Anti-metastatic Effects Of DNA Vaccine Encoding Single-chain Trimer Composed Of MHC I And Vascular Endothelial Growth Factor Receptor 2 Peptide

Objective: Vascular endothelial growth factor receptor 2(VEGFR2)-mediated signaling is the key rate-limiting step in angiogenesis. VEGFR2 serves as the most important target of anti-angiogenic therapy for cancers. Furthermore, it was identified that there were three H-2Db-restricted CD8+ T-cell epitopes in mouse VEGFR2, designated as KDR1, KDR2 and KDR3, respectively. KDR1 was derived from the intracellular domain of VEGFR2, while KDR2 and KDR3 were derived from the extracellular domain. Immunization with KDR2 or KDR3, but not KDR1 peptide, was able to break self-tolerance and induce a specific CD8+ cytotoxic T-lymphocyte(CTL) immune response in C57BL/6 mice, thereby inhibiting angiogenesis and tumor growth in a mouse model. In addition, it was found that KDR2 was more effiiently processed by proteasomes and/or transported to the endoplasmic reticulum by transporter-associated with antigen processing(TAP) rather than KDR3, offsetting the higher affiity of KDR3. Therefore, KDR2 is an attractive candidate of CD8+ T-cell epitopes suitable for active immunotherapy targeting tumor blood vessels in which the main aim is to break self-tolerance of the molecules that promote angiogenesis. It has been demonstrated that the single-chain trimer(SCT) composed of major histocompatibility complex(MHC) class I heavy chain, β2-microglobulin(β2m), and antigen peptide was a particularly powerful strategy used to increase the potency of DNA vaccine against tumor and infection. Therefore, the aim of the present study is to cnstruct an SCT-encoding VEGFR2 antigen peptide(aa400-408, KDR2), β2m, and mouse MHC class I heavy chain H-2Db(pc DNA3.1(+)-KDR2-β2m-H-2Db, or SCT-KDR2) and to test its antimetastatic effects in mouse metastatic models, as well as to explore its related mechanisms.Methods: c DNA encoding β2m and H-2Db were cloned from splencytes of C57BL/6 mouse by RT-PCR. c DNA encoding the H-2Db–restricted KDR2 epitope and OVA epitope were synthesized. The gene segments were linked by Linker1 and Linker 2, then, they were inserted into the eukaryotic expression plasmind pc DNA3.1(+) to form the corresponding recombinant plasmid, pc DNA3.1(+)-KDR2-β2m and H-2Db(SCT-KDR2) and pc DNA3.1(+)-OVA-β2m and H-2Db(SCT-OVA), respectively. The constructs were expressed in human embryonic kidney cell line A293 cells. SCT proteins were analysed by flow cytometry. CTL activity specific for VEGFR2 was dected by LDH release assay. Tumor induced angiogenesis was examined both by alginate bead analysis and immunohistochemstry. Antimetastatic effects of SCT-KDR2 vaccination were examined in both B16 melanoma mouse metastasis model and 3LL Lewis lung carcinoma metastasis mouse model.Results: Our results confimed that the SCT-encoded recombinant plasmids pc DNA3.1(+)-KDR2-β2mH-2Db and pc DNA3.1(+)-OVA-β2m-H-2Db were constructed successfully and they can be expressed in eukaryotic cells. CTL activity specific for VEGFR2 was induced by intradermal immunization of mice with SCT-KDR2. Alginate bead and immunohistochemical analyses of tumor cell-induced angiogenesis showed that tumor-induced neovascularization in mice vaccinated with SCT-KDR2 construct were inhibited. FITC-dextran concentration in alginate beads derived from mice immunized with SCT-KDR2 was signifiantly reduced when compared with that in beads derived from mice vaccinated with the vector or SCT-OVA(P<0.01). Furthermore, the immunohistochemical analysis of factor VIII-related antigen in B16 melanoma showed again that vaccination of mice with SCT-KDR2 inhibited tumorinduced vascularization. Tumors in the SCT-KDR2 group showed markedly decreased factor VIII-related antigen-positive cells compared with those in the vector and SCT-OVA groups. To examine the anti-metastatic effects of SCT-KDR2 vaccination, a murine metastatic model with B16 melanoma was initially employed. It was obvious that the numbers of the tumor nodules on the lung surfaces from the mice immunized with SCT-KDR2 were significantly reduced as compared to those from the mice vaccinated with vector or SCT-OVA. To confim this fiding, anti-metastatic effects in the mouse 3LL Lewis lung carcinoma metastatic model was also examined. Our data showed again that vaccination of mice with SCT-KDR2 markedly inhibited pulmonary metastasis.Conclusion: In the present study, we have introduced the SCT platform into our DNA vaccine that consists of KDR2, β2m and H-2Db and the constructed SCT DNA vaccine was effiiently expressed in eukaryotic cells. The results also show that SCT DNA vaccine has its ability to induce CTL response to VEGFR2 and its capacity to inhibit tumor-induced angiogenesis and metastasis in murine models of B16 melanoma and 3LL Lewis lung carcinoma.