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

分选酶介导在 T 细胞上安装识别模块以实现重定向 ki

• 批准号: 8683479
• 负责人: Hidde L. Ploegh
• 金额: $ 25.45万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8683479
• 关键词: Antibodies; Antigen Receptors; Antigens; Autologous; Autologous Transplantation; Biochemical Reaction; Biological Assay; CD19 gene; CD8B1 gene; Cancer Patient; Cancerous; Cell membrane; Cell surface; Cells; Clinical; Cytotoxic T-Lymphocytes; Cytotoxic agent; Engineering; Genetic; Genome; Glycine; Immune; Immune response; Immunoglobulin Fragments; Immunotherapy; In Vitro; Kinetics; Lymphocyte; MHC Class II Genes; Malignant - descriptor; Mammalian Cell; Mediating; Methods; Modification; Mus; Mutagenesis; N-terminal; Natural Killer Cells; Patients; Peptides; Peptidyltransferase; Proteins; Reaction; Recombinant Proteins; Risk; Safety; Site-Directed Mutagenesis; Specificity; Surface; T-Cell Receptor; T-Lymphocyte; Tumor Antigens; Variant; Viral Vector; adduct; anti-influenza; base; cancer immunotherapy; cytotoxicity; enzyme substrate; in vivo; in vivo Model; killings; macromolecule; neoplastic cell; novel strategies; public health relevance; receptor; response; sortase; transacylation; tumor; vector

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.

描述（由申请人提供）：本申请利用了最近的观察结果，即淋巴细胞在分选酶催化的酶促反应中容易被修饰。携带适当暴露的LPXTG基序的任何实体可以通过内源受体蛋白连接到细胞表面，所述内源受体蛋白必须展示暴露的甘氨酸残基以参与反应。我们建议通过安装特异性识别模块来重定向细胞毒性T细胞的特异性，这些模块最终可用于选择性靶向癌细胞。如果成功，这种方法将能够用识别肿瘤特异性抗原的抗体或其衍生物离体修饰细胞毒性T细胞或自然杀伤细胞。然后，这种修饰的自体细胞的再输注可以用作免疫疗法，具有几个优点：不需要对转移的细胞进行遗传修饰，转移的细胞是自体的，酶促修饰既快速又温和，并且引发的反应是自限性的。为了追求这一令人兴奋的新方法，我们提出了以下具体目标：目标1。通过分选酶A介导的缀合使淋巴细胞表面官能化。我们的研究结果表明，分选酶A可用于缀合（修饰）肽或蛋白质的分子表达在淋巴细胞的表面。我们将确定使修饰分子数量最大化的反应条件（例如分选酶变体、细胞和探针浓度、动力学）以及鉴别内源性底物。目标2.通过在已知特异性的CD8 T细胞上安装VHH赋予的体外细胞毒性。我们已经用分选酶A将单链抗体固定到小鼠淋巴细胞表面。我们将研究是否可以通过结合已知特异性的单域抗体片段（抗流感HA，抗II类MHC）来重定向CD8 T细胞的细胞毒性。将在体外细胞毒性试验中评估抗原特异性杀伤。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体内诱导杀死小鼠肿瘤模型中肿瘤细胞的潜力。如果成功，这些研究将提供一种新的免疫疗法，利用免疫识别特异性的优势，同时避免将自体细胞用作细胞毒性剂的遗传修饰。这将是癌症细胞免疫疗法的重大进展。