Construction Of Oral DNA Vaccines Based On TEM8 And Investigation Of Their Anti-tumor Efficacy

Angiogenesis is important for some normal physiologic progression such as embryonic development, wound healing, inflammation, but also important for some pathologic conditions such as tumor, rheumatic arthritis, retinopathies, etc. Direct and indirect evidences indicated that the growth and metastasis of tumor are angiogenesis-dependent. Therefore, tumor angiogenesis research has become a hot spot recently. Up to date, some specific anti-angiogenic drugs have been enrolled clinical trials and proved that anti-angiogenesis was a potential method for cancer therapy. Since anti-angiogenic therapy needs a persistent and long-term impact to suppress tumor growth, specific chemical or biological inhibitors of angiogenesis often require constant administration at a relatively high dose level that is very troublesome and costly. Compared with chemical therapy, active immunotherapy is more convenient, cost effective and worthy of intensive exploration. Recently, researchers have obtained some evidences that active immunotherapy can inhibit tumor growth in vivo, but some disadvantages such as the low specificity of the targeted antigen need to be improved.Tumor endothelial markers (TEMs) which dominantly expressed in endothelium of human colon cancer were observed by Croix and his colleagues in 2000. Following evidences indicated that TEM8 is one of the most specific antigens for anti-angiogenic tumor immunotherapy. However, whether TEM8 can act as an ideal target for anti-angiogenic therapy remains unclear. In this report, we tested our hypothesis that active immunotherapy targeting TEM8 could inhibit tumor angiogenesis and tumor growth.we firstly constructed the eukaryotic expression plasmid pTEM8-â… encoding TEM8-â… and bicistronic expression plasmid pTEM8/mGM-CSF containing human TEM8-â… and murine GM-CSF. After identified with restriction enzymes and sequencing, the two plasmids were used to transform an attenuated strain of murine Salmonella typhimurium (SL7207) to obtain oral vaccines based on TEM8. At the same time, eukaryotic expression plasmid pEGFP-N1 encoding green fluorescent protein (GFP) was used to transform SL7207 to obtain a tracing vaccine. Then, C57BL/6 mice were immunized with these oral vaccines. 3 days later, antigen presentation cells expressing GFP were found both in Peyer's patches and spleen, confirming that the vaccine was effectively processed and the immunization condition was optimal.We adopted B16F10 murine melanoma model and CT26 murine colon carcinoma model to estimate the vaccine's anti-tumor efficacy by protective setting or therapeutic setting. In protective experiment, mice were immunized three times at 2-wk intervals. Two week thereafter, mice were challenged with tumor cells by s.c. or i.v. injection to induce primary tumor or experimental pulmonary metastases. In therapeutic experiment, mice were challenged with tumor cells 3 days after the first immunization, then the other two times vaccinations were performed at 2-wk intervals as before. We found that immunotherapy with SL7207/pTEM8-â… or SL727/pTEM8/mGM-CSF primed effective anti-tumor immune response at both protective and therapeutic settings, resulting in markedly reduced growth of tumors and prolonged life span of immunized mice.We also used a monoclonal antibody specific for murine CD31 to detect the microvessel density of tumor tissues, and observed that MVD of tumors in mice immunized with TEM8 vaccines is obviously lower than the control group. The results of alginate assay in vivo and FITC-Dextran ingestion assay also indicated that angiogenesis in immunized mice was inhibited. To detect TEM8 specific CTLs, splenocytes were collected from mice immunized with pTEM8/mGM-CSF or control group, and tested by a standard 51Cr release assay. The results showed that only splenocytes isolated from the mice immunized with pTEM8/mGM-CSF killed the CT26 cells transfected with TEM8 gene. A multiclonal antibody specific for TEM8 was used to detect the expression profile of TEM8 in CT26 colon carcinoma tissues, the results showed that the TEM8 proteins only expressed in tumor endothelial cells. These results further demonstrated that the anti-tumor activity of TEM8 vaccines is resulted from destroying the tumor endothelial cells.At last, we explored the vaccine's anti-tumor mechanism and side effects. We did the experiment of protective immunization utilizing the C57BL/6J derived CD4 gene knockout mice and CD8 gene knockout mice. No obvious protective immune response was observed in mouse immunized with the vaccine in the absence of CD8 molecules; but the CD4 deficiency seemed do not affect the vaccine induced protective immune response. These results implied that CD8+ T cells play the essential role in the anti-tumor immune response primed by the vaccines. As to side effects, we didn't find obvious differences between the mice orally immunized with pTEM8/mGM-CSF with control group in body weight, major viscera appearance and general state. The skin vulneration assay in the two groups also showed no difference in skin wound healing and histologic characteristics.Based on the results above, we can draw following conclusions:â‘ Oral DNA vaccines delivered by attenuated salmonella have the ability to break self-tolerance and induce the immune response against self antigens;â‘¡The vaccines targeting the TEM8 can inhibit tumor growth by anti-angiogenesis effects;â‘¢CD8+ T cells play the critical role in tumor rejection immune response primed by oral DNA vaccine delivered by attenuated salmonella.