Mvegf Epitope Vaccine In The The Mice Xenografts Application,

Tumor growth is dependent on angiogenesis, many pro-or anti-angiogenic factors have been implicated in the process, the key factor of which is vascular endothelial growth factor (VEGF). VEGF have been found high-expressed in many kinds of tumor. Many studies demonstrated that inhibition of VEGF function may inhibit tumor growth.Now, VEGF antibody(AvastinTM antibody), has been used in the tumor therapy, and exhibited good effects, but the disadvantages still existed. Though AvastinTM is a humanized antibody, it still retains heterology. In clinical application, the antibody was generally used repeatedly, so enough attention should be paid to the heterology of antibody. In addition, the bigger clinical dosage and high production cost of antibody drug results in its high price of clinical therapy, and limits its clinical application to some extent.VEGF antibody treatment of tumor is a passive immunotherapy. If the active immunotherapy through VEGF vaccine is adopted in tumor treatment, the disadvantages of VEGF antibody treatment of tumor, including repeated use, heterology etc, will be avoided. But VEGF is a protein with its own normal physiological function, generally, the body has no response to its own normal protein. Obviously, VEGF protein can not be used as vaccine protein.The purpose of this article is to design a mVEGF vaccine to break or bypass the immune tolerance, making body producing neutralized antibody against mVEGF to inhibit angiogenesis and tumor growth.In order to achieve the above purpose, the design strategy for mVEGF vaccine was determined. Firstly, find the epitopes of mVEGF based on its advanced structure, then display the selected epitopes on a simple protein scaffold to construct new proteins, the new proteins are used as mVEGF vaccine candidates.The variable region of heavy chain of antibody (VH) was selected to be the simple protein scaffold. Because the antigen binding region of antibody is composed of the variable region of heavy chain and variable region of light chain (VL), the VH alone is unstable. To increase the stability of VH, three key amino acid residues were mutated. The mutated VH was easily expressed in E.coli, and was successfully refolded the stable protein. CDR3of VH was selected as the site for display of the antigen epitope, because of its high variability in peptide length and composition of amino acids.Three candidate neutralized epitopes of VEGF164were selected based on the structure of VEGF165and its receptor complex. They were epitopel (EYPDEIEYIFKP), epitope2(KSHEVIKFMDV) and epitope3(IMRIKPHQSQH).The amino acid sequence of CDR3was firstly replaced by that of epitope2of VEGF164to construct a new protein, which was designated as mFV2. The amino acid sequence of mFV3was then converted into its encoding sequence, and the encoding sequence was fully synthesized and cloned into pET-24a vector to construct epitope2expression vector. BL21(DE3) was transformed by the epitope2expression vector, and then induced by IPTG, mFV2was highly expressed as inclusion body. After washing, the inclusion body was dissolved in8mol/L urea, the pure mFV2protein was obtained by Sephacryl S-100size exclusion chromatography, and successfully refolded.The encoding sequences of epitopel and epitope3of VEGF164was inserted into the CDR3region by overlapping PCR, and then cloned into pET-24a vector. The mFV1and mFV3were expressed, purified and refolded using the same method.The purified mFV1, mFV2and mFV3were then used to immunize the female Balb/c mice. Five weeks later, three kinds of immune serum were obtained, which were designated pAb1, pAb2and pAb3。They all specifically recognized VEGF164but not unrelated proteins analyzed by ELISA. The titers of the three kinds of immune serum reached1:104. Western Blot analysis indicated that pAb1and pAb2could recognize VEGF164, but pAb3not. Probably, the epitope3is a conformational epitope, which lost its conformational structure in Western blot analysis. pAb1, pAb2and pAb3all recognized the expressed VEGF164in B16cells in immunofluorescence analysis. The expressed VEGF164of B16cells was located in cytoplasm.HUVEC cells were used to test the neutralized activities of the three kinds of immune serum. The abilities of proliferation, migration and tubular formation of HUVEC were analyzed. The results demonstrated that VEGF164could promote migration of HUVEC and stimulate its proliferation and tubular formation. pAbl, pAb2and pAb3all exhibited the activities of VEGF164inhibition to some extent, in which pAb3had the best inhibition activity.Based on the above results, the experimental study on tumor therapy of the three mVEGF vaccine was carried out.Firstly, three mVEGF vaccines were inoculated in mice to observe if they could inhibit H22tumor growth. BALB/c mice were used, and divided into four groups, including model group, mFVl group, mFV2group and mFV3group. The mouse in mFVl group, mFV2group and mFV3group was immunized three times with mFVl proein, mFV2protein and mFV3protein, respectively. Five weeks later, the immune serum was analyzed by ELISA, at the same time, all the mice in the four groups were inoculated with H22tumor cells. When the tumors could be touched, start to record the status of tumor growth everyday, and finally all the mice were put to death to separate and to weight the tumors. The results indicated that all the three vaccines exhibited the abilities to inhibit tumor growth on some extent. Among them, mFV3showed excellent effects, its inhibition rate of tumor reached83.8%.To learn the details of tumor inhibition of mVEGF vaccines, the tumor tissues were analyzed by immunochemistry. The results showed that micro-vessel densities(MVD) in vaccine groups were lower than that in model group, in which the MVD of tissue from mFV3group was lowest, this is the reason why mFV3exhibited best ability of tumor inhibition.In S180tumor model, the three vaccines showed the same effects as those in H22tumor model. mFV3still gave the best ability of tumor inhibition, the tumor inhibition rate reached80.3%.To mimic the usage in clinic practice, the study on the therapeutic inoculation of mVEGF vaccines in mouse tumor model was performed. The female BALB/c mice were divided into four groups, they were model group, mFV3group, cisplatin group and mFV3plus cisplatin group. H22tumor cells were inoculated in all the mice, at the same time, the mice were treated with vaccine or cisplatin. The mice in the model group was injected with physiological saline once a week for two weeks, the mice in mFV3group were immunized with mFV3protein in dayl and day14, the mice in cisplatin group were injected with cisplatin once a week for two weeks and immunized with mFV3protein in day1and day14. All the test mice were put to death in day28, the tumors were separated and measured. Finally, in mFV3group the rate of tumor inhibition was27.3%, in cisplatin group, the rate of tumor inhibition was35.7%, while in mFV3plus cisplatin group, the rate of tumor inhibition reached66.2%. Obviously, inoculation of vaccine was not conflict with chemical therapy, they can be combined in the application of the treatment of tumor. In brief, the epitope display system based on the variable region of antibody was established in this paper. Using this system, three mVEGF vaccines were constructed. The experiment results indicated the three vaccines had good antigenicity, and could induce mice to produce the neutralized antibodies. In tumor model, they all exhibited the abilities to inhibit tumor growth to some extent.