Trispecific CAR-T cells targeting CD19, CD20 and CD22 to treat B-cell malignancies

项目总结 CD19靶向嵌合抗原受体T细胞(CAR-ts)使细胞免疫治疗发生了革命性的变化 难治性B细胞恶性肿瘤，如B细胞非霍奇金淋巴瘤(B-NHL)和B细胞急性淋巴母细胞瘤 白血病(B-ALL)。尽管完全缓解率很高，复发，其中50%是在第一年内， 仍然是一个关键挑战，突显了对新型CAR-T细胞产品的需求。复发的两种模式是 观察到：(1)抗原阴性复发，由靶抗原丢失引起；(2)抗原阳性复发， 是由于汽车缺乏持久性或功能丧失所致。商业CD19汽车公司员工 然而，无论是CD28还是4-1BB共刺激结构域，一个新的OX-40结构域都被证明可以促进 持久性、细胞毒性和减少疲劳。最后，越来越多的证据表明，茎状结构 CAR-T产物中的记忆T细胞表型与更持久的反应有关。施耐德等人。阿尔 开发了具有OX-40共刺激结构域的三特异性CD19、CD20、CD22靶向小体，并 与CD19相比，在临床前淋巴瘤模型中显示出显著的活性。我们验证了这些 用内部制造的三特异性CAR的研究结果，证实了它们的特异性、细胞毒性和 临床前B细胞淋巴瘤模型的免疫表型适合性。我们的建议旨在解决这些限制 商业CD19汽车通过第一辆人类内部制造的带有牛的三种特定汽车- 40共刺激区域，用于复发、难治性B细胞恶性肿瘤。这些三合一的汽车是 俄亥俄州立大学细胞治疗实验室使用CliniMACS Prodigy设备 6天的制造工艺，允许输注茎状记忆汽车。我们假设 三特异性CART将(I)降低由抗原阴性克隆性逃逸介导的复发风险；(Ii)增强 CART的持久性，这将转化为更深入和更持久的临床反应。为了解决这个问题， 我们提出了两个目标。在目标1中，我们的目标是用内部制造的产品进行第一阶段的人体试验 复发性/难治性B-NHL、B-ALL、B-早幼粒细胞白血病和慢性白血病患者的三特异性基因片段 淋巴细胞性白血病。主要终点是三种特定汽车的可行性、安全性以及建立一个 推荐的第二阶段剂量。次要终点是疗效和反应持续时间。在目标2中，我们的目标是 确定对三特异性CART的疗效和抗药性的关键机制。具体来说，我们计划评估：(I) CAR-TS的持续性及其与完全缓解率、反应持续时间的相关性 不良事件的发生率和严重程度；(2)受损的免疫表型和转录特征 用最先进的高维光谱流式细胞术检测CAR-T和其他单个核细胞的功能 并进行引证排序。 在这个项目完成后，我们的工作将建立在内部生产三种规格的可行性 Car-ts，并对复发和疗效机制提供了见解。

Project summary CD19 targeted chimeric antigen receptor T cells (CAR-Ts) have revolutionized cellular immunotherapy in refractory B-cell malignancies such as B-cell Non-Hodgkin’s lymphoma (B-NHL) and B-cell acute lymphoblastic leukemia (B-ALL). Despite high rates of complete remissions, relapses, 50% of which within the first year, remain a critical challenge highlighting the need for novel CAR-T cell products. Two patterns of relapse are observed: (i) Antigen-negative relapses, caused by target antigen loss and (ii) antigen-positive relapses, which are mediated by lack of persistence or loss of function of the CAR-Ts. Commercial CD19 CAR-Ts employ either a CD28 or a 4-1BB costimulatory domain however, a novel OX-40 domain has been shown to promote persistence, cytotoxicity and decrease exhaustion. Finally, increasing evidence suggests that stem-like memory T cell phenotype in the CAR-T product is associated with more durable responses. Schneider et. al developed a Trispecific CD19, CD20, CD22-targeting CAR-Ts with an OX-40 costimulatory domain and showed significant activity in preclinical lymphoma models compared to CD19 CAR-Ts. We validated these findings with in-house manufactured Trispecific CAR-Ts and confirmed their specificity, cytotoxicity, and immunophenotypic fitness in preclinical B-cell lymphoma models. Our proposal seeks to address the limitations of commercial CD19 CAR-Ts through a first-in-human in-house manufactured Trispecific CAR-Ts with an OX- 40 costimulatory domain for relapsed, refractory B-cell malignancies. These trispecific CAR-Ts are manufactured in the Ohio State University Cell therapy laboratory using the CliniMACS Prodigy device over a 6-day manufacturing process which allows for infusion of stem-like memory CAR-Ts. We hypothesize that trispecific CARTs will (i) reduce the risk of relapse mediated by antigen negative clonal escape; (ii) enhance persistence of CAR-Ts which will translate into deeper and more durable clinical responses. To address this, we propose two Aims. In Aim 1, we aim to conduct a first-in-human phase I trial with in-house manufactured trispecific CAR-Ts in patients with relapsed/refractory B-NHL, B-ALL, B-prolymphocytic leukemia, and chronic lymphocytic leukemia. Primary endpoints are feasibility, safety of trispecific CAR-Ts along with establishing a recommended phase II dose. Secondary endpoints are efficacy and duration of response. In Aim 2, we aim to identify key mechanisms of efficacy and resistance to trispecific CAR-Ts. Specifically, we plan to assess: (i) persistence of CAR-Ts and its correlation with complete remission rates, duration of response as well as incidence and severity of adverse events; (ii) immunophenotypic and transcriptional features of impaired function of CAR-Ts and other mononuclear cells using state of the art high-dimensional spectral flow cytometry and CITE-sequencing. At completion of this project, our work will have established feasibility of in-house manufacturing of trispecific CAR-Ts and provided insights into mechanisms of relapse and efficacy.