Abstract 3999: Augmentation of antitumor effects by CAR-T cells secreting anti-PD-1 single-chain antibody fused with IL-21

Abstract

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy has shown remarkable responses in hematologic malignancies. However, many patients suffer from limited response and tumor relapse due to lack of persisting CAR-T cells and immune escape. Delivery of immunomodulatory cytokines is one of the strategies to improve immune function; however, the systemic effect of cytokines can be detrimental. Methods: To prevent the toxic effects of cytokines, we proposed the concept of targeting cytokines to tumor-specific T cells in vivo. To this end, we developed CAR-T cells secreting a fusion protein (PD-1Ab21) composed of anti-PD-1 single chain antibody and IL-21, which target IL-21 to CAR-T cells to promote their proliferation and differentiation into memory cells while blocking the PD-1 signal of CAR-T cells. The secretion and functions of PD-1Ab21 were identified. The proliferation and differentiation of CAR-T cells secreting PD-1Ab21 (PD-1Ab21-CAR-T) were compared in vitro with that of the conventional CAR-T cells. The therapeutic effects of CAR-T cells secreting PD-1Ab21 were studied in immunodeficient mice engrafted with human Burkitt lymphoma Daudi, multiple myeloma cell U266 and Her2/neu+ breast cancer cell BT474. Results: The results showed that the culture supernatant of PD-1Ab21-CAR-T cells, like anti-PD-1 antibody, blocked the binding of PD-1 to PD-L1 and stimulated STAT3 phosphorylation in human T lymphocytic leukemia cell Hut78 with the same activity as IL-21. In the conventional culture milieu with high dose of IL-2, the proliferation of the PD-1Ab21-CAR-T cells was not different from that of conventional CAR-T cells. While cultured with low dose of IL-2 that could not support the proliferation of conventional CAR-T cells, the PD-1Ab21-CAR-T cells still showed significant proliferation. A major fraction of the PD-1Ab21-CAR-T products was shown to co-express CCR7, CD62L and CD45RA, and express high level of TCF-1, indicating a memory stem-like phenotype. While the most of conventional CAR-T cells showed effector phenotype defined as CD45RA+CCR7-. Furthermore, adoptive transfer of PD-1Ab21-CAR-T cells exhibited significantly improved CAR-T cell expansion and resulted in superior tumor eradication in the xenograft models of human B-cell malignancy, multiple myeloma and Her2/neu+ breast cancer. The enhanced cellular function by secreting PD-1Ab21 was mediated through an autocrine and/or paracrine manners. Conclusion: We developed an enhanced CAR-T cells. Our data provide preclinical evidence to support the translation of this design for an improved CAR-T cell-mediated anti-tumor response. Citation Format: Ying Li, Jitao Zhao, Xiangrui Cheng, Hang Li, Ran Tian, Qianqian He, Shengdian Wang. Augmentation of antitumor effects by CAR-T cells secreting anti-PD-1 single-chain antibody fused with IL-21 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3999.