Superantigen-mutant-linked Tumor Cells Expressing Heat Shock Protein 70 Onto The Surface Elicit Potent Antitumor Specific Immunity

Superantigen-mutant-linked Tumor Cells Expressing Heat Shock Protein 70 Onto The Surface Elicit Potent Antitumor SpecificImmunityIn tumor immunotherapy, the vaccines made from modified tumor cells become attractive recently. It is known that potent tumor-antigen-specific cytotoxic lymphocytes (CTL) play a leading role in antitumor immunity. Therefore, in the development of the antitumor vaccine, it was intensively sought to reinforce antitumor CTL elicited by the vaccines. However, the identification and isolation of tumor CTL epitopes are too difficulty to be feasible for individual tumor therapy.Heat shock proteins (HSP), highly conserved and widely existing in all prokaryotes and eukaryotes, are known as a great family of molecular chaperone proteins stress-inducible in synthesis. Recently, some HSPs derived from tumor cells were found to be able effectively to initiate specific immunity against the tumor through the transportation of tumor antigen peptides to antigen presenting cells (APCs) on MHC class I presentation way to activate tumor-antigen-specific CTL. Inducing tumor-antigen-specific CTL without resorting to the identification of CTL epitopes from individual cancers, vaccines based on HSP become practical and attractive. Besides, some HSPs could efficiently improve the immunogenicity of the tumor cells, induce the maturation and activation of dendritic cells (DC) and amplify the secretion and transduction of cytokines (CKs). Hence, HSP was of unique advantage when used in the antitumor vaccine. Furthermore, some research showed that genetic expressing of some HSPs onto the vaccine cell surface, membrane-bound HSP (mbHSP), may probably facilitate this course, and thereby elicit more effective tumor-specific CTL in the tumor therapy. Nonetheless, the antitumor specific immunity induced by HSP alone is not strong enough to be satisfactory perhaps for the unfavorable context in which tumor antigens are delivered.Superantigens (SAg), typified by staphylococcal enterotoxin A (SEA), are a collection of bacterial and virus proteins capable of potent and extensive immune activation. SAg has been applied for antitumor therapy widely. However, it may be advisable to combine SAg with other therapies because of its heavy side-effect. In light of the mechanism by which HSP70 (a typicalHSP) elicits specific immunity, we hypothesize that, if SAg anchored on the tumor cells genetically modified with mbHSP could break immuno-tolerance and produce a potential and extensive immuno-activation around the cells, such a microsurrounding may be greatly propitious to the delivery of tumor antigen peptides and initiation of antitumor specific immunity: High local concentration of cytokines, recruited T lymphocytes and activated DCs would favor the transportation and DC intracellular process of HSP-peptide complexes, and promote "MHC I -peptide-HSP" complexes to activate tumor-antigen-specific CD8+ T. Meanwhile, an appropriate amount of superantigen anchored onto the cells could not activate the host's immunity overly, and thereby prevent immune inhibition occurring subsequently. Also, based on some studies, the mutant SEA D227A has lower toxicity and less side-effect than native SEA; So, the site-directed mutation will be applied in this study in favor of vaccine efficacy .To test the hypothesis, we first construct the expression vector of the fusion protein of SEA and its mutant SEA D227A with transmembrane sequence (TM), and obtained the proteins in vitro. After HSP70 or mbHSP70 expression vector was constructed and transfected into the melanoma B16 cells, the fusion proteins were anchored onto the cells, and the dual-modified vaccines were created in this study. Then, C57BL/6 mice were vaccinated before and after the melanoma cells B16 were inoculated, and its therapeutic effects and immune protection were investigated.Part I the mbHSP70 gene-modified vaccine were prepared and itsantitumor effect was investigated1. Construction of mbHSP70 expression vector in eukaryotesThe HSP7...