Novel anti-GPRC5D antibodies, bispecific antigen-binding molecules that bind to GPRC5D and CD3, and their use

Abstract

The present disclosure provides an anti-GPRC5D antibody or an antigen-binding fragment thereof, a bispecific antigen-binding molecule that binds to GPRC5D and CD3, an isolated polynucleotide encoding the same, a pharmaceutical composition 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 to GPRC5D and CD3, and their use.

Background Art

[0002] The G protein-coupled receptor, class C, group 5, member D (GPRC5D) is an atypical class C G protein-coupled receptor (GPCR) for rare diseases 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 related target on plasma cells in autoimmunity. GPRC5D has been reported to be associated with prognosis and tumor burden in patients with multiple myeloma (Atamaniuk, J., et al., Eur J Clin Invest, 42(9):953-960, 2012; and Cohen, Y., et al., Hematology, 18(6):348-351, 2013). Various anti-GPRC5D antibodies have been described in the art, for example, in WO2018017786A2 and WO2021018925A1.

[0003] The CD3 (Cluster of Differentiation 3) T cell coreceptor is a protein complex composed of four distinct chains, the CD3 gamma chain, the CD3 delta chain, and two CD3 epsilon chains. These chains associate with a molecule known as the T cell receptor (TCR) and the zeta chain to generate activation signals in T lymphocytes. The TCR, zeta chain, and CD3 molecules together form the TCR-CD3 complex. The TCR as a subunit recognizes and binds an antigen, and the CD3 as a subunit transmits and transduces antigen stimulation into the signaling pathway, ultimately regulating T cell activation. The CD3 protein is present in substantially all T cells. A more recent application of CD3 antibodies is in the form of bispecific antibodies, i.e., antibodies that bind to CD3 on one hand and to tumor cell antigens on the other. By binding to both of their targets simultaneously, such antibodies create a transient interaction between the target cells and T cells, triggering the activation of any cytotoxic T cells and the subsequent lysis of the target cells.

[0004] There is still a need for additional drugs for treating cancer, particularly multiple myeloma.

Summary of the Invention

[0005] Throughout this disclosure, the articles "a", "an", and "the" refer to one or more than one (i.e., at least one) of the grammatical objects of the articles herein. By way of example, "antibody" means one antibody or more than one antibody.

[0006] In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to GPRC5D and contains one, two, or three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and / or HCDR3) within any one of the heavy-chain variable (VH) region sequences selected from the group consisting of SEQ ID NOs: 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, two, or three light-chain complementarity-determining regions (LCDR1, LCDR2, and / or LCDR3) within any one of the light-chain variable (VL) region sequences selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184, and 192. The present disclosure provides an antibody or an antigen-binding fragment thereof that comprises

[0007] In some embodiments, the antibody or the 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 NOs: 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, 110, 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.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a VH region comprising one or two or three of HCDR1, HCDR2, and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a VL region comprising one or two or three of LCDR1, LCDR2, and LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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, 110, 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.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure i. an HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 97, 105, 113, 121, 129, 137, 145, 153, 169, 177, and 185; ii. an HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 98, 106, 114, 122, 130, 138, 146, 154, 170, 178, and 186; and iii. An HCDR3 comprising 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 comprising.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is: i. An 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. An 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. An LCDR3 comprising 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 comprising.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is: i. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; ii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; iii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19; iv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; v. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35; vi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; vii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51; viii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 57, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 58, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59; ix. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 67; x. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75; xi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99; xii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107; xiii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115; xiv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 122, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123; xv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 129, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 130, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 131; xvi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139; xvii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 146, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 147; xviii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 155; xix. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 169, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 170, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 171; xx. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 177, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 178, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 179; or xxi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 185; an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 186; an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 187 and comprising.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is: i. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; ii. An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 12, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 13, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 14; iii. An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 20, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 21, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 22; iv. An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 28, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 29, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 30; v. An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 36, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 37, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 38; vi. An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 44, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 45, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 46; vii. An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 52, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 53, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 54; viii. An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 60, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 61, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 62; ix. An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 68, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 69, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 70; x. An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 76, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 77, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 78; xi. An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 100, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 101, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 102; xii. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110; xiii. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118; xiv. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 124, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 125, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 126; xv. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 132, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 133, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 134; xvi. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 140, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 141, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 142; xvii. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150; xviii. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 156, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 157, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 158; xix. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 173, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 174; xx. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 180, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 181, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 182; or xxi. An LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 188, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 189, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 190 is included.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is: i. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; ii. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; iii. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; iv. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; v. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; vi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; vii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 53, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54; viii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 57, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 58, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 60, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 61, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 62; ix. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 67, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70; x. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78; xi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102; xii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110; xiii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118; xiv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 122, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 124, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 125, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 126; xv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 129, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 130, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 131, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 132, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 133, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 134; xvi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 140, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 141, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 142; xvii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 146, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 147, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150; xviii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 155, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 156, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 157, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 158; xix. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 169, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 170, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 171, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 173, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 174; xx. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 177, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 178, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 179, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 180, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 181, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 182; or xxi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 185, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 186, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 187, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 188, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 189, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 190 comprising.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a VH region having the amino acid sequence set forth 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 with 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.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a VL region having the amino acid sequence set forth 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 with 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.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure 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.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure further comprises one or more amino acid residue substitutions or modifications and still retains the binding affinity for GPRC5D. In some embodiments, at least one of the substitutions or modifications is present in one or more of the CDR sequences of the VH or VL region. In some embodiments, at least one of the substitutions or modifications is present in one or more of the non-CDR sequences of the VH or VL region. In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure further comprises one or more non-natural amino acid (NNAA) substitutions. In some embodiments, it is possible to conjugate NNAA.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure i. is cross-reactive with human, cynomolgus monkey, and mouse GPRC5D; ii. is capable of binding to human GPRC5D, cynomolgus monkey GPRC5D, or mouse GPRC5D as measured by FACS assay; iii. binds to human GPRC5D with an EC as a measured value of 10 nM or less when measured by FACS assay 50 ; iv. exhibits T cell-dependent cytotoxicity when measured by FACS assay and has one or more binding properties selected from the group consisting of.

[0020] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that competes with the above-described antibody or antigen-binding fragment thereof with respect to binding to GPRC5D.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is a chimeric, humanized, or human antibody, or antigen-binding fragment thereof.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is a labeled antibody, a bivalent antibody, an anti-idiotype antibody, or a fusion protein.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is a diabody, Fab, Fab’, F(ab’)2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv’), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), camelized single-domain antibody, nanobody, domain antibody, or bivalent domain antibody.

[0024] 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 a 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 NOs: 161 to 163.

[0025] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof of the present disclosure is a λ light chain or a κ light chain.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is a bispecific or multispecific antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is 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, 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.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is a bispecific antibody or antigen-binding fragment thereof that binds to GPRC5D and CD3. In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a GPRC5D-binding portion and a CD3-binding portion. In some embodiments, the CD3-binding portion is One or two or three heavy chain complementarity 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 One or two or three light chain complementarity determining regions (LCDR1, LCDR2, and / or LCDR3) contained within the light chain variable (VL) region sequence of SEQ ID NO: 88 or 96 comprises.

[0028] In some embodiments, the CD3 binding portion is HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81 or 89, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82 or 90, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83 or 91; and / or LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84 or 92, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 85 or 93, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 86 or 94 comprises.

[0029] In some embodiments, the CD3 binding portion is i. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 85, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 86; or ii. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 90, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 92, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 93, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94 comprises.

[0030] In some embodiments, the CD3 binding portion comprises the VH / VL amino acid sequence pair of SEQ ID NO: 87 / 88 or 95 / 96.

[0031] 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 exchanged with each other.

[0032] In some embodiments, the bispecific or multispecific antibody or antigen-binding fragment thereof of the present disclosure further comprises an Fc region. In some embodiments, the bispecific or multispecific antibody or antigen-binding fragment thereof of the present disclosure further comprises the Fc region of a human immunoglobulin (Ig). In some embodiments, the bispecific or multispecific antibody or antigen-binding fragment thereof of the present disclosure further comprises 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 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 the L234A and L235A (according to EU numbering) amino acid substitutions. In some embodiments, the Fc region of one heavy chain comprises the S354C and T366W (according to EU numbering) amino acid substitutions, and the Fc region of the other heavy chain comprises the Y349C, T366S, L368A, and Y407V (according to EU numbering) amino acid substitutions.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is linked to one or more conjugate moieties. In some embodiments, 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 binder, a purification moiety or other anti-cancer agent. In some embodiments, the conjugate moiety is covalently bound either directly or via a linker.

[0034] In another aspect, the present disclosure provides a chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof provided herein, a transmembrane region, and an intracellular signaling region. In some embodiments, the transmembrane region comprises the transmembrane region of CD3, CD4, CD8, or CD28. In some embodiments, the intracellular signaling region is selected from the group consisting of the intracellular signaling region sequences of CD3 (e.g., CD3ζ), FcγRI, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLR, or a combination thereof. In some embodiments, the antigen-binding fragment of the chimeric antigen receptor is a scFv. In some embodiments, the chimeric antigen receptor is grafted onto allogeneic cells, autologous cells, or xenogeneic cells. In some embodiments, the chimeric antigen receptor is grafted onto immune effector cells. In some embodiments, the chimeric antigen receptor is grafted onto T cells, natural killer cells, macrophage cells, or tumor-infiltrating lymphocytes.

[0035] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof of the present disclosure, and / or a chimeric antigen receptor, and one or more pharmaceutically acceptable carriers.

[0036] In another aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof of the present disclosure, and / or a chimeric antigen receptor.

[0037] In another aspect, the present disclosure provides a vector comprising the isolated polynucleotide of the present disclosure.

[0038] In another aspect, the present disclosure provides a host expression system comprising the vector of the present disclosure or having the polynucleotide of the present disclosure integrated into its genome. In some embodiments, the host expression system of the present disclosure is a microorganism, yeast, or mammalian cell. In some embodiments, the microorganism is selected from the group consisting of Escherichia coli (E. coli) and Bacillus subtilis (B. 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.

[0039] In another aspect, the present disclosure provides a virus comprising the vector of the present disclosure.

[0040] In another aspect, the present disclosure provides a kit comprising the antibody of the present disclosure or an antigen-binding fragment thereof and / or the chimeric antigen receptor of the present disclosure and / or the pharmaceutical composition of the present disclosure, and a second therapeutic agent.

[0041] In another aspect, the present disclosure provides a method for expressing the antibody of the present disclosure or an antigen-binding fragment thereof and / or the chimeric antigen receptor of the present disclosure, the method comprising culturing the host expression system of the present disclosure under conditions in which the antibody or antigen-binding fragment of the present disclosure is expressed.

[0042] In another aspect, the present disclosure provides a method for treating, preventing, or alleviating a disease, disorder, or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody of the present disclosure or an antigen-binding fragment thereof, and / or a chimeric antigen receptor, and / or a pharmaceutical composition.

[0043] In another aspect, the present disclosure provides the use of the antibody of the present disclosure or an antigen-binding fragment thereof, and / or a chimeric antigen receptor, and / or a pharmaceutical composition in the manufacture of a medicament for treating a GPRC5D-related disease, disorder, or condition in a subject.

[0044] In another aspect, the present disclosure provides for the use of an antibody or an antigen-binding fragment thereof, and / or a chimeric antigen receptor, and / or a pharmaceutical composition of the present disclosure in the manufacture of a diagnostic reagent for diagnosing a GPRC5D-related disease, disorder, or condition.

[0045] In some embodiments, the disease, disorder, or condition is cancer. In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the disease, disorder, or condition is a GPRC5D-expressing B-cell cancer. In some embodiments, the disease, disorder, or condition is lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lung adenocarcinoma, or squamous cell lung cancer), abdominal cancer, carcinoid cancer, bone cancer, pancreatic cancer, undifferentiated neuroectodermal tumor, skin cancer, gallbladder cancer, head or neck cancer, squamous cell carcinoma, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial cancer), anal region cancer (e.g., anal squamous cell carcinoma), gastric cancer, esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatic tumor, or liver cancer), cholangiocarcinoma, 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 ureteral cancer (e.g., renal oncocytoma), cutaneous T-cell lymphoma, medulloblastoma, nephroblastoma, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, choroid plexus papilloma, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) neoplasm, soft tissue sarcoma (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi sarcoma), spinal cord axial tumor, glioma (e.g., ependymoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, eye cancer (e.g., retinoblastoma), brainstem glioma, or mixed glioma, e.g., oligoastrocytoma), brain tumor (e.g., glioblastoma / multiform glioblastoma (GBM), non-glioblastoma brain tumor, or meningioma), skin or intraocular melanoma, thrombocytosis, mesothelioma, fungating polyposis, Sézary syndrome, idiopathic myelofibrosis, solitary plasmacytoma, vestibular schwannoma, Ewing sarcoma, chondrosarcoma, MYH-related polyposis, pituitary adenoma, pediatric cancer, e.g., pediatric sarcoma (e.g., neuroblastoma, rhabdomyosarcoma, and osteosarcoma), hematological 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),Selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), chronic lymphoblastic leukemia, acute lymphoblastic leukemia, hairy cell leukemia (HCL), Burkitt lymphoma (BL), multiple myeloma (e.g., relapsed or refractory multiple myeloma), T 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.

[0046] In some embodiments, the subject is human.

[0047] In some embodiments, administration is via a parenteral route including subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection; or via a non-parenteral route including transdermal, oral, intranasal, intraocular, sublingual, rectal, or topical surface.

[0048] In some embodiments, a method of 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, a contrast agent, a cytotoxic agent, and an angiogenesis inhibitor, a kinase inhibitor, a costimulatory molecule agonist, a coinhibitory molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or a functional fragment thereof, a detectable label or reporter, an antibacterial agent, a gene editing agent, 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 an antigen-binding fragment thereof, and / or the pharmaceutical composition of the present disclosure.

[0049] In another aspect, the present disclosure provides a method of inactivating GPRC5D-expressing cells in vivo or in vitro, the method comprising contacting the GPRC5D-expressing cells with an antibody or an antigen-binding fragment thereof of the present disclosure and / or a chimeric antigen receptor and / or a pharmaceutical composition.

[0050] In another aspect, the present disclosure provides a method of modulating GPRC5D activity in GPRC5D-expressing cells, the method comprising exposing the GPRC5D-expressing cells to an antibody or an antigen-binding fragment thereof of the present disclosure, and / or a chimeric antigen receptor, and / or a pharmaceutical composition.

[0051] In another aspect, the present disclosure provides a method of detecting the presence or amount of GPRC5D in a sample, the method comprising contacting the sample with an antibody or an antigen-binding fragment thereof of the present disclosure, and / or a chimeric antigen receptor, and / or a pharmaceutical composition, and determining the presence or amount of GPRC5D in the sample.

[0052] In another aspect, the present disclosure provides a method of diagnosing a GPRC5D-related disease, disorder, or condition in a subject, the method comprising: a) obtaining a sample from the subject; b) contacting the sample obtained from the subject with an antibody or an antigen-binding fragment thereof of the present disclosure and / or a chimeric antigen receptor and / or a pharmaceutical composition; c) determining the presence or amount of GPRC5D in the sample; and d) correlating the presence or amount of GPRC5D with the presence or situation of a GPRC5D-related disease, disorder, or condition in the subject.

[0053] In another aspect, the present disclosure provides a kit comprising an antibody or an antigen-binding fragment thereof of the present disclosure and / or a chimeric antigen receptor and / or a pharmaceutical composition, which is useful in the detection of GPRC5D, optionally recombinant GPRC5D, GPRC5D expressed on the cell surface, or GPRC5D-expressing cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0054]

Figure 1-1

Figure 1-2

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0055] The following description of the present disclosure is intended merely to illustrate various embodiments of the present disclosure. Therefore, the specific modifications considered should not be construed as limitations on the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and it is understood that embodiments of such equivalents are included herein. All references cited herein, including publications, patents, and patent applications, are hereby incorporated by reference in their entirety.

[0056] Definitions As used herein, the term "antibody" includes any immunoglobulin that binds specifically to an antigen, monoclonal antibodies, polyclonal antibodies, multivalent antibodies, bivalent antibodies, monovalent antibodies, multispecific antibodies, or bispecific antibodies. A native intact antibody includes two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, and each heavy chain includes a variable region (VH), as well as first, second, third, and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are classified as lambda or kappa, and each light chain includes a variable region (VL) and a constant region. An antibody has a "Y" shape, and the stem of the Y includes the second and third constant regions of the two heavy chains joined together via disulfide bonds. Each arm of the Y includes the variable region of a single heavy chain and the first constant region bound to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are involved in antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, and HCDR3). The three CDRs are interposed between adjacent stretches known as framework regions (FRs) (light chain FRs including LFR1, LFR2, LFR3, and LFR4, heavy chain FRs including HFR1, HFR2, HFR3, and HFR4) that are more highly conserved than the CDRs and form a scaffold structure that holds the highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are each characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major antibody classes are further classified into subclasses such as IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain).

[0057] In certain embodiments, the antibodies provided herein include any antigen-binding fragment thereof. As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody that contains 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 include the intact native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab’, F(ab’)2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv’), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bispecific antibodies, multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies. An antigen-binding fragment is capable of binding to the same antigen or epitope to which the parent antibody binds.

[0058] "Fab" with respect to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound by a disulfide bond to a single heavy chain variable region and the first constant region.

[0059] "Fab’" refers to a Fab fragment that includes a portion of the hinge region.

[0060] "F(ab’)2" refers to a dimer of Fab’.

[0061] "Fc" with respect to an antibody (e.g., an antibody of the IgG, IgA, or IgD isotype) refers to that portion of the antibody consisting of the second and third constant domains of the first heavy chain bound via a disulfide bond to the second and third constant domains of the second heavy chain. The Fc of IgM and IgE isotype antibodies further includes a fourth constant domain. The Fc portion of an antibody is involved in various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0062] "Fv" with respect to an antibody refers to the smallest fragment of an antibody that has a complete antigen-binding site. The Fv fragment consists of the variable region of a single heavy chain bound to the variable region of a single light chain.

[0063] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light-chain variable region and a heavy-chain variable region connected via a linker (e.g., a peptide sequence) or directly to each other (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0064] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv connected to the Fc region of an antibody.

[0065] "Camelized single-domain antibody", "heavy-chain antibody", or "HCAb" refers to an antibody that contains two VH domains and 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; US Patent No. 6,005,079). Heavy-chain antibodies originally come from camelids (camels, dromedaries, and llamas). Although lacking a light chain, camelized antibodies have a genuine antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK. et al., Immunogenetics. Apr;54(1):39-47 (2002); Nguyen VK. et al., Immunology. May;109(1):93-101 (2003)). The variable domain of the heavy-chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by the acquired immune response (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0066] "Nanobody" refers to an antibody fragment consisting of the VHH domain from a heavy-chain antibody, as well as two constant domains, CH2 and CH3.

[0067] "Diabody" or "dAb" includes small antibody fragments having two antigen-binding sites, and the fragment includes a VH domain connected to a VL domain within the same polypeptide chain (VH-VL or VL-VH) (see, for example, Holliger P. et al., Proc Natl Acad Sci USA. Jul 15;90(14):6444-8(1993); European Patent No. 404097; WO93 / 11161). By using a linker that is too short to pair between the two domains of the same chain, the domains are forced to pair with the complementary domain of another chain, thereby creating two antigen-binding sites. The antigen-binding sites can target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes).

[0068] "Domain antibody" refers to an antibody fragment containing only the variable region of the heavy chain or the variable region of the light chain. In certain examples, two or more VH domains are covalently linked by a peptide linker to create a bivalent or multivalent domain antibody. The two VH domains of the bivalent domain antibody can target the same or different antigens.

[0069] As used herein, the term "valence" refers to the presence of a specified number of antigen-binding sites on a given molecule. The term "monovalent" refers to an antibody or antigen-binding fragment having only a single antigen-binding site; the term "multivalent" refers to an antibody or antigen-binding fragment having multiple antigen-binding sites. Thus, the terms "bivalent", "tetravalent", and "hexavalent" indicate the presence of two, four, and six antigen-binding sites, respectively, on the antigen-binding molecule. In some embodiments, the antibody or its antigen-binding fragment is bivalent.

[0070] As used herein, a "bispecific" antibody refers to an engineered 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.

[0071] As used herein, a "multispecific" antibody refers to an antibody that specifically binds to at least two distinct antigens or at least two distinct epitopes within the same antigen. Multispecific antibodies can bind, for example, to 2, 3, 4, 5, or more distinct antigens or distinct epitopes within the same antigen.

[0072] In certain embodiments, a "scFv dimer" is a bivalent diabody or bispecific scFv (BsFv) comprising a VH-VL (linked by a peptide linker) that forms a dimer with another VH-VL such that the VH of one moiety cooperates with the VL of the other moiety to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, a "scFv dimer" is a bispecific diabody comprising VH1-VL2 (linked by a peptide linker) associated with VL1-VH2 (linked by a peptide linker) such that VH1 and VL1 cooperate and VH2 and VL2 cooperate, and each cooperating pair has a different antigen specificity.

[0073] "dsFv" refers to a disulfide-stabilized Fv fragment in which the bond 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 moieties are linked by a peptide linker (e.g., a long flexible linker) and each is bound to two VL moieties via a disulfide bridge. In some embodiments, dsFv-dsFv' is bispecific in that the heavy and light chains paired by each disulfide have different antigen specificities.

[0074] As used herein, the term "chimeric" means an antibody or antigen-binding fragment having portions of a heavy and / or light chain derived from one species and the remaining heavy and / or light chains derived from a different species. In an exemplary example, a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0075] As used herein, the term "humanized" means that an antibody or antigen-binding fragment includes CDRs derived from a non-human animal, FR regions derived from a human, and optionally a constant region derived from a human. The CDRs of the humanized antibodies provided in the present disclosure may contain mutations (s) as compared to the CDRs of their parental antibodies.

[0076] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or an antigen-binding fragment thereof and an antigen.

[0077] An antibody or antigen-binding fragment thereof that "specifically binds" or "is specifically bound" to a target (e.g., an epitope) is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to "specifically bind" if it reacts or associates more frequently, more rapidly, for a longer period of time, and / or with greater affinity with a particular cell or substance than with an alternative cell or substance. An antibody "specifically binds" to a target if it binds with greater affinity, avidity, more readily, and / or for a longer period of time 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, avidity, more readily, and / or for a longer period of time than it binds to other GPRC5D epitopes or non-GPRC5D epitopes. By reading this definition, it is also understood 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, "specifically bound" or "specifically binds" does not necessarily (although it may) require exclusive binding. Generally, although not necessarily, the reference to binding means specific binding.

[0078] The ability to "compete with respect to binding to GPRC5D", as used herein, 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, an antibody or antigen-binding fragment that competes with respect to 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, this inhibition can be greater than 95%, or greater than 99%.

[0079] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies can bind to the same or closely related epitopes within an antigen if they exhibit competitive binding with respect to the antigen. Epitopes can be linear or conformational (i.e., including spatially separated 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%, the antibody or antigen-binding fragment can be considered to bind to the same / closely related epitope as the reference antibody.

[0080] As used herein, the term "amino acid" refers to an organic compound containing amine (-NH2) and carboxyl (-COOH) functional groups, along with a side chain specific to each amino acid. The names of amino acids are represented in this disclosure by standard one-letter or three-letter notations, which are summarized below.

[0081]

Table A

[0082] "Conservative substitution" when referring to an amino acid sequence refers to the replacement of 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 among amino acid residues having hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), amino acid residues having neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), amino acid residues having acidic side chains (e.g., Asp, Glu), amino acid residues having basic side chains (e.g., His, Lys, and Arg), or amino acid residues having aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions generally do not cause significant changes in the three-dimensional structure of a protein and can thus retain the biological activity of the protein.

[0083] As used herein, the term "identical" refers to a nucleic acid sequence (or its complementary strand) or an amino acid sequence having 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 with another sequence when optimally aligned.

[0084] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the percent (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) that are identical when compared to the reference sequence to which it is being compared, by the total number of amino acid residues (or bases) in the shorter of the candidate sequence or the reference sequence. Conservative substitutions of amino acid residues may or may not be considered as identical residues. Alignments for the purpose of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the U.S. National Center for Biotechnology Information (NCBI), see also Altschul S.F. 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 D.G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art may use the default parameters provided by the tool or may generate, upon request, parameters that are appropriate for the alignment, for example, by selecting a suitable algorithm.

[0085] "Effector function", as used herein, refers to the biological activity resulting from the binding of the Fc region of an antibody to its effectors such as the C1 complex and Fc receptors. Exemplary effector functions include complement-dependent cytotoxicity (CDC) mediated by the interaction of an antibody with C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by the binding of the Fc region of an antibody to an Fc receptor on an effector cell; and phagocytosis. Effector function can be evaluated using various assays, such as Fc receptor binding assays, C1q binding assays, and cell lysis assays.

[0086] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC", as used herein, refers to a cell-mediated reaction in which effector cells expressing an Fc receptor (FcR) recognize an antibody or antigen-binding fragment bound on a target cell and subsequently cause lysis of the target cell. "ADCC activity" or "ADCC effect" refers to the ability of an antibody or antigen-binding fragment bound on a target cell to induce an ADCC reaction as described above.

[0087] "Complement-dependent cytotoxicity" or "CDC", as used herein, refers to a mechanism by which an antibody can mediate specific target cell lysis through activation of the complement system of an organism. In CDC, C1q binds to the antibody, this binding induces the complement cascade, and as a result of classical pathway complement activation, the formation of a membrane attack complex (MAC) (C5b to C9) on the surface of the target cell occurs. "CDC activity" or "CDC effect" refers to the ability of an antibody or antigen-binding fragment bound on a target cell to induce a CDC reaction as described above.

[0088] As used herein, "target cell" refers to a cell to which an antibody comprising an Fc region specifically binds via a protein moiety that is generally C-terminal to the Fc region. An "effector cell" is a leukocyte that expresses one or more Fc receptors and performs an effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred. Effector cells can be isolated from natural sources, such as blood or PBMCs, as is known in the art.

[0089] An "isolated" substance has been altered by human hand from its natural state. If an "isolated" composition or substance occurs naturally, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that occurs naturally in a living animal is not "isolated," but is "isolated" if it is sufficiently separated from coexisting materials in its natural state to exist 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 has 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% purity as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis, capillary electrophoresis) or chromatography methods (e.g., ion exchange chromatography, or reverse phase HPLC).

[0090] As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to effect expression of that protein. A vector can be used to transform, transduce, or transfect a host cell so as to effect expression of genetic elements carried within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. The category of animal viruses used as vectors includes retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and parvoviruses (e.g., SV40). A vector can contain various elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, selectable elements, and a reporter gene. In addition, a vector can contain an origin of replication. A vector can also include materials that aid its entry into cells, 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) containing a nucleic acid sequence provided herein encoding an antibody or an antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, parvoviruses (e.g., SV40), lambda phage, and M13 phage, plasmid 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, p15TV-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.

[0091] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide and / or vector can be introduced or has been introduced.

[0092] As used herein, the term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and insects. Except where noted, the terms "patient", "subject", or "individual" are used interchangeably herein.

[0093] The term "anti-tumor activity" means a reduction in tumor cell proliferation, survival rate, or metastatic activity. For example, anti-tumor activity can be indicated by a decrease in the abnormal cell growth rate that occurs during treatment, or the stability or reduction of tumor size, or longer survival due to treatment compared to a control that does not receive treatment. Such activity can be evaluated using accepted 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 for examining anti-tumor activity.

[0094] "Treating" or "treatment" of a disease, disorder, or condition, as used herein, includes preventing or alleviating the disease, disorder, or condition, slowing the rate of onset or occurrence of the disease, disorder, or condition, reducing the risk that the disease, disorder, or condition will occur, preventing or delaying the occurrence of symptoms associated with the disease, disorder, or condition, reducing or eliminating symptoms associated with the disease, disorder, or condition, producing complete or partial regression of the disease, disorder, or condition, curing the disease, disorder, or condition, or any combination thereof.

[0095] The terms "diagnosing," "diagnosis," or "diagnosed" refer to the identification of a pathological condition, disease, or condition, such as the identification of a GPRC5D-related disease, or the identification of a subject having a GPRC5D-related disease who may benefit from a particular treatment regimen. In some embodiments, the diagnosis includes the identification of an abnormal amount or activity of GPRC5D. In some embodiments, the diagnosis refers to the identification of cancer in a subject.

[0096] As used herein, the terms "biological sample" or "sample" refer to a biological composition obtained from or derived from a subject of interest that contains cell entities and / or other molecular entities that are characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological characteristics. 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 of skill 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 mucus and sputum), peritoneal fluid, pleural fluid, chest fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, abdominal fluid, ascites, or pericardial fluid. In some embodiments, the biological sample is tissue or cells obtained from the stomach, heart, liver, spleen, lung, kidney, skin, or blood vessels of a subject.

[0097] As used herein, the term "GPRC5D" refers to G protein-coupled receptor family C group 5 member D, including any variant, conformation, isoform, and species homolog of GPRC5D that is naturally expressed by cells or expressed by cells transfected with the GPRC5D gene. For example, GPRC5D as described herein can refer to G protein-coupled receptor family C group 5 member D proteins derived from any vertebrate origin, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). Exemplary sequences of the human GPRC5D protein are described, for example, 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, Biochim. Biophys. Acta 1518, 237-248). As used herein, the term "GPRC5D" encompasses any form of GPRC5D, for example, 1) a native unprocessed GPRC5D molecule, a "full-length" GPRC5D chain, or a naturally occurring variant of GPRC5D including, for example, splice variants or allelic variants; 2) any form of GPRC5D resulting from processing in cells; or 3) the full length, fragments (e.g., truncated forms, extracellular / membrane-spanning domains), or modified forms (e.g., mutant forms, glycosylated / PEGylated, His-tag / immunofluorescent fusion forms) of GPRC5D subunits produced through recombinant methods.

[0098] "CD3", as used herein, refers to the cluster of differentiation 3 protein and includes any variant, conformation, isoform, and species homolog of CD3 that is naturally expressed by cells or expressed by cells transfected with the CD3 gene. For example, CD3 as described herein can refer to the cluster of differentiation 3 protein derived from any vertebrate origin, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). In mammals, the CD3 molecule is a multi-protein complex of six chains, including the CD3 gamma chain, CD3 delta chain, two CD3 epsilon chains, and a homodimer of the CD3 zeta chain, where the CD3 zeta chain is the intracellular tail of the CD3 molecule, and the CD3 gamma, CD3 delta, and CD3 epsilon chains all contain an extracellular domain (ECD) expressed on the surface of T cells. Exemplary sequences of human CD3 include the human CD3 epsilon protein (NCBI reference sequence number NP_000724), the human CD3 delta protein (NCBI reference sequence number NP_000723), and the human CD3 gamma protein (NCBI reference sequence number NP_000064).Exemplary sequences of non-human CD3 include Macaca fascicularis (monkey) CD3 epsilon protein (NCBI reference sequence number NP_001270544), Macaca fascicularis (monkey) CD3 delta protein (NCBI reference sequence number NP_001274617), Macaca fascicularis (monkey) CD3 gamma protein (NCBI reference sequence number NP_001270839); Mus musculus (mouse) CD3 epsilon protein (NCBI reference sequence number NP_031674), Mus musculus (mouse) CD3 delta protein (NCBI reference sequence number NP_038515), Mus musculus domesticus (mouse) CD3 gamma protein (NCBI reference sequence number AAA37400); Rattus norvegicus (rat) CD3 epsilon protein (NCBI reference sequence number NP_001101610), Rattus norvegicus (rat) CD3 delta protein (NCBI reference sequence number NP_037301), Rattus norvegicus (rat) CD3 gamma protein (NCBI reference sequence number NP_001071114). In certain embodiments, the CD3 used herein can also be a recombinant CD3, such as a recombinant CD3 epsilon protein, a recombinant CD3 delta protein, and a recombinant CD3 gamma protein, which can optionally be expressed as a recombinant CD3 complex. The recombinant CD3 complex may be expressed on the cell surface or, alternatively, expressed as a soluble form that is not associated on the cell surface. In certain embodiments, CD3 is human CD3. The terms "CD3", "CD-3", "CD 3", and "cluster of differentiation 3" can be used interchangeably in the present disclosure.

[0099] The term "anti-GPRC5D antibody" refers to an antibody that binds to GPRC5D (e.g., human GPRC5D). The term "anti-human GPRC5D antibody" or "anti-hGPRC5D antibody" refers to an antibody that specifically binds to human GPRC5D.

[0100] 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 the CD3 gamma protein. In some embodiments, the anti-CD3 antibodies provided herein specifically bind to the CD3 delta protein. In some embodiments, the anti-CD3 antibodies provided herein specifically bind to the CD3 epsilon protein.

[0101] The term "CD3 gamma" as used herein is intended to encompass any form of CD3 gamma, such as 1) an unprocessed, native CD3 gamma molecule, a "full-length" CD3 gamma chain, or a naturally occurring variant of CD3 gamma, such as a splice variant or allelic variant; 2) any form of CD3 gamma resulting from processing in a cell; or 3) the full length, a fragment (e.g., a truncated form, extracellular / membrane-spanning domain), or a modified form (e.g., a mutant form, glycosylated / PEGylated, His-tag / immunofluorescent fusion form) of a CD3 gamma subunit produced through recombinant methods.

[0102] The term "CD3 delta" as used herein is intended to encompass any form of CD3 delta, such as 1) an unprocessed, native CD3 delta molecule, a "full-length" CD3 delta chain, or a naturally occurring variant of CD3 delta, such as a splice variant or allelic variant; 2) any form of CD3 delta resulting from processing in a cell; or 3) the full length, a fragment (e.g., a truncated form, extracellular / membrane-spanning domain), or a modified form (e.g., a mutant form, glycosylated / PEGylated, His-tag / immunofluorescent fusion form) of a CD3 delta subunit produced through recombinant methods.

[0103] The term "CD3 epsilon", as used herein, is intended to encompass any form of CD3 epsilon, such as 1) an unprocessed natural CD3 epsilon molecule, a "full-length" CD3 epsilon chain, or a naturally occurring variant of CD3 epsilon, such as a splice variant or an allelic variant; 2) any form of CD3 epsilon resulting from processing in a cell; or 3) a fragment (e.g., a truncated form, an extracellular / membrane-spanning domain) of a full-length, recombinantly produced CD3 epsilon subunit, or a modified form (e.g., a mutant form, a glycosylated / PEGylated form, a His-tag / immunofluorescent fusion form).

[0104] "GPRC5D-related" or "GPRC5D-associated" diseases, disorders, or conditions, as used herein, refer to any disease, disorder, or condition caused by, exacerbated by, or otherwise associated with an increase or decrease in the expression or activity of GPRC5D. In some embodiments, GPRC5D-related diseases, disorders, or conditions are disorders associated with excessive cell proliferation, such as cancer. In certain embodiments, a GPRC5D-related disease or condition is characterized by the expression or overexpression of GPRC5D and / or a GPRC5D-related gene.

[0105] The term "pharmaceutically acceptable" indicates that the designated carrier, medium, diluent, excipient(s), and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation and is physiologically compatible with the subject to which it is administered.

[0106] The term "GPRC5D-expressing cell", as used herein, refers to a cell that expresses GPRC5D on its surface.

[0107] Anti-GPRC5D antibody 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 (e.g., specifically binding) to GPRC5D (e.g., human GPRC5D).

[0108] The binding affinity of an antibody or antigen-binding fragment thereof provided herein is the ratio (k off / k on ) of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule has reached equilibrium, and can be represented by the K D value. The antigen-binding affinity (e.g., K D ) can be appropriately determined using suitable methods known in the art, including, for example, flow cytometry assays. In some embodiments, the binding of different concentrations of an antibody or antigen-binding fragment thereof to an antigen can be determined by flow cytometry, and the determined mean fluorescence intensity (MFI) can first be plotted against the antibody concentration, and then the K D value can be calculated by fitting the specific binding fluorescence intensity (Y) and the antibody concentration (X) dependence to a one-site saturation equation: Y = B max * X / (K D + X) using Prism version 5 (GraphPad Software, San Diego, CA), where B max refers to the maximum specific binding of the test antibody to the antigen.

[0109] The binding of an antibody or antigen-binding fragment thereof provided herein to GPRC5D can also be represented by the "half-maximal effective concentration" (EC 50 ) value, which refers to the antibody concentration at which 50% of its maximum binding is observed. The EC 50 value 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 assay, and other binding assays.

[0110] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of binding to human GPRC5D, cynomolgus GPRC5D, or mouse GPRC5D as measured by FACS assay. In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are cross-reactive with human, cynomolgus, and mouse GPRC5D.

[0111] As used herein, the term "cross-reactive" refers to the ability of a binding protein to bind to targets other than those against which it was generated. Generally, a binding protein binds to its target tissue(s) / antigen(s) with suitably high affinity, but exhibits suitably low affinity for non-target normal tissues / antigens. Individual binding proteins are generally selected to meet two criteria: (1) the antibody binds to tissues appropriate for the known expression of the antibody target when visualized using staining methods known in the art, and (2) the staining patterns are similar between human and tox species (e.g., mouse and cynomolgus) tissues from the same organ. These and other methods for assessing cross-reactivity are known to those of skill in the art (e.g., US Patent Application Publication No. 20090311253A1).

[0112] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to human GPRC5D with an EC 50 of 10 nM or less (e.g., 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less) as measured by FACS assay. In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to cynomolgus GPRC5D and / or mouse GPRC5D with an EC 50 of 20 nM or less (e.g., 15 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less) as measured by FACS assay.

[0113] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein exhibit T cell-dependent cytotoxicity (e.g., as measured by a FACS assay). In certain embodiments, T cell-dependent cytotoxicity is measured by the method described in Example 3 of the present disclosure.

[0114] Exemplary anti-GPRC5D antibodies In certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to GPRC5D and that comprises one or two or three heavy chain complementarity determining regions (HCDR1, HCDR2, and / or HCDR3) contained within any one of the heavy chain variable (VH) region sequences selected from the group consisting of SEQ ID NOs: 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 complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable (VL) region sequences selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184, and 192. The present disclosure provides an antibody or antigen-binding fragment thereof that comprises the foregoing.

[0115] Those skilled in the art can define or identify the CDR boundaries of the VH or VL regions by methods well known in the art as long as the amino acid sequences of the VH or VL regions are known. For example, the CDR boundaries of an antibody or its antigen-binding fragment can be defined or identified according to the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3):651-63(1985); Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901(1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83(1989); Kabat E.A. et al., Sequences of Proteins of immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27: 55-77(2003); Marie-Paule Lefranc et al., Immunome Research, 1(3),(2005); Marie-Paule Lefranc, Molecular Biology of B cells(second edition), chapter 26, 481-514,(2015)). In some embodiments, the CDR boundaries of the antibodies or their antigen-binding fragments provided herein are identified according to the Kabat convention. In some embodiments, the CDR boundaries of the antibodies or their antigen-binding fragments provided herein are identified according to the IMGT convention. In some embodiments, the CDR boundaries of the antibodies or their antigen-binding fragments provided herein are identified according to the Chothia convention.In some embodiments, the CDR boundaries of the antibodies or antigen-binding fragments thereof provided herein are identified according to the Al-Lazikani convention.

[0116] In certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to GPRC5D and 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.

[0117] The antibody "36A3G8", as used herein, 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.

[0118] The antibody "28H9C12", as used herein, 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.

[0119] The antibody "26E2H6", as used herein, 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.

[0120] The antibody "20C4E1", as used herein, 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.

[0121] The antibody "15D6G2", as used herein, 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.

[0122] When used herein, the 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.

[0123] When used herein, the 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.

[0124] When used herein, the 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.

[0125] When used herein, the 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.

[0126] When used herein, the 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.

[0127] When used herein, the 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.

[0128] When used herein, the 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.

[0129] When used herein, the 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.

[0130] When used herein, the 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.

[0131] When used herein, the 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.

[0132] When used herein, the 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.

[0133] When used herein, the 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.

[0134] When used herein, the 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.

[0135] When used herein, the 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.

[0136] When used herein, the 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.

[0137] The antibody "101B3G3", as used herein, 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.

[0138] The specific amino acid sequences of the heavy chain variable region and the light chain variable region of each of the above exemplary antibodies are shown in Table 3 below.

[0139] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence of SEQ ID NO: 7, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence of SEQ ID NO: 8.

[0140] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence of SEQ ID NO: 15, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence of SEQ ID NO: 16.

[0141] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence of SEQ ID NO: 23, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence of SEQ ID NO: 24.

[0142] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence of SEQ ID NO: 31, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence of SEQ ID NO: 32.

[0143] 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 set forth in SEQ ID NO: 39, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 40.

[0144] 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 set forth in SEQ ID NO: 47, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 48.

[0145] 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 set forth in SEQ ID NO: 55, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 56.

[0146] 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 set forth in SEQ ID NO: 63, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 64.

[0147] 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 set forth in SEQ ID NO: 71, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 72.

[0148] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 79, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 80.

[0149] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 103, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 104.

[0150] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 111, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 112.

[0151] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 119, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 120.

[0152] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 127, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 128.

[0153] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises three heavy-chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 135, and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 136.

[0154] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises three heavy-chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 143, and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 144.

[0155] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises three heavy-chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 151, and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 152.

[0156] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises three heavy-chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 159, and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 160.

[0157] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises three heavy-chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 175, and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 176.

[0158] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 183, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 184.

[0159] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the VH region sequence set forth in SEQ ID NO: 191, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the VL region sequence set forth in SEQ ID NO: 192.

[0160] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise at least one (e.g., 1, 2, or 3) heavy or light chain CDRs comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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, 110, 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.

[0161] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a VH region comprising one or two or three of HCDR1, HCDR2, and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.

[0162] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a VL region comprising one or two or three of LCDR1, LCDR2, and LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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, 110, 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.

[0163] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise an HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 97, 105, 113, 121, 129, 137, 145, 153, 169, 177, and 185; an HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 98, 106, 114, 122, 130, 138, 146, 154, 170, 178, and 186; and an HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 99, 107, 115, 123, 131, 139, 147, 155, 171, 179, and 187.

[0164] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise an LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 100, 108, 116, 124, 132, 140, 148, 156, 172, 180, and 188; an LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 101, 109, 117, 125, 133, 141, 149, 157, 173, 181, and 189; and an LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 102, 110, 118, 126, 134, 142, 150, 158, 174, 182, and 190.

[0165] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein i. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; ii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; iii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19; iv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; v. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35; vi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; vii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51; viii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 57, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 58, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59; ix. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 67; x. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75; xi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99; xii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107; xiii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115; xiv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 122, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123; xv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 129, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 130, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 131; xvi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139; xvii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 146, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 147; xviii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 155; xix. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 169, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 170, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 171; xx. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 177, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 178, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 179; or xxi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 185; an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 186; an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 187 comprising.

[0166] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are i. LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; ii. LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; iii. LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; iv. LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; v. LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; vi. LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; vii. LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 53, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54; viii. LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 60, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 61, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 62; ix. LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70; x. LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78; xi. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 100, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 101, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 102; xii. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 108, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 109, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 110; xiii. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 116, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 117, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 118; xiv. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 124, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 125, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 126; xv. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 132, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 133, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 134; xvi. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 140, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 141, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 142; xvii. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 148, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 149, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 150; xviii. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 156, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 157, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 158; xix. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 172, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 173, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 174; xx. LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 180, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 181, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 182; or LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 188, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 189, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 190 comprising.

[0167] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein i. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; ii. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; iii. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; iv. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; v. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; vi. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; vii. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 53, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54; viii. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 57, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 58, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 60, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 61, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 62; ix. an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 67, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70; x. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78; xi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102; xii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110; xiii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118; xiv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 122, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 124, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 125, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 126; xv. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 129, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 130, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 131, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 132, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 133, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 134; xvi. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 137, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 138, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 139, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 140, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 141, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 142; xvii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 146, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 147, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150; xviii. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 155, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 156, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 157, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 158; xix. An HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 169, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 170, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 171, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 173, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 174; HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 177, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 178, HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 179, LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 180, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 181, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 182; or xxi. HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 185, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 186, HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 187, LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 188, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 189, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 190 comprising.

[0168] The sequence numbers of the heavy chain (designated as "H") variable region, light chain (designated as "L") variable region, HCDR, and LCDR of each of the above 21 monoclonal antibodies are shown in Table 1 below. The amino acid sequences of each CDR of the 21 exemplary monoclonal antibodies are shown in Table 2 below. Unless otherwise indicated, the CDR boundaries described in Table 2 below were defined or identified according to the Kabat convention. The amino acid sequences of each VH and VL of the 21 exemplary monoclonal antibodies are shown in Table 3 below.

[0169] [Table 1]

[0170] [Table 2-1] [Table 2-2] [Table 2-3]

[0171] [Table 3-1]

Table 3-2

Table 3-3

Table 3-4

[0172] Assuming that each of the 21 exemplary monoclonal antibodies binds to GPRC5D and that the antigen-binding specificity is provided primarily by the CDR1, CDR2, and CDR3 regions, the HCDR1, HCDR2, and HCDR3 sequences as well as the LCDR1, LCDR2, and LCDR3 sequences 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 as well as LCDR1, LCDR2, and LCDR3) to generate an anti-GPRC5D antibody or an antigen-binding fragment thereof of the present disclosure. The GPRC5D binding of such "mixed and matched" antibodies can be tested using the binding assays described above and in the Examples. Preferably, when mixing and matching VH CDR sequences, the HCDR1, HCDR2, and / or HCDR3 sequences from a particular VH sequence are replaced with structurally similar CDR sequence(s). Similarly, when mixing and matching VL CDR sequences, the LCDR1, LCDR2, and / or LCDR3 sequences from a particular VL sequence are preferably replaced with structurally similar CDR sequence(s). For example, the HCDR1 of antibodies 26E2H6 and 71F8H2 share some structural similarity and are thus amenable to mixing and matching. It will be readily apparent to those 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 with respect to the 21 exemplary monoclonal antibodies.

[0173] It is known that CDRs are involved in antigen binding. However, it has been found that not all six CDRs are necessarily essential or non-exchangeable. In other words, it is possible to exchange, vary, or modify one or more CDRs of each of the 21 exemplary monoclonal antibodies and still retain specific binding affinity for GPRC5D.

[0174] 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 NOs: 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 thus is thought to contact the antigen most frequently and provide the most free energy to the affinity of the antibody for the antigen. Similarly, the heavy chain CDR3 is thought to be the most diverse CDR of the antigen-binding site with respect to length, amino acid composition, and conformation by virtue of multiple diversification mechanisms (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) as well as the desired antigen-binding affinity (Schier R, et al., J Mol Biol. 263:551-67).

[0175] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have a VH region having the amino acid sequence set forth 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% (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%, at least 99%) 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.

[0176] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have a VL region having the amino acid sequence set forth 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% (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%, at least 99%) 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.

[0177] 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.

[0178] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein include suitable framework region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof are capable of binding to GPRC5D. The CDR sequences provided in Table 2 above are obtained from murine antibodies, but they can be grafted onto 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 techniques.

[0179] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are humanized. Humanized antibodies or antigen-binding fragments thereof are desirable due to their reduced immunogenicity in humans. A humanized antibody is chimeric in its variable region because non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of an antibody or antigen-binding fragment can be essentially carried out by substituting a non-human (e.g., murine) CDR gene for the corresponding human CDR gene in a human immunoglobulin gene (see, e.g., Jones et al., (1986) Nature 321:522-525; Riechmann et al., (1988) Nature 332:323-327; Verhoeyen et al., (1988) Science 239:1534-1536).

[0180] Suitable human heavy and light chain variable domains can be selected to achieve this purpose using methods known in the art. In an exemplary example, a "best fit" approach can be used, where non-human (e.g., rodent) antibody variable domain sequences are screened or BLASTed against a database of known human variable domain sequences to identify the human sequence closest to the non-human query sequence and used as a human scaffold structure for grafting the non-human CDR sequences (see, e.g., Sims et al., (1993) J. Immunol. 151:2296; Chothia et al., (1987) J. Mol. Biol. 196:901). Alternatively, a framework derived from the consensus sequence of all human antibodies may be used for grafting non-human CDRs (see, e.g., Carter et al., (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al., (1993) J. Immunol., 151:2623).

[0181] In some embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein are humanized. In certain embodiments, the humanized antibodies or antigen-binding fragments thereof provided herein are composed of substantially all human sequences except for the CDR sequences that are non-human. In some embodiments, the variable region, FR, and, if present, the constant region are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and human constant region sequences may be derived from different human immunoglobulin genes. For example, the FR region may be derived from one human antibody and the constant region may be derived from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises human heavy chain HFR1, HFR2, HFR3, and HFR4, and / or light chain LFR1, LFR2, LFR3, and LFR4.

[0182] In some embodiments, the FR region derived from a human may contain the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are substituted with the corresponding residues from the parental non-human antibody. This may be desirable in certain embodiments to closely approximate the humanized antibody or fragment thereof to the non-human parental antibody structure and optimize the binding characteristics (e.g., increase the binding affinity). In certain embodiments, the humanized antibodies or antigen-binding fragments thereof provided herein contain 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer than 1 amino acid residue substitutions in each of the human FR sequences, or 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer than 1 amino acid residue substitutions in all of the FR sequences of the heavy or light chain variable domains. In some embodiments, such changes in amino acid residues may be present in the heavy chain FR region only, the light chain FR region only, or both chains. In certain embodiments, one or more amino acids of the human FR sequence are randomly mutated to increase the binding affinity. In certain embodiments, one or more amino acids of the human FR sequence are reverted to the corresponding amino acid(s) of the parental non-human antibody to increase the binding affinity.

[0183] In some embodiments, the anti-GPRC5D antibodies and antigen-binding fragments thereof provided herein comprise all or a portion of the heavy chain variable domain and / or all or a portion of the light chain variable domain. In one embodiment, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein is a single domain antibody consisting of all or a portion of the heavy chain variable domain provided herein. More information regarding such single domain antibodies is available in the art (see, e.g., U.S. Patent No. 6,248,516).

[0184] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein further comprise an Fc region. In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein further comprise the Fc region of a human immunoglobulin (Ig). In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein further comprise a constant region optionally further comprising heavy and / or light chain constant regions. In certain embodiments, the heavy chain constant region comprises CH1, hinge, and / or CH2-CH3 regions (or optionally CH2-CH3-CH4 regions). In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgM. In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise a lambda (λ) or kappa (κ) light chain. The constant region of the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein may be identical to the wild-type constant region sequence or may differ by one or more mutations.

[0185] In certain embodiments, the heavy chain constant region comprises an Fc region. The Fc region is known to mediate effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of antibodies. Fc regions of different Ig isotypes have different abilities to induce effector functions. For example, it is recognized that the Fc regions of IgG1 and IgG3 induce both ADCC and CDC more effectively than the Fc regions of IgG2 and IgG4. In certain embodiments, the anti-GPRC5D antibodies and antigen-binding fragments thereof provided herein comprise the Fc region of the IgG1 or IgG3 isotype capable of inducing ADCC or CDC; or alternatively the constant region of the IgG4 or IgG2 isotype that may reduce or abrogate effector function. In some embodiments, the Fc region is derived from human IgG1 with enhanced effector function. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 161. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 161)

[0186] 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 use.

[0187] The antibodies or antigen-binding fragments thereof provided herein can be monoclonal antibodies, polyclonal antibodies, humanized antibodies, human antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, multispecific antibodies, labeled antibodies, bivalent antibodies, anti-idiotypic antibodies, or fusion proteins. Recombinant antibodies are antibodies prepared in vitro using recombinant methods rather than in animals.

[0188] In certain embodiments, the disclosure provides an anti-GPRC5D antibody or an antigen-binding fragment thereof that competes with the antibodies or antigen-binding fragments thereof provided herein with respect to binding to GPRC5D. In certain embodiments, the disclosure provides an anti-GPRC5D antibody or an antigen-binding fragment thereof that competes with any one of antibody 36A3G8, 28H9C12, 26E2H6, 20C4E1, 15D6G2, 12G12C2, 71F8H2, 64G9C1, 98A3A8, 96C9E7, 90G6H3, 89F11E3, 87A3G8, 83A9B8, 82A1C10, 119E4B4, 116G1G4, 116F1G6, 110B4H5, 108C4C12, or 101B3G3 with respect to binding to human GPRC5D. In some embodiments, the disclosure provides an anti-GPRC5D antibody or an antigen-binding fragment thereof that competes with the antibodies or antigen-binding fragments thereof provided herein with respect to the same epitope.

[0189] The ability to "block binding" or "compete with respect to the same epitope," as used herein, refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human GPRC5D and an anti-GPRC5D antibody) to any detectable extent. In certain embodiments, an antibody or antigen-binding fragment thereof that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85% or at least 90%. In certain embodiments, this inhibition may be greater than 85% or greater than 90%.

[0190] One of ordinary skill in the art will recognize that it is possible to determine without undue experimentation whether a human monoclonal antibody binds to the same epitope as an antibody of the present disclosure (e.g., murine monoclonal antibodies 36A3G8, 28H9C12, 26E2H6, 20C4E1, 15D6G2, 12G12C2, 71F8H2, 64G9C1, 98A3A8, 96C9E7, 90G6H3, 89F11E3, 87A3G8, 83A9B8, 82A1C10, 119E4B4, 116G1G4, 116F1G6, 110B4H5, 108C4C12, or 101B3G3) by determining whether the former prevents the latter from binding to the GPRC5D antigen polypeptide. As shown by a decrease in binding to the GPRC5D antigen polypeptide by an antibody of the present disclosure, if a test antibody competes with an antibody of the present disclosure, the two antibodies bind to the same or closely related epitopes. Or if the binding of the test antibody to the GPRC5D antigen polypeptide is inhibited by an antibody of the present disclosure, the two antibodies bind to the same or closely related epitopes.

[0191] In certain embodiments, the present disclosure provides an anti-GPRC5D antibody or antigen-binding fragment thereof that competes with ATG596 for binding to GPRC5D. In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment that competes with an antibody or antigen-binding fragment provided herein for binding to GPRC5D is not ATG596.

[0192] "ATG596", as used herein, refers to an antibody or an antigen-binding fragment thereof that contains one or two or three heavy-chain CDRs contained within the heavy-chain variable region having the amino acid sequence of SEQ ID NO: 199, and one or two or three light-chain CDRs contained within the light-chain variable region having the amino acid sequence of SEQ ID NO: 200. In certain embodiments, ATG596 comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 193, HCDR2 comprising the amino acid sequence of SEQ ID NO: 194, HCDR3 comprising the amino acid sequence of SEQ ID NO: 195, LCDR1 comprising the amino acid sequence of SEQ ID NO: 196, LCDR2 comprising the amino acid sequence of SEQ ID NO: 197, and LCDR3 comprising the 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 according to the Kabat convention.

[0193]

Table 4

[0194] Antibody variant The antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.

[0195] In certain embodiments, the antibody variant comprises one or more amino acid residue substitutions or modifications, but still retains binding affinity for GPRC5D. In certain embodiments, at least one of the substitutions or modifications is present in one or more of the CDR sequences of the VH region or VL region. In certain embodiments, at least one of the substitutions or modifications is present in one or more of the non-CDR sequences of the VH region or VL region. In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise one or more non-natural amino acid (NNAA) substitutions. In certain embodiments, the NNAA can be conjugated.

[0196] For example, an antibody variant may contain one or more of the CDR sequences provided in Table 2 above, one or more non-CDR sequences of the heavy or light chain variable regions provided in Table 3 above, and / or one or more amino acid residue substitutions or modifications in the constant region (e.g., the Fc region). Such variants retain binding specificity for GPRC5D of their parental antibody but have one or more desired properties conferred by the modification(s) or substitution(s). For example, the antibody variant may have improved antigen-binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, enhanced effector function(s), improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., one or more introduced cysteine residues), among others.

[0197] The parental antibody sequence can be screened using methods known in the art, such as "alanine scan mutagenesis," to identify residues suitable or preferred for modification or substitution (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and exchanged for neutral or negatively charged amino acids (e.g., alanine or polyalanine), and the modified antibody can be produced and screened for the desired property. If substitution at a particular amino acid position demonstrates the desired functional change, that position can be identified as a potential residue for modification or substitution. The potential residue may be further evaluated by substituting it with different types of residues (e.g., cysteine residues, positively charged residues, etc.).

[0198] Affinity variant Antibody affinity variants can contain modifications or substitutions in one or more of the CDR sequences, one or more of the FR sequences provided in Table 2 above, or the heavy or light chain variable region sequences provided in Table 3 above. Since it is well known in the art that the CDR regions are adjacent to two FR regions in the variable region, the FR sequences can be readily identified by one of ordinary skill in the art based on the CDR sequences in Table 2 above and the variable region sequences in Table 3 above. The affinity variant retains the specific binding affinity for GPRC5D of the parental antibody or has an improved GPRC5D binding affinity as compared to the parental antibody. In certain embodiments, at least one (or all) of the substitution(s) in the CDR sequence, FR sequence, or variable region sequence comprises a conservative substitution.

[0199] One of ordinary skill in the art may substitute one or more amino acid residues in the CDR sequences provided in Table 2 above and the variable region sequences provided in Table 3 above and still obtain an antibody or antigen-binding fragment that retains the binding affinity or binding ability for GPRC5D or even retains an improved binding affinity or binding ability. This can be achieved using various methods known in the art. For example, a library of antibody variants (e.g., Fab or scFv variants) can be generated, expressed by phage display technology, and then screened for binding affinity to human GPRC5D. In another example, computer software can be used to virtually simulate the binding of an antibody to human GPRC5D and identify the amino acid residues on the antibody that form the binding interface. Such residues may be avoided in substitutions to prevent a reduction in binding affinity or may be targeted for substitution to provide stronger binding.

[0200] In certain embodiments, the humanized antibodies or antigen-binding fragments thereof provided herein include one or more amino acid residue substitutions in one or more of the CDR sequences and / or one or more of the FR sequences. In certain embodiments, the affinity variant includes a total of 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer substitutions in the CDR sequences and / or FR sequences.

[0201] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein 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 sequence(s) set forth in Table 2 above, and still retain specific binding affinity for GPRC5D at a level similar to or even higher than that of its parental antibody, and include 1, 2, or 3 CDR sequences.

[0202] In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the sequence(s) set forth in Table 3 above, and still retains specific binding affinity for GPRC5D at a level similar to or higher than that of its parental antibody, and includes one or more variable region sequences. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted in the variable region sequences set forth in Table 3 above. In some embodiments, the substitution, insertion, or deletion is present in a region outside the CDR (e.g., FR).

[0203] Glycosylation variant The anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein also include glycosylation variants that can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment thereof.

[0204] The antibodies or antigen-binding fragments thereof provided herein may include 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) can be attached. Glycosylation of an antibody is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine. Removal of a native glycosylation site can be readily achieved, for example, by altering the amino acid sequence such that one of the above tripeptide sequences (in the case of an N-linked glycosylation site) or serine or threonine residue (in the case of an O-linked glycosylation site) present in the sequence is substituted. A novel glycosylation site can be similarly created by introducing such a tripeptide sequence or serine or threonine residue.

[0205] Cysteine-engineered variant The anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein also include cysteine-engineered variants that include one or more introduced free cysteine amino acid residues.

[0206] A free cysteine residue is a residue that is not part of a disulfide bridge. Cysteine-engineered variants are useful for conjugating, for example, cytotoxic compounds and / or contrast compounds, labels, or radioisotopes through, for example, maleimide or haloacetyl at the engineered cysteine site. Methods for engineering an antibody or antigen-binding fragment thereof to introduce a free cysteine residue are known in the art; see, for example, WO2006 / 034488.

[0207] Fc variant The anti-GPRC5D antibodies and antigen-binding fragments provided herein also include Fc variants that contain one or more amino acid residue modifications or substitutions in their Fc region and / or hinge region.

[0208] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein include one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants may have an extended pharmacokinetic half-life since they bind to FcRn at acidic pH, which allows them to escape degradation in lysosomes and then traffic and be released from the cell. Methods for engineering antibodies or antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art and are described, for example, in Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 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., J. Immunology, 176:346-356 (2006).

[0209] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein include one or more amino acid substitutions (s) that alter ADCC. Certain amino acid residues in the CH domains of the Fc region can be substituted to provide enhanced ADCC activity. Alternatively or in addition, the carbohydrate structure of the antibody can be altered to enhance ADCC activity. Methods for altering ADCC activity by engineering of antibodies are described in the art, see, for example, Shields RL.et al., J Biol Chem. 2001. 276(9):6591-604; Idusogie EE.et al., J Immunol. 2000. 164(8):4178-84; Steurer W.et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE.et al., J Immunol. 2001, 166(4):2571-5; Lazar GA.et al., PNAS, 2006, 103(11):4005-4010; Ryan MC.et al., Mol.Cancer Ther., 2007, 6:3009-3018; Richards JO,.et al., Mol Cancer Ther. 2008, 7(8):2517-27; Shields R.L.et al., J.Biol.Chem, 2002, 277:26733-26740; Shinkawa T.et al., J.Biol.Chem, 2003, 278:3466-3473.

[0210] In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions (s) that alter CDC, e.g., by enhancing or impairing C1q binding and / or CDC (see, e.g., WO99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 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 can be replaced with different amino acid residues to alter Clq binding and / or enhance CDC (see U.S. Patent No. 6,194,551 by Idusogie et al.). One or more amino acid substitutions (s) can also be introduced to alter the complement fixation ability of the antibody (see PCT application WO94 / 29351 by Bodmer et al.).

[0211] In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein comprises one or more amino acid substitutions (s) at positions 234 and / or 235 of human immunoglobulin (according to EU numbering). In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein comprises two amino acid substitutions at positions 234 and 235 of human immunoglobulin (e.g., IgG1) (according to EU numbering). In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein comprises the L234A and L235A (according to EU numbering) amino acid substitutions.

[0212] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein include one or more amino acid substitution(s) (s) at the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include the introduction of a protrusion into the first Fc polypeptide and a cavity into the second Fc polypeptide, and the protrusion can be positioned in the cavity to facilitate the interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating antibodies with these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0213] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein include an amino acid substitution at position 366 (according to EU numbering) of the first Fc polypeptide and one, two, or three amino acid substitutions at one, two, or three positions of positions 366, 368, and 407 (according to EU numbering) of the second Fc polypeptide. In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein include the T366W substitution (according to EU numbering) of the first Fc polypeptide and the T366S+L368A+Y407V substitution (according to EU numbering) of the second Fc polypeptide.

[0214] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein further comprise one or more (optional) amino acid substitutions in one of the first Fc polypeptides and one or more (optional) amino acid substitutions in the second Fc polypeptide to introduce an unnatural disulfide bond between the two Fc polypeptides. For example, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise an amino acid substitution at position 354 (according to EU numbering) of the first Fc polypeptide and an amino acid substitution at position 349 (according to EU numbering) of the second Fc polypeptide. In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise an S354C substitution (according to EU numbering) of the first Fc polypeptide and a Y349C substitution (according to EU numbering) of the second Fc polypeptide.

[0215] 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 the S354C and T366W (according to EU numbering) amino acid substitutions of the first Fc polypeptide, and the Y349C, T366S, L368A, and Y407V (according to EU numbering) amino acid substitutions of the second Fc polypeptide.

[0216] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise a first Fc polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 162 and a second Fc polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 163. KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 162) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 163)

[0217] Antigen-binding fragment Also provided herein are anti-GPRC5D antigen-binding fragments. For example, various types of antigen-binding fragments, including exemplary antibodies whose CDRs are shown in Table 2 above and whose variable sequences are shown in Table 3 above, and different variants thereof (e.g., affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants, etc.), are known in the art and can be developed based on the anti-GPRC5D antibodies provided herein.

[0218] In certain embodiments, the anti-GPRC5D antigen-binding fragments provided herein are diabodies, Fabs, Fab’, F(ab’)2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv’), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), camelized single-domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

[0219] To produce such antigen-binding fragments, a variety of techniques can be used. Exemplary methods include enzymatic digestion of intact antibodies (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 in host cells such as E. coli (e.g., in the case of Fab, Fv and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., in the case of ScFv), and formation of F(ab’)2 fragments by chemical coupling of two Fab’-SH fragments (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0220] In certain embodiments, the antigen-binding fragment is an scFv. Generation of scFv is described, for example, in WO93 / 16185; U.S. Patent Nos. 5,571,894; and 5,587,458. The scFv may be fused to an effector protein at either the amino or carboxyl terminus to provide a fusion protein (see, e.g., Antibody Engineering, ed. Borrebaeck).

[0221] 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 valences is considered multivalent and includes, for example, trivalent, tetravalent, hexavalent, and so on.

[0222] A bivalent molecule can be monospecific if both of its binding sites are specific with respect to binding to the same antigen or the same epitope. In certain embodiments, this provides a stronger binding to the antigen or epitope than the monovalent counterpart. Similarly, a multivalent molecule can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, the first valence of the binding site and the second valence of the binding site are either structurally identical (i.e., having the same sequence) or structurally different (i.e., having different sequences despite having the same specificity).

[0223] 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 if two of its binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0224] Bispecific or multispecific antibodies In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein are bispecific or multispecific. In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein are further linked to a second binding moiety having a binding specificity different from that of the anti-GPRC5D antibody or antigen-binding fragment thereof. In some embodiments, the bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein have a first specificity for GPRC5D and 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.

[0225] 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 is present or can be present on the surface of tumor cells and that is located on or within tumor cells. In some embodiments, only tumor cells can present tumor-associated antigens, and normal cells, i.e., non-tumor cells, cannot. In some other embodiments, the tumor-associated antigen can be expressed exclusively on tumor cells or can represent a tumor-specific mutation as compared to non-tumor cells. In some other embodiments, the tumor-associated antigen can be found in both tumor cells and non-tumor cells, but is overexpressed in tumor cells as compared to non-tumor cells or is accessible for antibody binding in tumor cells because the tumor tissue architecture is less dense as compared to non-tumor tissue. In some embodiments, the tumor-associated antigen is located in the vasculature of the tumor.

[0226] In certain embodiments, the bispecific 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 other than 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), 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), 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.

[0227] In certain embodiments, the bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and CD3. In certain embodiments, the bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and BCMA. In certain embodiments, the bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and CD38. In certain embodiments, the bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and CD19. In certain embodiments, the bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and FcRH5. In certain embodiments, the bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and PD-L1. In certain embodiments, the bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein bind to GPRC5D and PD-1.

[0228] In certain embodiments, the bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein comprise a GPRC5D-binding portion and a CD3-binding portion.

[0229] As used herein, the term "GPRC5D-binding portion" with respect to a bispecific or multispecific antibody or antigen-binding fragment thereof refers to a portion capable of binding to GPRC5D (e.g., human GPRC5D, mouse GPRC5D, and cynomolgus monkey GPRC5D). The GPRC5D-binding portion can take any form that enables specific recognition of the target GPRC5D. For example, the GPRC5D-binding portion can 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.

[0230] The GPRC5D-binding portion of the bispecific or multispecific antibodies provided herein can be derived from any of the anti-GPRC5D antibodies described above. In certain embodiments, the GPRC5D-binding portion of the bispecific or multispecific antibodies provided herein comprises HCDR1, HCDR2, and / or HCDR3 contained within any one of the heavy chain variable region sequences set forth in Table 3 above, and LCDR1, LCDR2, and / or LCDR3 contained within any one of the light chain variable region sequences set forth in Table 3 above. In certain embodiments, the GPRC5D-binding portion of the bispecific or multispecific antibodies provided herein comprises HCDR1, HCDR2, and / or HCDR3 comprising the amino acid sequences set forth in Table 2 above. In certain embodiments, the GPRC5D-binding portion of the bispecific or multispecific antibodies provided herein comprises LCDR1, LCDR2, and / or LCDR3 comprising the amino acid sequences set forth 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 set forth in Table 2 above. In certain embodiments, the GPRC5D-binding portion of the bispecific or multispecific antibodies provided herein comprises HCDR1, HCDR2, and / or HCDR3 derived from the exemplary 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 described above.In certain embodiments, the GPRC5D-binding portion of the bispecific or multispecific antibodies provided herein comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 derived from the exemplary 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 described above.

[0231] In certain embodiments, the GPRC5D-binding portion of the bispecific or multispecific antibodies provided herein comprises a heavy chain variable region having the amino acid sequence set forth 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 set forth in Table 3 above. In certain embodiments, the GPRC5D-binding portion of the bispecific or multispecific antibodies provided herein comprises a light chain variable region having the amino acid sequence set forth 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 set forth 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 NOs: 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.

[0232] As used herein, the term "CD3 binding portion", when referring to a bispecific or multispecific antibody or an antigen-binding fragment thereof, refers to a portion capable of binding to CD3 (e.g., human CD3, mouse CD3, and cynomolgus CD3). The CD3 binding portion can take any form that enables specific recognition of the target CD3. For example, the CD3 binding portion can 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. In some embodiments, the CD3 binding portion of the bispecific or multispecific antibodies provided herein is derived from an anti-CD3 antibody that binds to and activates primary cultured T cells. The CD3 binding portion of the bispecific or multispecific antibodies provided herein can be derived from any of the anti-CD3 antibodies known in the art, such as those described in US Patent Application Publication No. 20200048348A1, WO2013186613A1, WO2015001085A1, etc.

[0233] In some embodiments, the CD3-binding portion of the bispecific or multispecific antibodies provided herein comprises one or two or three heavy chain complementarity 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 one or two or three light chain complementarity 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 or multispecific antibodies provided herein comprises HCDR1, HCDR2, and / or HCDR3 contained within the VH region sequence of SEQ ID NO: 95, and LCDR1, LCDR2, and / or LCDR3 contained within the VL region sequence of SEQ ID NO: 96. In some embodiments, the CDR boundaries of the CD3-binding portion are identified according to the Kabat convention. In some embodiments, the CDR boundaries of the CD3-binding portion are identified according to the IMGT convention. In some embodiments, the CDR boundaries of the CD3-binding portion are identified according to the Chothia convention. In some embodiments, the CDR boundaries of the CD3-binding portion are identified according to the Al-Lazikani convention.

[0234] In some embodiments, the CD3-binding portion of the bispecific or multispecific antibodies provided herein comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81 or 89, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82 or 90, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83 or 91; and / or LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84 or 92, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 85 or 93, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 86 or 94.

[0235] In some embodiments, the CD3 binding portion of the bispecific or multispecific antibodies provided herein is i. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 85, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 86; or ii. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 90, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 92, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 93, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94 and comprises.

[0236] In some embodiments, the CD3 binding portion of the bispecific or multispecific antibodies provided herein comprises the VH / VL amino acid sequence pair of SEQ ID NO: 87 / 88 or 95 / 96.

[0237] The amino acid sequences of the CDRs, VH region, and VL region of exemplary CD3 binding portions are shown in Table 5 below. The CDR boundaries described in Table 5 below were defined or identified according to the Kabat convention.

[0238]

Table 5

[0239] Exemplary bispecific antibodies or antigen-binding fragments thereof provided herein are (1) A GPRC5D-binding portion comprising HCDR1, HCDR2, and HCDR3 contained within the VH region sequence set forth in SEQ ID NO: 127, and LCDR1, LCDR2, and LCDR3 contained within the VL region sequence set forth in SEQ ID NO: 128; 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; (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; (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; (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; (5) A GPRC5D-binding portion comprising HCDR1, HCDR2, and HCDR3 contained within the VH region sequence set forth in SEQ ID NO: 39, and LCDR1, LCDR2, and LCDR3 contained within the VL region sequence set forth in SEQ ID NO: 40; 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; (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; (7) A GPRC5D-binding portion comprising HCDR1, HCDR2, and HCDR3 contained within the VH region sequence set forth in SEQ ID NO: 79, and LCDR1, LCDR2, and LCDR3 contained within the VL region sequence set forth in SEQ ID NO: 80; 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; (8) A GPRC5D-binding portion comprising HCDR1, HCDR2, and HCDR3 contained within the VH region sequence set forth in SEQ ID NO: 175, and LCDR1, LCDR2, and LCDR3 contained within the VL region sequence set forth in SEQ ID NO: 176; 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; or (9) A GPRC5D-binding portion comprising HCDR1, HCDR2, and HCDR3 contained within the VH region sequence set forth in SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 contained within the VL region sequence set forth in SEQ ID NO: 24; 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 comprising.

[0240] Another exemplary bispecific antibody or antigen-binding fragment thereof provided herein is (1) A GPRC5D-binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 set forth 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 set forth in SEQ ID NOs: 81, 82, 83, 84, 85, and 86, respectively; (2) A GPRC5D-binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 set forth 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 set forth in SEQ ID NOs: 81, 82, 83, 84, 85, and 86, respectively; (3) A GPRC5D-binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 set forth 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 set forth in SEQ ID NOs: 81, 82, 83, 84, 85, and 86, respectively; (4) A GPRC5D-binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 described 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 described in SEQ ID NOs: 81, 82, 83, 84, 85, and 86 respectively; (5) A GPRC5D-binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 described 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 described in SEQ ID NOs: 81, 82, 83, 84, 85, and 86 respectively; (6) A GPRC5D-binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 described 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 described in SEQ ID NOs: 81, 82, 83, 84, 85, and 86 respectively; (7) A GPRC5D-binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 described 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 described in SEQ ID NOs: 81, 82, 83, 84, 85, and 86 respectively; (8) A GPRC5D-binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 described 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 described in SEQ ID NOs: 81, 82, 83, 84, 85, and 86 respectively; or (9) A GPRC5D-binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 described 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 described in SEQ ID NOs: 81, 82, 83, 84, 85, and 86 respectively.

[0241] Another exemplary bispecific antibody or antigen-binding fragment thereof provided herein is (1) A GPRC5D-binding portion comprising HCDR1, HCDR2, and HCDR3 contained within the VH region sequence of SEQ ID NO: 183, and LCDR1, LCDR2, and LCDR3 contained within the VL region sequence of SEQ ID NO: 184; and a CD3-binding portion comprising HCDR1, HCDR2, and HCDR3 contained within the VH region sequence of SEQ ID NO: 95, and LCDR1, LCDR2, and LCDR3 contained within the VL region sequence of SEQ ID NO: 96; (2) A GPRC5D-binding portion comprising HCDR1, HCDR2, and HCDR3 contained within the VH region sequence of SEQ ID NO: 71, and LCDR1, LCDR2, and LCDR3 contained within the VL region sequence of SEQ ID NO: 72; and a CD3-binding portion comprising HCDR1, HCDR2, and HCDR3 contained within the VH region sequence of SEQ ID NO: 95, and LCDR1, LCDR2, and LCDR3 contained within the VL region sequence of SEQ ID NO: 96; or (3) A GPRC5D-binding portion comprising HCDR1, HCDR2, and HCDR3 contained within the VH region sequence of SEQ ID NO: 79, and LCDR1, LCDR2, and LCDR3 contained within the VL region sequence of SEQ ID NO: 80; and a CD3-binding portion comprising HCDR1, HCDR2, and HCDR3 contained within the VH region sequence of SEQ ID NO: 95, and LCDR1, LCDR2, and LCDR3 contained within the VL region sequence of SEQ ID NO: 96 comprises.

[0242] Another exemplary bispecific antibody or antigen-binding fragment thereof provided herein is (1) a GPRC5D-binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 set forth 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 set forth in SEQ ID NOs: 89, 90, 91, 92, 93, and 94, respectively; (2) a GPRC5D-binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 set forth 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 set forth in SEQ ID NOs: 89, 90, 91, 92, 93, and 94, respectively; or (3) a GPRC5D-binding portion comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 set forth 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 set forth in SEQ ID NOs: 89, 90, 91, 92, 93, and 94, respectively.

[0243] Different formats of bispecific or multispecific antibodies are described in the art, for example, in Chames and Baty (2009) Curr Opin Drug Disc Dev 12:276. The bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein are not limited to any particular bispecific format and may have all different formats known in the art.

[0244] For example, the bispecific antibodies or antigen-binding fragments thereof provided herein can have a typical full-length antibody structure, i.e., an antibody having two different full-length antibody heavy chains and two different full-length antibody light chains. The full-length antibody heavy chain includes a heavy chain variable region (VH) and constant domains CH1, CH2, CH3, and optionally CH4. The full-length antibody light chain includes a light chain variable region (VL) and a constant domain CL. For example, the bispecific antibodies or antigen-binding fragments thereof provided herein may include 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 pair to form a first antigen-binding site that binds to GPRC5D, and the second light chain and the second heavy chain pair to form a second antigen-binding site that binds to CD3. With respect to another example, the bispecific antibodies or antigen-binding fragments thereof provided herein may include 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 pair to form a first antigen-binding site that binds to CD3, and the second light chain and the second heavy chain pair to form a second antigen-binding site that binds to GPRC5D.

[0245] For another example, the bispecific or multispecific antibodies or antigen-binding fragments thereof provided herein may include, but are not limited to, bispecific antibodies having complementary CH3 domains that form heterodimers, knob-into-hole molecules (Genentech, WO9850431), CrossMAbs (Roche, WO2009080253), or electrostatically matched molecules (Amgen, European Patent No. 1870459 and WO2009089004; Chugai, US Patent Application Publication No. 201000155133; Oncomed, WO2010129304). For example, with respect to the CrossMab format, the CD3-binding portion of the bispecific or multispecific antibodies provided herein further includes the constant domain CL and the constant domain CH1, and in some embodiments, the constant domains CL and CH1 are exchanged with each other; or the GPRC5D-binding portion of the bispecific or multispecific antibodies provided herein further includes the constant domain CL and the constant domain CH1, and in some embodiments, the constant domains CL and CH1 are exchanged with each other.

[0246] In some embodiments, the bispecific or multispecific antibodies provided herein further include an Fc region. In some embodiments, the bispecific or multispecific antibodies provided herein further include the Fc region of a human immunoglobulin (Ig), or optionally the Fc region of a human IgG. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3, or IgG4.

[0247] For another example, the bispecific antibody or antigen-binding fragment thereof provided herein may be a bifunctional fusion protein that targets 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), and the CD3-binding portion is linked to the GPRC5D-binding portion directly or via a linker. The CD3-binding portion may be linked to the N-terminus of the variable region of the GPRC5D-binding portion, to the C-terminus of the Fc region of the GPRC5D-binding portion, or to the C-terminus of the CL constant domain of the GPRC5D-binding portion. In some embodiments, the CD3-binding portion may be or may comprise an scFv comprising a VH region linked to a VL region directly or via a linker, and the VH region of the CD3-binding portion is linked to the GPRC5D-binding portion directly or via a linker. In some embodiments, the CD3-binding portion may be or may comprise an scFv comprising a VH region linked to a VL region directly or via a linker, and the VL region of the CD3-binding portion is linked to the GPRC5D-binding portion directly or via a linker.

[0248] As another example, the bispecific antibodies or antigen-binding fragments thereof provided herein may be bifunctional fusion proteins that target 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), and the GPRC5D-binding portion is linked to the CD3-binding portion directly or via a linker. The GPRC5D-binding portion may be linked to the N-terminus of the variable region of the CD3-binding portion, to the C-terminus of the Fc region of the CD3-binding portion, or to the C-terminus of the CL constant domain of the CD3-binding portion. In some embodiments, the GPRC5D-binding portion may be or may comprise an scFv comprising a VH region linked directly or via a linker to a VL region, and the VH region of the GPRC5D-binding portion is linked to the CD3-binding portion directly or via a linker. In some embodiments, the GPRC5D-binding portion may be or may comprise an scFv comprising a VH region linked directly or via a linker to a VL region, and the VL region of the GPRC5D-binding portion is linked to the CD3-binding portion directly or via a linker.

[0249] The term "linker", as used herein, refers to an artificial amino acid sequence having a length between 1, 2, 3, 4 or 5 amino acid residues, or between 5 and 15, 20, 30, 50 or more amino acid residues, that is linked by peptide bonds and used to link 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).

[0250] 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 helical linker, and a non-helical linker. Any suitable linker known in the art can be used. In certain embodiments, the linker comprises a peptide linker. For example, peptide linkers useful in the present disclosure may be rich in glycine and serine residues. Examples include single or repeating sequences containing threonine / serine and glycine, such as TGGGG (SEQ ID NO: 164), GGGGS (SEQ ID NO: 165) or SGGGG (SEQ ID NO: 166) or tandem repeats thereof (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats). 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 repeats 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%, at least 99% sequence identity to any one of SEQ ID NOs: 164-168.

[0251] Conjugate In some embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein further comprise one or more conjugate moieties. The conjugate moieties can be linked to the antibody or antigen-binding fragment thereof. A conjugate moiety is a moiety that can bind to the antibody or antigen-binding fragment thereof. It is contemplated that a variety of conjugate moieties can be linked to the antibodies or antigen-binding fragments thereof provided herein (see, e.g., “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate moieties can be linked to the antibody or antigen-binding fragment thereof, among other ways, by covalent bonding (e.g., disulfide bonding), affinity binding, intercalation, cooperative binding, complex formation, association, admixture, or addition. In some embodiments, the antibody or antigen-binding fragment thereof can be linked to one or more conjugates via a linker or crosslinker. The linker or crosslinker comprises reactive chemical groups that can react with the anti-GPRC5D antibody or fragment thereof. The reactive chemical groups can be N-succinimidyl esters and N-sulfosuccinimidyl esters. In addition, the linker can comprise reactive chemical groups that can be dithiopyridyl groups that can react with a 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., Biochem. J., 173:723-737 (1978)), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Patent No. 4,563,304), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB) (see U.S. Patent Application Publication No. 20090274713), N-succinimidyl 4-(2-pyridyldithio)pentaenoate (SPP) (see, e.g., CAS Registry Number 341498-08-6), 2-iminothiolane, or succinic anhydride. For example, an antibody or cell binding agent can be modified with a crosslinking reagent, and the antibody or cell binding agent containing a free or protected thiol group thus derived is reacted with a disulfide or thiol-containing maytansinoid 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.

[0252] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein can be engineered to contain specific sites outside of the epitope-binding portion that can be utilized to bind 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 bonding with the conjugate moiety.

[0253] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein may be indirectly linked to the conjugate moiety or through another conjugate moiety. For example, the antibodies or antigen-binding fragments thereof provided herein may be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin. In some embodiments, the conjugate moiety includes a clearance modifier (e.g., a polymer such as PEG that extends the half-life), 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 binder, a purification moiety or other anti-cancer agent (e.g., an agonist of toll-like receptor 7 (TLR-7), TLR-8 and / or TLR-9, siRNA, an antibody or antigen-binding fragment thereof, a peptide (e.g., a small peptide), etc.).

[0254] A "toxin" can be any agent that is harmful to cells, or can damage or kill cells. Examples of toxins include, but are not limited to, taxol, taxoids, CC-1065 and CC-1065 analogs, duocarmycin and duocarmycin analogs, enediynes such as calicheamicin, dolastatin and dolastatin analogs including auristatin, tomatamycin derivatives, leptomycin derivatives, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil and morpholinodoxorubicin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, DM4, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mitramycin, actinomycin 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., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiammineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin and anthramycin (AMC)), mitotic inhibitors (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin binders.

[0255] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C, 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 P, other lanthanides), luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules or gold for detection may be mentioned.

[0256] In certain embodiments, the conjugate moiety can be a clearance modifier that helps increase the half-life of the antibody. Exemplary examples include water-soluble polymers such as PEG, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol / propylene glycol and the like. The polymer can be a polymer of any molecular weight and can be branched or unbranched. The number of polymers binding to the antibody can vary and, if more than one polymer is bound, they can be the same or different molecules.

[0257] In certain embodiments, the conjugate moiety can be a purification moiety such as magnetic beads.

[0258] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are used as a base for conjugates.

[0259] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are conjugated to a signal peptide. A signal peptide (sometimes called a signal sequence, leader sequence, or leader peptide) can be used to facilitate the secretion and isolation of the antibodies or antigen-binding fragments thereof provided herein. Signal peptides are typically characterized by a core of hydrophobic amino acids that are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature protein since they pass through the secretory pathway. Thus, the present invention relates to the polypeptides described having a signal sequence, as well as polypeptides in which the signal sequence has been proteolytically cleaved (i.e., cleavage products). In one embodiment, a nucleic acid sequence encoding the signal sequence can be operably linked in an expression vector to a protein of interest, such as a protein that is not normally secreted or is otherwise difficult to isolate. The signal sequence directs, for example, the secretion of the protein from a eukaryotic cell host into which the expression vector is transformed, and the signal sequence is cleaved thereafter or simultaneously. The protein can then be readily purified from the extracellular medium by methods recognized in the art. Alternatively, the signal sequence can be linked to the protein of interest using a sequence that facilitates purification, such as a GST domain.

[0260] Chimeric antigen receptor In certain embodiments, the present disclosure provides chimeric antigen receptors comprising the antibodies or antigen-binding fragments thereof provided herein, a transmembrane region, and an intracellular signaling region.

[0261] The terms "chimeric antigen receptor", "CAR", or "CARs", as used herein, refer to an engineered receptor that grafts antigen specificity onto a cell (e.g., a T cell, such as a naive T cell, central memory T cell, effector memory T cell, regulatory T cell, or a combination thereof). A CAR is also known as an artificial T cell receptor, chimeric T cell receptor, or chimeric immunoreceptor. In some embodiments, a CAR comprises an antigen-specific targeting region (e.g., an antigen-binding fragment of an anti-GPRC5D antibody provided herein), an extracellular region, a transmembrane region, one or more co-stimulatory regions, and an intracellular signaling region.

[0262] In some embodiments, the antigen-specific targeting region is a scFv. In some embodiments, the transmembrane region comprises the transmembrane region of CD3, CD4, CD8, or CD28. In some embodiments, the co-stimulatory region comprises the co-stimulatory domains of CD28, ICOS, CD27, 4-1BB, OX40, and CD40L. In some embodiments, the intracellular signaling region is selected from the group consisting of the intracellular signaling region sequences 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 the amino acid sequence set forth in SEQ ID NO: 203. In some embodiments, the 4-1BB intracellular signaling region comprises the amino acid sequence set forth in SEQ ID NO: 204. In some embodiments, the CD3ζ intracellular signaling region comprises the amino acid sequence set forth in SEQ ID NO: 205. In some embodiments, the intracellular signaling region comprises a 4-1BB intracellular signaling region comprising the amino acid sequence set forth in SEQ ID NO: 204, and a CD3ζ intracellular signaling region comprising the amino acid sequence set forth in SEQ ID NO: 205.

[0263] A CAR can be grafted into various cells, such as allogeneic cells, autologous cells, or xenogeneic cells.

[0264] The term "allogeneic cell", as used herein, refers to any cell derived from a different subject of the same species.

[0265] As used herein, the term "autologous cell" refers to any cell derived from the same subject that is later reintroduced.

[0266] As used herein, the term "heterologous cell" refers to any cell derived from a different subject of a different species.

[0267] In some embodiments, the CAR is grafted into immune effector cells such as T cells, natural killer cells, macrophage cells, tumor infiltrating lymphocytes, and the like.

[0268] In some embodiments, the CAR provided herein exhibits cytotoxicity equal to or better than that of a CAR known in the art that targets GPRC5D, such as the CAR disclosed in WO2020092854A2. In some embodiments, the CAR provided herein exhibits cytotoxicity equal to or better than that of BMK-203. The term "BMK-203" includes an scFv targeting GPRC5D, a CD8 transmembrane region, a 4-1BB intracellular signaling region, and a CD3ζ intracellular signaling region. The scFv targeting GPRC5D includes a VH region comprising the amino acid sequence set forth in SEQ ID NO: 201 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 202; the CD8 transmembrane region comprises the amino acid sequence set forth in SEQ ID NO: 203; the 4-1BB intracellular signaling region comprises the amino acid sequence set forth in SEQ ID NO: 204; and the CD3ζ intracellular signaling region comprises the amino acid sequence set forth in SEQ ID NO: 205. The amino acid sequences of SEQ ID NOs: 201, 202, 203, 204, and 205 are shown below.

[0269]

Table B

[0270] Polynucleotides and Recombinant Methods The present disclosure provides an isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof provided herein, and / or a chimeric antigen receptor. As used herein, the terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and polymers thereof in single-stranded or double-stranded form. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0271] The DNA encoding an antibody or an antigen-binding fragment thereof provided herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibody). The encoding DNA can also be obtained by synthetic methods.

[0272] The isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof and / or a chimeric antigen receptor provided herein can be inserted into a vector using recombinant techniques known in the art for further cloning (amplification of DNA) or expression. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0273] The present disclosure provides a vector comprising an isolated polynucleotide provided herein. In certain embodiments, the polynucleotide provided herein is operably linked to at least one promoter (e.g., SV40, CMV, EF-1α), and encodes at least one selectable marker, and an antibody or an antigen-binding fragment thereof and / or a chimeric antigen receptor provided herein. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage, and M13 phage, plasmid 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, p15TV-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.

[0274] A vector comprising a polynucleotide sequence encoding an antibody or an antigen-binding fragment thereof and / or a chimeric antigen receptor provided herein can be introduced into a host expression system (e.g., a host cell) for cloning or gene expression. In certain embodiments, the host expression systems provided herein are microorganisms, yeasts, or mammalian cells. In certain embodiments, the microorganisms are selected from the group consisting of Escherichia coli and Bacillus subtilis. In certain embodiments, the yeast is Saccharomyces. In certain embodiments, the mammalian cells are selected from the group consisting of COS, CHO-S, CHO-K1, HEK-293, and 3T3 cells.

[0275] Suitable host cells for cloning or expressing DNA in the vectors herein are the prokaryotic cells, yeasts, or higher eukaryotic cells described above. Suitable prokaryotic cells for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella as well as Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0276] In addition to prokaryotic cells, eukaryotic cell microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for anti-GPRC5D antibody-encoding vectors. Saccharomyces cerevisiae or baker's yeast is most commonly used among lower eukaryotic cell host microorganisms. However, many other genera, species, and strains, 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 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Trichopodium, and Aspergillus hosts, such as A. nidulans and A. niger are generally available and useful herein.

[0277] Suitable host cells for expressing the glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms. Examples of invertebrates include plant and insect cells. A number of baculovirus strains and variants, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm) have been identified. A variety of virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 variant of Bombyx mori NPV, have been published, and such viruses can be used as the viruses herein according to the invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as host cells.

[0278] However, of greatest interest are vertebrate cells, and growing vertebrate cells in culture (tissue culture) is a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human fetal kidney cell lines (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); dog 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, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; mouse pre-gastric cancer cells (MFC), SNU620 cells, and human hepatoma cell line (HepG2). In some embodiments, the host cells are mammalian cultured cell lines such as CHO, BHK, NS0, 293, MFC, SNU620 and derivatives thereof.

[0279] Transform the host cell with the above expression or cloning vector for antibody production and culture it in a conventional nutrient medium appropriately modified for inducing the promoter, selecting transformants, or amplifying the gene encoding the desired sequence. In another embodiment, the antibody may be produced by homologous recombination known in the art. In certain embodiments, the host cell is capable of producing the antibodies or antigen-binding fragments thereof provided herein.

[0280] The present disclosure also provides a method of expressing an antibody or an antigen-binding fragment thereof and / or a chimeric antigen receptor provided herein, the method comprising culturing a host expression system provided herein under conditions in which the antibody or an antigen-binding fragment thereof and / or the chimeric antigen receptor is expressed. The host expression systems used to produce the antibodies or antigen-binding fragments thereof and / or chimeric antigen receptors provided herein can be cultured in a variety of media. Commercially available media such as Ham’s F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco’s Modified Eagle Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90 / 03430; WO87 / 00195; or U.S. Patent No. 30,985 may be used as a culture medium for host cells. Any of these media may be supplemented 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™ drug), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source, as needed. Other optional supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions such as temperature, pH, and the like are those previously used for the host cells selected for expression and will be apparent to those of skill in the art.

[0281] Using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the medium. When antibodies are produced intracellularly, the first step is to remove particulate debris, which is either host cells or lysed fragments, for example, by centrifugation or ultracentrifugation. Carter et al., Bio / Technology 10:163-167 (1992) describes procedures for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed over about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When antibodies are secreted into the medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. To inhibit proteolysis, protease inhibitors, such as PMSF, may be included in any of the foregoing steps, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0282] Antibodies or antigen-binding fragments thereof and / or chimeric antigen receptors prepared from a host expression system can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0283] In certain embodiments, Protein A immobilized on a solid phase is used for the immunoaffinity purification of antibodies and their antigen-binding fragments and / or chimeric antigen receptors. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains can be purified using Protein A (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is bound is most often agarose, although other matrices are available. Mechanically stable matrices, such as porous glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABXTM resin (J.T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification, such as fractionation on an ion-exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, heparin chromatography, SEPHAROSE™ chromatography on an anion or cation exchange resin (e.g., polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0284] After any optional preliminary purification step(s), the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography, preferably at a low salt concentration (e.g., about 0-0.25 M salt), using an elution buffer between about pH 2.5 and 4.5.

[0285] Pharmaceutical composition The present disclosure further provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof and / or a chimeric antigen receptor provided herein, and one or more pharmaceutically acceptable carriers.

[0286] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquids, gels, or solid-phase carriers, aqueous media, non-aqueous media, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, metal ion sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0287] Suitable components can include, for example, antioxidants, bulking agents, binders, disintegrants, buffers, preservatives, lubricants, flavorants, 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 hydroxytoluene, and / or propyl gallate. As disclosed herein, including one or more antioxidants, such as methionine, in a composition comprising an antibody or an antigen-binding fragment thereof and a conjugate provided herein reduces the oxidation of the antibody or an antigen-binding fragment thereof. This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving the stability of the antibody and maximizing its shelf life. Thus, in certain embodiments, a pharmaceutical composition is provided that comprises one or more antibodies or antigen-binding fragments thereof disclosed herein and one or more antioxidants, such as methionine. Further, a method is provided for preventing oxidation of an antibody or antigen-binding fragment provided herein, extending its shelf life, and / or improving its effectiveness by mixing the antibody or antigen-binding fragment with one or more antioxidants, such as methionine.

[0288] To further illustrate, pharmaceutically acceptable carriers can include, for example, sodium chloride injection solution, Ringer's injection solution, isotonic dextrose injection solution, sterile water injection solution, or dextrose and lactate Ringer's injection solution, non-aqueous media such as fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffering agents 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, hydroxypropylmethylcellulose, or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), metal ion sequestering agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antibacterial agents used as carriers can be added to pharmaceutical compositions in multi-dose containers containing phenol or cresol, mercury compounds, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubilizing enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0289] 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, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0290] In certain embodiments, the pharmaceutical composition is formulated into an injectable preparation. The injectable pharmaceutical composition can be prepared in any convenient form, such as a liquid solution, suspension, emulsion, or a solid form suitable for generating a liquid solution, suspension, or emulsion. The preparation of the injection includes a sterile and / or pyrogen-free solution ready for injection, a sterile dry soluble product, such as an implantable tablet, a lyophilized powder ready to be mixed with a solvent immediately before use, a sterile suspension ready for injection, a sterile dry insoluble product ready to be mixed with a vehicle immediately before use, and a sterile and / or pyrogen-free emulsion. The solution can be either aqueous or non-aqueous.

[0291] In certain embodiments, the unit dose parenteral preparation is packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration are known and practiced in the art and must be sterile and pyrogen-free.

[0292] In certain embodiments, the sterile lyophilized powder is prepared by dissolving the antibodies or antigen-binding fragments disclosed herein in a suitable solvent. The solvent may contain excipients that improve stability, or other pharmacological components of the powder or the reconstitution solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain, for example in one embodiment, a buffer at approximately neutral pH, such as citric acid, sodium or potassium phosphate, or other such buffers known to those of skill in the art. The desired formulation is provided by lyophilization under standard conditions known to those of skill in the art after subsequent filter sterilization of the solution. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single dosage or multiple dosages of the antibody or its antigen-binding fragment or composition thereof. Overfilling the vial with a small amount (e.g., about 10%) in excess of the amount required for the dosage or dosage set is acceptable to facilitate accurate aspiration of the sample and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as from about 4°C to room temperature.

[0293] Reconstituting the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, sterile water and / or pyrogen-free water, or other suitable carriers for liquids, are added to the lyophilized powder for reconstitution. The exact amount depends on the chosen treatment being administered and can be determined empirically.

[0294] Kit In certain embodiments, the present 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 present 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, anti-cancer agents, radiation therapy agents, immunotherapy agents, anti-angiogenesis agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormone therapy agents, anti-viral agents, antibiotics, analgesics, antioxidants, metal chelating agents, and cytokines.

[0295] Such kits may further comprise, if desired, one or more pharmaceutically acceptable carriers, such as containers having one or more additional containers, etc., which are readily apparent to those skilled in the art, and various conventional pharmaceutical kit components. Instructions for use, either as an insert or label indicating the amount of components to be administered, guidelines for administration, and / or guidelines for mixing the components can also be included in the kit.

[0296] Method of Use The present disclosure also provides a method of treating, preventing, or alleviating a disease, disorder, or condition in a subject, the method 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 a human.

[0297] In some embodiments, the GPRC5D-related disease, disorder, or condition is characterized by the expression or overexpression of GPRC5D.

[0298] In certain embodiments, the disease, disorder, or condition is cancer. In certain embodiments, the cancer is a GPRC5D-expressing cancer. As used herein, a "GPRC5D-expressing" cancer is characterized by expressing the GPRC5D protein in cancer cells, tumor-infiltrating immune cells, or expressing GPRC5D at a level significantly higher than expected for normal cells in cancer cells, tumor-infiltrating immune cells. Various methods can be used to determine the presence and / or amount of GPRC5D in a test biological sample from a subject. For example, the test biological sample can be exposed to an anti-GPRC5D antibody or an antigen-binding fragment thereof that binds to and detects the expressed GPRC5D protein. Alternatively, GPRC5D can also be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, and the like. In some embodiments, the test sample is derived from cancer cells or tissue, 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 whom 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 certain embodiments, the cancer is a solid tumor or a hematological tumor. In certain embodiments, the cancer is a GPRC5D-expressing B cell cancer.

[0299] In certain embodiments, the disease, disorder, or condition is lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lung adenocarcinoma, or squamous cell lung cancer), abdominal cancer, carcinoid cancer, bone cancer, pancreatic cancer, undifferentiated neuroectodermal tumor, skin cancer, gallbladder cancer, head or neck cancer, squamous cell carcinoma, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial cancer), anal area cancer (e.g., anal squamous cell carcinoma), gastric cancer or stomach cancer (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatoma, or liver cancer), cholangiocarcinoma, 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 cancer or ureteral cancer (e.g., renal oncocytoma), cutaneous T-cell lymphoma, medulloblastoma, nephroblastoma, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, choroid plexus papilloma, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) neoplasms, soft tissue sarcomas (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi sarcoma), spinal cord tumors, gliomas (e.g., ependymoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, eye cancer (e.g., retinoblastoma), brainstem glioma, or mixed glioma, e.g., oligoastrocytoma), brain tumors (e.g., glioblastoma / multiform glioblastoma (GBM), non-glioblastoma brain tumors, or meningioma), skin or intraocular melanoma, thrombocytosis, mesothelioma, mycosis fungoides, Sézary syndrome, idiopathic myelofibrosis, solitary plasmacytoma, vestibular schwannoma, Ewing sarcoma, chondrosarcoma, MYH-associated polyposis, pituitary adenoma, pediatric cancer, e.g., pediatric sarcomas (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 myelogenous leukemia (CML), acute myelogenous leukemia (AML), chronic myelomonocytic leukemia (CMML), chronic lymphoblastic leukemia, acute lymphoblastic leukemia, hairy cell leukemia (HCL), Burkitt lymphoma (BL), multiple myeloma (e.g.,Selected from the group consisting of relapsed or refractory multiple myeloma, T 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.

[0300] In some embodiments, the subject is identified as having cancer cells or tumor-infiltrating immune cells that express GPRC5D at levels significantly higher than levels normally found in non-cancer cells, optionally.

[0301] In another aspect, a method of treating, preventing, or alleviating a disease, disorder, or condition in a subject that would benefit from modulation of GPRC5D activity, the method 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.

[0302] The therapeutically effective amount of the antibody or antigen-binding fragment provided herein depends on various factors known in the art, such as, for example, the subject's body weight, age, past medical history, current medications, health status, and potential for cross-reactivity, allergies, hypersensitivity, and adverse side effects, as well as the route of administration and the degree of disease onset. One of ordinary skill in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dosage as indicated by these and other circumstances or requirements.

[0303] In certain embodiments, the antibodies or antigen-binding fragments provided herein and / or the chimeric antigen receptors 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 vary over the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than subsequent administered doses. In certain embodiments, the administered dose may vary during the course of treatment in response to the subject's response.

[0304] The dosing regimen may be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0305] 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, administration can be by parenteral routes including subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection; or by non-parenteral routes including transdermal, oral, intranasal, intraocular, sublingual, rectal, or topical surface.

[0306] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein and / or the chimeric antigen receptors provided herein can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibodies or antigen-binding fragments thereof disclosed herein and / or the chimeric antigen receptors provided herein can be administered in combination with a second therapeutic agent such as a chemotherapeutic agent, an anti-cancer agent, a radiation therapy 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 chelating agent, a cytokine, an activator, a contrast 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 a functional fragment thereof, a detectable label or reporter, an antibacterial agent, a gene editing agent, a beta agonist, a viral RNA inhibitor, a polymerase inhibitor, an interferon, or a microRNA.

[0307] As used herein, the term "immunotherapy" refers to a type of treatment that stimulates the immune system to fight diseases such as cancer or enhances the immune system in a general way. Examples of immunotherapy include, but are not limited to, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

[0308] "Targeted therapy" is a type of treatment 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, thereby preventing the effect of the therapeutic agent from reaching normal tissues.

[0309] In certain of these embodiments, the antibodies or antigen-binding fragments thereof provided herein, and / or the chimeric antigen receptors provided herein, and / or the pharmaceutical compositions provided herein, which are administered in combination with one or more additional therapeutic agents, may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the antibodies or antigen-binding fragments thereof and / or pharmaceutical compositions provided herein, and the additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition. However, the antibodies or antigen-binding fragments thereof and / or chimeric antigen receptors and / or pharmaceutical compositions provided herein that are administered "in combination with" another therapeutic agent need not be administered simultaneously with or in the same composition as the agent. An antibody or antigen-binding fragment thereof, or a chimeric antigen receptor or pharmaceutical composition that is administered before or after another agent is considered to be administered "in combination with" that agent as the phrase is used herein, even if the antibody or antigen-binding fragment or pharmaceutical composition or chimeric antigen receptor and the second agent are administered via different routes. Where possible, additional therapeutic agents that are administered in combination with the antibodies or antigen-binding fragments thereof, chimeric antigen receptors or pharmaceutical compositions disclosed herein are administered according to the schedule set forth in the package insert for the additional therapeutic agent, or according to Physicians’ Desk Reference 2003 (Physicians’ Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)) or protocols well known in the art.

[0310] The present disclosure further provides a method of inactivating GPRC5D-expressing cells in vivo or in vitro, the method comprising contacting the GPRC5D-expressing cells 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.

[0311] The present disclosure further provides a method of modulating GPRC5D activity in GPRC5D-expressing cells, the method comprising exposing the GPRC5D-expressing cells 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.

[0312] In another aspect, the present disclosure provides a method of detecting the presence or amount of GPRC5D in a sample, the method comprising contacting the sample 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, and determining the presence or amount of GPRC5D in the sample.

[0313] In another aspect, the present disclosure provides a method of diagnosing a GPRC5D-related disease, disorder, or condition in a subject, the method 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) correlating the presence or amount of GPRC5D with the presence or status of a GPRC5D-related disease, disorder, or condition in the subject.

[0314] In another aspect, the present 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, optionally conjugated to a detectable moiety useful for detecting GPRC5D, optionally recombinant GPRC5D, GPRC5D expressed on the cell surface, or GPRC5D-expressing cells. The kit may further comprise instructions for use.

[0315] In another aspect, the present disclosure also provides for the use of the antibodies or antigen-binding fragments thereof provided herein and / or the chimeric antigen receptors provided herein and / or the pharmaceutical compositions provided herein in the manufacture of a medicament for treating, preventing, or alleviating a GPRC5D-related disease, disorder, or condition in a subject, and in the manufacture of a diagnostic agent for diagnosing a GPRC5D-related disease, disorder, or condition.

[0316] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials, and methods described below fall, in whole or in part, within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but are merely intended to illustrate specific embodiments that fall within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials, and methods without practicing the capabilities of the invention and without departing from the scope of the invention. It is understood that many variations can be made to the procedures described herein and still be within the scope of the invention. It is the intention of the inventors that such variations be included within the scope of the invention.

Examples

[0317] [Example 1] Generation of Antibodies 1.1 Immunization To generate antibodies against GPRC5D, Balb / c and SJL mice were immunized (as shown in Table 6 below) 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-expressing DNA construct pTT5-H5 (human GPRC5D), referred to as "gene immunization"), or GPRC5D protein (i.e., recombinant human GPRC5D virus-like particles (hGPRC5D VLP), referred to as "protein immunization") in each group. After the primary immunization, several booster immunizations were performed until the animals produced a satisfactory antiserum titer suitable for hybridoma production. To compare the results and immune responses at the serum level, all immunization strategies were carried out in parallel. The immunization protocols for each group of animals are shown in Table 6 below.

[0318]

Table 6

[0319]

Table 7

[0320]

Table 8

[0321]

Table 9

[0322] 1.2 Generation and Screening of Hybridomas Spleen cell fusion was performed on the mice that responded best to immunization as determined by test bleed FACS. Lymphocytes from the spleen and lymph nodes were fused to the mouse myeloma cell line (SP2 / 0-Ag14) using an optimized electrofusion protocol. Multiple fusions were performed to ensure the success of the project.

[0323] The fusions were seeded into multiple 96-well plates (1×10 4 ~10 5 cells / well). The plates were monitored for growth and the medium was changed weekly. Wells with cell growth were screened on days 10 - 14 by a primary screening assay using a Mirrorball assay and / or other feasible assays. Multiple fusions of each targeted antigen were performed and screened. From the primary screening, positive parental clones showing binding positivity to HEK293-hGPRC5D were expanded into 24-well plates for secondary screening. After the primary screening, the positive parental clones expanded into 24-well plates were rescreened by the assays described in the following hybridoma screening funnel. The desired hybridomas were selected and advanced to subcloning.

[0324] The parental hybridomas with the desired reactivity and isotype from the above screening funnel were subcloned by multiple rounds of limiting dilution or single cell sorting until monoclonal antibodies were obtained.

[0325] The subcloning plates were screened by the Mirrorball assay, and subclones with good binding ability were expanded to 24-well plates for confirmation tests. The specificity and cross-reactivity of these subclones were confirmed by FACS analysis. Briefly, parental HEK293T, HEK293T cells stably overexpressing human GPRC5D protein (i.e., HEK293T-hGPRC5D), HEK293T cells stably overexpressing cynomolgus GPRC5D protein (i.e., HEK293T-cynoGPRC5D), HEK293T cells stably overexpressing mouse GPRC5D protein (i.e., HEK293T-mGPRC5D), and OPM2 cells were each incubated with the antibodies produced by each subclone. A fluorescent dye-conjugated secondary antibody was used to detect the binding of the primary antibody to the cells. The median fluorescence intensity (MFI) was measured by FACS analysis.

[0326] The desired subclone cell lines were sequenced and further expanded to culture flasks for cryopreservation. 0.5 - 1.0×10 7 cells / vial were initially cryopreserved in 4 - 6 vials per cell line. If desired, master cell banks and working cell banks can be established for the most valuable cell lines selected.

[0327] As a result, 21 antibodies with unique sequences showing binding positivity to HEK293T-hGPRC5D and OPM2 cells were discovered. Among them, some clones were also able to bind to HEK293T-cynoGPRC5D and / or HEK293T-mGPRC5D. The MFI and positive percentage of antibodies staining OPM2 cells, parental HEK293T, HEK293T-hGPRC5D, HEK293T-cynoGPRC5D, and HEK293T-mGPRC5D detected by FACS were summarized in Tables 10A, 10B, and 10C below, suggesting that these antibodies were GPRC5D recognition antibodies. ATG596 was a human IgG positive control antibody containing a VH region with the amino acid sequence set forth in SEQ ID NO: 199 and a VL region with the amino acid sequence set forth in SEQ ID NO: 200.

[0328] [Table 10A]

[0329] [Table 10B]

[0330] [Table 10C]

[0331] [Example 2] Characterization of Antibodies: Binding Affinity for Human, Cynomolgus Monkey, and Mouse GPRC5D 2.1 Antibodies 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.

[0332] 2.2 Sequencing of Hybridomas Total RNA was isolated from hybridoma cells according to the technical manual of RNAiso Plus (TAKARA catalog number 9109). Next, the total RNA was reverse transcribed into cDNA using an isotype-specific antisense primer or a universal primer according to the technical manual of PrimeScript II 1st Strand cDNA Synthesis Kit (TAKARA catalog number 6210A). The VH and VL antibody fragments were amplified according to TaKaRa Taq (trademark) (catalog number R001A). The amplified antibody fragments were individually cloned into a standard cloning vector. Colony PCR was performed to screen for clones with inserts of the correct size. Five or more colonies with inserts of the correct size were sequenced for each fragment. The sequences of different clones were aligned to provide the consensus sequence of these clones. The variable region sequences of the hybridoma antibodies are provided in Table 3 above herein.

[0333] 2.3 Generation of Chimeric Antibodies All sequences of the 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) from Table 10A, Table 10B, and Table 10C and the reference antibody ATG596 were selected to generate and produce human IgG1 chimeric antibodies. DNA encoding the variable regions of the 21 hybridoma antibodies and the reference antibody ATG596 was synthesized and subcloned into an expression vector pre-containing the 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" indicates "chimeric" and the following indicates the hybridoma antibody clone. For example, ch36A3G8 indicates that it is a chimeric antibody derived from the hybridoma antibody clone 36A3G8.

[0334] 2.4 Characterization of Chimeric Antibodies (Cell-Based Binding Affinity for GPRC5D) The binding affinities of the chimeric antibodies and the reference antibody ATG596 to CHOS-hGPRC5D, HEK293T-cynoGPRC5D, and HEK293T-mGPRC5D were determined by FACS analysis. The protocol for FACS analysis is described below: (a) Cells were washed with staining buffer (1% BSA in PBS), and the cell density was adjusted to 2×10 cells with staining buffer5 Adjusted to per well. (b) 50 μL of the test antibody diluted in a two-fold stepwise manner and cells were added to each well and incubated at 4 °C for 1 hour. (c) Centrifuged at 300 g for 3 minutes and the supernatant was discarded. (d) The cells were washed with 200 μL of staining buffer and step (c) was repeated. (e) The cells were incubated with 100 μl of the secondary antibody (APC rat anti-human IgG Fc antibody, diluted 1:100) in staining buffer at 4 °C for 0.5 hour. (f) Centrifuged at 300 g for 3 minutes and the supernatant was discarded. (g) The cells were washed with 200 μL of staining buffer and step (f) was repeated. (h) The cells were resuspended with 120 μL of staining buffer and analyzed by FACS.

[0335] As shown in Figures 1A, 1B, 1C, and Table 11 below, 18, 13, and 15 of the chimeric antibodies each showed positive binding affinity for human, cynomolgus monkey, and mouse GPRC5D, respectively.

[0336]

Table 11-1

Table 11-2

[0337] [Example 3] Characterization of Antibodies: T Cell-Dependent Cytotoxicity 3.1 Generation of Bispecific hIgG-scFv Antibodies Bispecific antibodies were generated and the T cell-dependent cytotoxic ability mediated by all candidates was evaluated. hIgG-scFv was used as the format of the generated bispecific antibodies. Each bispecific antibody contained the chimeric antibody and CD3-targeted scFv generated in Example 2, and the CD3-targeted scFv containing the VH region with the amino acid sequence described in SEQ ID NO: 87 and the VL region with the amino acid sequence described in SEQ ID NO: 88 was linked to the C-terminus of the Fc domain of the chimeric antibody by a (GGGGS)3 linker. The LALA mutation (L234A, L235A, according to EU numbering) was introduced into each bispecific antibody to abolish Fc receptor binding ability. The obtained bispecific antibodies are herein referred to 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, and the prefix "Bis-" indicates "bispecific" and is followed by the hybridoma antibody clone from which the chimeric antibody is derived. For example, "Bis-83A9B8" indicates a bispecific antibody containing the chimeric antibody from hybridoma antibody clone 83A9B8 and a CD3-targeted scFv (containing the VH region with the amino acid sequence described in SEQ ID NO: 87 and the VL region with the amino acid sequence described in SEQ ID NO: 88).

[0338] 3.2 T cell-dependent cytotoxicity of bispecific hIgG-scFv antibodies against CHO S cells The T cell-dependent cytotoxicity of bispecific hIgG-scFv antibodies in CHO S cells was tested by FACS analysis. The protocol for FACS analysis is described below: (a) CD3 + T cells were isolated from healthy donors. (b) 1×10 4Individual target cells (CHOS-hGPRC5D-GFP) and 1×10 5 cells of T cells were added to the wells of a 96-well U-bottom plate (effector:target ratio of 10:1). (c) After mixing the target cells and T cells, a 1:3 serial dilution of the bispecific hIgG-scFv antibody was added to each well at a final concentration of 100 nM at seven data points. (d) The plate was incubated at 37 °C and 5% CO2 for 48 hours. (e) After 2 days (48 hours), the plate was centrifuged and analyzed by FACS.

[0339] As shown in Table 12 and Figure 2, some of the selected bispecific antibodies showed strong T cell-dependent cytotoxic effects against CHOS-hGPRC5D cells.

[0340]

Table 12

[0341] 3.3 Generation of bispecific Crossmab antibodies To further evaluate the cytotoxicity mediated by the antibody candidates, another series of bispecific antibodies were constructed using the Crossmab format as described in WO2009 / 080253. Generally, each of the generated bispecific Crossmab antibodies is an IgG-like bispecific antibody and contains an anti-GPRC5D Fab domain, an anti-CD3 Fab domain, and a constant domain. The anti-GPRC5D Fab domain contains a GPRC5D-binding portion that includes the VH and VL regions of each of the hybridoma antibody clones screened in Example 1; the anti-CD3 Fab domain contains a CD3-binding portion that includes a VH region containing the amino acid sequence set forth in SEQ ID NO: 95 and a VL region containing the amino acid sequence set forth in SEQ ID NO: 96, and the CH1 and CL regions of the anti-CD3 Fab domain are exchanged with each other; the constant domain is a human IgG1 constant domain, and the LALA mutation (L234A, L235A, Eu numbering) was introduced into the IgG1 Fc region to abolish Fc receptor binding. In addition, the knob-into-hole mutation (S354C, T366W on the CH3 of the first heavy chain, Y349C, T366S, L368A, Y407V on the CH3 of the second heavy chain) was introduced to avoid heavy chain mismatching. ATG596 was used to construct the bispecific antibody together with the Crossmab format as a positive control. The bispecific Crossmab ATG596 (i.e., Bis-ATG596-CM) differs from the bispecific Crossmab antibodies tested only in the amino acid sequences of the VH and VL regions of the anti-GPRC5D Fab domain. JNJ-64407564 was also used as a positive control. The construct and amino acid sequence of JNJ-64407564 can be obtained from WO2018017786A2. In particular, JNJ-64407564 contains a GPRC5D-binding portion that includes the VH region set forth in SEQ ID NO: 199 and the VL region set forth in SEQ ID NO: 200, as well as a CD3-binding portion that includes the VH region set forth in SEQ ID NO: 95 and the VL region set forth in SEQ ID NO: 96.

[0342] The obtained bispecific antibodies are named 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-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. Here, the prefix "Bis-" indicates "bispecific", the middle indicates the hybridoma antibody clone, and the suffix "-CM" indicates "Crossmab". For example, "Bis-108C4C12-CM" indicates a bispecific Crossmab antibody containing the GPRC5D-binding portion including the VH and VL regions of the hybridoma antibody clone 108C4C12.

[0343] 3.4 T cell-dependent cytotoxicity of bispecific Crossmab antibodies against MM.1S cells The cytotoxicity of bispecific Crossmab antibodies against MM.1S cells was tested by FACS analysis. The protocol for FACS analysis is described below: (a) CD3 - T cells were isolated from healthy donors. (b) 1×10 4 target cells (CFSE-labeled MM.1S) and 1×10 5 T cells were added to the wells of a 96-well U-bottom plate (effector:target ratio of 10:1). (c) After mixing the target and T cells, a 1:4 serial dilution of the bispecific Crossmab antibody was added to each well at a final concentration of 5 nM at 9 data points. (d) The plate was incubated overnight at 37 °C and 5% CO2. (e) After incubation, the plate was centrifuged and analyzed by FACS.

[0344] As shown in Table 13 and Figure 3, all of the selected antibodies showed potent T cell-dependent cytotoxicity against MM.1S cells. The selected antibodies showed comparable maximal cytotoxicity against MM.1S cells, and three clones (i.e., Bis-108C4C12-CM, Bis-98A3A8-CM, and Bis-96C9E7-CM) had lower EC 50 compared to the reference antibodies, JNJ-64407564 and Bis-ATG596-CM.

[0345] [Table 13]

[0346] [Example 4] Characterization of Antibodies: In vivo Efficacy 4.1 Generation of a human PBMC humanized MM.1S mouse model To evaluate the in vivo activity of the bispecific antibodies, a human PBMC humanized MM.1S model was used. Each NCG (NOD CRISPR Prkdc Il2r gamma) mouse was transplanted subcutaneously (SC) with 1 × 10 7 MM.1S human MM (multiple myeloma) cells into the right flank. When the tumor volume reached 60 - 80 mm 3 the animals were injected intravenously (I.V.) with 1 × 10 7 human PBMC. When the tumor volume reached 100 - 140 mm 3Once reached, the mice were randomized into groups with similar starting tumor volumes and each mouse was administered the test substance I.V. on the same day. The treatment was administered at one dose every three days. JNJ-64407564 and Bis-ATG596-CM were used as reference antibodies. The mean value of tumor growth inhibition was calculated using the following formula, where mean value (control) represents the mean value of the tumor volume of mice treated with the hIgG isotype control; mean value (control D0) represents the mean tumor volume of mice treated with the hIgG isotype control on day 0; mean value (test) represents the mean value of the tumor volume of mice treated with the tested bispecific antibody; mean value (test D0) represents the mean tumor volume of mice treated with the tested bispecific antibody on day 0.

[0347] [Number]

[0348] As shown in Table 14 and Figure 4, most of the candidates showed more potent in vivo antitumor efficacy than Bis-ATG596-CM in the same format, suggesting a large in vivo therapeutic potential. Moreover, Bis-108C4C12-CM showed even better efficacy than the reference antibody JNJ-64407564.

[0349] [Table 14]

[0350] [Example 5] Characterization of Antibodies: CAR-T To evaluate the cytotoxic efficacy of chimeric antigen receptor (CAR) T cells generated by the selected antibodies, CARs were constructed, transfected into T cells, and then incubated with NCI-H929 cells. BMK-203 was used as a reference control. The protocol is described below.

[0351] 5.1 Plasmid Transfection and Recombinant Virus Collection Day 0: When the cell confluence reached 80%, 293T cells were collected and counted. 8.2×10 6 cells of 293T cell suspension were seeded into a T75 flask. The cell plate was returned to a 37°C, 5% CO2 incubator.

[0352] Day 1: After 24-hour incubation, the cell confluence reached >90%. The medium was removed and replaced with 11 mL of fresh Opti-MEM medium.

[0353] Preparation of the transfection system: Tube A: 2 mL of Opti-MEM + a total of 25 μg of DNA (12.5 μg of CAR vector + 7.5 μg of pGP + 5 μg of VSV-G). Tube B: 2 mL of Opti-MEM + 75 μg of PEI. Tube A was added to Tube B and mixed well. The mixture was incubated at room temperature for 20 minutes. The mixture was slowly added to the 293T cells and incubated at 37°C and 5% CO2 for 6 hours. The medium was removed and replaced with 15 mL of fresh complete DMEM medium (10% FBS + 1% P / S).

[0354] Day 3: 48 hours after transfection, all the virus supernatants were collected, filtered through a 0.45 μm filter, and stored at 4°C. Next, the medium was removed and replaced with 15 mL of fresh complete DMEM medium and incubated at 37°C and 5% CO2 overnight.

[0355] Day 4: 72 hours after transfection, all the virus supernatants were collected, filtered through a 0.45 μm filter, and combined with the previous virus supernatant. The supernatant was centrifuged at 20,000×g at 4°C for 4 hours. The supernatant was discarded and resuspended in 400 μL of X-VIVO +++ medium (5 ng / mL of IL-2 + 10 ng / mL of IL-7 + 5 ng / mL of IL-15).

[0356] 5.2 Centrifugal infection method CD3 +T cells were isolated from 50 M frozen human PBMCs and stimulated at a ratio of 1:1 with CD3 / CD28 Dynabeads (Gibco, #11131D) and grown in X-VIVO +++ medium.

[0357] Day 1: After 3 days of activation, the T cells were centrifugally infected with the CAR lentivirus. The prepared CAR lentivirus expressed a CAR containing an extracellular antigen-binding region having the VH and VL region amino acid sequences of each selected antibody, a CD8 transmembrane region having the amino acid sequence of SEQ ID NO: 203, a 4-1BB intracellular signaling region having the amino acid sequence of SEQ ID NO: 204, and a CD3ζ intracellular signaling region having the amino acid sequence set forth in SEQ ID NO: 205.

[0358] Concentrated virus and polybrene reagent (final concentration 8 μg / ml) were added to 5 × 10 5 activated T cells (cultured in 2 ml of X-VIVO +++ medium) in a 12-well plate (MOI ≈ 100:1), and the total volume was adjusted to 700 - 800 μl volume / well.

[0359] The mixture was centrifuged at 1000 × g for 2 hours at room temperature and then returned to a 37°C, 5% CO2 incubator. After 24 hours of incubation, the medium was replaced with fresh X-VIVO +++ medium.

[0360] Day 5: Four days after infection, the CD3 / CD28 Dynabeads were removed and the transduction efficiency (GFP positive%) was detected.

[0361] The CAR-T cell cytotoxicity assay was performed on the target cells.

[0362] 5.3 Cytotoxicity Assay 3 × 10 4 Cell Trace-Far Red-labeled NCI-H929 cells were added to a 96-well U-bottom plate (100 μl / well).

[0363] 100 μl / well of CAR-T or vehicle CD3 T cells were added to the wells at a specific effector-to-tumor cell (E:T) ratio. E-GFP positive cells.

[0364] After 48 hours of co-culture, the number of viable tumor cells that were positive for Far Red signaling was detected by FACS.

[0365] Cytotoxicity was calculated as follows: % cytotoxicity / CD3 T = (number of Far Red + cells in the CD3 T group - number of Far Red + cells in the CAR-T group) / number of Far Red + cells in the CD3 T group * 100%

[0366] 5.4 Results As shown in Figure 5, all of the test antibodies of the present inventors showed strong CAR-T cell cytotoxicity against NCI-H929 cells and exhibited equivalent or better cell killing efficacy compared to the reference control BMK-203.

Claims

1. An antibody that binds to GPRC5D or an antigen-binding fragment thereof, One, two, or three heavy chain complementarity-determining regions (HCDR1, HCDR2, and / or HCDR3) contained within any one heavy chain variable (VH) region sequence selected from the group consisting of SEQ ID NOs: 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, two, or three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within one of the light chain variable (VL) region sequences selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184, and 192. An antibody containing an antigen-binding fragment thereof.

2. i. HCDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 97, 105, 113, 121, 129, 137, 145, 153, 169, 177, and 185; ii. HCDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 98, 106, 114, 122, 130, 138, 146, 154, 170, 178, and 186; iii. HCDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 99, 107, 115, 123, 131, 139, 147, 155, 171, 179, and 187; iv. LCDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 100, 108, 116, 124, 132, 140, 148, 156, 172, 180, and 188; v. LCDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 101, 109, 117, 125, 133, 141, 149, 157, 173, 181 and 189; and vi. LCDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 102, 110, 118, 126, 134, 142, 150, 158, 174, 182, and 190. The antibody or antigen-binding fragment thereof according to claim 1, comprising:

3. i. HCDR1 containing the amino acid sequence described in SEQ ID NO: 1, HCDR2 containing the amino acid sequence described in SEQ ID NO: 2, HCDR3 containing the amino acid sequence described in SEQ ID NO: 3, LCDR1 containing the amino acid sequence described in SEQ ID NO: 4, LCDR2 containing the amino acid sequence described in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 6; ii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 9, HCDR2 containing the amino acid sequence described in SEQ ID NO: 10, HCDR3 containing the amino acid sequence described in SEQ ID NO: 11, LCDR1 containing the amino acid sequence described in SEQ ID NO: 12, LCDR2 containing the amino acid sequence described in SEQ ID NO: 13, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 14; iii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 17, HCDR2 containing the amino acid sequence described in SEQ ID NO: 18, HCDR3 containing the amino acid sequence described in SEQ ID NO: 19, LCDR1 containing the amino acid sequence described in SEQ ID NO: 20, LCDR2 containing the amino acid sequence described in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 22; iv. HCDR1 containing the amino acid sequence described in SEQ ID NO: 25, HCDR2 containing the amino acid sequence described in SEQ ID NO: 26, HCDR3 containing the amino acid sequence described in SEQ ID NO: 27, LCDR1 containing the amino acid sequence described in SEQ ID NO: 28, LCDR2 containing the amino acid sequence described in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 30; v. HCDR1 containing the amino acid sequence described in SEQ ID NO: 33, HCDR2 containing the amino acid sequence described in SEQ ID NO: 34, HCDR3 containing the amino acid sequence described in SEQ ID NO: 35, LCDR1 containing the amino acid sequence described in SEQ ID NO: 36, LCDR2 containing the amino acid sequence described in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 38; vi. HCDR1 containing the amino acid sequence described in SEQ ID NO: 41, HCDR2 containing the amino acid sequence described in SEQ ID NO: 42, HCDR3 containing the amino acid sequence described in SEQ ID NO: 43, LCDR1 containing the amino acid sequence described in SEQ ID NO: 44, LCDR2 containing the amino acid sequence described in SEQ ID NO: 45, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 46; vii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 49, HCDR2 containing the amino acid sequence described in SEQ ID NO: 50, HCDR3 containing the amino acid sequence described in SEQ ID NO: 51, LCDR1 containing the amino acid sequence described in SEQ ID NO: 52, LCDR2 containing the amino acid sequence described in SEQ ID NO: 53, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 54; viiii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 57, HCDR2 containing the amino acid sequence described in SEQ ID NO: 58, HCDR3 containing the amino acid sequence described in SEQ ID NO: 59, LCDR1 containing the amino acid sequence described in SEQ ID NO: 60, LCDR2 containing the amino acid sequence described in SEQ ID NO: 61, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 62; ix. HCDR1 containing the amino acid sequence described in SEQ ID NO: 65, HCDR2 containing the amino acid sequence described in SEQ ID NO: 66, HCDR3 containing the amino acid sequence described in SEQ ID NO: 67, LCDR1 containing the amino acid sequence described in SEQ ID NO: 68, LCDR2 containing the amino acid sequence described in SEQ ID NO: 69, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 70; x. HCDR1 containing the amino acid sequence described in SEQ ID NO: 73, HCDR2 containing the amino acid sequence described in SEQ ID NO: 74, HCDR3 containing the amino acid sequence described in SEQ ID NO: 75, LCDR1 containing the amino acid sequence described in SEQ ID NO: 76, LCDR2 containing the amino acid sequence described in SEQ ID NO: 77, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 78; xi. HCDR1 containing the amino acid sequence described in SEQ ID NO: 97, HCDR2 containing the amino acid sequence described in SEQ ID NO: 98, HCDR3 containing the amino acid sequence described in SEQ ID NO: 99, LCDR1 containing the amino acid sequence described in SEQ ID NO: 100, LCDR2 containing the amino acid sequence described in SEQ ID NO: 101, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 102; xi. HCDR1 containing the amino acid sequence described in SEQ ID NO: 105, HCDR2 containing the amino acid sequence described in SEQ ID NO: 106, HCDR3 containing the amino acid sequence described in SEQ ID NO: 107, LCDR1 containing the amino acid sequence described in SEQ ID NO: 108, LCDR2 containing the amino acid sequence described in SEQ ID NO: 109, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 110; xiiii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 113, HCDR2 containing the amino acid sequence described in SEQ ID NO: 114, HCDR3 containing the amino acid sequence described in SEQ ID NO: 115, LCDR1 containing the amino acid sequence described in SEQ ID NO: 116, LCDR2 containing the amino acid sequence described in SEQ ID NO: 117, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 118; xiv. HCDR1 containing the amino acid sequence described in SEQ ID NO: 121, HCDR2 containing the amino acid sequence described in SEQ ID NO: 122, HCDR3 containing the amino acid sequence described in SEQ ID NO: 123, LCDR1 containing the amino acid sequence described in SEQ ID NO: 124, LCDR2 containing the amino acid sequence described in SEQ ID NO: 125, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 126; xv. HCDR1 containing the amino acid sequence described in SEQ ID NO: 129, HCDR2 containing the amino acid sequence described in SEQ ID NO: 130, HCDR3 containing the amino acid sequence described in SEQ ID NO: 131, LCDR1 containing the amino acid sequence described in SEQ ID NO: 132, LCDR2 containing the amino acid sequence described in SEQ ID NO: 133, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 134; xvi. HCDR1 containing the amino acid sequence described in SEQ ID NO: 137, HCDR2 containing the amino acid sequence described in SEQ ID NO: 138, HCDR3 containing the amino acid sequence described in SEQ ID NO: 139, LCDR1 containing the amino acid sequence described in SEQ ID NO: 140, LCDR2 containing the amino acid sequence described in SEQ ID NO: 141, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 142; xvii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 145, HCDR2 containing the amino acid sequence described in SEQ ID NO: 146, HCDR3 containing the amino acid sequence described in SEQ ID NO: 147, LCDR1 containing the amino acid sequence described in SEQ ID NO: 148, LCDR2 containing the amino acid sequence described in SEQ ID NO: 149, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 150; xviiii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 153, HCDR2 containing the amino acid sequence described in SEQ ID NO: 154, HCDR3 containing the amino acid sequence described in SEQ ID NO: 155, LCDR1 containing the amino acid sequence described in SEQ ID NO: 156, LCDR2 containing the amino acid sequence described in SEQ ID NO: 157, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 158; xix. HCDR1 containing the amino acid sequence described in SEQ ID NO: 169, HCDR2 containing the amino acid sequence described in SEQ ID NO: 170, HCDR3 containing the amino acid sequence described in SEQ ID NO: 171, LCDR1 containing the amino acid sequence described in SEQ ID NO: 172, LCDR2 containing the amino acid sequence described in SEQ ID NO: 173, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 174; xx. HCDR1 containing the amino acid sequence described in SEQ ID NO: 177, HCDR2 containing the amino acid sequence described in SEQ ID NO: 178, HCDR3 containing the amino acid sequence described in SEQ ID NO: 179, LCDR1 containing the amino acid sequence described in SEQ ID NO: 180, LCDR2 containing the amino acid sequence described in SEQ ID NO: 181, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 182; or xxi. HCDR1 containing the amino acid sequence described in SEQ ID NO: 185, HCDR2 containing the amino acid sequence described in SEQ ID NO: 186, HCDR3 containing the amino acid sequence described in SEQ ID NO: 187, LCDR1 containing the amino acid sequence described in SEQ ID NO: 188, LCDR2 containing the amino acid sequence described in SEQ ID NO: 189, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 190 The antibody or antigen-binding fragment thereof according to claim 1, comprising:

4. VH regions having the amino acid sequence described in SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183 or 191, or homologous sequences having at least 80% sequence identity with SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 103, 111, 119, 127, 135, 143, 151, 159, 175, 183 or 191; and / or A VL region having the amino acid sequence described in SEQ ID NOs: 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 with SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 104, 112, 120, 128, 136, 144, 152, 160, 176, 184, or 192. The antibody or antigen-binding fragment thereof according to claim 1, comprising:

5. The antibody or its antigen-binding fragment according to claim 1, comprising a VH / VL amino acid sequence pair selected from the group consisting of SEQ ID NOs: 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.

6. It further comprises one or more amino acid residue substitutions or modifications, but still retains binding affinity to GPRC5D. At least one of the substitutions or modifications is present in one or more CDR sequences or non-CDR sequences of the VH region or VL region. Optionally, further comprising one or more non-natural amino acid (NNAA) substitutions, It is possible to optionally conjugate the aforementioned NNAA, Optionally, the antibody or its antigen-binding fragment is i. Cross-reactivity with humans, cynomolgus monkeys, and mouse GPRC5D; ii. The drug must be able to bind to human GPRC5D, cynomolgus monkey GPRC5D, or mouse GPRC5D as measured by the FACS assay; iii. When measured by FACS assay, the measured value should be EC50 of 10 nM or less, and the substance should bind to human GPRC5D; iv. Exhibiting T cell-dependent cytotoxicity as measured by the FACS assay. Having one or more characteristics selected from the group consisting of, The antibody or its antigen-binding fragment according to claim 1.

7. The antibody or its antigen-binding fragment according to claim 1, which is a chimera, humanized or human antibody or its antigen-binding fragment, a labeled antibody, a bivalent antibody, an anti-idiotype antibody, a fusion protein, a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (dsdiabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), camelized single-domain antibody, nanobody, domain antibody, or bivalent domain antibody.

8. The Fc region, optionally further comprising the Fc region of human immunoglobulin (Ig), or optionally the Fc region of human IgG; The Fc region may optionally be derived from human IgG1, IgG2, IgG3, or IgG4; The Fc region optionally includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 161 to 163; The light chain is optionally a λ light chain or a κ light chain. The antibody or its antigen-binding fragment according to claim 1.

9. A bispecific or polyspecific antibody or its antigen-binding fragment; Optionally, it is possible to bind to one or more additional antigens other than GPRC5D, or to a second epitope on GPRC5D. The antibody or its antigen-binding fragment according to claim 1.

10. A bispecific antibody or its antigen-binding fragment that binds to GPRC5D and CD3; Optionally, the bispecific antibody or its antigen-binding fragment includes a GPRC5D binding portion and a CD3 binding portion. The antibody or its antigen-binding fragment according to claim 1.

11. The aforementioned CD3 coupling portion One, two, or three heavy chain complementarity-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 One, two, or three light chain complementarity-determining regions (LCDR1, LCDR2, and / or LCDR3) contained within the light chain variable (VL) region sequence of Sequence ID No. 88 or 96. The antibody or antigen-binding fragment according to claim 10, comprising:

12. The aforementioned CD3 coupling portion HCDR1 containing the amino acid sequence described in SEQ ID NO: 81 or 89, HCDR2 containing the amino acid sequence described in SEQ ID NO: 82 or 90, and HCDR3 containing the amino acid sequence described in SEQ ID NO: 83 or 91; and / or LCDR1 containing the amino acid sequence described in SEQ ID NO: 84 or 92, LCDR2 containing the amino acid sequence described in SEQ ID NO: 85 or 93, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 86 or 94. The antibody or antigen-binding fragment thereof according to claim 11, comprising:

13. The aforementioned CD3 coupling portion i. HCDR1 containing the amino acid sequence described in SEQ ID NO: 81, HCDR2 containing the amino acid sequence described in SEQ ID NO: 82, HCDR3 containing the amino acid sequence described in SEQ ID NO: 83, LCDR1 containing the amino acid sequence described in SEQ ID NO: 84, LCDR2 containing the amino acid sequence described in SEQ ID NO: 85, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 86; or ii. HCDR1 containing the amino acid sequence described in SEQ ID NO: 89, HCDR2 containing the amino acid sequence described in SEQ ID NO: 90, HCDR3 containing the amino acid sequence described in SEQ ID NO: 91, LCDR1 containing the amino acid sequence described in SEQ ID NO: 92, LCDR2 containing the amino acid sequence described in SEQ ID NO: 93, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 94 The antibody or antigen-binding fragment thereof according to claim 12, comprising:

14. The antibody or antigen-binding fragment according to claim 13, wherein the CD3 binding portion comprises the VH / VL amino acid sequence pair of SEQ ID NO: 87 / 88 or 95 / 96.

15. The CD3 binding portion further comprises a steady domain CL and a steady domain CH1; Optionally, the steady-state domains CL and CH1 are exchanged with each other. The antibody or its antigen-binding fragment according to claim 14.

16. The antibody or antigen-binding fragment according to claim 9, further comprising an Fc region, optionally an Fc region of human immunoglobulin (Ig), or optionally an Fc region of human IgG.

17. The antibody or antigen-binding fragment thereof according to claim 16, 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).

18. The antibody or its antigen-binding fragment according to claim 1, which is linked to one or more conjugate portions.

19. A chimeric antigen receptor comprising an antibody or its antigen-binding fragment, a transmembrane region, and an intracellular signaling region, as described in any one of claims 1 to 18.

20. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18, and / or a chimeric antigen receptor containing the antibody or the antigen-binding fragment thereof, and one or more pharmaceutically acceptable carriers.

21. An isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18, and / or a chimeric antigen receptor comprising the antibody or the antigen-binding fragment thereof.

22. A vector comprising an isolated polynucleotide as described in claim 21.

23. A host expression system comprising a vector containing the polynucleotide described in claim 21, or a host genome in which the polynucleotide is incorporated.

24. A pharmaceutical composition for use in a method of treating, preventing or mitigating a disease, disorder, or condition in a subject, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, or a chimeric antigen receptor containing the antibody or antigen-binding fragment thereof, or the antibody or antigen-binding fragment thereof and / or the chimeric antigen receptor, and one or more pharmaceutically acceptable carriers, The method comprises administering a therapeutically effective amount of the antibody or its antigen-binding fragment, or the chimeric antigen receptor, or the pharmaceutical composition to the subject. The disease, injury, or condition is cancer, and the antibody or its antigen-binding fragment, or the chimeric antigen receptor, or the pharmaceutical composition.

25. A method for detecting the presence or amount of GPRC5D in a sample, comprising contacting the sample with a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, and / or a chimeric antigen receptor containing the antibody or antigen-binding fragment thereof, and / or the antibody or antigen-binding fragment thereof and / or the chimeric antigen receptor, and one or more pharmaceutically acceptable carriers, and determining the presence or amount of GPRC5D in the sample.

26. 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 a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 18, and / or a chimeric antigen receptor containing the antibody or antigen-binding fragment thereof, and / or the antibody or antigen-binding fragment thereof and / or the chimeric antigen receptor, and one or more pharmaceutically acceptable carriers; c) determining the presence or amount of GPRC5D in the sample; and d) correlating the presence or amount of GPRC5D with the presence or condition of a GPRC5D-related disease, disorder, or condition in the subject.

27. A kit comprising a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, useful for detecting GPRC5D, optionally recombinant GPRC5D, GPRC5D expressed on the cell surface, or GPRC5D-expressing cells, and / or a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, and / or the antibody or antigen-binding fragment thereof and / or the chimeric antigen receptor, and one or more pharmaceutically acceptable carriers.

28. The antibody or antigen-binding fragment according to claim 1, comprising HCDR1 comprising the amino acid sequence described in SEQ ID NO: 65, HCDR2 comprising the amino acid sequence described in SEQ ID NO: 66, HCDR3 comprising the amino acid sequence described in SEQ ID NO: 67, LCDR1 comprising the amino acid sequence described in SEQ ID NO: 68, LCDR2 comprising the amino acid sequence described in SEQ ID NO: 69, and LCDR3 comprising the amino acid sequence described in SEQ ID NO:

70.

29. The antibody or antigen-binding fragment according to claim 1, comprising HCDR1 comprising the amino acid sequence described in SEQ ID NO: 73, HCDR2 comprising the amino acid sequence described in SEQ ID NO: 74, HCDR3 comprising the amino acid sequence described in SEQ ID NO: 75, LCDR1 comprising the amino acid sequence described in SEQ ID NO: 76, LCDR2 comprising the amino acid sequence described in SEQ ID NO: 77, and LCDR3 comprising the amino acid sequence described in SEQ ID NO:

78.

30. The antibody or antigen-binding fragment according to claim 1, comprising HCDR1 comprising the amino acid sequence described in SEQ ID NO: 177, HCDR2 comprising the amino acid sequence described in SEQ ID NO: 178, HCDR3 comprising the amino acid sequence described in SEQ ID NO: 179, LCDR1 comprising the amino acid sequence described in SEQ ID NO: 180, LCDR2 comprising the amino acid sequence described in SEQ ID NO: 181, and LCDR3 comprising the amino acid sequence described in SEQ ID NO: 182.

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