Chimeric antigen receptor T (CAR-T) cell therapy is regarded as an effective solution for relapsed or refractory tumors, particularly for hematological malignancies. Although the initially approved anti-CD19 CART therapy has produced impressive outcomes, setbacks such as high relapse rates and resistance were experienced, driving the need to discover engineered CART cells that are more effective for therapeutic use. Innovations in the structure and manufacturing of CART cells have resulted in significant improvements in efficacy and persistence, particularly with the development of fourth-generation CART cells. Paired with an immune modifier, the use of fourthgeneration and next-generation CART cells will not be limited because of cytotoxic effects and will be an efficient tool for overcoming the tumor microenvironment. In this review, we summarize the recent transformations in the ectodomain, transmembrane domain, and endodomain of the CAR structure, which, together with innovative manufacturing technology and improved cell sources, improve the prospects for the future development of CART cell therapy.

Introduction: Patients with T cell malignancies usually have high relapse and mortality rates. Due to shared common surface antigen and potential contamination by malignant cells, development of CAR-T therapies for r/r T-ALL has been lagged, regardless of the costly and lengthy process of autologous CAR-T production. To overcome these challenges, we developed a universal CAR-T platform (TruUCAR™) that displayed superior expansion in patients without using preconditioning biologics such as αCD52 antibody. Here we report results from a prospective study of GC027, the first-in-human, universal CAR-T therapy for treating adult patients with r/r T-ALL to evaluate the safety and clinical efficacy. Methods: TruUCART™ GC027 contains a second-generation CAR with genomic disruption of TCRα and CD7 by CRISPR/Cas9 system to avoid GvHD and fratricide. It is manufactured using lentivirus and leukopaks from HLA-mismatched healthy donors. A T-ALL xenograft murine model were used to assess anti-leukemic efficacy and expansion. Preliminary safety, anti-leukemic activity and expansion kinetics of GC027 are being evaluated in in a single-arm, open-label, multi-center, prospective study for treating adult r/r T-ALL. To date, a total of 5 patients (age 19-38 yrs, median 24 yrs) were enrolled with marrow tumor load 4-80.2% (median prior lines 5). All 5 pts have received a 6-day enhanced preconditioning chemotherapy followed by a single infusion of GC027. Adverse events, disease response, and expansion kinetics were evaluated in this study. Results: GC027 demonstrated robust anti-leukemic activity and expansion in a highly malignant CCRF-CEM xenograft murine model. All mice infused with GC027 exhibited significantly reduced tumor burden and prolonged survival compared to control groups. As of Feb. 6, 2020, 5 pts had received a single dose of GC027, including 1 at 0.6x107/kg, 3 at 1x107/kg and 1 at 1.5x107/kg. 4 pts achieved MRD negative complete responses (MRD- CR) at D28 evaluation: 3 of them remained MRD- at follow-up re-evaluations (D118, 61, 161, respectively, and none was bridged to HSCT); 1 just achieved MRD- CR at D28 and follow-up results will be updated at the meeting. 1 pt achieved MRD+ CR at D14 but had relapsed disease at D29. In all 4 pts with MRD- CR, peak expansions of GC027 in peripheral blood were observed between week 1-2. In 1 pt with CNS disease, GC027 was detected in specimens from his bone marrow and cerebrospinal fluid (CSF). 4 pts experienced Grade 3 cytokine release syndrome (CRS) and 1 pt had Grade 4 CRS (by ASBMT Consensus Grading) along with elevated levels of IL6, IFNγ and TNFα. CRS symptoms were manageable and resolved after treatment and supportive care. None developed neurotoxicity or GvHD. 1 pt had prolonged cytopenia due to fungal infection and required anti-fungal therapy. Conclusions: With a single infusion of GC027, 80% of the patients had robust CAR-T cell expansion and achieved persistent MRD- CR without using any biologics as part of the preconditioning therapy or bridging to HSCT. As the first-in-human, universal CAR-T therapy for adult r/r T-ALL, GC027 has demonstrated superior clinical efficacy and induced deep response in patients with acceptable safety profile. The trial enrollment is ongoing and updated data will be presented at the meeting. Citation Format: Xinxin Wang, Shiqi Li, Lei Gao, Zhongtao Yuan, Kun Wu, Lin Liu, Le Luo, Yao Liu, Cheng Zhang, Jia Liu, Chunhui Yang, Yu Li, Zhimin Li, Jiaping He, Duanpeng Wang, Xun Ye, Xu Tan, Ruihao Huang, Jianning Ge, Yu Han, Dingsong Zhang, Youcheng Wang, Lihua Fang, Yingnian Chen, Wei Cao, Sanbin Wang, Xi Zhang. Clinical safety and efficacy study of TruUCAR™ GC027: The first-in-human, universal CAR-T therapy for adult relapsed/refractory T-cell acute lymphoblastic leukemia (r/r T-ALL) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT052.

Chimeric antigen receptor T (CAR-T) cell therapy has significantly improved the life expectancy for patients with refractory or relapse B cell lymphoma. As for B cell acute lymphoblastic leukemia (B-ALL), although the primary response rate is promising, the high incidence of early relapse has caused modest long-term survival with CAR-T cell alone. One of the main challenges is the limited persistence of CAR-T cells. To further optimize the clinical effects of CAR-T cells, many studies have focused on modifying the CAR structure and regulating CAR-T cell differentiation. In this review, we focus on CAR-T cell persistence and summarize the latest progress and strategies adopted during the in vitro culture stage to optimize CAR-T immunotherapy by improving long-term persistence. Such strategies include choosing a suitable cell source, improving culture conditions, combining CAR-T cells with conventional drugs, and applying genetic manipulations, all of which may improve the survival of patients with hematologic malignancies by reducing the probability of recurrence after CAR-T cell infusion and provide clues for solid tumor CAR-T cell therapy development.