Gene-engineered stem cell memory T-cells with anti-HIV chimeric antigen receptors

项目概要 CD8 细胞毒性 T 淋巴细胞 (CTL) 可以暂时控制感染者体内的 HIV，但最终会因病毒感染而失败 突变等因素，导致持续或慢性感染。然而，CTL 抑制 HIV 的报道 少数患者的感染表明克服这些障碍将能够成功实现 CTL 介导的控制 艾滋病毒感染。过继免疫治疗策略旨在通过以下方式赋予定向和增强的 CTL 反应： 补充离体扩增的自体 CD8 T 细胞，表达所需的抗原特异性 T 细胞 受体（TCR）。然而，这些细胞大多由于增殖反应下降和有限的功能而功能障碍。 CTL 活动。此外，需要特定的人类白细胞抗原才能正确地将抗原呈递给 T 细胞限制了基于外源 TCR 的方法。因此，这些方法对减少 HIV病毒载量。 嵌合抗原受体 (CAR) 是人工设计的受体，可赋予抗原所需的特异性 免疫效应T细胞。近年来，CAR免疫疗法在抗癌领域得到广泛推广 临床试验。其中，过继转移的用抗 CD19 CAR 修饰的自体 T 细胞显示出显着的效果 对急性和慢性淋巴细胞白血病患者的 B 细胞淋巴瘤的影响。我们假设 CAR 针对 HIV 感染细胞的免疫疗法可以根除持续感染的细胞和 HIV 潜伏病毒库 重新激活后的患者。关于抗 HIV CAR，CD4ze CAR 由细胞外结构域组成 与 CD3 z 链相连的人类 CD4 分子已在体外和人体临床试验中得到充分研究。这个 CAR 已被证明可以在体外介导高效的抗 HIV 活性，但由于存活率较低，其效果有限 和转导细胞的功能。我们考虑了这种失败的潜在原因：1） CD4 T 细胞和 CD8 T 细胞表达 CD4 z CAR 来抵抗 HIV 感染，2）缺乏共刺激信号传导域 适当的效应器和记忆反应所需的，以及3）在基因之前T细胞的大规模离体扩增 修饰导致细胞老化，导致效应器和记忆功能较差。 我们的总体目标是通过以下方式赋予长期、增强的 HIV 特异性效应器和记忆反应： 移植抗 HIV CAR 工程 T 细胞。我们将1）利用HIV-开发一类新型抗HIV CAR- 靶向广泛中和抗体，2) 通过共同保护 CAR 转导的 T 细胞免受 HIV 介导的细胞毒性 转导抗HIV基因，3）将共刺激信号域引入抗HIV CAR中，以确保 卓越的效应和记忆反应，4) 使用新鉴定的保留干细胞特性的记忆 T 细胞 称为“干细胞记忆T（TSCM）细胞”作为抗HIV CAR的载体。 TSCM 细胞在体外也能自我更新 在体内并分化为效应T细胞。因此，TSCM 细胞被设计来表达 HIV 特异性 CAR，从而提供 HIV特异性免疫细胞的取之不尽、用之不竭的来源。这些研究将使用以下方法在体内进一步建模 人源化骨髓、肝脏和胸腺（BLT）小鼠系统。

PROJECT SUMMARY CD8+ cytotoxic T-lymphocytes (CTLs) transiently control HIV in infected persons, but eventually fail due to viral mutation and other factors, resulting in persistent or chronic infection. However, reports of CTLs suppressing HIV infection in a few patients indicate that overcoming these barriers would allow for successful CTL-mediated control of HIV infection. Adoptive immunotherapy strategies aim to confer directed and enhanced CTL responses via supplementation of ex vivo-expanded autologous CD8+ T cells expressing a desired antigen-specific T cell receptor (TCR). However, these cells are mostly dysfunctional due to decreased proliferative response and limited CTL activity. Moreover, the requirement for a particular human-leukocyte antigen to properly present antigen to the T cell limits exogenous TCR-based approaches. Thus, such approaches have had minimal effects on reducing HIV-viral load. Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity onto immune effector T cells. In recent years, CAR immunotherapies have been extensively promoted in anti-cancer clinical trials. Of these, adoptively transferred autologous T cells modified with anti-CD19 CAR showed a dramatic impact on B cell lymphomas in acute and chronic lymphocytic leukemia patients. We hypothesize that CAR immunotherapy against HIV-infected cells can eradicate persistently infected cells and HIV latent reservoirs in patients following reactivation. Regarding an anti-HIV CAR, CD4ζ CAR consisting of the extracellular domain of the human CD4 molecule linked to the CD3ζ-chain has been well-studied in vitro and in human clinical trials. This CAR has been shown to mediate highly potent anti-HIV activity in vitro, but had limited effects due to poor survival and functionality of the transduced cells. We considered potential reasons for this failure due to 1) susceptibility of CD4+ T cells and CD8+ T cells expressing CD4ζ CAR to HIV infection, 2) lack of costimulatory signaling domains required for proper effector and memory response, and 3) massive ex vivo expansion of T cells prior to gene modification leading to cellular aging, resulting in poor effector and memory function. Our overall goal is to confer long-term, enhanced HIV-specific effector and memory responses via transplantation of anti-HIV CAR-engineered T cells. We will 1) develop a new class of anti-HIV CAR using HIV- targeting broadly neutralizing antibodies, 2) protect CAR-transduced T cells from HIV-mediated cytotoxicity by co- transduction with anti-HIV genes, 3) introduce costimulatory-signaling domains into anti-HIV CAR to ensure superior effector and memory responses, and 4) use newly identified memory T cells retaining stem cell properties called “stem cell memory T (TSCM) cells” as a carrier vehicle for anti-HIV CAR. TSCM cells self-renew in vitro as well as in vivo and differentiate into effector T cells. Thus, TSCM cells engineered to express HIV-specific CAR provide an inexhaustible source of HIV-specific immune cells. These studies will be further modeled in vivo using the humanized bone marrow, liver and thymus (BLT) mouse system.