The success of T cell therapies for solid tumors has been limited by the scarcity of tumor-specific targets, the T cell exhaustion and immunosuppressive tumor microenvironment, and the limited persistence and T cell trafficking to the tumor, among others. To better understand how successful T cell responses induced by immune checkpoint blockade (ICB) eliminate metastatic tumors, we conducted a longitudinal landscape analysis of the neoepitope-specific T cells during treatment in patients with melanoma, with and without response to ICB. Using newly developed technologies to isolate neoepitope-specific T cells, we characterized the target of the T cell responses and their evolution over time in the peripheral blood and the tumor. Briefly, after computational prediction of patient-specific putative neoantigens, hundreds of capture reagents were made consisting of the patient HLA class I subtypes loaded with the corresponding predicted neoantigens; neoepitope-specific T cells were then isolated, and the TCR alpha and beta sequenced. Tumor reactivity of the neoantigen-specific TCRs was assessed upon co-culture of autologous melanoma cell lines from each patient with T cells gene-edited to replace their endogenous TCR with the isolated neoantigen-specific TCRs. In these studies, we showed that all patients presented neoantigen-specific T cell responses that were tumor-reactive and targeted a limited number of immunodominant epitopes. Interestingly, in patients with clinical benefit from ICB, these responses were polyclonal, with multiple TCR clonotypes specific for the same mutations, and recurrently detected over time. Our results show that even in patients without response to ICB, we could isolate neoantigen-specific TCRs that could potentially be used to engineer large numbers of T cells for adoptive T cell therapy.

Another alternative for patients that do not respond to ICB is adoptive T cell therapy with T cells genetically modified to target shared antigens. We propose targeting the surface expression of TYRP1 for the treatment of cutaneous and rare subtypes of melanoma. TYRP1 is mainly expressed intracellularly in the melanosomes, with a small fraction of the total protein localized on the cell surface. Using a fine-tuning engineering approach, we developed a highly-sensitive CAR-T cell therapy that detects the low amounts of TYRP1 localized on the cell surface of tumor cells with high TYRP1 intracellular overexpression. The TYRP1 CAR-T cell therapy presents robust antitumor activity without toxicity in murine and patient-derived cutaneous, acral, and uveal melanoma models. Based on the efficacy and safety profile of this therapy, we are currently planning the phase I clinical trial.