The Study Of Recombinant α1, 3-galactosyltransferase On Oral Cancer Immunotherapy

Background: The difficulty of cancer immunotherapy in autologous tumor vaccines is that tumor cell can not uptake by antigen-presenting cells ( APC ). This study is directed to methods for stimulating complement-mediated destruction of tumors cells and concomitant stimulation of tumor specific antibody production and tumor specific cytotoxic cells. α-gal epitope is produced in large amounts in nonprimate mammals and New World Monkeys due to the intracellular activity of the glycosylation enzyme α1,3-galactosyltransferase. And not such binding was detectable on cells of human, but since as much as 1% of circulating IgG antibodies in humans interact with this epitope. This natural antibody was designated "anti-Gal". Innate antibody specific for cell surface α-gal epitope was the basis for complement-mediated hyperacute xenograft rejection and antibody dependent cell mediated cytotoxicity. Recombinant α1,3-galactosyltransferase ( rα1,3-GT ) can synthesize α-gal epitopes in vitro on human tumor cell, which maybe increase the uptake of vaccinating autologous tumor cell membranes by APC. Together, the opsonized autologous tumor membranes carrying tumor-associated antigens ( TAA ) is expected to increase effective uptake of the vaccine by APC, which will solve a difficult problem of poor presentation of TAA. Object: The aim of our study was to determine whether the expression of α-gal epitope can modify the tumorigenicity of human oral cancer cells and search a new method to solve a difficulty that tumor cell could not uptake by antigen-presenting cells.. Method: We isolated a mouse cDNA for α1,3-GT. This cDNA was inserted into the expression vector pBAD/Thio-TOPO. The cloned gene sequence contained α1,3-GT gene and its downstream (His)6 tag coding sequence. This tag enables the simple affinity purification of rα1,3-GT on a nickel-Sepharose column and elution with imidazole. And subsequent transformation of LMG194 with recombinant vector, resulted in high-level expression of rα1,3-GT. Incubation of human oral cancer cells ( Tca8113 ), mixed together with rα1,3-GT, neuraminidase , and UDP-Gal , observed the synthesis of α-gal epitopes on membrane-bound carbohydrate chains at the cell surface by FACS. Resulte: In this study the purified enzyme appears in SDS-PAGE as one band with the size of 53 kD. rα1,3-GT resulted in the effective synthesis of α-gal epitopes on membrane-bound carbohydrate chains at the cell surface. Conclusion: The method in this study is very effective in the synthesis of many α-gal epitopes on tumor membranes. These epitopes readily bind the naturally occurring anti-Gal antibody. These dates provided evidence that rα1,3-GT was suitable for the synthesis of α-gal epitopes on bulk amounts of oral cancer cells required for the preparation of autologous tumor vaccines.