Immunogene therapy of tumors with vaccine based onXenopushomologous vascular endothelial growth factor as a model antigen

Abstract

Overcoming immune tolerance of the growth factors associated with tumor growth should be a useful approach to cancer therapy by active immunity. We used vascular endothelial growth factor (VEGF) as a model antigen to explore the feasibility of the immunogene tumor therapy with a vaccine based on a single xenogeneic homologous gene, targeting the growth factors associated with angiogenesis. To test this concept, we constructed a plasmid DNA encoding Xenopus homologous VEGF (XVEGF-p) and control vectors. We found that immunogene tumor therapy with a vaccine based on XVEGF was effective at both protective and therapeutic antitumor immunity in several tumor models in mice. VEGF-specific autoantibodies in sera of mice immunized with XVEGF-p could be found in Western blotting analysis and ELISA assay. The purified immunoglobulins were effective at the inhibition of VEGFmediated endothelial cell proliferation in vitro, and at antitumor activity and the inhibition of angiogenesis by adoptive transfer in vivo. The elevation of VEGF in the sera of the tumor-bearing mice could be abrogated with XVEGF-p immunization. The antitumor activity and production of VEGF-specific autoantibodies, significantly elevated IgG1 and IgG2b, could be abrogated by the depletion of CD4 ؉ T lymphocytes. The observations may provide a vaccine strategy for cancer therapy through the induction of autoimmunity against the growth factors associated with tumor growth in a cross reaction with single xenogeneic homologous gene and may be of importance in the further exploration of the applications of other xenogeneic homologous genes identified in human and other animal genome sequence projects in cancer therapy.

A gene therapy for cancer based on the angiogenesis inhibitor, vasostatin

“…The full-length sequence of vasostatin was confirmed by dideoxy sequence to be identical to that in Genebank (GI:5921996) and those reported. [13][14][15] The expression plasmid pSecTag2B-vaso and the empty vector pSecTag2B were purified by using two rounds of passage over EndoFree columns (Qiagen, Chatsworth, CA), as reported previously by us 36 and others. 37 Western blot analysis Western blot analysis was performed as described.…”

“…Blots were then washed and incubated with a biotinylated secondary antibody (biotinylated rabbit anti-goat IgG), followed by transfer to Vectastain ABC (Vector Laboratories, Burlingame, CA, USA). 36 In vitro biological assay for vasostatin Biological activity of vasostatin was determined using conditioned medium from transfected cells in endothelial cell proliferation assay. 36,38 Briefly, 3000 endothelial cells (HUVECs and SVEC4-10), non-endothelial cells (NIH/3T3 fibroblast and T/G HA-VSMCs) or tumor cells (Meth A and LL/2c) were plated on to 96-well culture plates in triplicate and incubated (37°C, 5% CO 2 ) for 24 h in 100 l medium containing bFGF.…”

“…36 In vitro biological assay for vasostatin Biological activity of vasostatin was determined using conditioned medium from transfected cells in endothelial cell proliferation assay. 36,38 Briefly, 3000 endothelial cells (HUVECs and SVEC4-10), non-endothelial cells (NIH/3T3 fibroblast and T/G HA-VSMCs) or tumor cells (Meth A and LL/2c) were plated on to 96-well culture plates in triplicate and incubated (37°C, 5% CO 2 ) for 24 h in 100 l medium containing bFGF. The medium was replaced with 20 or 40 l of vasostatin-containing supernatant from transfected COS cells mentioned above or control cells.…”

“…After 72 h, the cell number was determined by a trypan blue dye exclusion test, and the percentage inhibition was calculated by the following formula as described: inhibition % = [(NϪN T )/(NϪNo)] × 100, where N is the number of untreated cells cultured for n days, No is the cell number on day 0, and N T is the number of treated cells cultured for n days. 3,36,38 …”

“…3,18,36 Five mice in each group were anesthetized. Corneal micropockets were created with a cataract knife.…”