Abstract 39: Preclinical in vitro and in vivo evaluation of CD8αβ+ CD19 CAR iPSCs T cells generated in a TCR signal-independent manner using DLL4/VCAM-coupled microbeads
Authors: Belew Muluken S. , Carpenedo Richard , Pallaoro Alessia , Hoi Hiofan , Deyati Avisek , Kirouac Daniel C. , Chandrasekaran Siddarth , Wall Valerie , Abraham Libin , Apelu Sommer , Cadell Michael H. , AuYeung Amanda , Subedar Omar , Martinez Elisa , Joshi Shri , Csaszar Elizabeth , Woodside Steven , Titus Emily , Bond Christopher 
Published: March 2024

Abstract
The use of clonally-derived, induced pluripotent stem cells (iPSCs) as starting material for therapeutic T cell manufacturing would overcome many limitations of autologous Chimeric Antigen Receptor T cell (CAR-T) therapies. Complex, multi-stage genome engineering, large batch production and rigorous lot testing could provide a consistent source of off-the-shelf, functionally enhanced T cell products. However, this vision cannot be realized using existing T cell differentiation platforms, which fail to present Notch ligands with the precision and intensity required to control T cell differentiation in scalable, suspension culture.
We have previously shown that VCAM1 synergizes with DLL4 to enhance Notch signaling and progenitor T cell differentiation. We have extended the utility of this discovery through the creation of DLL4/VCAM1-coated magnetic microbeads, enabling precise and temporal control of Notch signaling in suspension culture. iPSC-derived CD34+ cells cultured with DLL4/VCAM1 beads demonstrate dose-responsive activation of Notch gene expression (e.g., Notch1, DTX1, TCF7), resultant T lineage commitment (e.g. CD5, CD7 expression) and maturation to CD4-CD8+ (single positive) cells in a TCR signal-independent manner. Removal of the endogenous TCR is necessary to prevent graft-versus-host-disease. Therefore, differentiation and maturation in a TCR-independent process presents substantial advantage in the development of safe, off the shelf iPSC T cell products.
Using a clonal iPSC line with a CD19 CAR knocked in at the TRAC locus, we apply this approach to generate CAR-expressing, CD8αβ+ functional T cells, capable of multiple rounds of in vitro tumor cell lysis and sustained tumor growth inhibition in vivo comparable to primary T cells. This advancement demonstrates proof-of-concept for generation of highly efficacious, off-the-shelf CAR-T cells using novel, small-footprint manufacturing in order to broaden the applicability and accessibility of T cell therapies for cancer patients.
Citation Format: Muluken S. Belew, Richard Carpenedo, Alessia Pallaoro, Hiofan Hoi, Avisek Deyati, Daniel C. Kirouac, Siddarth Chandrasekaran, Valerie Wall, Libin Abraham, Sommer Apelu, Michael H. Cadell, Amanda AuYeung, Omar Subedar, Elisa Martinez, Shri Joshi, Elizabeth Csaszar, Steven Woodside, Emily Titus, Christopher Bond. Preclinical in vitro and in vivo evaluation of CD8αβ+ CD19 CAR iPSCs T cells generated in a TCR signal-independent manner using DLL4/VCAM-coupled microbeads [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 39.