Construction Of CAR-T Guided By HIV Broadly Neutralizing Antibodies A16 And Y498

ObjectAcquire immunodeficiency syndrome(AIDS)has been a major concern for public health since the first AIDS patient was discovered.Although combined antiretrovirus therapy(cART)can effectively inhibit virus replication,lower incidence rate and mortality rate and prolong survival time,the latent HIV of AIDS patients who receive cART treatment for a long time will rebound rapidly once the drugs are stopped.On the other hand,the high frequency mutation of HIV virus genome leads to the emergence of drug-resistant strains,which brings severe challenges to the current drug treatment.And in addition,because of the toxic effect of long-term medication on patients and the exhaustion of immune system,it is urgent for us to explore a more safe and effective antiviral therapy.In recent years,the chimeric antigen receptor(CAR)immunotherapy has achieved great success in the treatment of B-cell tumor and leukemia.Chimeric antigen receptor is consists of extracellular antigen targeted binding domain,transmembrane domain and intracellular T cell activation signal domain fusion.After the introduction and expression of foreign genes on effector T cells,the specific antigen recognition ability is endowed.Tumor cells can be killed specifically in vivo,after the modification and reinfusion of autoimmune cells.Previous studies have shown that HIV specific CAR-T cells can be produced by connecting the HIV specific antibody’s scFv or the antigen recognition binding domain of natural CD4 molecules to the T cell activation domalin of CD8+ T cells,which can specifically kill the cells expressing HIV envelope protein.CAR-T therapy shows great potential in the treatment of HIV.Two monoclonal broad-spectrum neutralizing antibodies against HIV,A16 and Y498(Patent No.:z1201310154423.4,z1201110078167.6),were isolated from HIV infected patients by phage antibody screening technology,recent years.Previous SPR experiments showed that the affinity of these two antibodies to HIV were higher than that of VRC01,which are more suitable for CAR-T construction.-Therefore,in this study we will use VRC01 as a control and construct CAR-T using the HIV broad-spectrum neutralizing antibodies A16 and Y498 which screened in the previous stage of our Lab and then evaluate their killing effect in this study.ProcedureIn this report,we link the scFv sequences of VRC01,A16 and Y498 together with the intracellular and transmembrane domain sequences of the third generation CAR by molecular cloning.Then we used lentivirus vector to introduce the three CARs which are HIV specific into primary CD8+T cellsWe then transduced the three HIV-specific CAR into primary CD8+T lymphocytes by lentiviral vector.After co-culture of the three CAR-T lymphocytes with two HIV Env-expressing target cell lines(HXB2 and JRFL).We verified their killing activity of the three CAR-T cells.ResultsIn this study,three CAR molecules were successfully constructed.The protein expression of the three CAR molecules was verified by immunofluorescence and Western Blot experiments.Through continuous adjustment,the lentivirus titer was gradually increased,and the viral viability as determined,and then we successfully transduced CD8+T cells.The results showed that the killing effect of CAR-T cells constructed by scFv based on A16 and Y498 on target cells was similar to that of VRC01.ConclusionsOur results show that it is highly feasible to construct CAR-T cells based on the scFv sequences of the broad-spectrum neutralizing antibodies A16 and Y498 screened in our laboratory,which has the significance of further research.We further speculate that the two CAR-T cells can also clear the activated latent infection reservoir in the body of HIV-infected patients treated with cART,which deserves further theoretical study and technical optimization.