Structural Analysis , mutation and therapeutic use of TCRs from HIV-specific CTLs

描述(由申请人提供)：HIV-1特异性CD8细胞毒性T淋巴细胞(CTL)反应在控制HIV-1复制方面发挥关键作用。TCR的亲和力与CTL功能密切相关，而表达TCR的CTL由于其同源的MHC-病毒多肽复合体具有高亲和力，在体内对中和病毒感染、终止病毒感染和延缓艾滋病病毒从黏膜接种部位的传播具有重要作用。关于HIV特异性CTL表达的TCR识别HIV多肽的结构基础：MHC I类复合体，以及特定TCR残基对其同源HIV多肽TCR亲和力的贡献，人们知之甚少。阐明HIV特异性CD8+CTL表达的TCR识别HIV多肽的结构与功能的相关性，将有助于我们更好地理解某些TCR赋予CTL强大的抗HIV活性的机制。我们假设，在解析TCR结构和鉴定TCR-肽接触残基后，通过突变与同源肽接触的TCR氨基酸，可以显著增强HIV特异性CTL的功能活性，从而增加其与其同源肽：MHC复合体的亲和力。我们已经将HIV特异性CTL克隆中的TCR基因克隆到慢病毒载体中，该载体可以有效地将外周CD8T细胞转化为具有特定特异性的HIV特异性CTL。我们还将这些TCR基因作为连接的单链TCR(ScTCR)克隆到细菌、果蝇和酵母载体中，以表达足够的蛋白质来产生晶体来确定TCR的结构。通过测定人类白细胞抗原-A*0201限制性的HIV-SL9特异性TCR及其同源MHC I类：SL9-肽组件形成的复合体的X射线晶体结构，将获得其同源HIV多肽TCR亲和力的结构基础。这些结构将使我们能够1)确定与MHC有关的物理和化学决定因素：肽识别；2)确定/合理化靶向突变对CTL功能的影响；3)进行具有独特结合和生物活性的新型TCR的结构辅助设计。了解TCR识别HIV表位的结构基础可能有助于设计能够诱导CTL以更高的亲和力识别HIV表位并更有效地预防HIV感染的疫苗。我们最近报道，188Re连接的HIV包膜特异性人单抗可以在体内有效地清除HIV-1感染的细胞。我们将确定这种方法是否可以用于开发一种新的艾滋病毒治疗方法，使用可溶性TCR四聚体与放射性同位素偶联，无论是天然形式还是携带我们确定的旨在增加亲和力的突变，并检测它们在体外和体内靶向和消除艾滋病毒感染细胞的能力。 公共卫生相关性：我们建议通过解决HIV-SL9特异性TCR与同源MHC第I类：SL9-肽组件形成的复合体的X射线晶体结构，对HIV特异性CTL的TCR进行结构-功能分析。我们将定义负责MHC的物理和化学决定因素：肽识别，确定/合理化靶向突变对CTL功能的影响，并利用这些信息作为结构辅助设计的基础，该药物源于与放射性核苷酸相连的TCR。

DESCRIPTION (provided by applicant): HIV-1 specific CD8 cytotoxic T lymphocyte (CTL) responses play a critical role in controlling HIV-1 replication. TCR avidity correlates with CTL function, and CTLs expressing TCRs with high avidity for their cognate MHC-viral peptide complex play an important in vivo role in neutralizing virus infections, terminating virus infection and delaying systemic AIDS virus dissemination from the mucosal inoculation site. Little is known about the structural basis by which the TCR's expressed by HIV-specific CTL recognize the HIV peptide:MHC Class I complex and the contributions of specific TCR residues to the TCR avidity for its cognate HIV peptide. Elucidation of the structure- function correlates of HIV peptide recognition by the TCR expressed by HIV-specific CD8+ CTLs will increase our understanding of the mechanism by which some TCRs confer CTLs with potent anti-HIV activity. We hypothesize that after solving the TCR structure and identifying TCR-peptide contact residues, the functional activity of HIV- specific CTLs can be markedly enhanced by mutation of TCR amino acids that contact the cognate peptide thereby increasing its affinity for its cognate peptide:MHC complex. We have cloned the TCR genes from an HIV-specific CTL clone into lentiviral vectors that efficiently transform peripheral CD8 T cells into HIV-specific CTLs with defined specificities. We have also cloned these TCR genes as a linked single chain TCR (scTCR) into bacterial, drosophila and yeast vectors to express sufficient protein to generate crystals to determine the TCR structure. The structural basis of TCR avidity for its cognate HIV peptide will be obtained by determining the X-ray crystallographic structures of the complexes formed by HLA-A*0201-restricted HIV-SL9-specific TCRs and their cognate MHC Class I:SL9-peptide assemblies. These structures will permit us to 1) define the physical and chemical determinants responsible for MHC:peptide recognition; 2) determine/rationalize the effect of targeted mutations on CTL function and 3) perform structure-assisted design of novel TCRs with unique binding and biological activities. Understanding the structural basis of TCR recognition of HIV epitopes may facilitate the design of vaccines capable of inducing CTLs that recognize HIV epitopes with a higher avidity and that are more effective in preventing HIV infection. We have recently reported that 188Re-linked HIV envelope-specific human mAb can be used to efficiently eliminate HIV-1-infected cells in vivo. We will determine if this approach could be used to develop a new HIV therapy using soluble TCR tetramers coupled to radioisotopes, either in the native form or carrying mutations we identified designed to increase avidity, and examine their in vitro and in vivo capacity to target and eliminate HIV-infected cells. PUBLIC HEALTH RELEVANCE: We propose to perform structure-function analysis of the TCR of HIV-specific CTLs by solving the X-ray crystallographic structures of the complexes formed by HIV-SL9- specific TCRs and cognate MHC Class I:SL9-peptide assemblies. We will define the physical and chemical determinants responsible for MHC:peptide recognition, determine/rationalize the effect of targeted mutations on CTL function and use this information as the basis for the structure-assisted design of novel HIV therapeutics derived from TCRs linked to radionucleotides.