Antitumor Effects Of The Fusion Protein Transmembrane Superantigen SEA

Bacterial superantigens (SAgs) are microbial toxins that target the immune system causing massive T-cell activation, cytokine release and systemic shock. They represent some of the most potent bioactive molecules ever discovered. Despite wide variation in their protein structure, SAgs all share a common ability to simultaneously bind the class II major histocompatibility complex (MHC-II) molecules expressed on professional antigen presenting cells (APCs) and the variable region of the T-cell receptor P -chain (TCR v P). The result is massive stimulation of any cell that expresses the correct TCR v P element on its surface. Staphylococcal enterotoxin A (SEA) belong to a family of bacterial SAgs that has the capacity to active a large proportion of T cells to mediate cytotoxity and cytokine secretion. SAgs bind to MHC dass II molecules as unprocessed proteins and subsequently activate T cells expressing particular T cell receptor v P -chains (TCR v P). SEA stimulates T cells bearing certain TCR v$ elements when bound as an unprocessed protein outside the antigen groove of MHC II molecules. The subsequent polyclonal T cell expansion results in massive cytokines release. Local release of IL-2 or IFN-y by transfected tumor cells can elicit systemic antitumor immunity.SEA has been shown to induce antitumor cytotoxicity in human lymphocytes in vitro against the K562 leukemia and the Raji lymphoblastoid B cell lines as well as human colon carcinoma cells; SEA can also prevents the development of human hyper-nephoroma in the cheek pouch of golden hamsters, Lewis carcinoma, 38C13 lymphoma in vivo. As above mentioned, the biological effects of SAgs make them very attractive to usefor targeted T cells activation and elimination of tumor cells expressing MHC II molecules. Actually, MHC class II molecule are expressed on normal B-cells and monocytes. To target the SAg to the site of tumor cells exist, SEA was genetically fused to the human colon cancer-reactive monoclonal antibody C215, C242. The results showed that the recombinant fusion protein C215Fab-SEA can trigger cytotoxic T cells against tumor cells those bear the proper tumor antigen irrespective of MHC class II expression, produce excessive cytokines and suppress the tumor growth. Unfortunately, the antitumor effects of this fusion protein are hampered and limited. Such as ?not all the malignancies express C215 antigen, ?targeting of superantigenicity evoked a pseudo-specific immune attack other than a tumor-specific immune response.Introduction of immunostimulatory antigens such as MHC I, MHC II, or B7-1 onto the surface of tumor cells can induce antitumor immunity as demonstrated by rejection of parental tumor in vivo. Typically, these approaches involve transfection of the relevant genes. However, transfection is time consuming, and primary tumor cells often do not grow well in vitro. Therefore, the practical application of this strategy to human tumors may be limited.In order to explore the use and widen the anti-tumor range of SAgs for T cell-based tumor therapy, we have genetically engineered fusion proteins with SEA and the hydrophobic transmembrane (TM) fragment of C-erb-B-2 gene. In this study, we describe a novel and maybe effective methods for incorporating the SEA on the surface of tumor cells. Fusing a TM sequence onto SEA gene result in its spontaneous association with the planning melanoma cell Iine-B16, as we know, the hydrophobic TM sequence mediate this passive anchoring, TM-SEA fusion protein should associate with the membranes of most cells as well as tumor cells. This would permit it to passively anchor onto cell membranes of living cells, and constructed the expression vector of TM-SEA fusion gene. In the meantime, the production of TM-SEA fusion protein was investigated for its biological effects on tumor cells both in vitro and in vivo. Our approach was to facilitate association of SEA with cell membrane so as to elicit antitumor response by TM-SEA fusion protein anchor onto the membrane of tumor cells. This research project...