Background Clinically effective CAR T cell therapy for solid tumors may require substantial T cell engineering to increase specificity and potency. We have developed AB-2100, an autologous, integrated circuit T (ICT) cell that encodes multiple synthetic ‘modules’ to overcome challenges in the treatment of clear cell renal cell carcinoma (ccRCC). AB-2100 includes a sequential ‘AND’ logic gate designed to limit off-tumor toxicity through dual tumor antigen recognition, a short hairpin ribonucleic acid-microRNA (shRNA-miR) module for the constitutive expression of shRNA-miRs for knockdown of FAS and TGFBR2, and a constitutive synthetic pathway activator (SPA) that drives constitutive STAT3 signaling for enhanced T cell cytotoxicity and expansion.
Methods On-target, off-tumor toxicity was previously observed with constitutive CA9 CAR T cell therapy. To overcome this, the AB-2100 sequential ‘AND’ logic gate consists of a priming receptor (PrimeR) specific for PSMA and an inducible CA9-targeted CAR that is expressed upon PrimeR engagement with PSMA on the tumor neovasculature of ccRCC. This unique feature of the logic gate increases the safety profile of AB-2100 given that PSMA and CA9 are not expressed in the same normal tissues. Dual-antigen specificity of the logic gate was assessed in vitro and in vivo via CA9+ and PSMA+CA9+-786-O tumors established on contralateral flanks. To model vascular priming, AB-2100 cells were co-cultured with PSMA-expressing endothelial HUVEC cells and K562-CA9 cells. An in vitro FAS cross-linking assay was conducted to assess the impact of FAS knockdown on FAS-mediated apoptosis. The enhanced anti-tumor activity conferred by TGFBR2 shRNA and SPA modules were assessed in a subcutaneous 786-O xenograft model. Lastly, AB-2100 potency was measured in a subcutaneous renal A498 xenograft tumor model.
Results In vitro cytotoxicity against single or dual antigen expressing tumor cell lines, as well as a dual flank xenograft model demonstrate that AB-2100 selectively kills tumors that express both CA9 and PSMA, and not tumors that express CA9 alone. Furthermore, we confirmed that AB-2100 was able to prime off of PSMA-expressing endothelial HUVEC cells and kill K562-CA9 tumor cells. Finally, AB-2100 containing shRNA-miR and SPA modules demonstrated enhanced anti-tumor activity in xenograft RCC models (786-O and A498).
Conclusions Preclinical data demonstrate that AB-2100 can selectively target antigens that cannot be safely targeted by conventional CARs, and overcome multiple suppressive mechanisms in the tumor microenvironment. These results support the evaluation of AB-2100 in the clinic for the treatment of advanced or metastatic ccRCC.
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