 185; ii. a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 98, 106, 114, 122, 130, 138, 146, 154, 170, 178 and 186; and iii. HCDR3, wherein the HCDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 99, 107, 115, 123, 131, 139, 147, 155, 171, 179 and 187.

6. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising: i. LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 100, 108, 116, 124, 132, 140, 148, 156, 172, 180 and 188; ii. LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 101, 109, 117, 125, 133, 141, 149, 157, 173, 181 and 189; and iii. LCDR3, wherein the LCDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 102, 110, 118, 126, 134, 142, 150, 158, 174, 182 and 190.

7. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising: i. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; ii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11; iii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19; iv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; v. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:33, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:34, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:35; vi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:43; vii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:49, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:50, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:51; viii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 57, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 58, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 59; ix. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 65, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 66, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 67; x. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 73, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 74, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 75; xi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 97, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 98, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 99; xii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 105, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 106, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 107; xiii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 114, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 115; xiv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 121, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 122, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 123; xv. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 129, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 130, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 131; xvi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 138, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 139; xvii. HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises SEQ ID NO: The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 146, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 147; xviii. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 153, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 154, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 155; xix. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 169, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 170, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 171; xx. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 177, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 178, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 179; or xxi. HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:185, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:186, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

187.

8. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising: i. LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; ii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 12, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14; iii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; iv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:28, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:29, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:30; v. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:36, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:37, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:38; vi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:44, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:45, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:46; vii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:53, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:54; viii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:60, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:61, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:62; ix. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:68, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:69, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:70; x. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:76, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:77, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:78; xi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 100, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 101, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 102; xii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 108, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 109, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 110; xiii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 116, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 117, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 118; xiv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 124, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 125, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126; xv. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 132, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 133, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 134; xvi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 140, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 141, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 142; xvii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 148, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 149, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 150; xviii. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 156, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 157, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 158; xix. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 172, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 173, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 174; xx. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 180, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 181, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 182; or xxi. LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:188, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:189, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

190.

9. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising: i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:12, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:13, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:14; iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; iv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:28, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:29, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:30; v. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:33, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:34, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:35, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:36, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:37, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:38; vi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:41, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:43, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:44, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:45, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:46; vii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:49, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:50, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:51, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:53, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:54; viii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:57, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:58, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:59, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:60, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:61, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:62; ix. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:65, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:66, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:67, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:68, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:69, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:70; x. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:73, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:74, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:75, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:76, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:77, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:78; xi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:97, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:98, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:99, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:100, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:101, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:102; xii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 105, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 106, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 107, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 108, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 109, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 110; xiii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 114, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 115, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 116, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 117, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 118; xiv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 121, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 122, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 123, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 124, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 125, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126; xv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 129, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 130, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 131, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 132, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 133, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 134; xvi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 138, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 139, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 140, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 141, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 142; xvii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 145, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 146, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 147, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 148, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 149, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 150; xviii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 153, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 154, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 155, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 156, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 157, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 158; xix. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 169, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 170, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 171, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 172, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 173, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 174; xx. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 177, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 178, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 179, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 180, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 181, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 182; or xxi. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:185, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:186, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:187, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:188, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:189, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:

190.

10. The antibody or antigen-binding fragment thereof according to any of the preceding claims, comprising a VH region having an amino acid sequence as shown in SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183 or 191, or a homologous sequence having at least 80% sequence identity to SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183 or 191.

11. An antibody or antigen-binding fragment thereof according to any of the preceding claims, comprising a VL region having an amino acid sequence as shown in SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184 or 192, or a homologous sequence having at least 80% sequence identity to SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184 or 192.

12. An antibody or antigen-binding fragment thereof according to any of the preceding claims, comprising a VH / VL amino acid sequence pair selected from the group consisting of SEQ ID NO: 7 / 8, 15 / 16, 23 / 24, 31 / 32, 39 / 40, 47 / 48, 55 / 56, 63 / 64, 71 / 72, 79 / 80, 103 / 104, 111 / 112, 119 / 120, 127 / 128, 135 / 136, 143 / 144, 151 / 152, 159 / 160, 175 / 176, 183 / 184 or 191 / 192.

13. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, further comprising one or more amino acid residue substitutions or modifications, but still retaining binding affinity for GPRC5D.

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein at least one of the substitutions or modifications is located in one or more CDR sequences of the VH region or VL region.

15. The antibody or antigen-binding fragment thereof according to claim 13, wherein at least one of the substitutions or modifications is located in one or more non-CDR sequences of the VH region or VL region.

16. The antibody or antigen-binding fragment of the preceding claim, further comprising one or more non-natural amino acid (NNAA) substitutions.

17. The antibody or antigen-binding fragment of claim 16, wherein the NNAA is capable of being conjugated.

18. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, having one or more properties selected from the group consisting of: i. Cross-reactive to human, cynomolgus monkey and mouse GPRC5D; ii. capable of binding to human GPRC5D, cynomolgus monkey GPRC5D or mouse GPRC5D as measured by FACS assay; iii. With an EC no greater than 10 nM as measured by FACS assay 50 Binds to human GPRC5D; iv. exhibit T cell-dependent cytotoxic effects as measured by FACS assay.

19. An antibody or antigen-binding fragment thereof that competes for binding to GPRC5D with the antibody or antigen-binding fragment thereof according to any one of the preceding claims.

20. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a chimeric, humanized or human antibody or antigen-binding fragment thereof.

21. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a labeled antibody, a bivalent antibody, an anti-idiotypic antibody or a fusion protein.

22. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, Fv fragment stabilized by disulfide bonds (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), a diabody stabilized by disulfide bonds (ds diabody), a single-chain antibody molecule (scFv), a scFv dimer (bivalent diabody), a camelized single domain antibody, a nanobody, a domain antibody or a bivalent domain antibody.

23. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, further comprising an Fc region, optionally an Fc region of a human immunoglobulin (Ig), or optionally an Fc region of a human IgG.

24. The antibody or antigen-binding fragment thereof according to claim 23, wherein the Fc region is derived from human IgG1, IgG2, IgG3 or IgG4.

25. The antibody or antigen-binding fragment thereof according to claim 24, wherein the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 161-163.

26. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the light chain is a lambda light chain or a kappa light chain.

27. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a bispecific antibody or a multispecific antibody or antigen-binding fragment thereof.

28. The antibody or antigen-binding fragment thereof according to claim 27, which is capable of binding to one or more additional antigens in addition to GPRC5D, or a second epitope on GPRC5D.

29. The antibody or antigen-binding fragment thereof of claim 28, wherein the one or more additional antigens other than GPRC5D are selected from the group consisting of KRAS, ERK, XPO1, mTORC1 / 2, PAK4, NAMPT, ATR, EGFR, FGFR, VEGF, LILRB, c-MET, Her2, Her3, CTLA4, GITA, CD112R, CD2, CD3, CD7, CD16, C D19, CD20, CD24, CD27, CD30, CD34, CD37, CD38, CD39, CD70, CD73, CD83, CD28, CD80(B7-1), CD86(B7-2) , CD40, CD40L(CD154), CD47, SIRPα, CD122, CD137, CD137L, OX40(CD134), OX40L(CD252), BCMA (e.g., BCM A02), FcRH5, PSMA, CLDN18 (e.g., CLDN18.2), NKG2C, 4-1BB, LIGHT, PVRIG, SLAMF7, HVEM, BAFFR, ICAM-1, 2B4, LFA-1, GITR, ICOS(CD278), ICOSLG(CD275), LAG3(CD223), A2AR, B7-H3(CD276), B7-H4(VTCN1), B 7-H5, BTLA(CD272), BTLA, CD160, CTLA-4(CD152), IDO1, IDO2, ILT3, TDO, KIR, LAIR-1, NOX2, PD-1, PD- L1, PD-L2, TIM-3, VISTA, SIGLEC-7(CD328), SIGLEC-9(CD329), SIGLEC-15, TIGIT, PVR(CD155) and TGFβ.

30. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a bispecific antibody or antigen-binding fragment thereof that binds to GPRC5D and CD3.

31. The antibody or antigen-binding fragment thereof according to claim 30, wherein the bispecific antibody or antigen-binding fragment thereof comprises a GPRC5D binding portion and a CD3 binding portion.

32. The antibody or antigen-binding fragment thereof of claim 31, wherein the CD3 binding portion comprises: One or two or three heavy chain complementarity determining regions (HCDR1, HCDR2 and / or HCDR3) contained in the heavy chain variable (VH) region sequence of SEQ ID NO: 87 or 95; and / or One or two or three light chain complementarity determining regions (LCDR1, LCDR2 and / or LCDR3), which are contained in the light chain variable (VL) region sequence of SEQ ID NO: 88 or 96.

33. The antibody or antigen-binding fragment thereof of claim 32, wherein the CD3 binding portion comprises: HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 81 or 89, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 82 or 90, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 83 or 91; and / or LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:84 or 92, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:85 or 93, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86 or 94.

34. The antibody or antigen-binding fragment thereof of claim 33, wherein the CD3 binding portion comprises: i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:81, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:82, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:83, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:84, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:85, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86; or ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:89, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:90, the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:91, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO:92, the LCDR2 comprises the amino acid sequence as shown in SEQ ID NO:93, and the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO:

94.

35. The antibody or antigen-binding fragment thereof according to claim 34, wherein the CD3 binding portion comprises the VH / VL amino acid sequence pair of SEQ ID NO: 87 / 88 or 95 / 96.

36. The antibody or antigen-binding fragment thereof according to claim 35, wherein the CD3 binding portion further comprises a constant domain CL and a constant domain CH1.

37. The antibody or antigen-binding fragment thereof according to claim 36, wherein the constant domains CL and CH1 are replaced with each other.

38. The antibody or antigen-binding fragment thereof according to any one of claims 27 to 37, further comprising an Fc region, optionally an Fc region of a human immunoglobulin (Ig), or optionally an Fc region of a human IgG.

39. The antibody or antigen-binding fragment thereof according to claim 38, wherein the Fc region is derived from human IgG1, IgG2, IgG3 or IgG4.

40. The antibody or antigen-binding fragment thereof of claim 38 or 39, wherein the Fc region comprises one or more amino acid substitutions selected from the group consisting of L234A, L235A, S354C, T366W, Y349C, T366S, L368A and Y407V (according to EU numbering).

41. The antibody or antigen-binding fragment thereof of claim 40, wherein the Fc region comprises L234A and L235A (according to EU numbering) amino acid substitutions.

42. The antibody or antigen-binding fragment thereof of claim 40, wherein the Fc region of one heavy chain comprises S354C and T366W (according to EU numbering) amino acid substitutions, and the Fc region of the other heavy chain comprises Y349C, T366S, L368A and Y407V (according to EU numbering) amino acid substitutions.

43. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, linked to one or more conjugate moieties.

44. The antibody or antigen-binding fragment thereof of claim 43, wherein the conjugate moiety comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a detectable label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, a purification moiety, or other anti-cancer drug.

45. The antibody or antigen-binding fragment thereof of claim 43 or 44, wherein the conjugate moiety is covalently linked directly or via a linker.

46. ​​A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 45, a transmembrane region and an intracellular signaling region.

47. The chimeric antigen receptor of claim 46, wherein the transmembrane region comprises the transmembrane region of CD3, CD4, CD8 or CD28.

48. The chimeric antigen receptor of claim 46, wherein the intracellular signaling region is selected from the group consisting of: intracellular signaling region sequences of CD3 (e.g., CD3ζ), FcγRI, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLR, or a combination thereof.

49. The chimeric antigen receptor of any one of claims 46 to 48, wherein the antigen binding fragment is a scFv.

50. The chimeric antigen receptor of any one of claims 46 to 49, wherein the chimeric antigen receptor is transplanted into an allogeneic cell, an autologous cell, or a xenogeneic cell.

51. The chimeric antigen receptor of any one of claims 46 to 50, wherein the chimeric antigen receptor is transplanted to an immune effector cell.

52. The chimeric antigen receptor of any one of claims 46 to 51, wherein the chimeric antigen receptor is transplanted into a T cell, a natural killer cell, a macrophage, or a tumor infiltrating lymphocyte.

53. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 45, and / or the chimeric antigen receptor according to any one of claims 46 to 52, and one or more pharmaceutically acceptable carriers.

54. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 45, and / or the chimeric antigen receptor according to any one of claims 46 to 52.

55. A vector comprising the isolated polynucleotide according to claim 54.

56. A host expression system comprising the vector according to claim 55, or having the polynucleotide according to claim 54 integrated into its genome.

57. The host expression system according to claim 56, which is a microorganism, a yeast or a mammalian cell, wherein the microorganism is selected from the group consisting of Escherichia coli and Bacillus subtilis, wherein the yeast is Saccharomyces, and wherein the mammalian cell is selected from the group consisting of COS, CHO-S, CHO-K1, HEK-293 and 3T3 cells.

58. A virus comprising the vector according to claim 55.

59. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 45 and / or the chimeric antigen receptor according to any one of claims 46 to 52 and / or the pharmaceutical composition according to claim 53, and a second therapeutic agent.

60. A method of expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 45 or the chimeric antigen receptor according to any one of claims 46 to 52, comprising culturing the host expression system according to claim 56 under conditions where the antibody or antigen-binding fragment thereof according to any one of claims 1 to 45 is expressed.

61. A method for treating, preventing or alleviating a disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 45 and / or a chimeric antigen receptor according to any one of claims 46 to 52 and / or a pharmaceutical composition according to claim 53.

62. The method of claim 61, wherein the disease, disorder or condition is cancer.

63. The method of claim 62, wherein the cancer is a solid tumor or a hematological tumor.

64. The method of claim 63, wherein the disease, disorder or condition is a B-cell cancer expressing GPRC5D.

65. The method of any one of claims 61 to 64, wherein the disease, disorder or condition is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lung adenocarcinoma or lung squamous cell carcinoma), peritoneal cancer, carcinoid, bone cancer, pancreatic cancer, primitive neuroectodermal tumors, skin cancer, gallbladder cancer, cancer of the head or neck, squamous cell carcinoma, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial carcinoma), anal region cancer (e.g., anal squamous cell carcinoma), stomach cancer or gastric cancer (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatocellular carcinoma or hepatocellular carcinoma), Bile duct cancer, sarcoma, colorectal cancer, fallopian tube cancer, salivary gland cancer, cervical cancer, endometrial cancer or uterine cancer, osteosarcoma, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, nasopharyngeal cancer, soft tissue sarcoma, polycythemia vera, urethral cancer, penile cancer, kidney or ureter cancer (e.g., rhabdoid tumor of the kidney), cutaneous T-cell lymphoma, medulloblastoma, Wilms tumor, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, choroid plexus papilloma, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, soft tissue sarcomas (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi's sarcoma), spinal axis tumors, glioma ( For example, ependymoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, eye cancer (e.g., retinoblastoma), brain stem glioma or mixed glioma such as oligoastrocytoma), brain tumor (e.g., glioblastoma / glioblastoma multiforme (GBM), non-glioblastoma brain tumor or meningioma), skin or intraocular melanoma, thrombocythemia, mesothelioma, mycosis fungoides, Sezary syndrome, idiopathic myelofibrosis, solitary plasmacytoma, vestibular schwannoma, Ewing sarcoma, chondrosarcoma, MYH-associated polyposis, pituitary adenoma, pediatric cancer such as pediatric sarcoma (e.g., neuroblastoma, rhabdomyosarcoma and osteosarcoma), blood cancer, Hodgkin lymphoma, Non-Hodgkin lymphoma, leukemia (e.g., lymphocytic / lymphoblastic leukemia), chronic or acute leukemia, mast cell leukemia, lymphocytic lymphoma, primary CNS lymphoma, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia (HCL), Burkitt lymphoma (BL), multiple myeloma (e.g., relapsed or refractory multiple myeloma), T-cell lymphoma or B-cell lymphoma, mantle cell lymphoma (MCL) (e.g.,relapsed or refractory mantle cell lymphoma), malignant melanoma, diffuse large B-cell lymphoma (DLBCL), DLBCL arising from follicular lymphoma, high-grade B-cell lymphoma, primary mediastinal large B-cell lymphoma, follicular lymphoma (FL), and primary mediastinal B-cell lymphoma.

66. The method of claim 65, wherein the disease, disorder or condition is multiple myeloma.

67. The method of any one of claims 61 to 66, wherein the subject is a human.

68. The method of any one of claims 61 to 67, wherein the administration is by a parenteral route, including subcutaneous, intraperitoneal, intravenous, intramuscular or intradermal injection; or a non-parenteral route, including transdermal, oral, intranasal, intraocular, sublingual, rectal or topical.

69. The method of any one of claims 61 to 68, wherein the method further comprises administering an additional therapeutic agent to the subject in need thereof.

70. The method of claim 69, wherein the additional therapeutic agent is selected from the group consisting of active agents, imaging agents, cytotoxic agents and angiogenesis inhibitors, kinase inhibitors, co-stimulatory molecule agonists, co-inhibitory molecule blockers, adhesion molecule blockers, anti-cytokine antibodies or functional fragments thereof, detectable markers or reporter molecules, antimicrobial agents, gene editing agents, beta agonists, viral RNA inhibitors, polymerase inhibitors, interferons and microRNAs.

71. The method of claim 69, wherein the additional therapeutic agent is administered to the subject in need thereof before, after, or simultaneously with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 45 and / or the chimeric antigen receptor according to any one of claims 46 to 52 and / or the pharmaceutical composition according to claim 53.

72. A method for inactivating a cell expressing GPRC5D in vivo or in vitro, comprising contacting the cell expressing GPRC5D with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 45 and / or a chimeric antigen receptor according to any one of claims 46 to 52 and / or a pharmaceutical composition according to claim 53.

73. A method for modulating GPRC5D activity in a cell expressing GPRC5D, comprising exposing the cell expressing GPRC5D to an antibody or antigen-binding fragment thereof according to any one of claims 1 to 45 and / or a chimeric antigen receptor according to any one of claims 46 to 52 and / or a pharmaceutical composition according to claim 53.

74. A method for detecting the presence or amount of GPRC5D in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 45 and / or the chimeric antigen receptor according to any one of claims 46 to 52 and / or the pharmaceutical composition according to claim 53, and determining the presence or amount of GPRC5D in the sample.

75. A method of diagnosing a GPRC5D-related disease, disorder or condition in a subject, comprising: a) obtaining a sample from the subject; b) contacting the sample obtained from the subject with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 45 and / or the chimeric antigen receptor according to any one of claims 46 to 52 and / or the pharmaceutical composition according to claim 53; c) determining the presence or amount of GPRC5D in the sample; and d) establishing a correlation between the presence or amount of GPRC5D in the subject and the presence or status of the GPRC5D-related disease, disorder or condition.

76. Use of an antibody or antigen binding fragment thereof according to any one of claims 1 to 45 and / or a chimeric antigen receptor according to any one of claims 46 to 52 and / or a pharmaceutical composition according to claim 53 in the preparation of a medicament for treating a GPRC5D-related disease, disorder or condition in a subject.

77. Use of an antibody or antigen binding fragment thereof according to any one of claims 1 to 45 and / or a chimeric antigen receptor according to any one of claims 46 to 52 and / or a pharmaceutical composition according to claim 53 in the preparation of a diagnostic agent for diagnosing a GPRC5D-related disease, disorder or condition.

78. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 45 and / or the chimeric antigen receptor according to any one of claims 46 to 52 and / or the pharmaceutical composition according to claim 53 for detecting GPRC5D, optionally recombinant GPRC5D, GPRC5D expressed on the surface of a cell, or a cell expressing GPRC5D.

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