Construction Of Peptide Vaccines Based On The EGFR Dimerization Targeting Strategy

Epidermal growth factor recepor (EGFR) was the first receptor to be proposed as thetarget for cancer therapy,however,the strategies for targeting were not enough successfulclinically."Dimerization" plays a key role in the activation of EGFR, so targeting theinterface domain of the dimerization maybe the key for improving the responses ofanti-EGFR clinically. A B-cell epitope peptide from the interface domain was found, by ourlaboratory recently, to be immunogenicity and potentiality in the clinical targeting of EGFR.This study will focus on the feasibility of targeting dimerization in anti-EGFR therapy,furthermore, a novel monoclonal antibody (mAb) and a therapeutic peptide vaccine will bedeveloped based on the strategy. The feasibility of targeting dimerization of EGFR will beevaluated by a series of in vitro experiments with the novel mAb and epithelioma cell linesof distinct subtypes of EGFR. Neisseria meningitidis P64k (P64k) and a promiscuous T cellepitope from the measles virus fusion protein (MVF) will be selected as theimunnopotentiating protein or peptide,and C57BL6mice employed as the poor antibodyresponder (PAR) animal for screening antigen peptides. The effectiveness of vaccine will beevaluated by Lewis lung cancer (LLC) exnografts grown in C57BL6mice.We hope that ourwork on the strategy for targeting EGFR should be helpful to tap the potential of anti-EGFRtherapy for cancer.Methods1. Preparation of B cell epitope of reorganization antigen peptide MVF-EMVF-E plasmid was imported into E.coli BL21(DE3) expression system.EDTA andPMSF were added to inhibit the degradation of endogenous proteases. The degraded proteinwere removed by anion exchange chromatography source30Q, The fusion protein was purified by Ni affinity chromatography. The antigen peptide was obtained by kinaseenzyme digestion.2.Preparation of chemical coupling B cell epitope P64K*E and P64K*MVF-EP64K protein was expressed in E.coli expression system. And purified through the Q,source30Q and PEI ion exchange column. The carrier protein P64K was obtained at the purity ofmore than90%. B cell epitopes E was obtained by chemical synthesis and MVF-E wasobtained via method1. The carrier protein, B-cell epitope E and MVF-E was dissolved inPBS at the molar ration of1:10respectively. Glutaricaldehyde was added at the finalconcentration of0.5%. After45min,0.2M glycine was added to over the reaction. At last,Sephadex G25was used to remove the small molecules.3. Preparation of B cell epitope of reorganization antigen peptide P64K-E and E-P64KP64K-E and E-P64K plasmid was introduced into E.coli expression system,the fusionprotein was purified by Ni affinity chromatography.4. Immunological analysis of B cell epitope antigen peptides60C57BL/6mice were randomly divided into six groups, group I: adjuvant controlgroup; Ⅱ group: MVF-E group; III Group: P64K*E group; Ⅳ group: P64K*MVF-Egroup; Ⅴ: P64K-E group; the Ⅵ groups: E-P64K group. After the initial immunization thebooster immunization was given every two weeks. Serum titer was assayed after10days ofeach immunization.5. Analysis of antitumor activity in vivoThe tumors was transplanted in dorsal scapular subcutaneously. The tumorgrowth,physical condition were observed in mice. The tumor was measured from the7thday after transplantation with the vernier caliper. After the21th day,the peel mousetumor,weighing, antitumor of vaccines and lung metastasis were observed.Results:1. Five kinds building form of MVF-E、P64K*E、P64K*MVF-E、P64K-E and E-P64K wereprepared successfully.2. Five peptide vaccine was produced high titers of antibody in mice, and wherein theMVF-E titers of up to4000, higher antibody titers generated by the P64K*E, up to32,000, while the P64K-E and E-P64K group titer of about8000to about16000. P64K of*theMVF-E of low titer, about2000to about4000.3. P64K*E, the P64K-E and E-P64K group with the control group of tumor suppressioncompared with the case having a statistically significant difference, wherein P64K*Einhibited tumor is most evident. The MVF-E,P64K*MVF-E inhibited tumor growth wasnot obvious. In21days, the mice in each group were not lung metastases.Conclusions:The form of vaccines of P64K*E was screened successfully, which can produce hightiters of anti-peptide antibody,and in vivo antitumor experiments showed with a strongtumor inhibitory.