Abstract PR14: Fibroblast activation protein as a universal target for chimeric antigen receptor T cell therapy in solid tumors.
Authors: Wang Liang-Chuan S. , Lo Albert , Scholler John , June Carl H. , Pure Ellen , Albelda Steven M. 
Published: August 2014

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 matrix (collagen, fibronectin), less M2 macrophages and fewer blood vessels, in comparison with the untreated and control T cell-treated tumors. To assure specificity, similar in vivo studies were also carried out in FAP knockout mice. In the KO mice, all the anti-tumor activity of FAP-CAR T cells was lost.

Conclusion: Here, we demonstrate the feasibility of inhibiting tumor growth by targeting tumor stroma, instead of tumor cells, with adoptively transferred CAR T cells. We detected no safety issues with these stroma-targeted CAR T cells. We also showed that the anti-tumor effects induced by our T cells were specific to FAP. We are currently evaluating different CAR constructs against human FAP with a goal of conducting a clinical trial.

This abstract is also presented as Poster A5.

Citation Format: Liang-Chuan S. Wang, Albert Lo, John Scholler, Carl H. June, Ellen Pure, Steven M. Albelda. Fibroblast activation protein as a universal target for chimeric antigen receptor T cell therapy in solid tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR14.