PROJECT SUMMARY Solid cancers are a leading cause of cancer related death in children and there is great interest in harnessing recent progress in immunotherapy for the treatment of pediatric solid tumors. Immune checkpoint inhibition (ICI) is the most active form of immunotherapy for adult solid cancers, but ICI is not effective in pediatric solid tumors. This discrepancy is explained by the low mutational burden of pediatric solid tumors, since neoantigens arising from tumor specific mutations are the target of the most potent ICI induced immune responses. Overexpressed non-mutated self-antigens, that are not expressed on normal vital tissues, can serve as the basis for effective immune therapies, but immune tolerance must be overcome to induce potent immune responses to this class of molecules. This project focuses on Ewing Sarcoma (EWS) a prototype low mutation burden solid tumor, for which progress has stalled. Standard therapies for EWS rely on dose intensive regimens largely developed in the 1970s and 80s which leave survivors with severe, lifelong late effects. No targeted therapeutics have been demonstrated to be effective. Few patients with metastatic or recurrent EWS survive. Using immunopeptidome profiling, we discovered novel peptides from lipase-1 (LIPI) and IGF2 binding protein 1 (IGF2BP1) that are presented by HLA-A2+ on EWS. These non-mutant proteins are overexpressed at high levels in the vast majority of EWS and are essentially absent from vital normal tissues, thereby demonstrating a very favorable profile for immune targeting. To translate this discovery into a therapeutic application for EWS, this project applies a workflow we developed to discover, characterize, and engineer T cells receptors (TCRs) targeting these peptides. The major overarching challenge that the project addresses is determining the optimal approach to identify and/or engineer high potency TCRs capable of targeting self-antigens without incurring cross-reactivity that would result in unacceptable toxicity. In Aim 1, we test the hypothesis that TCRs targeting LIPI- and IGF2BP1-derived peptides will be identified in HLA-A2+ hosts but will manifest low potency due to immune tolerance. We will simultaneously discover and compare antigen reactive TCRs present in HLA-A2– hosts, which we predict will be more potent, but may be unsafe due to cross-reactivity. In Aim 2, we use next generation approaches to engineer natural TCRs, identified in HLA-A2+ hosts, into more potent, but safe antigen-specific TCRs, through affinity maturation or catch bond engineering. Given the known risks for cross reactivity of high potency TCRs, next generation engineered TCRs developed here will be closely vetted across several platforms for cross-reactivity. In Aim 3, we use fitness enhancements developed in the Mackall lab to enhance the potency of CAR T cells to enhance the potency of T cells expressing our lead candidate LIPI- and IGF2BP1-reactive TCRs. The work conducted in this project will deliver state-of-the-art therapeutics ready for clinical testing in EWS and provide general understanding regarding the optimal approach to engineer TCRs targeting self- antigens, which will provide value in pediatric oncology and low mutation burden cancers beyond EWS.

PROJECT NARRATIVE This proposal is relevant to public health since Ewing sarcoma (EWS) is the second most common bone tumor of children and treatment remains suboptimal, with survival rates for patients who present without metastatic disease plateaued at 70% and less than 30% for patients who present with metastatic disease. Due to the dose intensity of current standard therapies, patients who survive EWS experience an unacceptable incidence and severity of late effects. The proposed research is highly relevant to the NIH mission, as it seeks to discover new approaches to harness the immune system for the treatment of EWS, which will deliver novel therapeutics for testing in this disease and will also provide generalizable knowledge regarding optimal approaches to develop immunotherapies for low mutational burden cancers.