Figure 2.

The specificity of EGFR recognition by EGFR scFv and the construction of EGFR-CXCR5 chimeric antigen receptor (CAR)-Ts

(A) Graphical representation of the CAR designed using the anti-EGFR scFv, CD8a hinge, and transmembrane domain, 4-1BB and CD3zeta endodomain. EGFR-CXCR5 was constructed with an additional CXCR5 sequence after the CD3zeta endodomain. (B) ELISA of anti-EGFR scFv with recombinant human immunoglobulin G1 (IgG1) Fc-conjugated EGFR (ErbB1), HER2 (ErbB2), HER3 (ErbB3), MUC1, Flk1 (VEGFR2), and FLT4 (VEGFR3). Recombinant proteins were coated in the plate wells at 5 μg/mL. Anti-EGFR scFv concentration started from 5,000 pg/mL and was diluted 5-fold repeatedly until 8 pg/mL. (C) FACs analysis of A549 and PC9 (LUAD cell lines), H929 (myeloma cell line), Raji (human Burkitt’s lymphoma cell line), and K562 (human myelogenous leukemia cell line) stained with anti-EGFR scFv. Concentration started from 20,000 ng/mL and was diluted 10-fold repeatedly until 0.2 ng/mL. (D) The expression of transgenes in lentivirus-transduced T cells was analyzed by flow cytometry using protein L and anti-CXCR5 antibody. Single dot represents individual sample. Error bars represent mean ± SD for each T cell population (n = 4).