| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2004: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
During the granted period, the results were obtained as follows : Molecular modeling of intermedilysin(ILY) and its relative toxins was carried out and valuable molecular information of ILY to identify the cell membrane binding site of ILY was obtained. Based on this information, various peptides with partial primary structure of the cell membrane binding domain of ILY(ILY4D) were synthesized and tested for their competitive inhibitory action. Moreover, the minimum structure necessary for human-specific binding was determined using chimeras of ILY and its relatives, then the structure was found to locate in the latter part of ILY4D with 56 residues except for the undecapeptide region. However, since the tertiary structure of ILY4D was required for membrane recognition of ILY, short peptides with its partial structure and the C-terminal 56mer peptide of ILY4D were not satisfactory for the application to cell membrane binding modules. On the other hand, though the molecular size was larg
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er than such peptides, two recombinants of ILY4D with individual size of spacer and a non-toxic mutant of ILY with a disulfide bridge fixing its conformation (ILY-SS), possessing the complete ILY4D structure and an anchor residue (Cys) at the N-terminal side of each molecule were successfully prepared as cell membrane binding modules. ILY4D modules were conjugated with (Fab')_2 fragment of anti-carcinoembryonic antigen (CEA) monoclonal antibody and it was confirmed that human erythrocytes coated with the conjugates could stably, efficiently and specifically bind to CEA-positive cancer cells. Therefore, it was thought that these molecules were applicable to missile cellular immunotherapy against cancer, ILY-SS module was also suggested to be useful for cell membrane modification technique. Moreover, the vector systems expressing desirable protein fused with both types of module at their N-terminal were developed. Development of missile cellular immunotherapy and Drug delivery system for genetherapy using these modules is expected hereafter. Less
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