Engineering T Cell Receptors for Adoptive Cell Therapies

描述（由申请人提供）：过继性T细胞疗法是治疗癌症的一种有效且高度特异性的方法。引入识别癌症抗原的受体提供了足够数量的具有适当特异性的T细胞来摧毁大的、已建立的肿瘤。迄今为止使用的受体包括针对pepMHC的T细胞受体（TCRs）或针对常规细胞表面癌症抗原的抗体（scFv）定向嵌合抗原受体（CARs）。本提案的目的是改进当前的治疗方法，并开发一种结合每种靶向方法优势的稳健策略。该策略涉及单链TCR （V¿-linker-V¿，称为scTv）融合到CD28（或4-1BB）和CD3的信号结构域。当被赋予高亲和力的scTv时，这种新的信号受体，我们称之为TCR-SCS （tcr -单链信号融合），可以重定向CD4和CD8 T细胞的活性。TCR-SCS可以在没有传统tcr相关风险的情况下使用。例如，TCR- scs受体限制了自肽脱靶反应，并避免了与内源性TCR链的错配。脱靶反应水平的降低是由于单链信号蛋白无法与CD8协同作用，这一过程增强了CD8 T细胞的敏感性，但也增加了其他pepMHC的刺激水平。为了在人类治疗中快速使用这些TCR-SCS受体，我们还将使用结构引导设计来设计针对特定pep/HLA-A2抗原的高亲和力tcr。我们的主要假设是TCR-SCS （tcr -单链信号融合物）可以快速地针对许多不同的pepMHC靶抗原进行工程设计，并且TCR-SCS将介导有效的CD4和CD8 T细胞活性，而不会脱靶交叉反应。因此，具体目标是：目标1。利用单个TCR支架分离抗多种肽/HLA-A2抗原的人T细胞受体。该方法将结合基于结构的设计，利用计算分析构建文库和先进的酵母展示技术来快速分离和进化tcr。目标2。确定TCR-SCS （tcr -单链信号融合）是否影响小鼠T细胞的持久性和功能。由于TCR-SCS格式是一种全新的方法，我们将使用涉及肽SIY的模型系统，以及针对SIY/Kb的高亲和力m33 TCR-SCS，进一步研究转导的CD4和CD8 T细胞的体内特性（与Hans Schreiber博士合作）。肿瘤模型将包括表达SIY/Kb的诱导B-Raf/PTEN-/-黑色素瘤（与Tom Gajewski博士合作）。目标3。利用高亲和力的TCR-SCS （tcr -单链信号融合物）靶向WT1的人tcr靶向肿瘤。已建立的移植的WT1/HLA-A2人类肿瘤将用于检测CD4和CD8 T细胞的有效性，这些细胞被WT1特异性TCR-SCS受体转导（与Philip Greenberg博士合作）。在Aim 1中分离的tcr的初步研究也将把结果扩展到其他人类肿瘤抗原。

DESCRIPTION (provided by applicant): Adoptive T cell therapy can be an effective and highly specific approach to the treatment of cancer. Introduction of receptors that recognize cancer antigens provides sufficient numbers of T cells with the appropriate specificity to destroy large, established tumors. The receptors used to date include either ¿¿ T cell receptors (TCRs) against pepMHC or antibody (scFv)-directed chimeric antigen receptors (CARs) against conventional cell surface cancer antigens. The purpose of this proposal is to improve upon current therapies and to develop a robust strategy that combines the advantages of each of these targeting approaches. The strategy involves a single-chain TCR (V¿-linker-V¿, called scTv) fused to signaling domains of CD28 (or 4-1BB) and CD3?. When endowed with a high-affinity scTv, this novel signaling receptor, which we call TCR-SCS (TCR-Single-Chain Signaling fusions), redirects activity of both CD4 and CD8 T cells. The TCR-SCS can be used without some of the risks associated with conventional ¿¿ TCRs. For example, TCR-SCS receptors limit self-peptide, off-target reactivities and they avoid mis-pairing with endogenous TCR chains. The reduced levels of off-target reactivities arise from the inability of the single-chain signaling protein to synergize with CD8, a process that enhances sensitivity in CD8 T cells but also increases the level of stimulation by other pepMHC. To enable the rapid use of these TCR-SCS receptors in human therapies, we will also use structure-guided design to engineer high-affinity TCRs against specific pep/HLA-A2 antigens. Our central hypotheses are that TCR-SCS (TCR-Single-Chain Signaling fusions) can be rapidly engineered against many different pepMHC target antigens, and that the TCR-SCS will mediate effective CD4 and CD8 T cell activity without off-target cross-reactivity. Accordingly, the specific aims are: Aim 1. To isolate human T cell receptors against diverse peptide/HLA-A2 antigens using a single TCR scaffold. The approach will involve a combination of structure-based design, taking advantage of computational analyses for library construction and advanced techniques of yeast display for the rapid isolation and evolution of the TCRs. Aim 2. To determine if a TCR-SCS (TCR-Single-Chain Signaling fusion) influences T cell persistence and function in mice. Because the TCR-SCS format is a completely novel approach, we will use the model system involving the peptide SIY, and the high-affinity m33 TCR-SCS against SIY/Kb, to further examine in vivo properties of transduced CD4 and CD8 T cells (collaboration with Dr. Hans Schreiber). Tumor models will include an inducible B-Raf/PTEN-/- melanoma that expresses SIY/Kb (collaboration with Dr. Tom Gajewski). Aim 3. To use TCR-SCS (TCR-Single-Chain Signaling fusions) with high-affinity, human TCRs against WT1 to target tumors. Established, transplanted WT1/HLA-A2 human tumors will be used to examine effectiveness of CD4 and CD8 T cells, transduced with WT1-specific TCR-SCS receptors (collaboration with Dr. Philip Greenberg). Preliminary studies with TCRs isolated in Aim 1 will also extend results to other human tumor antigens.