Abstract PR14: Fibroblast activation protein as a universal target for chimeric antigen receptor T cell therapy in solid tumors

Rationale: The recent success of chimeric antigen receptor (CAR) T cell therapy in treating leukemia has brought new enthusiasm to this strategy. While the majority of CAR research has been primarily focused on targeting tumor cells, our group is developing a new CAR construct to target Fibroblast Activation Protein (FAP), a molecule that is highly and selectively expressed on cancer-associated fibroblasts (CAFs). It is well known that these CAFs play an important role in tumor development and tumor progression. Thus, we hypothesized that the immune-mediated destruction of CAFs by adoptively transferred FAP-CAR T cells would inhibit tumor growth in pre-clinical tumor models. Experimental procedures: The 73.3 anti-mouse FAP hybridoma was sequenced to obtain the single chain Fv to insert into our existing CAR construct with the 4-1BB and CD3ζ intracellular signaling domains. Different combinations of FAP-CAR constructs were synthesized and transduced into human T cells first to screen for optimal structure-activity response. The best two FAP-CAR constructs were then selected to transduce into mouse T cells for in vitro cytotoxicity and cytokine assays, as well as in vivo studies. To evaluate the in vivo efficacy, 2 tumor cell lines, AE17 (mouse mesothelioma) and TC1 (mouse lung cancer), were inoculated subcutaneously into flanks of syngeneic C57Bl6 mice. When the flank tumors reached 150 mm3, 10 million CAR T cells were injected through tail vein and tumors were then monitored. Tumor tissues were collected at the end of the study to evaluate the effect of CAR T cells on tumor microenvironment by flow cytometry and/or by immunohistochemistry. Results: All 8 different combinations of anti-mouse FAP CAR constructs expressed on human T cells showed target-specific activities against 3T3parental (FAP-null) versus 3T3FAP cells, in terms of cytotoxicity and cytokine production. The two best FAP-CAR constructs were then transduced into mouse T cells and re-tested for cytotoxicity and cytokine production against 3T3parental and 3T3-FAP fibroblasts, and again showed target-specific killing and cytokine production. When FAP-CAR T cells were adoptively transferred into tumor bearing mice, tumor growth inhibition (~50%) was observed in both flank tumor models while control T cells did not show any efficacy. No toxicity was detected in the animal studies. We found that FAP-CAR T cell-treated tumors had significantly more apoptotic and less proliferating tumor cells, less