融合CD3ζ分子的TCR基因转染T细胞的信号通路及其抗肿瘤功能的研究

Adoptive transfer of genetically T-cell receptor (TCR) has been demonstrated the overall feasibility and clinical potential as treatments for different types of cancer. However, this promising clinical approach is limited by a serious potential consequence: the exogenous TCR mismatching with endogenous TCR chains may lead to the risk of self-reactivity. Therefore, it is urgent to reduce or avoid the generation of the mispairing TCR. In our previous study, we generated a TCRαζβζ which modified by fusing the original constant domains downstream of the extracellular cysteine of TCRα- and β- chains to complete human CD3ζ. And we had determined that the modified TCR improved expression and avoid mis-pairing on T cell surface. In this study, we will transfer the TCRαζβζ into T cells, then we will study the capacity of TCRαζβζ forming complexes with CD3 molecules. We will further characterize this TCRαζβζ in gene-engineered T cells and assess whether this receptor is able to interact with surface molecules and drive correct synapse formation in T cells. We would elucidate the signal transduction pathways of this TCRαζβζ molecule in T cells. We would elucidate the signal transduction pathways of this TCRαζβζ molecule in T cells.After that, we will test and analyze the ability of signal transduction of the TCRαζβζ. Finally, we will detect and assess the anti-tumor function of this TCR gene-engineered T cells in vitro.Taken together, this study may explain the anti-tumor effect and its molecular mechanism of this TCRαζβζ. Taken together, this study may provide further understanding of the TCRαζβζ in adoptive transfer of TCR gene therapy.

TCR基因过继性免疫治疗在治疗恶性肿瘤和病毒性疾病取得了一些成功。然而，这种有效的治疗策略却存在一个亟需解决的问题，即导入的外源TCR能与内源TCR形成杂合的异二聚体（错配TCR），这种错配的TCR可能会导致自身免疫性疾病。因此有必要设法避免或减少这种错配TCR的产生。前期研究中我们利用结构域替换和三维建模方法对TCR分子进行改造，设计并构建了融合CD3ζ分子的TCRαζβζ分子，并证实该TCRαζβζ分子在T细胞表面表达增强并能避免错配。在本研究中，我们将在此基础上将该TCRαζβζ转入T细胞，对该TCRαζβζ分子在T细胞中与CD3分子的相互作用、在T细胞膜形成免疫突触的情况以及向T细胞内传递活化信号的能力进行全面的检测与分析，阐明该TCRαζβζ分子在T细胞中的信号转导通路。最后检测转TCRαζβζ基因T细胞的抗肿瘤作用，为该TCRαζβζ修饰T细胞的过继性治疗的临床应用奠定基础。

TCR基因修饰的T细胞过继性转移回输患者体内的TCR-T疗法在治疗恶性肿瘤和病毒性疾病的临床研究中取得了一些成功。然而这种TCR基因治疗所引入的外源TCRα和 β链不仅仅是互相配对成针对肿瘤抗原的TCRαβ二聚体，还能与内源性的TCR α 和β链错配形成杂合TCR二聚体（错配的TCR）。这种错配的TCR可能会导致自身免疫性疾病。前期我们证实融合了CD3ζ的TCRαζβζ分子在T细胞表面表达增强并能避免错配。本研究中，我们克隆并鉴定了携带荧光蛋白的融合TCR双表达载体pIRES-TCRβζ-TCRαζCFP和CD3ζ蛋白的表达载体pIRES-CD3ζ-YFP，共转染T淋巴细胞，证实了TCRαζβζ定位于细胞膜上，并且依然与CD3ζ分子具有一定的相互作用，在T淋巴细胞中信号初始状态可控。另外我们成功构建了不含荧光蛋白的腺病毒穿梭质粒pDC315-TCRβζ-IRES-TCRαζ，成功包装出重组腺病毒并大量扩增和纯化。接着将重组TCRαζβζ腺病毒感染HLA-A2阳性的健康人T淋巴细胞，结果证实相比于野生型的TCR分子(wtTCR)，TCRαζβζ在T淋巴细胞表面的表达增强，经TCRαζβζ修饰的T淋巴细胞能更有效杀伤HLA-A2+的肿瘤细胞，并且依然保持HLA-A2限制性。在此基础上，为了将TCRαζβζ基因的TCR-T免疫疗法有针对性的应用于临床，我们针对HBV的特异性T淋巴细胞，采用单细胞技术分别获得特异性TCRVα26、TCRVα1/5基因和TCRVβ5基因，并成功构建了融合CD3ζ的双表达载体pIRES2-TCRβ5-ζ-TCRα1/5-ζ和pIRES2-TCRβ5-ζ-TCRα26-ζ。并探讨了终末分化的效应性T淋巴细胞经过重编程逆分化为早期T细胞（如初始T细胞和干细胞样记忆性T细胞）可行性。进而为TCRαζβζ基因的TCR-T 疗法的临床应用提供更有效的理论基础。

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