Preparation Of MB7.1-GPI And SEA-TM Dual-anchored Tumor Cell Vaccine And Its Antitumor Effect

The mechanisms for tumor cells to escape immune surveillance of the body include: weak tumor specific or associated antigens, defective antigen presentation process, down-regulated surface MHC molecules and lack or costimulatory molecules. Introduction of immunostimulatory molecules such as MHC I, MHCII and B7.1. to the tumor cells, and use of these surface-modified tumor cells as vaccine, can induce antilumor immunity as demonstrated by rejection of parental tumor in vivo. Typically, these approaches involving transfecu'on of gene is time consuming, gene expression is instability and primary tumor cells often do not grow well in vitro. Therefore, the practical application of this strategy to human tumors may be limited.Alternatively, surface molecules can be introduced onto tumor cells through the following approach: the DNA sequence of target protein is fused with a glycosyl-phosphalidylinositol(GPI) signal sequence, the purified, recombinant GPI-linkcd fusion proteins are incubated with tumor cells so the target protein can be anchored onto extracellular membranes of the tumor cells. In vitro studies applying this approach demonstrated that GPI-anchored MHC I molecules effectively promoted T cell-mediated cytotoxicity, and GPI-anchored B7.1 co-stimulated lymphocyte proliferation.However, as a prokaryotic protein such as bacteria superanligen is not well suited to a GPI-signal-sequence-fusion strategy for cell membrane anchoring because GPI linkage requires eukaryotic processing. Wahlsten developed a strategy for passively attaching the superanligen toxic shock syndrome toxin-1 (TSST1) onto tumor cells, this strategy was fusing TSST1 coding region to the transmembranc region (TM) sequence of the proto-oncogene c-erb-B2. TSST1-TM was expressed in bacteria. Purified TSSTI-TM can be anchored onto tumor cells, Ihe tumor vaccine derived from these tumor cells can stimulate proliferation of lymphocytes in vitro and induce a systemic anlitumor immunity. Ma ct al also reported that mice immunized with tumor cell vaccine, which was anchored by fusion protein slaphylococcal cnleroloxin A- transmembrane (SEA-TM), developed specific antilumor immunity.The mechanisms of tumor escape from the immune surveillance indicate thai the tumor development is associate with lack or down-regulation of various immunological factors and the multiple-surface modified tumor cell vaccine may induce immune response more effectively.Many studies have showed the tumor cell vaccine of multiple-gene transfection, can induce much stronger antitumor immunity than that of single-gene transfection. However, multiple-gene transfection is time consuming and less practicable.We are intended to develop a new approach for passively attaching mB7.1-GPI and SEA-TM onto tumor cells by protein transfer, with this approach the tumor cell vaccine membrane-anchored with the two different immunologic molecule is prepared. Using this model, we will explore the feasibility for preparation of immune molecule dual-anchored lumor cell vaccine and observe the effectiveness of this novel approach in treatment and immnopretection of parental murine tumor growth in the experimental animals.The objectives of the study includes: (1) to construct mB7.1-GPI fusion gene, to express and purify mB7.1-GPI fusion protein and incorporate it on lumor cells and to prepare mB7.1-GPI-anchoring tumor vaccine; (2) to prepare the vaccine of lumor cells membrane- anchored with SEA-TM; (3) to prepare SEA-TM and mB7.1-GPI dual-anchored tumor cell vaccine; (4) to observe the antitumor immunity induced by the prepared single or dual-anchored lumor cell vaccine in tumor-bearing mice.The sludy is reported as follows:Part 1: Cloning and expression of mB7.1-GPl fusion geneA DNA fragment encoding the firsl 247 amino acids of mB7.1 and a DNA fragment encoding the signal for GPI-anchor allachmenl of hPLAP-1 were amplified by PCR. The two amplified gene sequence were annealed to form a chimeric GPI-anchored mB7.1 molecule by gene SOEing. The resulting chimera was clone...