Preparation Of Xenogeneic Egfr Extracellular Domain Of Recombinant Dna And Protein Vaccine And Anti-tumor Effect In Mice

Epidermal growth factor receptor(EGFR) is a 170kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity encoded by the oncogene c-erbB. Mutation and overexpression of EGFR are common in many human epithelial tumors,which have been correlated with bad prognosis.The constitutive activation of the EGFR signal pathway can drive cancer cell proliferation,invasion and survival.Therefore,EGFR can be considered as a tumor-associated antigen(TAA) and become an attractive target for cancer therapy.In this study,we constructed a DNA vaccine and a protein vaccine based on regionⅡ-Ⅲof xenogeneic extracellular domain of EGFR(EGFR-ECD) fused with Fc portion of IgG(IgG-Fc).Their active anti-tumor immune effects on mouse Lewis lung cancer were evaluated,cEGFR-Fc recombinant gene was amplified by Splicing by overlap extension by PCR(SOE by PCR) and then cloned into pVAX1 plasmid as DNA vaccine.Meanwhile,we constructed secretory expression plasmid pET-28a(+)-S which carried the signal sequence of lysostaphin and then cloned cEGFR-Fc fragment into it.pET-28a(+)-S-cEGFR-Fc was transformed into E.coli stain BL21(DE3) followed by induction with IPTG.The expressed fusion proteins were purified by Protein A affinity chromatography and then used as protein vaccine. 48 C57BL/6J mice were randomized into four groups:DNA vaccine alone,protein vaccine alone,combination immunization with DNA vaccine and protein vaccine, and normal saline(NS) groups.Mice were challenged with Lewis lung cancer cells after the fourth immunization.Tumor growth,mice's status and potential toxicity of regimens above were observed.The weight of tumors was measured after 19 days to investigate the inhibition rate of tumor growth.The sera from immunized mice were assayed for the presence of anti-EGFR antibodies by Western blot and the antibody levels measured by ELISA.Splenic lymphocytes were isolated to determine the immunotoxic effect against A431 cells.Both pVAXI-cEGFR-Fc and pET-28a(+)-S-cEGFR-Fc were successfully constructed.After transformed with pET-28a(+)-S-cEGFR-Fc,E.coli BL21(DE3) expressed about 30-35%secretory protein after induction.The purification efficiency of protein A affinity chromatography was over 85%.Animal experiment results indicated that tumor volumes of mice in the three vaccine group were significantly smaller than those in the NS group(P＜0.01).And the tumor volumes of mice in combination immunization group were significantly smaller than those in the other two vaccine groups(P＜0.01).The tumor inhibition rate of DNA vaccine,protein vaccine group and combination immunization group was 37%,49%and 63% separately.Serum anti-EGFR antibodies were induced in all three vaccine groups. The antibody titer was highest in combination immunization group followed by protein vaccine and DNA vaccine group.The splenic lymphocytes were shown to be immunotoxic against A431 cells.The killing rates of the vaccine group were higher than that in the NS group(P＜0.01).This study demonstrated that recombinant DNA and protein vaccine based on the regionⅡ-Ⅲof xenogeneic EGFR-ECD can break immune tolerance against self-EGFR and induce specific anti-tumor autoimmunity.The combination use of these two vaccines may further enhance antitumor effect.