Gene therapy of malignant solid tumors by targeting erbB2 receptors and by activating T cells.

One of the strategies to improve the outcome of anti-erbB2-mediated immunotherapy is to combine anti-erbB2 antibodies with T-cell-based adoptive immunotherapy, which can be achieved by expressing anti-erbB2 mAb on the surface of T cells. A single-chain variable fragment (scFv) from an anti-erbB2 mAb has been expressed on T cell surface to bind to erbB2-positive cells, and CD3ζ has been expressed as a fusion partner at C terminus of this scFv to transduce signals. T cells grafted with this chimeric scFv/CD3ζ were able to specifically attack target tumor cells with no MHC/Ag restriction. To test the effects of CD28 signal on cellular activation and antitumor effectiveness of chimeric scFv/CD3ζ-modified T cells, we constructed a recombinant anti-erbB2 scFv/Fc/CD28/CD3ζ gene in a retroviral vector. T cells expressing anti-erbB2 scFv/Fc/CD28/CD3ζ specifically lyzed erbB2-positive target tumor cells and secreted not only interferon-γ (IFN-γ) but also IL-2 after binding to their target cells. Our data indicate that CD3 and CD28 signaling can be delivered in one molecule, which is sufficient for complete T cell activation without exogenous B7/CD28 co-stimulation.

[Construction of recombinant retroviruses expressing anti-CD20 scFv/CD80 /CD28/zeta gene and expression in Jurkat cell line].

AIM: To construct recombinant retroviruses expressing anti-CD20 scFv/CD80/CD28/zeta gene and detect its expression in Jurkat cells. METHOD: CD28-zetacDNA were amplified from plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/CD80 gene. The recombinant plasmids were transfected into PA 317 cells. Retroviruses were harvested from culture medium of PA 317 cells. Then NIH 3T3 were transfected with retroviruses. Objective gene expression was determined by PCR and FACS. Jurkat cells were transfected with high titer of retroviruses and resistant clones were obtained by G418 selection. Objective mRNA was determined by RT- PCR. RESULTS: The recombinant eukaryotic vector was constructed successfully by PCR and enzyme digestion analysis and objective gene was amplified from NIH 3T3 cells transfected with retroviruses by PCR; FACS showed that objective protein could be expressed in NIH 3T3 cells. Objective gene was amplified from Jurkat cells transfected with retroviruses by RT-PCR. CONCLUSION: Recombinant retrovirus expressing anti-CD20 scFv/CD80/CD28/zeta gene was successfully constructed and objective protein could be expressed in Jurkat cells.

[Construction of anti-cD20scFv/CD80/CD28/zeta recombinant gene modified T cell and research on its targeting cytotoxicity].

OBJECTIVE: To construct anti-CD20scFv/CD80/CD28/zeta recombinant gene modified T cells, test its effectiveness of eradicating CD20+ lymphoma cells and provide a probably new approach to tumor adoptive immunotherapy. METHODS: CD28-zeta cDNA were amplified from vector pBULLET and inserted into pLNCX vector that contained anti-CD20scFv/CD80 gene. The recombinant vectors were transduced into PA317 cells and high titer retroviruses were obtained to infect human peripheral blood T lymphocytes. Resistant T cells were obtained by G418 selection at one week. Then transduced T lymphocytes and lymphoma cell lines Daudi Raji were cocultured. The cytotoxicity and cytokine production of transduced T cells were determined by non-radio-activation cytotoxicity assay and ELISA respectively. RESULTS: The recombinant eukaryotic vector was constructed successfully as proved by enzyme digestion analysis and sequencing. These T cells were able to lyse CD20+ target cells and secrete high levels of IL-2 and IFN-gamma in vitro. CONCLUSION: Recombinant gene modified T cells can be constructed successfully. It can specially kill CD20 positive lymphoma cells in vitro.