Abstract 4809: Preclinical characterization of an iPSC-derived allogeneic CD19/CD20 CAR-T cell designed to address multiple limitations of current CAR-Ts

Abstract In this work, we present the differentiation and in vitro characterization of iPSC-derived dual antigen targeting CD19/CD20 CAR-T cells as a milestone towards enabling iPSC-derived allogeneic cell therapies. While autologous CAR-T cell therapy has become an established model in treating B cell malignancies, its application is limited by manufacturing constraints, the necessity of bridging therapy, and heterogeneity of cell product. Allogeneic CAR-T cells derived from iPSCs present an opportunity to overcome these challenges through the scalable production of homogeneous cell products that are genetically engineered and precisely differentiated. Additionally, tumor antigen loss or downregulation remains a driver of disease relapse in patients treated with CD19 CAR T-cells. Accordingly, multi-antigen targeting CAR strategies are being developed to overcome these challenges and elicit more complete and durable responses to CAR-T cell therapy. Multiple expression strategies have been employed to express CARs for multi-targeting applications, including use of bicistronic and tandem formats. We demonstrate strong functional CAR expression from both formats dependent upon the composition of the CAR hinge and transmembrane domains. Based on expression level and antigen binding of dual-targeting CAR formats and designs in Primary T cells, lead CARs were nominated for screening via iPSC-T cell differentiation and in vitro functional testing. These lead CARs were site-specifically integrated into the genome of iPSCs using a streamlined workflow for identification of appropriate insertion site and promoter. iPSCs engineered with CD19/CD20 CARs were successfully differentiated while preserving the anticipated CD8 T cell phenotype. The dual-targeting CAR-T cells successfully eliminate disease-relevant tumor cells expressing both or single antigen targets in vitro. The development of dual-targeting iPSC-derived allogeneic CD19/CD20 CAR-T cells using a readily scalable manufacturing platform represents a formidable advance in overcoming multiple hurdles faced by established CAR therapies and increasing accessibility of potent and persistent off-the-shelf treatments for cancer and autoimmune disease patients. Citation Format: Justin Yoo, Jen F. Lai, Laura Prochazka, Elisa Martinez, Chris Saxby, Michael H. Cadell, Sommer Apelu, Libin Abraham, Dylan Lee, Zahra Sahaf, Charlie Liu, Laxsha Thaya, Elizabeth Csaszar, Siddarth Chandrasekaran, Emily Titus, Chris Bond. Preclinical characterization of an iPSC-derived allogeneic CD19/CD20 CAR-T cell designed to address multiple limitations of current CAR-Ts [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 4809.