The Research About Establishment Of The Humanized PSMA-specific Chimeric Antigen Receptor(CAR)-modified T Cell

Objective: To design and construct the third-generation humanized PSMA-specific chimeric antigen receptor(CAR)gene,infecting Jurkat and Molt-4 cell with lentivirus to establish humanized PSMA-specific CAR T cells and identify their CAR expression and cytotoxicity.Methods:(1)According to the design of CD19-specific CAR invented by professor Carl H.June and others,design and construct the third-generation humanized PSMA-specific CAR by searching NCBI,PDB and IMGT database.The CAR are composed of a humanized PSMA single-chain antibody as antigen recognition domain,hinge and transmembrane domain of CD8α,and CD28、4-1BB/CD3ζ co-stimulation domain.Organize and synthetize the whole sequence of CAR gene,construct the recombinant CAR gene lentiviral vector and check them preliminarily;(2)Produce the recombinant lentiviral particles by the calcium phosphate transfection system.Collect lentivirus supernatants and infect Jurkat and Molt-4 cell to make CAR gene modification.Validate the efficiency of infection by flow cytometry and identify the most suitable multiplicity of infection of lentivirus,establish the humanized PSMA-specific CAR T cells finally;(3)Detect the expression of CAR gene via real-time PCR and protein-L indirect labeling method by flow cytometry;(4)Check the cytotoxicity of humanized PSMA-specific CAR T cell to target tumor cell via LDH releasing test kit.Results:(1)The whole sequence of CAR gene are synthetized successfully and recombinant CAR lentiviral vectors are confirmed by Electrophoresis;(2)The infection efficiency of the lentivirus for Jurkat cell and Molt-4 cells are high,and the efficiency is about 30% with one infection and can reach to 50% with another infection after 24h;(3)The expression of CAR gene in the Jurkat and Molt-4 cells infected by lentivirus has a significant difference compared with untreated groups(p <0.05);(4)CAR gene modified Molt-4 cells have the cytotoxicity to some extent,while CAR gene modified Jurkat cells do not have any cytotoxicity.Conclusion:1.Lentivirus for Jurkat and Molt-4 cell lines are infected with high efficiency,lentivirus supernatants produced by the calcium phosphate transfection system can be used for gene transduction in the T cell line;2.The constructed humanized PSMA-specific CAR can express in and on the surface of T cells successfully;3.Compared with Jurkat cell,humanized PSMA-specific CAR modified Molt-4 cell showed the cytotoxicity to some extent,which confirmed the successful design of CAR gene preliminarily;Molt-4 cell can be used as a tool cell for the research of CAR T cell technology..