A novel switchable car-T cell therapy for tumor retargeting and improved safety

Abstract

Background & Aim Chimeric antigen receptor (CAR) T cells have proven effective for treatment of B cell malignancies. However, conventional CAR T cell therapy is still hindered by such issues as relapse of refractory disease —due in part to tumor heterogeneity and antigen escape— cytokine release syndrome (CRS), on-target, off-tumor toxicity and costly manufacturing. To address all these issues, we developed a switchable immune receptor that can attach to different antigen recognition molecules (ARMs) and allow CAR T cells to switch their target antigens. Methods, Results & Conclusion Sortases are transpeptidases that can covalently bind proteins that have a short polypeptide recognition motif. We designed a sortase CAR (Srt.bbz) that consists of an extracellular sortase domain, a transmembrane domain and an intracellular signaling domain. Our preliminary tests showed that Srt.bbz T cells were capable of binding sortase-compatible probes in the picomolar range. Similarly, sortase-compatible Her2-ARMs attached to Srt.bbz T cells, and once “ARMed,” the T cells could recognize Her2 antigen and become activated. Next, we demonstrated tumor cytolysis using human primary Srt.bbz T cells that were directed to Her2+ tumor cells using Her2-ARMs and showed that the magnitude of cytolysis was dependent on the Her2-ARM dosage. To address whether immune escape by antigen loss could be overcome by Srt.bbz retargeting, we demonstrated that Srt.bbz T cells could kill Her2+EGFR+ tumor cells using either an EGFR-ARM or Her2-ARM. Next, we established that Srt.bbz T cells could provide improved safety by a combined Her2 and EGFR-ARM dosing, which increased tumor cytolysis compared to using either ARM alone. Finally, we tested Srt.bbz T cells in vivo using an intraperitoneal (IP) tumor model with an infusion of 10e6 Srt.bbz T cells and Her2-ARM injections every other day for 4 weeks. This resulted in complete tumor clearance in all mice (n = 17). In summary, we describe a novel switchable CAR T cell therapy with the potential for treating refractory disease and for improving CAR T cell safety.