Difunctional Fusion Protein Of IP10-scFv Oriented Collection Lymphocyte Targeted To Treat Mouse Malignant Glioma

In recent years, more and more evidence have indicated that the central nervous system are not immune privilege. Specifically, activated T lymphocytes, have the ability to penetrate the blood-brain barrier (BBB) under normal physiological conditions. So, it is possible to treat glioma with immune adjuvant therapy. As a specific glioma antigen identification and detection, glioma specific cytotoxic T lymphocytes(CTL) adoptive immunotherapy became the most remarkable treatment of the biological treatment of research field. However, adoptive treatment of the poor in vivo efficacy of the important reasons is targeting to raise glioma parts of the limited number of CTL, and because of tumor immune escape and reach the tumor site of CTL targeting and biological activity subject to greater inhibition, it is difficult to efficiently eradicate tumor cells. Therefore, it must overcome obstacle for us to how to target to raise enough glioma specific CTL into tumor and improve the ability of local targeting to clear tumor cell of the biological activity. Our strategy is in accordance with the classic dendritic cells(DC) as the antigen presenting cell(APC), incubation with glioma lysate to induce enough glioma specific CTL. Simultaneously, we construct difunctional fusion protein IP10-scFv, which not only can specific binding glioma surface antigen EGFRv Ⅲ, but also allow targeting of tumor cells to obtain high concentrations of IP10. It will achieve the dual adoptive cellular target of the CTL to the tumor cell enrichment and to enhance their cytotoxic activity on tumor cells through using of IP10on T cells directed chemotaxis and promote proliferation of lymphocyte activation and anti-tumor effect. So, this will greatly enhance the CTL attack the tumor cells in vivo targeting and effectiveness, it is a major step towards clinical application in future.Chapter I Construction and Expression of Fusion Protein IP10-scFvObjective To construct cytokine fusion protein IP10-scFv,then establish and evaluate stable expression fusion protein in NIH3T3cells. Methods To amplify by RT-PCR gene in mice IP10and synthesize entire genome of EGFRvⅢscFv by artificial, respectivel.Then, we introduced the sequences encoding a flexible linker of (Gly4Ser)3to connect mouse IP10and EGFRvⅢscFv genes and subcloned sequentially into downstream of promoter of CMV in expression vector pcDNA3.1(-). mouse fibroblasts cell line NIH3T3was transfected with IP10-EGFRvⅢscFv and positive clones were screened in the presence of G418. The expression level of IP10-scFv was examined by real-time PCR and Western blot. Also, Growth kinetics of IP10-scFv-NIH3T3cells was observed in vitro. Results Expression vector of pcDNA3.1(-)IP10-scFv was successfully constructed and NIN3T3stable cell line with high expression IP10-scFv was screened out by G418with the concentration of200mg/L. Simultaneously the growth of IP10-scFv-NIH3T3cells was not infected by transfected gene in vitro. Then, we evaluated the expression of IP10-scFv by fluorescence, real-time PCR and Western-blot. Though the expression of gene and protein were highest on72h by transient transfection, there were no significant diffference compared to clone cell line stable expression IP10-scFv (P＞0.05). Conclusion A novel cytokine fusion protein can express stably in cell line NIH3T3, So it lay the fudation for the latter part of the fusion protein extraction and functional studies. Chapter Ⅱ A large number of fusion protein preparation and research in vitro function Objective The use of PET efficient prokaryotic expression system, and the use of in vitro refolding technology, a large number of preparation of active fusion protein for functional studies and in vivo basis for the study. Methods The target gene IP10-scFv in eukaryotic expression vector was cut by restriction endonuclease and cloned into T7promoter downstream of the EcoR Ⅰ and Xho Ⅰ sites of the PET30a (Invitrogen) to generate a plasmid of pET-IP10-scFv, and efficiently expressed recombinant protein under IPTG induction. Then protein was purificated by Ni-His tag Fusion Protein Purification Kit and refolded by dialysis, in order to improve the yields of refolded protein, we introduced the oxidation conditions by adding of1mM oxidized glutathione(GSSG) and1mM reduced glutathione(GSH) to the dialysis buffer. At last, we used refolded protein to process functional study in vitro, containing competitive ELISA, cell migration assays, antigen binding assay. It is helpful for evaluation the chemiotaxis and specific binding. Results By PET efficient expression system, we got a lot of fusion protein(inclusion body expression), and obtained functional activity of the recombinant protein through successful using of refolding dialysis in vitro. However, the affinity constant of fusion protein was1.32±0.05×10-8M by ELISA assay, chemiotaxis was the same as IP10by cell migration assays and specific binding was68.6%by immunofluorescence and flow cytometry. Conclusion A novel fusion protein of IP10-scFv have the ability of specific binding EGFRvⅢ antigen, and chemiotaxis as the same to IP10. Chapter Ⅲ Fusion protein IP10-scFv combining CTL in vivo researchObject Founctional fusion protein that was harvested by in vitro dialysis refolding technology has been verified antigen-specific antibody binding capacity and CD8+T lymphocyte chemotaxis in vitro. So it is vital to do further research on the effect of anti-tumor in vivo. Methods Cytotoxic lymphocyte(CTL) was harvested by glioma cell lysates-pulsed dendritic cells con-cultured with CD8+T lymphocyte and injected by tail intravenous combining with intracranial injection IP10-scFv fusion protein. By synergistic action, we observed the change of tumor volume and the survival time of tumor-bearing mice. Also, we evaluated the quantitative extent of tumor infiltrating lymphocytes, duration of tumor infiltrating lymphocytes and detection of apoptosis of tumor cell by double staining with PI/Annexin V by flow cytometry as well as the time duration of chemotaxis and effect of chemotaxis by in vivo imaging. Results We successfully prepared glioma-specific cytotoxic T lymphocyte(CTL) by DC co-cultured techniques. However, it could pass through blood brain barrier and enriched in the tissue surrounding the tumor lasting for more than a week under the chemotaxis of IP10-scFv fusion protein. Comparing to alone IP10-scFv fusion protein that is only limited to mobilize CTL with itself, combining IP10-scFv and CTL could significantly reduce the tumor volume, accelerated apoptosis of tumor cells and prolong the survival time of tumor-bearing mice. Otherwise, In vivo imaging technology comfirmed that CTL reached the maximum concentration of infiltration in tumor between3and5days and gradually decreased after a week under the chemotaxis of IP10-scFv fusion protein. Conclusion Founctional fusion protein IP10-scFv that is obtained by prokaryotic expression, dialysis refolding technology in vitro combining with glioma-specific cytotoxic T lymphocyte(CTL) has the powerful anti-tumor effect, further more it could last for one week in vivo.