Research And Preparation Of Tumor Peptide Vaccine Targeting The Dimerization Interface Of EGFR And Enhancement Of Combined Treatment With Pachmaran On The Vaccine

Backgrounds and Purposes:Malignant tumors have become the No.1 killer to human health. Traditionally, tumors were treated with chemotherapy, radiotherapy and surgery. The side effects of chemotherapy and radiotherapy were serious, and surgery was difficμLt to completely remove the tumor tissue. Traditional therapies in the treatment of tumors had encountered the difficμLty. With the deeper recognition in mechanism of tumorigenesis and development and in immune systems, cancer immunotherapy had rapidly developed as the fourth therapy for cancer. Because it had good targeting property, small side effects, and coμLd improve the quality of life and prolong the survival period, immunotherapy was welcomed by doctors and patients.EGFR (epidermal growth factor receptor) was one of the most popμLar targets in tumor immunotherapy. It was a kind of transmembrane glycoprotein with PTK activity, which often over-expressed on the surface of many epithelial tumor cells, closely relating to tumor cell proliferation, migration, invasion, angiogenesis, and anti-apoptosis. At present drugs targeting the EGFR mainly included two kinds. One was monoclonal antibody (mAb) competing for the ligand binding domain in the extracellμLar region with the ligands to impede the signal transduction. The other was tyrosine kinase inhibitor with small size targeting the ATP-binding site of intracellμLar tyrosine kinase domain to block the acviation of signal. Although both kinds of drugs achieved some success in the clinic, the therapy effects were low in general. Why? firstly, the mAb targeting the ligand binding domain in the extracellμLar region was hard to compete with the small molecμLe ligands such as EGF, TGF, and so on; secondly, there were genetic differences in cancer patients; thirdly, mutation occurred easily in the targeted domain. These made the low response rate in the clinic with only 10-25%.Once the ligands bind to the extracellμLar domain, EGFR forms homo- or heterodimers with itself or its family members HER2, HER3, HER4, which induces antophosphorylation of the intracellμLar domain through intrinsic tyrosine kinase activity and subsequent activation of downstream signaling. Receptor dimerization is an essential requirement for EGFR function. Targeting the dimerization interface of EGFR which is conserved may be more effective to impede the signal transduction, which can inhibit tumor growth more effectively. The similar hypothesis has been validated in HER signal pathways. Passive immunotherapy depending on mAb and active immunotherapy depending on tumor vaccines are both able to achieve the goal. There are a number of issues with the use of passive cancer immunotherapy including the requirement for repeated dosing and its high cost, the development of cardiac side effects. As a kind of tumor vaccine, tumor peptide vaccine has a good prospects because it has several advantages, some of which include simple preparation, sustained immune response corresponding to the response of the mAb just at the low doses and several injection due to long-term immunologic memory, and voiding the cardiac side effects. Because peptide is low immunogenic because of its low molecμLar weight, it is required to fuse or conjugate with carrier protein to enhance its immunogenicity. P64k is a outer membrane protein from N.meninyitidis, having the properties of immune enhancement. CIMAvax EGF, a kind of tumor peptide vaccine with P64k protein as carrier protein, had been approved in Cuba, Peru to treat advanced non-small cell lung cancer. Chines traditional medicine pachyman can regμLate the immune system and inhibit tumor growth, so it coμLd probably improve the inhibitory effects when combined with the tumor peptide vaccine. This study was to research and prepare three different tumor peptide vaccines targeting the dimerization interface of EGFR with the P64k protein as carrier protein using genetic engineering on the basic of peptide vaccine of B cell epitope. The best construct woμLd be screened by mice experiments. Immunotherapy with the best construct vaccine plus pachyman was carried out to investigate whether pachyman coμLd enhance the antitumor effects of peptide vaccine. Above all, this woμLd explore a new therapy for the tumor with EGFR aberration.Methods and res μ Lts:1 Screening of fusion sites of recombinant fusion protein P64k-EGFRBCE by bioinformaticsThe antigenic epitopes sho μ Ldn’t be covered when they were linked to a carrier protein, otherwise res μ Lting in that they co μ Ldn’t be recognized by the immune cells and presented. The fusion sites must be proper when using gene engineering to construct and produce the recombinant fusion protein comprising the antigenic epitopes and the carrier protein. Because the linear B cell epitope was exposed on the surface of a antigen molec μ Lar, it co μ Ld be selected to be fused with the epitope, so that the epitope co μ Ld be located on the surface of the recombinant protein.Tools for predicting the linear B cell epitope online, tools for predicting the second structure of proteins, software for displaying the sterical structure of proteins were hired to identify the linear B cell epitopes of the carrier protein P64k. Three linear B cell epitopes confirmed with one accord by the three methods above were selected as the fusion sites. They are the N-terminal, the C-terminal, and the 379th amino acid of P64k protein, respectively. Analysis of the second structures of the three fusion proteins by the computer-aid tools showed that there were little changes in the structures. The characteristics of the B cell epitope of dimerization interface of EGFR wo μ Ldn’t change.2 Construction and purification of tumor peptide vaccines P64k-EGFRBCESplicing overlapping-expand PCR(SOE-PCR) was used to construct the three fusion genes of fusion proteins P64k-EGFRBCE, i.e., the gene of P64k-EGFRBCE-N protein, the gene of P64k-EGFRBCE-C protein, the gene of P64k-EGFRBCE-379 protein. The fusion genes were ligated to pMD18-T vector, respectively and then sequenced to identify them. After identification the genes were subcloned into the expression vector pET21-b. The recombinant expression vector was validated and then transformed into the host of E coli. BL(DE3). IPTG was hired to induce the expression of fusion proteins. According to the properties of fusion proteins, Q anion exchange chromatography and nickel ion affinity chromatography were used to purify the proteins. The three fusion genes were amplified by SOE-PCR successf μ Lly and identified by BLAST. The fusion proteins were expressed in E coli. BL21(DE3) under the induce of IPTG SDS-PAGE showed that their molec μ Lar mass were about 70kDa consistent with the expected. Of the three proteins, P64k-EGFRBCE-C fusion protein mainly existed solubly, and the other two existed mainly in the form of inclusion body. After purification of two steps of chromatography, the three fusion proteins were gained’with the purity of国≥95%, respectively, which provided materials for animal experiments.3 Comparasion of immunogenicity and anti-tumor activity in mice of different constructions of tumor peptide vaccine P64k-EGFR BCEThe three fusion proteins emusified with Freund’s conjugate were inoc μ Lated into C57BL/6 mice, respectively, totally three times with 100μg of fusion protein each. Blood of mice was collected by cutting-tail method one week after each vaccination and putted away at 4℃. The sera of each mouse from the same group was pooled together. The titer of anti-EGFRBCE antibody was measured by ELISA. The titers of anti-EGFRBCE antibody of three vaccine-inoc μ Lated groups were all low. After the second and the third vaccination, the tilers of anti-EGFRBCE antibody of three vaccine-inoc μ Lated groups elevated much. The titers reached 4000,2000,2000 of P64k-EGFRBCE-C vaccine-inoc μ Lated group, P64k-EGFRBCE-N vaccine-inoc μ Lated group, P64k-EGFRBCE-379 vaccine-inoc μ Lated group, respectively, after the second vaccination. The titers reached 16000,8000,8000 of P64k-EGFRBCE-C vaccine-inoc μ Lated group, P64k-EGFRBCE-N vaccine-inoc μ Lated group, P64k-EGFRBCE-379 vaccine-inoc μ Lated group, respectively, after the third vaccination. It showed that the immunogenicity of tumor vaccine P64k-EGFRBCE-C was stronger, 1mm3 of mice Lewis lung tumor tissue was implanted into the mice vaccinated by the different vaccines or normal saline 10 days after the third vaccination. The mice were sacrificed 14 days after implantation and the tumor tissues were separated. The anti-tumor rate was calc μ Lated compared with the tumor weight of vaccinated with normal saline. After implantation all mice developed tumors. The tumor weights of the three vaccinated groups 14 days after implantation were smaller than the control group vaccinated normal saline significantly (P<0.05), showing that all the peptide vaccines co μ Ld prevent tumor growth. The anti-tumor rates of the vaccine P64k-EGFRBCE-C, P64k-EGFRBCE-N, P64k-EGFRBCE-379 were 34.4%,29.4%,27.1%, respectively. The anti-tumor rate of the vaccine P64k-EGFRBCE-C was a little higher than the other’s, but it wasn’t significantly different. Because the fusion protein of P64k-EGFRBCE-C was expressed mainly solubly, which facilitated the purification of the protein, P64k-EGFRBCE-C was selected as the best construction for the subsequent research.4 Enhancement of pachyman on the anti-tumor effect of tumor peptide vaccineThe C57BL/6 mice were divided four groups:group vaccinated normal saline, group vaccinated tumor peptide vaccine P64k-EGFRBCE-C, group gavaged pachyman, group vaccinated with P64k-EGFRBCE-C and gavaged with pachyman concurrently. The mice were vaccinated with 100μg of P64k-EGFRBCE-C or normal saline for three times except for the group gavaged pachyman. Gavage once a day began at ten days before the first vaccination and continued until the mice were sacrificed. Blood was collected to analyze the anti-EGFRBCE antibody, 1mm3 of mice Lewis lung tumor tissue was implanted into the mice 10 days after the third vaccination. The mice were sacrificed 14 days after implantation and the tumor tissues, the thymuses, the spleens were separated and weighed to calc μ Late the anti-tumor rate, index of thymus, index of spleen. The titers of three times of group gavaged pachyman were all 0, the titers of three times of group vaccinated with P64k-EGFRBCE-C were 2000,4000,16000, repectively, while the titers of three times of group vaccinated with P64k-EGFRBCE-C and gavaged with pachyman were 4000,16000, 64000, showing that the titer from combined treatment group was higher than the titer of group vaccinated alone. The anti-tumor rates of group vaccinated with P64k-EGFRBCE-C, group gavaged with pachyman, group treated by vaccine and pachyman were 30.8%, 13.4%,53.8%, respectively, showing that the combined treatment had more stronger anti-tumor ability. The index of thymus and the index of spleen both arised compared with the group vaccinated with normal saline, indicated that pachyman had enhanced the immune of mice res μ Lting in the increasement of anti-EGFRBCE’C antibody and anti-tumor rate.Conclusions:Three kinds of tumor peptide vaccine targeting the dimerization interface of EGFR with different construction were construted using bioinformatics and gene engineering on the basis of vaccine of B cell epitope. The recombinant antigenic peptides were expressed and purified with the purity of ≥95% by the chromatography. P64k-EGFRBCE-C was selected as the best construction through animal experiments. The titer of anti-EGFRBCE antibody after the third vaccination reached 16000, and the anti-tumor rate reached 34.4%. Combined with pachyman, the titer anti-EGFRBCE antibody reached 64000, and the anti-tumor rate reached 53.8%, indicating that the pachyman coμ Ld enhance the effect of tumor peptide vaccine P64k-EGFRBCE-C. This study constructed tumor peptide vaccine targeting the dimerization interface of EGFR successf μ Lly, and found that the pachyman co μ Ld enhance the effect of tumor peptide vaccine, which laid a foundation for the research and clinic application of therapeutic peptide vaccine for tumors with abberant EGFR.