PROJECT SUMMARY/ABSTRACT Melanoma (MEL) is a model malignancy for studying the mechanisms of cancer immunotherapy. Antibodies that block negative regulators of T cell function, termed immune checkpoint blockade (ICB), have transformed the treatment of MEL and other solid cancers. Although some patients have durable disease control, many fail to respond or progress after initially experiencing tumor regression. Therapeutic resistance is enriched in two molecularly defined MEL subtypes. Twenty eight percent of MELs possess activating mutations (Mut) in the driver oncogene NRAS, the second most common Mut RAS isoform. Beyond MEL, Mut NRAS occurs in other prevalent malignancies, including colorectal cancer (CRC). We and others recently discovered that patients with Mut NRAS MEL and CRC have a significantly shorter time to treatment failure. Separately, ~30% of MELs acquire mutations in beta-2-microglobulin (B2M), an essential component of the human leukocyte antigen class I (HLA-I) complex, following ICB progression. Cancers with Mut B2M are intrinsically resistant to CD8+ T cell killing. Thus, two major gaps in knowledge that limit the potential of immunotherapy in MEL and other common cancers include: (1) identification of immunogenic antigens expressed by Mut NRAS tumors, and (2) therapeutic strategies to overcome genetic loss of HLA-I presentation. We hypothesize that cancers with Mut NRAS or Mut B2M can be therapeutically targeted using T cell receptor (TCR)-based immunotherapies. In support of our hypothesis, we discovered using a mass spectrometry (MS) screen that the three most common NRAS hotspot substitutions generate shared (or “public”) neoantigens (NeoAgs) presented by a prevalent HLA allele. Using a unique collection of biospecimens from patients who express an NRAS public NeoAg, we generated T cells specific for these epitopes, retrieved their TCR gene sequences, and transferred public NeoAg reactivity to polyclonal T cells. These results confirm the immunogenicity of screen-identified NRAS public NeoAgs and enable the development of TCR-based therapies. We further discovered that a significant proportion of MELs undergo direct killing by T cells that express an HLA class II (HLA-II) restricted TCR. Using a genome-scale CRISPR screen, we found that cancer eradication is preserved when B2M and other HLA-I genes are disrupted. Building on these preliminary data, we propose in Aim 1 to develop a novel therapeutic approach for cancers expressing an NRAS public NeoAg using TCR genetic engineering and adoptive cell transfer. In Aim 2, we will study the physical mechanisms underlying NRAS public NeoAg TCR specificity, including the unique capacity of some TCRs to accommodate multiple hotspot substitutions. In Aim 3, we will define the molecular basis for direct cancer cell killing by HLA class II-restricted TCRs and test combinations to enhance the antitumor efficacy of adoptively transferred CD4+ T cells. By completing these aims, we will de...