Targeting alternative splicing for TCR discovery in small cell carcinomas

摘要 我们提出了一个合作的免疫治疗发现计划，利用替代前mRNA 剪接作为癌症特异性表位的来源，用于T细胞受体（TCR）治疗小细胞癌， 前列腺和肺小细胞癌起源于许多不同的上皮组织，但通常是 具有侵袭性，没有治愈性治疗，并且具有可怕的预后。小细胞肺癌（SCLC）是最常见的肺癌。 常见亚型小细胞前列腺癌（SCPC）是一种罕见的原发性疾病，但正在成为越来越多的 常见的是对剥夺性剥夺治疗的反应的晚期表型转变。新兴研究 表明尽管它们的起源组织不同，但SCPC和SCLC在行为上高度相似， 分子表型这表明有效的靶向治疗可以解决这两种恶性肿瘤。 我们的策略是确定在小细胞中通过选择性前mRNA剪接产生的癌症特异性表位， 然后使用这些靶点来开发基于TCR的治疗方法。嵌合抗原受体t细胞 已经开发了靶向细胞表面蛋白的CAR-T（CAR-T）疗法用于一些血液恶性肿瘤， 但是这种策略对于上皮肿瘤不成功。靶表位的有限癌症特异性 在人体试验中导致了显著的靶向、非肿瘤毒性。我们选择了TCR来扩展 细胞表面之外的可用目标池。我们假设利用额外的蛋白质组 通过对可变剪接外显子的详细分析揭示的多样性将提供更好的靶点。 我们的主要研究人员团队包括选择性剪接的计算生物学专家 (Yi Xing），癌细胞生物学和免疫学（Owen Witte），以及造血细胞发育和 Gay Crooks（同性恋骗子）我们正在收集来自小细胞癌和正常组织的RNA-Seq数据， 通过慢病毒从良性细胞衍生的SCPC和SCLC的公共数据集和新的人类细胞系模型 转导这个组合数据集是我们发现工作的基础。我们计划把它和 总蛋白质组学分析，以确定影响蛋白质组成的剪接异构体。这些数据将进一步 与确定呈递给免疫系统的肽库的免疫肽组学测定相结合 被靶向癌细胞杀死。衍生自选择性剪接事件的表位显示出高癌症特异性， 蛋白质表达和预测或观察到的表位呈递将优先用于TCR开发。 我们将使用这些表位从幼稚的人类T细胞群体中选择TCR，使用高度器官型的免疫组织化学方法。 Crooks实验室开发的体外人工人胸腺培养系统。

ABSTRACT We present a collaborative immunotherapeutics discovery program that exploits alternative pre-mRNA splicing as a source of cancer-specific epitopes for T-cell receptor (TCR) therapy of small cell carcinomas of the prostate and lung. Small cell carcinomas arise from many different epithelial tissues but are generally aggressive, have no curative treatment, and carry a dire prognosis. Small cell lung cancer (SCLC) is the most common subtype. Small cell prostate cancer (SCPC) is rare as a primary disease but is becoming increasingly common as a late-stage phenotypic transition in response to hormone-deprivation therapy. Emerging research indicates that despite their disparate tissues of origin, SCPC and SCLC are highly similar in behavior and molecular phenotype. This suggests effective targeted therapies could address both malignancies. Our strategy is to define cancer-specific epitopes created by alternative pre-mRNA splicing in small cell carcinomas and then use these targets to develop TCR-based therapeutics. Chimeric antigen receptor T-cell (CAR-T) therapies targeting cell surface proteins have been developed for some hematological malignancies, but this strategy has been unsuccessful for epithelial tumors. The limited cancer specificity of the target epitope has led to significant on-target, off-tumor toxicities in human trials. We have chosen to pursue TCRs to expand the pool of available targets beyond the cell surface. We hypothesize that tapping into the additional proteomic diversity revealed by a detailed analysis of alternatively spliced exons will provide better targets. Our team of principal investigators includes experts in the computational biology of alternative splicing (Yi Xing), cancer cell biology and immunology (Owen Witte), and hematopoietic cell development and immunology (Gay Crooks). We are compiling RNA-Seq data on small cell cancers and normal tissues from public datasets and new human cell line models of SCPC & SCLC derived from benign cells by lentiviral transduction. This combined dataset serves as the foundation for our discovery effort. We plan to pair this with total proteomics analysis to identify spliced isoforms that affect protein composition. This data will be further integrated with immunopeptidomics assays that define the pool of peptides presented to the immune system by the target cancer cells. Epitopes derived from alternative splicing events that show high cancer specificity, protein expression, and predicted or observed epitope presentation will be prioritized for TCR development. We will use these epitopes to select TCRs from naïve human T-cell populations using a highly organotypic in vitro artificial human thymic culture system developed in the Crooks laboratory.