Sortase-mediated installation of recognition modules on T cells for redirected ki

描述（由申请人提供）：这项申请利用了最近的观察，即淋巴细胞在由分选酶催化的酶促反应中很容易被修饰。任何携带适当暴露的LPXTG基模的实体都可以通过内源性受体蛋白连接到细胞表面，而内源性受体蛋白必须显示暴露的甘氨酸残基才能参与反应。我们建议通过安装特定的识别模块来重新定向细胞毒性T细胞的特异性，这些识别模块最终可用于选择性地靶向癌细胞。如果成功，这种方法将使细胞毒性T细胞或自然杀伤细胞的抗体或其衍生物能够识别肿瘤特异性抗原的体外修饰成为可能。再输注这种修饰过的自体细胞可以用作免疫治疗，有几个优点：不需要对移植的细胞进行遗传修饰，移植的细胞是自体的，酶修饰既快速又温和，引起的反应是自我限制的。为了实现这一令人兴奋的新方法，我们提出以下具体目标：通过分类酶a介导的偶联对淋巴细胞表面的功能化。我们的研究结果表明，分选酶A可用于偶联（修饰）肽或蛋白到淋巴细胞表面表达的分子。我们将确定使修饰分子数量最大化的反应条件（例如，分选酶变异、细胞和探针浓度、动力学），并确定内源性底物。目标2。体外细胞毒性通过安装vhs对CD8 T细胞已知的特异性。我们已经使用排序酶a将单链抗体安装到小鼠淋巴细胞表面。我们将研究CD8 T细胞的细胞毒性是否可以通过偶联已知特异性的单域抗体片段（抗流感HA，抗II类MHC）来重定向。抗原特异性杀伤将在体外细胞毒性试验中进行评估。目标3。重定向淋巴细胞的体内功能。基于目标2的结果，我们将研究这种方法在小鼠肿瘤模型中诱导肿瘤细胞杀伤的潜力。如果成功，这些研究将提供一种新的免疫治疗形式，利用免疫识别的特异性优势，同时避免对自体细胞进行遗传修饰，以用作细胞毒性药物。这将是癌症细胞免疫疗法的重大进展。

DESCRIPTION (provided by applicant): This application exploits the recent observation that lymphocytes are readily modified in an enzymatic reaction catalyzed by sortase. Any entity bearing a suitably exposed LPXTG motif can be ligated to the cell surface by means of endogenous acceptor proteins, which must display (an) exposed glycine residue(s) to participate in the reaction. We propose to redirect the specificity of cytotoxic T cells through installation of specific recognition modules that can ultimately be used to selectively target cancerous cells. If successful, this approach would enable the modification ex vivo of cytotoxic T cells or natural killer cells with antibodies or their derivatives that recognize tumor-specific antigens. Reinfusion of such modified autologous cells might then be used as immunotherapy, with several advantages: no genetic modification of the transferred cells is required, the transferred cells are autologous, the enzymatic modification is both rapid and mild, and the elicited response is self-limiting. To pursue this exciting new approach we propose the following specific aims: Aim 1. Functionalization of the lymphocyte cell surface by sortase A-mediated conjugation. Our results show that sortase A can be used to conjugate (modified) peptides or proteins to molecules expressed at the surface of lymphocytes. We shall determine reaction conditions that maximize the number of modified molecules (e.g. sortase variants, cell and probe concentrations, kinetics) as well as identity the endogenous substrates. Aim 2. In vitro cytotoxicity conferred by installation of VHHs on CD8 T cells of known specificity. We have already achieved the installation of single chain antibodies to the murine lymphocyte cell surface using sortase A. We shall investigate whether CD8 T cell cytotoxicity can be redirected by conjugating a single domain antibody fragment of known specificity (anti flu HA, anti Class II MHC). Antigen-specific killing will be assessed in in vitro cytotoxicity assays. Aim 3. In vivo function of redirected lymphocytes. Based on the results from aim 2, we will investigate the potential of this approach to induce killing of tumor cells in a murine tumor model in vivo. If successful, these studies will provide a new form of immunotherapy that exploits the advantages of the specificity of immune recognition, while avoiding genetic modification of the autologous cells to be used as cytotoxic agents. This would be a major advance in cellular immunotherapy of cancer.