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

项目摘要 靶向CD 19的嵌合抗原受体T细胞（CAR-Ts）已经彻底改变了细胞免疫疗法， 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性淋巴瘤 白血病（B-ALL）。尽管完全缓解率很高，复发率，其中50%在第一年内， 仍然是一个关键的挑战，突出了对新型CAR-T细胞产品的需求。复发的两种模式是 观察到：（i）由靶抗原丢失引起的抗原阴性复发和（ii）抗原阳性复发， 是由CAR-T缺乏持久性或功能丧失介导的。商业CD 19 CAR-T采用 无论是CD 28或4-1BB共刺激结构域，然而，一种新的OX-40结构域已经显示出促进 持久性、细胞毒性和减少疲劳。最后，越来越多的证据表明， CAR-T产品中的记忆T细胞表型与更持久的应答相关。施奈德等al 开发了具有OX-40共刺激结构域的三特异性CD 19、CD 20、CD 22靶向CAR-T， 与CD 19 CAR-T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 内部生产的三特异性CAR-T的结果，并证实了其特异性、细胞毒性和 临床前B细胞淋巴瘤模型中的免疫表型适应性。我们的建议旨在解决 商业CD 19 CAR-T通过首次在人体内部生产的三特异性CAR-T与OX- 40共刺激结构域用于复发性、难治性B细胞恶性肿瘤。这些三特异性CAR-T是 在俄亥俄州州立大学细胞治疗实验室使用CliniMACS Prodigy设备制造， 6-一天的制造过程，允许注入干样记忆CAR-T。我们假设 三特异性CART将（i）降低由抗原阴性克隆逃逸介导的复发风险;（ii）增强免疫应答;（iii）增强免疫应答。 CAR-T的持久性将转化为更深入和更持久的临床反应。为了解决这个问题， 我们提出两个目标。在目标1中，我们的目标是进行首次人体I期试验， 复发性/难治性B-NHL、B-ALL、B-幼淋巴细胞白血病和慢性 淋巴细胞白血病主要终点是三特异性CAR-T沿着的可行性、安全性以及建立 II期推荐剂量次要终点是疗效和缓解持续时间。在目标2中，我们旨在 确定对三特异性CAR-T的功效和抗性的关键机制。具体而言，我们计划评估：（i） CAR-Ts的持续性及其与完全缓解率、缓解持续时间以及 不良事件的发生率和严重程度;（ii）受损的免疫表型和转录特征 使用最先进的高维光谱流式细胞术检测CAR-T和其他单核细胞的功能 和CITE-sequencing。 在这个项目完成后，我们的工作将建立内部生产三特异性的可行性， CAR-Ts并提供了对复发和疗效机制的见解。

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.