Effective anti-tumor immunity in humans has been associated with the presence of T cells directed at cancer neoantigens1, which are T cell epitopes with tumor-specific expression arising from non-silent somatic mutations. They are highly immunogenic because they are not expressed in normal tissues and hence bypass central thymic tolerance. Although neoantigens were long-envisioned as optimal targets for an anti-tumor immune response2, their systematic discovery and evaluation only became feasible with the recent availability of massively-parallel sequencing for detection of all coding mutations within tumors, and of machine learning approaches to reliably predict those mutated peptides with high-affinity binding of autologous HLA molecules. We hypothesized that vaccination with neoantigens can both expand pre-existing neoantigen-specific T cell populations and induce a broader repertoire of new T cell specificities in cancer patients, tipping the intra-tumoral balance in favor of enhanced tumor control. Here we demonstrate the feasibility, safety and immunogenicity of a vaccine that targets up to 20 predicted personal tumor neoantigens. Vaccine-induced polyfunctional CD4+ and CD8+ T cells targeted 58 (60%) and 15 (16%), respectively, of the 97 unique neoantigens used across patients. These T cells discriminated mutated from wildtype antigens, and in some cases, directly recognized autologous tumor. Of 6 vaccinated patients, 4 had no recurrence at 25 months post-vaccination, while 2 with progressive disease were subsequently treated with anti-PD-1 therapy and experienced complete tumor regression, with expansion of the repertoire of neoantigen-specific T cells. These data provide a strong rationale for further development of this approach, alone and in combination with checkpoint therapies.

SUMMARY Immune checkpoint inhibitors (ICIs) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA sequencing (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients. CDK4/6-inhibition represses this program in individual malignant cells, induces senescence, and reduces melanoma tumor outgrowth in mouse models in vivo when given in combination with immunotherapy. Our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and suggests new therapeutic strategies to overcome immunotherapy resistance.