Characterizing head and neck tumor neoantigensand T cells: looking beyond the usual suspects

项目摘要 靶向由主要组织相容性呈递的癌症特异性抗原（“新抗原”）的免疫疗法 MHC复合物具有提高长期无病生存率的高潜力。T细胞工程师- 在有限数量的临床试验中，新兴技术最近取得了成功，并承诺将迎来一个新的EF时代。 有效的治疗，大大减少了不良反应。与黑色素瘤不同， 头颈部鳞状细胞癌（HNSCC）具有中度至低度的肿瘤浸润性， 负担因此，他们不太可能在他们的MHC分子上呈递相同类型的携带突变的抗原。 通过先前的研究所追求的cules。然而，新的HNSCC诊断越来越多地与人类相关。 乳头瘤病毒（HPV）感染，提供多种类型的替代的、治疗上有用的新抗原， 是可以合理定位的。与多种癌症新抗原类别兼容的广泛发现平台， 包括由HPV诱导的那些，将T细胞工程方法扩展到HNSCC。最终 本研究的目的是确定患者肿瘤在MHC上呈现的广泛的HNSCC新抗原， 以及识别它们的特异性T细胞受体。本建议的主要目的是：（一）比较 （ii）测试HPV依赖性HNSCC抗原与其他癌症特异性抗原结合的程度; 目前的HNSCC疗法可以诱导新抗原的呈递;和（iii）表征新抗原的演变- 接受检查点阻断（PD 1）的患者中的特异性效应T细胞及其T细胞受体库 疗法在目标1中，我们将扩展我们以前用于识别淋巴瘤特异性新抗原的方法 来源于免疫球蛋白基因座的重排。通过评估细胞系和原代HNSCC， 肿瘤DNA测序（外显子组和HPV聚焦）和蛋白质组（蛋白质表达，MHC抗原 发现）技术，我们将评估HPV作为新抗原可靠来源的程度， 其他潜在来源。我们开发的从头肽测序方法将使我们能够发现新抗- 这些基因将逃脱传统的蛋白质组学搜索方法。在目标2中，我们将用ra-1处理HNSCC细胞。 在培养物和患者来源的小鼠异种移植物中的放射、顺铂和干扰素γ。我们会监察 Aim中使用的蛋白质组学技术治疗诱导的新抗原呈递的变化 1.在目标3中，我们将生产从我们的初步数据中鉴定的优先新抗原的合成版本 并通过目标1和2所揭示的内容予以加强。我们会把它们和相应的MHC分子- cules并以多重方式，产生条形码化的MHC四聚体组。这些将用于 分析来自HNSCC患者的T淋巴细胞群，将从其对克隆T细胞受体进行测序。 因此，我们的目标是缓解目前限制新抗原靶向免疫治疗的两个主要障碍： 将提供高通量的方法来鉴定HNSCC新抗原及其同源T细胞受体。这个ap- 该方法应广泛适用于其他癌症。

PROJECT SUMMARY Immunotherapies targeting cancer-specific antigens (“neoantigens”) presented by Major Histocompatibility Complexes (MHC) have high potential for improving rates of long-term, disease-free survival. T cell engineer- ing technologies have found recent success in a limited number of clinical trials, and promise a new era of ef- fective treatments with greatly reduced adverse responses. Unlike melanoma, which neoantigen-targeting therapies have focused, head and neck squamous cell carcinomas (HNSCCs) have moderate-to-low tumor burdens. Thus they are less likely to present the same types of mutation-bearing antigens on their MHC mole- cules pursued by prior studies. However, new HNSCCs diagnoses are increasingly associated with human papilloma virus (HPV) infection, providing multiple types of alternative, therapeutically useful neoantigens that can be rationally targeted. A broad discovery platform compatible with multiple cancer neoantigen categories, including those induced by HPV, stands to extend T cell engineering approaches to HNSCC. The ultimate goal of this research is to define the broad range of HNSCC neoantigens a patient’s tumor presents on MHC, and the specific T cell receptors that recognize them. The main objectives of this proposal are (i) to compare HPV-dependent HNSCC antigens to other cancer-specific antigens; (ii) to test the extent to which neoantigen presentation can be induced by current HNSCC therapies; and (iii) to characterize the evolution of neoantigen- specific effector T cells and their T cell receptor repertoires in patients undergoing checkpoint blockade (PD1) therapy. In Aim 1, we will extend the approach we previously used to identify lymphoma-specific neoantigens derived from rearrangements of the immunoglobulin gene locus. By evaluating cell lines and primary HNSCC tumors with both DNA sequencing (exome and HPV-focused) and proteomic (protein expression, MHC antigen discovery) technologies, we will evaluate the extent to which HPV is a reliable source of neoantigens relative to other potential sources. De novo peptide sequencing methods we developed will allow us to discover neoanti- gens that would escape conventional proteomic search methods. In Aim 2, we will treat HNSCC cells with ra- diation, cisplatin, and interferon gamma in culture and in patient-derived mouse xenografts. We will monitor changes in neoantigen presentation induced by these treatments with the proteomic technologies used in Aim 1. In Aim 3, we will produce synthetic versions of prioritized neoantigens identified from our preliminary data and augmented by those revealed by Aims 1 and 2. We will assemble these with corresponding MHC mole- cules and in a multiplexed fashion, create panels of barcoded MHC tetramer panels. These will be used to profile T lymphocyte populations from HNSCC patients, from which clonal T cell receptors will be sequenced. Thus, our aims relieve the two major obstacles that currently limit neoantigen-targeting immunotherapies: they will provide high-throughput ways to identify HNSCC neoantigens and their cognate T cell receptors. This ap- proach should be broadly applicable to other cancers.