Abstract 3198: Fully human CD20 and CD19 dual-targeting CAR-T cells mediate long-term remissions and protection from tumor rechallenge in vivo

Abstract

Abstract Introduction: Despite significant progress in the chimeric antigen receptor (CAR)-T cell therapy of B cell malignancies, the 5-year progression-free survival rate in large B cell lymphoma following CD19-targeting CAR-T cell treatments remains below 50%. While some treatment failures were attributed to CD19 antigen loss, immunogenicity of murine single-chain fragment variable (scFv) elements has been implicated in relapse and resistance to CAR-T cell re-treatment. In this study, we evaluated fully human CD20 and CD19 dual-targeting CAR constructs (h20.19 CARs) combining human scFvs and distinct co-stimulatory domains and architectures, to mitigate tumor antigen escape, reduce immunogenicity, and improve CAR functionality. Methods: The h20.19 CAR constructs were assembled from fully human scFvs against lymphoma antigens CD19 and CD20, with hinge, transmembrane and co-stimulatory domains derived from 4-1BB, CD28, OX40 or ICOS. Tandem second generation (T8/8BBz), tandem third generation (T28/28BBz, T8/28BBz), and bicistronic (D28z/BBz, DBBz/28z, DOXz/ICz) CARs were evaluated. CAR T cells were derived from healthy donors’ CD4+ and CD8+ T cells by lentiviral transduction, and CAR expression was evaluated by flow cytometry. CAR-mediated target cell killing was assessed by co-culture assays, and cytokine elaboration by ELISA. In vivo CAR-T cell function was evaluated in a Raji lymphoma NSG xenograft model using low CAR-T dose (2x106/mouse). Tumor-free survivors were rechallenged with Raji cells to compare CAR-T cell potency and persistence. Results: The hCD20.19 CAR-T cells lysed CD19+CD20+ tumor lines Raji, NALM-6 and REH, and single-antigen lines 293T CD19 and 293T CD20, but not the parental CD19-CD20- 293T cells, indicating antigen-specific efficacy. Cytokines TNFα, IL2 and IFNγ were strongly upregulated in response to Raji cells. During repeat co-culture with Raji cells, the third-generation tandem hCD20.19 CARs and the bicistronic CARs showed greater cytotoxicity than the second-generation CAR T8/8BBz. In vivo, all third-generation tandem and bicistronic CARs induced rapid and durable Raji tumor rejection by study day 7 post CAR-T, with no apparent toxicity. Upon long rest and rechallenge with Raji cells on study day 50, all h20.19 CARs attenuated tumor progression as compared to age-matched untreated mice cohort. CARs T8/28BBz and D28z/BBz afforded the least protection from Raji rechallenge, whereas CAR DOXz/ICz mediated rapid tumor rejection, but mice met euthanasia criteria by study day 71 due to weight loss. The tandem CAR T28/28BBz and bicistronic CAR DBBz/28z, demonstrated tumor rejection, persistence and safety after rechallenge, up to study day 86. Conclusions: In summary, the fully human dual-targeting h20.19 CARs exhibit robust anti-tumor function and persistence in vitro and in vivo, mediate long-term remissions and resist tumor rechallenge. Citation Format: Peirong Hu, Brittany Steimle, Ngoc Tran, Pradyot Dash, Dina Schneider. Fully human CD20 and CD19 dual-targeting CAR-T cells mediate long-term remissions and protection from tumor rechallenge in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3198.