Elimination of melanoma by sortase A-generated TCR-like antibody-drug conjugates (TL-ADCs) targeting intracellular melanoma antigen MART-1.

Most tumor-associated proteins are located inside tumor cells and thus are not accessible to current marketed therapeutic monoclonal antibodies or their cytotoxic conjugates. Human leukocyte antigen (HLA) class I can present peptides derived from intracellular tumor-associated proteins and somatically mutated proteins on the cell's surface, forming an HLA/peptide complex as tumor-specific antigens for T cell receptor (TCR) recognition. Therefore, HLA-mediated presentation of intracellular tumor antigen peptides provides a viable way to distinguish tumor cells from normal cells, which is important for broadening antigen selection, especially for antibody-drug conjugates (ADCs) regarding their highly cytotoxic payload. We applied sortase A-mediated conjugation to develop TCR-like ADCs (i.e., EA1 HL-vcMMAE) targeting intracellular MART-1 protein, a melanocyte-differentiating antigen specific for metastatic melanomas, via the cell surface HLA-A2/MART-126-35 peptide complex. Homogenous EA1 HL-vcMMAE (drug to antibody ratio of 4) efficiently eliminated melanoma cells in xenograft mouse models with no obvious toxicity at the therapeutic dosage. Trametinib, an MEK inhibitor serving as an HLA expression enhancing agent, augmented the TL-ADCs' efficacy both in vitro and in vivo by upregulating MART-126-35 peptide presentation, thus providing a strategy for overcoming the limitation of antigen presentation level for TL-ADCs. Hence, our findings validate the strategy of using sortase A-generated TL-ADCs to target tumor-specific intracellular proteins, with or without agents present, to increase presenting TCR epitope peptides.