Dual-Vector-Transduction with Single-Step Magnetic Selection (Zip-sort) Increases Transgenic Payloads to Enhance CAR T Cell Activity

Introduction: Chimeric antigen receptor (CAR) T cells have demonstrated potent activity in hematologic malignancies and are showing promise in treating solid tumors. However, tumor cells can escape from CAR T cells through multiple mechanisms including (1) insufficient or heterogeneous target antigen expression which promotes antigen low or antigen-negative escape, and (2) upregulation of inhibitory ligands or receptors on tumor cells and T cells, which impairs T cell persistence and function. CAR T cells can also (3) become exhausted in response to repetitive antigen challenges in the tumor microenvironment. While many innovations in CAR design and manufacturing have been developed to overcome individual tumor escape mechanisms, addressing multiple immune evasion strategies simultaneously will likely require engineering T cells with multiple countermeasures. However, engineering T cells with multiple transgenes is limited by packaging constraints and efficiencies of current vector systems. To overcome these challenges, we engineered a leucine zipper-based cell purification method, called Zip-sort, that enables selective single-step magnetic purification of T cells transduced with two vectors to increase transgene payload.

Methods: We constructed a system whereby each of two vectors co-expresses a heterodimerizing leucine zipper pair, resulting in affinity-tag expression only on the surface of dual-transduced cells, and enabling selective immunomagnetic purification. Leveraging the increased packaging capacity afforded by the Zip-sort methodology, we co-expressed multiple CARs and switch receptors to enable T cells to overcome antigenic heterogeneity, resist exhaustion, and persist and proliferate in response to inhibitory ligand expression. We also included dual safety switches to enable CAR T cell elimination in case of toxicity. We dual-transduced T cells to express up to four CARs targeting CD19, CD20, CD79b, and BAFF-R and up to three switch receptors including Fas-4-1BB, CD200R-CD27, and PD-1-OX40. We characterized these multi-CAR multi-Switch receptor T cells in vitro with live cell imaging evaluating NFkB signaling, T cell proliferation, and target cell elimination, with single-cell RNA sequencing, and with multiparameter flow cytometry. We also established and tested syngeneic mouse models of pre-B-cell and acute myeloid leukemia with antigen heterogeneity and inhibitory ligand overexpression.

Results: Zip-sort methodology enabled production of highly purified dual-transduced T cells over a range of initial transduction efficiencies without significantly affecting patterns of genomic vector integration compared with single-transduced T cells. Integrated vector copy number was on average two-fold higher in dual-transduced vs. single-transduced T cells. By engineering Zip-sorting technology into safety switches and switch receptors, we further extended the functions of this system while limiting vector insert size. Following single-step Zip-sort purification, dual-transduced T cells expressed multiple CARs and switch receptors with uniformly high purity and receptor expression. These modifications enabled T cells to eliminate heterogeneous leukemia populations expressing up to four target antigens and up to three inhibitory ligands. Dual-CAR T cells targeting CD19 and CD20 and co-expressing Fas-4-1BB, CD200R-CD27, and PD-1-OX40 switch receptors survived and eliminated targets with high FasL expression and proliferated in response to CD200 and PD-L1 expression on leukemia cells. Co-expression of multiple switch receptors reduced transcriptomic signatures of exhaustion in dual-CAR T cells, upregulated gene pathways related to proliferation and metabolic activity, enhanced the capacity of T cells to lyse target cells and release cytokines following repeated antigen challenges, and promoted leukemia clearance in vivo.

Conclusion: Zip-sort purification of dual-transduced T cells facilitates production of complex cell products incorporating multiple transgenic payloads. Our proof of principle experiments demonstrate that T cells can be engineered to overcome multiple tumor resistance mechanisms simultaneously. The Zip-sort system provides a powerful methodology to construct and compare novel cellular therapies and is compatible with clinical translation based on immunomagnetic selection principles.