The Construction, Expression And Generation Of Targeting T Cells To HCC Cells By A Chimeric Receptor With A Highly Specific Humanized Disulfide-stabilized ScFv-CD3ζ

Hepatocellular carcinoma (HCC)> one of the most common malignant tumors, often happens with high incidence and mortality in China.Clinical diagnosis and treatment are very difficult.With the development of molecular biology, genetic engineering and protein engineering, genetically engineered antibody is a new way for diagnosis and treatment to tumors. Above all, the application of single chain Fv is a hot point in immunogenetic therapy. Our group has successfully constructed a humanized disulfide-stabilized scFv (hdsFv), which can reduce its immunogenicity of mouse-derived scFv and keep the high stability, specificity and affinity of its parent antibody to some extent. The CD3 is present on mature human T cells. It is associated with the T cell receptor (TCR) and is responsible for the signal transduction of the TCR. The 4 chain of CD3 plays a crucial role in the signal transduction of T cell activation. We made a basic study on HCC-specific T cell by constructing a chimeric receptor which was comprised of the CD3ζ gene fused to the anti-HCC hdsFv, putting it into a eukaryotic expression vector and transfecting it into Jurkat cells and PBMC.1.The construction of a eukaryotic expression vector pcDNA3-hdsFv-CD3ζ. The transmembrane and intracellular domains of CD3ζ cDNA was amplified from human T lymphocytes by RT-PCR. The hdsFv gene was cloned from pET-22b(+) - hdsFv by PCR.The CD3ζ fragments were ligated to thedownstream of the anti-HCC hdsFv cDNA and sequence were verified. The sequence of hdsFv was in accordance with the known sequence. The sequence of cDNA of the transmembrane and intracellular domains of CD3ζ was conformed to the sequence of Genebank.2. The expression and location of hdsFv-CD3ζ in transfected Jurkat cells. The pcDNA3-hdsFv-CD3ζ was transfected into human CD4+ Jurkat cells through lipofectamine. The positive cells was filtrated by G418. The protein of the hdsFv-CD3ζ was confirmed by Western blotting. Immunocytochemical staining was done with anti-CD3ζ mAb before and after transfection. We observed the expression of fused protein on cell membrane and the increment of CD3ζ expression after the transfection and detected hdsFv in transfected Jurkat cells by RT-PCR.3. The detection of function of transfected Jurkat cells and PBMC. When transfected Jurkat cells were incubated with HepG2 cells, the Jurkat cells adhered to the tumor cells, which indicated the cell-specific targeting of Jurkat T cells. The pcDNA3-hdsFv-CD3ζ was transfected into PBMC through lipofectamine. Immunocytochemical staining was done with anti-CD3ζ mAb before and after transfection. We observed increment of CD3ζ expression after the transfection, which confirmed hdsFv-CD3ζ in PBMC. Activated T cell revealed by CD25 up-regulation, which was proved in transfected PBMC by FCM. Robust IL-2 secretion were observed when the hdsFv-CD3ζ PBMC were stimulated by incubated with HepG2 cells. We cocultured transfected and untransfected cells with HepG2 cells and detected the apoptosis of HepG2 cells by Annexin V immunofluorscence.The results showed that transfected PBMC could induce the apoptosis of HepG2 cells better.Transfect pcDNA3-hdsFv-CD3ζ and pcDNA3-hdsFv into PBMC, and the PBMC were cocultured with HepG2 cells and Hela cells.Untransfected PBMC were cocultured with HepG2 cells and Hela cells as a control group. As a result,hdsFv-CD3ζ and hdsFv grafted PBMC could lyse HepG2 cells distinctly. The mortality of HepG2 reached 75.85 % and 62.39 % separately. hdsFv-CD3ζ grafted PBMC could lyse Hela cells, too.The mortality reached 63.13%. The high mortality of Hela cells by cocultured with hdsFv-CD3ζ PBMC is probably related with function of CD3ζ and presence of IL-2 which was put into PBMC. But such killing is unspecific.In our study, we have successfully cloned the transmembrane and intracellular domains of CD3ζ from T cell, constructed a eukaryotic expression vector pcDNA3-hdsFv-CD3ζ which was successfully expressed in Jurkat cells...