Attacking the Immunopeptidome of Ewing Sarcoma

项目摘要 实体癌是儿童癌症相关死亡的主要原因， 儿童实体瘤免疫治疗的最新进展。免疫检查点抑制（ICI） ICI是成人实体瘤最有效的免疫治疗形式，但ICI对儿童实体瘤无效。 这种差异可以通过儿科实体瘤的低突变负担来解释，因为新生抗原的产生是由于肿瘤细胞的增殖而引起的。 来自肿瘤特异性突变的靶基因是最有效的ICI诱导的免疫应答的靶标。过表达 在正常的重要组织上不表达的非突变的自身抗原可以作为有效治疗的基础。 免疫疗法，但必须克服免疫耐受性，以诱导对这类 分子。该项目的重点是尤文肉瘤（EWS）的原型低突变负荷的实体瘤， 进展已经停滞。EWS的标准疗法依赖于剂量密集型方案，主要是在 20世纪70年代和80年代，给幸存者留下了严重的终身后遗症。目前还没有针对性的治疗方法 证明是有效的。很少有转移性或复发性EWS患者存活。使用免疫肽组 通过分析，我们发现了来自脂肪酶-1（LIPI）和IGF 2结合蛋白1（IGF 2BP 1）的新肽， 由HLA-A2+在EWS上呈现。这些非突变蛋白在绝大多数细胞中高水平过表达， EWS和基本上不存在于重要的正常组织，从而证明了一个非常有利的概况， 免疫靶向为了将这一发现转化为EWS的治疗应用，该项目应用了 我们开发了一个工作流程，以发现，表征和工程化T细胞受体（TCR），靶向这些 缩氨酸该项目应对的主要总体挑战是确定最佳方法， 鉴定和/或工程化能够靶向自身抗原而不引起交叉反应性高效TCR 会导致不可接受的毒性在目标1中，我们检验了靶向LIPI-和LIPI-的TCR的假设。 IGF 2BP 1衍生肽将在HLA-A2+宿主中鉴定，但由于免疫缺陷， 宽容我们将同时发现和比较HLA-A2宿主中存在的抗原反应性TCR， 我们预测将更有效，但由于交叉反应性可能不安全。在目标2中，我们使用下一代 将在HLA-A2+宿主中鉴定的天然TCR工程化为更有效但安全的抗原特异性TCR的方法 TCR，通过亲和力成熟或捕获键工程。考虑到已知的高剂量的交叉反应性风险， 这里开发的下一代工程TCR将在几个平台上进行密切审查 用于交叉反应性。在目标3中，我们使用Mackall实验室开发的健身增强功能来增强效力 增强表达我们的主要候选物LIPI-和IGF 2BP 1-反应性的T细胞的效力 TCR。在这个项目中进行的工作将提供最先进的治疗准备临床试验， EWS，并提供关于工程TCR靶向自我的最佳方法的一般理解， 抗原，这将在儿科肿瘤学和EWS以外的低突变负荷癌症中提供价值。

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.