Construction And Evaluation Of Carcinoembryonic Antigen-specific Chimeric Receptor

Backgroud and ObjectiveAdoptive cell therapy,which involves the ex vivo expansion of autologous immune cells to large numbers that are ultimately transferred back to the patients.It has been widely applied in clinical with lower toxicity,security and elimination microscopic residual in cancer patients. Non-specific adoptive cell therapy used the presently in clinical,such as tumor infiltrating lymphocytes(TILs),lymphokine activated killer cell(LAK), cytokine induced killer cell(CIK). As a result of these cells are not identication of tumor-specific,TCR randomly recognize the tumor antigens and tumor cells often downregulation MHC molecules inhibiting T cells recognition and anti-tumor antigen.chimeric antigen receptors(CARs) are contain three distinct modules: an extracellular target binding, a transmembrane module which anchors the molecule into the cell membrane, and an intracellular signaling domain that transmits activation signals. Single chain variable fragment(sc Fv) constructed from the variable heavy(VH) and variable light(VL) sequences of a monoclonal antibody(m Ab) specific for a tumor cell surface molecule.Intracellular signaling domain usually is CD3ζ chain or Fc Rγ, or link to one or more costimulatory molecules, such as OX-40/CD134, CD137/4-1BB,CD28.The exodomains of CARs have also been fashioned from antigen(ligands) to redirect specificity to antibodies(acceptors). This direct binding ideally provides the genetically modified T cell with a fully-competent activation signal, activated effector cells,proliferation, and cytokine production,such as TNF-α, IFN-γ, perforin,granzyme killing tumor cells. It has the several advantages.First,antibodies(acceptors) specific bind to antigen(ligands).Second,non-MHC-restricted and overcome Immune escape of tumor cells do not express or low expression of tumor antigens and tumor microenvironment downregulation MHC-Ⅰmolecules.Third, it will not occur which transgenic TCR mismatch TCR endogenous in patients.At last, antigen can be carbohydrates, lipids and proteins.Carcinoembryonic antigen(CEA) is a glycoprotein with a molecular weight of approximately 200 k Da.CEA is overexpressed on tumors in 60–94% of patients with metastatic and recurrent colorectal cancer. Extensively used in diagnosis, prognosis and monitoring of recurrence in patients with colorectal cancer.Therefore, our study based on the studies,construct lentiviral vectors LV-EF1α-sc Fv(CEA)-CD3-2A-GFP-WPRE containing chimeric antigen receptor targeted an anti-CEA single-chain antibody by molecular cloning technology. Its intracellular signal is CD3ζ chain. Packaging the lentivirus vector, infection of human T cells, preliminary explore the chimeric antigen receptor expression and anti-tumor cytotoxicity of T cells to colorectal cancer CEA positive tumor cell lines when the vector infected into T cells.It laid the basis for follow up further research and colorectal cancer therapy.Methods1. The lentiviral vector LV-EF1α-sc Fv(CEA)-2A-GFP-WPRE constructed by molecular cloning techniques.2. Packaging lentivirus vector used calcium phosphate-mediated transfection method.3. The lentiviral vector was infected into human peripheral blood mononuclear cells(PBMC).4. Flow cytometry and Native western blot were used to analyze the expression scFv(CEA)-CAR of T lymphocytes.5. Cytotoxicity against the colorectal cancer CEA positive, CEA-negative tumor cell lines detected by cytotoxic assay in vitro.Results1. lentiviral vector LV-EF1α-scFv(CEA)-2A-GFP-WPRE was constructed by PCR, enzyme digestion, ligation and the nucleotide sequence detection result indicated it was correct.2. Packaging lentivirus vector used calcium phosphate method and flow cytometry detecting the titer of lentivirus was 3×107TU/ml.3. 10 MOI lentivirus infected peripheral blood mononuclear cells.After 14 days of incubation, fluorescence microscope observed infected T cells,it is showing the typical morphology of aggregate growth and expression of GFP, which indicating T cells infected with lentivirus vector successfully.4. Expression of scFv(CEA)-CAR value in T cells were 62.8% analyzing by flow cytometry. Native western blot demonstated T cells expression of anti-CEA antibody correctly after infection.5. CAR-modified T cells efficiently specific killed the colorectal cancer CEA-positive tumor cell lines, with CEA-negative tumor cell lines almost no cytotoxicity.ConclusionChimeric antigen receptor could express in T cells properly and produce specific killing effect on CEA positive tumor cell lines. Our study laid the foundation for future research in vivo and used in colorectal cancer therapy.