Antibodies and conjugates for PMEL17

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 List This application includes a sequence listing submitted electronically in ASCII format and incorporated herein by reference in its entirety. The ASCII copy was created on 6 December 2019, named PAT058359-WO-PCT_SL.txt, and is 285,253 bytes in size.

[0002] The present invention generally relates to anti-PMEL17 antibodies, or fragments thereof, their conjugates, for example, their GNAQ / GNA11 inhibitor conjugates, 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 maturation, PMEL17 is transiently expressed on the cell surface before being trafficked into melanosomes, where it is degraded into various domains, which then multimerize to form fibrous sheets. These patterns then function as a support for capturing melanin. Melanosome PMEL17 expression has been found to be regulated by the lineage oncogene MITF and upregulated in various primary and metastatic subcutaneous and uveal melanomas. 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 PMEL17 is extensively processed by proprotein convertases during its maturation. The protein is cleaved between V467 / K468, forming two subdomains, Mα at the N-terminus and Mβ at the C-terminus, which are thought to be maintained via disulfide crosslinks. Some PMEL17 molecules are transiently expressed on the cell surface after leaving the Golgi apparatus. Subsequently, most PMEL17 is thought to be redirected to melanocytes for further maturation, while some PMEL17 is reduced. After additional enzymatic cleavage, PMEL17 is degraded into various domains, which reorganize to form fibrous sheets on which melanin polymerizes (Non-Patent Literature 1; Non-Patent Literature 2).

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

[0006] GNAQ / GNA11 and cancer The GNAQ and GNA11 genes are nearly ubiquitously expressed and encode the alpha subunit of the heterotrimeric G protein Gq / 11, which acts as a binary molecular switch that cycles 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 numerous signaling pathways via the generation of second messengers IP3 and DAG. Signaling termination is induced by GTP hydrolysis mediated by the intrinsic GTPase activity of their Gα proteins. Gq and G11 have been shown to be involved in a wide range of physiological functions, e.g., 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 fixation of GNAQ / 11 to its active state. Q209 GNAQ / 11 is a preferential-acting oncogene that transforms melanocytes by inducing activation of multiple pathways, e.g., PKC / MAPK, Rho / Rac, β-catenin, and YAP. While the PKC / MAPK pathway has been shown as one contributing factor to GNAQ-mediated oncogenesis, multiple lines of evidence suggest that mutated GNAQ / 11 may also control additional pathways (i.e., YAP, β-catenin) that likely play a similar role in UM tumorigenesis. Interestingly, another somatically activated mutation in GNAQ (R183Q) has recently been described as the cause of Sturge-Weber syndrome (SWS), a neurocutaneous disorder characterized by capillary malformations (port-wine spots) as well as choroidal and leptomeningeal vascular malformations. Therefore, GNAQ and GNA11 constitute potential therapeutic targets for the treatment of uveal and cutaneous melanomas.

[0008] Antibody-drug conjugates Antibody-drug conjugates ("ADCs") are used for the local delivery of cytotoxic agents in cancer treatment (see, for example, Non-Patent Document 6). ADCs enable targeted delivery of the drug portion, achieving maximum efficacy with minimal toxicity. An ADC contains an antibody conjugated to a drug selected for its ability to bind to cells targeted for therapeutic intervention and for its ability to inhibit cell proliferation or for its cytotoxic activity. Thereafter, the binding of the antibody to the targeted cells delivers the drug to the site where its therapeutic effect is needed.

[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 influence the therapeutic efficacy of ADCs (beyond target-specific characteristics) include various aspects requiring customized fine-tuning, such as optimal antibody affinity as a balance between target-mediated disposition (TMDD) and the exposure that drives efficacy, evaluation of Fc-mediated function (antibody-dependent cell-mediated cytotoxicity, ADCC), method of binding (site-specific or non-site-specific), drug / payload ratio ("DAR" or "drug-antibody ratio") that binds to each antibody, linker cleavage or stability, ADC stability, and ADC aggregation tendency.

[0010] Antibodies, application methods, and cytotoxic payloads with improved properties for use as effective ADC therapeutic compositions and methods are still needed. [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

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[0012] In one embodiment, the present application relates to an antibody or antigen-binding fragment thereof that binds to PMEL17, a. Heavy chain variable regions including heavy chain CDR1 (complementarity determination region 1) of SEQ ID NO: 1, 4, 5, or 7, heavy chain CDR2 (complementarity determination region 2) of SEQ ID NO: 2, 6, or 8, and heavy chain CDR3 (complementarity determination region 3) of SEQ ID NO: 3 or 9; and light chain variable regions including light chain CDR1 (complementarity determination region 1) of SEQ ID NO: 14, 17, or 20, light chain CDR2 (complementarity determination region 2) of SEQ ID NO: 15 or 18, and light chain CDR3 (complementarity determination region 3) of SEQ ID NO: 16 or 19; b. Heavy chain variable region including heavy chain CDR1 of SEQ ID NOs. 33, 36, 37, or 39, heavy chain CDR2 of SEQ ID NOs. 34, 38, or 40; heavy chain CDR3 of SEQ ID NOs. 35 or 41; light chain CDR1 of SEQ ID NOs. 46, 49, or 52; light chain CDR2 of SEQ ID NOs. 47 or 50; and light chain CDR3 of SEQ ID NOs. 48 or 51; c. Heavy chain variable region including heavy chain CDR1 of SEQ ID NOs. 5, 7, 57, or 60; heavy chain CDR2 of SEQ ID NOs. 58, 61, or 62; heavy chain CDR3 of SEQ ID NOs. 59 or 63; light chain CDR1 of SEQ ID NOs. 68, 71, or 74; light chain CDR2 of SEQ ID NOs. 69 or 72; and light chain CDR3 of SEQ ID NOs. 70 or 73; d. Heavy chain variable region including heavy chain CDR1 of SEQ ID NOs. 79, 82, 83, or 85; heavy chain CDR2 of SEQ ID NOs. 80, 84, or 86; heavy chain CDR3 of SEQ ID NOs. 81 or 87; light chain CDR1 of SEQ ID NOs. 92, 95, or 98; light chain CDR2 of SEQ ID NOs. 93 or 96; and light chain CDR3 of SEQ ID NOs. 94 or 97; e. Heavy chain variable region including heavy chain CDR1 of SEQ ID NOs. 103, 106, 107, or 109; heavy chain CDR2 of SEQ ID NOs. 104, 108, or 110; heavy chain CDR3 of SEQ ID NOs. 105 or 111; light chain CDR1 of SEQ ID NOs. 49, 52, or 116; light chain CDR2 of SEQ ID NOs. 47 or 50; and light chain CDR3 of SEQ ID NOs. 117 or 118; f. Heavy chain variable region including heavy chain CDR1 of SEQ ID NOs. 123, 126, 127, or 129; heavy chain CDR2 of SEQ ID NOs. 124, 128, or 130; heavy chain CDR3 of SEQ ID NOs. 125, or 131; light chain CDR1 of SEQ ID NOs. 136, 139, or 142; light chain CDR2 of SEQ ID NOs. 137, or 140; and light chain CDR3 of SEQ ID NOs. 138, or 141; g. Heavy chain variable region including heavy chain CDR1 of SEQ ID NOs. 123, 126, 127, or 129, heavy chain CDR2 of SEQ ID NOs. 124, 128, or 130; heavy chain CDR3 of SEQ ID NOs. 147 or 148; light chain CDR1 of SEQ ID NOs. 153, 156, or 158; light chain CDR2 of SEQ ID NOs. 50 or 154; and light chain CDR3 of SEQ ID NOs. 155 or 157; h. Heavy chain variable region including heavy chain CDR1 of SEQ ID NOs. 103, 106, 107, or 109; heavy chain CDR2 of SEQ ID NOs. 104, 108, or 110; heavy chain CDR3 of SEQ ID NOs. 163 or 164; light chain CDR1 of SEQ ID NOs. 49, 52, or 116; light chain CDR2 of SEQ ID NOs. 47 or 50; and light chain CDR3 of SEQ ID NOs. 169 or 170; i. Heavy chain variable region including heavy chain CDR1 of SEQ ID NOs. 175, 178, 179 or 181, heavy chain CDR2 of SEQ ID NOs. 176, 180 or 182; heavy chain CDR3 of SEQ ID NOs. 177 or 183; light chain CDR1 of SEQ ID NOs. 49, 52 or 116; light chain CDR2 of SEQ ID NOs. 47 or 50; and light chain CDR3 of SEQ ID NOs. 188 or 189; j. Heavy chain variable region including heavy chain CDR1 of SEQ ID NOs. 103, 106, 107, or 109; heavy chain CDR2 of SEQ ID NOs. 104, 108, or 110; heavy chain CDR3 of SEQ ID NOs. 194 or 195; light chain CDR1 of SEQ ID NOs. 49, 52, or 116; light chain CDR2 of SEQ ID NOs. 47 or 50; and light chain CDR3 of SEQ ID NOs. 200 or 201; k. Heavy chain variable region including heavy chain CDR1 of SEQ ID NOs. 206, 209, 210, or 212; heavy chain CDR2 of SEQ ID NOs. 207, 211, or 213; heavy chain CDR3 of SEQ ID NOs. 208 or 214; light chain CDR1 of SEQ ID NOs. 153, 156, or 158; light chain CDR2 of SEQ ID NOs. 50 or 154; and light chain CDR3 of SEQ ID NOs. 219 or 220; l. Heavy chain variable region including heavy chain CDR1 of SEQ ID NOs. 206, 209, 210, or 212; heavy chain CDR2 of SEQ ID NOs. 207, 211, or 213; heavy chain CDR3 of SEQ ID NOs. 225, or 226; light chain CDR1 of SEQ ID NOs. 136, 139, or 142; light chain CDR2 of SEQ ID NOs. 137, or 140; and light chain CDR3 of SEQ ID NOs. 231, or 232; m. Heavy chain variable region including a heavy chain variable region containing HCDR1 of sequence number 206, 209, 210, or 212, HCDR2 of sequence number 207, 211, or 213, and HCDR3 of sequence number 237 or 238; and light chain variable region including LCDR1 of sequence number 243, 245, or 247, LCDR2 of sequence number 47 or 50, and LCDR3 of sequence number 244 or 246; n. Heavy chain variable region including a heavy chain variable region containing HCDR1 of sequence number 206, 209, 210, or 212, HCDR2 of sequence number 207, 211, or 213, and HCDR3 of sequence number 252 or 253; and light chain variable region including LCDR1 of sequence number 153, 156, or 158, LCDR2 of sequence number 50 or 154, and LCDR3 of sequence number 258 or 259; o. Heavy chain CDR1 of SEQ ID NO: 1, 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; 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. Heavy chain CDR1 of SEQ ID NO: 5, heavy chain CDR2 of SEQ ID NO: 6, heavy chain CDR3 of SEQ ID NO: 3, light chain CDR1 of SEQ ID NO: 17, light chain CDR2 of SEQ ID NO: 18, and light chain CDR3 of SEQ ID NO: 19; r. 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; s. Heavy chain CDR1 of SEQ ID NO: 33, heavy chain CDR2 of SEQ ID NO: 34, heavy chain CDR3 of SEQ ID NO: 35, light chain CDR1 of SEQ ID NO: 46, light chain CDR2 of SEQ ID NO: 47, and light chain CDR3 of SEQ ID NO: 48; t. Heavy chain CDR1 of SEQ ID NO: 36, heavy chain CDR2 of SEQ ID NO: 34, heavy chain CDR3 of SEQ ID NO: 35, light chain CDR1 of SEQ ID NO: 46, light chain CDR2 of SEQ ID NO: 47, and light chain CDR3 of SEQ ID NO: 48; u. Heavy chain CDR1 of SEQ ID NO: 37, heavy chain CDR2 of SEQ ID NO: 38, heavy chain CDR3 of SEQ ID NO: 35, light chain CDR1 of SEQ ID NO: 49, light chain CDR2 of SEQ ID NO: 50, and 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. Heavy chain CDR1 of SEQ ID NO: 57, heavy chain CDR2 of SEQ ID NO: 58, heavy chain CDR3 of SEQ ID NO: 59, light chain CDR1 of SEQ ID NO: 68, light chain CDR2 of SEQ ID NO: 69, and light chain CDR3 of SEQ ID NO: 70; x. Heavy chain CDR1 of SEQ ID NO: 60, heavy chain CDR2 of SEQ ID NO: 58, heavy chain CDR3 of SEQ ID NO: 59, light chain CDR1 of SEQ ID NO: 68, light chain CDR2 of SEQ ID NO: 69, and light chain CDR3 of SEQ ID NO: 70; y. Heavy chain CDR1 of SEQ ID NO: 5, heavy chain CDR2 of SEQ ID NO: 61, heavy chain CDR3 of SEQ ID NO: 59, light chain CDR1 of SEQ ID NO: 71, light chain CDR2 of SEQ ID NO: 72, and 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. Heavy chain CDR1 of SEQ ID NO: 123, 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; 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. Heavy chain CDR1 of SEQ ID NO: 129, heavy chain CDR2 of SEQ ID NO: 130, heavy chain CDR3 of SEQ ID NO: 131, light chain CDR1 of SEQ ID NO: 142, light chain CDR2 of SEQ ID NO: 140, and 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. Heavy chain CDR1 of SEQ ID NO: 103, 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; 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. Heavy chain CDR1 of SEQ ID NO: 107, heavy chain CDR2 of SEQ ID NO: 108, heavy chain CDR3 of SEQ ID NO: 163, light chain CDR1 of SEQ ID NO: 49, light chain CDR2 of SEQ ID NO: 50, and 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. Heavy chain CDR1 of SEQ ID NO: 103, 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; 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. Heavy chain CDR1 of SEQ ID NO: 210, heavy chain CDR2 of SEQ ID NO: 211, heavy chain CDR3 of SEQ ID NO: 225, light chain CDR1 of SEQ ID NO: 139, light chain CDR2 of SEQ ID NO: 140, and 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. Heavy chain variable region including HCDR1 of SEQ ID NO: 206, HCDR2 of SEQ ID NO: 207, and HCDR3 of SEQ ID NO: 237, and light chain variable region including LCDR1 of SEQ ID NO: 243, LCDR2 of SEQ ID NO: 47, and LCDR3 of SEQ ID NO: 244; III. Heavy chain variable region including HCDR1 of SEQ ID NO: 209, HCDR2 of SEQ ID NO: 207, and HCDR3 of SEQ ID NO: 237, and light chain variable region including LCDR1 of SEQ ID NO: 243, LCDR2 of SEQ ID NO: 47, and LCDR3 of SEQ ID NO: 244; mmm. Heavy chain variable region including HCDR1 of SEQ ID NO: 210, HCDR2 of SEQ ID NO: 211, and HCDR3 of SEQ ID NO: 237, and light chain variable region including LCDR1 of SEQ ID NO: 245, LCDR2 of SEQ ID NO: 50, and LCDR3 of SEQ ID NO: 246; nnn. Heavy chain variable region including HCDR1 of SEQ ID NO: 212, HCDR2 of SEQ ID NO: 213, and HCDR3 of SEQ ID NO: 238; and light chain variable region including LCDR1 of SEQ ID NO: 247, LCDR2 of SEQ ID NO: 50, and LCDR3 of SEQ ID NO: 244; ooo. A heavy chain variable region including HCDR1 of sequence number 206, HCDR2 of sequence number 207, and HCDR3 of sequence number 252, and a light chain variable region including LCDR1 of sequence number 153, LCDR2 of sequence number 154, and LCDR3 of sequence number 258; ppp. Heavy chain variable region including HCDR1 of SEQ ID NO: 209, HCDR2 of SEQ ID NO: 207, and HCDR3 of SEQ ID NO: 252, and light chain variable region including LCDR1 of SEQ ID NO: 153, LCDR2 of SEQ ID NO: 154, and LCDR3 of SEQ ID NO: 258; qqq. A heavy chain variable region including HCDR1 of SEQ ID NO: 210, HCDR2 of SEQ ID NO: 211, and HCDR3 of SEQ ID NO: 252, and a light chain variable region including LCDR1 of SEQ ID NO: 156, LCDR2 of SEQ ID NO: 50, and LCDR3 of SEQ ID NO: 259; or rrr. Heavy chain variable region including HCDR1 of SEQ ID NO: 212, HCDR2 of SEQ ID NO: 213, and HCDR3 of SEQ ID NO: 253; and light chain variable region including LCDR1 of SEQ ID NO: 158, LCDR2 of SEQ ID NO: 50, and LCDR3 of SEQ ID NO: 258 Disclosed are antibodies or antigen-binding fragments thereof containing [the specified substance].

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

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

[0015] The antibodies or antigen-binding fragments described herein may contain one or more cysteine ​​substitutions. In one embodiment, the antibody or antigen-binding fragment contains one or more cysteine ​​substitutions in the heavy chain of the antibody or antigen-binding fragment, selected from S152C, S375C, or both S152C and S375C, the positions of which are 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 is of formula (C): Ab-(L A -(D) n ) y (C) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of both GNAQ and GNA11; Ab is an antibody or its antigen-binding fragment that binds to the human PMEL17 protein; L A It 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 an antibody or an antigen-binding fragment thereof) is a conjugate 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 of formula (C-1):

Chemical formula

Chemical formula

[0019] This application also discloses pharmaceutical compositions comprising the antibody disclosed herein, or its antigen-binding fragment, and a pharmaceutically acceptable carrier. This application also discloses pharmaceutical compositions comprising the antibody-drug conjugate and a pharmaceutically acceptable carrier disclosed herein.

[0020] The application also discloses a method for treating or preventing cancer in a patient requiring treatment or prevention of cancer, 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 methods for treating or preventing cancer, an antibody-drug conjugate or pharmaceutical composition is administered to a patient in combination with one or more additional therapeutic compounds. In one embodiment, one or more additional therapeutic compounds are selected from standard therapeutic chemotherapeutic agents, MDM2 inhibitors, MRC2 inhibitors, PKC inhibitors, MAPK inhibitors, costimulatory molecules, or checkpoint inhibitors. In one embodiment, the costimulatory molecule is selected from 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 agonists. In another embodiment, the checkpoint inhibitor is selected from the inhibitors PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and / or TGFR beta.

[0022] This application also discloses antibody-drug conjugates or pharmaceutical compositions disclosed herein for use as pharmaceuticals. In one embodiment, the antibody-drug conjugates or pharmaceutical compositions disclosed herein are for use in the treatment or prevention of PMEL17-expressing cancer or cancers containing mutations in the GNAQ or GNA11 genes in patients requiring treatment or prevention of such cancers.

[0023] In one embodiment, the application discloses the use of an antibody or its antigen-binding fragment, antibody-drug conjugate or pharmaceutical composition disclosed herein for treating or preventing PMEL17-expressing cancer in patients requiring treatment or prevention of PMEL17-expressing cancer.

[0024] In one embodiment, this application discloses the use of the antibodies or their antigen-binding fragments, antibody-drug conjugates, or pharmaceutical compositions disclosed herein for treating or preventing PMEL17-expressing cancer or cancer containing mutations in the GNAQ or GNA11 genes in patients requiring treatment or prevention of cancer. In one embodiment, this application discloses the use of the antibodies or their antigen-binding fragments, antibody-drug conjugates, or pharmaceutical compositions disclosed herein in the manufacture of pharmaceuticals.

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

[0026] This application also discloses nucleic acids encoding antibodies or antigen-binding fragments disclosed herein. In one embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NOs: 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. This application also discloses vectors comprising nucleic acids, and host cells comprising vectors or nucleic acids. This application also discloses a method for producing antibodies or antigen-binding fragments disclosed herein, comprising culturing host cells and recovering antibodies from the cell culture. In one embodiment, the method for recovering antibodies from a cell culture is: a) Remove cells and filter the culture; b) A step of purifying the culture by affinity chromatography; c) Inactivate all viruses in the culture by adjusting the pH to 3.4-3.6, then readjust the pH to 5.8-6.2 and filter the culture; d) A step in which the culture is purified by cation exchange chromatography and the culture is subjected to on-column reduction; e) Performing anion exchange chromatography on the culture; f) A step of removing the virus by nanofiltration; g) filtering the culture containing the antibody; and h) Step to obtain purified antibodies Includes.

[0027] This application also relates to a method for producing an anti-PMEL17 antibody-drug conjugate, (a) The following equation (B): R 8 -L B -(D) n (B) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of both GNAQ and GNA11; R 8 is a reactive group; L B These are cleavable or non-cleavable linkers, n is 1, 2, 3, or 4. Pre-forming the linker-drug portion; (b) Conjugating the linker-drug portion to an antibody recovered from a cell culture using a method for producing an antibody or antigen-binding fragment as disclosed herein to produce an antibody-drug conjugate; and (c) Purify the antibody-drug conjugate. A method including the following is disclosed.

[0028] This application also discloses diagnostic reagents comprising an antibody or its antigen-binding fragment disclosed herein. In some embodiments, the antibody or its antigen-binding fragment is labeled with radiolabeling, fluorescent labeling, a chromophore, an imaging agent, or a metal ion. [Brief explanation of the drawing]

[0029] [Figure 1] Exemplary data on the in vitro anti-UM activity of GNAQ / 11 inhibitor compounds (A1) and (A2) are shown. [Figure 2] Exemplary data on the activity of GNAQ / 11 inhibitor compounds (A1) and (A2) in inducing apoptosis in uveal melanoma cells are presented. [Figure 3] Exemplary data on GNAQ / 11 inhibition by compound (A1) and compound (A2) are shown. Compounds (A1) and (A2) reduced IP1 levels (Figure 3A) and relative proliferation rate (Figure 3B) in 92.1 cells. Immunoblots of 92.1 cells treated with compound (A1) and (A2) showed reduced ERK signaling (Figure 3C). [Figure 4]Exemplary data regarding the metabolic stability and PK properties of compound (A1) are shown. Both the disappearance of compound (A1) (Figure 4A) and the appearance of the ring-opened compound (A8) (Figure 4B) were monitored over 24 hours. Except in rats, the sum of % residual compound (A1) and % formed compound (A8) showed stoichiometric values ​​over 24 hours (Figure 4C). The PK of compound (A1) after intravenous administration in mice was characterized by extremely high clearance and moderate to high volume of distribution (Figure 4D). [Figure 5] Exemplary data regarding the metabolic stability and PK properties of compounds (A1) and (A2) are shown. The in vitro stability of compound (A2) was tested in plasma and blood from different species (Figure 5A). Compound (A2) showed good chemical stability in three different systems (Figure 5B). The PK of compound (A2) in female balb / c mice showed high clearance and a short excretion half-life (Figure 5C). Compounds (A1) and (A2) were stable for 4 hours in buffer at pH 5.6 and in lysosomes (Figure 5D). [Figure 6] Exemplary data on the in vitro anti-uveal melanoma activity of anti-PMEL17-(B1)ADC are presented. Data are presented as the mean of three independent replicates compared to PBS-treated cells (control). [Figure 7] Exemplary data on anti-PMEL17-(B1)ADCs that induce apoptosis in uveal melanoma cells are presented. Data are presented as the average of three independent replicates. [Figure 8] Exemplary data on the in vitro anti-uveal melanoma activity of anti-PMEL17-(B2)ADCs and anti-PMEL17 mAbs are presented. Data are presented as the mean of three independent replicates compared to PBS-treated cells (control). [Figure 9] Exemplary data on GNAQ / 11 inhibition by anti-PMEL17-(B1) and anti-PMEL17-(B2)ADCs in uveal melanoma cells are presented. IP1 levels (nM) are presented as the mean of three independent replicates. [Figure 10]Exemplary data on the binding activity of anti-PMEL17 antibodies to intact platelets and uveal melanoma cells are presented. [Figure 11] Exemplary data on the effects of compound (A1) and anti-PMEL17-(B1)ADC on human platelet aggregation are shown. [Figure 12] Exemplary data on the in vivo antitumor activity of anti-PMEL17-(B1)ADCs are shown. 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 at day 0 was approximately 200-250 mm3. No weight loss was observed until 14 days after treatment (Figure 12B). Values ​​are mean ± SEM; sample size (n=4 mice per group). G1-(B1) treatment resulted in inhibition of GNAQ signaling and tumor cell proliferation, as indicated by reductions in pERK and Ki67 levels, respectively (Figure 12C). Furthermore, G1-(B1) induced cell apoptosis compared to vehicle and isotype control 3207-(B1) treated mice, which correlated with tumor cell accumulation of G1-(B1)ADCs as detected by IgG staining (Figure 12C). No changes were observed at MITF and PMEL17 levels after GNAQ inhibition (Figure 12C). Platelet aggregation inhibition was not observed in G1-(B1) treated mice up to 7 days (Figures 12D and E). [Figure 13A] Exemplary data on the effects of G1-(B1)ADC on a mouse model of liver and lung metastases of uveal melanoma are presented. Individual photographs from each mouse are shown 45 days after IV injection of 92.1-luciferase cells (immediately before the start of treatment) and 12 days after treatment (Figures 13A and 13B); sample size, (n=6 mice per group). [Figure 13B]Exemplary data on the effects of G1-(B1)ADC on a mouse model of uveal melanoma with liver and lung metastases are presented. Individual photographs from each mouse are shown 45 days after IV injection of 92.1-luciferase cells (immediately before the start of treatment) and 12 days after treatment (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. Lung tumors (bioluminescence signal) in Figure 13B are indicated by black arrows. Corresponding weight change (%) relative to day 15 was evaluated 2-3 times per week before and after treatment with G1-(B1) 20 mg / kg (gray circles). [Figure 13C] The values ​​in Figure 13C are mean ± SEM; sample size (n=5-6 mice per group). The initial body weight on day 15 was approximately 21g. [Figure 14] Exemplary data regarding the PK properties of G1-(B1)ADC are shown. The pharmacokinetic profile (total IgG levels) of G1-(B1) showed a slightly more than positive increase with exposure to doses of 7.5 to 30 mg / kg in nude mice (Figure 14A). In tumor-bearing mice, free payload concentrations were measured after administration of either targeted-bound G1-(B1) or isotype control 3207-(B1). A clear (>4-fold) increase in tumor delivery of compound (A1) payload could be observed using targeted ADCs (Figure 14B). Conversion of the antibody-bound compound (A1) (white circles) to its ring-opened form, compound (A8) (black circles), was demonstrated in vivo in mice (Figure 14C). In an in vivo efficacy study comparing two different DAR2 formats with the G1-(B1) DAR4 format and the DAR4 Fc silent format, exposure to DAR2(E152C) and DAR4 Fc silent ADCs resulted in minimal clearance, while DAR2(S375C) exposure led to a faster decline (Figure 14D). Figure 14E shows the concentrations of 3207(isotype control antibody)-(B1)DAR4(E152C, S375C) and 3207(isotype control antibody)-(B1)DAR4 Fc silent conjugates over time. [Figure 15] Exemplary data on the in vitro stability of anti-PMEL17-GNAQ / 11i ADC in buffer, mouse, rat, and human plasma and the in vivo stability of anti-PMEL17-GNAQ / 11i ADC in mice are shown. [Figure 16] Exemplary data on the in vivo efficacy of G1-E152C-DAR2-(B1), G1-S375C-DAR2-(B1), and Fc silent G1-(B1) in a xenograft model of uveal melanoma are shown. Values represent mean ± SEM; sample size, (n = 5 - 6 mice per group). The initial tumor volume on day 0 was approximately 300 - 325 mm3. [Figure 17] Exemplary data on the in vitro anti-uveal melanoma activity of anti-PMEL17-(B1) ADC are shown. The data are presented as the mean of three independent replicates relative to PBS-treated cells (control). [Figure 18] Exemplary data on the in vivo anti-tumor activity of anti-PMEL17-(B1) ADC are shown. [Figure 19] Exemplary data on the immunohistochemical analysis of tumor biopsies from patients with metastatic uveal melanoma are shown. [Figure 20] Exemplary sensorgrams for evaluating epitope binding of anti-PMEL antibodies are shown. Figure 20A illustrates the binding step. Figure 20B shows the sensorgram when antibody G1 3J LC is immobilized first and 17A9 is flowed. Figure 20C shows the 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.

Mode for Carrying Out the Invention

[0030] Definitions Unless otherwise specified, the following terms and phrases used in this specification are intended to have the following meanings.

[0031] The term "alkyl" refers to a monovalent saturated hydrocarbon chain having a specific number of carbon atoms. For example, C1-C6 alkyl refers to an alkyl group having from 1 to 6 carbon atoms. The alkyl group may be straight-chain or branched-chain. Representative branched-chain alkyl groups have 1, 2, or 3 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 component that connects two portions by a covalent linkage but decomposes under physiologically relevant conditions to cleave the covalent linkage between the portions. Typically, a cleavable linking group is cleaved more rapidly in the intracellular environment than extracellularly in vivo, causing preferential generation of the released payload inside the targeted cell. Cleavage may be enzymatic or non-enzymatic but generally releases the payload without degrading the antibody. Cleavage may leave a portion of the linking group or linker component attached to the payload, or it may release the payload having no residues of the linking group.

[0033] As used herein, "non-cleavable" refers to a linking group or linker component that is not particularly sensitive to degradation under physiological conditions. For example, it is at least as stable as the antibody or antigen-binding fragment portion of the conjugate. Such linking groups may be referred to as "stable" and are sufficiently resistant to degradation to retain the payload linked to the antibody or antigen-binding fragment until the antibody or antigen-binding fragment is at least partially degraded itself, 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 from the antibody attached to the payload or drug portion delivered in vivo, e.g., one or more amino acid groups.

[0034] The term "antibody" refers to a polypeptide of the immunoglobulin family that can bind noncovalently, reversibly, and specifically to a corresponding antigen. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and 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 binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors (such as 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-idiotype (anti-Id) antibodies (for example, anti-Id antibodies against the antibodies of the present invention). Antibodies may 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 "complementarity-determining domain" or "complementarity-determining region" ("CDR") interchangeably refers to the hypervariable regions of the VL and VH. The CDR is the target protein binding site of the antibody chain, and it possesses specificity for such target proteins. Three CDRs (numbered sequentially from the N-terminus, CDR1-3) are present in each human VL or VH, constituting approximately 15-20% of the variable domain. Structurally, the CDR is complementary to the target protein epitope and therefore directly contributes to binding specificity. The remaining range of the VL or VH, the so-called framework region, shows little variation in its amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. WH Freeman & Co., New York, 2000).

[0037] The location of CDRs and framework areas can be determined using various well-known definitions in the relevant technical field, such as Kabat, Chothia, the International ImMunoGeneTics Database (IMGT) (www.imgt.org / on the World Wide Web), and AbM (see, for example, 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 antigen-binding sites is also described in the following: 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 the light and heavy chains are divided into structurally homologous and functionally homologous regions. The terms "constant" and "variable" are used functionally. In this regard, it goes without saying 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, and complement binding. According to notation rules, the numbering of constant region domains increases distal to 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 domain and CL domain actually contain the carboxyl-terminal domains of the heavy and light chains, respectively.

[0039] As used herein, the term “antigen-binding fragment” refers to one or more portions of an antibody that possess the ability to specifically interact with an antigen epitope (e.g., by binding, steric hindrance, stabilization / destabilization, or spatial distribution). Examples of binding fragments include, but are not limited to, single-chain Fv(scFv), camel antibodies, disulfide-crosslinked Fv(sdFv), Fab fragments, F(ab') fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by disulfide crosslinking at the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of single-arm VL and VH domains of an antibody; dAb fragments consisting of a 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 the Fv fragment, VL and VH, are encoded by separate genes, they can be linked together by a synthetic linker using recombination methods. This allows the two domains to be paired, with the VL and VH regions forming a single protein chain that creates a monovalent molecule (known as 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 included within the term "antigen-binding fragment." These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and such fragments are screened for practicality in the same manner as intact antibodies.

[0041] Antigen-binding fragments may also be incorporated into single-domain antibodies, maxibodies, minibodies, 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. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies).

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

[0043] As used herein, the terms “monoclonal antibody” or “monoclonal antibody composition” refer to polypeptides, including antibodies and antigen-binding fragments having substantially the same amino acid sequence or originating from the same genetic source. The term also includes preparations of antibody molecules with a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

[0044] As used herein, the term "human antibody" includes antibodies in which both the framework and CDR regions have variable regions derived from human sequences. Furthermore, if the antibody contains a constant region, the constant region also derives from a human sequence, such as, for example, a human germline sequence, a mutant version of a human germline sequence, or a human sequence such as the antibody-containing consensus framework sequence derived from human framework sequence analysis, as described, for example, Knappik et al., J.Mol.Biol.296:57-86, 2000. Antibodies derived from human sequences in which one or more CDRs have been mutated for affinity maturation or for manufacturing / payload binding purposes are also included. 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 present invention may contain amino acid residues not encoded by human sequences (for example, mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutations in vivo, or conservative substitutions that promote stability or production).

[0046] As used herein, the term “recognize” means that an antibody or its antigen-binding fragment finds its epitope and interacts with (e.g., binds to) it, whether the epitope is linear or conformational. The term “epitope” refers to a site on an antigen to which the antibody or antigen-binding fragment of the present invention specifically binds. Epitopes can be formed from both adjacent amino acids or non-adjacent amino acids arranged by the tertiary folding of the protein. Epitopes formed from adjacent amino acids are typically retained immediately after exposure to a denaturing solvent, while epitopes formed by tertiary folding are typically lost immediately after treatment with the denaturing solvent. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a specific spatial conformation. Methods for determining the spatial conformation of an epitope include the techniques described in this art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)). A "paratope" is the part of the antibody that recognizes the 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 region of the antibody "arm" interacts with the antigen at numerous sites via weak non-covalent bonds; the more interactions there are, the stronger the affinity.

[0048] The term "isolated antibody" refers to an antibody that is substantially free from other antibodies with different antigenic specificity. However, an isolated antibody that specifically binds to a particular antigen may exhibit cross-reactivity to other antigens. Furthermore, an isolated antibody may be substantially free from other cellular substances 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 by comparison with all other known variable region amino acid sequences encoded by the immunoglobulin variable region sequence of the human germline. The corresponding human germline sequence may also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence, as determined by comparison with all other evaluated variable region amino acid sequences. The corresponding human germline sequence may be the framework region only, the complementarity-determining region only, the framework and complementarity-determining region, the variable segment (as defined above), or any other combination of sequences or subsequences containing the variable region. Sequence identity may 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 sequence or amino acid sequence may have sequence identity with the reference variable region nucleic acid sequence or amino acid sequence of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The corresponding human germline sequence 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 binds" or "selectively binds," 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 biological agents, such as in a biological sample, e.g., blood, serum, plasma, or tissue sample. Thus, under specified particular immunoassay conditions, an antibody or binding agent having a particular binding specificity binds to a particular antigen at least 2-fold above background and substantially does not bind in large amounts to other antigens present in the sample. In one embodiment, under specified immunoassay conditions, an antibody or binding agent having a particular binding specificity binds to a particular antigen at least 10-fold above background and substantially does not bind in large amounts to other antigens present in the sample. Specific binding of an antibody or binding agent under such conditions may require that the antibody or agent be selected for its specificity for that particular protein. If desired or appropriate, this selection can be achieved by removing antibodies that cross-react with the molecule from other species (e.g., mouse or rat) or other subtypes. Additionally, in some embodiments, an antibody or antibody fragment that cross-reacts with a particular desired molecule is selected.

[0051] A variety of immunoassay formats may be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that are specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for descriptions of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction will result in a signal that is at least 2-fold, more typically at least 10-100-fold above background signal.

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

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

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

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

[0056] The term “conservatively modified variant” is used for both amino acid sequences and nucleic acid sequences. For a given nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that codes for the same or essentially the same amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, for an essentially the same sequence. Due to the degeneracy of genetic coding, many functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Therefore, at all positions where alanine is identified by a codon, the codon can be changed to one of the corresponding codons described, without changing the coded polypeptide. Such nucleic acid variants are “silent variants,” and these are a type of conservatively modified variant. Furthermore, all nucleic acid sequences in this specification that code for polypeptides describe all possible silent variants of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, usually the sole codon for methionine, and TGG, usually the sole codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silent mutation in the nucleic acid encoding the polypeptide is latent within each described sequence.

[0057] With respect to polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions, or additions to the polypeptide sequence that substitute amino acids with chemically similar amino acids. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are also included in, and are not excluded from, the polymorphic variants, interspecies homologs, and alleles of the present invention. The following eight groups contain amino acids that are conserved substitutions with each other: 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 modification” is used to refer to amino acid modifications that do not significantly affect or alter the binding properties of an antibody containing an amino acid sequence.

[0058] As used herein, the term “optimized” refers to a nucleotide sequence modified to encode an amino acid sequence using preferred codons in a producing cell or organism, typically 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. The optimized nucleotide sequence is manipulated to retain, to the greatest extent possible, the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the “parent” sequence.

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

[0060] In sequence comparison, typically one sequence functions as the reference sequence compared to the test sequence. The test and reference sequences are input into a computer using a sequence comparison algorithm, and, if necessary, the subsequent coordinates and sequence algorithm program parameters are specified. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence to the reference sequence based on the program parameters.

[0061] As used herein, the “comparison window” includes a reference to one segment of adjacent positions selected from the group consisting of 20 to 600, typically about 50 to about 200, and typically about 100 to about 150, where the sequences can be compared to a reference sequence with the same number of adjacent positions after the two sequences have been optimally aligned. Sequence alignment methods 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, for example, 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, 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 analysis is generally available from the National Center for Biotechnology Information. This algorithm first involves identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with a word of the same length in the database sequence, match or satisfy a certain positive threshold score T. T is called the neighbor word score threshold (Altschul et al., op. cit.). These initial neighbor word hits serve as a seed to initiate a search for longer HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction stops if: the cumulative alignment score falls by X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of one or more negative scoring residue alignments; or it reaches the end of either sequence. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word length of 11 (W), a prediction of 10 (E), 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 10 predictions (E), while the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) uses 50 alignments (B), 10 predictions (E), M=5, N=-4, and 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 minimum sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the minimum 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] Percent identicality 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 the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, percentage identicality between two amino acid sequences can also 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 from www.gcg.com)), using the BLOSUM62 matrix or 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 indicator of substantial identity between two nucleic acid sequences or polypeptides, beyond the previously noted percentage of sequence identity, is that the polypeptide encoded by the first nucleic acid may cross-reactive immunologically with an enhanced antibody against the polypeptide encoded by the second nucleic acid, as described below. Therefore, the polypeptide is typically substantially identical to the second polypeptide if, for example, the two peptides differ only by conservative substitutions. Another indicator of substantial identity between two nucleic acid sequences is that the two molecules or their complements may hybridize under stringent conditions, as described below. Yet another indicator of substantial identity is that the same primers may be used to amplify the sequences.

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

[0067] Unless otherwise specified, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. Specifically, as detailed below, degenerate codon substitutions can be achieved by producing sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (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] In the context of nucleic acids, the term "operably bound" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. A typical example is the functional relationship between a transcriptional regulatory sequence and the sequence being transcribed. For example, a promoter or enhancer sequence is operably bound to a coding sequence if it stimulates or modulates the transcription of the coding sequence in a suitable host cell or other expression system. Typically, a promoter transcriptional regulatory sequence operably bound to a transcriptional sequence is physically adjacent to the transcriptional sequence, i.e., cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically adjacent to or positioned in close proximity to the coding sequence they enhance transcription of.

[0069] The terms “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms are used for amino acid polymers, which are artificial chemical mimics of corresponding naturally occurring amino acids, and for naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly includes its conservatively modified variants.

[0070] As used herein, the terms “antibody-drug conjugate” or “immunoconjugate” refer to the binding of an antibody or its antigen-binding fragment to another agent, such as a chemotherapeutic agent, toxin, immunotherapy agent, or imaging probe. The binding may be covalent or non-covalent, such as via electrostatic interaction. 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 fusion proteins that can be expressed from polynucleotides encoding the immunoconjugate. As used herein, “fusion protein” refers to a protein produced by the binding of two or more genes or gene fragments that originally encoded distinct 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 humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise indicated, the terms "patient" and "subject" are used interchangeably herein.

[0072] As used herein, the terms “cytotoxin” or “cytotoxic agent” refer to any agent that is harmful to the growth and proliferation of cells and that can act to reduce, inhibit, or destroy cells or malignant tumors.

[0073] As used herein, the term “anticancer agent” means any agent that can be used to treat or prevent cell proliferation disorders, such as cancer, including but not limited to cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, and immunotherapeutic agents.

[0074] As used herein, the terms “drug portion” or “payload” refer to the chemical portion bound to the antibody or antigen-binding fragment of the present invention, which may be any therapeutic or diagnostic agent, e.g., anticancer, anti-inflammatory, anti-infective (e.g., antifungal, antibacterial, antiparasitic, antiviral), or anesthetic. For example, the drug portion may be an anticancer agent, e.g., a cytotoxin. In certain embodiments, the drug portion is a target inhibitor compound. Furthermore, the payload may be a biophysical probe, fluorescent label, spin label, infrared probe, affinity probe, chelator, spectroscopic probe, radioactive probe, lipid molecule, polyethylene glycol, polymer, spin label, DNA, RNA, protein, peptide, surface, antibody, antibody fragment, nanoparticle, quantum dot, liposome, PLGA particle, sugar, or polysaccharide.

[0075] In some embodiments, the drug portion or payload is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of both 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-responsive 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 to prevent GDP release, or binds to an active GTP-bound state to prevent GNAQ / 11 interaction with downstream effectors. In some embodiments, the GNAQ / 11 inhibitor functions by inhibiting mutant GNAQ and / or GNA11, for example, those containing the Q209L / P mutation. Methods for attaching such drug portions to a linker compatible with the target portion are described herein, along with methods known in the art. See, for example, 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 subunit 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), resulting in 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-trisphosphate (IP3) and diacylglycerol (DAG). 2+ Following a transient increase, IP3 is rapidly converted to IP2, IP1, and myo-inositol. On the other hand, DAG activates protein kinase C (PKC), leading to a cascade of phosphorylation of RAF, MEK, and ERK, which then translocate to the nucleus to 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 publicly available in GenBank under the following accession numbers. NP_002063 (Sequence ID 268) [ka] NM_002072 (Sequence ID 269) [ka] [ka] [ka]

[0078] The nucleic acid and amino acid sequence of human GNA11 are publicly available in GenBank under the following accession numbers. NP_002058 (Sequence ID 270) [ka] NM_002067 (Sequence ID 271) [ka] [ka] [ka]

[0079] "Tumor" refers to the growth and proliferation of new cells, and includes all precancerous and cancerous cells and tissues, regardless of whether they are malignant or benign.

[0080] The term “antitumor activity” refers to a reduction in the rate of tumor cell proliferation, survival, or metastatic activity. For example, antitumor activity can be demonstrated by a decrease in the growth rate of abnormal cells occurring during treatment, or by stability or reduction in tumor size, or by a longer treatment-induced survival period compared to an untreated control. Such activity can be evaluated using approved 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 antitumor 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 dysregulation or uncontrolled cell growth. Exemplary cancers include carcinomas, sarcomas, leukemias, and lymphomas.

[0082] The term "cancer" includes primary malignancies (for example, those in which cells do not migrate to any other part of the body of the target other than the site of the initial tumor) and secondary malignancies (for example, those resulting from metastasis, which is the migration of tumor cells to a secondary site different from the site of the initial tumor).

[0083] The term "PMEL17" (also known as premelanosome 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 are publicly available 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). The term "PMEL17" as used herein refers collectively to all naturally occurring isoforms of the PMEL17 protein, or their variants. NP_008859 (Sequence ID 272) [ka] NP_001307050 (Sequence ID 273) [ka] NP_001307051 (Sequence ID 274) [ka] NP_001186982 (Sequence ID 275) [ka] NP_001186983 (Sequence ID 276) [ka] NM_006928 (Sequence ID 277) [ka] NM_001200053 (Sequence ID 278) [ka] NM_001200054 (Sequence ID 279) [ka] NM_001320121 (Sequence ID 280) [ka] NM_001320122 (Sequence ID 281) [ka]

[0084] The term "mutant" 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 possess one or more of the activities of the reference polypeptide. For example, a mutant may have approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the reference polypeptide, while retaining one or more of the activities of the reference polypeptide.

[0085] As used herein, “to treat,” “to treat,” or “treatment” of any disease or disorder means, in one embodiment, improving the disease or disorder (i.e., slowing, inhibiting, or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, “to treat,” “to treat,” or “treatment” means reducing or improving at least one physical parameter, including one that is not identifiable by the patient. In yet another embodiment, “to treat,” “to treat,” or “treatment” means modulating the disease or disorder physically (e.g., stabilizing identifiable symptoms), physiologically (e.g., stabilizing a physical parameter), or both.

[0086] As used herein, the terms “prevent,” “prevention,” and “prevention” refer to preventive measures for any disease or disorder, or to delaying the progression of a disease or disorder.

[0087] The terms “therapeutably acceptable dose” or “therapeutably effective dose” interchangeably refer to a dose sufficient to produce the desired outcome (i.e., reduction of 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 dose does not induce or cause any undesirable side effects. In some embodiments, a therapeutically acceptable dose induces or causes side effects, but only those that are acceptable to the healthcare provider in terms of the patient’s condition. A therapeutically acceptable dose may be determined by administering a low dose first and then gradually increasing the dose until the desired effect is achieved. The “prophylactically effective dose” and “therapeutably effective dose” of the molecule of this invention can prevent the onset of symptoms, e.g., cancer-related symptoms, or reduce the severity of symptoms, respectively.

[0088] The term "simultaneous administration" refers to the simultaneous presence of two activators in an individual's bloodstream. The activators 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 internalize upon such binding. The antibodies and antibody fragments (e.g., antigen-binding fragments) of the present invention can be used in the production of antibody-drug conjugates. Furthermore, the present invention provides antibody-drug conjugates having desired pharmacokinetic characteristics and other desired attributes, and therefore usable for the treatment or prevention of cancer expressing PMEL17. The present invention further provides pharmaceutical compositions comprising the antibody-drug conjugates of the present invention, as well as methods for preparing such pharmaceutical compositions for the treatment or prevention of cancer and methods for using the same.

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

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

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

[0093] In another embodiment, the drug portion (D) of the antibody-drug conjugate of the present invention is a GNAQ and GNA11 inhibitor (GNAQ / GNA11 inhibitor).

[0094] In another embodiment, the drug portion of the antibody-drug conjugate of the present invention is Structure of equation (A): [ka] (In the formula, R0 is methyl or ethyl, R1 is methyl or i-propyl, and R2 is methyl or ethyl.) It is a compound that has [a certain characteristic].

[0095] In another embodiment, the drug portion of the antibody-drug conjugate of the present invention has the following structure: [ka] Compound (A1) has the following properties.

[0096] In another embodiment, the drug portion of the antibody-drug conjugate of the present invention has the following structure: [ka] Compound (A2) has the following properties.

[0097] In another embodiment, the drug portion of the antibody-drug conjugate of the present invention has the following structure: [ka] This is compound (A3) which has [the characteristic].

[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 portion (L A -(D) n ) In a second aspect, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) is Linker (L A The system comprises one or more drug moieties covalently attached to the ), each of which is independently selected from a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of both GNAQ and GNA11 (GNAQ / GNA11 inhibitor).

[0101] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) is Linker (L A The system comprises one or more drug moieties covalently attached to the ) and each of the one or more drug moieties is independently selected from the GNAQ inhibitor.

[0102] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) is Linker (L A It comprises one or more drug portions attached to ), each of which is independently selected from a GNA11 inhibitor.

[0103] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) is Linker (L A The system comprises one or more drug moieties covalently attached to ), each of which is independently selected from GNAQ and GNA11 inhibitors (GNAQ / GNA11 inhibitors).

[0104] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) is Linker (L A It contains one or more drug moieties covalently attached to the linker (L A The linker is a cleavable linker, and one or more drug portions are independently selected from a GNAQ inhibitor, a GNA11 inhibitor, or a GNAQ and GNA11 inhibitor (GNAQ / GNA11 inhibitor).

[0105] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) is Linker (L A It contains one or more drug moieties covalently attached to the linker (L A) is a cleavable linker, and one or more drug portions are independently selected from GNAQ inhibitors.

[0106] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) is Linker (L A It contains one or more drug moieties covalently attached to the linker (L A ) is a cleavable linker, and one or more drug portions are independently selected from GNA11 inhibitors.

[0107] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) is Linker (L A It contains one or more drug moieties covalently attached to the linker (L A The linker is a cleavable linker, and one or more drug portions are independently selected from GNAQ and GNA11 inhibitors (GNAQ / GNA11 inhibitors).

[0108] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) is Linker (L A It contains one or more drug moieties covalently attached to the linker (L A The linker is non-cleavable, and one or more drug portions are independently selected from a GNAQ inhibitor, a GNA11 inhibitor, or a GNAQ and GNA11 inhibitor (GNAQ / GNA11 inhibitor).

[0109] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) is Linker (L A It contains one or more drug moieties covalently attached to the linker (L A) is a non-cleavable linker, and one or more drug moieties are independently selected from GNAQ inhibitors.

[0110] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) is Linker (L A It contains one or more drug moieties covalently attached to the linker (L A ) is a non-cleavable linker, and one or more drug portions are independently selected from GNA11 inhibitors.

[0111] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) is Linker (L A It contains one or more drug moieties covalently attached to the linker (L A The linker is non-cleavable, and one or more drug portions are independently selected from GNAQ and GNA11 inhibitors (GNAQ / GNA11 inhibitor).

[0112] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention ((L A -(D) n ))) Linker (L A ) is expressed by the following formula: [ka] (In the formula, X1 is a divalent bond group; X2 is a self-sacrificing spacer; Y1 is [ka] And in the formula, Y1 * This indicates the attachment point to X2, and Y1 ** This indicates other attachment points; L1 is a divalent peptide linker, L2 is either a linker or a coupling agent. It has.

[0113] In another embodiment, the linker-drug portion of the antibody-drug conjugate of the present invention, ((L A -(D) n ))) is expressed by the following formula: [ka] (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of both GNAQ and GNA11; X1 is a divalent bond group; X2 is a self-sacrificing spacer; Y1 is [ka] And in the formula, Y1 * This indicates the attachment point to X2, and Y1 ** This indicates the attachment point to D; L1 is a divalent peptide linker, L2 is either a linker or a coupling agent. 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), or its stereoisomer or pharmaceutically acceptable salt. R 8 -L B -(D) n (B) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of both GNAQ and GNA11; R 8 is a reactive group; L B These are either cleavable or non-cleavable linkers. n is 1, 2, 3, or 4. It is a compound that has [a certain characteristic].

[0115] In one aspect, in the formula, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of both GNAQ and GNA11; R 8 is a reactive group; L B This is a cleavable linker comprising one or more linker components selected from a self-sacrificing spacer, a phosphate group, a carbonate group, and a divalent peptide linker. n is 1, 2, 3, or 4. The linker-drug of the present invention having the structure of formula (B), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0116] In one embodiment, the linker-drug of the present invention has the structure of formula (B-1), or its stereoisomer or pharmaceutically acceptable salt. [ka] (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of both GNAQ and GNA11; R 8 is a reactive group; X2 is a self-sacrificing spacer; Y1 is [ka] And in the formula, Y1 * This indicates the attachment point to X2, and Y1 ** This indicates the attachment point to D; L1 is a divalent peptide linker, L2 is either a linker or a coupling agent. It is a compound that has [a certain characteristic].

[0117] Specific embodiments and examples of the 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. In the formula, D is a compound of formula (B) or formula (B-1) which is a GNAQ inhibitor, or a stereoisomer or pharmaceutically acceptable salt thereof.

[0119] Embodiment 2. In the formula, D is a compound of formula (B) or formula (B-1) which is a GNA11 inhibitor, or a stereoisomer or pharmaceutically acceptable salt thereof.

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

[0121] Embodiment 4. In the formula, D is [Chemical formula] (In the formula, R 0 is methyl or ethyl, R 1 is methyl or isopropyl, R 2 is methyl or ethyl, *** is L B or the attachment point to Y1) is a compound of formula (B) or formula (B-1), or a stereoisomer or pharmaceutically acceptable salt thereof.

[0122] Embodiment 5. In the formula, D is [Chemical formula] (In the formula, *** is L B or the attachment point to Y1) is a compound of formula (B) or formula (B-1), or a stereoisomer or pharmaceutically acceptable salt thereof.

[0123] Embodiment 6. In the formula, D is [Chemical formula] (In the formula, *** is LB (Or indicates the attachment point to Y1) A compound of formula (B) or formula (B-1), or its stereoisomer or pharmaceutically acceptable salt.

[0124] Embodiment 7. In the formula, D is [ka] (In the formula, *** L B (Or indicates the attachment point to Y1) A compound of formula (B) or formula (B-1), or its stereoisomer or pharmaceutically acceptable salt.

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

[0126] Embodiment 9. Structure of formula (B-2a), or a pharmaceutically acceptable salt thereof. [ka] (In the formula, X2, L1, L2 and R 8 (This is defined as in the compound of formula (B-1) above.) Compounds of formula (B), formula (B-1), or formula (B-2) having the same, or stereoisomers thereof or pharmaceutically acceptable salts thereof.

[0127] Embodiment 10. Structure of formula (B-2b), or a pharmaceutically acceptable salt thereof. [ka] (In the formula, X2, L1, L2 and R 8 (This is defined as in the compound of formula (B-1) above.) Compounds of formula (B), formula (B-1), or formula (B-2) having the same, or stereoisomers thereof or pharmaceutically acceptable salts thereof.

[0128] Embodiment 11. Structure of formula (B-2c), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, X2, L1, L2 and R 8 (This is defined as in the compound of formula (B-1) above.) Compounds of formula (B), formula (B-1), or formula (B-2) having the same, or stereoisomers thereof or pharmaceutically acceptable salts thereof.

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

[0130] Embodiment 13. Structure of formula (B-3a), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, X2, L1, L2 and R 8 (This is defined as in the compound of formula (B-1) above.) Compounds of formula (B), formula (B-1), or formula (B-3) having the same, or stereoisomers thereof or pharmaceutically acceptable salts thereof.

[0131] Embodiment 14. Structure of formula (B-3b), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, X2, L1, L2 and R 8 (This is defined as in the compound of formula (B-1) above.) Compounds of formula (B), formula (B-1), or formula (B-3) having the same, or stereoisomers thereof or pharmaceutically acceptable salts thereof.

[0132] Embodiment 15. Structure of formula (B-3c), or a pharmaceutically acceptable salt thereof: [ka] (In the formula, X2, L1, L2 and R 8 (This is defined as in the compound of formula (B-1) above.) Compounds of formula (B), formula (B-1), or formula (B-3) having the same, or stereoisomers thereof or pharmaceutically acceptable salts thereof.

[0133] Embodiment 16. In formula, R 0 It is methyl or ethyl; R 1 It is methyl or isopropyl; R 2 It is methyl or ethyl; X2 is [ka] A self-sacrificing spacer selected from, where X2 * This indicates the attachment point to L1, and X2 ** teeth, [ka] Attachment point to the substrate, or [ka] Indicate the attachment point to the substrate; L1 is a divalent peptide linker containing 2 to 4 amino acid residues; L2 is the 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] Selected from, in the formula, Each R 4 These are independently selected from H and C1-C6 alkyl groups; Each R 5 These are independently selected from H, C1-C6 alkyl, F, Cl, and -OH; Each R 6These are independently selected from H, C1-C6 alkyl, F, Cl, -NH2, -OCH3, -OCH2CH3, -N(CH3)2, -CN, -NO2, and -OH. Each R 7 H and C are independent of each other. 1~6 Benzyloxy substituted with alkyl, fluoro, -C(=O)OH, benzyl substituted with -C(=O)OH, C substituted with -C(=O)OH 1~4 C substituted with alkoxy and -C(=O)OH 1~4 Selected from alkyl groups, Compounds of formula (B-2) of Embodiment 8, compound (B-3) of Embodiment 12, or pharmaceutically acceptable salts thereof.

[0134] Embodiment 17. In formula X2, [ka] A self-sacrificing spacer selected from, where X2 * This indicates the attachment point to L1, and X2 ** The attachment point to Y1 is [ka] Attachment point to the substrate, or [ka] A compound from any one of Embodiments 1 to 16, showing an attachment site to a group.

[0135] Embodiment 18. In formula, X2 is [ka] And in the formula, X2 * This indicates the attachment point to L1, and X2 ** The attachment point to Y1 is [ka] Attachment point to the substrate, or [ka] A compound from any one of Embodiments 1 to 17, showing an attachment site to a group.

[0136] Embodiment 19. A compound from any one of Embodiments 1 to 18, wherein L1 is a divalent peptide linker comprising 2 to 4 amino acid residues.

[0137] Embodiment 20. A compound from any one of Embodiments 1 to 18, wherein L1 is a divalent 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. A compound from any one of Embodiments 1 to 18, wherein L1 is a divalent peptide linker comprising at least one valine (Val) or citrulline (Cit) residue.

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

[0140] Embodiment 23. In the formula, L1 is [ka] A divalent dipeptide linker selected from, where L1 is * This indicates the attachment point to L2, and L1 ** This is one of the compounds from Embodiments 1 to 18, which indicates the attachment site to X2.

[0141] Embodiment 24. A compound from any one of Embodiments 1 to 18, wherein L1 is ValCit.

[0142] Embodiment 25. In the formula, L1 is [ka] And in the formula, L1 * This indicates the attachment point to L2, and L1 ** This is one of the compounds from Embodiments 1 to 18, which indicates the attachment site to X2.

[0143] Embodiment 26. A compound from 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 ** - A linker selected from, in the formula L2 * This indicates the attachment point to L1, and L2 ** This indicates the attachment point to R8; During the ceremony, Each R3 is independently selected from H and C1-C6 alkyl groups; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, one of any one of the compounds in Embodiments 1 to 25.

[0145] Embodiment 28. In the formula, L2 is - * C(=O)((CH2) m O) p (CH2) m ** -or- * C(=O)(CH2) m ** - and in the formula, L2 * This indicates the attachment point to L1, and L2 ** This indicates the point of attachment to R8. One compound from 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] And in the formula, L2 * This indicates the attachment point to L1, and L2 ** This is one of the compounds from Embodiments 1 to 25, which indicates the attachment site to R8.

[0147] Embodiment 30. In 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] And in the formula, Each R 4 These are independently selected from H and C1-C6 alkyl groups; Each R 5 These are independently selected from H, C1-C6 alkyl, F, Cl, and -OH; Each R 6 These are independently selected from H, C1-C6 alkyl, F, Cl, -NH2, -OCH3, -OCH2CH3, -N(CH3)2, -CN, -NO2, and -OH. Each R 7 H and C are independent of each other. 1-6Benzyloxy substituted with alkyl, fluoro, -C(=O)OH, benzyl substituted with -C(=O)OH, C substituted with -C(=O)OH 1~4 C substituted with alkoxy and -C(=O)OH 1~4 A compound selected from alkyl groups, one of any one of embodiments 1 to 29.

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

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

[0150] Embodiment 33. In the formula, R 0 It is methyl or ethyl; R 1 It is methyl or isopropyl; R 2 It is methyl or ethyl; X2 is [ka] And in the formula, X2 * This indicates the attachment point to L1, and X2 ** teeth, [ka] Indicate the attachment point to the substrate; L1 is [ka] And in the formula, L1 * This indicates the attachment point to L2, and L1** This indicates the attachment point to X2; L2 is [ka] And in the formula, L2 * This indicates the attachment point to L1, and L2 ** This indicates the point of attachment to R8. R 8 teeth, [ka] That is, A compound of formula (B-2) of Embodiment 8, or a pharmaceutically acceptable salt thereof.

[0151] Embodiment 34. In the formula, R 0 It is methyl or ethyl; R 1 It is methyl or isopropyl; R 2 It is methyl or ethyl; X2 is [ka] And in the formula, X2 * This indicates the attachment point to L1, and X2 ** teeth, [ka] Indicate the attachment point to the substrate; L1 is [ka] And in the formula, L1 * This indicates the attachment point to L2, and L1 ** This indicates the attachment point to X2; L2 is [ka] And in the formula, L2 * This indicates the attachment point to L1, and L2 **This indicates the point of attachment to R8. R 8 teeth, [ka] That is, A compound of formula (B-3) of 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 is of formula (C): Ab-(L A -(D)n ) y (C) (In the formula, D is the drug portion; Ab is an antibody or its antigen-binding fragment that binds to the human PMEL17 protein; L A It is a linker; n is 1, 2, 3, or 4. y is 1, 2, 3, or 4. Linker - Drug portion (L A -(D) n (These are covalently attached to the antibody or its antigen-binding fragment.) It is a combination of the two.

[0165] In one aspect, in the formula, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of both GNAQ and GNA11; Ab is an antibody or its antigen-binding fragment that binds to the human PMEL17 protein; L A This is a cleavable linker comprising one or more linker components selected from a self-sacrificing spacer, a phosphate group, a carbonate group, and a divalent peptide linker; n is 1, 2, 3, or 4. y is 1, 2, 3, or 4. Linker - Drug portion (L A -(D) n ) is an antibody-drug conjugate of the present invention having the structure of formula (C), which is covalently attached to an antibody or its antigen-binding fragment.

[0166] In one 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 both GNAQ and GNA11; Ab is an antibody or its antigen-binding fragment that binds to the human PMEL17 protein; X1 is a divalent bond group; X2 is a self-sacrificing spacer; Y1 is [ka] And in the formula, Y1 * This indicates the attachment point to X2, and Y1 ** This indicates the attachment point to D; L1 is a divalent peptide linker; L2 is a linker or coupling. (y is 1, 2, 3, or 4) It is a combination of the two.

[0167] In the compound of formula (C), one or more linker-drug moieties (L B -(D) n ) can be covalently attached to an antibody or its antigen-binding fragment Ab, thereby linking one or more drug portions D to the antibody or its antigen-binding fragment Ab via a linker L A It is attached covalently via L. A A is any chemical moiety capable of binding an antibody or its antigen-binding fragment Ab to one or more drug moieties D. A conjugate of formula (C), in which one or more drug moieties D are covalently bound to an antibody or its antigen-binding fragment Ab, can be formed using a bifunctional or polyfunctional linker reagent having one or more identical or different reactive functional groups. A linker L is formed by reacting one of the reactive functional groups of the bifunctional or polyfunctional linker reagent with a group on the antibody or its antigen-binding fragment Ab, for example, a thiol or amine (e.g., cysteine, N-terminus or amino acid side chain, e.g., lysine). A It forms a covalent bond with one end of the linker L. Examples of such reactive functional groups in a bifunctional or polyfunctional linker reagent include, but are not limited to, maleimides, thiols, and NHS esters. One or more other reactive functional groups in a bifunctional or polyfunctional linker reagent link one or more drug moieties D to linker L. AIt is used to attach to something covalently.

[0168] In one embodiment, L A is a cleavable linker. In another embodiment, L A It is a non-cleaving linker. In some embodiments, L A These include acid-unstable linkers, photo-unstable linkers, peptidase-cleavable linkers, esterase-cleavable linkers, glycosidase-cleavable linkers, phosphodiesterase-cleavable linkers, disulfide bond-reducing linkers, hydrophilic linkers, or dicarboxylic acid-based linkers.

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

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

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

[0172] In one embodiment, L A This is a cleavable linker comprising one or more linker components selected from a self-sacrificing spacer, a phosphate group, a divalent peptide linker, and a divalent bonding group.

[0173] In another embodiment, linker (L A ) is expressed by the following formula: [ka] (In the formula, X1 is a divalent bond group; X2 is a self-sacrificing spacer; Y1 is [ka] And in the formula, Y1 * This indicates the attachment point to X2, and Y1 ** This indicates other attachment points; L1 is a divalent peptide linker, L2 is either a linker or a coupling agent. It has.

[0174] In another embodiment, linker (L A ) is expressed by the following formula: [ka] (In the formula, X1 is a divalent bond group; X2 is a self-sacrificing spacer; Y1 is [ka] And in the formula, Y1 * This indicates the attachment point to X2; L1 is a divalent peptide linker, L2 is either a linker or a coupling agent. It has.

[0175] In another embodiment, linker (L A ) is expressed by the following formula: [ka] (In the formula, X1 is a divalent bond group; X2 is a self-sacrificing spacer; Y1 is [ka] And in the formula, Y1 * This indicates the attachment point to X2; L1 is a divalent peptide linker, L2 is either a linker or a coupling agent. It has.

[0176] The drug-antibody ratio is an exact integer value for a particular conjugate molecule (e.g., the product of n and y in formula (C)), while it is understood that the value is often an average value when used to describe a sample containing many molecules, typically due to some degree of heterogeneity associated with the conjugation step. 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 about 1 to about 5, typically about 1, 2, 3, or 4. In some embodiments, at least 50% of the sample by weight are compounds having an average DAR plus or minus 2, and preferably at least 50% of the sample are conjugates having an average DAR plus or minus 1. In other embodiments, conjugates have a DAR of about 2. In some embodiments, a DAR of "about y" means that the measured value for 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 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 mean value of the product of n and y, also known as the drug-antibody ratio (DAR)) is approximately 1 to approximately 10, and approximately 1 to approximately 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) 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), approximately 1 to approximately 5, approximately 1.5 to approximately 4.5, or approximately 2 to approximately 4.

[0178] In one embodiment provided by this disclosure, the conjugate is substantially high in purity and has one or more of the following characteristics: (a) more than 90% of the conjugate species (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100%), preferably more than 95%, are monomers; (b) the level of unconjugated linkers in the conjugate preparation is less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or 0%) (relative to the total linker); (c) less than 10% of the conjugate species are crosslinked. (d) the amount of free drug in the conjugate preparation (ADP-induced platelet aggregation inhibitor, e.g., GNAQ inhibitor, GNA11 inhibitor, or GNAQ and GNA11 inhibitor) is less than approximately 2% (e.g., approximately 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%) (mol / mol relative to total drug).

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

[0180] Embodiment 47. In the formula, D is a compound of formula (C) or formula (C-1), which is a GNAQ inhibitor.

[0181] Embodiment 48. In the formula, D is a compound of formula (C) or formula (C-1), wherein D is a GNA11 inhibitor.

[0182] Embodiment 49. In the formula, D is a compound of formula (C) or formula (C-1), which is an inhibitor of GNAQ and GNA11.

[0183] Embodiment 50. In formula, D is [ka] (In the formula, R 0 R1 is methyl or ethyl, R1 is methyl or isopropyl, and R2 is methyl or ethyl. *** L A (Or indicates the attachment point to Y1) A combination of formula (C) or formula (C-1).

[0184] Embodiment 51. In the formula, D is [ka] (In the formula, *** L A (Or indicates the attachment point to Y1) A combination of formula (C) or formula (C-1).

[0185] Embodiment 52. In the formula, D is [ka] (In the formula, *** L A (Or indicates the attachment point to Y1) A combination of formula (C) or formula (C-1).

[0186] Embodiment 53. In formula, D is [ka] (In the formula, *** L A (Or indicates the attachment point to Y1) A combination of formula (C) or formula (C-1).

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

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

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

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

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

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

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

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

[0195] Embodiment 62. In formula, R 0 It is methyl or ethyl; R 1 It is methyl or isopropyl; R 2 It is methyl or ethyl; Ab is an antibody or its antigen-binding fragment that binds to the human PMEL17 protein; X2 is [ka] A self-sacrificing spacer selected from, where X2 * This indicates the attachment point to L1, and X2 ** teeth, [ka] Attachment point to the substrate, or [ka] Indicate the attachment point to the substrate; L1 is a divalent peptide linker containing 2 to 4 amino acid residues; L2 is the 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] , ** -A divalent bond group selected from, where X1 * This indicates the attachment point to L2, and X1 ** This indicates the attachment point to Ab; During the ceremony, Each R 4 These are independently selected from H and C1-C6 alkyl groups; Each R 5 These are independently selected from H, C1-C6 alkyl, F, Cl, and -OH; Each R 6 These are independently selected from H, C1-C6 alkyl, F, Cl, -NH2, -OCH3, -OCH2CH3, -N(CH3)2, -CN, -NO2, and -OH. Each R 7 H and C are independent of each other. 1~6 Benzyloxy substituted with alkyl, fluoro, -C(=O)OH, benzyl substituted with -C(=O)OH, C substituted with -C(=O)OH 1~4 C substituted with alkoxy and -C(=O)OH 1~4 Selected from alkyl groups, y is 1, 2, 3, or 4. The composite of formula (C-2) of Embodiment 54 or the composite of formula (C-3) of Embodiment 58.

[0196] Embodiment 63. In formula X2, [ka] A self-sacrificing spacer selected from, where X2 * This indicates the attachment point to L1, and X2 ** is the attachment point to Y1, or [ka] Attachment point to the substrate, or [ka] A composite body from any one of embodiments 54 to 62, showing an attachment point to a substrate.

[0197] Embodiment 64. In formula, X2 is [ka] And in the formula, X2 * This indicates the attachment point to L1, and X2 ** is the attachment point to Y1, or [ka] Attachment point to the substrate, or [ka] A composite body from any one of embodiments 54 to 63, showing an attachment point to a substrate.

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

[0199] Embodiment 66. A conjugate of any one of Embodiments 54 to 65, wherein L1 is a divalent 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. A conjugate of any one of Embodiments 54 to 64, wherein L1 is a divalent peptide linker comprising at least one valine (Val) or citrulline (Cit) residue.

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

[0202] Embodiment 69. In formula, L1 is [ka] A divalent dipeptide linker selected from, where L1 is * This indicates the attachment point to L2, and L1 ** This is one of the bonding bodies from embodiments 54 to 64, showing the attachment point to X2.

[0203] Embodiment 70. A combination of any one of Embodiments 54 to 64, wherein L1 is ValCit.

[0204] Embodiment 71. In formula, L1 is [ka] And in the formula, L1 * This indicates the attachment point to L2, and L1 ** This is one of the bonding bodies from embodiments 54 to 64, showing the attachment point to X2.

[0205] Embodiment 72. In the formula, L2 is a linker, and the combination is one of any one of Embodiments 54 to 71.

[0206] Embodiment 73. In 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 ** - A linker selected from, in the formula L2 * This indicates the attachment point to L1, and L2 ** This indicates the attachment point to X1; During the ceremony, Each R3 is independently selected from H and C1-C6 alkyl groups; Each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Each p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14, forming one of the combinations from any one of embodiments 54 to 71.

[0207] Embodiment 74. In formula, L2 is - * C(=O)((CH2) m O) p (CH2) m ** -or- * C(=O)(CH2) m ** - and in the formula, L2 * This indicates the attachment point to L1, and L2 ** This indicates the attachment point to X1. In the formula, 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, one of the combinations of any one of embodiments 54 to 71.

[0208] Embodiment 75. In formula, L2 is [ka] And L2 * This indicates the attachment point to L1, and L2 ** This is one of the bonding bodies from embodiments 54 to 71, showing the attachment point to X1.

[0209] Embodiment 76. In formula X1, [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] , ** -A divalent bond group selected from, where X1 * This indicates the attachment point to L2, and X1 ** This indicates the attachment point to Ab; During the ceremony, Each R 4 These are independently selected from H and C1-C6 alkyl groups; Each R 5 These are independently selected from H, C1-C6 alkyl, F, Cl, and -OH; Each R 6 These are independently selected from H, C1-C6 alkyl, F, Cl, -NH2, -OCH3, -OCH2CH3, -N(CH3)2, -CN, -NO2, and -OH. Each R 7 H and C are independent of each other. 1~6 Benzyloxy substituted with alkyl, fluoro, -C(=O)OH, benzyl substituted with -C(=O)OH, C substituted with -C(=O)OH 1~4 C substituted with alkoxy and -C(=O)OH 1~4 Selected from alkyl groups, A composite body from any one of embodiments 54 to 75.

[0210] Embodiment 77. In formula X1, [ka] A divalent bond group selected from, where X1 * This indicates the attachment point to L2, and X1 ** This indicates the attachment point to Ab. A composite body from any one of embodiments 54 to 75.

[0211] Embodiment 78. In formula X1, [ka] A divalent bond group selected from, where X1 * This indicates the attachment point to L2, and X1 ** This indicates the attachment point to Ab. A composite body from any one of embodiments 54 to 75.

[0212] Embodiment 79. In formula, Ab is an antibody or its antigen-binding fragment that binds to the human PMEL17 protein; R 0 It is methyl or ethyl; R 1 It is methyl or isopropyl; R 2 It is methyl or ethyl; X1 is [ka] A divalent bond group selected from, where X1 * This indicates the attachment point to L2, and X1 ** This indicates the attachment point to Ab, X2 is [ka] And in the formula, X2 * This indicates the attachment point to L1, and X2 ** teeth, [ka] Indicate the attachment point to the substrate; L1 is [ka] And in the formula, L1 * This indicates the attachment point to L2, and L1 ** This indicates the attachment point to X2; L2 is [ka] And in the formula, L2 * This indicates the attachment point to L1, and L2 ** This indicates the point of attachment to R8. A compound of formula (C-2) of Embodiment 54, where y is 1, 2, 3, or 4.

[0213] Embodiment 80. In formula, Ab is an antibody or its antigen-binding fragment that binds to the human PMEL17 protein; R 0 It is methyl or ethyl; R 1 It is methyl or isopropyl; R 2 It is methyl or ethyl; X1 is [ka] A divalent bond group selected from, where X1 * This indicates the attachment point to L2, and X1 ** This indicates the attachment point to Ab, X2 is [ka] And in the formula, X2 * This indicates the attachment point to L1, and X2 ** teeth, [ka] Indicate the attachment point to the substrate; L1 is [ka] And in the formula, L1 * This indicates the attachment point to L2, and L1 ** This indicates the attachment point to X2; L2 is [ka] And in the formula, L2 * This indicates the attachment point to L1, and L2 ** This indicates the point of attachment to R8. A compound of formula (C-3) of Embodiment 58, where y is 1, 2, 3, or 4.

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

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

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

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

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

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

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

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

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

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

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

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

[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 interpreted as being limited to classical chemotherapeutic agents. For example, the drug moiety may be a protein, peptide, or polypeptide having the desired biological activity. Examples of such proteins include toxins, such as abrin, lysine 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, the antibody, antibody fragment (e.g., antigen-binding fragment), or functional equivalent of the present invention is bound to a drug moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radiotoxin. Examples of cytotoxins include, but are not limited to, taxanes (see, e.g., International Publication No. 01 / 38318 and PCT / US03 / 02675), DNA alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulicin analogs, duocalmycin analogs, auristatin E, auristatin F, meitansinoids, 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. al., Blood (2003) (electronic publication prior to print publication), U.S. Patent No. 5,475,092, No. 6,340,701, No. 6,372,738, and No. 6,436,931, U.S. Patent Application Publication No. 2001 / 0036923A1, pending U.S. Patent Applications No. 10 / 024,290 and No. 10 / 116,053, and International Publication No. 01 / 49698 (see pamphlet), Taxone, Cytochalasin B, Gramicidal Examples include 1-D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, t.colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogs or homologs.Examples of therapeutic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), ablating agents (e.g., mechloretamine, thiotepa chlorambucil, meiphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP)). Other examples include cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine) (see, for example, Seattle Genetics U.S. Patent Application Publication No. 20090304721).

[0228] Other examples of cytotoxins that can be conjugated to the antibodies, antibody fragments (antigen-binding fragments), or functional equivalents of the present invention include duocalmycin, calicheamicin, meitansine, and auristatin, and their derivatives.

[0229] Methods for conjugating various types of cytotoxins, linkers, and therapeutic agents to antibodies are known in the art; see, for example, 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 known 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, such as Zevalin® (DEC Pharmaceuticals) and Bexxar® (Corixa Pharmaceuticals), and radioimmunoconjugates can be prepared using the antibodies of the present invention by similar methods. In certain embodiments, 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 by reference 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, respectively.

[0231] The antibody, antibody fragment (e.g., antigen-binding fragment), or functional equivalent of the present 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 present invention provides a fusion protein comprising the antibody fragment (e.g., antigen-binding fragment) described herein (e.g., 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 techniques such as gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to modify the activity of the antibody or its fragment of the present invention (e.g., an antibody or its fragment having higher affinity and a lower dissociation rate). Generally, see U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patent 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 those patents and publications is incorporated herein by reference as a whole). Antibodies or their fragments, or antibodies or their fragments that encode them, can be modified before recombination by subjecting them to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods. Polynucleotides encoding antibodies or their fragments that specifically bind to an antigen can be recombined with one or more components, motifs, sections, parts, 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 marker sequences, such as peptides, to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexahistidine peptide (SEQ ID NO: 267), for example, a tag provided for pQE vectors (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), 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 a convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag (Wilson et al., (1984) Cell 37:767) corresponding to an epitope derived from influenza hemagglutinin protein, 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 efficacy.

[0234] In other embodiments, the antibody, antibody fragment (e.g., antigen-binding fragment), or functional equivalent of the present invention is conjugated to a diagnostic or detection agent. Such immunoconjugates may be useful for monitoring or prognostic diagnosis of the onset, occurrence, progression, and / or severity of disease and / or disorder as part of a clinical trial procedure, for example, in determining the effectiveness of a particular treatment. Such diagnosis and detection may involve detecting antibodies using detectable substances, such as various enzymes, for example, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; artificial groups, for example, streptavidin / biotin and avidin / biotin; and fluorescent materials, for example, Alexa Fluor350, Alexa Fluor405, Alexa Fluor430, Alexa Fluor488, Alexa Fluor500, Alexa Fluor514, Alexa Fluor532, Alexa Fluor546, Alexa Fluor555, Alexa Fluor568, Alexa Fluor594, Alexa Fluor610, Alexa Fluor633, Alexa Fluor647, Alexa Fluor660, Alexa Fluor680, Alexa Fluor700, Alexa Fluor Fluor750, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, e.g., luminol, but not limited to the following; bioluminescent materials, e.g., luciferase, luciferin, and aequorin, but not limited to the following; radioactive materials, e.g., iodine, but not limited to the following 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 Enjoy, 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 Sn, as well as positron-emitting metals used in various positron emission tomography techniques, 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 particularly useful for immunoassays or the purification of target antigens. Examples of such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0236] 3. Bonding and preparation of ADCs Method for preparing antibody conjugates of formula (C), formula (C-1), and formula (C-2). A general reaction scheme for the formation of the compound of formula (C) is shown in Scheme 1 below. [ka] (In the formula, RG1 is a compatible reactive group R attached to the linker-drug compound) 8 A reactive group on the antibody or its antigen-binding fragment Ab, such as a thiol, amine, or ketone, which reacts with the antibody or its antigen-binding fragment Ab, thereby covalently bonding the antibody or its antigen-binding fragment Ab to one or more linker-drug moieties. RG1 and R 8 A non-limiting example of such a reaction of the group is maleimide (R1), which reacts with thiol (RG1) to produce a succinimide ring. 8 ), or hydroxylamine (R) that reacts with ketone (RG1) to produce an oxime. 8 ) In one embodiment, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of both GNAQ and GNA11 (GNAQ / GNA11 inhibitor), and L a RG1 and R 8 A linker further comprising a divalent bond group formed when reacting, where n is 1, 2, 3, or 4, and y is 1, 2, 3, or 4.

[0237] A general reaction scheme for the formation of the compound of formula (C-1) is shown in Scheme 2 below. [ka] (In the formula, RG1 is a compatible reactive group R attached to the linker-drug portion) 8 A reactive group on the antibody or its antigen-binding fragment Ab, such as a thiol, amine, or ketone, which reacts with the antibody or its antigen-binding fragment Ab, thereby covalently bonding the antibody or its antigen-binding fragment Ab to one or more linker-drug moieties. RG1 and R 8 A non-limiting example of such a reaction of the group is maleimide (R1), which reacts with thiol (RG1) to produce a succinimide ring. 8 ), or hydroxylamine (R) that reacts with ketone (RG1) to produce an oxime. 8 ) In one embodiment, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of both GNAQ and GNA11 (GNAQ / GNA11 inhibitor), and X1 is RG1 and R 8 The divalent bonding group (e.g., succinimide ring or oxime) formed when reacting is Y1, the phosphate group is X2, the self-sacrificing spacer is L1, the divalent peptide linker is L2, the bond or linker is y is 1, 2, 3 or 4.

[0238] A general reaction scheme for the formation of the compound of formula (C-2) is shown in Scheme 3 below. [ka] (In the formula, RG1 is a compatible reactive group R attached to the linker-drug portion) 8 A reactive group on the antibody or its antigen-binding fragment Ab, such as a thiol, amine, or ketone, which reacts with the antibody or its antigen-binding fragment Ab, thereby covalently bonding the antibody or its antigen-binding fragment Ab to one or more linker-drug moieties. RG1 and R 8 A non-limiting example of such a reaction of the group is maleimide (R1), which reacts with thiol (RG1) to produce a succinimide ring. 8 ), or hydroxylamine (R) that reacts with ketone (RG1) to produce an oxime. 8 ) Here, R 0 is methyl or ethyl, and R 1 is methyl or isopropyl, and R 2 is methyl or ethyl, and X1 is RG1 and R 8 The divalent bonding group (e.g., succinimide ring or oxime) formed when reacting is X2, a self-sacrificing spacer is X2, a divalent peptide linker is L1, a bond or linker is L2, and y is 1, 2, 3, or 4.

[0239] A general reaction scheme for the formation of the compound of formula (C-2) is shown in Scheme 4 below. [ka] (In the formula, RG1 is a compatible reactive group R attached to the linker-drug portion) 8 A reactive group on the antibody or its antigen-binding fragment Ab, such as a thiol, amine, or ketone, which reacts with the antibody or its antigen-binding fragment Ab, thereby covalently bonding the antibody or its antigen-binding fragment Ab to one or more linker-drug moieties. RG1 and R 8 A non-limiting example of such a reaction of the group is maleimide (R1), which reacts with thiol (RG1) to produce a succinimide ring. 8 ), or hydroxylamine (R) that reacts with ketone (RG1) to produce an oxime. 8 ) Here, R 0 is methyl or ethyl, and R 1 is methyl or isopropyl, and R 2 is methyl or ethyl, and X1 is RG1 and R 8 The divalent bonding group (e.g., succinimide ring or oxime) formed when reacting is X2, a self-sacrificing spacer is X2, a divalent peptide linker is L1, a bond or linker is L2, and y is 1, 2, 3, or 4.

[0240] Method for binding to manipulated cysteine ​​antibody residues The conjugates of the present invention can be prepared, for example, using cysteine ​​residues manipulated in antibodies by site-directed mutagenesis. Such site-directed conjugates are homogeneous and possess 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] Since the modified cysteine ​​in antibodies expressed in mammalian cells is modified by adducts (disulfides), such as glutathione (GSH) and / or cysteine, during their biosynthesis (Chen et al. 2009), the modified cysteine ​​residues in the initially expressed product are nonreactive with thiol-reactive reagents, such as maleimide or bromo- or iodoacetamide groups. To conjugate the payload to the modified cysteine ​​after expression, the glutathione or cysteine ​​adducts must be removed by reducing their disulfide adducts, which generally also involves reducing the native disulfides in the expressed protein. Deprotection of adducted modified cysteine ​​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 re-oxidation of all native disulfide bonds of the antibody to restore and / or stabilize the functional antibody structure.

[0242] Antibodies containing manipulated cysteine ​​residues can be reduced and reoxidized using several methods for the preparation of antibody-drug conjugates. Attempts following reoxidation protocols already described in the literature 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 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 re-oxidizing antibodies containing manipulated cysteine ​​residues for the preparation of antibody-drug conjugates. Freshly prepared DTT is added to 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 changes to remove DTT and re-oxidize the antibody's native disulfide bonds. An alternative method is to remove the reducing agent via a desalting column, e.g., Sephadex G-25 equilibrated with PBS. Once the protein is completely reduced, 1 mM oxidized ascorbate (dehydroascorbic acid) is optionally added to the desalted sample, and a re-oxidation incubation is performed for 20–24 hours.

[0244] In another exemplary method, deprotection of the operational Cys residue is achieved by adding fully reduced cysteine ​​at a concentration of 20 mM to an antibody conjugated to a protein A-Sepharose resin. Reduction of the Cys adduct is achieved by incubation at room temperature for approximately 30–60 minutes, after which the reducing agent is rapidly removed by washing the resin with 50 beds of PBS. Reoxidation of the reductive antibody is achieved by incubating the washing slurry at room temperature with or without the addition of 50–2000 nM CuCl2 as an accelerator. Except for the use of copper sulfate, the examples herein use each of the protocols described herein, and the results are similar. Reoxidation restores the intrachain disulfide, while dialysis, desalting, or protein A chromatography removes the reducing agent as well as the cysteine ​​and glutathione initially bound to the operational cysteine ​​in the antibody. Typically, the reoxidation process is monitored using HPLC reverse-phase chromatography. The antibody was 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 0.1% TFA-containing water at 1.5 mL / min, and peak detection at 215, 254, and 280 nm.

[0245] After reoxidation, the antibody is conjugated to a linker-drug compound, for example, a compound of formula (B), formula (B-1), formula (B-2), or formula (B-3) (see Scheme 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 a volume of 5-10 molar equivalents relative to the antibody. Incubation is performed for 1-2 hours. The conjugation process is monitored by reverse-phase HPLC capable of separating the conjugated antibody from the 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 column elution is performed by a linear gradient of 30-60% acetonitrile in water containing 0.1% TFA at a flow rate of 1.5 ml / min. Protein elution from the column is monitored at 280 nm, 254 nm, and 215 nm.

[0246] Alternatively, for antibodies bound to protein A resin, after reoxidizing the antibody, wash the resin with 10 column volume of PBS, then resuspend the resin in an equal volume of PBS, add an 8-fold excess of compound (B), (B-1), (B-2), or (B-3) (in DMSO), and incubate at room temperature for 2 hours. Then wash the resin with 50 column volume of PBS to elute the resulting antibody-drug conjugate from the protein A resin, neutralize with 1 / 10 volume of 1 M Tris pH 9.0, exchange buffer in a suitable buffer, and perform preparative size exclusion chromatography (if necessary).

[0247] Immunoconjugates are also characterized by the average loading of the drug portion to the antibody-binding portion, commonly referred to as the drug-antibody ratio (DAR). The DAR value is estimated, for example, from LC-MS data for reduced and deglycosylated samples. LC / MS allows for the quantification of the average number of payload molecules attached to the antibody in the ADC. HPLC separates the antibody into light and heavy chains, and further separates the 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. The average DAR for the ADC can be calculated from the average loading of the LC and HC chains. 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] In the event of any inconsistency between the text of this specification and the sequence listings throughout the entire text of this application, the text of this specification shall prevail.

[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 present invention include those in which an amino acid or nucleic acid encoding an amino acid has been mutated, but which have at least 60, 70, 80, 90, or 95 percent identity with the sequences listed in Table 2. In some embodiments, this includes mutant amino acid sequences in which one, two, three, four, or five or fewer amino acids are mutated within the variable region compared to the variable region shown in the sequences listed in Table 2, while retaining substantially the same therapeutic activity as the antibodies shown in Table 2.

[0315] Since each of these antibodies can bind to PMEL17, the sequences of the VH, VL, full-length light chain, and full-length heavy chain (amino acid sequences and nucleotide sequences encoding the amino acid sequences) can be “mixed and matched” to produce other PMEL17-binding antibodies of the present invention. Such “mixed and matched” PMEL17-binding antibodies can be tested using binding assays known in the art (e.g., ELISA and other assays described in the Examples section). When these chains are mixed and matched, the VH sequence from a particular VH / VL pair 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 pair should be replaced with a structurally similar full-length heavy chain sequence. Similarly, the VL sequence from a particular VH / VL pair 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 pair should be replaced with a structurally similar full-length light chain sequence. Accordingly, in one embodiment, the present 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 NOs: 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 NOs: 21, 25, 29, 53, 75, 99, 119, 143, 159, 171, 190, 202, 221, 233, 248 or 260; the antibody specifically binds to PMEL17.

[0316] In another embodiment, the present invention provides (i) a full-length heavy chain comprising an amino acid sequence optimized for expression in mammalian expression cells 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 an isolated monoclonal antibody having a full-length light chain comprising an amino acid sequence optimized for expression in mammalian cells 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 the antigen-binding moiety thereof.

[0317] In another embodiment, the present invention provides a PMEL17-conjugated antibody comprising heavy and light chain CDR1, CDR2, and CDR3, or combinations thereof, as listed in Table 2. The amino acid sequences of the antibody's VH CDR1 are shown, 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 the antibody's 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 the antibody's 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 the antibody's VL CDR1 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 the antibody's VL CDR2 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 antibody's VL CDR3 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] If each of these antibodies can bind to PMEL17 and its antigen-binding specificity is primarily provided by the CDR1, 2, and 3 regions, then the sequences of VH CDR1, CDR2, and CDR3, as well as the sequences of VL CDR1, CDR2, and CDR3, can be “mixed and matched” (i.e., CDRs from various antibodies can be mixed and matched). Such “mixed and matched” PMEL17-binding antibodies can be tested using binding assays known in the art and assays described in the examples (e.g., ELISA). When VH CDR sequences are mixed and matched, the sequences of CDR1, CDR2, and / or CDR3 derived from a particular VH sequence should be replaced with structurally similar CDR sequences. Similarly, when VL CDR sequences are mixed and matched, the sequences of CDR1, CDR2, and / or CDR3 derived 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 can be generated by substituting the sequences of one or more VH and / or VL CDR regions of the monoclonal antibody of the present invention with structurally similar sequences derived from the CDR sequences shown herein.

[0319] Therefore, 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; SEQ ID NOs: 2, 6, 8, 34, 38, 40, 58, 61, 62, 80, 84, 86 Heavy chain CDR2 containing an amino acid sequence selected from the group consisting of 104, 108, 110, 124, 128, 130, 176, 180, 182, 207, 211, and 213; 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 Heavy chain CDR3 containing the amino acid sequence; Light chain CDR1 containing the 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; Light chain CDR1 containing the amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 18, 47, 50, 69, 72, 93, 96, 137, 140, and 154 2; and provides an isolated monoclonal antibody or its antigen-binding region containing a light chain CDR3 containing 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; the antibody specifically binds to PMEL17.

[0320] In a specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to PMEL17 includes 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, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to PMEL17 includes heavy chain CDR1 of SEQ ID NO: 33, 36, 37, or 39; heavy chain CDR2 of SEQ ID NO: 34, 38, or 40; heavy chain CDR3 of SEQ ID NO: 35 or 41; light chain CDR1 of SEQ ID NO: 46, 49, or 52; light chain CDR2 of SEQ ID NO: 47 or 50; and light chain CDR3 of SEQ ID NO: 48 or 51.

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

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

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

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

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

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

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

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

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

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

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

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

[0334] In a specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to PMEL17 is: a) Heavy chain CDR1 of SEQ ID NO: 1, 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; b) 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; c) Heavy chain CDR1 of SEQ ID NO: 5, heavy chain CDR2 of SEQ ID NO: 6, heavy chain CDR3 of SEQ ID NO: 3, light chain CDR1 of SEQ ID NO: 17, light chain CDR2 of SEQ ID NO: 18, and 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 Includes a CDR sequence selected from the above.

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

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

[0337] In a specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to PMEL17 is: a) 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; b) 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; c) 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; or d) 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 Includes a CDR sequence selected from the above.

[0338] In a specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to PMEL17 is: a) 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; b) 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; c) 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; 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 Includes a CDR sequence selected from the above.

[0339] In a specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to PMEL17 is: a) Heavy chain CDR1 of SEQ ID NO: 123, 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; b) 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; c) 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; or d) Heavy chain CDR1 of SEQ ID NO: 129, heavy chain CDR2 of SEQ ID NO: 130, heavy chain CDR3 of SEQ ID NO: 131, light chain CDR1 of SEQ ID NO: 142, light chain CDR2 of SEQ ID NO: 140, and light chain CDR3 of SEQ ID NO: 138 Includes a CDR sequence selected from the above.

[0340] In a specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to PMEL17 is: a) 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; b) 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; c) 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; or d) 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 Includes a CDR sequence selected from the above.

[0341] In a specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to PMEL17 is: a) Heavy chain CDR1 of SEQ ID NO: 103, 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; b) 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; c) Heavy chain CDR1 of SEQ ID NO: 107, heavy chain CDR2 of SEQ ID NO: 108, heavy chain CDR3 of SEQ ID NO: 163, light chain CDR1 of SEQ ID NO: 49, light chain CDR2 of SEQ ID NO: 50, and light chain CDR3 of SEQ ID NO: 170; 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: 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 Includes a CDR sequence selected from the above.

[0342] In a specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to PMEL17 is: a) 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; b) 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; c) 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; 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 Includes a CDR sequence selected from the above.

[0343] In a specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to PMEL17 is: a) Heavy chain CDR1 of SEQ ID NO: 103, 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; b) 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; c) 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; 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: 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 Includes a CDR sequence selected from the above.

[0344] In a specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to PMEL17 is: a) 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; b) 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; c) 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; 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: 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 Includes a CDR sequence selected from the above.

[0345] In a specific embodiment, the antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to PMEL17 is: a) 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; b) 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; c) Heavy chain CDR1 of SEQ ID NO: 210, heavy chain CDR2 of SEQ ID NO: 211, heavy chain CDR3 of SEQ ID NO: 225, light chain CDR1 of SEQ ID NO: 139, light chain CDR2 of SEQ ID NO: 140, and 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 Includes a CDR sequence selected from the above.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0380] In certain embodiments, the antibody that specifically binds to PMEL17 is one of the antibodies or antibody fragments (e.g., antigen-binding fragments) 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 epitopes as the anti-PMEL17 antibodies listed in Table 2, or that cross-compete with the antibodies listed in Table 2. Therefore, additional antibodies and antibody fragments (e.g., antigen-binding fragments) can be identified based on their ability to cross-compete (e.g., statistically significantly inhibit the binding) with other antibodies of the present invention in PMEL17 binding assays via BIACORE or assays known to those skilled in the art to measure binding. The ability of a test antibody to inhibit the binding of an antibody or antibody fragment (e.g., antigen-binding fragment) of the present invention to PMEL17 (e.g., human PMEL17) demonstrates that the test antibody may compete with its antibody or antibody fragment (e.g., antigen-binding fragment) for binding to PMEL17; such an antibody may, according to a non-limiting theory, bind to the same or related (e.g., structurally similar or spatially proximal) epitopes on PMEL17 as the competing antibody or antibody fragment (e.g., antigen-binding fragment). In certain embodiments, the antibody that binds to the same epitope on PMEL17 as the antibody or antibody fragment (e.g., antigen-binding fragment) of the present invention, as 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 changes to the Fc domain framework The immunoconjugates of the present invention include a modified antibody or its antigen-binding fragment, further comprising modifications of framework residues within VH and / or VL to enhance the properties of the antibody. In some embodiments, the framework modification is made to reduce the immunogenicity of the antibody. For example, one approach involves “reverse-mutating” one or more framework residues to the corresponding germline sequences. More specifically, a somatically mutated antibody may contain framework residues different from those of the germline sequence from which the antibody originates. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody originates. To return the framework region sequences to the configuration of their germlines, somatic mutations can be “reverse-mutated” to germline sequences, for example, by site-directed mutagenesis. Such “reverse-mutated” antibodies are also intended to be included in the present invention.

[0383] Another type of framework modification involves reducing the potential immunogenicity of an antibody by mutating one or more residues within a framework region or within one or more CDR regions to remove a T cell epitope. This approach, also known as “deimmunization,” is described in more 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 region, the antibodies of the present invention may typically be manipulated to include modifications within the Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity (ADCC). Furthermore, the antibodies of the present invention may also be chemically modified to alter one or more functional properties of the antibody (e.g., one or more chemical moieties may be attached to 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, for example, 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 the 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 include modified or manipulated amino acid residues, such as one or more cysteine ​​residues, as sites for binding to a drug moiety (Junutula JR, et al., Nat Biotechnol 2008, 26:925-932). In one embodiment, the present invention provides a modified antibody or antibody fragment comprising the substitution of one or more amino acids by cysteine ​​at the positions described herein. The sites for cysteine ​​substitution are located within the constant region of the antibody or antibody fragment and are therefore applicable to a variety of antibodies or antibody fragments, and the sites are selected to provide a stable, homogeneous conjugate. The modified antibody or fragment may have one or more cysteine ​​substitutions, and these substitutions can be used in combination with other modification and binding methods described herein. Methods for inserting cysteine ​​at specific locations in antibodies are known in the art, see, for example, Lyons et al., (1990) Protein Eng., 3:703-708, International Publication No. 2011 / 005481, International Publication No. 2014 / 124316, and International Publication No. 2015 / 138615. In certain embodiments, the modified antibody comprises the 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 antibody's heavy chain, the positions being numbered according to the EU system.In some embodiments, the modified antibody or antibody fragment comprises one or more amino acid substitutions by cysteine ​​on 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, the positions numbered according to the EU system, and the light chain is a human kappa light chain. In certain embodiments, the modified antibody or its antibody fragment comprises a combination of two or more amino acid substitutions by cysteine ​​on its constant region, the combination comprising 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, the positions numbered according to the EU system. In certain embodiments, the modified antibody or antibody fragment comprises a single amino acid substitution by cysteine ​​in its constant region, the substitution being at position 375, 152, or 360 of the antibody heavy chain, position 107, 165, or 159 of the antibody light chain, the positions numbered according to the EU system, and the light chain is a kappa chain. In certain embodiments, the modified antibody or antibody fragment comprises a combination of two amino acid substitutions by cysteine ​​in its constant region, the combination comprising substitutions at position 375 and 152 of the antibody heavy chain, the positions numbered according to the EU system. In certain embodiments, the modified antibody or antibody fragment comprises a single amino acid substitution by cysteine ​​at position 360 of the antibody heavy chain, the positions numbered according to the EU system. In other specific embodiments, the modified antibody or antibody fragment comprises a single amino acid substitution by cysteine ​​at position 107 of the antibody light chain, the position is numbered according to the EU system, and the light chain is a kappa chain.

[0387] In additional embodiments, examples of antibodies or antibody fragments (e.g., antigen-binding fragments) useful in the immunoconjugates of the present invention include modified or manipulated antibodies, such as antibodies modified to introduce one or more other reactive amino acids (other than cysteine), for example, Pcl, pyrrolicin, peptide tags (e.g., S6, A1, and ybbR tags), and non-natural amino acids in place of at least one amino acid in the natural sequence, thereby providing a reactive site on the antibody or antigen-binding fragment for binding to a drug moiety or linker-drug moiety having complementary reactivity. For example, antibodies or antibody fragments can be modified to incorporate Pcl or pyrrolicin (W. Ou, et al., (2011) PNAS 108(26), 10437-10442; International Publication No. 2014124258) or unnatural amino acids (JYAxup, et al., Proc Natl Acad Sci USA, 109(2012), pp. 16101-16106; for an overview, see CCLiu and PGSchultz (2010) Annu Rev Biochem 79, 413-444; CHKim, et al., (2013) Curr Opin Chem Biol. 17, 412-419) as a site for drug binding. 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). One other example is the use of 4'-phosphopantetheinyltransferase (PPTase) for the conjugation of coenzyme A analogs (International Publication No. 2013184514 pamphlet), and (Gruenewald et al., (2015) Bioconjugate Chem. 26(12), 2554-62). Methods for conjugating such modified or manipulated antibodies to payloads or linker-payload combinations are known in the art.

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

[0389] In yet another embodiment, the Fc region is modified 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 may be substituted with different amino acid residues so that the antibody has a modified affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand whose affinity is modified may be, for example, the Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patent No. 5,624,821 and No. 5,648,260, both by Winter et al.

[0390] In another embodiment, one or more amino acids selected from amino acid residues may be substituted with different amino acid residues so that the antibody has modified C1q binding and / or reduced or terminated 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 modified to alter the complement-immobilizing antibody's ability. This approach is described, for example, in international publication brochure 94 / 29351 by Bodmer et al. Allotype amino acid residues include, but are not limited to, the constant regions of the heavy chains of IgG1, IgG2, and IgG3 subclasses and the constant regions of the light chains of kappa isotypes, as described by Jefferis et al., MAbs.1:332-338 (2009).

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

[0393] In another embodiment, one or more amino acid residues are modified to alter the complement-immobilizing antibody's ability. This approach is described, for example, in Bodmer et al.'s International Publication No. 94 / 29351. In a specific embodiment, one or more amino acids of the antibody of the present invention or its antigen-binding fragment are substituted with one or more allotype amino acid residues. Allotype amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses, as well as the constant regions of the light chains of kappa isotypes, 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, a non-glycosylated antibody can be produced (i.e., the antibody lacks glycosylation). Glycosylation may be modified, for example, to increase the antibody's affinity for an "antigen". Such carbohydrate modification may be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, glycosylation at one or more variable region framework glycosylation sites can be eliminated by making one or more amino acid substitutions that eliminate that site. Such nonglycosylation may increase the antibody's affinity for an 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: T252L, T254S, T256F may be introduced, as described in Ward's U.S. Patent No. 6,277,375. In addition, to increase the biological half-life, the antibody may be modified within the CH1 or CL region to contain a salvage receptor-binding epitope taken from the two loops of the CH2 domain in the Fc region of IgG, as described in Presta et al.'s U.S. Patents No. 5,869,046 and No. 6,121,022.

[0396] 3. Generation of anti-PMEL17 antibodies Anti-PMEL17 antibodies and their antibody fragments (e.g., antigen-binding fragments) may be produced by any means known in the art, including but not limited to recombinant expression, chemosynthesis, and enzymatic digestion of antibody tetramers, where full-length monoclonal antibodies may be obtained by hybridoma generation or recombinant generation. Recombinant expression may 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 antibodies as described herein, for example, polynucleotides encoding a variable region or variable segment of a heavy chain or light chain containing a complementarity-determining region 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 with 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 with the polynucleotides 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 with the polynucleotides 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 with the polynucleotides 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 a polypeptide containing the variable regions of both the heavy and light chains of an exemplary mouse anti-PMEL17 antibody. Some other polynucleotides encode two polypeptide segments that are substantially identical to the variable regions of the heavy and light chains of a mouse antibody, respectively.

[0400] Polynucleotide sequences can be generated by de novo solid-phase DNA synthesis of existing sequences encoding anti-PMEL17 antibodies or their binding fragments (e.g., sequences 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. Patent No. 4,458,066. The introduction of mutations into polynucleotide sequences by PCR can be carried out as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, HAErlich (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] Furthermore, the present invention provides expression vectors and host cells for generating the anti-PMEL17 antibody described above. Various expression vectors can be used to express polynucleotides encoding the anti-PMEL17 antibody chain or its binding fragment. Both virus-based and non-viral expression vectors can be used to generate antibodies in mammalian host cells. Examples of non-viral vectors and non-viral systems include plasmids or episomal vectors (typically having expression cassettes for expressing proteins or RNA) 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 this technique for expressing other proteins. Useful viral vectors include retrovirus, adenovirus, adeno-associated virus, herpesvirus-based vectors, SV40, papillomavirus, HBP Epstein-Barr virus-based vectors, vaccinia virus vectors, and Semryki Forest virus (SFV). See Brent et al., op. cit.; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[0402] The selection of an expression vector depends on the host cell in which the vector is intended to be expressed. Typically, an expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably bound to a polynucleotide encoding a chain or fragment of anti-PMEL17 antibody. In some embodiments, an inducing promoter is used to prevent the expression of the inserted sequence outside of induction conditions. Examples of inducing promoters include arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can grow under non-inducing conditions without biasing the expression product toward a coding sequence 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. Typical examples include the ATG start codon and adjacent ribosome binding sites or other sequences. In addition, the efficiency of expression can be enhanced by including an enhancer suitable for the cell system being used (see, for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, expression in mammalian host cells can be increased using SV40 enhancers or CMV enhancers.

[0403] Expression vectors can also provide secretion signal sequence locations to form a fusion protein with the polypeptide encoded by the inserted anti-PMEL17 antibody sequence. Often, the inserted anti-PMEL17 antibody sequence is bound to the signal sequence before being incorporated into the vector. Additionally, vectors used to accept sequences encoding the variable domains of the light and heavy chains of the anti-PMEL17 antibody occasionally encode a constant region or a portion thereof. Such vectors can produce an intact antibody or a fragment thereof by expressing the variable region as a fusion protein with the constant region. Typically, such a constant region is human.

[0404] The host cells for possessing and expressing the anti-PMEL17 antibody chain may be prokaryotes or eukaryotes. Escherichia coli (E. coli) is a useful prokaryotic host for cloning and expressing the polynucleotide of the present invention. Other suitable microbial hosts include rods, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, those skilled in the art can construct expression vectors, which typically contain expression regulatory sequences (e.g., origins of replication) compatible with the host cell. In addition, there are any number of various well-known promoters, such as lactose promoters, tryptophan (trp) promoters, beta-lactamase promoters, or phage-lambda-derived promoters. The promoter typically controls expression, sometimes along with the operator sequence, and also contains ribosome-binding site sequences, etc., to initiate and complete transcription and translation. Furthermore, other microorganisms, such as yeast, can be used to express the anti-PMEL17 polypeptide of the present invention. Insect cells combined with baculovirus vectors can also be used.

[0405] In some preferred embodiments, the anti-PMEL17 polypeptide of the present invention can be expressed and produced using mammalian host cells. For example, the mammalian host cells may be hybridoma cell lines expressing endogenous immunoglobulin genes (e.g., myeloma hybridoma clones described in the examples) or mammalian cell lines possessing exogenous expression vectors (e.g., SP2 / 0 myeloma cells illustrated below). These include any animal or human cells, whether standard or immortal, that lead to death or are immortal. 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 cultures for polypeptide expression is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells may contain expression regulatory sequences, such as origins of replication, promoters, and enhancers (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 typically contain promoters derived from mammalian genes or mammalian viruses. Appropriate promoters may be constitutive, cell type-specific, stage-specific, and / or tunable or moduloable. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-induced MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPSV promoters, tetracycline-induced CMV promoters (e.g., human immediate early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

[0406] The method for introducing an expression vector containing the target polynucleotide sequence varies depending on the type of cell host. For example, calcium chloride transduction is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. in general). Other methods include electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to the herpesvirus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. In many cases, stable expression is desired for long-term high-yield production of recombinant proteins. For example, a cell line that stably expresses the chain or binding fragment of an anti-PMEL17 antibody can be prepared using the expression vector and selection marker gene of the present invention, which contain a viral replication origin or endogenous expression element. After introducing the vector, the cells can be grown in enriched medium for 1-2 days before being switched to a selective medium. The purpose of the selection marker is to confer resistance to selection, and its presence enables the growth of cells that successfully express the introduced sequence in the selective medium. Stable, transfected cells exhibiting resistance 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 present 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 present invention are useful for inhibiting tumor growth, inducing differentiation, reducing tumor volume, and / or reducing tumorigenesis. The method of use may be in vitro, ex vivo, or in vivo.

[0408] In one embodiment, 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 "detection" includes quantitative or qualitative detection. In certain embodiments, the biological sample includes 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 embodiment, the present invention provides a method for detecting the presence of PMEL17 or hepatitis B in a biological sample. In a particular embodiment, the method includes contacting the biological sample with an anti-PMEL17 antibody under conditions that allow the antibody to bind to the antigen, and detecting whether a complex is formed between the antibody and the antigen.

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

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

[0412] The binding of anti-PMEL17 antibodies to PMEL17 can be detected using certain other methods. Such methods include, but are not limited to, antigen-binding assays well known in the art, such as Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, fluorescence immunoassay, protein A immunoassay, and immunohistochemistry (IHC).

[0413] In certain embodiments, the anti-PMEL17 antibody is labeled. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescence, color development, high electron density, chemiluminescence, and radiation labeling), and indirectly detectable moieties, such as enzymes or ligands, via enzymatic reactions or molecular interactions.

[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 immobilization of the anti-PMEL17 antibody before the assay procedure by adsorption to an insoluble matrix or surface (Bennich et al., U.S. Patent No. 3,720,760), or by covalent bonding (e.g., using glutaraldehyde crosslinking), or by immobilization of the anti-PMEL17 antibody after the formation of a complex between the anti-PMEL17 antibody and the PMEL17 protein, for example, by immunoprecipitation.

[0415] Any of the above-described diagnostic or detection embodiments may be carried out using the antibody-drug conjugate of the present invention in place of, or in addition to, the anti-PMEL17 antibody.

[0416] In one embodiment, the present invention provides a method for treating or preventing a disease, comprising administering the antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention to a patient. The present invention also provides the use of the antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention for treating or preventing a disease in a patient. In some embodiments, the present invention provides the antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention for use in treating or preventing a disease in a patient. In further embodiments, the present invention provides the use of the antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention in the manufacture of a pharmaceutical product 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, 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, cancer is characterized by increased expression of PMEL17 compared to healthy patients. In some embodiments, PMEL17 expression can be measured by an increase in PMEL17 RNA. In other embodiments, cancer is characterized by an increase in the 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, cancer is characterized by mutations, e.g., 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 their metastatic forms.

[0418] The present invention provides a method for treating or preventing cancer, comprising administering a therapeutically effective amount of the 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 human. In certain embodiments, the cancer is drug-resistant and / or recurrent cancer.

[0419] In certain embodiments, the present invention provides a method for inhibiting tumor growth, comprising administering a therapeutically effective amount of the antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention to a subject. In certain embodiments, the tumor is a solid tumor, such as melanoma, uveal melanoma, hepatocellular carcinoma, or a metastatic cancer thereof. In certain embodiments, the subject is 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 an 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 mutations, for example, activating mutations affecting Q209 or R183 in the GNAQ and / or GNA11 genes.

[0421] The present invention also provides a method for selecting a patient for treatment with the 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 a particular embodiment of the present invention, the method comprises selecting a patient by measuring the expression of PMEL17. In a particular embodiment of the present invention, the method comprises selecting a patient by identifying mutations, for example, activating mutations affecting Q209 or R183 in the GNAQ or GNA11 gene. In a particular embodiment, the method comprises measuring the level of PMEL17 expression in a patient and detecting the GNAQ and / or GNA11 gene.

[0422] For the treatment or prevention of disease, the appropriate dosage of the antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention depends on various factors, such as the type of disease to be treated, the severity and progression of the disease, the response to the disease, prior treatment, and the patient's medical history. The antibody or drug may be administered in a single dose or over a series of treatments lasting from several days to several months, or until a cure is achieved or a reduction in the disease state (e.g., a reduction in tumor size) is achieved. The optimal administration schedule 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 the repetition rate of administration based on the measured residence time and the concentration of the drug in body fluids or tissues.

[0423] Combination therapy In certain cases, the antibodies, antibody fragments (e.g., antigen-binding fragments), or antibody-drug conjugates of the present invention are used in combination with other therapeutic procedures, such as surgery and radiotherapy, and therapeutic agents, such as other anticancer agents, anti-allergic agents, antiemetics (or anti-emetics), analgesics, cytoprotective agents, and combinations thereof.

[0424] In one embodiment, the antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention is combined with a second compound having anticancer properties in a combination drug formulation or administration regimen as a combination therapy. The second compound in the combination drug formulation or administration regimen may have complementary activity to the combined antibody or immunoconjugate in such a way that they do not adversely affect each other. For example, the antibody, antibody fragment (e.g., antigen-binding fragment), or antibody-drug conjugate of the present invention can be administered in combination with, but are 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] As used herein, the term "pharmaceutical combination" refers to a fixed combination in a single-dose unit form, or a non-fixed combination or kit of parts for concomitant administration, where two or more therapeutic agents may be administered simultaneously or independently, or separately within a certain time interval, and in particular, depending on the time interval, the combination partners may exhibit a synergistic effect.

[0426] The term “combination therapy” refers to the administration of two or more therapeutic agents to treat or prevent the symptoms or disorders of the treatment described herein. Such administrations include the simultaneous administration of the therapeutic agents substantially concurrently, for example, in a single capsule in which the ratio of the active ingredients is fixed. In addition, such administrations include the simultaneous administration of each active ingredient multiple times, 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 administrations also include the sequential use of each type of therapeutic agent, approximately concurrently or at different time points. In any case, the treatment regimen will provide the beneficial effect of the drug combination in treating or preventing the symptoms or disorders described herein.

[0427] Combination therapy can provide a "synergistic effect" and can be demonstrated to be "synergistic," that is, the effect achieved when the active ingredients are used together is greater than the sum of the effects derived from using the compounds separately. A synergistic effect can be achieved when the active ingredients are: (1) simultaneously administered or delivered in a combined, unit-dose formulation; (2) simultaneously delivered alternately or as separate formulations; or (3) in some other regimen. In the case of alternating therapy, a synergistic effect can be achieved when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. Generally, during alternating therapy, effective doses of each active ingredient are administered sequentially, i.e., consecutively, but in combination therapy, effective doses 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®), and carmustine (BiCNU®). ), 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 triphosphate liposomal 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®), Idamycin (Idamycin®), Ifosfamide (IFEX®), Irinotecan (Camptosar®), L-asparaginase (ELSPAR®), Leucovorin calcium, Melphalan (Alkeran®), 6-Mercaptopurine (Purinethol®), Methotrexate (Folex®), Mitoxantrone (Novantrone®), Mylotarg,Examples include paclitaxel (Taxol®), phoenix (yttrium 90 / MX-DTPA), pentostatin, polyfeprosan 20 (Gliadel®) with carmustine implant, tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), vinorelbine (Navelbine®), and pemetrexed.

[0429] In one embodiment, the present invention provides a method for treating or preventing cancer by administering the antibody-drug conjugate of the present invention in combination with one or more MDM2 inhibitors, PKC inhibitors, PRC2 inhibitors, MAPK inhibitors, GPCR inhibitors, tyrosine kinase inhibitors, and, for example, but not limited to, BTK inhibitors, EGFR inhibitors, Her2 inhibitors, Her3 inhibitors, IGFR inhibitors, and Met inhibitors to subjects requiring treatment or prevention of cancer.

[0430] For example, examples of MDM2 inhibitors include, but are not limited to, RG7112 (RO5045337); RG7388 (RO5503781, idasanutrin); 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, varanol; riluzole; staurosporine; enzastaurin; δV1-1 (KAI-9803 or delcasertib); εV1-2 (KAI-1678); aprinocarcene; midostaurin (PKC412); UCN-01 (7-hydroxy-staurosporine); rottrelin (5,7,dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzyl)-8-cinnamoyl-1,2-chromene); and briostatin 1.

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

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

[0434] For example, as tyrosine kinase inhibitors, but not limited to, ibrutinib (PCI-32765); erlotinib hydrochloride (Tarceva®); linifanib (N-[4-(3-amino-1H-indazole-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 Examples include ]-6-methoxy-7-[3-(4-methylpropyl-1-yl)propoxy]quinoline-3-carbonitrile, also known as SKI-606 and described in U.S. Patent No. 6,780,996); dasatinib (Sprycel®); pazopanib (Votrient®); sorafenib (Nexavar®); zactima (ZD6474); and imatinib or imatinib mesylate (Gilvec® and Gleevec®).

[0435] As epidermal growth factor receptor (EGFR) inhibitors, but not limited to the following, are 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]triazine-5-yl)methyl)piperidine-3-ol (BMS690514); Canertinib dihydrochloride (CI-1033); 6-[4-[(4-ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidine-4-ol Min (AEE788, CAS497839-62-0); Mbritinib (TAK165); Peritinib (EKB569); Afatinib (BIBW2992); Neratinib (HKI-272); N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazole-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-yl]-carbamic acid, (3S)-3-morpholinyl methyl ester (BMS599626); Examples include N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrole-5-yl]methoxy]-4-quinazolinamine (XL647, CAS781613-23-8); and 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidine-6-yl]phenol (PKI166, CAS187724-61-4).

[0436] Examples of EGFR antibodies include, but are not limited to, cetuximab (Erbitux®); panitumumab (Vectibix®); matsuzumab (EMD-72000); nimotuzumab (hR3); zaltumumab; 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®); and neratinib (HKI-272, (2E)-N-[4-[[3-chloro-4-[(pyridine-2-yl)methoxy]phenyl] [Amino]-3-cyano-7-ethoxyquinoline-6-yl]-4-(dimethylamino)buto-2-enamide, as described in International Publication No. 05 / 028443; lapatinib or lapatinib nitosylate (Tykerb®); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazine-5-yl)methyl)piperidine-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, CAS850140-72-6);N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazole-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-yl]-carbami Examples include quinazolinamine, (3S)-3-morpholinyl methyl ester (BMS599626, CAS714971-09-2); canertinib dihydrochloride (PD183805 or CI-1033); and N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrole-5-yl]methoxy]-4-quinazolinamine (XL647, CAS781613-23-8).

[0438] Examples of 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] As MET inhibitors, but not limited to, cabozantinib (XL184, CAS 849217-68-1); foretinib (GSK1363089, formerly XL880, CAS 849217-64-7); tivantinib (ARQ197, CAS 1000873-98-2); 1-(2-hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinoline-4-yloxy)pyridine-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-indole-1-ylsulfonyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrole-2-yl}methylene)-1,3-dihydro-2H-indole-2-one (SU11271); (3Z)-N-(3-chlorophenyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrole-2-yl}methylene) -N-methyl-2-oxoindoline-5-sulfonamide(SU11274);(3Z)-N-(3-chlorophenyl)-3-{[3,5-dimethyl-4-(3-morpholine-4-ylpropyl)-1H-pyrrole-2-yl]methylene}-N-methyl-2-oxoindoline-5-sulfonamide(SU11606);6-[difluoro[6-(1-methyl-1H-pyrazole-4-yl)-1,2,4-triazolo[4,3-b]pyridazine-3-yl]methyl]-quinoline(JNJ38877605, CAS943540- 75-8); 2-[4-[1-(quinoline-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazine-6-yl]-1H-pyrazole-1-yl]ethanol (PF04217903, CAS956905-27-4); N-((2R)-1,4-dioxan-2-ylmethyl)-N-methyl-N'-[3-(1-methyl-1H-pyrazole-4-yl)-5-oxo-5H-benzo[4,5]cyclopenta[1,2-b]pyridine-7-yl]sulfamide (MK2461, CAS917879-39-1);Examples include 6-[[6-(1-methyl-1H-pyrazole-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-pyrrole-2-yl]methylene]-1,3-dihydro-2H-indole-2-one (PHA665752, CAS 477575-56-7).

[0440] Examples of 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 a general overview, see, for example, Yee, JNCI, 104;975 (2012).

[0441] In another embodiment, the present invention provides a method for treating or preventing cancer by administering the 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, β-arrestin inhibitors, GRK inhibitors, MAPK inhibitors, PI3K inhibitors, JAK inhibitors, etc., to subjects requiring treatment or prevention of cancer.

[0442] For example, as phosphoinositide 3-kinase (PI3K) inhibitors, there are, but are not limited to, idelalisib (Zydelig, GS-1101, Cal-101), 4-[2-(1H-indazole-4-yl)-6-[[4-(methylsulfonyl)piperazine-1-yl]methyl]thieno[3,2-d]pyrimidine-4-yl]morpholine (also known as GDC0941, as seen in International Publication No. 09 / 036082 and International Publication No. 09 / 055). As described in Pamphlet No. 730); 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinoline-3-yl)-2,3-dihydroimidazo[4,5-c]quinoline-1-yl]phenyl]propionnitrile (also known as BEZ235 or NVP-BEZ235, as described in International Publication No. 06 / 122806); 4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidine-4-yl)pyridine-2-amine (Also known as BKM120 or NVP-BKM120, as described in International Publication No. 2007 / 084786); Tozacertib (VX680 or MK-0457, CAS 639089-54-6); (5Z)-5-[[4-(4-pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione (GSK 1059615, 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-(morpholine-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6).

[0443] In yet another embodiment, the present invention provides a method for treating or preventing cancer by administering the antibody-drug conjugate of the present invention in combination with one or more apoptosis promoters, for example, but not limited to, IAP inhibitors, Bcl2 inhibitors, MCl1 inhibitors, Trail agents, and Chk inhibitors, to subjects requiring treatment or prevention of cancer.

[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 International Publication 04 / 005284, International Publication 04 / 007529, International Publication 05 / 097791, International Publication 05 / 069894, International Publication 05 / 069888, International Publication 05 / 094818, U.S. Patent Application Publication 2006 / 0014700, U.S. Patent Application Publication 2006 / 0025347, International Publication 06 / 069063, International Publication 06 / 010118, International Publication 06 / 017295, and International Publication 08 / 134679, all of which are incorporated herein by reference.

[0445] As a BCL-2 inhibitor, the following are some examples, 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 [L]benzamide (also known as ABT-263 and described in International Publication No. 09 / 155386); tetrocalcin A; antimycin; gossypol ((-)BL-193); ovatocrax; ethyl-2-amino-6-cyclopentyl-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromone-3-carboxylate (HA14-1); oblimercen (G3139, Genasense®); Bak Examples include BH3 peptide; (-)-gossypol acetate (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 Navitocrax (ABT-263, CAS 923564-51-6).

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

[0447] As checkpoint kinase (CHK) inhibitors, the following but not limited to: 7-hydroxystaurosporine (UCN-01); 6-bromo-3-(1-methyl-1H-pyrazole-4-yl)-5-(3R)-3-piperidinyl-pyrazolo[1,5-a]pyrimidine-7-amine (SCH900776, CAS891494-63-6); 5-(3-fluorophenyl)-3-ureidothiophene- 2-Carboxylic acid N-[(S)-piperidine-3-yl]amide (AZD7762, CAS860352-01-8); 4-[((3S)-1-azabicyclo[2.2.2]octo-3-yl)amino]-3-(1H-benzimidazole-2-yl)-6-chloroquinoline-2(1H)-one (CHIR124, CAS405168-58-3); 7-aminodactinomycin (7-AAD), isogranulate Mido, debromohymenialdisine; N-[5-bromo-4-methyl-2-[(2S)-2-morpholinylmethoxy]-phenyl]-N'-(5-methyl-2-pyradinyl)urea (LY2603618, CAS911222-45-2); sulforaphane (CAS4478-93-7, 4-methylsulfinylbutyl isothiocyanate); 9,10,11,12-tetrahydro-9,12-epoxy-1H- Examples include diindro[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazosin-1,3(2H)-dione (SB-218078, CAS135897-06-2); 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 for treating or preventing cancer by administering the antibody-drug conjugate of the present invention in combination with one or more immunomodulators (e.g., one or more activators of costimulatory molecules or inhibitors of immune checkpoint molecules) to a subject requiring treatment or prevention of cancer.

[0449] In certain embodiments, the immunomodulator is an activator of the costimulatory molecule. In one embodiment, the agonist of the costimulatory molecule is selected from agonists 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 ligands (e.g., agonist antibodies or their antigen-binding fragments, or soluble fusions).

[0450] In certain embodiments, the immunomodulator is an inhibitor of an immune checkpoint molecule. In one embodiment, the immunomodulator is an inhibitor of PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and / or TGFR beta. 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 terms “inhibition” or “inhibitor” include reductions in the activity of a specific parameter, e.g., an immune checkpoint inhibitor. For example, inhibition of at least 5%, 10%, 20%, 30%, 40%, or 50% or more of activity, e.g., inhibition of PD-1 or PD-L1 activity, is included by this term. Thus, inhibition does not need to be 100%.

[0451] Inhibition of inhibitory molecules can be carried out at the DNA, RNA, or protein level. In some embodiments, the expression of the inhibitory molecule can be inhibited using inhibitory nucleic acids (e.g., dsRNA, siRNA, or shRNA). In other embodiments, the inhibitor of the inhibitory signal is a polypeptide, for example, a soluble ligand (e.g., PD-1-Ig or CTLA-4 Ig), or an antibody or its antigen-binding fragment that binds to the inhibitory molecule; for example, an antibody or its fragment 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 (also referred to herein as “antibody molecule”).

[0452] In one embodiment, the antibody molecule is a complete antibody or a fragment thereof (e.g., Fab, F(ab')2, Fv, or a single-chain Fv fragment (scFv)). In yet another embodiment, the antibody molecule has a heavy chain constant region (Fc) selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; in particular, one selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, and more specifically, 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 modified and mutated, for example, to alter the properties of the antibody molecule (e.g., to increase or decrease one or more of the Fc receptor binding, antibody glycosylation, 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, the bispecific antibody molecule has a first binding specificity to PD-1 or PD-L1 and a second binding specificity, for example, 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 yet 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 yet another embodiment, the bispecific antibody molecule binds to TIM-3 and LAG-3. Any combination of the above molecules can be produced in a multispecific antibody molecule, for example, a triplicate antibody having a first binding specificity to PD-1 or PD-L2, and second and third binding specificities to two or more of TIM-3, LAG-3, or PD-L2.

[0454] In certain embodiments, the immunomodulator is a PD-1 inhibitor, for example, a human PD-1 inhibitor. In another embodiment, the immunomodulator is a PD-L1 inhibitor, for example, a human PD-L1 inhibitor. In one embodiment, the PD-1 or PD-L1 inhibitor is an antibody molecule against PD-1 or PD-L1. The PD-1 or PD-L1 inhibitor can be administered alone or in combination with other immunomodulators, for example, in combination with LAG-3, TIM-3, or CTLA4 inhibitors. In an exemplary embodiment, the PD-1 or PD-L1 inhibitor, for example, an anti-PD-1 or PD-L1 antibody molecule, is administered in combination with a LAG-3 inhibitor, for example, an anti-LAG-3 antibody molecule. In another embodiment, a PD-1 or PD-L1 inhibitor, such as an anti-PD-1 or PD-L1 antibody molecule, is administered in combination with a TIM-3 inhibitor, such as an anti-TIM-3 antibody molecule. In yet another embodiment, a PD-1 or PD-L1 inhibitor, such as an anti-PD-1 antibody molecule, is administered in combination with a LAG-3 inhibitor, such as an anti-LAG-3 antibody molecule, and a TIM-3 inhibitor, such as an anti-TIM-3 antibody molecule. Other combinations of immunomodulators with 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 antibody molecule known in the art or disclosed herein can be used in the above combinations of checkpoint molecule inhibitors.

[0455] In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody selected from nivolumab, pembrolizumab, or pizilizumab. 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 International Publication No. 2006 / 121168.

[0456] In other embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (trade name KEYTRUDA, formerly known as lambrolizumab, 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, International Publication No. 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 International Publication No. 2009 / 101611. Other anti-PD1 antibodies are disclosed in U.S. Patent No. 8,609,089, U.S. Patent Application Publication No. 2010028330, and / or U.S. Patent Application Publication No. 20120114649. Another example of an anti-PD-1 antibody is 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 International Publication No. 2015 / 112900.

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

[0460] In some embodiments, the PD-Ll inhibitor is an anti-PD-Ll antibody. In some embodiments, the anti-PD-Ll inhibitor is selected from YW243.55.S70, MPDL3280A, MEDI-4736, or MDX-1105MSB-0010718C (also known as A09-246-2), which have sequences disclosed in, for example, International Publication No. 2013 / 0179174 (or sequences substantially identical or similar thereto, e.g., sequences identical by at least 85%, 90%, or 95% or more to a particular 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 International Publication No. 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 International Publication No. 2010 / 077634 (the heavy chain and light chain variable region sequences are shown 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 other embodiments, the PD-L2 inhibitor is AMP-224, a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1 (B7-DCIg; Amplimmune; disclosed, for example, in International Publication No. 2010 / 027827 and International Publication No. 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 anti-LAG3 antibody disclosed in International Publication No. 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 anti-TIM3 antibody disclosed in International Publication No. 2015 / 117002.

[0467] Pharmaceutical composition To prepare a pharmaceutical composition or sterile composition containing an 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 (for example, but not limited to, subcutaneous melanoma, uveal melanoma, hepatocellular carcinoma, and their metastatic cancers).

[0468] Therapeutic and diagnostic formulations may 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 (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: Disperse Systems, Marcel Dekker, NY, 1990; see Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY, 2000).

[0469] The selection of a therapeutic administration regimen depends on several factors, including the turnover rate of the entity's serum or tissue, the severity of the disease, the entity's immunogenicity, and the accessibility of target cells within the biological matrix. In certain embodiments, the administration regimen maximizes the amount of therapeutic agent delivered to the patient while maintaining an acceptable level of side effects. Therefore, the amount of biologic delivered depends in part on the specific entity and the severity of the condition being treated. Guidance is available for selecting appropriate doses of antibodies, cytokines, and small molecules (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 a clinician, for example, using parameters or factors known or presumed in the technique that affect or are expected to affect the treatment or prevention. Typically, the dose is started somewhat less than the appropriate dose and then increased in small increments, taking into account any negative side effects, until the desired or optimal effect is achieved. Important diagnostic measures include, for example, measures of symptoms of the infusion response. Important diagnostic measures include, for example, symptoms of inflammation or the level of inflammatory cytokines produced.

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

[0472] Compositions comprising the antibody or fragment thereof of the present invention may be provided by continuous infusion, or by administration at intervals of, for example, one day, one week, or one to seven times per week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, or once every eight weeks. Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation. Specific dose protocols should include the maximum dose or dose frequency to avoid undesirable major side effects.

[0473] The dosage of the immunoconjugate of the present invention administered to a patient may be 0.0001 mg / kg of the patient's body weight to 100 mg / kg. The dosage may be 0.0001 mg / kg of patient's body weight 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. The dosage of the antibody or fragment thereof according to the present invention can be calculated by multiplying the patient's body weight in kilograms (kg) by the dose to be administered in mg / kg.

[0474] The immunoconjugate of the present invention may be administered repeatedly, and the administration may be less than once a day, or divided into intervals of at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, 4 months, 5 months, or at least 6 months. In some embodiments, the immunoconjugate of the present invention can be administered twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, or less frequently. In specific embodiments, the administration of the immunoconjugate of the present invention is repeated every two weeks.

[0475] The effective dose for a particular patient may vary depending on factors such as the symptoms being treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects (see, for example, 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, topical application or skin application, subcutaneous injection or subcutaneous infusion, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal fluid, or intrafocal administration, or by a continuous release system or implant (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 (See 77:4030-4034, 1980; U.S. Patent Nos. 6,350,466 and 6,316,024). If necessary, the composition may also contain a solubilizer or a local anesthetic, such as lidocaine to relieve pain at the injection site, or both. In addition, pulmonary administration may also be used, for example, by the use of an inhaler or nebulizer, and by formulation with an aerosolizing agent. For example, see U.S. Patent 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 see International Publication Nos. 92 / 19244, 97 / 32572, 97 / 44013, 98 / 31346, and 99 / 66903 (each of these applications is incorporated herein by reference in its entirety).

[0477] The compositions of the present invention may also be administered via one or more routes of administration using one or more of the various methods known in the art. As will be recognized by those skilled in the art, the route and / or method of administration will vary depending on the desired outcome. Routes of administration for selected immunoconjugates of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral administration routes, e.g., by injection or infusion. Parenteral administration may typically refer to administration methods other than intestinal and topical administration by injection, and may include, but are not limited to, intravenous, intramuscular, intra-arterial, intra-shearing, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injections and infusions. In addition, the compositions of the present invention may be administered via parenteral routes, e.g., topical, epidermal, or mucosal administration routes, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical. In one embodiment, the immunoconjugate of the present invention is administered by infusion. In another embodiment, the immunoconjugate of the present invention is administered subcutaneously.

[0478] When the immunoconjugate of the present invention is administered via a controlled-release or sustained-release system, controlled-release or sustained-release can be achieved using a pump (see Langer, op. cit.; 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). The controlled release or sustained release of the therapeutic agent of the present invention can be achieved using polymer materials (see, for example, 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 See also 1:105,1989; U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; International Publication No. 99 / 15154; and International Publication No. 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), polyanhydride, 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 eluting impurities, stable during storage, sterile, and biodegradable. Controlled-release or sustained-release systems may be positioned close to the prophylactic or therapeutic target and therefore require only small systemic doses (see, e.g., Goodson, in Medical Applications of Controlled Release, op. cit., vol. 2, pp. 115-138, 1984).

[0479] The controlled-release system is discussed in the overview by Langer (Science 249:1527-1533, 1990). A sustained-release formulation containing one or more immunoconjugates of the present invention can be produced using any technique known to those skilled in the art. See, for example, U.S. Patent No. 4,526,938, International Publication No. 91 / 05548, International Publication No. 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 may be formulated in the form of ointments, creams, transdermal patches, lotions, gels, sprays, aerosols, liquids, emulsions, or other forms well 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 topical dosage forms that cannot be sprayed, a carrier or one or more excipients suitable for topical application are typically used, and in some examples, a viscous semi-solid or solid form having a dynamic viscosity greater than that of water is typically used. Suitable formulations include, but are not limited to, liquids, suspensions, emulsions, creams, ointments, powders, liniments, plasters, etc., which may be sterilized or mixed with excipients (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) to affect various properties, such as osmotic pressure. Other suitable topical dosage forms include, in some examples, sprayable aerosol preparations in which the active ingredient, combined with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile substance (e.g., a gaseous propellant, e.g., freon) or in a squeeze bottle. Moisturizers or humectants may also be added to the pharmaceutical composition and dosage form if desired. Examples of such additional components are well known in the art.

[0481] When a composition containing an immunoconjugate is administered intranasally, it may be formulated in the form of an aerosol, spray, mist, or drops. In particular, prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of aerosol spray dispensers from a pressurized pack or nebulizer by the 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 for delivering a measured amount. Capsules and cartridges (e.g., composed of gelatin) for use in an inhaler or inhaler may be formulated containing the compound and a suitable powder base, e.g., a powder mixture of lactose or starch.

[0482] Methods of co-administration or concurrent treatment with a second therapeutic agent, such as cytokines, steroids, chemotherapeutic agents, antibiotics, 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, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective amount of therapeutic agent may reduce symptoms by at least 10%; at least 20%; at least about 30%; at least 40%; or at least 50%.

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

[0484] In certain embodiments, the immunoconjugates of the present invention may be formulated to ensure appropriate in vivo distribution. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the immunoconjugates of the present invention cross the BBB (optionally), they may be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patent No. 4,522,811; U.S. Patent No. 5,374,548; and U.S. Patent No. 5,399,331. The liposomes may contain one or more moieties that are selectively transported into specific cells or organs and thus enhance targeted drug delivery (see, for example, Ranade, (1989) J. Clin. Pharmacol. 29:685). Examples of targeting moieties include phorate or biotin (see, e.g., U.S. Patent No. 5,416,016 by Low et al.); mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibody (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. Laukkanen (1994) FEBS See also Lett.346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0485] The present invention provides an administration protocol for a pharmaceutical composition, comprising the immunoconjugate of the present invention alone or in combination with other therapies, to a subject requiring such therapies. The combination therapy (preventive or therapeutic) of the present invention may be administered to the subject simultaneously or sequentially. The combination therapy (preventive or therapeutic) of the present invention may also be administered cyclically. Cycling therapy involves administering a first therapy (first preventive or therapeutic) for a period of time, then a second therapy (second preventive or therapeutic) for a period of time, and repeating this sequential administration (i.e., a cycle) to reduce the development of resistance to one of the therapys (e.g., agents), to avoid or mitigate the side effects of one of the therapys (e.g., agents), and / or to improve the efficacy of the therapy.

[0486] The combination therapy (prevention or treatment) of the present invention may be administered to the subject simultaneously.

[0487] The term "simultaneously" is not limited to administering treatments precisely at the same time, but rather means that the antibody-drug conjugate of the present invention is administered to the subject in an order and within a time interval such that it acts in conjunction with other treatments to enhance the benefits compared to ordinary administration. For example, each therapy may be administered to the subject simultaneously or sequentially in any order at different time points; however, if not administered simultaneously, they should be administered at times close enough to produce the desired therapeutic or preventive effect. Each therapy may be administered to the subject separately in any appropriate form and by any appropriate route. In various embodiments, the treatment (preventive or therapeutic agent) is administered to the subject at intervals of less than 5 minutes, less than 15 minutes, less than 30 minutes, less than 1 hour, about 1 hour, about 1 to 2 hours, about 2 to 3 hours, about 3 to 4 hours, about 4 to 5 hours, about 5 to 6 hours, about 6 to 7 hours, about 7 to 8 hours, about 8 to 9 hours, about 9 to 10 hours, about 10 to 11 hours, about 11 to 12 hours, 24 hours, 48 ​​hours, 72 hours, or 1 week apart. In other embodiments, two or more treatments (preventive or therapeutic agents) are administered during the same patient visit.

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

[0489] Example 1: Exemplary Linker-Drug Compound Synthesis 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-pentazacyclodocosane-6- Synthesis of (B1) methylpropyl(2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamide pentanoate 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-pentazacyclodocosan-6-yl)-2-methylpropyl(2S,3R)-3-((hydroxyhydrophosphoryl)oxy)-4-methyl-2-propionamidepentanoate(1-1): [ka] Imidazole (102 mg, 1.49 mmol, 15 equivalents) was dissolved in acetonitrile (ACN) (1.4 mL) and cooled in an ice bath at low temperature (crushing of ImH was observed, and the mixture was removed from the ice bath to dissolve ImH). Then, phosphorus trichloride (1.0 M in ACN) (499 μl, 0.499 mmol, 5 equivalents) was added dropwise (resulting in a white suspension), and the mixture was stirred for 10 minutes. Next, add triethylamine (250 μl, 1.796 mmol, 18 equivalents), stir the mixture for 40 minutes, and then add (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-pentazacyclodocosan-6-yl)-2-methylpropyl(2S,3R)-3-hydroxy-4-methyl-2-propionamidepentanoate (A1) (100 mg, 0.100 mmol, 1.0 equivalent; compound (A1) was obtained using the method described in Example 3-1) was added. The yellow-orange heterogeneous mixture was heated 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 g C18 column, neutral mobile phase). The product fraction was recovered and freeze-dried to yield H-phosphonate (1-1) as a yellow-white amorphous powder. LCMS: MH+=1066.3, 0.78 min (Acquity UPLC BEH C18 1.7um column, 2-minute runs at 2-98% 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-pentazacyclodocosan-6- Synthesis of (B1)-2-methylpropyl(2S,3R)-3-((((4-((S)-2-((S)-2-(3((2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamide pentanoate: [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-pentazacyclodocosan-6-yl)-2-methylpropyl(2S,3R)-3-((hydroxyhydrophosphoryl)oxy)-4-methyl-2-propionamide penta Noate (1-1) (100 mg, 0.094 mmol, 1.0 equivalent) and (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (108 mg, 0.188 mmol, 2.0 equivalents, CAS number 2055041-37-5) (both lyophilized powders were transferred to 10 mL vials) were dissolved in pyridine (4 mL). Then, pivaloyl chloride (0.058 mL, 0.469 mmol, 5 equivalents) was added dropwise to obtain a pale yellow solution. The mixture was stirred at room temperature for 10 minutes, and then an additional 1.0 equivalent of pivaloyl chloride was added. A freshly prepared iodine solution (47.6 mg, 0.188 mmol, 2.0 equivalents) was added to pyridine-water (14:1, 750 uL) to obtain a dark brown, clear solution.The mixture was stirred for 25 minutes and prepared directly by reverse-phase flash chromatography (40 g C-18 column, 0% Ac / MeCN for 3 minutes, then 0-60% ACN / water for 15 minutes, neutral method) to (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-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamide pentanoate (B-1) was produced. HRMS;MH+ = 1638.7700, 2.84 mins.

[0491] Examples 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-pentazacyclodocosan-6-yl) Synthesis of -2-methylpropyl(2S,3R)-2-acetamide-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-5-ureidopentanamide)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-pentazacyclodocosan-6-yl)-2-methylpropyl(2S,3R)-2-acetamido-3-((hydroxyhydrophosphoryl)oxy)-4-methylpentanoate(1-2): [ka] Imidazole (85 mg, 1.25 mmol, 15 equivalents) was dissolved in acetonitrile (ACN) (2.5 mL) and cooled in an ice bath at low temperature (crushing of ImH was observed, and the mixture was removed from the ice bath to dissolve ImH). Then, phosphorus trichloride (36.4 μl, 0.417 mmol, 5 equivalents dissolved in 0.5 mL of MeCN) was added dropwise (resulting in a white suspension), and the mixture was stirred for 10 minutes. Next, add triethylamine (174 μl, 1.25 mmol, 15 equivalents), stir the mixture for 40 minutes, and then add (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-pentazacyclodocosan-6-yl)-2-methylpropyl(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate (A2) (80 mg, 0.084 mmol, 1.0 equivalent; compound (A2) was obtained using the method described in Example 3-2) was added. The yellow-orange heterogeneous mixture was heated 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 g C18 column, neutral mobile phase). The product fraction was recovered and freeze-dried to yield H-phosphonate (1-2) as a yellow-white amorphous powder. LCMS: MH+=1024.3, 0.78 min (Acquity UPLC BEH C18 1.7um column, 2-minute runs at 2-98% NH4OH with water / MeCN+0.1%, 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-pentazacyclodocosan-6-yl Synthesis of )-2-methylpropyl(2S,3R)-2-acetamide-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-5-ureidopentanamide)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-pentazacyclodocosan-6-yl)-2-methylpropyl(2S,3R)-2-acetamido-3-((hydroxyhydrophosphoryl)oxy)-4-methylpentanoyl (1-2) (50 mg, 0.049 mmol, 1.0 equivalent) and (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS#2055041-37-5) (33.7 mg, 0.059 mmol, 1.2 equivalents) (both lyophilized powders were transferred to 10 mL vials) were dissolved in pyridine (1 mL). Then, pivaloyl chloride (0.042 mL, 0.342 mmol, 7 equivalents) was added dropwise to obtain a pale yellow solution. The mixture was stirred at room temperature for 30 minutes. A freshly prepared iodine solution (49.6 mg, 0.195 mmol, 4 equivalents) was added to pyridine-water (20:1, 500 uL) to obtain a dark brown, clear solution.The mixture was stirred for 30 minutes and prepared directly by reverse-phase flash chromatography (40 g C-18 column, 0% Ac / MeCN for 3 minutes, followed by 0-70% ACN / water for 15 minutes, neutral method) to (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-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamide pentanoate (B2) was produced. HRMS;MH+ = 1595.7200, 2.25 mins.

[0493] Examples 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-pentazacyclodocosan-6-yl)- Synthesis of 2-methylpropyl(2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamide pentanoate (B3) [ka] Compound (B3) can be obtained using the same procedure as described in Example 1-1, but in step 1, compound (A3) (from Example 3-3) is used instead of compound (A1).

[0494] Examples 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-pentazacyclodocosa Synthesis of (B4) 6-yl)-2-methylpropyl(2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamide pentanoate [ka] Compound (B4) was obtained using the same procedure as described in Example 1-1, except that in step 2, (S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)-3-methylbutanamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS#1949793-46-7) was replaced with (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS#2055041-37-5). HRMS; MH+ = 1594.5400, 2.88 min.

[0495] Examples 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-pentazacyclodocosane-6 Synthesis of -yl)-2-methylpropyl(2S,3R)-2-acetamide-3-((((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)-3-methylbutanamide)-5-ureidopentanamide)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-pyrrole-1-yl)propanamide)-3-methylbutanamide)-N-(4-(hydroxymethyl) (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS# is 2055041-37-5).

[0496] Examples 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-pentazacyclodocosane-6- Synthesis of (B6) 2-methylpropyl(2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl)oxy)(hydroxy)phosphoryl)oxy)-4-methyl-2-propionamide pentanoate [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-pyrrole-1-yl)propanamide)-3-methylbutanamide)-N-(4-(hydroxymethyl)fer (S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS# is 2055041-37-5).

[0497] Examples 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-pentazacyclodocosane Synthesis of -6-yl)-2-methylpropyl(2S,3R)-3-((((4-((S)-2-((S)-2-(3((2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl)oxy)carbonyl)oxy)4-methyl-2-propionamide pentanoate (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-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CAS# is 2055041-37-5). Chloroformate (1-3) can be obtained by reacting compound (A1) with phosgene.

[0498] Examples 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-pentazacyclodocosane-6 Synthesis of -yl)-2-methylpropyl(2S,3R)-2-acetamide-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl)oxy)carbonyl)oxy)4-methylpentanoate (B8) [ka] Compound (B8) can be obtained using the method described in Examples 1-7, but compound (A2) is used in place of compound (A1).

[0499] Examples 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-pentazacyclodocosane-6- Synthesis of (B9) 2-methylpropyl(2S,3R)-3-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)propanamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl)oxy)carbonyl)oxy)4-methyl-2-propionamide pentanoate [ka] Compound (B9) can be obtained using the method described in Examples 1-7, but compound (A3) is used in place of compound (A1).

[0500] Examples 1-10: (R)-1-((3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-22-isopropyl-3-((R)-1-methoxyeth...

Claims

1. An antibody or antigen-binding fragment thereof that binds to the human PMEL17 protein, a. A heavy chain variable region (VH) including heavy chain complementarity determination region 1 (CDR1) of SEQ ID NO: 79, heavy chain complementarity determination region 2 (CDR2) of SEQ ID NO: 80, and heavy chain complementarity determination region 3 (CDR3) of SEQ ID NO: 81; and a light chain variable region (VL) including 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; b. VH containing heavy chain CDR1 of SEQ ID NO: 82, heavy chain CDR2 of SEQ ID NO: 80, and heavy chain CDR3 of SEQ ID NO: 81; and VL containing 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; c. VH containing heavy chain CDR1 of SEQ ID NO: 83, heavy chain CDR2 of SEQ ID NO: 84, and heavy chain CDR3 of SEQ ID NO: 81; and VL containing 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; or d. VH containing heavy chain CDR1 of SEQ ID NO: 85, heavy chain CDR2 of SEQ ID NO: 86, and heavy chain CDR3 of SEQ ID NO: 87; and VL containing 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 An antibody containing an antigen-binding fragment thereof.

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

99.

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

101.

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

5. The antibody or antigen-binding fragment according to claim 4, wherein the one or more cysteine ​​substitutions are selected from E152C, S375C, or both E152C and S375C of the heavy chain of the antibody or antigen-binding fragment, 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 both GNAQ and GNA11; Ab is an antibody or antigen-binding fragment thereof that binds to the human PMEL17 protein according to any one of claims 1 to 6; L Ａ It is a linker; n is 1, 2, 3, or 4. (y is 1, 2, 3, or 4) Antibody-drug conjugates containing such substances.

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

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

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

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

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

13. D 【Chemistry 1】 The antibody-drug conjugate according to any one of claims 7 to 11.

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

15. The following structure 【Transformation 3】 An antibody-drug conjugate according to any one of claims 7 to 13, having the following characteristics.

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

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

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

19. A pharmaceutical composition according to claim 18 for use in the treatment or prevention of cancer in patients requiring treatment, wherein the cancer contains a mutation in the GNAQ gene or GNA11 gene expressing PMEL17, or contains a mutation in the GNAQ gene, the GNA11 gene, or both the GNAQ and GNA11 genes expressing PMEL17.

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

21. The pharmaceutical composition according to claim 20, wherein the one or more additional therapeutic compounds are selected from standard therapeutic chemotherapeutic agents, MDM2 inhibitors, MRC2 inhibitors, PKC inhibitors, MAPK inhibitors, costimulatory molecules, or checkpoint inhibitors.

22. The pharmaceutical composition according to 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 a CD83 ligand agonist.

23. The pharmaceutical composition according to claim 21, wherein the checkpoint inhibitor is selected from inhibitors 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 metastatic cancer thereof.

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

26. The nucleic acid according to claim 25, wherein the nucleic acid comprises the nucleotide sequences of sequence numbers 91 and 102.

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

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

29. A method for producing an antibody or antigen-binding fragment, comprising culturing the host cells described in claim 28 and recovering the antibody or its antigen-binding fragment from the cell culture.

30. The ability to recover the antibody or its antigen-binding fragment from cell cultures a) A step of removing cells and filtering the culture; b) A step of purifying the culture by affinity chromatography; c) Inactivating all viruses in the cell culture by adjusting the pH to 3.4 to 3.6, then readjusting the pH to 5.8 to 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) The step of performing anion exchange chromatography on the cell culture; f) A step of removing the virus by nanofiltration; g) filtering the cell culture containing the antibody or its antigen-binding fragment; and h) Step of obtaining a purified antibody or its antigen-binding fragment. The method according to claim 29, including the method described in claim 29.

31. A method for producing an anti-PMEL17 antibody drug conjugate, (a) The following equation (B): R ８ -L Ｂ -(D) ｎ (B) (In the formula, D is a GNAQ inhibitor, a GNA11 inhibitor, or an inhibitor of both GNAQ and GNA11; R ８ is a reactive group; L Ｂ It is a cleavable or non-cleavable linker, n is 1, 2, 3, or 4. Pre-forming the linker-drug portion; (b) Conjugating the linker-drug portion to the antibody or its antigen-binding fragment recovered from the cell culture as described in claim 29 or 30 to produce an antibody-drug conjugate; and (c) Purify the antibody-drug conjugate. Methods that include...

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

33. The diagnostic reagent according to claim 32, wherein the antibody or its antigen-binding fragment is labeled with radiolabeling, fluorescent labeling, a chromophore, an imaging agent, or a metal ion.

34. The following structure: 【Transformation 6】 Including; Here, Ab is a. VH containing heavy chain CDR1 of SEQ ID NO: 79, heavy chain CDR2 of SEQ ID NO: 80, and heavy chain CDR3 of SEQ ID NO: 81; and VL containing 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; b. VH containing heavy chain CDR1 of SEQ ID NO: 82, heavy chain CDR2 of SEQ ID NO: 80, and heavy chain CDR3 of SEQ ID NO: 81; and VL containing 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; c. VH containing heavy chain CDR1 of SEQ ID NO: 83, heavy chain CDR2 of SEQ ID NO: 84, and heavy chain CDR3 of SEQ ID NO: 81; and VL containing 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; or d. VH containing heavy chain CDR1 of SEQ ID NO: 85, heavy chain CDR2 of SEQ ID NO: 86, and heavy chain CDR3 of SEQ ID NO: 87; and VL containing 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 It includes, and y is 2. An antibody or its antigen-binding fragment that binds to the human PMEL17 protein, Antibody-drug conjugate.

35. The following structure: 【Transformation 7】 Including; Here, Ab is, It comprises VH containing the amino acid sequence of SEQ ID NO: 88 and VL containing the amino acid sequence of SEQ ID NO: 99; and y is 2. An antibody or its antigen-binding fragment that binds to the human PMEL17 protein, Antibody-drug conjugate.

36. The following structure: 【Transformation 8】 It comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 90, except that position 375 is S when numbered according to the SEQ ID NO: 90 sequence or EU system, and a light chain containing the amino acid sequence of SEQ ID NO: 101; and y is 2. An antibody or its antigen-binding fragment that binds to the human PMEL17 protein, Antibody-drug conjugate.

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

38. A pharmaceutical composition according to claim 37 for use in the treatment or prevention of cancer in patients requiring treatment, wherein the cancer contains a mutation in the GNAQ gene or the GNA11 gene that expresses PMEL17, or expresses PMEL17 and contains a mutation in the GNAQ gene, the GNA11 gene, or both the GNAQ gene and the GNA11 gene.

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