Liquid biopsy-based toolkits for neoantigen and cognate TCR discovery for cancer immunotherapy

项目总结/摘要 来源于肿瘤特异性突变的新抗原是当前癌症的主要驱动力。 免疫疗法新抗原和抗原特异性T细胞之间的相互作用使T细胞识别成为可能 和肿瘤杀伤。解析新抗原和抗原反应性T细胞通常需要新鲜（或速冻） 肿瘤材料，可以从其中对肿瘤组织进行测序，并且可以分离肿瘤浸润淋巴细胞 并且扩大了。不幸的是，新鲜（或快速冷冻）肿瘤组织活检的可用性是一个重要的问题。 个性化免疫治疗的限制因素。石蜡包埋病理标本的测序质量 对于新抗原发现来说常常是次优的。来自单个肿瘤病变的小活检的测序数据可以 不能代表肿瘤的全部克隆谱。更重要的是，肿瘤的分子特征 随着时间的推移而动态地演变。但是对于患有多种癌症的患者来说，重复活检取样很少可行 需要进行侵入性活检 循环肿瘤细胞（CTC）和匹配的血液淋巴细胞是新抗原和同源抗原的良好替代物。 TCR发现。它们使临床医生能够反复和非侵入性地询问突变景观 和抗肿瘤免疫的动态演变。一个重大的技术 这里的挑战是开发高通量，低样本量，微创工具来解决 新抗原和同源TCR动力学以及TCR基因与特异性新抗原的匹配。我们建议 通过战略整合开发基于液体活检的新抗原和同源TCR发现工具包 新型芯片上图像细胞仪平台和高度模块化的纳米颗粒条形码单分子 肽-MHC四聚体细胞分选测定。我们将进行先进的开发和严格的验证， 工具包在这项工作中我们将研究在免疫过程中新抗原和TCR库的动态演变。 使用从黑素瘤患者连续收集的血液样品进行免疫检查点阻断。一旦成功 完成后，它将为基于液体活检的新抗原和同源物提供一种简单的非侵入性方法。 TCR的发现对于那些手术活检不可用或无法实现的患者，具有变革性 个性化新抗原疫苗和基于T细胞的免疫疗法的潜力。新抗原和同源物 TCR动力学将为理解免疫治疗反应和耐药性提供重要见解。

Project Summary/Abstract Neoantigens derived from tumor-specific mutations are a major driving force behind the current cancer immunotherapies. The interactions between neoantigens and antigen-specific T cells enable T cell recognition and tumor killing. Resolving neoantigens and antigen-reactive T cells normally requires fresh (or snap frozen) tumor materials from which the tumor tissue can be sequenced and tumor-infiltrating lymphocytes can be isolated and expanded. Unfortunately, the availability of fresh (or snap frozen) tumor tissue biopsies is a significant limiting factor for personalized immunotherapy. Sequencing quality of paraffin-embedded pathological specimen is often suboptimal for neoantigen discovery. Sequencing data of a small biopsy from a single tumor lesion may not be representative of the tumor's full clonal spectrum. More importantly, the molecular profile of tumors evolves dynamically over time. But repeated biopsy sampling is seldom feasible for patients with many cancers that require invasive biopsy procedures. Circulating tumor cells (CTCs) and match blood lymphocytes are good surrogates for neoantigen and cognate TCR discovery. They enable clinicians to repeatedly and non-invasively interrogate the mutational landscape and dynamic evolution of the anti-tumor immunity via serially collected blood samples. A major technical challenge here is to develop high-throughput, low sample volume, minimally invasive tools to resolve the neoantigen and cognate TCR dynamics and to match the TCR genes with specific neoantigens. We propose to develop a toolkit for liquid biopsy-based neoantigen and cognate TCR discovery through a strategic integration of a novel on-chip image cytometry platform and a highly modular nanoparticle-barcoded single-molecule peptide-MHC tetramer cell sorting assay. We will perform advanced development and rigorous validation of the toolkit in this proposed work. We will investigate the dynamic evolution of neoantigen and TCR repertoires during immune checkpoint blockade using serially collected blood samples from melanoma patients. Upon successful completion, it will deliver a simple and noninvasive approach for liquid biopsy-based neoantigen and cognate TCR discovery for patients whose surgical biopsies are not available or not attainable, with transformative potentials on personalized neoantigen vaccines and T-cell based immunotherapies. The neoantigen and cognate TCR dynamics will provide critical insights into the understanding of immunotherapy response and resistance.