Recent advances in targeted therapy and immunotherapy have substantially improved the treatment of melanoma. However, therapeutic strategies are still needed for unresponsive or treatment-relapsed melanoma patients. To discover antibody-drug conjugate (ADC)-tractable cell surface targets for melanoma, we developed an atlas of melanoma cell surface binding antibodies (pAbs) using a proteome-scale antibody array platform (PETAL). Target identification of pAbs led to development of melanoma cell killing ADCs against LGR6, TRPM1, ASAP1, and MUC18, among others. MUC18 was overexpressed in both tumor cells and tumor-infiltrating blood vessels across major melanoma subtypes, making it a potential dual-compartment and universal melanoma therapeutic target. AMT-253, an MUC18-directed ADC based on topoisomerase I inhibitor exatecan and a self-immolative T moiety, had a higher therapeutic index compared to its microtubule inhibitor-based counterpart and favorable pharmacokinetics and tolerability in monkeys. AMT-253 exhibited MUC18-specific cytotoxicity through DNA damage and apoptosis and a strong bystander killing effect, leading to potent antitumor activities against melanoma cell line and patient-derived xenograft models. Tumor vasculature-targeting by a mouse MUC18-specific antibody-T1000-exatecan conjugate inhibited tumor growth in human melanoma xenografts. Combination therapy of AMT-253 with an anti-angiogenic agent generated higher efficacy than single agent in a mucosal melanoma model. Beyond melanoma, AMT-253 was also efficacious in a wide range of MUC18-expressing solid tumors. Efficient target/antibody discovery in combination with the T moiety-exatecan linker-payload exemplified here may facilitate discovery of new ADC to improve cancer treatment.

PETAL monoclonal antibody arrays (60,000 mAb printed on 2 x slides) were prepared as previously reported. Melanoma cells GAK, HMVII and A375 (1-2x10⁷) suspended in 2 ml 1x PBS were first labeled with DNA dye Syto-62 (ThermoFisher, S11344) at 1:10000 ratio for 10 minutes. Labeled cells were then incubated with PETAL array for 30 minutes at 37°C without shaking (0.5-1x10⁷ cells per array). After a gentle washing step in 1x PBS buffer to remove unbound cells, the slides were scanned by GenePix 4200A (Modecular Device). Images were analyzed by GenePix Pro software (GenePix Pro, RRID:SCR_010969).