Development and optimization of Eva1 () targeting chimeric antigen receptor T cells.

Background: Whereas chimeric antigen receptor gene modified T (CAR-T) cell therapy has been clinically applied to malignant lymphomas and multiple myeloma, CAR-T cell therapy for solid tumors has so far not reached clinical application. Epithelial V-like antigen 1 (Eva1), transcribed from is a small surface protein highly expressed on various tumor cells. We selected Eva1 as a novel solid tumor-target antigen because of its broad expression across various tumor types. The purpose of the present study is to develop and optimize CAR-T cells targeting Eva1.

Method: We prepared various humanized single chain variable fragment sequences based on a mouse anti-human Eva1 monoclonal antibody. We constructed six humanized Eva1CAR-Ts and selected one that maintained specificity and good cellular proliferation after antigen stimulation. We further optimized the length of the extracellular spacer domain and the choice of the intracellular domain in vitro and in two different xenograft mouse models.

Results: We confirmed Eva1 expression on various tumor cell lines by flow cytometry and analysis of public database, but we also observed that normal monocytes weakly expressed Eva1. A combination of short spacer domain and 4-1BB or CD79A/CD40 intracellular domain provided higher treatment efficacy both in vitro and in vivo. The cytokine release on autologous monocyte stimulation to Eva1CAR-T cells was comparable to that on autologous B cell stimulation to CD19CAR-T cells. Humanized Eva1CAR-T cells demonstrated excellent therapeutic efficacy by infusing a single dose of Eva1CAR-T cells (1×10) in both NCI-H1975 lung cancer and CFPAC-1 pancreatic cancer cell line grafted model.

Conclusions: In summary, these data suggest that humanized Eva1CAR-T has promising therapeutic potential for the treatment of various Eva1-positive solid tumors. Regarding on-target/off-tumor recognition, further detailed analyses of the Eva1CAR-T cell responses to normal tissues are needed.