Engineering T Cell Receptors for Adoptive Cell Therapies

描述(由申请人提供)：采用T细胞疗法可以是一种有效和高度特异的癌症治疗方法。识别癌症抗原的受体的引入提供了足够数量的具有适当特异性的T细胞，以摧毁大的、已建立的肿瘤。到目前为止，使用的受体包括针对PepMHC的��T细胞受体(TCR)或针对常规细胞表面癌症抗原的抗体(ScFv)导向的嵌合抗原受体(CARS)。这项提议的目的是改进目前的治疗方法，并制定一种结合这些靶向方法各自优点的强有力的战略。该策略包括一个单链TcR(V�-Linker-V�，简称SCTV)，融合到CD2 8(或4-1BB)和CD3？的信号域。当被赋予高亲和力的SCTV时，这种新的信号受体，我们称为TCR-SCS(TCR-单链信号融合)，可以重定向CD4和CD8T细胞的活性。TCR-SCS的使用没有与传统��TCR相关的一些风险。例如，TCR-SCS受体限制了自身多肽的非靶标反应，并避免了与内源性TCR链的错误配对。脱靶反应性水平的降低是由于单链信号蛋白无法与CD8协同作用，这一过程提高了CD8 T细胞的敏感性，但也增加了其他多肽MHC的刺激水平。为了能够在人类治疗中快速使用这些TCR-SCS受体，我们还将使用结构指导设计来设计针对特定PEP/HLA-A2抗原的高亲和力TCR。我们的中心假设是，TCR-SCS(TCR-单链信号融合)可以针对许多不同的多肽MHC靶抗原快速构建，并且TCR-SCS将介导有效的CD4和CD8T细胞活性，而不会发生靶外交叉反应。因此，本研究的具体目的是：1.用单一的TCR支架分离针对不同多肽/HLA-A2抗原的人T细胞受体。该方法将结合基于结构的设计，利用构建文库的计算分析和先进的酵母展示技术来快速分离和进化TCRs。目的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，我们将使用包含多肽SIY和针对SIY/KB的高亲和力M33 TCR-SCS的模型系统来进一步检测转导的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的诱导性B-Raf/PTEN-/-黑色素瘤(与Tom Gajeski博士合作)。目的3.利用高亲和力的人TCR抗WT1单链信号融合蛋白(TCR-SCS)靶向肿瘤。已建立的、移植的WT1/HLA-A2人类肿瘤将用于检验通过WT1特异性TCR-SCS受体转导的CD4和CD8 T细胞的有效性(与Philip Greenberg博士合作)。对AIM 1中分离的TCRs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.