Immune Treatment On Human Digestive Tract Tumor By Using α-gal Epitopes

Main idea of this research describes a method by which the human natural anti-gal antibody can be exploited as an endogenous adjuvant for targeting autologous tumor vaccines to antigen-presenting cells (APCs). Tumor cells remaining in the patient after completion of surgery, radiation, and chemotherapy are the cause of tumor relapse. These residual tumor cells can not be detected by imaging, but their destruction may be feasible by active immunotherapy.Since specific tumor-associated antigens (TAAs) have not been identified for the majority of cancers, and it is believed that their expression varies from one type of tumor to the other, and in different patients that have the same type of tumor irradiated autologous tumor vaccines have been considered as an immunotherapy treatment that may elicit an immune response against the residual tumor cells expressing TAAs. However, tumor cells evolve in cancer patients in a stealthy way, i.e., they are not detected by APCs, even in the form of vaccine. Effective targeting of tumor vaccines for uptake by APCs is a prerequisite for eliciting an effective immune response which requires transport of the vaccine by APCs from the vaccination site to the draining lymph nodes. In the lymph nodes, the APCs transporting the vaccine process and present peptides, including the autologous TAA peptides for activation of the tumor-specific T cells. The required targeting of vaccines to APCs is feasible in humans by the use of anti-gal. Solid tumors obtained from surgery are homogenized and their membranes subjected toα-gal epitope synthesis. Administration of irradiated autologous tumor vaccines processed to express-gal epitopes results in in situ opsonization of the vaccinating cells or cell membranes due to anti-gal binding to these epitopes. The bound antibody serves to target the autologous tumor vaccine to APCs because the Fc portion of the antibody interacts with Fc receptors on APCs. Since patients receive their own TAAs, the vaccine is customized for autologous TAAs in the individual patient. The repeated vaccination with such autologous tumor vaccines provides the immune system of each patient with an additional opportunity to be effectively activated by the autologous TAAs. In some of the immunized patients this activation may be potent enough to induce an immune-mediated eradication of the residual tumor cells expressing these TAAs.The difficulty of cancer immunotherapy in autologous tumor vaccines is that tumor cell can not uptake by antigen-presenting cells (APC). Recombinantα1,3-galactosy- ltransferase (rα1,3-GT) can synthesizeα-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) in vitro on human tumor cell, which maybe increase the uptake of vaccinating autologous tumor cell membranes by APC. This epitope is produced in large amounts in nonprimate mammals and New World Monkeys due to the intracellular activity of the glycosylation enzymeα1,3-galactosyltransferase. And not such binding was detectable on cells of human, Old World monkeys and apes, but since as much as 1% of circulating IgG antibodies in humans interact with this epitope. This natural antibody was designated"anti-gal". Anti-gal is the most abundant naturally occurring antibody in humans, constituting 1% of serum IgG (20-100g/ml serum). Anti-gal interacts specifically with theα-gal epitope (Galα1-3Galβ1-4GlcNAc-R) on glycolipids and glycoproteins and is produced throughout life as a result of antigenic stimulation by bacteria of the gastrointestinal flora. Innate antibody specific for cell surfaceα-gal epitope was the basis for complement-mediated hyperacute xenograft rejection and antibody dependent cell mediated cytotoxicity.The main protocol is to express and purify catalytic domain of murineα-1,3-galactosyltransferase in a soluble form in E.coli and to utilize the enzyme to synthesizeα-gal epitopes on digestive tract tumor cells as a novel tumor vaccine. Firstly, according to the sequence of genebank, design a pair of primers and use RT-PCR technique to acquire the aim DNA sequence with two digested sites of restriction enzymes (NdeⅠand XhoⅠ). Digest both the PCR products and pET-15b with NdeⅠand XhoⅠ. Purify them with a reuse kit. Ligate them with T4 ligatease overnight. Establish expression system in pET-15b to express catalytic domain of murineα-1, 3-galactosyltransferase. IPTG induce expression of the aim protein. 12% SDS-PAGE analyze the results of purification. Identify its expression with PCR, expression test and western blotting for it takes (His) 6 tag.Α-1, 3-galactosyltransferase can transfer gal structure to N-Acetyllactosamine receptors. UDP-gal takes 2 negative charges while UDP takes 3 and both of them have absorbency at 260 nm so we can identify its activity by HPLC with anion exchange column. Moreover use catalytic domain ofα-1, 3-galactosyltransferase to synthesisα-gal epitopes on human digestive tract tumor cells including cancer of esophagus and stomach. Then incubate the cells with human serum and with ovine anti-human IgG coupled with FITC as the secondary antibody. Analyze fluorescent intensity by flow cytometry. Incubate the cancer cells withα-gal epitopes with its own serum to test the ability of their interaction through numbers of cell death. Acquire DNA sequence for expression ofα-1,3GTcd with RT-PCR. Efficiently express and purifyα-1,3-galactosyltransferase with His-tag in a soluble form in E. coli BL21.Identify its activity by HPLC with anion exchange column. The results of flow cytometry give the evidence of the successful expression of theα-gal epitopes on human digestive tract tumor cells. Then separately culture the tumor cells withα-gal epitopes and those withoutα-gal epitopes in their autologous human serum, and observe the difference of numbers of death cells.This research synthesizes recombinant murineα-1, 3GT-pET-15b vector and expressesα-1, 3-galactosyltransferase in a soluble form in E. coli BL21. Western blotting, PCR and expression test identify its expression. Test its catalytic activity with HPLC. Then successfully utilizes it synthesizeα-gal epitopes on digestive tract tumor cells including two cases of esophagus cancer and two cases of stomach cancer. The difference of numbers of death cells between cell groups withα-gal epitopes and groups withoutα-gal epitopes has statistics meaning. It gives an evidence of interaction ofα-gal epitopes and anti-gal. This research makes a stable foundation for use of this method in clinical tumor patient immunotherapy.