Exploring human T lymphocytes expressing a CAR directed to the tumor-neoantigen EGFRvIII for adoptive cellular therapy to non-small cell lung cancer
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Abstract
Adoptive cellular therapy (ACT) using genetically engineered T cells expressing a chimeric-antigen-receptor (CAR) to redirect their specificity holds great promise in tumor immunotherapy. However, in contrast to remarkable clinical responses observed with CD19-CAR T cells for B-cell leukemias, efficacy of ACT in solid tumors is often hampered by insufficient and non-durable immunity of T cells facing an immunosuppressive tumor microenvironment. Moreover, identifying tumor-specific neoantigens with low off-target activity is difficult. The epithelial growth factor variant III (EGFRvIII) is only found on neoplastic tissue and represents a promising candidate target for ACT in e.g. glioblastoma as already shown in clinical trials and has also been described for non-small cell lung cancer (NSCLC). EGFRvIII-CAR T cells have not yet been tested in NSCLC, we therefore aimed in this preclinical study to evaluate antitumor immunity of EGFRvIII-CAR T cells against NSLCL in vitro and in vivo using a xenograft mouse model. Human, preactivated T cells were retrovirally transduced, and CAR expression was analyzed by flow cytometry 7 days (d) post transduction. Stably transfecetd EGFRvIII+ cells of a HLA class I/II-deficient melanoma cell line (Ma-Mel-86) or the NSLCL cell line A549 were used as targets. EGFRvIII-CAR T cells were examined for IFN-γ/Granzyme B release and cytotoxicity using ELISpot-, 51Cr-release- and 48h co-culture assays. To evaluate the therapeutic potential in vivo, intratumoral T-cell transfer into NSG mice displaying palpable subcutaneous tumor engraftment was performed. Upon retroviral transduction >85% T cells expressed the EGFRvIII-CAR as confirmed by flow cytometry. EGFRvIII-CAR T cells induced vigorous responses to both EGFRvIII+ tumor targets in vitro as measured by IFN-γ and Granzyme B release and elicited potent cytotoxicity (>60%) upon short-term 5h coculture (51Cr-release) and >90% in co-culture assays after 2d. Moreover, we generated EGFRvIII-CAR redirected T cells derived from naïve, CD45RA+ precursors displaying a stem cell and central memory phenotype, which have been previously reported to elicit superior antitumor immunity as compared to more differentiated T cells. The less differentiated EGFRvIII-CAR redirected T cells showed comparable anti-EGFRvIII responses when compared to EGFRvIII-CAR T cells obtained from bulk cultures regarding stimulation activity and cytokine response. In this study we could prove effective reactivity of EGFRvIII-CAR T cells towards EGFRvIII-expressing melanoma and lung cancer cells in vitro. To evaluate the therapeutic potential of EGFRvIII-CAR T cells in vivo, further exploitation and optimization regarding the mouse model is required to overcome obstacles we encountered in preliminary studies. Although EGFRvIII appears not to be a dominant target among the different EGFR mutations found in NSCLC patients, EGFRvIII-CAR ACT might provide valuable information as a proof of concept study for immunotherapy in lung cancer and might contribute to improve ACT for solid tumors.