Construction Of Chimeric Antigen Receptor T Cells (CAR-T) Targeting HER2 And Its Killing Activity Against Tumor Cells In Vitro

Objective:Chimeric Antigen Receptor T Cell（CAR-T）therapy is a kind of cellular immunotherapy based on chimeric antigen receptor（CAR）.Although significant progress has been made in the treatment of hematologic tumors,many challenges remain in the treatment of solid tumors,including the complex tumor microenvironment of solid tumors,various immunosuppressive mechanisms and lack of specific tumor antigens,which may hinder the treatment of solid tumors by CAR-T cells.In addition,the effective transmission of CAR intracellular signals is also an important prerequisite for the complete activation of CAR-T cells and their optimal cytotoxic activity.The activation and proliferation of T cells require multiple signals,including T cell receptor（TCR）（signal 1）,co-stimulatory signal（signal 2）and cytokines（signal 3）.Therefore,optimizing the structure of CAR is the key to enhance the function of CAR-T cells.In this study,we used the second-generation CAR containing CD28 co-stimulatory signals as the main framework to construct three different CARs targeting HER2:1)H28ξ（classical second-generation CAR containing CD3ξand CD28 intracellular region）;2)H28ξ（YRHQ）（the YRHQ motif is introduced at the end of the CD3ξchain）;3)H28-△IL-2Rγ-ξ（YRHQ）（the truncated IL-2Rγis inserted between CD28 intracellular region and CD3ξintracellular domain）.We constructed three different HER2-CAR-T cells by these three kinds of HER2-CAR,which included different signal molecule domains,namely H28ξ-CAR-T,H28ξ（YRHQ）-CAR-T,H28-△IL-2Rγ-ξ（YRHQ）-CAR-T.Using HER2-positive SKOV3,MDA-MB-453 and HER2-negative MCF-7 tumor cells as target cells,we observed the recognition and killing activity of three kinds of HER2-CAR-T cells against target cells and compared phenotypic differences of different HER2-CAR-T cells.The purpose is to explore whether YRHQ motif and/or△IL-2Rγsignaling molecules can improve the intracellular signal of HER2-CAR-T cells and their killing activity.Methods:1.Recombinant lentiviruses expression vector pCDH-HER2-CAR was constructed.The three DNA fragments encoding three CARs（H28ξ,H28ξ（YRHQ）and H28-△IL-2Rγ-ξ（YRHQ））synthesized by Nanjing Gen Script Biotechnology Co.,Ltd.were cloned into lentiviral expression vector pCDH-CMV-GFP by double digestion with Eco R I and Bam H I and then identified by double digestion.2.Packaging pCDH-HER2-CAR lentiviral particles.The packaging plasmids（p LP-1,p LP-2 and p LP-VSVG）and the expression plasmid（pCDH-HER2-CAR）were co-transfected into 293T cells.The supernatant was collected at 48h and 72h after infection and the lentivirus was concentrated by ultracentrifugation.Then 293T cells were infected with lentiviruses in different volumes.The GFP positive rate of 293T cells infected by each lentivirus was detected by flow cytometry and the virus titer was calculated.3.Isolation and activation of human peripheral blood T lymphocytes.Human PBMCs were selected from the peripheral blood of healthy people by density gradient centrifugation and human CD3 positive T lymphocytes were selected from PBMCs by CD3 magnetic beads.The purity of the sorted CD3positive T cells was determined by flow cytometry.4.The activation condition,infection condition and multiple of infection were optimized.T cells were cultured in RPMI 1640 complete medium containing 5μg/ml of cured CD3m Ab（OKT3）,0.5μg/ml of free CD28m Ab,10ng/ml of rh IL-2 and rh IL-7 for 48h.T cells were infected with HER2-CAR lentivirus by centrifugating at 24℃,2500rpm for 90min and incubating in37℃,5%CO₂ incubator for 12-16 hours.The culturing medium was changed afterwards.Multiple of infection（MOI）was fifty.5.Construction of HER2-CAR-T cells.The activated T lymphocytes were infected with HER2-CAR lentivirus（MOI=50）,4 days later,three kinds of HER2-CAR-T cells were stained with Biotin-Protein L（which binds to theκchain in sc Fv）and PE Streptavidin.The expression rate of CAR in HER2-CAR-T cells was detected by flow cytometry.6.Detection of HER2 expression in tumor cells.The SKOV3,MDA-MB-453 and MCF-7 cells were cultured in vitro.The expression of HER2 on the cell surface was detected by cell immunofluorescence.7.Determination of T cells proliferation.HER2-CAR-T lymphocytes and non-transduced T cells were counted at the 1st,3rd,5th and 7th days respectively after T lymphocytes were infected with lentivirus by cell counting plate to understand the proliferation of T cells.8.The killing activity of HER2-CAR-T cells against tumor cells.8.1 The cytotoxic activity of HER2-CAR-T cells.HER2-CAR-T cells were cultured in vitro for 8 days and rested for 2 days.Then they were co-cultured with SKOV3,MDA-MB-453 and MCF-7 target cells at effect-target ratios of 20:1,10:1,5:1 and 1:1 for 16 hours.The killing activity of HER2-CAR T cells was detected by LDH release assay.8.2 The levels of IL-2,IFN-γand GZMB secreted by HER2-CAR-T cells.HER2-CAR-T cells were cultured in vitro for 8 days and rested for 2 days.Then they were co-cultured with SKOV3,MDA-MB-453 and MCF-7 target cells at effect-target ratios of 20:1,10:1,5:1 and 1:1 for 24 hours,respectively.The levels of IL-2,IFN-γand GZMB in the cultured supernatant were detected by ELISA.8.3 Detection the phosphorylation level of transcription factor STAT3 in HER2-CAR-T cells.HER2-CAR-T cells were cultured in vitro for 8 days and rested for 2 days.Then they were co-cultured with SKOV3 cells at effect-target ratios of 5:1 for 4 hours.The phosphorylation level of transcription factor STAT3 was detected by Western blot.8.4 Detection of immune checkpoint molecules TIM-3 and PD-1 in HER2-CAR-T cells.HER2-CAR-T cells were cultured in vitro for 8 days and rested for 2 days.Then they were co-cultured with SKOV3 cells at effect-target ratios of 5:1 for 24 hours.The expression levels of checkpoint molecules TIM-3 and PD-1 in HER2-CAR-T cells were detected by Western blot.9.Phenotype analysis of HER2-CAR-T cells.HER2-CAR-T cells were cultured in vitro for 8 days and rested for 2 days.Flow cytometry was used to detect the expression of CCR7 and CD45RO on the surface of HER2-CAR-T cells to analyze the distribution of different subtypes of T cells,such as:（Na（?）ve T cells,T_N）CCR7⁺CD45RO^-,(Effector T cells,T_EFF)CCR7^-CD45RO^-,(Central memory T cells,T_CM)CCR7^-CD45RO^-and(Effector memory T cells,T_EM)CCR7^-CD45RO⁺.Meanwhile,the above-mentioned rested HER2-CAR-T cells were co-cultured with SKOV3 cells for 24 hours at an effective target ratio of 5:1 and the phenotypic changes of HER2-CAR-T cells were detected as above.Results:1.Three recombinant expression vectors targeting HER2,pCDH-H28ξ,pCDH-H28ξ（YRHQ）and pCDH-H28-△IL-2Rγ-ξ（YRHQ）were successfully constructed.2.The results of flow cytometry showed that 293T cells were infected with 1μl concentrated lentivirus carrying H28ξ-CAR,H28ξ（YRHQ）-CAR or H28-△IL-2Rγ-ξ（YRHQ）-CAR,the expression rates of GFP were 34.35%,33%,38.99%,respectively.The calculated virus titers were 1.72×10⁸,1.65×10⁸,1.95×10⁸,respectively.3.The results of flow cytometry showed that the purity of CD3 positive T lymphocytes reached 94.3%.4.Flow cytometry showed that the expression rates of CAR in H28ξ-CAR-T,H28ξ（YRHQ）-CAR-T and H28-△IL-2Rγ-ξ（YRHQ）-CAR-T cells were 33.3%±2.85%,28.30%±3.2%and 34.57%±3.3%,respectively.5.Cell immunofluorescence results showed that SKOV3 cells and MDA-MB-453 cells expressed HER2 antigen on their surface,while MCF-7cells did not.6.According to cell count,there was no statistical difference in the proliferation of the three HER2-CAR T cells in vitro.7.The killing activity of HER2-CAR T cells against tumor cells.7.1 Compared with NT T,H28ξ-CAR-T,H28ξ（YRHQ）-CAR-T and H28-△IL-2Rγ-ξ（YRHQ）-CAR-T exhibited significant cytotoxic activity against HER2-positive SKOV3 or MDA-MB-453 cells at effect-to-target ratios of 5:1,10:1 and 20:1（P<0.05 or P<0.01）.Among them,H28ξ（YRHQ）-CAR-T showed the highest cytotoxicity compared with the other two HER2-CAR-T cells（P<0.05）,while the cytotoxicity of H28-△IL-2Rγ-ξ（YRHQ）-CAR T cells was significantly lower than that of H28ξ-CAR-T cells（P<0.05）.For HER2-negative MCF-7 cells,the killing activity of the three HER2-CAR-T cells was low and there was no significant difference.7.2 ELISA results showed that compared with NT T cells,the levels of IL-2 and IFN-γwere significantly increased after the three HER2-CAR-T cells were co-cultured with SKOV3 or MDA-MB-453 cells（P<0.05 or P<0.01）;H28ξ-CAR-T and H28ξ（YRHQ）-CAR-T cells secreted higher levels of GZMB than H28-△IL-2Rγ-ξ（YRHQ）-CAR-T and NT T cells（P<0.05 or P<0.01）.7.3 Western blot results showed that the phosphorylation level of transcription factor STAT3 was significantly increased in both H28ξ（YRHQ）-CAR-T cells and H28-△IL-2Rγ-ξ（YRHQ）-CAR-T cells with YRHQ motif after HER2 antigen stimulation（P<0.01）.7.4 Western blot results showed that there was no significant difference in the expression of TIM-3 in the three HER2-CAR-T cells stimulated by HER2antigen;the PD-1 expression level of H28-△IL-2Rγ-ξ（YRHQ）-CAR-T cells was significantly higher than that of NT T or the other two HER2-CAR-T cells（P<0.05）.8.Flow cytometry results showed that there was no significant difference in CCR7 and CD45RO expression among the three HER2-CAR-T cells activated by CD3/CD28 double antibody after resting for 2 days（P>0.05 for all）.After co-culture with HER2-positive SKOV3 tumor cells,the number of T_EM cells in H28ξ（YRHQ）-CAR-T and H28-△IL-2Rγ-ξ（YRHQ）-CAR-T cells was significantly increased（P<0.05）,while the number of central memory cells was significantly decreased compared with NT T and H28ξ-CAR-T cells（P<0.05）.There was no significant difference between the H28ξ-CAR-T cell subsets and NT T cells.Conclusion:1.HER2-CAR-T cells show specific killing activity against HER2-positive SKOV3 or MDA-MB-453 tumor cells.2.The CD3ξ（YRHQ）motif can induce phosphorylation of the transcription factor STAT3 in HER2-CAR-T cells,thereby enhancing the killing activity of HER2-CAR-T cells.3.H28-△IL-2Rγ-ξ（YRHQ）-CAR-T cells express higher levels of PD-1,which may be the cause of the cell failure.4.After H28ξ（YRHQ）-CAR-T cells are stimulated by HER2-positive SKOV3 cells,the proportion of T_EM cell subsets increases,which can increase the proliferation and killing potential of CAR-T cells to a certain extent.