Abstract 3173: JL-lightning CAR-T, a next generation non-viral CAR-T without <i>in-vitro</i> culturing procedure, shows high clinical efficacy, good safety with low treatment dosage

Abstract Background: CAR-T with high in vivo proliferation and long persistency could benefit hematologic malignancy patients with low relapse and long survival. Previous studies reveal that CAR-T stemness (T-stem) and exhaustion level are major determinants of CAR-T quality that contribute to clinical outcome. However, conventional manufacturing process with 9-14 days in vitro culture sacrifices T-stem and prolong the vein-to-vein process. Researchers have tried to shorten this process, but many failed due to T-cell activation insufficiency, transfection toxicity, and poor quality control, especially in non-viral transfection. Methods: Two versions of non-viral JL-Lightning-CAR-T fast manufacturing process have been developed to shorten in vitro culture process, V-30h and V-6h. Of note, there is no culture after transfection to enhance CAR-T-stem and in vivo expansion. JL-transposon has been developed and proved with high transfection efficiency and low toxicity on both activated and resting T cells. We generated a tri-targeting CAR-T (BZE2204) using VHHs against CD19, CD22 and BCMA, then evaluated its safety and efficacy with both V-30h and V-6h in vitro and in vivo using Raji orthotropic model. One investigator-initiated trial (IIT) is designed to assess the safety and preliminary efficacy of BZE2204 CAR-T in relapsed/refractory Non-Hopkins Lymphoma (r/r NHL) patients. Results and Conclusion: In vitro experiments showed both versions of BZE2204 exerted strong cytotoxicity on CD19-, CD22- and BCMA-positive tumor cells with high CAR-T cell expansion rates, superior stemness and low exhaustion phenotype compared with conventional CAR-T. V-6h, which has the shortest process, displayed the highest T-stem and proliferation among all groups. In vivo models showed V-30h BZE2204 inhibited tumor growth and significantly prolonged mice lifespan under super low dosage (1E5 CAR-T/mouse). Three r/r NHL patients with high tumor burden were enrolled and infused with the dosage of 40-fold lower than conventional CAR-T. The data showed that CAR-T BZE2204 had promising clinical response with manageable safety profile, two achieved complete remission (CR) within 1 month, and the third patient with a single tumor over 7000mm2 in-size, achieved partial remission (PR) with &amp;gt;80% of tumor shrinkage. Pharmacokinetics data showed high CAR-T proliferation with Cmax of 5066 CAR-T cells/ul and AUC0-28 of 46, 486 cells*d. Our results show JL-Lightning CAR-T achieves high anti-tumor efficacy and good safety in large tumor-burden r/r NHL patients with super low dosage, likely due to short non-viral manufacturing process, high stemness and proliferation, and VHH-based multi-targeting design in CAR-T product. It offers a more effective, affordable and short vein-to-vein process option for hematologic malignancies CAR-T treatment. Citation Format: Shuya Wang, Yan Sun, Jinxing Lou, Hongfan Zhu, Zhicai Lin, Dan Sun, Jiaguo Li, Lijie Rong, Jun Guo, Jinjing Zhang, Yi Liu, Wenfeng Xu, Qijun Qian. JL-lightning CAR-T, a next generation non-viral CAR-T without in-vitro culturing procedure, shows high clinical efficacy, good safety with low treatment dosage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3173.