Targeting alternative splicing for TCR discovery in small cell carcinomas

摘要 我们提出了一个合作的免疫疗法发现计划，该计划利用了替代的前-mRNAs 剪接作为T细胞受体(TCR)治疗小细胞癌的肿瘤特异性表位来源 前列腺和肺。小细胞癌起源于许多不同的上皮组织，但通常 攻击性强，没有根治方法，预后极差。小细胞肺癌(SCLC)是最常见的 公共子类型。小细胞前列腺癌(Scpc)是一种少见的原发病，但其发病率正日益上升。 常见为激素剥夺治疗反应的晚期表型转变。新兴研究 表明尽管它们起源的组织不同，但SCPC和SCLC在行为和 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。 我们的策略是确定通过在小细胞中选择性地剪接前mRNA而产生的癌症特异性表位 然后利用这些靶点来开发基于TCR的疗法。嵌合抗原受体T细胞 针对细胞表面蛋白的(CAR-T)疗法已被开发用于一些血液系统恶性肿瘤， 但这一策略在治疗上皮性肿瘤方面并不成功。靶标表位的肿瘤特异性有限 在人体试验中导致了显著的靶向和肿瘤外毒性。我们已经选择了TCR来扩张 细胞表面以外的可用靶子池。我们假设，利用额外的蛋白质组 通过对选择性剪接外显子的详细分析揭示的多样性将提供更好的靶标。 我们的主要研究团队包括选择性剪接的计算生物学专家 (易星)，癌细胞生物学和免疫学(Owen Witte)，以及造血细胞发育和 免疫学(Gay Crooks)。我们正在汇编小细胞癌和正常组织的RNA-Seq数据 慢病毒从良性细胞衍生的SCPC和SCLC的公开数据集和新的人类细胞系模型 转导。这个组合的数据集是我们发现工作的基础。我们计划将这一点与 总蛋白质组学分析，以确定影响蛋白质组成的剪接异构体。这一数据将进一步 与定义呈现给免疫系统的多肽库的免疫表位学分析相结合 通过靶标癌细胞。来自表现出高癌症特异性的选择性剪接事件的表位， TCR的开发将优先考虑蛋白质表达、预测或观察到的表位呈现。 我们将使用这些表位从幼稚的人类T细胞群体中选择TCR，使用的是高度器官型的 克鲁克斯实验室开发的体外人工人类胸腺培养系统。

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.