Antibodies to PMEL17 and conjugates thereof

Abstract

To provide antibodies, attachment methods, and cytotoxic payloads with improved properties for use as effective antibody drug conjugate therapeutic compositions and methods.SOLUTION: This application discloses anti-PMEL17 antibodies, antigen binding fragments thereof, and antibody drug conjugates of the antibodies or antigen binding fragments conjugated to a GNAQ / GNA11 inhibitor. The invention also relates to methods of treating or preventing cancer using the antibodies, antigen binding fragments, and antibody drug conjugates. Also disclosed herein are methods of making the antibodies, antigen binding fragments, and antibody drug conjugates, and methods of using the antibodies and antigen binding fragments as diagnostic reagents.SELECTED DRAWING: Figure 18

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on December 6, 2019, is named PAT058359-WO-PCT_SL.txt, and is 285,253 bytes in size.

[0002] The present invention relates generally to anti-PMEL17 antibodies, or fragments thereof, conjugates thereof, including GNAQ / GNA11 inhibitor conjugates thereof, and their use for the treatment or prevention of cancer. [Background technology]

[0003] PMEL17 (also known as gp100 and SILV) is a type I single-pass transmembrane protein produced by melanocytes and involved in melanin synthesis. During its maturation, PMEL17 is transiently expressed on the cell surface and then trafficked to melanosomes, where it is disassembled into various domains that multimerize to form fibrillar sheets. Such patterns then function as supports for capturing melanin. Melanosomal PMEL17 expression is regulated by the family oncogene MITF and has been found to be upregulated in various primary and metastatic subcutaneous and uveal melanomas. The transient cell surface expression and subsequent internalization of PMEL17 make it a suitable target for the development of antibody-drug conjugates (ADCs) for the treatment of melanoma.

[0004] PMEL17 and cancer During its maturation, PMEL17 is extensively processed by proprotein convertases. The protein is cleaved between V467 and K468 to form two subdomains, Mα at the N-terminus and Mβ at the C-terminus, which are thought to be maintained via disulfide bridges. Some PMEL17 molecules are transiently expressed on the cell surface after exiting the Golgi apparatus. Most PMEL17 is then redirected to melanocytes for further maturation, while some PMEL17 is shed. After additional enzymatic cleavage, PMEL17 is degraded into various domains, which reorganize to form fibrous sheets into which melanin polymerizes (Non-Patent Document 1; Non-Patent Document 2).

[0005] PMEL17 constitutes a potential therapeutic target for the treatment of melanoma. PMEL17 is a direct transcriptional target of the MITF oncogene in melanoma, as observed by mRNA expression studies (Non-Patent Document 3). PMEL17 expression is restricted to melanocyte lineages, such as dermal melanocytes, hair bulb melanocytes, retinal pigment epithelium, pigmented ciliary epithelium, and possibly choroidal melanocytes in the retina. PMEL17 is also highly expressed in melanocytic lineage tumors, such as subcutaneous and uveal melanoma. In contrast, mRNA studies have demonstrated that PMEL17 expression is restricted to other tumor types and normal tissues (Non-Patent Document 4). In addition, ADC and ImmTAC compounds targeting PMEL17 have already been described to specifically induce melanoma killing in vivo and in vitro and are currently being evaluated in clinical trials (Non-Patent Document 5).

[0006] GNAQ / GNA11 and cancer The GNAQ and GNA11 genes encode the alpha subunits of the heterotrimeric G protein Gq / 11, which is nearly ubiquitously expressed and acts as a binary molecular switch, cycling between an active guanosine triphosphate (GTP)-bound state and an inactive guanosine diphosphate (GDP)-bound state. GTP-bound Gαq and Gα11 activate the β-isoform of phospholipase C, which triggers multiple signaling pathways through the generation of the second messengers IP3 and DAG. Signaling termination is triggered by GTP hydrolysis mediated by the intrinsic GTPase activity of the Gα proteins. Gq and Gα11 have been shown to be involved in a wide range of physiological functions, including platelet activation, myocardial hypertrophy, and smooth muscle tone.

[0007] Oncogenic mutations in either GNAQ or GNA11 occur in up to 90% of uveal melanoma (UM) cases and approximately 2-3% of cutaneous melanomas. Approximately 95% of these mutations affect codon 209 (Q209) in the Ras-like domain, resulting in complete or partial loss of GTPase activity and thereby locking GNAQ / 11 in its active state. Q209 GNAQ / 11 is a preferentially acting oncogene that transforms melanocytes by inducing activation of multiple pathways, including PKC / MAPK, Rho / Rac, β-catenin, and YAP. While the PKC / MAPK pathway has been shown to be one contributing factor to GNAQ-mediated oncogenesis, multiple lines of evidence suggest that mutant GNAQ / 11 governs additional pathways (i.e., YAP, β-catenin) that likely play a role in UM tumorigenesis as well. Interestingly, another somatic activating mutation in GNAQ (R183Q) was recently described to be responsible for Sturge-Weber syndrome (SWS), a neurocutaneous disorder characterized by capillary malformations (port-wine spots) and choroidal and leptomeningeal vascular malformations. Thus, GNAQ and GNA11 constitute potential therapeutic targets for the treatment of uveal and cutaneous melanoma.

[0008] antibody-drug conjugates Antibody-drug conjugates ("ADCs") have been used for the local delivery of cytotoxic agents in the treatment of cancer (see, for example, Non-Patent Document 6). ADCs allow for targeted delivery of drug moieties, achieving maximum efficacy with minimal toxicity. ADCs comprise an antibody selected for its ability to bind to cells targeted for therapeutic intervention and conjugated to a drug selected for cytostatic or cytotoxic activity. Binding of the antibody to the targeted cells thereby delivers the drug to the site where its therapeutic effect is required.

[0009] Many antibodies that recognize and selectively bind to targeted cells, such as cancer cells, have been disclosed for use in ADCs. Despite intensive research on ADCs, antibody binding to a specific target of interest is not sufficient to predict success in ADC applications. Examples of factors that may affect the therapeutic efficacy of ADCs (in addition to target-specific characteristics) include various aspects that require customized fine-tuning, such as optimal antibody affinity as a balance between target-mediated disposition (TMDD) and efficacy-driving exposure, evaluation of Fc-mediated function (antibody-dependent cell-mediated cytotoxicity, ADCC), method of conjugation (site-specific or not), ratio of drug / payload molecules conjugated to each antibody ("DAR" or "drug-antibody ratio"), linker cleavability or stability, ADC stability, and ADC aggregation tendency.

[0010] There remains a need for antibodies, attachment methods, and cytotoxic payloads with improved properties for use as effective ADC therapeutic compositions and methods. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Valencia JC,et al.Sorting of Pmel17 to melanosomes through the plasma membrane by AP1 and AP2:evidence for the polarized nature of melanocytes.J Cell Sci.2006 Mar 15;119(Pt 6):1080-91

Outdoor Tool2

Outdoor Tools3

Outdoor Tools 4

Direct Environment 5

[0012] In one embodiment, the present application provides an antibody or antigen-binding fragment thereof that binds to PMEL17, a. a heavy chain variable region comprising a heavy chain CDR1 (complementarity determining region 1) of SEQ ID NO: 1, 4, 5, or 7, a heavy chain CDR2 (complementarity determining region 2) of SEQ ID NO: 2, 6, or 8, and a heavy chain CDR3 (complementarity determining region 3) of SEQ ID NO: 3 or 9; and a light chain variable region comprising a light chain CDR1 (complementarity determining region 1) of SEQ ID NO: 14, 17, or 20, a light chain CDR2 (complementarity determining region 2) of SEQ ID NO: 15 or 18, and a light chain CDR3 (complementarity determining region 3) of SEQ ID NO: 16 or 19; b. A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 33, 36, 37, or 39, a heavy chain CDR2 of SEQ ID NO: 34, 38, or 40, a heavy chain CDR3 of SEQ ID NO: 35 or 41, a light chain CDR1 of SEQ ID NO: 46, 49, or 52, a light chain CDR2 of SEQ ID NO: 47 or 50, and a light chain CDR3 of SEQ ID NO: 48 or 51; c. A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 5, 7, 57, or 60, a heavy chain CDR2 of SEQ ID NO: 58, 61, or 62, a heavy chain CDR3 of SEQ ID NO: 59 or 63; a light chain CDR1 of SEQ ID NO: 68, 71, or 74; a light chain CDR2 of SEQ ID NO: 69 or 72; and a light chain CDR3 of SEQ ID NO: 70 or 73; d. A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 79, 82, 83, or 85, a heavy chain CDR2 of SEQ ID NO: 80, 84, or 86, a heavy chain CDR3 of SEQ ID NO: 81 or 87, a light chain CDR1 of SEQ ID NO: 92, 95, or 98, a light chain CDR2 of SEQ ID NO: 93 or 96, and a light chain CDR3 of SEQ ID NO: 94 or 97; e. a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 103, 106, 107, or 109, a heavy chain CDR2 of SEQ ID NO: 104, 108, or 110, a heavy chain CDR3 of SEQ ID NO: 105 or 111; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 117 or 118; f. a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 123, 126, 127, or 129, a heavy chain CDR2 of SEQ ID NO: 124, 128, or 130, a heavy chain CDR3 of SEQ ID NO: 125 or 131; a light chain CDR1 of SEQ ID NO: 136, 139, or 142; a light chain CDR2 of SEQ ID NO: 137 or 140; and a light chain CDR3 of SEQ ID NO: 138 or 141; g. A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 123, 126, 127, or 129, a heavy chain CDR2 of SEQ ID NO: 124, 128, or 130, a heavy chain CDR3 of SEQ ID NO: 147 or 148, a light chain CDR1 of SEQ ID NO: 153, 156, or 158, a light chain CDR2 of SEQ ID NO: 50 or 154, and a light chain CDR3 of SEQ ID NO: 155 or 157; h. a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 103, 106, 107, or 109, a heavy chain CDR2 of SEQ ID NO: 104, 108, or 110, a heavy chain CDR3 of SEQ ID NO: 163 or 164, a light chain CDR1 of SEQ ID NO: 49, 52, or 116, a light chain CDR2 of SEQ ID NO: 47 or 50, and a light chain CDR3 of SEQ ID NO: 169 or 170; i. a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 175, 178, 179, or 181, a heavy chain CDR2 of SEQ ID NO: 176, 180, or 182, a heavy chain CDR3 of SEQ ID NO: 177 or 183; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 188 or 189; j. a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 103, 106, 107, or 109, a heavy chain CDR2 of SEQ ID NO: 104, 108, or 110, a heavy chain CDR3 of SEQ ID NO: 194 or 195, a light chain CDR1 of SEQ ID NO: 49, 52, or 116, a light chain CDR2 of SEQ ID NO: 47 or 50, and a light chain CDR3 of SEQ ID NO: 200 or 201; k. A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 206, 209, 210, or 212, a heavy chain CDR2 of SEQ ID NO: 207, 211, or 213, a heavy chain CDR3 of SEQ ID NO: 208 or 214; a light chain CDR1 of SEQ ID NO: 153, 156, or 158; a light chain CDR2 of SEQ ID NO: 50 or 154; and a light chain CDR3 of SEQ ID NO: 219 or 220; l. A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 206, 209, 210, or 212, a heavy chain CDR2 of SEQ ID NO: 207, 211, or 213, a heavy chain CDR3 of SEQ ID NO: 225 or 226; a light chain CDR1 of SEQ ID NO: 136, 139, or 142; a light chain CDR2 of SEQ ID NO: 137 or 140; and a light chain CDR3 of SEQ ID NO: 231 or 232; m. a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, 209, 210, or 212, an HCDR2 of SEQ ID NO: 207, 211, or 213, and an HCDR3 of SEQ ID NO: 237 or 238; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 243, 245, or 247, an LCDR2 of SEQ ID NO: 47 or 50, and an LCDR3 of SEQ ID NO: 244 or 246; n. A heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, 209, 210, or 212, an HCDR2 of SEQ ID NO: 207, 211, or 213, and an HCDR3 of SEQ ID NO: 252 or 253; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 153, 156, or 158, an LCDR2 of SEQ ID NO: 50 or 154, and an LCDR3 of SEQ ID NO: 258 or 259; o. a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 14, a light chain CDR2 of SEQ ID NO: 15, and a light chain CDR3 of SEQ ID NO: 16; p. heavy chain CDR1 of SEQ ID NO: 4, heavy chain CDR2 of SEQ ID NO: 2, heavy chain CDR3 of SEQ ID NO: 3, light chain CDR1 of SEQ ID NO: 14, light chain CDR2 of SEQ ID NO: 15, and light chain CDR3 of SEQ ID NO: 16; q. a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 6, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 17, a light chain CDR2 of SEQ ID NO: 18, and a light chain CDR3 of SEQ ID NO: 19; r. a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 8, a heavy chain CDR3 of SEQ ID NO: 9, a light chain CDR1 of SEQ ID NO: 20, a light chain CDR2 of SEQ ID NO: 18, and a light chain CDR3 of SEQ ID NO: 16; s. a heavy chain CDR1 of SEQ ID NO: 33, a heavy chain CDR2 of SEQ ID NO: 34, a heavy chain CDR3 of SEQ ID NO: 35, a light chain CDR1 of SEQ ID NO: 46, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 48; t. a heavy chain CDR1 of SEQ ID NO: 36, a heavy chain CDR2 of SEQ ID NO: 34, a heavy chain CDR3 of SEQ ID NO: 35, a light chain CDR1 of SEQ ID NO: 46, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 48; u. a heavy chain CDR1 of SEQ ID NO: 37, a heavy chain CDR2 of SEQ ID NO: 38, a heavy chain CDR3 of SEQ ID NO: 35, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 51; v. heavy chain CDR1 of SEQ ID NO: 39, heavy chain CDR2 of SEQ ID NO: 40, heavy chain CDR3 of SEQ ID NO: 41, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 48; w. a heavy chain CDR1 of SEQ ID NO: 57, a heavy chain CDR2 of SEQ ID NO: 58, a heavy chain CDR3 of SEQ ID NO: 59, a light chain CDR1 of SEQ ID NO: 68, a light chain CDR2 of SEQ ID NO: 69, and a light chain CDR3 of SEQ ID NO: 70; x. a heavy chain CDR1 of SEQ ID NO: 60, a heavy chain CDR2 of SEQ ID NO: 58, a heavy chain CDR3 of SEQ ID NO: 59, a light chain CDR1 of SEQ ID NO: 68, a light chain CDR2 of SEQ ID NO: 69, and a light chain CDR3 of SEQ ID NO: 70; y. a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 61, a heavy chain CDR3 of SEQ ID NO: 59, a light chain CDR1 of SEQ ID NO: 71, a light chain CDR2 of SEQ ID NO: 72, and a light chain CDR3 of SEQ ID NO: 73; z. heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO: 62, heavy chain CDR3 of SEQ ID NO: 63, light chain CDR1 of SEQ ID NO: 74, light chain CDR2 of SEQ ID NO: 72, and light chain CDR3 of SEQ ID NO: 70; aa. heavy chain CDR1 of SEQ ID NO: 79, heavy chain CDR2 of SEQ ID NO: 80, heavy chain CDR3 of SEQ ID NO: 81, light chain CDR1 of SEQ ID NO: 92, light chain CDR2 of SEQ ID NO: 93, and light chain CDR3 of SEQ ID NO: 94; bb. heavy chain CDR1 of SEQ ID NO: 82, heavy chain CDR2 of SEQ ID NO: 80, heavy chain CDR3 of SEQ ID NO: 81, light chain CDR1 of SEQ ID NO: 92, light chain CDR2 of SEQ ID NO: 93, and light chain CDR3 of SEQ ID NO: 94; cc. heavy chain CDR1 of SEQ ID NO: 83, heavy chain CDR2 of SEQ ID NO: 84, heavy chain CDR3 of SEQ ID NO: 81, light chain CDR1 of SEQ ID NO: 95, light chain CDR2 of SEQ ID NO: 96, and light chain CDR3 of SEQ ID NO: 97; dd. heavy chain CDR1 of SEQ ID NO: 85, heavy chain CDR2 of SEQ ID NO: 86, heavy chain CDR3 of SEQ ID NO: 87, light chain CDR1 of SEQ ID NO: 98, light chain CDR2 of SEQ ID NO: 96, and light chain CDR3 of SEQ ID NO: 94; ee. Heavy chain CDR1 of SEQ ID NO: 103, heavy chain CDR2 of SEQ ID NO: 104, heavy chain CDR3 of SEQ ID NO: 105, light chain CDR1 of SEQ ID NO: 116; light chain CDR2 of SEQ ID NO: 47; and light chain CDR3 of SEQ ID NO: 117; ff. heavy chain CDR1 of SEQ ID NO: 106, heavy chain CDR2 of SEQ ID NO: 104, heavy chain CDR3 of SEQ ID NO: 105, light chain CDR1 of SEQ ID NO: 116, light chain CDR2 of SEQ ID NO: 47, and light chain CDR3 of SEQ ID NO: 117; gg. heavy chain CDR1 of SEQ ID NO: 107, heavy chain CDR2 of SEQ ID NO: 108, heavy chain CDR3 of SEQ ID NO: 105, light chain CDR1 of SEQ ID NO: 49, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 118; hh. heavy chain CDR1 of SEQ ID NO: 109, heavy chain CDR2 of SEQ ID NO: 110, heavy chain CDR3 of SEQ ID NO: 111, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 117; ii. a heavy chain CDR1 of SEQ ID NO: 123, a heavy chain CDR2 of SEQ ID NO: 124, a heavy chain CDR3 of SEQ ID NO: 125, a light chain CDR1 of SEQ ID NO: 136, a light chain CDR2 of SEQ ID NO: 137, and a light chain CDR3 of SEQ ID NO: 138; jj. heavy chain CDR1 of SEQ ID NO: 126, heavy chain CDR2 of SEQ ID NO: 124, heavy chain CDR3 of SEQ ID NO: 125, light chain CDR1 of SEQ ID NO: 136, light chain CDR2 of SEQ ID NO: 137, and light chain CDR3 of SEQ ID NO: 138; kk. heavy chain CDR1 of SEQ ID NO: 127, heavy chain CDR2 of SEQ ID NO: 128, heavy chain CDR3 of SEQ ID NO: 125, light chain CDR1 of SEQ ID NO: 139, light chain CDR2 of SEQ ID NO: 140, and light chain CDR3 of SEQ ID NO: 141; ll. A heavy chain CDR1 of SEQ ID NO: 129, a heavy chain CDR2 of SEQ ID NO: 130, a heavy chain CDR3 of SEQ ID NO: 131, a light chain CDR1 of SEQ ID NO: 142, a light chain CDR2 of SEQ ID NO: 140, and a light chain CDR3 of SEQ ID NO: 138; mm. heavy chain CDR1 of SEQ ID NO: 123, heavy chain CDR2 of SEQ ID NO: 124, heavy chain CDR3 of SEQ ID NO: 147, light chain CDR1 of SEQ ID NO: 153, light chain CDR2 of SEQ ID NO: 154, and light chain CDR3 of SEQ ID NO: 155; nn. heavy chain CDR1 of SEQ ID NO: 126, heavy chain CDR2 of SEQ ID NO: 124, heavy chain CDR3 of SEQ ID NO: 147, light chain CDR1 of SEQ ID NO: 153, light chain CDR2 of SEQ ID NO: 154, and light chain CDR3 of SEQ ID NO: 155; oo. heavy chain CDR1 of SEQ ID NO: 127, heavy chain CDR2 of SEQ ID NO: 128, heavy chain CDR3 of SEQ ID NO: 147, light chain CDR1 of SEQ ID NO: 156, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 157; pp. heavy chain CDR1 of SEQ ID NO: 129, heavy chain CDR2 of SEQ ID NO: 130, heavy chain CDR3 of SEQ ID NO: 148, light chain CDR1 of SEQ ID NO: 158, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 155; qq. A heavy chain CDR1 of SEQ ID NO: 103, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 163, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 169; rr. heavy chain CDR1 of SEQ ID NO: 106, heavy chain CDR2 of SEQ ID NO: 104, heavy chain CDR3 of SEQ ID NO: 163, light chain CDR1 of SEQ ID NO: 116, light chain CDR2 of SEQ ID NO: 47, and light chain CDR3 of SEQ ID NO: 169; ss. a heavy chain CDR1 of SEQ ID NO: 107, a heavy chain CDR2 of SEQ ID NO: 108, a heavy chain CDR3 of SEQ ID NO: 163, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 170; tt. heavy chain CDR1 of SEQ ID NO: 109, heavy chain CDR2 of SEQ ID NO: 110, heavy chain CDR3 of SEQ ID NO: 164, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 169; uu. heavy chain CDR1 of SEQ ID NO: 175, heavy chain CDR2 of SEQ ID NO: 176, heavy chain CDR3 of SEQ ID NO: 177, light chain CDR1 of SEQ ID NO: 116, light chain CDR2 of SEQ ID NO: 47, and light chain CDR3 of SEQ ID NO: 188; vv. heavy chain CDR1 of SEQ ID NO: 178, heavy chain CDR2 of SEQ ID NO: 176, heavy chain CDR3 of SEQ ID NO: 177, light chain CDR1 of SEQ ID NO: 116, light chain CDR2 of SEQ ID NO: 47, and light chain CDR3 of SEQ ID NO: 188; ww. heavy chain CDR1 of SEQ ID NO: 179, heavy chain CDR2 of SEQ ID NO: 180, heavy chain CDR3 of SEQ ID NO: 177, light chain CDR1 of SEQ ID NO: 49, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 189; xx. Heavy chain CDR1 of SEQ ID NO: 181, heavy chain CDR2 of SEQ ID NO: 182; heavy chain CDR3 of SEQ ID NO: 183, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 188; yy. a heavy chain CDR1 of SEQ ID NO: 103, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 194, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 200; zz. heavy chain CDR1 of SEQ ID NO: 106, heavy chain CDR2 of SEQ ID NO: 104, heavy chain CDR3 of SEQ ID NO: 194, light chain CDR1 of SEQ ID NO: 116, light chain CDR2 of SEQ ID NO: 47, and light chain CDR3 of SEQ ID NO: 200; aaa. heavy chain CDR1 of SEQ ID NO: 107, heavy chain CDR2 of SEQ ID NO: 108, heavy chain CDR3 of SEQ ID NO: 194, light chain CDR1 of SEQ ID NO: 49, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 201; bbb. heavy chain CDR1 of SEQ ID NO: 109, heavy chain CDR2 of SEQ ID NO: 110, heavy chain CDR3 of SEQ ID NO: 195, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 200; ccc. heavy chain CDR1 of SEQ ID NO: 206, heavy chain CDR2 of SEQ ID NO: 207, heavy chain CDR3 of SEQ ID NO: 208, light chain CDR1 of SEQ ID NO: 153, light chain CDR2 of SEQ ID NO: 154, and light chain CDR3 of SEQ ID NO: 219; ddd. heavy chain CDR1 of SEQ ID NO: 209, heavy chain CDR2 of SEQ ID NO: 207, heavy chain CDR3 of SEQ ID NO: 208, light chain CDR1 of SEQ ID NO: 153, light chain CDR2 of SEQ ID NO: 154, and light chain CDR3 of SEQ ID NO: 219; eee. heavy chain CDR1 of SEQ ID NO: 210, heavy chain CDR2 of SEQ ID NO: 211, heavy chain CDR3 of SEQ ID NO: 208, light chain CDR1 of SEQ ID NO: 156, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 220; fff. heavy chain CDR1 of SEQ ID NO: 212, heavy chain CDR2 of SEQ ID NO: 213, heavy chain CDR3 of SEQ ID NO: 214, light chain CDR1 of SEQ ID NO: 158, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 219; ggg. heavy chain CDR1 of SEQ ID NO: 206, heavy chain CDR2 of SEQ ID NO: 207, heavy chain CDR3 of SEQ ID NO: 225, light chain CDR1 of SEQ ID NO: 136, light chain CDR2 of SEQ ID NO: 137, and light chain CDR3 of SEQ ID NO: 231; hhh. heavy chain CDR1 of SEQ ID NO: 209, heavy chain CDR2 of SEQ ID NO: 207, heavy chain CDR3 of SEQ ID NO: 225, light chain CDR1 of SEQ ID NO: 136, light chain CDR2 of SEQ ID NO: 137, and light chain CDR3 of SEQ ID NO: 231; iii. a heavy chain CDR1 of SEQ ID NO: 210, a heavy chain CDR2 of SEQ ID NO: 211, a heavy chain CDR3 of SEQ ID NO: 225, a light chain CDR1 of SEQ ID NO: 139, a light chain CDR2 of SEQ ID NO: 140, and a light chain CDR3 of SEQ ID NO: 232; jjj. Heavy chain CDR1 of SEQ ID NO: 212, heavy chain CDR2 of SEQ ID NO: 213, heavy chain CDR3 of SEQ ID NO: 226, light chain CDR1 of SEQ ID NO: 142; light chain CDR2 of SEQ ID NO: 140; and light chain CDR3 of SEQ ID NO: 231; kkk. a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 243, an LCDR2 of SEQ ID NO: 47, and an LCDR3 of SEQ ID NO: 244; lll. A heavy chain variable region comprising an HCDR1 of SEQ ID NO: 209, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 243, an LCDR2 of SEQ ID NO: 47, and an LCDR3 of SEQ ID NO: 244; mmm. a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 210, an HCDR2 of SEQ ID NO: 211, and an HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 245, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 246; nnn. a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 212, an HCDR2 of SEQ ID NO: 213, and an HCDR3 of SEQ ID NO: 238; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 247, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 244; ooo. A heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 252, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 153, an LCDR2 of SEQ ID NO: 154, and an LCDR3 of SEQ ID NO: 258; ppp. a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 209, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 252, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 153, an LCDR2 of SEQ ID NO: 154, and an LCDR3 of SEQ ID NO: 258; qqq. A heavy chain variable region comprising an HCDR1 of SEQ ID NO: 210, an HCDR2 of SEQ ID NO: 211, and an HCDR3 of SEQ ID NO: 252, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 156, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 259; or rrr. a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 212, an HCDR2 of SEQ ID NO: 213, and an HCDR3 of SEQ ID NO: 253; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 158, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 258.

[0010] Disclosed is an antibody or antigen-binding fragment thereof comprising:

[0013] The antibody or antigen-binding fragment thereof that binds to PMEL17 of the present application is a. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 21; b. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 25; c. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 29; d. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 53; e. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 64, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 75; f. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 88, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 99; g. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 112, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 119; h. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 132, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 143; i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 149, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 159; j. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 165, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 171; k. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 184, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 190; l. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 196, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 202; m. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 215, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 221; n. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 227, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 233; o. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 239, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 248; or p. A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 254, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 260. It may also include.

[0014] In another embodiment, the antibody or antigen-binding fragment thereof that binds to PMEL17 a. a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain comprising the amino acid sequence of SEQ ID NO: 23; b. a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain comprising the amino acid sequence of SEQ ID NO: 27; c. a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain comprising the amino acid sequence of SEQ ID NO: 31; d. a heavy chain comprising the amino acid sequence of SEQ ID NO: 44, and a light chain comprising the amino acid sequence of SEQ ID NO: 55; e. a heavy chain comprising the amino acid sequence of SEQ ID NO: 66, and a light chain comprising the amino acid sequence of SEQ ID NO: 77; f. a heavy chain comprising the amino acid sequence of SEQ ID NO: 90, and a light chain comprising the amino acid sequence of SEQ ID NO: 101; g. A heavy chain comprising the amino acid sequence of SEQ ID NO: 114, and a light chain comprising the amino acid sequence of SEQ ID NO: 121; h. a heavy chain comprising the amino acid sequence of SEQ ID NO: 134, and a light chain comprising the amino acid sequence of SEQ ID NO: 145; i. a heavy chain comprising the amino acid sequence of SEQ ID NO: 151, and a light chain comprising the amino acid sequence of SEQ ID NO: 161; j. a heavy chain comprising the amino acid sequence of SEQ ID NO: 167, and a light chain comprising the amino acid sequence of SEQ ID NO: 173; k. a heavy chain comprising the amino acid sequence of SEQ ID NO: 186, and a light chain comprising the amino acid sequence of SEQ ID NO: 192; l. a heavy chain comprising the amino acid sequence of SEQ ID NO: 198, and a light chain comprising the amino acid sequence of SEQ ID NO: 204; m. a heavy chain comprising the amino acid sequence of SEQ ID NO: 217, and a light chain comprising the amino acid sequence of SEQ ID NO: 223; n. A heavy chain comprising the amino acid sequence of SEQ ID NO: 229, and a light chain comprising the amino acid sequence of SEQ ID NO: 235; o. a heavy chain comprising the amino acid sequence of SEQ ID NO: 241 and a light chain comprising the amino acid sequence of SEQ ID NO: 250; or p. a heavy chain comprising the amino acid sequence of SEQ ID NO: 256, and a light chain comprising the amino acid sequence of SEQ ID NO: 262 Includes:

[0015] The antibodies or antigen-binding fragments thereof described herein may contain one or more cysteine ​​substitutions. In one embodiment, the antibody or antigen-binding fragment thereof contains one or more cysteine ​​substitutions selected from S152C, S375C, or both S152C and S375C in the heavy chain of the antibody or antigen-binding fragment thereof, positions numbered according to the EU system. The antibodies disclosed herein may be monoclonal antibodies.

[0016] In one embodiment, the antibody drug conjugate of the present invention has formula (C): Ab-(L A -(D) n ) y (C) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; L A is a linker; n is 1, 2, 3 or 4; y is 1, 2, 3 or 4; Linker-drug moiety-(L A -(D) n ) is covalently attached to the antibody or antigen-binding fragment thereof It is a combination of

[0017] In another embodiment of the antibody drug conjugate of Formula (C), L A is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, a carbonate group, and a bivalent peptide linker.

[0018] In another embodiment, the antibody drug conjugate of formula (C) is Formula (C-1): [ka] (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2, and Y1 ** indicates the point of attachment to D; L1 is a bivalent peptide linker; L2 is a bond or a linker; y is 1, 2, 3 or 4 It is a combination of

[0019] The present application also discloses a pharmaceutical composition comprising the antibody, or antigen-binding fragment thereof, disclosed herein and a pharmaceutically acceptable carrier. The present application also discloses a pharmaceutical composition comprising the antibody-drug conjugate disclosed herein and a pharmaceutically acceptable carrier.

[0020] The present application also discloses a method of treating or preventing cancer in a patient in need thereof, comprising administering to the patient an antibody drug conjugate or pharmaceutical composition disclosed herein, wherein the cancer expresses PMEL17 and contains a mutation in the GNAQ or GNA11 gene, or the cancer expresses PMEL17 and contains a mutation in GNAQ, GNA11, or both.

[0021] In some embodiments of the methods for treating or preventing cancer, the antibody-drug conjugate or pharmaceutical composition is administered to the patient in combination with one or more additional therapeutic compounds. In one embodiment, the one or more additional therapeutic compounds are selected from a standard of care chemotherapeutic agent, an MDM2 inhibitor, an MRC2 inhibitor, a PKC inhibitor, a MAPK inhibitor, a costimulatory molecule, or a checkpoint inhibitor. In one embodiment, the costimulatory molecule is selected from an agonist of OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, STING, or CD83 ligand. In another embodiment, the checkpoint inhibitor is selected from an inhibitor of PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and / or TGFRbeta.

[0022] The present application also discloses an antibody-drug conjugate or pharmaceutical composition disclosed herein for use as a medicament. In one embodiment, the antibody-drug conjugate or pharmaceutical composition disclosed herein is for use in treating or preventing a PMEL17-expressing cancer or a cancer containing a mutation in the GNAQ or GNA11 gene in a patient in need of such treatment or prevention.

[0023] In one embodiment, the present application discloses the use of an antibody or antigen-binding fragment thereof, antibody-drug conjugate, or pharmaceutical composition disclosed herein for treating or preventing a PMEL17-expressing cancer in a patient in need of such treatment or prevention.

[0024] In one embodiment, the present application discloses the use of an antibody or antigen-binding fragment thereof, antibody-drug conjugate, or pharmaceutical composition disclosed herein for treating or preventing a PMEL17-expressing cancer or a cancer containing a mutation in the GNAQ or GNA11 gene in a patient in need of such treatment or prevention. In one embodiment, the present application discloses the use of an antibody or antigen-binding fragment thereof, antibody-drug conjugate, or pharmaceutical composition disclosed herein in the manufacture of a medicament.

[0025] In one embodiment, the cancer expresses PMEL17 or contains a mutation in the GNAQ or GNA11 gene. In one embodiment, the cancer is uveal melanoma, subcutaneous melanoma, hepatocellular carcinoma, or a metastatic form thereof.

[0026] The present application also discloses nucleic acids encoding the antibodies or antigen-binding fragments disclosed herein. In one embodiment, the nucleic acids comprise the nucleotide sequence of SEQ ID NO: 13, 24, 28, 32, 45, 56, 67, 78, 91, 102, 115, 122, 135, 146, 152, 162, 168, 174, 187, 193, 199, 205, 218, 224, 230, 236, 242, 251, 257, or 26. The present application also discloses vectors comprising the nucleic acids, and host cells comprising the vectors or nucleic acids. The present application also discloses methods for producing the antibodies or antigen-binding fragments disclosed herein, comprising culturing host cells and recovering the antibody from the cell culture. In one embodiment, the method for recovering the antibody from the cell culture comprises: a) removing the cells and filtering the culture; b) purifying the culture by affinity chromatography; c) inactivating any viruses in the culture by adjusting the pH to 3.4-3.6, then readjusting the pH to 5.8-6.2 and filtering the culture; d) purifying the culture by cation exchange chromatography and performing on-column reduction of the culture; e) performing anion exchange chromatography on the culture; f) removing viruses by nanofiltration; g) filtering the antibody-containing culture; and h) Obtaining purified antibodies Includes:

[0027] The present application also provides a method for producing an anti-PMEL17 antibody drug conjugate, comprising: (a) Formula (B) below: R 8 -L B -(D) n (B) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; R 8 is a reactive group; L B is a cleavable or non-cleavable linker, n is 1, 2, 3 or 4 preforming the linker-drug moiety; (b) attaching the linker-drug moiety to an antibody recovered from the cell culture using the methods for producing an antibody or antigen-binding fragment disclosed herein to produce an antibody drug conjugate; and (c) purifying the antibody-drug conjugate A method is disclosed that includes:

[0028] The present application also discloses diagnostic reagents comprising the antibodies or antigen-binding fragments thereof disclosed herein. In some embodiments, the antibodies or antigen-binding fragments thereof are labeled with a radiolabel, a fluorescent label, a chromophore, an imaging agent, or a metal ion. [Brief explanation of the drawings]

[0029] [Figure 1] 1 shows exemplary data regarding the in vitro anti-UM activity of GNAQ / 11 inhibitors Compound (A1) and Compound (A2). [Figure 2] 1 shows exemplary data regarding the activity of GNAQ / 11 inhibitors Compound (A1) and Compound (A2) to induce apoptosis in uveal melanoma cells. [Figure 3] Exemplary data for GNAQ / 11 inhibition by Compound A1 and Compound A2 are shown. Compound A1 and Compound A2 reduced IP1 levels (FIG. 3A) and relative proliferation rates (FIG. 3B) in 92.1 cells. Immunoblot analysis of 92.1 cells treated with Compound A1 and Compound A2 showed reduced ERK signaling (FIG. 3C). [Figure 4]Exemplary data regarding the metabolic stability and PK characteristics of compound A1 are shown. Both the disappearance of compound A1 (FIG. 4A) and the appearance of the open-ring form compound A8 (FIG. 4B) were monitored over 24 hours. Except in rats, the sum of % remaining compound A1 and % formed compound A8 shows stoichiometry over 24 hours (FIG. 4C). The PK of compound A1 after intravenous administration in mice is characterized by extremely high clearance and a moderate to high volume of distribution (FIG. 4D). [Figure 5] Exemplary data on the metabolic stability and PK properties of Compound A1 and Compound A2 are shown. The in vitro stability of Compound A2 was tested in plasma and blood from different species (FIG. 5A). Compound A2 showed good chemical stability in three different systems (FIG. 5B). PK of Compound A2 in female balb / c mice showed high clearance and a short elimination half-life (FIG. 5C). Compound A1 and Compound A2 were stable in buffer at pH 5.6 and in lysosomes for 4 hours (FIG. 5D). [Figure 6]

[0023] Figure 1 shows exemplary data for the in vitro anti-uveal melanoma activity of anti-PMEL17-(B1) ADC. Data are presented as the average of three independent replicates relative to PBS-treated cells (control). [Figure 7]

[0023] Figure 1 shows exemplary data for anti-PMEL17-(B1) ADCs inducing apoptosis in uveal melanoma cells. Data are presented as the average of three independent replicates. [Figure 8]

[0023] Figure 1 shows exemplary data on the in vitro anti-uveal melanoma activity of anti-PMEL17-(B2) ADC and anti-PMEL17 mAb. Data are presented as the average of three independent replicates relative to PBS-treated cells (control). [Figure 9]

[0023] Figure 1 shows exemplary data for GNAQ / 11 inhibition by anti-PMEL17-(B1) and anti-PMEL17-(B2) ADCs in uveal melanoma cells. IP1 levels (nM) are presented as the average of three independent replicates. [Figure 10]1 shows exemplary data regarding the binding activity of anti-PMEL17 antibodies to intact platelets and uveal melanoma cells. [Figure 11] 1 shows exemplary data regarding the effects of compound (A1) and anti-PMEL17-(B1) ADC on human platelet aggregation. [Figure 12] Exemplary data are shown for the in vivo antitumor activity of anti-PMEL17-(B1) ADC. G1-(B1) inhibited tumor growth in a dose-dependent manner (Figure 12A). Values ​​are mean ± SEM; sample size, n = 5-12 mice per group. Initial tumor volume on day 0 was approximately 200-250 mm. Body weight loss was not observed until 14 days after treatment (Figure 12B). Values ​​are mean ± SEM; sample size, n = 4 mice per group. G1-(B1) treatment resulted in GNAQ signaling inhibition and tumor cell proliferation inhibition, as indicated by reduced levels of pERK and Ki67, respectively (Figure 12C). Furthermore, G1-(B1) induced cell apoptosis compared with vehicle- and isotype control 3207-(B1)-treated mice, which correlated with tumor cell accumulation of G1-(B1) ADC as detected by IgG staining (Figure 12C). No changes were observed in MITF and PMEL17 levels after GNAQ inhibition (Figure 12C). Platelet aggregation inhibition was not observed in G1-(B1)-treated mice for up to 7 days (Figures 12D and E). [Figure 13A] Exemplary data are shown for the effect of the G1-(B1) ADC on a mouse model of uveal melanoma liver and lung metastasis. Individual photographs from each mouse are shown 45 days after iv injection of 92.1-luciferase cells (just before the start of treatment) and 12 days after treatment (FIGS. 13A and 13B); sample size, n = 6 mice per group. [Figure 13B]Exemplary data are shown for the effect of G1-(B1) ADC on liver and lung metastases in a mouse model of uveal melanoma. Individual photographs from each mouse are presented 45 days (just before the start of treatment) and 12 days after iv injection of 92.1-luciferase cells (Figures 13A and 13B); sample size (n = 6 mice per group). The initial BLI for liver metastases on day 0 was approximately 2.8*109 p / sec / cm2. The lung tumor (bioluminescence signal) in Figure 13B is indicated by a black arrow. Corresponding body weight modulation (% relative to day 15) was assessed 2-3 times weekly before and after treatment with G1-(B1) 20 mg / kg (gray circles). [Figure 13C] Values ​​in Figure 13C are mean ± SEM; sample size, (n = 5-6 mice per group). Initial body weight on day 15 was approximately 21 g. [Figure 14] Exemplary data on the PK characteristics of the G1-(B1) ADC are shown. The pharmacokinetic profile (total IgG levels) of G1-(B1) showed a slightly more than linear increase in exposure with doses from 7.5 to 30 mg / kg in nude mice (Figure 14A). In tumor-bearing mice, free payload concentrations were measured after administration of either target-bound G1-(B1) or the isotype control 3207-(B1). A clear (>4-fold) increase in tumor delivery of compound (A1) payload could be observed using the targeted ADC (Figure 14B). Conversion of compound (A1) (open circles) while still bound to the antibody to its open-ring form, compound (A8) (filled circles), was demonstrated in vivo in mice (Figure 14C). Exposure in an in vivo efficacy study comparing two different DAR2 formats with the G1-(B1) DAR4 format and the DAR4 Fc-silent format showed minimal clearance for the DAR2(E152C) and DAR4 Fc-silent ADCs, while DAR2(S375C) exposure declined more quickly (Figure 14D). Figure 14E shows the concentration of 3207 (isotype control antibody)-(B1)DAR4(E152C, S375C) and 3207 (isotype control antibody)-(B1)DAR4 Fc-silent conjugates over time. [Figure 15] 1 shows exemplary data regarding the in vitro stability of anti-PMEL17-GNAQ / 11i ADCs in buffer, mouse, rat, and human plasma, and the in vivo stability of anti-PMEL17-GNAQ / 11i ADCs in mice. [Figure 16] Exemplary data are shown for the in vivo efficacy of G1-E152C-DAR2-(B1), G1-S375C-DAR2-(B1), and Fc-silenced G1-(B1) in a xenograft model of uveal melanoma. Values ​​represent mean ± SEM; sample size, n = 5–6 mice per group. Initial tumor volume on day 0 was approximately 300–325 mm3. [Figure 17]

[0023] Figure 1 shows exemplary data for the in vitro anti-uveal melanoma activity of anti-PMEL17-(B1) ADC. Data are presented as the average of three independent replicates relative to PBS-treated cells (control). [Figure 18] 1 shows exemplary data regarding the in vivo anti-tumor activity of anti-PMEL17-(B1) ADCs. [Figure 19] 1 shows exemplary data regarding immunohistochemical analysis of tumor biopsies from patients with metastatic uveal melanoma. [Figure 20] Exemplary sensorgrams for evaluating epitope binning of anti-PMEL antibodies are shown. Figure 20A illustrates the binding step. Figure 20B shows a sensorgram when antibody G1 3J LC is immobilized first and 17A9 is flowed. Figure 20C shows a sensorgram when 17A9 is immobilized first and G1 3J LC is flowed. In both cases, binding is observed when the second antibody is flowed, suggesting that G1 3J LC and 17A9 bind to different epitopes of human PMEL. DETAILED DESCRIPTION OF THE INVENTION

[0030] definition Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings:

[0031] The term "alkyl" refers to a monovalent saturated hydrocarbon chain having the specified number of carbon atoms. For example, C1-C6 alkyl refers to an alkyl group having from 1 to 6 carbon atoms. Alkyl groups can be straight or branched. Representative branched alkyl groups have one, two, or three branches. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, sec-butyl, and t-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl.

[0032] As used herein, "cleavable" refers to a linking group or linker moiety that connects two moieties by a covalent bond but degrades under physiologically relevant conditions to sever the covalent bond between the moieties; typically, a cleavable linking group is cleaved more rapidly in the intracellular environment than outside the cell in vivo, causing release of the payload to occur preferentially inside the targeted cell. Cleavage can be enzymatic or non-enzymatic, but generally releases the payload from the antibody without degrading the antibody. Cleavage can leave some portion of the linking group or linker moiety attached to the payload, or it can release the payload without any residue of the linking group.

[0033] As used herein, "non-cleavable" refers to a linking group or linker moiety that is not particularly susceptible to degradation under physiological conditions, e.g., it is at least as stable as the antibody or antigen-binding fragment portion of the conjugate. Such linking groups are sometimes referred to as "stable," meaning that they are sufficiently resistant to degradation to retain the payload linked to the antibody or antigen-binding fragment until the antibody or antigen-binding fragment itself is at least partially degraded, i.e., degradation of the antibody or antigen-binding fragment precedes cleavage of the linking group in vivo. Degradation of the antibody portion of an ADC having a stable or non-cleavable linking group may leave some or all of the linking group, e.g., one or more amino acid groups, from the antibody attached to the payload or drug moiety that is delivered in vivo.

[0034] The term "antibody" refers to a polypeptide of the immunoglobulin family that can non-covalently, reversibly, and specifically bind to a corresponding antigen. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). VH and VL each consist of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0035] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (e.g., anti-Id antibodies to an antibody of the invention). Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0036] The terms "complementarity-determining domain" or "complementarity-determining region" ("CDR") interchangeably refer to the hypervariable regions of VL and VH. CDRs are the target protein-binding sites of antibody chains, which confers specificity for such target proteins. Three CDRs (numbered sequentially from the N-terminus, CDR1 to CDR3) are present in each human VL or VH, constituting approximately 15 to 20% of the variable domain. CDRs are structurally complementary to the epitope of the target protein and are therefore directly responsible for binding specificity. The remaining regions of VL or VH, the so-called framework regions, show less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).

[0037] The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, the International ImMunoGeneTics database (IMGT) (on the World Wide Web at www.imgt.org / ), and AbM (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)). The definition of an antigen-binding site is also described in Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).

[0038] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light (CL) and heavy (CH1, CH2, or CH3) chains confer important biological properties, such as secretion, transplacental motility, Fc receptor binding, complement binding, etc. By convention, the numbering of constant region domains increases distally from the antigen-binding site or amino-terminus of the antibody. The N-terminus is the variable region, and the C-terminus is the constant region; the CH3 and CL domains actually comprise the carboxy-terminal domains of the heavy and light chains, respectively.

[0039] The term "antigen-binding fragment" as used herein refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of binding fragments include, but are not limited to, single-chain Fv (scFv), camelid antibody, disulfide-bridged Fv (sdFv), Fab fragment, F(ab') fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragment consisting of the VH and CH1 domains; Fv fragment consisting of the VL and VH domains of a single arm of an antibody; dAb fragment consisting of the VH domain (Ward et al., Nature 341:544-546, 1989); and isolated complementarity-determining regions (CDRs) or other epitope-binding fragments of an antibody.

[0040] Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods with a synthetic linker, allowing the two domains to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv ("scFv"); see, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883, 1988). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment." These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0041] Antigen-binding fragments may also be incorporated into single domain antibodies, maxibodies, minibodies, single domain antibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments may be grafted into polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).

[0042] The antigen-binding fragment may be incorporated into a single-chain molecule comprising a pair of tandem Fv segments (VH-CH1-VH-CH1), which, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8:1057-1062, 1995; and U.S. Pat. No. 5,641,870).

[0043] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to polypeptides, including antibodies and antigen-binding fragments, having substantially the same amino acid sequence or derived from the same genetic source. The terms also include preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0044] As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from a human sequence, e.g., a human germline sequence, or a mutated version of a human germline sequence, or an antibody-containing consensus framework sequence derived from human framework sequence analysis, e.g., as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000. Also included are antibodies derived from human sequences in which one or more CDRs have been mutated for affinity maturation or manufacturing / payload-binding purposes. See Kilpatrick et al., "Rapid development of affinity matured monoclonal antibodies using RIMMS," Hybridoma. 1997 Aug;16(4):381-9.

[0045] The human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or conservative substitutions that enhance stability or manufacturing).

[0046] As used herein, the term "recognize" refers to an antibody or antigen-binding fragment thereof finding and interacting with (e.g., binding to) its epitope, whether linear or conformational. The term "epitope" refers to the site on an antigen to which an antibody or antigen-binding fragment of the present invention specifically binds. Epitopes can be formed both from contiguous amino acids or from noncontiguous amino acids arranged by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include techniques in the art, such as x-ray crystallography and two-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)). A "paratope" is the portion of an antibody that recognizes an epitope of an antigen.

[0047] The term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen at multiple sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0048] The term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. However, an isolated antibody that specifically binds to an antigen may have cross-reactivity to other antigens. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0049] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence, as determined in comparison with all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. Corresponding human germline sequence may also refer to the human variable region amino acid sequence or subsequence that shares the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence, as determined in comparison with all other variable region amino acid sequences evaluated. The corresponding human germline sequence may be framework regions only, complementarity determining regions only, framework and complementarity determining regions, variable segments (as defined above), or other combinations of sequences or subsequences that comprise variable regions. Sequence identity can be determined using methods described herein, such as aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid or amino acid sequence of the reference variable region. Corresponding human germline sequences can be determined, for example, through the publicly available International ImMunoGeneTics database (IMGT) (on the World Wide Web at www.imgt.org / ) and V-base (on the World Wide Web at vbase.mrc-cpe.cam.ac.uk).

[0050] The phrases "specifically bind" or "selectively bind," when used in the context of describing the interaction of an antigen (e.g., a protein) with an antibody, antibody fragment, or antibody-derived binding agent, refer to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologics, e.g., in a biological sample, such as a blood, serum, plasma, or tissue sample. Thus, under specified, specific immunoassay conditions, an antibody or binding agent with a specific binding specificity will bind to the specific antigen at least twice as much as background and will not significantly bind to other antigens present in the sample. In one embodiment, under specified, specific immunoassay conditions, an antibody or binding agent with a specific binding specificity will bind to the specific antigen at least 10 times as much as background and will not significantly bind to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require that the antibody or agent be selected for its specificity for a particular protein. If desired or appropriate, this selection can be achieved by subtracting out antibodies from other species (e.g., mouse or rat) or other subtypes that cross-react with the molecule. Additionally, in some embodiments, antibodies or antibody fragments are selected that cross-react with a particular molecule of interest.

[0051] A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, *Using Antibodies*, *A Laboratory Manual* (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction will produce a signal at least two times the background signal, more typically at least 10-100 times the background signal.

[0052] The term "equilibrium dissociation constant (Kd, M)" refers to the dissociation rate constant (kd, time-1) divided by the association rate constant (ka, time-1, M-1). The equilibrium dissociation constant can be measured using any method known in the art. Antibodies of the present invention typically have an equilibrium dissociation constant of about 10 -7 or 10 -8 Less than M, e.g., about 10 -9 M or 10 -10 less than about 10 M, in some embodiments -11 M, 10 -12 M or 10 -13 It will be less than M.

[0053] The term "bioavailability" refers to the systemic availability (i.e., blood / plasma levels) of a given amount of drug administered to a patient. Bioavailability is an absolute value that indicates measurement of both the time (rate) and total amount (extent) of drug that reaches the systemic circulation from an administered dosage form.

[0054] As used herein, the phrase "consisting essentially of" refers to the genus or species of active agent included in a method or composition, and any excipients that are inert for the intended use of the method or composition. In some embodiments, the phrase "consisting essentially of" explicitly excludes the inclusion of one or more additional active agents other than the antibody drug conjugate of the invention. In some embodiments, the phrase "consisting essentially of" explicitly excludes the inclusion of one or more additional active agents other than the antibody drug conjugate of the invention and a co-administered second agent.

[0055] The term "amino acid" refers to naturally occurring, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. An amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a compound that has a structure different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

[0056] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode the same or essentially the same amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, to essentially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variation. Additionally, all nucleic acid sequences herein that encode a polypeptide describe all possible silent variations of the nucleic acid. Those of skill in the art will recognize that each codon in a nucleic acid (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0057] With respect to polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions, or additions to a polypeptide sequence that substitute an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence.

[0058] As used herein, the term "optimized" refers to a nucleotide sequence that has been altered to encode an amino acid sequence using codons preferred in a production cell or organism, usually a eukaryotic cell, such as a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese hamster ovary cell (CHO), or a human cell. An optimized nucleotide sequence has been engineered to retain, entirely, or as closely as possible, the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the "parent" sequence.

[0059] The terms "percent identical" or "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refer to the degree to which two or more sequences or subsequences are the same. Two sequences are "identical" if the amino acid or nucleotide sequence is the same over the region being compared. Two sequences are "substantially identical" if a specified percentage of amino acid residues or nucleotides are the same (i.e., 60% identity, or in some cases 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, if not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using one of the sequence comparison algorithms described below or by manual alignment and visual inspection. In some cases, identity exists over a region that is at least about 30 nucleotides (or 10 amino acids) in length, more preferably over a region that is 100-500 nucleotides or 1000 nucleotides or more (or 20, 50, 200 amino acids or more) in length.

[0060] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. Using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0061] As used herein, a "comparison window" includes reference to any one segment of the number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150, within which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, 2003).

[0062] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is halted when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program defaults to a word length of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) uses an alignment (B) of 50, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

[0063] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0064] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17 (1988), which is incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453 (1970), which is incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a BLOSUM62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0065] Another indication that two nucleic acid sequences or polypeptides are substantially identical, other than the percentage of sequence identity noted above, is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0066] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including synthetic, naturally occurring, and non-naturally occurring nucleic acids, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0067] Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly set forth. Specifically, as detailed below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91-98).

[0068] The term "operably linked" in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence with a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Typically, a promoter transcriptional regulatory sequence that is operably linked to a transcribed sequence is physically contiguous to the transcribed sequence, i.e., cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically adjacent to or positioned closely adjacent to the coding sequence whose transcription they enhance.

[0069] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0070] As used herein, the term "antibody-drug conjugate" or "immunoconjugate" refers to the attachment of an antibody or antigen-binding fragment thereof to another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, etc. The attachment can be via a covalent bond or a non-covalent interaction, such as electrostatic forces. Various linkers known in the art can be used to form antibody-drug conjugates. Furthermore, antibody-drug conjugates can be provided in the form of a fusion protein that can be expressed from a polynucleotide encoding the immunoconjugate. As used herein, a "fusion protein" refers to a protein created through the joining of two or more genes or gene fragments that originally encoded separate proteins (e.g., peptides and polypeptides). Translation of the fusion gene results in a single protein possessing functional properties derived from each of the original proteins.

[0071] The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where indicated, the terms "patient" or "subject" are used interchangeably herein.

[0072] The term "cytotoxin" or "cytotoxic agent" as used herein refers to any agent that is detrimental to the growth and proliferation of cells and can act to reduce, inhibit, or destroy cells or malignancies.

[0073] As used herein, the term "anti-cancer agent" refers to any agent that can be used to treat or prevent a cell proliferative disorder, e.g., cancer, including, but not limited to, cytotoxic agents, chemotherapeutic agents, radiation therapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0074] As used herein, the term "drug moiety" or "payload" refers to a chemical moiety attached to an antibody or antigen-binding fragment of the invention and can include any therapeutic or diagnostic agent, such as an anti-cancer, anti-inflammatory, anti-infective (e.g., anti-fungal, anti-bacterial, anti-parasitic, anti-viral), or anesthetic agent. For example, the drug moiety can be an anti-cancer agent, such as a cytotoxin. In certain embodiments, the drug moiety is a target inhibitor compound. Furthermore, the payload can be a biophysical probe, a fluorescent label, a spin label, an infrared probe, an affinity probe, a chelator, a spectroscopic probe, a radioactive probe, a lipid molecule, polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein, a peptide, a surface, an antibody, an antibody fragment, a nanoparticle, a quantum dot, a liposome, a PLGA particle, a sugar, or a polysaccharide.

[0075] In some embodiments, the drug moiety or payload is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor). In some embodiments, the GNAQ / 11 inhibitor is a molecule that inhibits GNAQ / 11-mediated production of IP3 and / or exhibits a dose-response antiproliferative effect in cells dependent on GNAQ / 11 signaling (i.e., GNAQ / 11-mutant uveal melanoma cells). In some embodiments, the GNAQ / 11 inhibitor is a compound that stabilizes GNAQ / 11 in an inactive GDP-bound state, preventing GDP release, or binds to an active GTP-bound state, preventing GNAQ / 11 interaction with downstream effectors. In some embodiments, the GNAQ / 11 inhibitor functions by inhibiting mutant GNAQ and / or GNA11, e.g., those containing the Q209L / P mutation. Methods for attaching such drug moieties to linkers compatible with targeting moieties are listed in this disclosure along with methods known in the art, see, e.g., Singh et al., (2009) Therapeutic Antibodies: Methods and Protocols, vol. 525, 445-457.

[0076] GNAQ (guanine nucleotide-binding protein G(q) subunit alpha, also known as CMC1, G-ALPHA-q, GAQ, SWS, and G protein subunit alpha q) and GNA11 (guanine nucleotide-binding protein subunit alpha-11, also known as FBH, FBH2, FHH2, GNA-11, HHC2, HYPOC2, and G protein subunit alpha 11) are closely related GTPases that constitute the α subunits of heterotrimeric G proteins that act downstream of G protein-coupled receptors (GPCRs). The α subunit acts as a switch for G protein activation by exchanging guanosine diphosphate (GDP) for guanosine triphosphate (GTP), leading to the activation of distinct downstream effectors. Activation is terminated by intrinsic GTPase activity. This is because GTP is hydrolyzed to GDP (Van Raamsdonk et al., 2010, N Engl J Med.;363(23):2191-9). Classical activation of the Gq protein cascade occurs via phospholipase C-β (PLC-β), which hydrolyzes the phospholipid phosphatidylinositol 4,5-bisphosphate to release two potent second messengers: D-myo-inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Intracellular Ca 2+ After a transient increase in DAG, IP3 is rapidly converted to IP2, IP1, and myo-inositol. Meanwhile, DAG activates protein kinase C (PKC), leading to a cascade of phosphorylation of RAF, MEK, and ERK, which translocate to the nucleus and regulate cell proliferation and survival (Krantz et al., 2017, Clin Ophthalmol.;11:279-289).

[0077] The nucleic acid and amino acid sequences of human GNAQ are published in GenBank under the following accession numbers: NP_002063 (sequence number 268) [ka] NM_002072 (sequence number 269) [ka] [ka] [ka]

[0078] The nucleic acid and amino acid sequences of human GNA11 are published in GenBank under the following accession numbers: NP_002058 (SEQ ID NO: 270) [ka] NM_002067 (sequence number 271) [ka] [ka] [ka]

[0079] "Tumor" refers to neoplastic cell growth and proliferation, whether malignant or benign, including all pre-cancerous and cancerous cells and tissues.

[0080] The term "anti-tumor activity" refers to a reduction in the rate of tumor cell proliferation, survival, or metastatic activity. For example, anti-tumor activity can be demonstrated by a decrease in the rate of growth of abnormal cells or a stable or reduced tumor size during treatment, or a longer survival period resulting from treatment compared to a control without treatment. Such activity can be assessed using recognized in vitro or in vivo tumor models, including, but not limited to, xenograft models, allograft models, MMTV models, and other known models known in the art for investigating anti-tumor activity.

[0081] The term "malignant tumor" refers to a non-benign tumor or cancer. As used herein, the term "cancer" includes malignant tumors characterized by dysregulated or uncontrolled cell growth. Exemplary cancers include carcinomas, sarcomas, leukemias, and lymphomas.

[0082] The term "cancer" includes primary malignant tumors (e.g., those whose cells have not migrated to sites in a subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., those resulting from metastasis, which is the migration of tumor cells to secondary sites different from the site of the original tumor).

[0083] The term "PMEL17" (also known as pre-melanosome protein (PMEL), D12S53E, ME20, ME20-M, ME20M, P1, P100, gp100, SI, SIL, and silver locus protein homolog (SILV)) refers to a type I single-pass transmembrane protein produced by melanocytes and involved in melanin synthesis. The nucleic acid and amino acid sequences of human PMEL17 have been published in GenBank under the following accession numbers: NP_008859, NP_001307050, NP_001307051, NP_001186982, NP_001186983 (amino acid sequences), and NM_006928, NM_001200053, NM_001200054, NM_001320121, NM_001320122 (nucleotide sequences). As used herein, the term "PMEL17" is used to collectively refer to all naturally occurring isoforms of the PMEL17 protein, or variants thereof. NP_008859 (SEQ ID NO: 272) [ka] NP_001307050 (sequence number 273) [ka] NP_001307051 (sequence number 274) [ka] NP_001186982 (sequence number 275) [ka] NP_001186983 (sequence number 276) [ka] NM_006928 (sequence number 277) [ka] NM_001200053 (sequence number 278) [ka] NM_001200054 (sequence number 279) [ka] NM_001320121 (sequence number 280) [ka] NM_001320122 (sequence number 281) [ka]

[0084] The term "variant" refers to a polypeptide that has substantially the same amino acid sequence as a reference polypeptide, or is encoded by substantially the same nucleotide sequence, and may have one or more activities of the reference polypeptide. For example, a variant may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the reference polypeptide, while retaining one or more activities of the reference polypeptide.

[0085] As used herein, the terms "treat," "treating," or "treatment" of any disease or disorder refer, in one embodiment, to ameliorating the disease or disorder (i.e., slowing, inhibiting, or alleviating the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treat," "treating," or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treat," "treating," or "treatment" refers to modulating the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of a physical parameter), or both.

[0086] As used herein, the terms "prevent," "preventing," and "prevention" of any disease or disorder refer to prophylactic treatment of a disease or disorder or delaying the progression of a disease or disorder.

[0087] The terms "therapeutically acceptable amount" or "therapeutically effective dose" refer interchangeably to an amount sufficient to produce a desired result (i.e., reduction in tumor size, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal, or parasitic infection). In some embodiments, a therapeutically acceptable amount does not induce or cause undesirable side effects. In some embodiments, a therapeutically acceptable amount induces or causes side effects, but only those side effects that are tolerable by a healthcare provider in light of the patient's condition. A therapeutically acceptable amount may be determined by administering an initial low dose and gradually increasing the dose until the desired effect is achieved. A "prophylactically effective dose" and a "therapeutically effective dose" of a molecule of the invention may prevent the onset of or reduce the severity of disease symptoms, for example, cancer-related disease symptoms, respectively.

[0088] The term "co-administration" refers to the simultaneous presence of two active agents in the blood of an individual. The active agents may be delivered simultaneously or sequentially.

[0089] The present invention provides antibodies, antibody fragments (e.g., antigen-binding fragments), and drug conjugates thereof, i.e., antibody-drug conjugates or ADCs, that bind to PMEL17. In particular, the present invention provides antibodies and antibody fragments (e.g., antigen-binding fragments) that bind to PMEL17 and are internalized upon such binding. The antibodies and antibody fragments (e.g., antigen-binding fragments) of the present invention can be used to produce antibody-drug conjugates. Furthermore, the present invention provides antibody-drug conjugates that have desirable pharmacokinetic characteristics and other desirable attributes and can therefore be used to treat or prevent cancers that express PMEL17. The present invention further provides pharmaceutical compositions comprising the antibody-drug conjugates of the present invention, as well as methods of making and using such pharmaceutical compositions for the treatment or prevention of cancer.

[0090] Drug Moiety (D) In one embodiment, the drug moiety (D) of the antibody drug conjugate of the invention is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor).

[0091] In another embodiment, the drug moiety (D) of the antibody drug conjugate of the invention is a GNAQ inhibitor.

[0092] In another embodiment, the drug moiety (D) of the antibody drug conjugate of the invention is a GNA11 inhibitor.

[0093] In another embodiment, the drug moiety (D) of the antibody drug conjugate of the invention is an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor).

[0094] In another embodiment, the drug moiety of the antibody drug conjugate of the invention is Structure of formula (A): [ka] (wherein R0 is methyl or ethyl, R1 is methyl or i-propyl, and R2 is methyl or ethyl) It is a compound having the formula:

[0095] In another embodiment, the drug moiety of the antibody drug conjugate of the invention has the following structure: [ka] The compound (A1) has the formula:

[0096] In another embodiment, the drug moiety of the antibody drug conjugate of the invention has the following structure: [ka] The compound (A2) has the formula:

[0097] In another embodiment, the drug moiety of the antibody drug conjugate of the invention has the following structure: [ka] The compound (A3) has the formula:

[0098] Table 1 shows the inhibitory activity of compounds (A1), (A2) and (A3) obtained using the assay described in Example 5.

[0099] [Table 1]

[0100] Linker-drug moiety (L A -(D) n ) In a second embodiment, the linker-drug moiety (L A -(D) n ))) is the linker (L A ), wherein the one or more drug moieties are each independently selected from a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor).

[0101] In another embodiment, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), wherein the one or more drug moieties are each independently selected from a GNAQ inhibitor.

[0102] In another embodiment, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ) attached to said antibody, wherein said one or more drug moieties are each independently selected from a GNA11 inhibitor.

[0103] In another embodiment, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), wherein the one or more drug moieties are each independently selected from inhibitors of GNAQ and GNA11 (GNAQ / GNA11 inhibitors).

[0104] In another embodiment, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a cleavable linker, and the one or more drug moieties are each independently selected from a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor).

[0105] In another embodiment, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A) is a cleavable linker and the one or more drug moieties are each independently selected from a GNAQ inhibitor.

[0106] In another embodiment, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a cleavable linker, and the one or more drug moieties are each independently selected from a GNA11 inhibitor.

[0107] In another embodiment, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a cleavable linker and the one or more drug moieties are each independently selected from inhibitors of GNAQ and GNA11 (GNAQ / GNA11 inhibitors).

[0108] In another embodiment, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a non-cleavable linker, and the one or more drug moieties are each independently selected from a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor).

[0109] In another embodiment, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A) is a non-cleavable linker and the one or more drug moieties are each independently selected from a GNAQ inhibitor.

[0110] In another embodiment, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a non-cleavable linker and the one or more drug moieties are each independently selected from a GNA11 inhibitor.

[0111] In another embodiment, the linker-drug moiety ((L A -(D) n ))) is the linker (L A ), and one or more drug moieties covalently attached to a linker (L A ) is a non-cleavable linker and the one or more drug moieties are each independently selected from inhibitors of GNAQ and GNA11 (GNAQ / GNA11 inhibitors).

[0112] In another embodiment, the linker-drug moiety ((L A -(D) n ))) linker (L A ) is expressed as: [ka] (In the formula, X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2, and Y1 ** indicates other attachment points; L1 is a bivalent peptide linker, L2 is a bond or a linker It has.

[0113] In another embodiment, the linker-drug moiety, ((L A -(D) n ))) is the following formula: [ka] (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2, and Y1 ** indicates the point of attachment to D; L1 is a bivalent peptide linker, L2 is a bond or a linker It has.

[0114] Linker-drug compound (L B -(D) n ) In one embodiment, the linker-drug of the present invention has the structure of Formula (B): R 8 -L B -(D) n (B) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; R 8 is a reactive group; L B is a cleavable or non-cleavable linker, n is 1, 2, 3 or 4 It is a compound having the formula:

[0115] In one embodiment, wherein: D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; R 8 is a reactive group; L B is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, a carbonate group, and a bivalent peptide linker; n is 1, 2, 3 or 4; A linker-drug of the present invention having the structure of Formula (B):

[0116] In one aspect, the linker-drug of the present invention has the structure of formula (B-1): [ka] (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; R 8 is a reactive group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2, and Y1 ** indicates the point of attachment to D; L1 is a bivalent peptide linker, L2 is a bond or a linker It is a compound having the formula:

[0117] Specific aspects and examples of linker-drug compounds of the present invention are provided in the following list of additional enumerated embodiments. It is recognized that the features specified in each embodiment can be combined with other specific features to provide further embodiments of the present invention.

[0118] Embodiment 1. A compound of Formula (B) or Formula (B-1), or a stereoisomer or pharmaceutically acceptable salt thereof, wherein D is a GNAQ inhibitor.

[0119] Embodiment 2. A compound of Formula (B) or Formula (B-1), or a stereoisomer or pharmaceutically acceptable salt thereof, wherein D is a GNA11 inhibitor.

[0120] Embodiment 3. A compound of Formula (B) or Formula (B-1), or a stereoisomer or pharmaceutically acceptable salt thereof, wherein D is an inhibitor of GNAQ and GNA11.

[0121] Embodiment 4. In the formula, D is [ka] (In the formula, R 0 is methyl or ethyl, and R 1 is methyl or isopropyl, and R 2 is methyl or ethyl, *** L B or Y1) or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0122] Embodiment 5. In the formula, D is [ka] (In the formula, *** L B or Y1) or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0123] Embodiment 6. In the formula, D is [ka] (In the formula, *** LB or Y1) or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0124] Embodiment 7. In the formula, D is [ka] (In the formula, *** L B or Y1) or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0125] Embodiment 8. A structure of formula (B-2), or a pharmaceutically acceptable salt thereof [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl, X2, L1, L2 and R 8 is as defined in the compound of formula (B-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof.

[0126] Embodiment 9. A structure of formula (B-2a), or a pharmaceutically acceptable salt thereof [ka] (In the formula, X2, L1, L2 and R 8 is as defined in the compound of formula (B-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof.

[0127] Embodiment 10. A structure of formula (B-2b): or a pharmaceutically acceptable salt thereof [ka] (In the formula, X2, L1, L2 and R 8 is as defined in the compound of formula (B-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof.

[0128] Embodiment 11. A structure of formula (B-2c), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, X2, L1, L2 and R 8 is as defined in the compound of formula (B-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof.

[0129] Embodiment 12. A structure of formula (B-3), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl, X2, L1, L2 and R 8 is as defined in the compound of formula (B-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof.

[0130] Embodiment 13. A structure of formula (B-3a), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, X2, L1, L2 and R 8 is as defined in the compound of formula (B-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof.

[0131] Embodiment 14. A structure of formula (B-3b), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, X2, L1, L2 and R 8 is as defined in the compound of formula (B-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof.

[0132] Embodiment 15. A structure of formula (B-3c), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, X2, L1, L2 and R 8 is as defined in the compound of formula (B-1) above) or a stereoisomer or pharmaceutically acceptable salt thereof.

[0133] Embodiment 16. wherein: R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; X2 is [ka] wherein X2 is a self-immolative spacer selected from * indicates the attachment point to L1, and X2 ** teeth, [ka] the point of attachment to the group, or [ka] indicates the point of attachment to the group; L1 is a bivalent peptide linker containing 2 to 4 amino acid residues; L2 is a linker, R 8 teeth, [ka] , -N3, -ONH2, -NR 4 C(=O)CH=CH2, SH, -SSR 13 , -S(=O)2(CH=CH2), -NR 4 S(=O)2(CH=CH2), -NR 4 C(=O)CH2Br, -NR 4 C(=O)CH2I, -NHC(=O)CH2Br, -NHC(=O)CH2I, -C(=O)NHNH2, [ka] , -CO2H, -NH2, [ka] is selected from: Each R 4 is independently selected from H and C1-C6 alkyl; Each R 5 are independently selected from H, C1-C6 alkyl, F, Cl, and —OH; Each R 6are independently selected from H, C1-C6 alkyl, F, Cl, —NH2, —OCH3, —OCH2CH3, —N(CH3)2, —CN, —NO2, and —OH; Each R 7 are independently H, C 1~6 Alkyl, fluoro, benzyloxy substituted by -C(=O)OH, benzyl substituted by -C(=O)OH, C substituted by -C(=O)OH 1~4 C substituted by alkoxy and -C(=O)OH 1~4 selected from alkyl, A compound of formula (B-2) of embodiment 8, formula (B-3) of embodiment 12, or a pharmaceutically acceptable salt thereof.

[0134] Embodiment 17. In the formula, X2 is [ka] wherein X2 is a self-immolative spacer selected from * indicates the attachment point to L1, and X2 ** is the attachment point to Y1, [ka] the point of attachment to the group, or [ka] 17. The compound of any one of embodiments 1 to 16, showing the point of attachment to the group.

[0135] Embodiment 18. In the formula, X2 is [ka] In the formula, X2 * indicates the attachment point to L1, and X2 ** is the attachment point to Y1, [ka] the point of attachment to the group, or [ka] 18. The compound of any one of embodiments 1 to 17, showing the point of attachment to the group.

[0136] Embodiment 19. The compound of any one of embodiments 1 to 18, wherein L1 is a bivalent peptide linker comprising 2 to 4 amino acid residues.

[0137] Embodiment 20. The compound of any one of embodiments 1-18, wherein L1 is a bivalent peptide linker comprising an amino acid residue selected from valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

[0138] Embodiment 21. The compound of any one of embodiments 1 to 18, wherein L1 is a bivalent peptide linker comprising at least one valine (Val) or citrulline (Cit) residue.

[0139] Embodiment 22. The compound of any one of embodiments 1 to 18, wherein L1 is a bivalent dipeptide linker selected from ValCit, PheLys, ValAla, and ValLys.

[0140] Embodiment 23. In the formula, L1 is [ka] wherein L1 is a divalent dipeptide linker selected from * indicates the attachment point to L2, and ** 19. The compound of any one of embodiments 1 to 18, wherein indicates the point of attachment to X2.

[0141] Embodiment 24. The compound of any one of embodiments 1 to 18, wherein L1 is ValCit.

[0142] Embodiment 25. In the formula, L1 is [ka] where L1 * indicates the attachment point to L2, and ** 19. The compound of any one of embodiments 1 to 18, wherein indicates the point of attachment to X2.

[0143] Embodiment 26. The compound of any one of embodiments 1 to 25, wherein L2 is a linker.

[0144] Embodiment 27. In the formula, L2 is - * C(=O)((CH2) m O) p (CH2) m ** -,- * C(=O)(CH2) m ** -,- * C(=O)(CH2) n NHC(=O)(CH2) m ** -,- * C(=O)(CH2) m NHC(=O)((CH2) m O) p (CH2) m ** -,- * ((CH2) m O) p (CH2) m ** -,- * ((CH2) m O) p (CH2) m ** -, -(CH2) m -,- * (CH2) m NHC(=O)(CH2) m ** -,- * (CH2) m NHC(=O)(CH2) m C(=O)NH(CH2) m ** -,- * ((CH2) m O) p (CH2) mNHC(=O)(CH2) m ** -,- ** ((CH2) m O) p CH2) m C(=O)NH(CH2) m ** -,- * (CH2) m C(R3)2 ** -and- * (CH2) m C(R3)2SS(CH2) m NHC(=O)(CH2) m ** -, wherein L2 is a linker selected from * indicates the attachment point to L1, and ** indicates the attachment point to R8; During the ceremony, each R3 is independently selected from H and C1-C6 alkyl; each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; 26. The compound of any one of embodiments 1 to 25, wherein each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.

[0145] Embodiment 28. In the formula, L2 is - * C(=O)((CH2) m O) p (CH2) m ** -or- * C(=O)(CH2) m ** -, where L2 * indicates the attachment point to L1, and ** indicates the attachment point to R8, 26. The compound of any one of embodiments 1 to 25, wherein each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0146] Embodiment 29. In the formula, L2 is [ka] where L2 * indicates the attachment point to L1, and ** 26. The compound of any one of embodiments 1 to 25, wherein indicates the point of attachment to R8.

[0147] Embodiment 30. In the formula, R 8 teeth, [ka] , -N3, -ONH2, -NR 4 C(=O)CH=CH2, SH, -SSR 13 , -S(=O)2(CH=CH2), -NR 4 S(=O)2(CH=CH2), -NR 4 C(=O)CH2Br, -NR 4 C(=O)CH2I, -NHC(=O)CH2Br, -NHC(=O)CH2I, -C(=O)NHNH2, [ka] , -CO2H, -NH2, [ka] where: Each R 4 is independently selected from H and C1-C6 alkyl; Each R 5 are independently selected from H, C1-C6 alkyl, F, Cl, and —OH; Each R 6 are independently selected from H, C1-C6 alkyl, F, Cl, —NH2, —OCH3, —OCH2CH3, —N(CH3)2, —CN, —NO2, and —OH; Each R 7 are independently H, C 1-6Alkyl, fluoro, benzyloxy substituted by -C(=O)OH, benzyl substituted by -C(=O)OH, C substituted by -C(=O)OH 1~4 C substituted by alkoxy and -C(=O)OH 1~4 30. The compound of any one of embodiments 1 to 29, wherein the alkyl is selected from:

[0148] Embodiment 31. In the formula, R 8 teeth, [ka] 30. The compound of any one of embodiments 1 to 29, wherein

[0149] Embodiment 32. In the formula, R 8 teeth, [ka] 30. The compound of any one of embodiments 1 to 29, wherein

[0150] Embodiment 33. In the formula: R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; X2 is [ka] In the formula, X2 * indicates the attachment point to L1, and X2 ** teeth, [ka] indicates the point of attachment to the group; L1 is [ka] where L1 * indicates the attachment point to L2, and** indicates the attachment point to X2; L2 is [ka] where L2 * indicates the attachment point to L1, and ** indicates the attachment point to R8, R 8 teeth, [ka] That is, The compound of formula (B-2) of embodiment 8, or a pharmaceutically acceptable salt thereof.

[0151] Embodiment 34. wherein: R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; X2 is [ka] In the formula, X2 * indicates the attachment point to L1, and X2 ** teeth, [ka] indicates the point of attachment to the group; L1 is [ka] where L1 * indicates the attachment point to L2, and ** indicates the attachment point to X2; L2 is [ka] where L2 * indicates the attachment point to L1, and **indicates the attachment point to R8, R 8 teeth, [ka] That is, 13. A compound of formula (B-3) according to embodiment 12, or a pharmaceutically acceptable salt thereof.

[0152] Embodiment 35. [ka] A compound of formula (B), formula (B-1) or formula (B-2),

[0153] Embodiment 36. [ka] A compound of formula (B), formula (B-1) or formula (B-2),

[0154] Embodiment 37. [ka] A compound of formula (B), formula (B-1) or formula (B-2),

[0155] Embodiment 38. [ka] A compound of formula (B), formula (B-1) or formula (B-2),

[0156] Embodiment 39. [ka] A compound of formula (B), formula (B-1) or formula (B-2),

[0157] Embodiment 40. [ka] A compound of formula (B), formula (B-1) or formula (B-2),

[0158] Embodiment 41. [ka] A compound of formula (B), formula (B-1) or formula (B-3),

[0159] Embodiment 42. [ka] A compound of formula (B), formula (B-1) or formula (B-3),

[0160] Embodiment 43. [ka] A compound of formula (B), formula (B-1) or formula (B-3),

[0161] Embodiment 44. [ka] A compound of formula (B), formula (B-1) or formula (B-3),

[0162] Embodiment 45. [ka] A compound of formula (B), formula (B-1) or formula (B-3),

[0163] Embodiment 46. [ka] A compound of formula (B), formula (B-1) or formula (B-3),

[0164] antibody-drug conjugates In one embodiment, the antibody drug conjugate of the present invention has formula (C): Ab-(L A -(D)n ) y (C) (In the formula, D is a drug moiety; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; L A is a linker; n is 1, 2, 3 or 4; y is 1, 2, 3 or 4; Linker-drug moiety (L A -(D) n ) is covalently attached to the antibody or antigen-binding fragment thereof It is a combination of

[0165] In one embodiment, wherein: D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; L A is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, a carbonate group, and a bivalent peptide linker; n is 1, 2, 3 or 4; y is 1, 2, 3 or 4; Linker-drug moiety (L A -(D) n ) is covalently attached to the antibody or antigen-binding fragment thereof, an antibody-drug conjugate of the present invention having the structure of formula (C):

[0166] In one embodiment, the antibody drug conjugate of formula (C) has the formula (C-1): [ka] (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor or an inhibitor of GNAQ and GNA11; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2, and Y1 ** indicates the point of attachment to D; L1 is a bivalent peptide linker; L2 is a bond or a linker; y is 1, 2, 3 or 4 It is a combination of

[0167] In the conjugate of formula (C), one or more linker-drug moieties (L B -(D) n ) can be covalently attached to an antibody or antigen-binding fragment thereof Ab, thereby attaching one or more drug moieties D to the antibody or antigen-binding fragment thereof Ab via a linker L A Covalently attached via L A is any chemical moiety capable of linking an antibody or antigen-binding fragment thereof Ab to one or more drug moieties D. Conjugates of formula (C), in which one or more drug moieties D are covalently attached to an antibody or antigen-binding fragment thereof Ab, can be formed using bifunctional or polyfunctional linker reagents having one or more reactive functional groups, which may be the same or different. One of the reactive functional groups of the bifunctional or polyfunctional linker reagent is used to react with a group on the antibody or antigen-binding fragment thereof Ab, for example, a thiol or amine (e.g., cysteine, the N-terminus, or an amino acid side chain, e.g., lysine), to form the linker L. A Such reactive functional groups of a bifunctional or polyfunctional linker reagent include, but are not limited to, maleimide, thiol, and NHS ester. One or more other reactive functional groups of a bifunctional or polyfunctional linker reagent may connect one or more drug moieties D to a linker L. AIt is used to covalently attach to

[0168] In one embodiment, L A is a cleavable linker. A is a non-cleavable linker. A is an acid labile linker, a photolabile linker, a peptidase cleavable linker, an esterase cleavable linker, a glycosidase cleavable linker, a phosphodiesterase cleavable linker, a disulfide bond reducible linker, a hydrophilic linker, or a dicarboxylic acid-based linker.

[0169] In one embodiment, L A is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, a carbonate group, and a bivalent peptide linker.

[0170] In one embodiment, L A is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, a carbonate group, a bivalent peptide linker, and a bivalent linking group.

[0171] In one embodiment, L A is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, and a bivalent peptide linker.

[0172] In one embodiment, L A is a cleavable linker comprising one or more linker components selected from a self-immolative spacer, a phosphate group, a bivalent peptide linker, and a bivalent linking group.

[0173] In another embodiment, the linker (L A ) is expressed as: [ka] (In the formula, X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2, and Y1 ** indicates other attachment points; L1 is a bivalent peptide linker, L2 is a bond or a linker It has.

[0174] In another embodiment, the linker (L A ) is expressed as: [ka] (In the formula, X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2; L1 is a bivalent peptide linker, L2 is a bond or a linker It has.

[0175] In another embodiment, the linker (L A ) is expressed as: [ka] (In the formula, X1 is a divalent linking group; X2 is a self-sacrificing spacer; Y1 is [ka] wherein Y1 * indicates the attachment point to X2; L1 is a bivalent peptide linker, L2 is a bond or a linker It has.

[0176] While the drug-antibody ratio has a precise integer value for a particular conjugate molecule (e.g., the product of n and y in Formula (C)), it is understood that when used to describe a sample containing many molecules, due to the degree of heterogeneity typically associated with the conjugation step, the value is often an average value. The average loading for a sample of conjugates is referred to herein as the drug-antibody ratio, or "DAR." In some embodiments, the DAR is from about 1 to about 5, and typically about 1, 2, 3, or 4. In some embodiments, at least 50% of the sample by weight is a compound having an average DAR plus or minus 2, and preferably at least 50% of the sample is a conjugate containing an average DAR plus or minus 1. Other embodiments include conjugates with a DAR of about 2. In some embodiments, a DAR of "about y" means that the measured value for the DAR is within 20% of the product of n and y in Formula (I). In some embodiments, a DAR of "about n" means that the measured value for the DAR is within 20% of n in Formula (II).

[0177] In one embodiment, the average molar ratio of drug to antibody in the conjugate of Formula (C) (i.e., the average value of the product of n and y, also known as the drug-antibody ratio (DAR)) is from about 1 to about 10, from about 1 to about 6 (e.g., 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.10, 3.11, 3.12, 3.13, 3.14, 3.15, 3.16, 3.17, 3.18, 3.19, 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.30, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, 4.40, 4.41, 4.42, 4.43, 4.44, 4.45, 4.46, 4.47, 4.48, 4.49, 4.50, 4.51, 4.52, 4.53, 4.54, 4.55, 4.56, 4.57, 4.58, 4.59, 4.60, 4.61, 4.62, 4. 0.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0), about 1 to about 5, about 1.5 to about 4.5, or about 2 to about 4.

[0178] In one embodiment provided by the present disclosure, the conjugates have a substantially high degree of purity and have one or more of the following characteristics: (a) greater than about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100%), preferably greater than about 95%, of the conjugate species are monomeric; (b) the conjugate preparation has an unconjugated linker level of less than about 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or 0%) (of total linker); and (c) less than 10% of the conjugate species are crosslinked. (d) the level of free drug (ADP-induced platelet aggregation inhibitor, e.g., GNAQ inhibitor, GNA11 inhibitor, or GNAQ and GNA11 inhibitor) in the conjugate preparation is less than about 2% (e.g., about 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0% or less) (mol / mol of total drug).

[0179] Specific aspects and examples of antibody drug conjugates of the invention are provided in the list of additional enumerated embodiments below. It is recognized that the features specified in each embodiment can be combined with other specific features to provide further embodiments of the invention.

[0180] Embodiment 47. A conjugate of Formula (C) or Formula (C-1), wherein D is a GNAQ inhibitor.

[0181] Embodiment 48. A conjugate of Formula (C) or Formula (C-1), wherein D is a GNA11 inhibitor.

[0182] Embodiment 49. A conjugate of formula (C) or formula (C-1), wherein D is an inhibitor of GNAQ and GNA11.

[0183] Embodiment 50. wherein D is [ka] (In the formula, R 0 is methyl or ethyl, R1 is methyl or isopropyl, and R2 is methyl or ethyl; *** L A or Y1) A conjugate of formula (C) or formula (C-1):

[0184] Embodiment 51. In the formula, D is [ka] (In the formula, *** L A or Y1) A conjugate of formula (C) or formula (C-1):

[0185] Embodiment 52. wherein D is [ka] (In the formula, *** L A or Y1) A conjugate of formula (C) or formula (C-1):

[0186] Embodiment 53. Wherein D is [ka] (In the formula, *** L A or Y1) A conjugate of formula (C) or formula (C-1):

[0187] Embodiment 54. Structure of formula (C-2): [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2is methyl or ethyl; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or a linker; y is 1, 2, 3 or 4 A conjugate of formula (C) or formula (C-1) having the formula:

[0188] Embodiment 55. Structure of Formula (C-2a): [ka] (In the formula, Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or a linker; y is 1, 2, 3 or 4 A conjugate of formula (C) or formula (C-1) having the formula:

[0189] Embodiment 56. Structure of Formula (C-2b): [ka] (In the formula, Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or a linker; y is 1, 2, 3 or 4 A conjugate of formula (C) or formula (C-1) having the formula:

[0190] Embodiment 57. Structure of Formula (C-2c): [ka] (In the formula, Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or a linker; y is 1, 2, 3 or 4 A conjugate of formula (C) or formula (C-1) having the formula:

[0191] Embodiment 58. Structure of formula (C-3): [ka] (In the formula, R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or a linker; y is 1, 2, 3 or 4 A conjugate of formula (C) or formula (C-1) having the formula:

[0192] Embodiment 59. Structure of Formula (C-3a): [ka] (In the formula, Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or a linker; y is 1, 2, 3 or 4 A conjugate of formula (C) or formula (C-1) having the formula:

[0193] Embodiment 60. Structure of Formula (C-3b): [ka] (In the formula, Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or a linker; y is 1, 2, 3 or 4 A conjugate of formula (C) or formula (C-1) having the formula:

[0194] Embodiment 61. Structure of Formula (C-3c): [ka] (In the formula, Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X1 is a divalent linking group; X2 is a self-sacrificing spacer; L1 is a bivalent peptide linker; L2 is a bond or a linker; y is 1, 2, 3 or 4 A conjugate of formula (C) or formula (C-1) having the formula:

[0195] Embodiment 62. wherein: R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; X2 is [ka] wherein X2 is a self-immolative spacer selected from * indicates the attachment point to L1, and X2 ** teeth, [ka] the point of attachment to the group, or [ka] indicates the point of attachment to the group; L1 is a bivalent peptide linker containing 2 to 4 amino acid residues; L2 is a linker; X1 is [ka] ,- * NR 4 C(=O)CH2 ** -,- * NHC(=O)CH2 ** -,- * S(=O)2CH2CH2 ** -,- * (CH2)2S(=O)2CH2CH2 ** -,- * NR 4 S(=O)2CH2CH2 ** -,- * NR 4 C(=O)CH2CH2 ** -, -NH-, -C(=O)-, - * NHC(=O) ** -,- *CH2NHCH2CH2 ** -,- * NHCH2CH2 ** -, -S-, [ka] , ** -, wherein X1 is a divalent linking group selected from * indicates the attachment point to L2, and X1 ** indicates the point of attachment to Ab; During the ceremony, Each R 4 is independently selected from H and C1-C6 alkyl; Each R 5 are independently selected from H, C1-C6 alkyl, F, Cl, and —OH; Each R 6 are independently selected from H, C1-C6 alkyl, F, Cl, —NH2, —OCH3, —OCH2CH3, —N(CH3)2, —CN, —NO2, and —OH; Each R 7 are independently H, C 1~6 Alkyl, fluoro, benzyloxy substituted by -C(=O)OH, benzyl substituted by -C(=O)OH, C substituted by -C(=O)OH 1~4 C substituted by alkoxy and -C(=O)OH 1~4 alkyl, y is 1, 2, 3 or 4; A conjugate of formula (C-2) of embodiment 54 or a conjugate of formula (C-3) of embodiment 58.

[0196] Embodiment 63. In the formula, X2 is [ka] wherein X2 is a self-immolative spacer selected from * indicates the attachment point to L1, and X2 ** is the attachment point to Y1, or [ka] the point of attachment to the group, or [ka] 63. The conjugate of any one of embodiments 54 to 62, showing the point of attachment to the group.

[0197] Embodiment 64. In the formula, X2 is [ka] In the formula, X2 * indicates the attachment point to L1, and X2 ** is the attachment point to Y1, or [ka] the point of attachment to the group, or [ka] 64. The conjugate of any one of embodiments 54 to 63, showing the point of attachment to the group.

[0198] Embodiment 65. The conjugate of any one of embodiments 54 to 64, wherein L1 is a bivalent peptide linker comprising 2 to 4 amino acid residues.

[0199] Embodiment 66. The conjugate of any one of embodiments 54 to 65, wherein L1 is a bivalent peptide linker comprising an amino acid residue selected from valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

[0200] Embodiment 67. The conjugate of any one of embodiments 54 to 64, wherein L1 is a bivalent peptide linker comprising at least one valine (Val) or citrulline (Cit) residue.

[0201] Embodiment 68. The conjugate of any one of embodiments 54 to 64, wherein L1 is a bivalent dipeptide linker selected from ValCit, PheLys, ValAla, and ValLys.

[0202] Embodiment 69. In the formula, L1 is [ka] wherein L1 is a divalent dipeptide linker selected from * indicates the attachment point to L2, and ** The conjugate of any one of embodiments 54 to 64, wherein indicates the point of attachment to X2.

[0203] Embodiment 70. The conjugate of any one of embodiments 54 to 64, wherein L1 is ValCit.

[0204] Embodiment 71. wherein: L1 is [ka] where L1 * indicates the attachment point to L2, and ** The conjugate of any one of embodiments 54 to 64, wherein indicates the point of attachment to X2.

[0205] Embodiment 72. The conjugate of any one of embodiments 54 to 71, wherein L2 is a linker.

[0206] Embodiment 73. In the formula, L2 is - * C(=O)((CH2) m O) p (CH2) m ** -,- * C(=O)(CH2) m ** -,- * C(=O)(CH2) n NHC(=O)(CH2) m ** -,- *C(=O)(CH2) m NHC(=O)((CH2) m O) p (CH2) m ** -,- * ((CH2) m O) p (CH2) m ** -,- * ((CH2) m O) p (CH2) m ** -, -(CH2) m -,- * (CH2) m NHC(=O)(CH2) m ** -,- * (CH2) m NHC(=O)(CH2) m C(=O)NH(CH2) m ** -,- * ((CH2) m O) p (CH2) m NHC(=O)(CH2) m ** -,- ** ((CH2) m O) p CH2) m C(=O)NH(CH2) m ** -,- * (CH2) m C(R3)2 ** -and- * (CH2) m C(R3)2SS(CH2) m NHC(=O)(CH2) m ** -, wherein L2 is a linker selected from * indicates the attachment point to L1, and ** indicates the attachment point to X1; During the ceremony, each R3 is independently selected from H and C1-C6 alkyl; each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; 72. The conjugate of any one of embodiments 54 to 71, wherein each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.

[0207] Embodiment 74. In the formula, L2 is - * C(=O)((CH2) m O) p (CH2) m ** -or- * C(=O)(CH2) m ** -, where L2 * indicates the attachment point to L1, and ** indicates the attachment point to X1, 72. The conjugate of any one of embodiments 54 to 71, wherein each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0208] Embodiment 75. wherein L2 is [ka] and L2 * indicates the attachment point to L1, and ** The conjugate of any one of embodiments 54 to 71, wherein indicates the point of attachment to X1.

[0209] Embodiment 76. wherein X1 is [ka] ,- * NR 4 C(=O)CH2 ** -,- * NHC(=O)CH2 ** -,- * S(=O)2CH2CH2 ** -,- * (CH2)2S(=O)2CH2CH2** -,- * NR 4 S(=O)2CH2CH2 ** -,- * NR 4 C(=O)CH2CH2 ** -, -NH-, -C(=O)-, - * NHC(=O) ** -,- * CH2NHCH2CH2 ** -,- * NHCH2CH2 ** -, -S-, [ka] , ** -, wherein X1 is a divalent linking group selected from * indicates the attachment point to L2, and X1 ** indicates the point of attachment to Ab; During the ceremony, Each R 4 is independently selected from H and C1-C6 alkyl; Each R 5 are independently selected from H, C1-C6 alkyl, F, Cl, and —OH; Each R 6 are independently selected from H, C1-C6 alkyl, F, Cl, —NH2, —OCH3, —OCH2CH3, —N(CH3)2, —CN, —NO2, and —OH; Each R 7 are independently H, C 1~6 Alkyl, fluoro, benzyloxy substituted by -C(=O)OH, benzyl substituted by -C(=O)OH, C substituted by -C(=O)OH 1~4 C substituted by alkoxy and -C(=O)OH 1~4 selected from alkyl, The conjugate of any one of embodiments 54 to 75.

[0210] Embodiment 77. wherein X1 is [ka] wherein X1 is a divalent linking group selected from * indicates the attachment point to L2, and X1 ** indicates the point of attachment to Ab, The conjugate of any one of embodiments 54 to 75.

[0211] Embodiment 78. wherein X1 is [ka] wherein X1 is a divalent linking group selected from * indicates the attachment point to L2, and X1 ** indicates the point of attachment to Ab, The conjugate of any one of embodiments 54 to 75.

[0212] Embodiment 79. wherein: Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; X1 is [ka] wherein X1 is a divalent linking group selected from * indicates the attachment point to L2, and X1 ** indicates the attachment point to Ab, X2 is [ka] In the formula, X2 * indicates the attachment point to L1, and X2 ** teeth, [ka] indicates the point of attachment to the group; L1 is [ka] where L1 * indicates the attachment point to L2, and ** indicates the attachment point to X2; L2 is [ka] where L2 * indicates the attachment point to L1, and ** indicates the attachment point to R8, 55. The conjugate of formula (C-2) of embodiment 54, wherein y is 1, 2, 3 or 4.

[0213] Embodiment 80. wherein: Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein; R 0 is methyl or ethyl; R 1 is methyl or isopropyl; R 2 is methyl or ethyl; X1 is [ka] wherein X1 is a divalent linking group selected from * indicates the attachment point to L2, and X1 ** indicates the attachment point to Ab, X2 is [ka] In the formula, X2 * indicates the attachment point to L1, and X2 ** teeth, [ka] indicates the point of attachment to the group; L1 is [ka] where L1 * indicates the attachment point to L2, and ** indicates the attachment point to X2; L2 is [ka] where L2 * indicates the attachment point to L1, and ** indicates the attachment point to R8, 59. The conjugate of formula (C-3) of embodiment 58, wherein y is 1, 2, 3 or 4.

[0214] Embodiment 81. Structure: [ka] (In the formula, y is 2 or 4; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-2) having the formula:

[0215] Embodiment 82. Structure: [ka] (In the formula, y is 2 or 4; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-2) having the formula:

[0216] Embodiment 83. Structure: [ka] (In the formula, y is 2 or 4; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-2) having the formula:

[0217] Embodiment 84. Structure: [ka] (In the formula, y is 2 or 4; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-2) having the formula:

[0218] Embodiment 85. Structure: [ka] (In the formula, y is 2 or 4; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-2) having the formula:

[0219] Embodiment 86. Structure: [ka] (In the formula, y is 2 or 4; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-2) having the formula:

[0220] Embodiment 87. Structure: [ka] (In the formula, y is 2 or 4; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-3) having the formula:

[0221] Embodiment 88. Structure: [ka] (In the formula, y is 2 or 4; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-3) having the formula:

[0222] Embodiment 89. Structure: [ka] (In the formula, y is 2 or 4; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-3) having the formula:

[0223] Embodiment 90. Structure: [ka] (In the formula, y is 2 or 4; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-3) having the formula:

[0224] Embodiment 91. Structure: [ka] (In the formula, y is 2 or 4; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), (C-1) or (C-3) having the formula:

[0225] Embodiment 92. Structure: [ka] (In the formula, y is 2 or 4; Ab is an antibody or antigen-binding fragment thereof that binds to human PMEL17 protein A conjugate of formula (C), formula (C-1) or formula (C-3) having the formula:

[0226] Furthermore, the antibodies, antibody fragments (e.g., antigen-binding fragments), or functional equivalents of the present invention can be conjugated to a drug moiety that modifies a given biological response. The drug moiety should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein, peptide, or polypeptide possessing a desired biological activity. Such proteins can include, for example, toxins such as abrin, ricin A, Pseudomonas aeruginosa exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, cytokines, apoptotic agents, anti-angiogenic agents, or biological response modifiers such as lymphokines.

[0227] In one embodiment, an antibody, antibody fragment (eg, antigen-binding fragment) or functional equivalent of the invention is conjugated to a drug moiety, such as a cytotoxin, a drug (eg, an immunosuppressant) or a radiotoxin. Examples of cytotoxins include, but are not limited to, taxanes (see, e.g., WO 01 / 38318 and PCT / US03 / 02675), DNA alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycin analogs, auristatin E, auristatin F, maytansinoids, pyrrolobenzodiazepines (PBDs), and cytotoxic agents containing reactive polyethylene glycol moieties (e.g., Sasse et al., J. Antibiot. (Tokyo), 53, 879-85 (2000); Suzawa et al., Bioorg. Med. Chem., 8, 2175-84 (2000); Ichimura et al., J. Antibiot. (Tokyo), 44, 1045-53 (1991); Francisco et al., J. Antibiot. (Tokyo), 44, 1045-53 (1991)). al., Blood (2003) (electronic publication ahead of print publication), U.S. Patent Nos. 5,475,092, 6,340,701, 6,372,738, and 6,436,931, U.S. Patent Application Publication No. 2001 / 0036923A1, pending U.S. Patent Applications Nos. 10 / 024,290 and 10 / 116,053, and WO 01 / 49698), taxon, cytochalasin B, gramicidin Examples of antihistamines include methadone D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof.Therapeutic agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), ablating agents (e.g., mechlorethamine, thiotepa, chlorambucil, meiphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) Also included are cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine) (see, e.g., U.S. Patent Application Publication No. 20090304721 to Seattle Genetics).

[0228] Other examples of cytotoxins that can be conjugated to the antibodies, antibody fragments (antigen-binding fragments) or functional equivalents of the invention include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof.

[0229] Methods for conjugating various types of cytotoxins, linkers, and therapeutic agents to antibodies are known in the art; see, e.g., Saito et al., (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail et al., (2003) Cancer Immunol. Immunother. 52:328-337; Payne, (2003) Cancer Cell 3:207-212; Allen, (2002) Nat. Rev. Cancer 2:750-763; Pastan and Kreitman, (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter and Springer, (2001) Adv. Drug Deliv. Rev. 53:247-264.

[0230] The antibodies, antibody fragments (e.g., antigen-binding fragments), or functional equivalents of the present invention can also be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, e.g., Zevalin™ (DEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the present invention. In a specific embodiment, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are generally known in the art and are described in Denardo et al., (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al., (1999) Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., (1999) Nucl. Med. Biol. 26(8):943-50, each of which is incorporated by reference in its entirety.

[0231] An antibody, antibody fragment (e.g., antigen-binding fragment), or functional equivalent of the invention can also be conjugated to a heterologous protein or polypeptide (or a fragment thereof, preferably a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids) to produce a fusion protein. In particular, the invention provides fusion proteins comprising an antibody fragment (e.g., antigen-binding fragment) described herein (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, VH domain, VH CDR, VL domain, or VL CDR) and a heterologous protein, polypeptide, or peptide.

[0232] Additional fusion proteins can be generated through the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of an antibody or fragment thereof of the invention (e.g., an antibody or fragment thereof with higher affinity and lower dissociation rate). See generally U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998) Trends Biotechnol. 16(2):76-82; Hansson et al., (1999) J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, (1998) Biotechniques 24(2):308-313 (each of which patents and publications is incorporated herein by reference in its entirety). Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, can be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. Polynucleotides encoding antibodies or fragments thereof that specifically bind to an antigen can be recombined with one or more components, motifs, sections, moieties, domains, fragments, etc., of one or more heterologous molecules.

[0233] Furthermore, the antibodies, antibody fragments (e.g., antigen-binding fragments), or functional equivalents of the present invention can be conjugated to a marker sequence, e.g., a peptide, to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexahistidine peptide (SEQ ID NO: 267), such as the tag provided in pQE vectors (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA 91311), among others, many of which are commercially available. As described in Gentz ​​et al., (1989) Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistidine (SEQ ID NO: 267) provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., (1984) Cell 37:767), and the "FLAG" tag (A. Einhauer et al., J. Biochem. Biophys. Methods 49:455-465, 2001). According to the present invention, antibodies or antigen-binding fragments can also be conjugated to tumor-penetrating peptides to enhance their effectiveness.

[0234] In other embodiments, the antibodies, antibody fragments (e.g., antigen-binding fragments), or functional equivalents of the invention are conjugated to a diagnostic or detection agent. Such immunoconjugates may be useful for monitoring or prognosing the onset, development, progression, and / or severity of diseases and / or disorders as part of a clinical testing procedure, e.g., determining the effectiveness of a particular treatment. Such diagnosis and detection can be accomplished by coupling antibodies to detectable substances, including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; artificial groups, such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials, such as, but not limited to, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 666, Alexa Fluor 688, Alexa Fluor 700, Alexa Fluor 668, Alexa Fluor 68 ... Fluor 750, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, for example, but not limited to, luminol; bioluminescent materials, for example, but not limited to, luciferase, luciferin, and aequorin; radioactive materials, for example, but not limited to, iodine ( 131 I, 125 I, 123 I, and 121 I,), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115 In, 113 In, 112 In, and 111 In,), technetium ( 99Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F). 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 64 Cu, 113 Sn, and 117 This can be achieved by binding to Sn; as well as various positron-emitting metals using positron emission tomography, and non-radioactive paramagnetic metal ions.

[0235] The antibodies, antibody fragments (e.g., antigen-binding fragments), or functional equivalents of the present invention can also be attached to solid supports, which are particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0236] 3. Conjugation and Preparation of ADC Methods for producing antibody conjugates of formula (C), formula (C-1) and formula (C-2) A general reaction scheme for the formation of conjugates of formula (C) is shown below in Scheme 1. [ka] where RG is a compatible reactive group R attached to the linker-drug compound. 8 A reactive group, by way of example only, a thiol, amine, or ketone, on the antibody or antigen-binding fragment thereof Ab that reacts with, thereby covalently linking the antibody or antigen-binding fragment thereof Ab to one or more linker-drug moieties. 8 A non-limiting example of such a reaction of a group is a maleimide (R 8 ), or hydroxylamine (R 8 ) is) In one embodiment, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor), and L a are RG1 and R 8 wherein n is 1, 2, 3, or 4; and y is 1, 2, 3, or 4.

[0237] A general reaction scheme for the formation of conjugates of formula (C-1) is shown below in Scheme 2. [ka] where RG is a compatible reactive group R attached to the linker-drug moiety. 8 A reactive group, by way of example only, a thiol, amine, or ketone, on the antibody or antigen-binding fragment thereof Ab that reacts with, thereby covalently linking the antibody or antigen-binding fragment thereof Ab to one or more linker-drug moieties. 8 A non-limiting example of such a reaction of a group is a maleimide (R 8 ), or hydroxylamine (R 8 ) is) In one embodiment, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11 (GNAQ / GNA11 inhibitor), and X1 is a combination of RG1 and R 8 reacts, Y is a phosphate group, X is a self-immolative spacer, L is a divalent peptide linker, L is a bond or linker, and y is 1, 2, 3, or 4.

[0238] A general reaction scheme for the formation of conjugates of formula (C-2) is shown below in Scheme 3. [ka] where RG is a compatible reactive group R attached to the linker-drug moiety. 8 A reactive group, by way of example only, a thiol, amine, or ketone, on the antibody or antigen-binding fragment thereof Ab that reacts with, thereby covalently linking the antibody or antigen-binding fragment thereof Ab to one or more linker-drug moieties. 8 A non-limiting example of such a reaction of a group is a maleimide (R 8 ), or hydroxylamine (R 8 ) is) where R 0 is methyl or ethyl, and R 1 is methyl or isopropyl, and R 2 is methyl or ethyl, and X1 is R1 and R2 8 is a divalent linking group (e.g., a succinimide ring or oxime) formed when X reacts with X; X is a self-immolative spacer; L is a divalent peptide linker; L is a bond or linker; and y is 1, 2, 3, or 4.

[0239] A general reaction scheme for the formation of conjugates of formula (C-2) is shown below in Scheme 4. [ka] where RG is a compatible reactive group R attached to the linker-drug moiety. 8 A reactive group, by way of example only, a thiol, amine, or ketone, on the antibody or antigen-binding fragment thereof Ab that reacts with, thereby covalently linking the antibody or antigen-binding fragment thereof Ab to one or more linker-drug moieties. 8 A non-limiting example of such a reaction of a group is a maleimide (R 8 ), or hydroxylamine (R 8 ) is) where R 0 is methyl or ethyl, and R 1 is methyl or isopropyl, and R 2 is methyl or ethyl, and X1 is R1 and R2 8 is a divalent linking group (e.g., a succinimide ring or oxime) formed when X reacts with X; X is a self-immolative spacer; L is a divalent peptide linker; L is a bond or linker; and y is 1, 2, 3, or 4.

[0240] Methods for conjugation to engineered cysteine ​​antibody residues Conjugates of the invention can be prepared, for example, by engineered cysteine ​​residues into antibodies by site-directed mutagenesis. Such site-directed conjugates are homogeneous and have improved properties (Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS, Lu Y, Meng YG, Ng C, Yang J, Lee CC, Duenas E, Gorrell J, Katta V, Kim A, McDorman K, Flagella K, Venook R, Ross S, Spencer SD, Lee Wong W, Lowman HB, Vandlen R, Sliwkowski MX, Scheller RH, Polakis P, Mallet W. (2008) Nature Biotechnology 26:925-932).

[0241] Because engineered cysteines in antibodies expressed in mammalian cells are modified with adducts (disulfides), such as glutathione (GSH) and / or cysteine, during their biosynthesis (Chen et al. 2009), the engineered cysteine ​​residues in the initially expressed product are unreactive with thiol-reactive reagents, such as maleimides or bromo- or iodoacetamide groups. To conjugate a payload to the engineered cysteine ​​after expression, the glutathione or cysteine ​​adducts must be removed by reducing their disulfide adducts, which generally also involves reducing native disulfides in the expressed protein. Deprotection of the adducted engineered cysteines can be achieved by first exposing the antibody to a reducing agent, such as dithiothreitol (DTT), TCEP, or reduced cysteine, followed by a procedure that allows for the reoxidation of all native disulfide bonds in the antibody to restore and / or stabilize functional antibody structure.

[0242] Several methods can be used to reduce and reoxidize antibodies with engineered cysteine ​​residues for the preparation of antibody-drug conjugates. Attempts to follow previously described reoxidation protocols using high concentrations of CuSO4 resulted in protein precipitation (Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS, Lu Y, Meng YG, Ng C, Yang J, Lee CC, Duenas E, Gorrell J, Katta V, Kim A, McDorman K, Flagella K, Venook R, Ross S, Spencer SD, Lee Wong W, Lowman HB, Vandlen R, Sliwkowski MX, Scheller RH, Polakis P, Mallet W. (2008) Nature Biotechnology 26:925). The inventors have successfully prepared and obtained antibody drug conjugates using several different methods of reduction and antibody reoxidation.

[0243] The following describes a method for reducing and reoxidizing an antibody with engineered cysteine ​​residues for the preparation of antibody-drug conjugates. Freshly prepared DTT is added to the purified Cys mutant antibody to a final concentration of 10 mM. After incubation with DTT at room temperature for 1 hour, the mixture is dialyzed against PBS at 4°C for 3 days, with daily buffer exchanges to remove DTT and reoxidize the antibody's native disulfide bonds. An alternative method is to remove the reducing reagent through a desalting column, such as Sephadex G-25 equilibrated with PBS. Once the protein is fully reduced, 1 mM oxidized ascorbate (dehydroascorbic acid) is optionally added to the desalted sample, and the reoxidation incubation is carried out for 20–24 hours.

[0244] In another exemplary method, deprotection of engineered Cys residues is achieved by adding fully reduced cysteine ​​at a concentration of 20 mM to antibody bound to Protein A-Sepharose resin. Reduction of the Cys adduct is achieved by incubation for approximately 30-60 minutes at room temperature, followed by rapid removal of the reducing agent by washing the resin with 50 beds of PBS. Reoxidation of the reduced antibody is achieved by incubating the washed slurry at room temperature with or without the addition of 50-2000 nM CuCl2 as a promoter. Except for the use of copper sulfate, examples herein use each of the protocols described herein with similar results. Reoxidation restores intrachain disulfides, while dialysis, desalting, or Protein A chromatography removes the reducing agent and glutathione originally linked to the cysteine ​​and engineered cysteine ​​of the antibody. Typically, the reoxidation process is monitored using HPLC reverse-phase chromatography. The antibody is loaded onto a PLRP-S column (4000 Å, 50 mm × 2.1 mm, Agilent) heated to 80 °C and eluted using a linear gradient of 30-45% CH3CN in water containing 0.1% TFA at 1.5 mL / min with peak detection at 215, 254, and 280 nm.

[0245] After reoxidation, the antibody is conjugated to a linker-drug compound, e.g., a compound of Formula (B), Formula (B-1), Formula (B-2), or Formula (B-3) (see Schemes 1-4). For example, a compound of Formula (B), Formula (B-1), Formula (B-2), or Formula (B-3) is added to the reoxidized Cys mutant antibody in PBS buffer (pH 7.2) at 5-10 molar equivalents relative to the antibody. Incubation is carried out for 1-2 hours. The conjugation process is monitored by reverse-phase HPLC, which allows separation of conjugated from unconjugated antibody. The conjugation reaction mixture is analyzed on a PRLP-S column (4000 Å, 50 mm × 2.1 mm, Agilent) heated to 80°C, and the column is eluted with a linear gradient of 30-60% acetonitrile in water containing 0.1% TFA at a flow rate of 1.5 ml / min. Elution of the protein from the column is monitored at 280 nm, 254 nm and 215 nm.

[0246] Alternatively, for antibodies bound to Protein A resin, once the antibody is reoxidized, the resin is washed with 10 column volumes of PBS, then the resin is resuspended in an equal volume of PBS and an 8-fold excess of a compound of Formula (B), Formula (B-1), Formula (B-2) or Formula (B-3) (in DMSO) is added and incubated for 2 hours at room temperature. The resin is then washed with 50 column volumes of PBS and the resulting antibody-drug conjugate is eluted from the Protein A resin, neutralized with 1 / 10 volume of 1 M Tris pH 9.0, buffer exchanged into an appropriate buffer and subjected to preparative size exclusion chromatography (if required).

[0247] Immunoconjugates are also characterized in terms of the average loading of drug moieties relative to antibody-binding moieties, commonly referred to as the drug-antibody ratio (DAR). DAR values ​​are estimated, for example, from LC-MS data on reduced and deglycosylated samples. LC / MS allows for quantification of the average number of payload (drug moiety) molecules attached to the antibody in an ADC. HPLC separates antibodies into light and heavy chains, and also separates heavy chains (HC) and light chains (LC) according to the number of linker-payload groups per chain. Mass spectrometry data allows for the identification of component species in the mixture, such as LC, LC+1, LC+2, HC, HC+1, HC+2, etc. From the average loading of LC and HC chains, the average DAR for the ADC can be calculated. The DAR for a given immunoconjugate sample represents the average number of drug (payload) molecules attached to a tetrameric antibody containing two light chains and two heavy chains.

[0248] Throughout the text of this application, in the event of any conflict between the text of the specification and the sequence listing, the text of the specification shall control.

[0249] [Table 2]

[0250] [Table 3]

[0251]

Table 4

[0252]

Table 5

[0253]

Table 6

[0254]

Table 7

[0255]

Table 8

[0256]

Table 9

[0257]

Table 10

[0258]

Table 11

[0259]

Table 12

[0260]

Table 13

[0261]

Table 14

[0262]

Table 15

[0263] Table 16

[0264]

Table 17

[0265]

Table 18

[0266]

Table 19

[0267] Table 20

[0268] Table 21

[0269] Table 22

[0270] Table 23

[0271] Table 24

[0272] Table 25

[0273] Table 26

[0274] Table 27

[0275] Table 28

[0276] Table 29

[0277]

Table 30

[0278] Table 31

[0279] Table 32

[0280] Table 33

[0281] Table 34

[0282] Table 35

[0283] Table 36

[0284] Table 37

[0285] Table 38

[0286] Table 39

[0287] Table 40

[0288] Table 41

[0289] Table 42

[0290] Table 43

[0291] Table 44

[0292] Table 45

[0293] Table 46

[0294] Table 47

[0295] Table 48

[0296] Table 49

[0297] Table 50

[0298] Table 51

[0299] Table 52

[0300] Table 53

[0301] Table 54

[0302] Table 55

[0303] Table 56

[0304] Table 57

[0305] Table 58

[0306] Table 59

[0307] Table 60

[0308] Table 61

[0309] Table 62

[0310] Table 63

[0311] Table 64

[0312] Table 65

[0313] [Table 66]

[0314] Other antibodies of the invention include those in which the amino acids or nucleic acids encoding the amino acids have been mutated, but which have at least 60, 70, 80, 90, or 95 percent identity to the sequences set forth in Table 2. In some embodiments, this includes mutant amino acid sequences in which no more than 1, 2, 3, 4, or 5 amino acids have been mutated in the variable regions when compared to the variable regions set forth in the sequences set forth in Table 2, while retaining substantially the same therapeutic activity as the antibodies set forth in Table 2.

[0315] Because each of these antibodies can bind to PMEL17, the VH, VL, full-length light chain, and full-length heavy chain sequences (amino acid sequences and nucleotide sequences encoding the amino acid sequences) can be "mixed and matched" to generate other PMEL17-binding antibodies of the invention. Such "mixed and matched" PMEL17-binding antibodies can be tested using binding assays known in the art (e.g., ELISAs and other assays described in the Examples section). When these chains are mixed and matched, the VH sequence from a particular VH / VL pairing should be replaced with a structurally similar VH sequence. Similarly, the full-length heavy chain sequence from a particular full-length heavy chain / full-length light chain pairing should be replaced with a structurally similar full-length heavy chain sequence. Similarly, the VL sequence from a particular VH / VL pairing should be replaced with a structurally similar VL sequence. Similarly, the full-length light chain sequence from a particular full-length heavy chain / full-length light chain pairing should be replaced with a structurally similar full-length light chain sequence. Thus, in one aspect, the invention provides an isolated antibody or antibody fragment (e.g., Fab or Fab') having: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 42, 64, 88, 112, 132, 149, 165, 184, 196, 215, 227, 239 or 254; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 25, 29, 53, 75, 99, 119, 143, 159, 171, 190, 202, 221, 233, 248 or 260; wherein the antibody specifically binds to PMEL17.

[0316] In another aspect, the present invention provides an isolated monoclonal antibody having: (i) a full-length heavy chain comprising an amino acid sequence optimized for expression in a cell of a mammalian expression system selected from the group consisting of SEQ ID NOs: 12, 44, 66, 90, 114, 134, 151, 167, 186, 198, 217, 229, 241, or 256; and a full-length light chain comprising an amino acid sequence optimized for expression in a mammalian cell selected from the group consisting of SEQ ID NOs: 23, 27, 31, 55, 77, 101, 121, 145, 161, 173, 192, 204, 223, 235, 250, or 262; or (ii) a functional protein comprising an antigen-binding portion thereof.

[0317] In another aspect, the invention provides PMEL17-binding antibodies comprising the heavy and light chain CDR1, CDR2, and CDR3, or a combination thereof, set forth in Table 2. The amino acid sequences of the VH CDR1 of the antibodies are set forth, for example, in SEQ ID NOs: 1, 4, 5, 7, 33, 36, 37, 39, 57, 60, 79, 82, 83, 85, 103, 106, 107, 109, 123, 126, 127, 129, 175, 178, 179, 181, 206, 209, 210, and 212. The amino acid sequences of antibody VH CDR2 are shown, for example, in SEQ ID NOs: 2, 6, 8, 34, 38, 40, 58, 61, 62, 80, 84, 86, 104, 108, 110, 124, 128, 130, 176, 180, 182, 207, 211, and 213. The amino acid sequences of antibody VH CDR3 are shown, for example, in SEQ ID NOs: 3, 9, 35, 41, 59, 63, 81, 87, 105, 111, 125, 131, 147, 148, 163, 164, 177, 183, 194, 195, 208, 214, 225, 226, 237, 238, 252, and 253. The amino acid sequences of VL CDR1 of the antibody are shown, for example, in SEQ ID NOs: 14, 17, 20, 46, 49, 52, 68, 71, 74, 92, 95, 98, 116, 136, 139, 142, 153, 156, 158, 243, 245, and 247. The amino acid sequences of VL CDR2 of the antibody are shown, for example, in SEQ ID NOs: 15, 18, 47, 50, 69, 72, 93, 96, 137, 140, and 154. The amino acid sequences of the VL CDR3 of the antibodies are shown, for example, in SEQ ID NOs: 16, 19, 48, 51, 70, 73, 94, 97, 117, 118, 138, 141, 155, 157, 169, 170, 188, 189, 200, 201, 219, 220, 231, 232, 244, 246, 258, and 259.

[0318] Each of these antibodies can bind to PMEL17, and provided that its antigen-binding specificity is primarily provided by the CDR1, 2, and 3 regions, the VH CDR1, CDR2, and CDR3 sequences and the VL CDR1, CDR2, and CDR3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched). Such "mixed and matched" PMEL17-binding antibodies can be tested using binding assays known in the art and those described in the Examples (e.g., ELISAs). When VH CDR sequences are mixed and matched, the CDR1, CDR2, and / or CDR3 sequence from a particular VH sequence should be replaced with structurally similar CDR sequences. Similarly, when VL CDR sequences are mixed and matched, the CDR1, CDR2, and / or CDR3 sequence from a particular VL sequence should be replaced with structurally similar CDR sequences. It will be readily apparent to those skilled in the art that novel VH and VL sequences may be generated by replacing the sequences of one or more VH and / or VL CDR regions with structurally similar sequences derived from the CDR sequences set forth herein for the monoclonal antibodies of the invention.

[0319] Thus, in some embodiments, the present invention provides a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5, 7, 33, 36, 37, 39, 57, 60, 79, 82, 83, 85, 103, 106, 107, 109, 123, 126, 127, 129, 175, 178, 179, 181, 206, 209, 210, and 212; , 104, 108, 110, 124, 128, 130, 176, 180, 182, 207, 211, and 213; heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 35, 41, 59, 63, 81, 87, 105, 111, 125, 131, 147, 148, 163, 164, 177, 183, 194, 195, 208, 214, 225, 226, 237, 238, 252, and 253; a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, 20, 46, 49, 52, 68, 71, 74, 92, 95, 98, 116, 136, 139, 142, 153, 156, 158, 243, 245, and 247; and a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 18, 47, 50, 69, 72, 93, 96, 137, 140, and 154. and a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 19, 48, 51, 70, 73, 94, 97, 117, 118, 138, 141, 155, 157, 169, 170, 188, 189, 200, 201, 219, 220, 231, 232, 244, 246, 258, and 259; wherein the antibody specifically binds to PMEL17.

[0320] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 1, 4, 5, or 7; a heavy chain CDR2 of SEQ ID NO: 2, 6, or 8; a heavy chain CDR3 of SEQ ID NO: 3 or 9; a light chain CDR1 of SEQ ID NO: 14, 17, or 20; a light chain CDR2 of SEQ ID NO: 15 or 18; and a light chain CDR3 of SEQ ID NO: 16 or 19.

[0321] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 33, 36, 37, or 39; a heavy chain CDR2 of SEQ ID NO: 34, 38, or 40; a heavy chain CDR3 of SEQ ID NO: 35 or 41; a light chain CDR1 of SEQ ID NO: 46, 49, or 52; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 48 or 51.

[0322] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 5, 7, 57, or 60; a heavy chain CDR2 of SEQ ID NO: 58, 61, or 62; a heavy chain CDR3 of SEQ ID NO: 59 or 63; a light chain CDR1 of SEQ ID NO: 68, 71, or 74; a light chain CDR2 of SEQ ID NO: 69 or 72; and a light chain CDR3 of SEQ ID NO: 70 or 73.

[0323] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 79, 82, 83, or 85; a heavy chain CDR2 of SEQ ID NO: 80, 84, or 86; a heavy chain CDR3 of SEQ ID NO: 81 or 87; a light chain CDR1 of SEQ ID NO: 92, 95, or 98; a light chain CDR2 of SEQ ID NO: 93 or 96; and a light chain CDR3 of SEQ ID NO: 94 or 97.

[0324] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 103, 106, 107, or 109; a heavy chain CDR2 of SEQ ID NO: 104, 108, or 110; a heavy chain CDR3 of SEQ ID NO: 105 or 111; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 117 or 118.

[0325] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 123, 126, 127, or 129; a heavy chain CDR2 of SEQ ID NO: 124, 128, or 130; a heavy chain CDR3 of SEQ ID NO: 125 or 131; a light chain CDR1 of SEQ ID NO: 136, 139, or 142; a light chain CDR2 of SEQ ID NO: 137 or 140; and a light chain CDR3 of SEQ ID NO: 138 or 141.

[0326] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 123, 126, 127, or 129, a heavy chain CDR2 of SEQ ID NO: 124, 128, or 130; a heavy chain CDR3 of SEQ ID NO: 147 or 148; a light chain CDR1 of SEQ ID NO: 153, 156, or 158; a light chain CDR2 of SEQ ID NO: 50 or 154; and a light chain CDR3 of SEQ ID NO: 155 or 157.

[0327] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 103, 106, 107, or 109, a heavy chain CDR2 of SEQ ID NO: 104, 108, or 110; a heavy chain CDR3 of SEQ ID NO: 163 or 164; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 169 or 170.

[0328] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 175, 178, 179, or 181; a heavy chain CDR2 of SEQ ID NO: 176, 180, or 182; a heavy chain CDR3 of SEQ ID NO: 177 or 183; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 188 or 189.

[0329] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 103, 106, 107, or 109, a heavy chain CDR2 of SEQ ID NO: 104, 108, or 110; a heavy chain CDR3 of SEQ ID NO: 194 or 195; a light chain CDR1 of SEQ ID NO: 49, 52, or 116; a light chain CDR2 of SEQ ID NO: 47 or 50; and a light chain CDR3 of SEQ ID NO: 200 or 201.

[0330] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 206, 209, 210, or 212; a heavy chain CDR2 of SEQ ID NO: 207, 211, or 213; a heavy chain CDR3 of SEQ ID NO: 208 or 214; a light chain CDR1 of SEQ ID NO: 153, 156, or 158; a light chain CDR2 of SEQ ID NO: 50 or 154; and a light chain CDR3 of SEQ ID NO: 219 or 220.

[0331] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain CDR1 of SEQ ID NO: 206, 209, 210, or 212, a heavy chain CDR2 of SEQ ID NO: 207, 211, or 213; a heavy chain CDR3 of SEQ ID NO: 225 or 226; a light chain CDR1 of SEQ ID NO: 136, 139, or 142; a light chain CDR2 of SEQ ID NO: 137 or 140; and a light chain CDR3 of SEQ ID NO: 231 or 232.

[0332] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, 209, 210 or 212, an HCDR2 of SEQ ID NO: 207, 211 or 213, and an HCDR3 of SEQ ID NO: 237 or 238; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 243, 245 or 247, an LCDR2 of SEQ ID NO: 47 or 50, and an LCDR3 of SEQ ID NO: 244 or 246.

[0333] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, 209, 210 or 212, an HCDR2 of SEQ ID NO: 207, 211 or 213, and an HCDR3 of SEQ ID NO: 252 or 253; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 153, 156 or 158, an LCDR2 of SEQ ID NO: 50 or 154, and an LCDR3 of SEQ ID NO: 258 or 259.

[0334] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 14, a light chain CDR2 of SEQ ID NO: 15, and a light chain CDR3 of SEQ ID NO: 16; b) a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 14, a light chain CDR2 of SEQ ID NO: 15, and a light chain CDR3 of SEQ ID NO: 16; c) a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 6, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 17, a light chain CDR2 of SEQ ID NO: 18, and a light chain CDR3 of SEQ ID NO: 19; or d) heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO: 8, heavy chain CDR3 of SEQ ID NO: 9, light chain CDR1 of SEQ ID NO: 20, light chain CDR2 of SEQ ID NO: 18, and light chain CDR3 of SEQ ID NO: 16 The CDR sequences are selected from:

[0335] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain CDR1 of SEQ ID NO: 33, a heavy chain CDR2 of SEQ ID NO: 34, a heavy chain CDR3 of SEQ ID NO: 35, a light chain CDR1 of SEQ ID NO: 46, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 48; b) a heavy chain CDR1 of SEQ ID NO: 36, a heavy chain CDR2 of SEQ ID NO: 34, a heavy chain CDR3 of SEQ ID NO: 35, a light chain CDR1 of SEQ ID NO: 46, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 48; c) a heavy chain CDR1 of SEQ ID NO: 37, a heavy chain CDR2 of SEQ ID NO: 38, a heavy chain CDR3 of SEQ ID NO: 35, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 51; or d) a heavy chain CDR1 of SEQ ID NO: 39, a heavy chain CDR2 of SEQ ID NO: 40, a heavy chain CDR3 of SEQ ID NO: 41, a light chain CDR1 of SEQ ID NO: 52, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 48 The CDR sequences are selected from:

[0336] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain CDR1 of SEQ ID NO: 57, a heavy chain CDR2 of SEQ ID NO: 58, a heavy chain CDR3 of SEQ ID NO: 59, a light chain CDR1 of SEQ ID NO: 68, a light chain CDR2 of SEQ ID NO: 69, and a light chain CDR3 of SEQ ID NO: 70; b) a heavy chain CDR1 of SEQ ID NO: 60, a heavy chain CDR2 of SEQ ID NO: 58, a heavy chain CDR3 of SEQ ID NO: 59, a light chain CDR1 of SEQ ID NO: 68, a light chain CDR2 of SEQ ID NO: 69, and a light chain CDR3 of SEQ ID NO: 70; c) a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 61, a heavy chain CDR3 of SEQ ID NO: 59, a light chain CDR1 of SEQ ID NO: 71, a light chain CDR2 of SEQ ID NO: 72, and a light chain CDR3 of SEQ ID NO: 73; or d) a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 62, a heavy chain CDR3 of SEQ ID NO: 63, a light chain CDR1 of SEQ ID NO: 74, a light chain CDR2 of SEQ ID NO: 72, and a light chain CDR3 of SEQ ID NO: 70 The CDR sequences are selected from:

[0337] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain CDR1 of SEQ ID NO: 79, a heavy chain CDR2 of SEQ ID NO: 80, a heavy chain CDR3 of SEQ ID NO: 81, a light chain CDR1 of SEQ ID NO: 92, a light chain CDR2 of SEQ ID NO: 93, and a light chain CDR3 of SEQ ID NO: 94; b) a heavy chain CDR1 of SEQ ID NO: 82, a heavy chain CDR2 of SEQ ID NO: 80, a heavy chain CDR3 of SEQ ID NO: 81, a light chain CDR1 of SEQ ID NO: 92, a light chain CDR2 of SEQ ID NO: 93, and a light chain CDR3 of SEQ ID NO: 94; c) a heavy chain CDR1 of SEQ ID NO: 83, a heavy chain CDR2 of SEQ ID NO: 84, a heavy chain CDR3 of SEQ ID NO: 81, a light chain CDR1 of SEQ ID NO: 95, a light chain CDR2 of SEQ ID NO: 96, and a light chain CDR3 of SEQ ID NO: 97; or d) a heavy chain CDR1 of SEQ ID NO: 85, a heavy chain CDR2 of SEQ ID NO: 86, a heavy chain CDR3 of SEQ ID NO: 87, a light chain CDR1 of SEQ ID NO: 98, a light chain CDR2 of SEQ ID NO: 96, and a light chain CDR3 of SEQ ID NO: 94 The CDR sequences are selected from:

[0338] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain CDR1 of SEQ ID NO: 103, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 105, a light chain CDR1 of SEQ ID NO: 116; a light chain CDR2 of SEQ ID NO: 47; and a light chain CDR3 of SEQ ID NO: 117; b) a heavy chain CDR1 of SEQ ID NO: 106, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 105, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 117; c) a heavy chain CDR1 of SEQ ID NO: 107, a heavy chain CDR2 of SEQ ID NO: 108, a heavy chain CDR3 of SEQ ID NO: 105, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 118; or d) heavy chain CDR1 of SEQ ID NO: 109, heavy chain CDR2 of SEQ ID NO: 110, heavy chain CDR3 of SEQ ID NO: 111, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 117 The CDR sequences are selected from:

[0339] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain CDR1 of SEQ ID NO: 123, a heavy chain CDR2 of SEQ ID NO: 124, a heavy chain CDR3 of SEQ ID NO: 125, a light chain CDR1 of SEQ ID NO: 136, a light chain CDR2 of SEQ ID NO: 137, and a light chain CDR3 of SEQ ID NO: 138; b) a heavy chain CDR1 of SEQ ID NO: 126, a heavy chain CDR2 of SEQ ID NO: 124, a heavy chain CDR3 of SEQ ID NO: 125, a light chain CDR1 of SEQ ID NO: 136, a light chain CDR2 of SEQ ID NO: 137, and a light chain CDR3 of SEQ ID NO: 138; c) a heavy chain CDR1 of SEQ ID NO: 127, a heavy chain CDR2 of SEQ ID NO: 128, a heavy chain CDR3 of SEQ ID NO: 125, a light chain CDR1 of SEQ ID NO: 139, a light chain CDR2 of SEQ ID NO: 140, and a light chain CDR3 of SEQ ID NO: 141; or d) a heavy chain CDR1 of SEQ ID NO: 129, a heavy chain CDR2 of SEQ ID NO: 130, a heavy chain CDR3 of SEQ ID NO: 131, a light chain CDR1 of SEQ ID NO: 142, a light chain CDR2 of SEQ ID NO: 140, and a light chain CDR3 of SEQ ID NO: 138 The CDR sequences are selected from:

[0340] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain CDR1 of SEQ ID NO: 123, a heavy chain CDR2 of SEQ ID NO: 124, a heavy chain CDR3 of SEQ ID NO: 147, a light chain CDR1 of SEQ ID NO: 153, a light chain CDR2 of SEQ ID NO: 154, and a light chain CDR3 of SEQ ID NO: 155; b) a heavy chain CDR1 of SEQ ID NO: 126, a heavy chain CDR2 of SEQ ID NO: 124, a heavy chain CDR3 of SEQ ID NO: 147, a light chain CDR1 of SEQ ID NO: 153, a light chain CDR2 of SEQ ID NO: 154, and a light chain CDR3 of SEQ ID NO: 155; c) a heavy chain CDR1 of SEQ ID NO: 127, a heavy chain CDR2 of SEQ ID NO: 128, a heavy chain CDR3 of SEQ ID NO: 147, a light chain CDR1 of SEQ ID NO: 156, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 157; or d) a heavy chain CDR1 of SEQ ID NO: 129, a heavy chain CDR2 of SEQ ID NO: 130, a heavy chain CDR3 of SEQ ID NO: 148, a light chain CDR1 of SEQ ID NO: 158, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 155 The CDR sequences are selected from:

[0341] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain CDR1 of SEQ ID NO: 103, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 163, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 169; b) a heavy chain CDR1 of SEQ ID NO: 106, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 163, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 169; c) a heavy chain CDR1 of SEQ ID NO: 107, a heavy chain CDR2 of SEQ ID NO: 108, a heavy chain CDR3 of SEQ ID NO: 163, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 170; or d) a heavy chain CDR1 of SEQ ID NO: 109, a heavy chain CDR2 of SEQ ID NO: 110, a heavy chain CDR3 of SEQ ID NO: 164, a light chain CDR1 of SEQ ID NO: 52, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 169 The CDR sequences are selected from:

[0342] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain CDR1 of SEQ ID NO: 175, a heavy chain CDR2 of SEQ ID NO: 176, a heavy chain CDR3 of SEQ ID NO: 177, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 188; b) a heavy chain CDR1 of SEQ ID NO: 178, a heavy chain CDR2 of SEQ ID NO: 176, a heavy chain CDR3 of SEQ ID NO: 177, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 188; c) a heavy chain CDR1 of SEQ ID NO: 179, a heavy chain CDR2 of SEQ ID NO: 180, a heavy chain CDR3 of SEQ ID NO: 177, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 189; or d) heavy chain CDR1 of SEQ ID NO: 181, heavy chain CDR2 of SEQ ID NO: 182; heavy chain CDR3 of SEQ ID NO: 183, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 188 The CDR sequences are selected from:

[0343] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain CDR1 of SEQ ID NO: 103, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 194, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 200; b) a heavy chain CDR1 of SEQ ID NO: 106, a heavy chain CDR2 of SEQ ID NO: 104, a heavy chain CDR3 of SEQ ID NO: 194, a light chain CDR1 of SEQ ID NO: 116, a light chain CDR2 of SEQ ID NO: 47, and a light chain CDR3 of SEQ ID NO: 200; c) a heavy chain CDR1 of SEQ ID NO: 107, a heavy chain CDR2 of SEQ ID NO: 108, a heavy chain CDR3 of SEQ ID NO: 194, a light chain CDR1 of SEQ ID NO: 49, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 201; or d) a heavy chain CDR1 of SEQ ID NO: 109, a heavy chain CDR2 of SEQ ID NO: 110, a heavy chain CDR3 of SEQ ID NO: 195, a light chain CDR1 of SEQ ID NO: 52, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 200 The CDR sequences are selected from:

[0344] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain CDR1 of SEQ ID NO: 206, a heavy chain CDR2 of SEQ ID NO: 207, a heavy chain CDR3 of SEQ ID NO: 208, a light chain CDR1 of SEQ ID NO: 153, a light chain CDR2 of SEQ ID NO: 154, and a light chain CDR3 of SEQ ID NO: 219; b) a heavy chain CDR1 of SEQ ID NO: 209, a heavy chain CDR2 of SEQ ID NO: 207, a heavy chain CDR3 of SEQ ID NO: 208, a light chain CDR1 of SEQ ID NO: 153, a light chain CDR2 of SEQ ID NO: 154, and a light chain CDR3 of SEQ ID NO: 219; c) a heavy chain CDR1 of SEQ ID NO: 210, a heavy chain CDR2 of SEQ ID NO: 211, a heavy chain CDR3 of SEQ ID NO: 208, a light chain CDR1 of SEQ ID NO: 156, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 220; or d) a heavy chain CDR1 of SEQ ID NO: 212, a heavy chain CDR2 of SEQ ID NO: 213, a heavy chain CDR3 of SEQ ID NO: 214, a light chain CDR1 of SEQ ID NO: 158, a light chain CDR2 of SEQ ID NO: 50, and a light chain CDR3 of SEQ ID NO: 219 The CDR sequences are selected from:

[0345] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain CDR1 of SEQ ID NO: 206, a heavy chain CDR2 of SEQ ID NO: 207, a heavy chain CDR3 of SEQ ID NO: 225, a light chain CDR1 of SEQ ID NO: 136, a light chain CDR2 of SEQ ID NO: 137, and a light chain CDR3 of SEQ ID NO: 231; b) a heavy chain CDR1 of SEQ ID NO: 209, a heavy chain CDR2 of SEQ ID NO: 207, a heavy chain CDR3 of SEQ ID NO: 225, a light chain CDR1 of SEQ ID NO: 136, a light chain CDR2 of SEQ ID NO: 137, and a light chain CDR3 of SEQ ID NO: 231; c) a heavy chain CDR1 of SEQ ID NO: 210, a heavy chain CDR2 of SEQ ID NO: 211, a heavy chain CDR3 of SEQ ID NO: 225, a light chain CDR1 of SEQ ID NO: 139, a light chain CDR2 of SEQ ID NO: 140, and a light chain CDR3 of SEQ ID NO: 232; or d) heavy chain CDR1 of SEQ ID NO: 212, heavy chain CDR2 of SEQ ID NO: 213, heavy chain CDR3 of SEQ ID NO: 226, light chain CDR1 of SEQ ID NO: 142; light chain CDR2 of SEQ ID NO: 140; and light chain CDR3 of SEQ ID NO: 231 The CDR sequences are selected from:

[0346] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 243, an LCDR2 of SEQ ID NO: 47, and an LCDR3 of SEQ ID NO: 244; b) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 209, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 243, an LCDR2 of SEQ ID NO: 47, and an LCDR3 of SEQ ID NO: 244; c) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 210, an HCDR2 of SEQ ID NO: 211, and an HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 245, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 246; or d) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 212, an HCDR2 of SEQ ID NO: 213, and an HCDR3 of SEQ ID NO: 238; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 247, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 244. The CDR sequences are selected from:

[0347] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 is a) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 206, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 252, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 153, an LCDR2 of SEQ ID NO: 154, and an LCDR3 of SEQ ID NO: 258; b) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 209, an HCDR2 of SEQ ID NO: 207, and an HCDR3 of SEQ ID NO: 252, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 153, an LCDR2 of SEQ ID NO: 154, and an LCDR3 of SEQ ID NO: 258; c) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 210, an HCDR2 of SEQ ID NO: 211, and an HCDR3 of SEQ ID NO: 252, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 156, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 259; or d) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 212, an HCDR2 of SEQ ID NO: 213, and an HCDR3 of SEQ ID NO: 253; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 158, an LCDR2 of SEQ ID NO: 50, and an LCDR3 of SEQ ID NO: 258. The CDR sequences are selected from:

[0348] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 21.

[0349] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 25.

[0350] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 29.

[0351] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 53.

[0352] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 64, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 75.

[0353] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 88, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 99.

[0354] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 112, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 119.

[0355] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 132, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 143.

[0356] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 149, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 159.

[0357] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 165, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 171.

[0358] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 184, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 190.

[0359] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 196 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 202.

[0360] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 215, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 221.

[0361] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 227 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 233.

[0362] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 239, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 248.

[0363] In a specific embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 254 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 260.

[0364] In a specific embodiment, the antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:12 and a light chain comprising the amino acid sequence of SEQ ID NO:23.

[0365] In a specific embodiment, an antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:12 and a light chain comprising the amino acid sequence of SEQ ID NO:27.

[0366] In a specific embodiment, the antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:12 and a light chain comprising the amino acid sequence of SEQ ID NO:31.

[0367] In a specific embodiment, the antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:44 and a light chain comprising the amino acid sequence of SEQ ID NO:55.

[0368] In a specific embodiment, an antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:66 and a light chain comprising the amino acid sequence of SEQ ID NO:77.

[0369] In a specific embodiment, an antibody or antibody fragment (eg, an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:90 and a light chain comprising the amino acid sequence of SEQ ID NO:101.

[0370] In a specific embodiment, an antibody or antibody fragment (eg, an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:114 and a light chain comprising the amino acid sequence of SEQ ID NO:121.

[0371] In a specific embodiment, an antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:134 and a light chain comprising the amino acid sequence of SEQ ID NO:145.

[0372] In a specific embodiment, an antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:151 and a light chain comprising the amino acid sequence of SEQ ID NO:161.

[0373] In a specific embodiment, an antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:167 and a light chain comprising the amino acid sequence of SEQ ID NO:173.

[0374] In a specific embodiment, an antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:186 and a light chain comprising the amino acid sequence of SEQ ID NO:192.

[0375] In a specific embodiment, an antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:198 and a light chain comprising the amino acid sequence of SEQ ID NO:204.

[0376] In a specific embodiment, an antibody or antibody fragment (eg, an antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:217 and a light chain comprising the amino acid sequence comprising SEQ ID NO:223.

[0377] In a specific embodiment, an antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:229 and a light chain comprising the amino acid sequence of SEQ ID NO:235.

[0378] In a specific embodiment, an antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:241 and a light chain comprising the amino acid sequence of SEQ ID NO:250.

[0379] In a specific embodiment, an antibody or antibody fragment (eg, antigen-binding fragment) that specifically binds to PMEL17 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:256 and a light chain comprising the amino acid sequence of SEQ ID NO:262.

[0380] In certain embodiments, the antibody that specifically binds to PMEL17 is an antibody or antibody fragment (eg, an antigen-binding fragment) listed in Table 2.

[0381] 1. Identification of epitopes and antibodies that bind to the same epitopes The present invention also provides antibodies and antibody fragments (e.g., antigen-binding fragments) that specifically bind to the same epitope as the anti-PMEL17 antibodies described in Table 2, or that cross-compete with the antibodies described in Table 2. Accordingly, additional antibodies and antibody fragments (e.g., antigen-binding fragments) can be identified based on their ability to cross-compete (e.g., statistically significantly competitively inhibit the binding of) other antibodies of the invention in, for example, PMEL17 binding assays via BIACORE or assays known to those of skill in the art that measure binding. The ability of a test antibody to inhibit the binding of antibodies and antibody fragments (e.g., antigen-binding fragments) of the invention to PMEL17 (e.g., human PMEL17) demonstrates that the test antibody can compete with that antibody or antibody fragment (e.g., antigen-binding fragment) for binding to PMEL17; that such an antibody, according to non-limiting theory, can bind to the same or a related (e.g., structurally similar or spatially proximal) epitope on PMEL17 as the competing antibody or antibody fragment (e.g., antigen-binding fragment). In certain embodiments, an antibody that binds to the same epitope on PMEL17 as an antibody or antibody fragment (e.g., antigen-binding fragment) of the invention shown in Table 2 is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0382] 2. Further modifications to the Fc region framework Immunoconjugates of the present invention include modified antibodies or antigen-binding fragments thereof that further comprise modifications of framework residues within VH and / or VL that improve the properties of the antibody. In some embodiments, the framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived. To return the framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence, for example, by site-directed mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0383] Another type of framework modification involves mutating one or more residues within the framework regions, or within one or more CDR regions, to reduce the potential immunogenicity of the antibody by removing T-cell epitopes. This approach, also known as "deimmunization," is described in further detail in U.S. Patent Application Publication No. 20030153043 by Carr et al.

[0384] In addition to, or instead of, modifications made within the framework or CDR regions, antibodies of the invention may be engineered to include modifications, typically within the Fc region, to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity (ADCC). Furthermore, antibodies of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody), again to alter one or more functional properties of the antibody, or modified to alter its glycosylation. Each of these embodiments is described in further detail below.

[0385] In one embodiment, the hinge region of CH1 is modified such that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0386] In some embodiments, the antibodies or antibody fragments disclosed herein contain modified or engineered amino acid residues, such as one or more cysteine ​​residues, as sites for conjugation to a drug moiety (Junutula JR, et al., Nat Biotechnol 2008, 26:925-932). In one embodiment, the present invention provides modified antibodies or antibody fragments comprising substitutions of one or more amino acids with cysteine ​​at the positions described herein. The sites for cysteine ​​substitutions are located in the constant region of the antibody or antibody fragment and are therefore applicable to a variety of antibodies or antibody fragments, with the sites selected to provide stable, homogeneous conjugates. The modified antibodies or fragments can have one, two, or more cysteine ​​substitutions, which can be used in combination with other modifications and conjugation methods described herein. Methods for inserting cysteines at specific positions in antibodies are known in the art, see, for example, Lyons et al., (1990) Protein Eng., 3:703-708, WO 2011 / 005481, WO 2014 / 124316, and WO 2015 / 138615. In certain embodiments, the modified antibody comprises a substitution of one or more amino acids by cysteine ​​in its constant region selected from positions 117, 119, 121, 124, 139, 152, 153, 155, 157, 164, 169, 171, 174, 189, 191, 195, 197, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, ​​390, 392, 398, 400 and 422 of the heavy chain of the antibody, positions numbered according to the EU system.In some embodiments, the modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine ​​in its constant region selected from positions 107, 108, 109, 114, 129, 142, 143, 145, 152, 154, 156, 159, 161, 165, 168, 169, 170, 182, 183, 197, 199, and 203 of the light chain of the antibody or antibody fragment, where positions are numbered according to the EU system, and the light chain is a human kappa light chain. In certain embodiments, the modified antibody or antibody fragment thereof comprises a combination of substitutions of two or more amino acids with cysteine ​​in its constant region, where combinations include substitutions at position 375 of the antibody heavy chain, position 152 of the antibody heavy chain, position 360 of the antibody heavy chain, or position 107 of the antibody light chain, where positions are numbered according to the EU system. In certain embodiments, the modified antibody or antibody fragment thereof comprises a substitution of one amino acid by cysteine ​​in its constant region, the substitution being at position 375 of the antibody heavy chain, position 152 of the antibody heavy chain, position 360 of the antibody heavy chain, position 107 of the antibody light chain, position 165 of the antibody light chain, or position 159 of the antibody light chain, where positions are numbered according to the EU system, and the light chain is a kappa chain. In certain embodiments, the modified antibody or antibody fragment thereof comprises a combination of substitutions of two amino acids by cysteine ​​in its constant region, the combination comprising substitutions at position 375 of the antibody heavy chain and position 152 of the antibody heavy chain, where positions are numbered according to the EU system. In certain embodiments, the modified antibody or antibody fragment thereof comprises a substitution of one amino acid by cysteine ​​at position 360 of the antibody heavy chain, where positions are numbered according to the EU system. In other particular embodiments, the modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine ​​at position 107 of the antibody light chain, where positions are numbered according to the EU system and the light chain is a kappa chain.

[0387] In additional embodiments, antibodies or antibody fragments (e.g., antigen-binding fragments) useful in the immunoconjugates of the invention include modified or engineered antibodies, e.g., antibodies that have been modified to introduce one or more other reactive amino acids (other than cysteine), such as Pc1, pyrrolysine, peptide tags (e.g., S6, A1, and ybbR tags), and unnatural amino acids, in place of at least one amino acid of the native sequence, thereby providing a reactive site on the antibody or antigen-binding fragment for attachment to a drug moiety or linker-drug moiety of complementary reactivity. For example, antibodies or antibody fragments can be modified to incorporate Pcl or pyrrolysine (W. Ou, et al., (2011) PNAS 108(26), 10437-10442; WO 2014124258) or unnatural amino acids (JY Axup, et al., Proc Natl Acad Sci USA, 109 (2012), pp. 16101-16106; for reviews, see CC Liu and PG Schultz (2010) Annu Rev Biochem 79, 413-444; CH Kim, et al., (2013) Curr Opin Chem Biol. 17, 412-419) as sites for conjugation to drugs. Similarly, peptide tags for enzymatic conjugation can be introduced into antibodies (Strop P., et al., Chem Biol. 2013, 20(2):161-7; Rabuka D., Curr Opin Chem Biol. 2010 Dec; 14(6):790-6; Rabuka D, et al., Nat Protoc. 2012, 7(6):1052-67). Another example is the use of 4'-phosphopantetheinyl transferase (PPTase) for the attachment of coenzyme A analogs (WO 2013184514), and (Gruenewald et al., (2015) Bioconjugate Chem. 26(12), 2554-62). Methods for conjugating such modified or engineered antibodies with payloads or linker-payload combinations are known in the art.

[0388] In another embodiment, the Fc-hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment so that the antibody has impaired Staphylococcus protein A (SpA) binding compared to the native Fc-hinge domain. This approach is described in further detail in U.S. Pat. No. 6,165,745 by Ward et al.

[0389] In yet other embodiments, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be substituted with a different amino acid residue so that the antibody has altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0390] In another embodiment, one or more selected amino acids can be substituted with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0391] In another embodiment, one or more amino acid residues are altered to alter the antibody's ability to fix complement. This approach is described, for example, in WO 94 / 29351 by Bodmer et al. Allotypic amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses and the constant region of the light chain of the kappa isotype, as described by Jefferis et al., MAbs.1:332-338 (2009).

[0392] Antibody fusion protein complexes containing such mutations may or may not mediate reduced antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In some embodiments, amino acid residues L234 and L235 of the IgG1 constant region are substituted with A234 and A235. In some embodiments, amino acid residue N267 of the IgG1 constant region is substituted with A267. In some embodiments, amino acid residues D265 and P329 of the IgG1 constant region are substituted with A265 and A329. Other antibody Fc silencing mutations may also be used.

[0393] In another embodiment, one or more amino acid residues are altered to alter the antibody's ability to fix complement. This approach is described, for example, in WO 94 / 29351 by Bodmer et al. In a specific embodiment, one or more amino acids of an antibody or antigen-binding fragment thereof of the invention are substituted with one or more allotypic amino acid residues. Allotypic amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses and the constant region of the light chain of the kappa isotype, as described by Jefferis et al., MAbs.1:332-338 (2009).

[0394] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be produced (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an "antigen." Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such approaches are described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0395] In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase biological half-life, the antibody can be altered in the CH1 or CL region to contain salvage receptor binding epitopes taken from two loops of the CH2 domain of the IgG Fc region, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al.

[0396] 3. Generation of Anti-PMEL17 Antibodies Anti-PMEL17 antibodies and antibody fragments thereof (e.g., antigen-binding fragments) can be produced by any means known in the art, including, but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, where full-length monoclonal antibodies can be obtained by hybridoma production or recombinant production. Recombinant expression can be derived from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0397] The present invention further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding the heavy or light chain variable regions or variable segments comprising complementarity determining regions, as described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 11, 43, 65, 89, 113, 133, 150, 166, 185, 197, 216, 228, 240, and 255. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to the polynucleotide of SEQ ID NOs: 22, 26, 30, 54, 76, 100, 120, 144, 160, 172, 191, 203, 222, 234, 249, and 261.

[0398] In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to the polynucleotide of SEQ ID NOs: 13, 45, 67, 91, 115, 135, 152, 168, 187, 199, 218, 230, 242, and 257. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to the polynucleotide of SEQ ID NOs: 24, 28, 32, 56, 78, 102, 122, 146, 162, 174, 193, 205, 224, 236, 251, and 263.

[0399] The polynucleotides of the present invention may encode only the variable region sequence of an anti-PMEL17 antibody. They may also encode both the variable and constant regions of the antibody. Some polynucleotide sequences encode polypeptides containing both the heavy and light chain variable regions of one exemplary murine anti-PMEL17 antibody. Some other polynucleotides encode two polypeptide segments substantially identical to the heavy and light chain variable regions, respectively, of one murine antibody.

[0400] Polynucleotide sequences can be generated by de novo solid-phase DNA synthesis of existing sequences encoding anti-PMEL17 antibodies or binding fragments thereof (e.g., sequences as shown in the Examples below) or by PCR mutagenesis. Direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid-support method of U.S. Pat. No. 4,458,066. Introduction of mutations into polynucleotide sequences by PCR can be carried out, for example, as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0401] Also provided herein are expression vectors and host cells for producing the above-described anti-PMEL17 antibodies. Various expression vectors can be used to express polynucleotides encoding the anti-PMEL17 antibody chains or binding fragments thereof. Both viral and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmid or episomal vectors (typically carrying expression cassettes for protein or RNA expression) and human artificial chromosomes (see, e.g., Harrington et al., Nat. Genet. 15:345, 1997). For example, non-viral vectors useful for expressing anti-PMEL17 polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B & C, pcDNA™3.1 / His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, SV40, papilloma viruses, HBP Epstein-Barr virus-based vectors, vaccinia virus vectors, and Semliki Forest virus (SFV). See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[0402] The choice of expression vector depends on the intended host cell in which the vector will be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the anti-PMEL17 antibody chain or fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except under inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population toward coding sequences whose expression products are better tolerated by the host cell. In addition to promoters, other regulatory elements may be required or desired for efficient expression of the anti-PMEL17 antibody or fragment. These elements typically include an ATG initiation codon and adjacent ribosome binding sites or other sequences. In addition, the efficiency of expression can be enhanced by the inclusion of enhancers appropriate for the cell system used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0403] The expression vector may also provide a secretory signal sequence site to form a fusion protein with the polypeptide encoded by the inserted anti-PMEL17 antibody sequence. Often, the inserted anti-PMEL17 antibody sequence is conjugated to a signal sequence before being included in the vector. The vector used to receive the sequences encoding the anti-PMEL17 antibody light and heavy chain variable domains also sometimes encodes constant regions or portions thereof. Such vectors can produce intact antibodies or fragments thereof by expressing the variable regions as fusion proteins with the constant regions. Typically, such constant regions are human.

[0404] Host cells for harboring and expressing the chains of anti-PMEL17 antibodies can be prokaryotic or eukaryotic. Escherichia coli (E. coli) is one prokaryotic host useful for cloning and expressing the polynucleotides of the invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one skilled in the art can also construct expression vectors, which typically contain expression control sequences (e.g., an origin of replication) compatible with the host cell. In addition, any number of well-known promoters will be available, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or promoter systems derived from phage lambda. The promoter typically controls expression, optionally with an operator sequence, and contains ribosome binding site sequences and the like, to initiate and complete transcription and translation. Other microorganisms, such as yeast, can also be used to express the anti-PMEL17 polypeptides of the invention. Insect cells in combination with baculovirus vectors can also be used.

[0405] In some preferred embodiments, mammalian host cells can be used to express and produce the anti-PMEL17 polypeptides of the present invention. For example, mammalian host cells can be hybridoma cell lines expressing endogenous immunoglobulin genes (e.g., myeloma hybridoma clones described in the Examples) or mammalian cell lines harboring exogenous expression vectors (e.g., SP2 / 0 myeloma cells exemplified below). These include any mortal, normal, or immortal normal or abnormal animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell culture to express polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), as well as essential processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters can be constitutive, cell-type specific, stage-specific, and / or tunable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (e.g., the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0406] Methods for introducing expression vectors containing polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed.). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic transfer, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to herpesvirus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced DNA uptake, and ex vivo transduction. In many cases, stable expression will be desired for long-term, high-yield production of recombinant proteins. For example, cell lines stably expressing anti-PMEL17 antibody chains or binding fragments thereof can be prepared using expression vectors of the invention containing viral origins of replication or endogenous expression elements and a selectable marker gene. After introduction of the vector, cells can be grown for 1-2 days in an enriched medium before being switched to a selective medium. The purpose of the selectable marker is to confer resistance to the selection, and its presence allows growth of cells that successfully express the introduced sequences in a selective medium. Resistant, stably transfected cells can be grown using tissue culture techniques appropriate to the cell type.

[0407] therapeutic use The antibodies, antibody fragments (e.g., antigen-binding fragments), and antibody-drug conjugates of the invention are useful in a variety of applications, including, but not limited to, the treatment or prevention of cancer, such as solid tumors or heme malignancies. In certain embodiments, the antibodies, antibody fragments (e.g., antigen-binding fragments), and antibody-drug conjugates of the invention are useful for inhibiting tumor growth, inducing differentiation, reducing tumor volume, and / or reducing tumor formation. Methods of use can be in vitro, ex vivo, or in vivo methods.

[0408] In one aspect, the antibodies, antibody fragments (e.g., antigen-binding fragments), and antibody-drug conjugates of the present invention are useful for detecting the presence of PMEL17 in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues. In certain embodiments, such tissues include normal and / or cancerous tissues that express higher levels of PMEL17 compared to other tissues.

[0409] In one aspect, the invention provides a method for detecting the presence of PMEL17 or Hepatitis B in a biological sample. In certain embodiments, the method comprises contacting the biological sample with an anti-PMEL17 antibody under conditions that allow binding of the antibody to the antigen, and detecting whether a complex forms between the antibody and the antigen.

[0410] In one aspect, the present invention provides methods for diagnosing a disorder associated with increased expression of PMEL17. In certain embodiments, the method comprises contacting a test cell with an anti-PMEL17 antibody; determining (either quantitatively or qualitatively) the level of expression of PMEL17 on the test cell by detecting binding of the anti-PMEL17 antibody to the PMEL17 antigen; and comparing the level of expression of PMEL17 in the test cell with the level of expression of PMEL17 on a control cell (e.g., a normal cell of the same tissue origin as the test cell or a cell that expresses PMEL17 at a level comparable to such a normal cell), wherein a higher level of expression of PMEL17 on the test cell compared to the control cell indicates the presence of a disorder associated with increased expression of PMEL17. In certain embodiments, the test cell is obtained from an individual suspected of having a disorder associated with increased expression of PMEL17. In certain embodiments, the disorder is a cell proliferation disorder, e.g., cancer or tumor. In certain embodiments, the method comprises measuring the copy number of the PMEL17 gene in the test cell.

[0411] In certain embodiments, diagnostic or detection methods, such as those described above, involve detecting binding of an anti-PMEL17 antibody to PMEL17 expressed on the surface of a cell or in a membrane preparation obtained from a cell expressing PMEL17 on its surface. An exemplary assay for detecting binding of an anti-PMEL17 antibody to PMEL17 expressed on the surface of a cell is a "FACS" assay.

[0412] Certain other methods can be used to detect binding of anti-PMEL17 antibodies to PMEL17, including, but not limited to, antigen-binding assays well known in the art, such as Western blots, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry (IHC).

[0413] In certain embodiments, the anti-PMEL17 antibody is labeled, including, but not limited to, labels or moieties that are directly detected (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radiolabels) and moieties that are indirectly detected, e.g., via an enzymatic reaction or molecular interaction, such as an enzyme or ligand.

[0414] In certain embodiments, the anti-PMEL17 antibody is immobilized on an insoluble matrix. Immobilization involves separating the anti-PMEL17 antibody from any PMEL17 protein that remains free in solution. This is conventionally achieved by insolubilizing the anti-PMEL17 antibody prior to the assay procedure by adsorption to a water-insoluble matrix or surface (Bennich et al., U.S. Pat. No. 3,720,760) or by covalent binding (e.g., using glutaraldehyde cross-linking), or by insolubilizing the anti-PMEL17 antibody after formation of a complex between the anti-PMEL17 antibody and PMEL17 protein, for example, by immunoprecipitation.

[0415] Any of the above diagnostic or detection embodiments may be performed using an antibody drug conjugate of the invention instead of, or in addition to, an anti-PMEL17 antibody.

[0416] In one embodiment, the invention provides a method of treating or preventing a disease, the method comprising administering to a patient an antibody, antibody fragment (e.g., an antigen-binding fragment), or antibody-drug conjugate of the invention. The invention also provides use of an antibody, antibody fragment (e.g., an antigen-binding fragment), or antibody-drug conjugate of the invention for treating or preventing a disease in a patient. In some embodiments, the invention provides an antibody, antibody fragment (e.g., an antigen-binding fragment), or antibody-drug conjugate of the invention for use in treating or preventing a disease in a patient. In a further embodiment, the invention provides use of an antibody, antibody fragment (e.g., an antigen-binding fragment), or antibody-drug conjugate of the invention in the manufacture of a medicament for treating or preventing a disease in a patient.

[0417] In certain embodiments, the disease treated by the antibodies, antibody fragments (e.g., antigen-binding fragments), and antibody-drug conjugates of the present invention is cancer. In certain embodiments, the cancer is characterized by PMEL17-expressing cells to which the antibodies, antibody fragments (e.g., antigen-binding fragments), and antibody-drug conjugates of the present invention bind. In certain embodiments, the cancer is characterized by increased expression of PMEL17 relative to healthy patients. In some embodiments, PMEL17 expression can be measured by increased PMEL17 RNA. In other embodiments, the cancer is characterized by increased DNA copy number of PMEL17. Other methods for measuring or determining the level of PMEL17 expression are known to those skilled in the art. In certain embodiments, the cancer is characterized by mutations, such as activating mutations affecting Q209 or R183 in the GNAQ and / or GNA11 genes. Examples of diseases that can be treated and / or prevented include, but are not limited to, melanoma, uveal melanoma, hepatocellular carcinoma, and metastatic cancers thereof.

[0418] The present invention provides methods for treating or preventing cancer, comprising administering a therapeutically effective amount of an antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention. In certain embodiments, the cancer is a solid tumor, such as melanoma, uveal melanoma, hepatocellular carcinoma, or a metastatic form thereof. In certain embodiments, the subject is a human. In certain embodiments, the cancer is a resistant cancer and / or a recurrent cancer.

[0419] In certain embodiments, the invention provides a method of inhibiting tumor growth, comprising administering to a subject a therapeutically effective amount of an antibody, antibody fragment (e.g., an antigen-binding fragment), or antibody-drug conjugate of the invention. In certain embodiments, the tumor is a solid tumor, such as melanoma, uveal melanoma, hepatocellular carcinoma, or a metastatic form thereof. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or has had a tumor removed.

[0420] In certain embodiments, the tumor expresses PMEL17 to which the anti-PMEL17 antibody binds. In certain embodiments, the tumor overexpresses human PMEL17. In certain embodiments, the tumor has an increased copy number of the PMEL17 gene. In certain embodiments, the tumor is characterized by a mutation, for example, an activating mutation affecting Q209 or R183 in the GNAQ and / or GNA11 genes.

[0421] The present invention also provides methods for selecting a patient for treatment with an antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention, comprising administering a therapeutically effective amount of the antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate. In certain aspects of the invention, the method comprises selecting the patient by measuring expression of PMEL17. In certain aspects of the invention, the method comprises selecting the patient by identifying a mutation, e.g., an activating mutation affecting Q209 or R183 in the GNAQ or GNA11 gene. In certain embodiments, the method comprises measuring the level of PMEL17 expression in the patient and detecting the GNAQ and / or GNA11 gene.

[0422] The appropriate dosage of an antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention for the treatment or prevention of disease depends on various factors, such as the type of disease to be treated, the severity and course of the disease, the response of the disease, previous treatments, the patient's medical history, etc. The antibody or drug can be administered once or over a series of treatments lasting from a few days to several months, or until a cure occurs or a diminution of the disease state (e.g., a decrease in tumor size) is achieved. Optimal administration schedules can be calculated from measurements of drug accumulation in the patient's body and will vary depending on the relative potency of the individual antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate. The treating physician can estimate repetition rates for administration based on measured residence times and drug concentrations in body fluids or tissues.

[0423] Combination therapy In certain instances, the antibodies, antibody fragments (e.g., antigen-binding fragments), or antibody-drug conjugates of the invention are used in combination with other therapeutic treatments, such as surgery and radiation therapy, therapeutic agents, such as other anti-cancer agents, anti-allergic agents, anti-emetic agents (or anti-nausea agents), analgesics, cytoprotective agents, and combinations thereof.

[0424] In one embodiment, an antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention is combined with a second compound having anti-cancer properties in a pharmaceutical combination formulation or administration regimen as a combination therapy. The second compound of the pharmaceutical combination formulation or administration regimen may have complementary activities to the antibody or immunoconjugate of the combination so that they do not adversely affect each other. For example, an antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention may be administered in combination with, but not limited to, chemotherapeutic agents, immunomodulators, tyrosine kinase inhibitors, GNAQ / GNA11 downstream signaling pathway inhibitors, IAP inhibitors, Bcl2 inhibitors, Mcl1 inhibitors, and other GNAQ / GNA11 inhibitors.

[0425] The term "pharmaceutical combination" as used herein refers to a fixed combination in the form of a single dosage unit, or to a non-fixed combination or kit of parts for co-administration, wherein two or more therapeutic agents can be administered independently at the same time or separately within a time interval, particularly where the combination partners can exhibit a synergistic, e.g., synergistic, effect.

[0426] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat or prevent a therapeutic condition or disorder described in this disclosure. Such administration includes co-administration of the therapeutic agents substantially simultaneously, e.g., in a single capsule in which the ratio of active ingredients is fixed. Alternatively, such administration includes co-administration of each active ingredient in multiple doses or in separate containers (e.g., capsules, powders, and liquids). The powders and / or liquids may be reconstituted or diluted to the desired dose before administration. In addition, such administration also includes sequential use of each type of therapeutic agent, either at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of the drug combination in treating or preventing the condition or disorder described herein.

[0427] Combination therapy can provide a "synergistic effect" and may prove to be "synergistic," i.e., the effect achieved when active ingredients are used together is greater than the sum of the effects resulting from using the compounds separately. Synergistic effects can be achieved when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered simultaneously as alternating or separate formulations; or (3) by some other regimen. When delivered in alternation therapy, synergistic effects can be achieved when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. Generally, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.

[0428] Common chemotherapy agents considered for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-dioxa-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), and riboflavin (Ricin®). ), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine ( Registered Trademark), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), Mylotarg,Paclitaxel (Taxol®), phoenix (yttrium 90 / MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®), and pemetrexed.

[0429] In one aspect, the present invention provides a method of treating or preventing cancer by administering to a subject in need thereof an antibody drug conjugate of the invention in combination with one or more MDM2 inhibitors, PKC inhibitors, PRC2 inhibitors, MAPK inhibitors, GPCR inhibitors, tyrosine kinase inhibitors, including, but not limited to, BTK inhibitors, EGFR inhibitors, Her2 inhibitors, Her3 inhibitors, IGFR inhibitors, and Met inhibitors.

[0430] For example, MDM2 inhibitors include, but are not limited to, RG7112 (RO5045337); RG7388 (RO5503781, idasanutlin); MI-77301 (SAR405838); MK-8242 (SCH-900242); AMG232; CGM097; DS3032b; HDM201; and ALRN-6924.

[0431] For example, PKC inhibitors include, but are not limited to, balanol; riluzole; staurosporine; enzastaurin; δV1-1 (KAI-9803 or delcasertib); εV1-2 (KAI-1678); aprinocarsen; midostaurin (PKC412); UCN-01 (7-hydroxy-staurosporine); rottlerin (5,7,dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzyl)-8-cinnamoyl-1,2-chromene); and bryostatin 1.

[0432] For example, PRC2 inhibitors include, but are not limited to, EI1; EPZ011989; EPZ005687; tetramethylpiperidinylbenzamide; UNC1999; and GSK126.

[0433] For example, MAPK inhibitors include, but are not limited to, vemurafenib (Zelboraf); dabrafenib (Tafinlar); encorafenib (Braftovi); trametinib (Mekinist); cobimetinib (Cotellic); binimetinib (Mektovi); and ulixertinib.

[0434] For example, tyrosine kinase inhibitors include, but are not limited to, ibrutinib (PCI-32765); erlotinib hydrochloride (Tarceva®); linifanib (N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N'-(2-fluoro-5-methylphenyl)urea, also known as ABT869 and available from Genentech); sunitinib malate (Sutent®); bosutinib (4-[(2,4-dichloro-5-methoxyphenyl)amino ]-6-methoxy-7-[3-(4-methylpropyl-1-yl)propoxy]quinoline-3-carbonitrile, also known as SKI-606 and described in U.S. Pat. No. 6,780,996); dasatinib (Sprycel®); pazopanib (Votrient®); sorafenib (Nexavar®); Zactima (ZD6474); and imatinib or imatinib mesylate (Gilvec® and Gleevec®).

[0435] Epidermal growth factor receptor (EGFR) inhibitors include, but are not limited to, erlotinib hydrochloride (Tarceva®), gefitinib (Iressa®); N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3"S")-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide, Tovok®); vandetanib (Caprelsa®) ); lapatinib (Tykerb®); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); canertinib dihydrochloride (CI-1033); 6-[4-[(4-ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-ol amine (AEE788, CAS497839-62-0); mubritinib (TAK165); pelitinib (EKB569); afatinib (BIBW2992); neratinib (HKI-272); N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbamic acid, (3S)-3-morpholinylmethyl ester (BMS599626); N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8); and 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol (PKI166, CAS 187724-61-4).

[0436] EGFR antibodies include, but are not limited to, cetuximab (Erbitux®); panitumumab (Vectibix®); matuzumab (EMD-72000); nimotuzumab (hR3); zalutumumab; TheraCIM h-R3; MDX0447 (CAS339151-96-1); and ch806 (mAb-806, CAS946414-09-1).

[0437] Human epidermal growth factor receptor 2 (Her2 receptor) (also known as Neu, ErbB-2, CD340, or p185) inhibitors include, but are not limited to, trastuzumab (Herceptin®); pertuzumab (Omnitarg®); trastuzumab emtansine (Kadcyla®); neratinib (HKI-272, (2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl] amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide, as described in WO 05 / 028443; lapatinib or lapatinib ditosylate (Tykerb®); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514) (2E)-N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butenamide (BIBW-2992, CAS 850140-72-6); N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbaminium canertinib dihydrochloride (PD183805 or CI-1033); and N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8).

[0438] Her3 inhibitors include, but are not limited to, LJM716, MM-121, AMG-888, RG7116, REGN-1400, AV-203, MP-RM-1, MM-111, and MEHD-7945A.

[0439] MET inhibitors include, but are not limited to, cabozantinib (XL184, CAS849217-68-1); foretinib (GSK1363089, formerly known as XL880, CAS849217-64-7); tivantinib (ARQ197, CAS1000873-98-2); 1-(2-hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG458); Crizotinib (Xalkori®, PF-02341066); (3Z)-5-(2,3-dihydro-1H-indol-1-ylsulfonyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-1,3-dihydro-2H-indol-2-one (SU11271); (3Z)-N-(3-chlorophenyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene) -N-Methyl-2-oxoindoline-5-sulfonamide (SU11274); (3Z)-N-(3-chlorophenyl)-3-{[3,5-dimethyl-4-(3-morpholin-4-ylpropyl)-1H-pyrrol-2-yl]methylene}-N-methyl-2-oxoindoline-5-sulfonamide (SU11606); 6-[difluoro[6-(1-methyl-1H-pyrazol-4-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]methyl]-quinoline (JNJ38877605, CAS 943540- 75-8); 2-[4-[1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl]-1H-pyrazol-1-yl]ethanol (PF04217903, CAS956905-27-4); N-((2R)-1,4-dioxan-2-ylmethyl)-N-methyl-N'-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclopenta[1,2-b]pyridin-7-yl]sulfamide (MK2461, CAS917879-39-1);6-[[6-(1-methyl-1H-pyrazol-4-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]thio]-quinoline (SGX523, CAS 1022150-57-7); and (3Z)-5-[[(2,6-dichlorophenyl)methyl]sulfonyl]-3-[[3,5-dimethyl-4-[[(2R)-2-(1-pyrrolidinylmethyl)-1-pyrrolidinyl]carbonyl]-1H-pyrrol-2-yl]methylene]-1,3-dihydro-2H-indol-2-one (PHA665752, CAS 477575-56-7).

[0440] IGF1R inhibitors include, but are not limited to, BMS-754807, XL-228, OSI-906, GSK0904529A, A-928605, AXL1717, KW-2450, MK0646, AMG479, IMCA12, MEDI-573, and BI836845. For review, see, e.g., Yee, JNCI, 104;975 (2012).

[0441] In another aspect, the present invention provides a method for treating or preventing cancer by administering to a subject in need thereof an antibody-drug conjugate of the present invention in combination with one or more GNAQ / GNA11 downstream signaling pathway inhibitors, for example, but not limited to, a β-arrestin inhibitor, a GRK inhibitor, a MAPK inhibitor, a PI3K inhibitor, a JAK inhibitor, etc.

[0442] For example, phosphoinositide 3-kinase (PI3K) inhibitors include, but are not limited to, idelalisib (Zydelig, GS-1101, Cal-101), 4-[2-(1H-indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC0941, and described in WO 09 / 036082 and WO 09 / 05554), and 730); 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile (also known as BEZ235 or NVP-BEZ235 and described in WO 06 / 122806); 4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridine-2-amine (also known as BKM120 or NVP-BKM120 and described in WO 2007 / 084786); tozasertib (VX680 or MK-0457, CAS 639089-54-6); (5Z)-5-[[4-(4-pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione (GSK1059615, CAS 958852-01-2); (1E,4S,4aR,5R,6aS,9aR)-5-(acetyl oxy)-1-[(di-2-propenylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione (PX866, CAS 502632-66-8); and 8-phenyl-2-(morpholin-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6).

[0443] In yet another aspect, the present invention provides a method of treating or preventing cancer by administering to a subject in need thereof an antibody-drug conjugate of the present invention in combination with one or more pro-apoptotic agents, including, but not limited to, an IAP inhibitor, a Bcl2 inhibitor, an MCl1 inhibitor, a Trail agent, or a Chk inhibitor.

[0444] For example, IAP inhibitors include, but are not limited to, LCL161, GDC-0917, AEG-35156, AT406, and TL32711. Other examples of IAP inhibitors include, but are not limited to, those disclosed in WO 04 / 005284, WO 04 / 007529, WO 05 / 097791, WO 05 / 069894, WO 05 / 069888, WO 05 / 094818, U.S. Patent Application Publication No. 2006 / 0014700, U.S. Patent Application Publication No. 2006 / 0025347, WO 06 / 069063, WO 06 / 010118, WO 06 / 017295, and WO 08 / 134679, all of which are incorporated herein by reference.

[0445] BCL-2 inhibitors include, but are not limited to, venetoclax (also known as GDC-0199, ABT-199, RG7601); 4-[4-[[2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(4-morpholinyl)-1-[(phenylthio)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl

[0023] [1-methyl]benzamide (also known as ABT-263 and described in WO 09 / 155386); tetrocarcin A; antimycin; gossypol ((-)BL-193); obatoclax; ethyl-2-amino-6-cyclopentyl-4-(1-cyano-2-ethoxy-2-oxoethyl)-4Hchromone-3-carboxylate (HA14-1); oblimersen (G3139, Genasense®); Bak These include BH3 peptides; (-)-gossypol acetic acid (AT-101); 4-[4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]-benzamide (ABT-737, CAS 852808-04-9); and navitoclax (ABT-263, CAS 923564-51-6).

[0446] Pro-apoptotic receptor agonists (PARA), such as DR4 (TRAILR1) and DR5 (TRAILR2), including, but not limited to, dulanermin (AMG-951, RhApo2L / TRAIL); mapatumumab (HRS-ETR1, CAS658052-09-6); lexatumumab (HGS-ETR2, CAS845816-02-6); Apomab (Apomab®); conatumumab (AMG655, CAS896731-82-1); and tigatuzumab (CS1008, CAS946415-34-5, available from Daiichi Sankyo).

[0447] Checkpoint kinase (CHK) inhibitors include, but are not limited to, 7-hydroxystaurosporine (UCN-01); 6-bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-(3R)-3-piperidinyl-pyrazolo[1,5-a]pyrimidin-7-amine (SCH900776, CAS891494-63-6); 5-(3-fluorophenyl)-3-ureidothiophene- 2-Carboxylic acid N-[(S)-piperidin-3-yl]amide (AZD7762, CAS 860352-01-8); 4-[((3S)-1-azabicyclo[2.2.2]oct-3-yl)amino]-3-(1H-benzimidazol-2-yl)-6-chloroquinolin-2(1H)-one (CHIR124, CAS 405168-58-3); 7-aminodactinomycin (7-AAD), isogranulati N-[5-bromo-4-methyl-2-[(2S)-2-morpholinylmethoxy]-phenyl]-N'-(5-methyl-2-pyrazinyl)urea (LY2603618, CAS 911222-45-2); Sulforaphane (CAS 4478-93-7, 4-methylsulfinylbutylisothiocyanate); 9,10,11,12-tetrahydro-9,12-epoxy-1H- diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-1,3(2H)-dione (SB-218078, CAS 135897-06-2); and TAT-S216A (YGRKKRRQRRRLYRSPAMPENL (SEQ ID NO: 282)), and CBP501 ((d-Bpa)sws(d-Phe-F5)(d-Cha)rrrqrr).

[0448] In a further embodiment, the present invention provides a method of treating or preventing cancer by administering to a subject in need thereof an antibody drug conjugate of the invention in combination with one or more immunomodulatory agents (e.g., one or more activators of costimulatory molecules or inhibitors of immune checkpoint molecules).

[0449] In certain embodiments, the immunomodulatory agent is an activator of a costimulatory molecule. In one embodiment, the costimulatory molecule agonist is selected from an agonist (e.g., an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion) of OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, STING, or CD83 ligand.

[0450] In certain embodiments, the immunomodulatory agent is an inhibitor of an immune checkpoint molecule. In one embodiment, the immunomodulatory agent is an inhibitor of PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and / or TGFRbeta. In one embodiment, the inhibitor of an immune checkpoint molecule inhibits PD-1, PD-L1, LAG-3, TIM-3, or CTLA4, or any combination thereof. The term "inhibition" or "inhibitor" includes a reduction in a particular parameter, e.g., the activity of a given molecule, e.g., an immune checkpoint inhibitor. For example, inhibition of at least 5%, 10%, 20%, 30%, 40%, 50%, or more of activity, e.g., inhibition of PD-1 or PD-L1 activity, is encompassed by this term. Thus, inhibition need not be 100%.

[0451] Inhibition of inhibitory molecules can be carried out at the DNA, RNA, or protein level. In some embodiments, inhibitory nucleic acids (e.g., dsRNA, siRNA, or shRNA) can be used to inhibit expression of inhibitory molecules. In other embodiments, the inhibitor of an inhibitory signal is a polypeptide, such as a soluble ligand (e.g., PD-1-Ig or CTLA-4 Ig), or an antibody or antigen-binding fragment thereof that binds to an inhibitory molecule; for example, an antibody or fragment thereof (also referred to herein as an "antibody molecule") that binds to PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and / or TGFR beta, or a combination thereof.

[0452] In one embodiment, the antibody molecule is a complete antibody or a fragment thereof (e.g., Fab, F(ab')2, Fv, or single-chain Fv fragment (scFv)). In yet other embodiments, the antibody molecule has a heavy chain constant region (Fc) selected from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, e.g., one selected from the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, more particularly, one selected from the heavy chain constant region of IgG1 or IgG4 (e.g., human IgG1 or IgG4). In one embodiment, the heavy chain constant region is human IgG1 or human IgG4. In one embodiment, the constant region is altered, e.g., mutated, to modify the properties of the antibody molecule (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function).

[0453] In certain embodiments, the antibody molecule is in the form of a bispecific or multispecific antibody molecule. In one embodiment, a bispecific antibody molecule has a first binding specificity for PD-1 or PD-L1 and a second binding specificity, e.g., a second binding specificity for TIM-3, LAG-3, or PD-L2. In one embodiment, the bispecific antibody molecule binds to PD-1 or PD-L1 and TIM-3. In another embodiment, the bispecific antibody molecule binds to PD-1 or PD-L1 and LAG-3. In another embodiment, the bispecific antibody molecule binds to PD-1 and PD-L1. In yet another embodiment, the bispecific antibody molecule binds to PD-1 and PD-L2. In another embodiment, the bispecific antibody molecule binds to TIM-3 and LAG-3. Any combination of the above molecules can be made into a multispecific antibody molecule, e.g., a trispecific antibody comprising a first binding specificity for PD-1 or PD-L1 and second and third binding specificities for two or more of TIM-3, LAG-3, or PD-L2.

[0454] In a specific embodiment, the immunomodulatory agent is an inhibitor of PD-1, e.g., human PD-1. In another embodiment, the immunomodulatory agent is an inhibitor of PD-L1, e.g., human PD-L1. In one embodiment, the PD-1 or PD-L1 inhibitor is an antibody molecule directed against PD-1 or PD-L1. PD-1 or PD-L1 inhibitors can be administered alone or in combination with other immunomodulatory agents, e.g., in combination with inhibitors of LAG-3, TIM-3, or CTLA4. In an exemplary embodiment, an inhibitor of PD-1 or PD-L1, e.g., an anti-PD-1 or PD-L1 antibody molecule, is administered in combination with an LAG-3 inhibitor, e.g., an anti-LAG-3 antibody molecule. In another embodiment, an inhibitor of PD-1 or PD-L1, e.g., an anti-PD-1 or PD-L1 antibody molecule, is administered in combination with a TIM-3 inhibitor, e.g., an anti-TIM-3 antibody molecule. In yet another embodiment, an inhibitor of PD-1 or PD-L1, e.g., an anti-PD-1 antibody molecule, is administered in combination with a LAG-3 inhibitor, e.g., an anti-LAG-3 antibody molecule, and a TIM-3 inhibitor, e.g., an anti-TIM-3 antibody molecule. Other combinations of immunomodulatory agents and PD-1 inhibitors (e.g., one or more of PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and / or TGFR) are also within the scope of the present invention. Any of the antibody molecules known in the art or disclosed herein can be used in the above combinations of inhibitors of checkpoint molecules.

[0455] In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody selected from nivolumab, pembrolizumab, or pidilizumab. In some embodiments, the anti-PD-1 antibody is nivolumab. Alternative names for nivolumab include MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in U.S. Patent No. 8,008,449 and PCT Publication WO 2006 / 121168.

[0456] In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (trade name KEYTRUDA, formerly known as lambrolizumab, also known as Merck3745, MK-3475, or SCH-900475) is a humanized IgG4 monoclonal antibody that binds to PD1. Pembrolizumab is disclosed, for example, in Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134-44, WO 2009 / 114335, and U.S. Patent No. 8,354,509.

[0457] In some embodiments, the anti-PD-1 antibody is pidilizumab. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO 2009 / 101611. Other anti-PD1 antibodies are disclosed in U.S. Pat. No. 8,609,089, U.S. Patent Application Publication No. 2010028330, and / or U.S. Patent Application Publication No. 20120114649. Other anti-PD1 antibodies include AMP514 (Amplimmune).

[0458] In some embodiments, the PD-1 inhibitor is PDR001, also known as spartalizumab, or any other anti-PD-1 antibody disclosed in WO 2015 / 112900.

[0459] In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising the extracellular or PD-1-binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224.

[0460] In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 inhibitor is selected from, for example, YW243.55.S70, MPDL3280A, MEDI-4736, or MDX-1105MSB-0010718C (also referred to as A09-246-2) having a sequence disclosed therein (or a sequence substantially identical or similar thereto, e.g., at least 85%, 90%, 95% or more identical to the specified sequence).

[0461] In one embodiment, the PD-L1 inhibitor is MDX-1105. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO 2007 / 005874.

[0462] In one embodiment, the PD-L1 inhibitor is YW243.55.S70. The YW243.55.S70 antibody is an anti-PD-L1 antibody described in WO 2010 / 077634 (the heavy and light chain variable region sequences are set forth in SEQ ID NOs: 20 and 21, respectively).

[0463] In one embodiment, the PD-L1 inhibitor is MDPL3280A (Genentech / Roche). MDPL3280A is a human Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies against PD-L1 are disclosed in U.S. Patent No. 7,943,743 and U.S. Patent Application Publication No. 20120039906.

[0464] In another embodiment, the PD-L2 inhibitor is AMP-224, a PD-L2 Fc-fused soluble receptor that blocks the interaction between PD1 and B7-H1 (B7-DCIg; Amplimmune; disclosed, for example, in WO 2010 / 027827 and WO 2011 / 066342).

[0465] In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule. In one embodiment, the LAG-3 inhibitor is BMS-986016. In one embodiment, the LAG-3 inhibitor is LAG525 or any of the anti-LAG3 antibodies disclosed in WO 2015 / 138920.

[0466] In one embodiment, the TIM-3 inhibitor is an anti-TIM3 antibody molecule. In one embodiment, the TIM-3 inhibitor is MBG453 or any of the anti-TIM3 antibodies disclosed in WO 2015 / 117002.

[0467] Pharmaceutical Composition To prepare a pharmaceutical or sterile composition comprising the immunoconjugate, the immunoconjugate of the present invention is mixed with a pharmaceutically acceptable carrier or excipient. The composition may further contain one or more other therapeutic agents suitable for the treatment or prevention of PMEL17-expressing cancers (including, but not limited to, subcutaneous melanoma, uveal melanoma, hepatocellular carcinoma, and metastatic cancers thereof).

[0468] Formulations of therapeutic and diagnostic agents can be prepared by mixing them with physiologically acceptable carriers, excipients, or stabilizers, for example, in the form of lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY, 2001; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY, 2000; Avis, et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY, 1993; Lieberman, et al. (eds.), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY, 1990; Lieberman, et al. (eds.), Pharmaceutical Dosage Forms: Dispersions Systems, Marcel Dekker, NY, 1990; see Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY, 2000).

[0469] The choice of dosing regimen for a therapeutic agent depends on several factors, including the serum or tissue turnover rate of the entity, the level of disease symptoms, the immunogenicity of the entity, and the accessibility of target cells within the biological matrix. In certain embodiments, the dosing regimen maximizes the amount of therapeutic agent delivered to the patient with an acceptable level of side effects. Thus, the amount of biologic delivered will depend in part on the particular entity and the severity of the condition being treated. Guidance for selecting appropriate doses of antibodies, cytokines, and small molecules is available (e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK, 1996; Kresina (ed.), Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY, 1991; Bach (ed.), Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY, 1993; Baert et al., New Engl. J. Med. 348:601-608, 2003; Milgrom et al., New Engl. J. Med. 341:1966-1973, 1999; Slamon et al., New Engl.J.Med.344:783-792,2001;Beniaminovitz et al.,New Engl.J.Med.342:613-619,2000;Ghosh et al.,New Engl.J.Med.348:24-32,2003;Lipsky et al.,New See Engl.J.Med.343:1594-1602, 2000).

[0470] The determination of the appropriate dose is made by the clinician, for example, using parameters or factors known or suspected in the art that affect or are predicted to affect treatment or prevention. Typically, the dose is started somewhat less than optimal and then increased in small increments for any negative side effects until the desired or optimal effect is achieved. Important diagnostic measures include measures of disease symptoms, for example, of infusion reactions. Important diagnostic measures include, for example, those of inflammation or levels of inflammatory cytokines produced.

[0471] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention or its ester, salt, or amide used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors known in the medical arts.

[0472] Compositions containing the antibodies or fragments thereof of the present invention can be provided by continuous infusion or by administration, for example, at daily, weekly intervals, or 1 to 7 times per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks. Doses can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation. Specific dosing protocols include the maximum dose or dose frequency that avoids significant undesirable side effects.

[0473] For the immunoconjugates of the invention, the dosage administered to a patient may be from 0.0001 mg / kg to 100 mg / kg of the patient's body weight. Dosages may be 0.0001 mg / kg to 30 mg / kg, 0.0001 mg / kg to 20 mg / kg, 0.0001 mg / kg to 10 mg / kg, 0.0001 mg / kg to 5 mg / kg, 0.0001 to 2 mg / kg, 0.0001 to 1 mg / kg, 0.0001 mg / kg to 0.75 mg / kg, 0.0001 mg / kg to 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 to 0.15 mg / kg, 0.0001 to 0.01 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to 0.25 mg / kg, or 0.01 to 0.10 mg / kg of the patient's body weight. Dosage of the antibodies or fragments thereof of the invention can be calculated by multiplying the patient's weight in kilograms (kg) by the dose to be administered in mg / kg.

[0474] Doses of the immunoconjugates of the invention may be repeated, and administration may be less than daily, or may be separated by at least 1, 2, 3, 5, 10, 15, 30, 45 days, 2 months, 75 days, 3 months, 4 months, 5 months, or at least 6 months. In some embodiments, the immunoconjugates of the invention may be given twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, or less frequently. In a specific embodiment, administration of the immunoconjugates of the invention is repeated every two weeks.

[0475] The amount effective for a particular patient may vary depending on factors such as the condition being treated, the patient's overall health, the method, route and dose of administration, and the severity of side effects (see, e.g., Maynard et al., A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Publ., London, UK, 2001).

[0476] The route of administration may be, for example, by topical or dermal application, subcutaneous injection or infusion, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, or intralesional administration, or by sustained-release systems or implants (e.g., Sidman et al., Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985). 77:4030-4034, 1980; U.S. Patent Nos. 6,350,466 and 6,316,024). Where necessary, the composition may also include a solubilizing agent or a local anesthetic, such as lidocaine to ease pain at the site of the injection, or both. In addition, pulmonary administration can be employed, such as by use of an inhaler or nebulizer, and by formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903 (each of these applications is incorporated herein by reference in its entirety).

[0477] Compositions of the present invention can also be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be recognized by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. The selected route of administration for the immunoconjugates of the present invention can include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. Parenteral administration can refer to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, intrathecal, epidural, and intrasternal injection and infusion. Alternatively, compositions of the present invention can be administered via a parenteral route, such as a topical, epidermal, or mucosal route of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical. In one embodiment, the immunoconjugates of the present invention are administered by injection. In another embodiment, the immunoconjugates of the invention are administered subcutaneously.

[0478] If the immunoconjugates of the invention are administered in a controlled- or sustained-release system, the controlled- or sustained-release can be achieved using a pump (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:20, 1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:574, 1989). Polymeric materials can be used to achieve controlled or sustained release of the therapeutic agents of the invention (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1983; Levy et al., Science 228:190, 1985; During et al., Ann. Neurol. 25:351, 1989; Howard et al., J. Neurosurg. 7:197, 1989). 1:105, 1989; U.S. Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; WO 99 / 15154; and WO 99 / 20253. Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters.In one embodiment, the polymer used in the sustained-release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. Controlled- or sustained-release systems can be placed in close proximity to the prophylactic or therapeutic target, thus requiring only a small systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).

[0479] Controlled-release systems are discussed in the review by Langer, Science 249:1527-1533, 1990. Any technique known to those of ordinary skill in the art can be used to produce sustained-release formulations containing one or more immunoconjugates of the invention. See, e.g., U.S. Pat. No. 4,526,938, WO 91 / 05548, WO 96 / 20698, Ning et al., Radiotherapy & Oncology 39:179-189, 1996; Song et al., PDA Journal of Pharmaceutical Science & Technology 50:372-397, 1995; Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, 1997; and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, 1997 (each of these applications is incorporated herein by reference in its entirety).

[0480] When the immunoconjugates of the present invention are administered topically, they can be formulated in the form of ointments, creams, transdermal patches, lotions, gels, sprays, aerosols, solutions, emulsions, or other forms known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, viscous semi-solid or solid forms containing a carrier or one or more excipients compatible with topical application, in some cases having a dynamic viscosity greater than that of water, are typically used. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) to affect various properties, such as osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations, in some cases, the active ingredient combined with solid or liquid inert carrier is mixed with pressurized volatile substance (for example, gaseous propellant, for example, freon) or packaged in squeeze bottle.If desired, moisturizer or humectant can also be added to pharmaceutical compositions and dosage forms.Examples of such additional components are well known in the art.

[0481] When a composition containing an immunoconjugate is administered intranasally, it can be formulated in aerosol form, spray, mist, or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer by use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, for example, gelatin) for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, for example, lactose or starch.

[0482] Methods of co-administration or co-treatment with a second therapeutic agent, such as a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation, are known in the art (see, for example, Hardman et al., (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Philadelphia, Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Philadelphia, Pa.). An effective amount of a therapeutic agent can reduce disease symptoms by at least 10%; at least 20%; at least about 30%; at least 40%; or at least 50%.

[0483] Additional therapies (e.g., prophylactic or therapeutic agents) that can be administered in combination with the immunoconjugates of the invention may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 to about 3 hours apart, about 3 to about 4 hours apart, about 4 to about 5 hours apart, about 5 to about 6 hours apart, about 6 to about 7 hours apart, about 7 to about 8 hours apart, about 8 to about 9 hours apart, about 9 to about 10 hours apart, about 10 to about 11 hours apart, about 11 to about 12 hours apart, about 12 to 18 hours apart, 18 to 24 hours apart, 24 to 36 hours apart, 36 to 48 hours apart, 48 to 52 hours apart, 52 to 60 hours apart, 60 to 72 hours apart, 72 to 84 hours apart, 84 to 96 hours apart, or 96 to 120 hours apart from the immunoconjugates of the invention. Two or more treatments may be administered during the same patient visit.

[0484] In certain embodiments, immunoconjugates of the invention can be formulated to ensure proper in vivo distribution. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that immunoconjugates of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that selectively transport into specific cells or organs, thus improving targeted drug delivery (see, e.g., Ranade, (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include phorate or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (Bloeman et al., (1995) FEBS Lett. 357:140; Owais et al., (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al., (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al., (1994) J. Biol. Chem. 269:9090); K. Keinanen; M.L. Laukkanen (1994) FEBS See also Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0485] The present invention provides administration protocols for pharmaceutical compositions comprising an immunoconjugate of the present invention, alone or in combination with other treatments, to a subject in need thereof. The treatments (prophylactic or therapeutic) of the combination therapy of the present invention may be administered simultaneously or sequentially to a subject. The treatments (prophylactic or therapeutic) of the combination therapy of the present invention may also be administered cyclically. Cycling therapy involves administering a first treatment (first prophylactic or therapeutic agent) for a period of time, followed by a second treatment (second prophylactic or therapeutic agent) for a period of time, and repeating this sequential administration (i.e., cycling) to reduce the development of resistance to one of the therapies (e.g., agents), avoid or alleviate side effects of one of the therapies (e.g., agents), and / or improve the efficacy of the treatment.

[0486] The treatments (prophylactic or therapeutic) of the combination therapies of the present invention can be administered simultaneously to a subject.

[0487] The term "concurrently" is not limited to administration of therapies at exactly the same time, but rather means that pharmaceutical compositions comprising the antibodies or fragments thereof of the present invention are administered to a subject in an order and within a time interval such that the antibody-drug conjugates of the present invention act together with other therapies to provide increased benefit beyond that normally achieved. For example, each therapy may be administered to a subject simultaneously or sequentially in any order at different times; however, if not administered simultaneously, they should be administered sufficiently close in time to provide the desired therapeutic or prophylactic effect. Each therapy may be administered to a subject separately, in any suitable form, and by any suitable route. In various embodiments, the therapies (prophylactic or therapeutic agents) are administered to a subject less than 5 minutes apart, less than 15 minutes apart, less than 30 minutes apart, less than 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 to about 3 hours apart, about 3 to about 4 hours apart, about 4 to about 5 hours apart, about 5 to about 6 hours apart, about 6 to about 7 hours apart, about 7 to about 8 hours apart, about 8 to about 9 hours apart, about 9 to about 10 hours apart, about 10 to about 11 hours apart, about 11 to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart. In other embodiments, two or more therapies (prophylactic or therapeutic agents) are administered during the same patient visit.

[0488] The prophylactic or therapeutic agents of the combination therapies may be administered to a subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies may be administered to a subject simultaneously in separate pharmaceutical compositions. The prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration. The prophylactic or therapeutic agents of the combination therapies may be administered to a subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies may be administered to a subject simultaneously in separate pharmaceutical compositions. The prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration. [Example]

[0489] Example 1: Synthesis of exemplary linker-drug compounds Example 1-1: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-22-isopropyl-3-((R)-1-methoxyethyl)-4,9,10,12,16-pentamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosane-6- Synthesis of (2S,3R)-3-(((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamidopentanoate (B1) Step 1: Synthesis of (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-22-isopropyl-3-((R)-1-methoxyethyl)-4,9,10,12,16-pentamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl)-2-methylpropyl(2S,3R)-3-((hydroxyhydrophosphoryl)oxy)-4-methyl-2-propionamidopentanoate (1-1): [ka] Imidazole (102 mg, 1.49 mmol, 15 equiv) was dissolved in acetonitrile (ACN) (1.4 mL) and cooled in an ice bath (a crash of ImH was observed, and the mixture was removed from the ice bath to dissolve the ImH). Phosphorus trichloride (1.0 M in ACN) (499 μl, 0.499 mmol, 5 equiv) was then added dropwise (resulting in a white suspension), and the mixture was stirred for 10 min. Triethylamine (250 μl, 1.796 mmol, 18 equiv) was then added and the mixture was stirred for 40 min, followed by (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-22-isopropyl-3-((R)-1-methoxyethyl)-4,9,10,12,16-pentamethyl-15-methylene- 2,5,8,11,14,17,20-Heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl)-2-methylpropyl (2S,3R)-3-hydroxy-4-methyl-2-propionamidopentanoate (A1) (100 mg, 0.100 mmol, 1.0 equiv.; compound (A1) was obtained using the method described in Example 3-1) was added. The yellow-orange heterogeneous mixture was warmed to room temperature and stirred for a total of 60 minutes. The mixture was treated with water (0.2 mL), and the material was purified by reverse-phase flash chromatography (0-100% ACN / water, 40 gram C18 column, neutral mobile phase). The product fractions were collected and lyophilized to give H-phosphonate (1-1) as a pale yellow amorphous powder. LCMS: MH+ = 1066.3, 0.78 min (Acquity UPLC BEH C18 1.7 um column, 2-98% 2 min run with water / MeCN + 0.1% NH4OH, basic method).

[0490] Step 2: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-22-isopropyl-3-((R)-1-methoxyethyl)-4,9,10,12,16-pentamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosane-6-yl) Synthesis of (2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamidopentanoate (B1): [ka] (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-22-isopropyl-3-((R)-1-methoxyethyl)-4,9,10,12,16-pentamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl)-2-methylpropyl(2S,3R)-3-((hydroxyhydrophosphoryl)oxy)-4-methyl-2-propionamidopenta Noate (1-1) (100 mg, 0.094 mmol, 1.0 equiv.) and (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (108 mg, 0.188 mmol, 2.0 equiv., CAS number 2055041-37-5) (both lyophilized powders were transferred to a 10 mL vial) were dissolved in pyridine (4 mL). Pivaloyl chloride (0.058 mL, 0.469 mmol, 5 equiv.) was then added dropwise to give a pale yellow solution. The mixture was stirred at room temperature for 10 minutes, and then 1.0 equiv. of additional pivaloyl chloride was added. A freshly prepared solution of iodine (47.6 mg, 0.188 mmol, 2.0 equiv) in pyridine-water (14:1, 750 uL) was added to give a clear dark brown solution.The mixture was stirred for 25 min and directly prepared by reverse-phase flash chromatography (40 g C-18 column, 0% Ac / MeCN for 3 min, then 0 to 60% ACN / water over 15 min, neutral method) to give (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-22-isopropyl-3-((R)-1-methoxyethyl)-4,9,10,12,16-pentamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-

[0047] This gave pentaazacyclodocosan-6-yl)-2-methylpropyl(2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamidopentanoate (B-1). HRMS; MH+ = 1638.7700, 2.84 min.

[0491] Example 1-2: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-((R)-1-methoxyethyl)-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl) Synthesis of 2-methylpropyl(2S,3R)-2-acetamido-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methylpentanoate (B2) Step 1: Synthesis of (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-((R)-1-methoxyethyl)-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl)-2-methylpropyl(2S,3R)-2-acetamido-3-((hydroxyhydrophosphoryl)oxy)-4-methylpentanoate (1-2): [ka] Imidazole (85 mg, 1.25 mmol, 15 equiv) was dissolved in acetonitrile (ACN) (2.5 mL) and cooled in an ice bath while still cold (a crash of ImH was observed, and the mixture was removed from the ice bath to dissolve the ImH). Phosphorus trichloride (36.4 μl, 0.417 mmol, 5 equiv dissolved in 0.5 mL MeCN) was then added dropwise (resulting in a white suspension), and the mixture was stirred for 10 min. Triethylamine (174 μl, 1.25 mmol, 15 equiv) was then added and the mixture was stirred for 40 min, followed by (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-((R)-1-methoxyethyl)-4,9,10,12,16,22-hexamethyl-15-methylene-2, 5,8,11,14,17,20-Heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl)-2-methylpropyl (2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate (A2) (80 mg, 0.084 mmol, 1.0 equiv.; compound (A2) was obtained using the method described in Example 3-2) was added. The yellow-orange heterogeneous mixture was warmed to room temperature and stirred for a total of 30 minutes. The mixture was treated with water (1 mL), and the material was purified by reverse-phase flash chromatography (0-100% ACN / water, 40 gram C18 column, neutral mobile phase). The product fractions were collected and lyophilized to give H-phosphonate (1-2) as a pale yellow amorphous powder. LCMS: MH+ = 1024.3, 0.78 min (Acquity UPLC BEH C18 1.7 um column, 2-98% 2 min run with water / MeCN + 0.1% NH4OH, basic method).

[0492] Step 2: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-((R)-1-methoxyethyl)-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl Synthesis of 2S,3R)-2-methylpropyl(2S,3R)-2-acetamido-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methylpentanoate [ka] (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-((R)-1-methoxyethyl)-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl)-2-methylpropyl(2S,3R)-2-acetamido-3-((hydroxyhydrophosphoryl)oxy)-4-methylpentanoate (1-2) (50 mg, 0.049 mmol, 1.0 equiv.) and (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS#2055041-37-5) (33.7 mg, 0.059 mmol, 1.2 equiv.) (both lyophilized powders were transferred to a 10 mL vial) were dissolved in pyridine (1 mL). Pivaloyl chloride (0.042 mL, 0.342 mmol, 7 equiv.) was then added dropwise to give a pale yellow solution. The mixture was stirred at room temperature for 30 min. A freshly prepared solution of iodine (49.6 mg, 0.195 mmol, 4 equiv) in pyridine-water (20:1, 500 uL) was added to give a clear dark brown solution.The mixture was stirred for 30 min and directly prepared by reverse-phase flash chromatography (40 g C-18 column, 0% Ac / MeCN for 3 min, then 0-70% ACN / water over 15 min, neutral method) to give (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-22-isopropyl-3-((R)-1-methoxyethyl)-4,9,10,12,16-pentamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-

[0110] Pentaazacyclodocosan-6-yl)-2-methylpropyl(2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamidopentanoate (B2). HRMS; MH+ = 1595.7200, 2.25 min.

[0493] Example 1-3: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-((R)-1-methoxyethyl)-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl)- Synthesis of 2-methylpropyl(2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamidopentanoate (B3) [ka] Compound (B3) can be obtained using a procedure similar to that described in Example 1-1, except that in step 1, compound (A3) (from Example 3-3) is used instead of compound (A1).

[0494] Example 1-4: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-22-isopropyl-3-((R)-1-methoxyethyl)-4,9,10,12,16-pentamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosa Synthesis of (2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamidopentanoate (B4) [ka] Compound (B4) was obtained using a procedure similar to that described in Example 1-1, except that in step 2, (S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS# 1949793-46-7) was used instead of (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS# is 2055041-37-5). HRMS; MH+ = 1594.5400, 2.88 min.

[0495] Example 1-5: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-((R)-1-methoxyethyl)-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosane-6 Synthesis of (2S,3R)-2-acetamido-3-((((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methylpentanoate (B5) [ka] Compound (B5) can be obtained using the same procedure as described in Example 1-1, except that in step 1, compound (A2) (from Example 3-2) is used instead of compound (A1), and in step 2, (S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-2- ... (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS# is 2055041-37-5) is used instead of (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS# is 2055041-37-5).

[0496] Example 1-6: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-((R)-1-methoxyethyl)-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosane-6- Synthesis of (2S,3R)-3-(((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamidopentanoate (B6) [ka] Compound (B6) can be obtained using the same procedure as described in Example 1-1, except that in step 1, compound (A3) (from Example 3-3) is used instead of compound (A1), and in step 2, (S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-2- ... (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS# is 2055041-37-5) is used instead of (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS# is 2055041-37-5).

[0497] Example 1-7: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-22-isopropyl-3-((R)-1-methoxyethyl)-4,9,10,12,16-pentamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosane Synthesis of (4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)oxy)-4-methyl-2-propionamidopentanoate (B7) [ka] Compound (B7) can be obtained by reacting chloroformate (1-3) with (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS# is 2055041-37-5). Chloroformate (1-3) can be obtained by reacting compound (A1) with phosgene.

[0498] Example 1-8: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-((R)-1-methoxyethyl)-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosane-6 Synthesis of (2S,3R)-2-acetamido-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)oxy)-4-methylpentanoate (B8) [ka] Compound (B8) can be obtained by the method described in Examples 1-7, except that compound (A2) is used in place of compound (A1).

[0499] Example 1-9: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-((R)-1-methoxyethyl)-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosane-6- Synthesis of (2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)oxy)-4-methyl-2-propionamidopentanoate (B9) [ka] Compound (B9) can be obtained by the method described in Examples 1-7, except that compound (A3) is used in place of compound (A1).

[0500] Example 1-10: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-22-isopropyl-3-((R)-1-methoxyethyl)-4,9,10,12,16-pentamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclo Synthesis of docosan-6-yl)-2-methylpropyl(2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)oxy)-4-methyl-2-propionamidopentanoate (B10) [ka] Compound (B10) can be obtained using the method described in Examples 1-7, except that (S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS# 1949793-46-7) is replaced with (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS# is 2055041-37-5).

[0501] Example 1-11: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-((R)-1-methoxyethyl)-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodextrin Synthesis of (2S,3R)-2-acetamido-3-((((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)oxy)-4-methylpentanoate (B11) [ka] Compound (B11) can be obtained by the method described in Examples 1-10, except that compound (A2) is used in place of compound (A1).

[0502] Example 1-12: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-((R)-1-methoxyethyl)-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosa Synthesis of (2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)oxy)-4-methyl-2-propionamidopentanoate (B12) [ka] Compound (B12) can be obtained by the method described in Examples 1-10, except that compound (A3) is used in place of compound (A1).

[0503] Example 2: Generation of anti-PMEL17 antibodies Example 2-1: Preparation of cell lines expressing PMEL17 Full-length human, cynomolgus monkey, and rat PMEL17 genes were synthesized based on amino acid sequences from the GenBank or Uniprot databases. All synthetic DNA fragments were cloned into appropriate expression vectors.

[0504] Engineering Stable PMEL17-expressing cell lines were generated and cultured under appropriate selective conditions to produce stable PMEL17-expressing cell lines.

[0505] Example 2-2: Whole cell panning against PMEL17 The phagemid library is based on the HuCAL PLATINUM® (Knappik et al., 2000) and Ylanthia concepts (Tiller et al., 2013) and uses CysDisplay™ technology (Lohning, 2001) for the display of Fabs on the phage surface.

[0506] For each panning, approximately 4 x 10 13 HuCAL PLATINUM® or approximately 1 x 10 14 In parallel, 0.5-1.0 x 10 Ylanthia® phage antibodies expressing the antigen PMEL17 were injected per phage pool. 7 0.5-1.0 x 10 target cells and no expression of the antigen PMEL17 7Adsorbed cells were resuspended in 1 ml of PBS / 5% FCS for blocking on ice. Blocked target cells were spun down, resuspended in preblocked phage particles, and incubated for 2 hours at 4°C on a rotator. Phage-cell complexes were washed three times in PBS / 5% FCS. Elution of specifically bound phage from target cells was performed by acidic elution using 0.1 M glycine-HCl / 0.5 M NaCl, pH 2.2, for 10 minutes. After centrifugation, the supernatant (eluate) was neutralized by adding 2 M unbuffered Tris. To remove phage binding to cell surface molecules other than the target antigen, post-adsorption was performed at 0.5–1.0 × 10 cells, respectively. 7 Three replicates were performed using 10 ...

[0507] Example 2-3: Subcloning, expression and screening of Fab fragments To facilitate rapid expression of soluble Fab, the Fab-encoding insert of the selected HuCAL PLATINUM® phage was subcloned from the pMORPH®30 display vector into the pMORPH®x11_FH expression vector. Subcloning was performed by triple digestion with EcoRI, XbaI, and BmtI. After transformation of E. coli TG1-F, single clone expression and preparation of periplasmic extracts containing HuCAL®-Fab fragments were performed as previously described (Rauchenberger et al., 2003).

[0508] The Fab-encoding inserts of selected Ylanthia® phages were subcloned from the pYPdis10 display vector into the pYBex10_Fab_FH expression vector. Subcloning was performed by triple digestion with XbaI, EcoRI-HF, and PstI-HF. After transformation of E. coli TG1-F, single clone expression and preparation of periplasmic extracts containing Ylanthia®-Fab fragments were perform...

Claims

1. An antibody or antigen-binding fragment thereof that binds to human PMEL17 protein, a. a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (CDR1) of SEQ ID NO: 79, 82, 83, or 85, a heavy chain complementarity determining region 2 (CDR2) of SEQ ID NO: 80, 84, or 86, and a heavy chain complementarity determining region 3 (CDR3) of SEQ ID NO: 81 or 87; and a light chain variable region (VL) comprising a light chain CDR1 of SEQ ID NO: 92, 95, or 98, a light chain CDR2 of SEQ ID NO: 93 or 96, and a light chain CDR3 of SEQ ID NO: 94 or 97; b. A VH comprising a heavy chain CDR1 of SEQ ID NO: 79, a heavy chain CDR2 of SEQ ID NO: 80, and a heavy chain CDR3 of SEQ ID NO: 81; and a VL comprising a light chain CDR1 of SEQ ID NO: 92, a light chain CDR2 of SEQ ID NO: 93, and a light chain CDR3 of SEQ ID NO: 94; c. A VH comprising a heavy chain CDR1 of SEQ ID NO: 82, a heavy chain CDR2 of SEQ ID NO: 80, and a heavy chain CDR3 of SEQ ID NO: 81; and a VL comprising a light chain CDR1 of SEQ ID NO: 92, a light chain CDR2 of SEQ ID NO: 93, and a light chain CDR3 of SEQ ID NO: 94; d. a VH comprising a heavy chain CDR1 of SEQ ID NO: 83, a heavy chain CDR2 of SEQ ID NO: 84, and a heavy chain CDR3 of SEQ ID NO: 81; and a VL comprising a light chain CDR1 of SEQ ID NO: 95, a light chain CDR2 of SEQ ID NO: 96, and a light chain CDR3 of SEQ ID NO: 97; or e. A VH comprising a heavy chain CDR1 of SEQ ID NO: 85, a heavy chain CDR2 of SEQ ID NO: 86, and a heavy chain CDR3 of SEQ ID NO: 87; and a VL comprising a light chain CDR1 of SEQ ID NO: 98, a light chain CDR2 of SEQ ID NO: 96, and a light chain CDR3 of SEQ ID NO:

94.

1. An antibody or antigen-binding fragment thereof comprising:

2. An antibody or antigen-binding fragment thereof that binds to human PMEL17 protein, comprising a VH having the amino acid sequence of SEQ ID NO: 88 and a VL having the amino acid sequence of SEQ ID NO:

99.

3. An antibody or antigen-binding fragment thereof that binds to human PMEL17 protein, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 90 and a light chain having the amino acid sequence of SEQ ID NO:

101.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof comprises one or more cysteine ​​substitutions in the constant region of the antibody or antigen-binding fragment thereof.

5. 5. The antibody or antigen-binding fragment thereof of claim 4, wherein the one or more cysteine ​​substitutions are selected from E152C, S375C, or both E152C and S375C in the heavy chain of the antibody or antigen-binding fragment thereof, and the one or more positions are numbered according to the EU system.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is a monoclonal antibody.

7. Formula (C): Ab-(L Ａ -(D) ｎ ) ｙ (C) (In the formula, D is a guanine nucleotide binding protein G(q) subunit alpha (GNAQ) inhibitor, a guanine nucleotide binding protein subunit alpha-11 (GNA11) inhibitor, or an inhibitor of GNAQ and GNA11; Ab is an antibody or an antigen-binding fragment thereof that binds to the human PMEL17 protein of any one of claims 1 to 6; L Ａ is a linker; n is 1, 2, 3 or 4; y is 1, 2, 3 or 4 10. An antibody-drug conjugate comprising:

8. The antibody-drug conjugate of claim 7, wherein n is 1.

9. The antibody-drug conjugate of claim 7 or 8, wherein y is 2.

10. The antibody-drug conjugate of any one of claims 7 to 9, wherein the linker is a cleavable linker or a non-cleavable linker.

11. The antibody-drug conjugate of claim 10 , wherein the linker comprises a ValCit peptide linker.

12. The antibody-drug conjugate of any one of claims 7 to 11, wherein D is an inhibitor of GNAQ and GNA11.

13. D is 【Chemical 1】 The antibody-drug conjugate according to any one of claims 7 to 11, wherein

14. D is 【Chemistry 2】 The antibody-drug conjugate according to any one of claims 7 to 11, wherein

15. The following structure 【Chemistry 3】 The antibody-drug conjugate of any one of claims 7 to 13, having the formula:

16. The following structure 【Chemistry 4】 The antibody-drug conjugate of any one of claims 7 to 12 or 14, having the following structure:

17. The following formula (C-2): 【Chemistry 5】 (In the formula, R ０ is methyl or ethyl; R １ is methyl or isopropyl; R ２ is methyl or ethyl; Ab is an antibody or an antigen-binding fragment thereof that binds to the human PMEL17 protein of any one of claims 1 to 6; X １ is a divalent linking group; X ２ is a self-immolative spacer; L １ is a bivalent peptide linker; L ２ is a bond or linker, y is 1, 2, 3 or 4 1. An antibody-drug conjugate comprising:

18. A pharmaceutical composition comprising the antibody of any one of claims 1 to 6, or an antigen-binding fragment thereof, or the antibody-drug conjugate of any one of claims 7 to 17, and a pharmaceutically acceptable carrier.

19. 20. The pharmaceutical composition of claim 18 for use in treating or preventing cancer in a patient in need of treatment, wherein the cancer expresses PMEL17, contains a mutation in the GNAQ gene or the GNA11 gene, or expresses PMEL17 and contains a mutation in the GNAQ gene, the GNA11 gene, or both the GNAQ and GNA11 genes.

20. 20. The pharmaceutical composition of claim 19, wherein the composition is administered to a patient in combination with one or more additional therapeutic compounds.

21. 21. The pharmaceutical composition of claim 20, wherein the one or more additional therapeutic compounds are selected from a standard of care chemotherapeutic agent, an MDM2 inhibitor, an MRC2 inhibitor, a PKC inhibitor, a MAPK inhibitor, a costimulatory molecule, or a checkpoint inhibitor.

22. 22. The pharmaceutical composition of claim 21, wherein the costimulatory molecule is selected from OX40, CD2, CD27, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, STING, or an agonist of a CD83 ligand.

23. 22. The pharmaceutical composition of claim 21, wherein the checkpoint inhibitor is selected from an inhibitor of PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and / or TGFR beta.

24. The pharmaceutical composition according to any one of claims 19 to 23, wherein the cancer is uveal melanoma, subcutaneous melanoma, hepatocellular carcinoma, or a metastatic cancer thereof.

25. A nucleic acid encoding the antibody, or antigen-binding fragment thereof, of any one of claims 1 to 6.

26. 26. The nucleic acid of claim 25, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 91 or 102.

27. A vector comprising the nucleic acid of claim 25 or 26.

28. 28. A host cell comprising a nucleic acid according to claim 25 or 26, or a vector according to claim 27.

29. 30. A method for producing an antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 28 and recovering the antibody or antigen-binding fragment thereof from the cell culture.

30. recovering the antibody or antigen-binding fragment thereof from the cell culture. a) removing cells and filtering the culture; b) purifying the culture by affinity chromatography; c) inactivating any viruses in the cell culture by adjusting the pH to 3.4-3.6, then readjusting the pH to 5.8-6.2 and filtering the culture; d) purifying the culture by cation exchange chromatography and performing on-column reduction of the cell culture supernatant; e) subjecting the cell culture to anion exchange chromatography; f) removing viruses by nanofiltration; g) filtering the cell culture containing the antibody or antigen-binding fragment thereof; and h) Obtaining the purified antibody or antigen-binding fragment thereof 30. The method of claim 29, comprising:

31. 1. A method for producing an anti-PMEL17 antibody drug conjugate, comprising: (a) Formula (B) below: R ８ -L Ｂ -(D) ｎ (B) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of GNAQ and GNA11; R ８ is a reactive group; L Ｂ is a cleavable or non-cleavable linker, n is 1, 2, 3 or 4 preforming the linker-drug moiety of (b) attaching the linker-drug moiety to the antibody or antigen-binding fragment thereof recovered from the cell culture as claimed in claim 29 or 30 to produce an antibody drug conjugate; and (c) purifying the antibody-drug conjugate. A method comprising:

32. A diagnostic reagent comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

33. 33. The diagnostic reagent of claim 32, wherein the antibody or antigen-binding fragment thereof is labeled with a radiolabel, a fluorescent label, a chromophore, an imaging agent, or a metal ion.

34. The following structure: 【Chemistry 6】 Including; Here, Ab is a. A VH comprising a heavy chain CDR1 of SEQ ID NO:79, a heavy chain CDR2 of SEQ ID NO:80, and a heavy chain CDR3 of SEQ ID NO:81; and a VL comprising a light chain CDR1 of SEQ ID NO:92, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:94; b. A VH comprising a heavy chain CDR1 of SEQ ID NO: 82, a heavy chain CDR2 of SEQ ID NO: 80, and a heavy chain CDR3 of SEQ ID NO: 81; and a VL comprising a light chain CDR1 of SEQ ID NO: 92, a light chain CDR2 of SEQ ID NO: 93, and a light chain CDR3 of SEQ ID NO: 94; c. A VH comprising a heavy chain CDR1 of SEQ ID NO: 83, a heavy chain CDR2 of SEQ ID NO: 84, and a heavy chain CDR3 of SEQ ID NO: 81; and a VL comprising a light chain CDR1 of SEQ ID NO: 95, a light chain CDR2 of SEQ ID NO: 96, and a light chain CDR3 of SEQ ID NO: 97; or d. A VH comprising a heavy chain CDR1 of SEQ ID NO: 85, a heavy chain CDR2 of SEQ ID NO: 86, and a heavy chain CDR3 of SEQ ID NO: 87; and a VL comprising a light chain CDR1 of SEQ ID NO: 98, a light chain CDR2 of SEQ ID NO: 96, and a light chain CDR3 of SEQ ID NO:

94. and y is 2, an antibody or an antigen-binding fragment thereof that binds to human PMEL17 protein; Antibody drug conjugates.

35. The following structure: 【Chemistry 7】 Including; Here, Ab is comprising a VH comprising the amino acid sequence of SEQ ID NO: 88 and a VL comprising the amino acid sequence of SEQ ID NO: 99; and y is 2, an antibody or an antigen-binding fragment thereof that binds to human PMEL17 protein; Antibody drug conjugates.

36. The following structure: 【Chemistry 8】 wherein the Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 90 or the amino acid sequence of SEQ ID NO: 90 except that position 375, numbered according to the EU system, is S, and a light chain comprising the amino acid sequence of SEQ ID NO: 101; and y is 2, an antibody or an antigen-binding fragment thereof that binds to human PMEL17 protein; Antibody drug conjugates.

37. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 34 to 36 and a pharmaceutically acceptable carrier.

38. 38. The pharmaceutical composition of claim 37 for use in treating or preventing cancer in a patient in need of treatment, wherein the cancer expresses PMEL17, contains a mutation in the GNAQ gene or the GNA11 gene, or expresses PMEL17 and contains a mutation in the GNAQ gene, the GNA11 gene, or both the GNAQ gene and the GNA11 gene.

Images