| Objective: To construct chimeric T cell receptor(cTCR) binding selectively to tumor vascular endothelial cell and tumor cells which express the VEGF receptor(KDR), and to detect the cell activity of T cell transfected with cTCR. Methods: The hinger-FcRγ was synthesized and amplified by PCR-based gene assembly. The cTCR(VEGF121-hinger-FcRγ) was constructed by splicing VEGF121 amplified by PCR with hinger-FcRγusing ligation methods and its retroviral vector was constructed. The packaging cell was transfected by calcium phosphate and virus with high titer was used to infect tumor infiltrating lymphocyte(TIL) isolated from liver cancer tissues. RT-PCR was used to indentify the expression of exogenous gene in infected TIL. The cytotoxicty of infected TIL on NIH3T3, ECV304, HepG2 and A375 was detected with MTT colorimetric assay. Green fluorescence protein(GFP) gene was cloned to MSCVhyg to detect fluorescence in infected TIL. Results: Both VEGF121 and hinger-FcRγ have a different base compared with the reported sequence, but the deducted amino sequence was correct. Green fluorescence could be seen in packaging cell and part of TIL transfected with GFP. RT-PCR analysis indicated that mRNA of exogenous gene was present in cells infected with recombinant retrovirus. The cytotoxity of TIL infected with MSCVneo on target cell was similar to that of uninfected TIL；both of them only have some cytotoxity on cancer cell line. No significant cytotoxity of TIL infected with MSCVneo-cTCR on NIH3T3 could be found, but cytotoxity on ECV304 expressing KDR was significant；the cytotoxityon HepG2 was similar to that of MSCVneo-TIL and uninfected TIL, but cytotoxity on A375 expressing KDR was higher significantly. Conclusion: Chimeric T cell receptor permanently grafts TIL cell with predefined new specific recognization. TIL expressing VEGF121-hinger-FcRγcan recognize and kill selectively to vascular endothelial cell and tumor cells which express the VEGF receptor KDR.
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