Preclinical Study On The Mechanism Of PD-1 Gene Knockout T Cells In Colorectal Cancer And Safety Evaluation In-vivo

Targeting T cell checkpoint is the hottest topic in cancer immunotherapy today.Clinical studies have demonstrated that PD1 monoclonal antibody drugs directly acting on blocking the inhibitory immune point,activate T cells,enhance the function of T cells,and achieve the purpose of treating tumors.The intestine is considered to be the largest immune organ in the body due to the largest surface area exposed to antigens and the large number of immune cells,and the occurrence of colorectal cancer is closely related to tumor immune escape.This study intends to establish an in vitro isolation and culture technique of peripheral blood T lymphocytes induced by CD3 antibody,knock out PD-1 gene of T cells by CRISPR/Cas9 gene editing technology,use mouse colon cancer model to study the in vivo anti-tumor effect of PD-1 knockout T cells and its possible mechanism,and evaluate the safety of PD-1 knockout T cells in cynomolgus monkeys,in order to establish a new strategy of T cell immunotherapy in tumor.Chapter ?:Study on the therapeutic effect and mechanism of PD-1 gene knockout in T cells in mouse colorectal cancerPart ?:Establishment of CRISPR/Cas9-mediated mouse PD-1 knockout T cells and their role in inhibiting tumors in vitro[Methods]Mice PD-1 gene sequences were searched in the GENEBANK database,using the gRNA online design tool(Fig.http://crispr.mit.edu),sgRNAs targeting PD-1 gene were designed and selected to construct sgRNA recombinant knockout vectors,then identified and verified it by sequencing.Isolate mice peripheral blood PBMCs and coculture with colorectal cancer cell line CT-26.The groups divided into:CT-26 cell group,PD-1 knockout T cell group,CT-26 co-cultured with PD-1 knockout T cells group,CT-26 co-cultured with PD-1 knockout T cells and PD-1 monoclonal antibody group.The cell growth curve of each group was drawn.The killing ability of PD-1 knockout T cells was detected by CCK-8.The changes in the proportion of T lymphocytes,cell cycle and apoptosis were detected by flow cytometry.The secretion of cytokines secreted in vitro was detected by ELISA.[Results]The targeted mouse PD-1 knockout sgRNA was successfully obtained,the targeted mouse PD-1 gene sgRNA/Cas9 expression vector was constructed,the mouse PD-1 knockout T cells were established,and their in vitro characteristics were tested.The results showed that:there was no significant change in the surface phenotype of PD-1KO T cells compared with mock T cells.In vitro tumor inhibition effect of PD-1KOT cells and CT-26 at different target ratios,compared with the other CT-26 and PD-1KOT cells separately,the cell survival rate of CT-26 co-cultured with PD-1 knockout T cells,CT-26 co-cultured with PD-1 knockout T cells and PD-1 monoclonal antibody group was significantly decreased at 72 hours;also same as IFN-?,TNF-?,IL-2 cytokine secretion levels are increased in th groups.[Conclusion]In vitro experiments showed that it could inhibit the growth of tumor cells,laying a foundation for in vivo anti-tumor research.Part ?:In vivo effect-and-mechanism of CRISPR/Cas9-mediated-mice-PD-1 knockout T cells in the treatment of metastatic colorectal cancer[Methods]Colorectal cancer cell line CT-26 was cultured to prepare mice colorectal cancer xenograft models.The experimental groups were model were divided into four groups such as:control group(injection of PBS via tail vein),negative control group(injection of mock T cells via tail vein),PD-1 gene knockout T cell group(injection of PD-1 gene knockout T cells via tail vein)and PD-1 monoclonal antibody group(injection of PD-1 monoclonal antibody via tail vein).The body weight,activity and survival time of mice in each group in colorectal cancer xenograft model were observed,and the survival rate curve of mice was drawn.After sacrificed,the liver,lung and intestine tissues were taken for pathological analysis.Mesenteric lymph nodes and spleen lymphocytes were isolated for the CD4+,CD8+T and Treg cell ratios detection by flow cytometry,and the secretion levels of tumor immune-related cytokines were detected by ELISA.[Results]Compared with the model control group,allogeneic PD-1 knockout T cells inhibited the growth of orthotopic xenografts and metastasis to the liver and lungs in colorectal cancer-bearing mice,and prolonged the survival time of mice.PD-1 knockout T cells infused into colorectal tumor-bearing mice inhibited colorectal cancer metastasis by increasing interferon-?,tumor necrosis factor-? and interleukin-12 secretion,decreasing interleukin-6,interleukin-17 and transforming growth factor-? secretion in mesenteric lymph nodes and spleen,and increasing CD44+CD62L-memory T cells and decreasing CD4+FoxP3+regulatory T cells.[Conclusion]Allogeneic PD-1 knockout T cells show an inhibitory effect on tumor metastasis in vivo in colorectal cancer,and play an anti-tumor role in colorectal cancer by relying on CD8+T cells.Chapter ?:Safety Evaluation of T Cell PD-1 Gene Knockout in the Treatment of Metastatic Colorectal CancerPart ?:Establishment and biological characteristics of CRISPR/Cas9-mediated PD-1 knockout T cells in cynomolgus monkeys[Methods]The cynomolgus monkey PD-1 gene sequence was searched in the GENEBANK database,using the gRNA online design tool(Fig.http://crispr.mit.edu)sgRNA targeting PD-1 gene was designed and selected;Cynomolgus monkey PBMCs were isolated and cultured,the transfection efficiency was detected 48 hours later,and the silencing of PD-1 gene was verified using PCR technology.PHA and anti-humanCD3Ab?IL-2 were added to induce T cell proliferation,cell morphology and growth were observed and cell growth curves were drawn,cell proliferation was detected by CCK-8 assay.Cell cycle,apoptosis,and the expression of characteristic molecules on the surface of cynomolgus PD-1 ???? T cells were detected by flow cytometry,and IFN-? and IL-2 secreted by cynomolgus PD-1 ???? T cells were detected by ELISA.[Results]Compared with T cells without PD-1 gene knockout,the proliferation of PD-1 knockout T cells was not inhibited,and the growth curve was basically the same as that of T cells without PD-1 gene knockout;there was no statistical difference in the proportion of CD3,CD4 and CD8 detected by flow cytometry;the results of cell cycle and apoptosis assay showed that compared with T cells without PD-1 gene knockout,there was no statistical difference in the proportion of T cells in G0/G1 phase,S phase and G2/M phase,and also in the proportion of early apoptotic.The ELISA results showed that there was no statistical difference in the amount of cytokines released by PD-1 knockout T cells compared with that released by T cells without PD-1 knockout under the same culture conditions.[Conclusion]The disruption of inhibitory checkpoint gene PD-1 leads no affect on the viability of PD-1 knockout T cells laying a foundation for its in vivo safety evaluation.Part ?:In vivo safety evaluation of CRISPR/Cas9-mediated PD-1 knockout T cells in cynomolgus monkeys[Methods]PD-1 knockout T cells were enriched and infused into cynomolgus monkeys by intravenous.Referring to the effective dose of human cytokine-induced lymphocyte reinfusion(2×107/kg),they were dissolved in 100 ml 0.9%sodium chloride injection.The control group was intravenously injected with 100 ml/(kg·time)0.9%sodium chloride injection once at the same time every 3 days in the morning for a total of 3 doses.Peripheral blood was drawn at 4 h,24 h,48 h,1 w,2 w,3 w,and 4 w until 100 days after infusion.The expression of CD3 and PD-1 was detected by flow cytometry:CD3(+),PD-1(-)were PD-1 knockout T cells still alive in vivo,CD3(+),PD-1(+)were PD-1 knockout T cells no longer alive in vivo.The body weight and activity of cynomolgus monkeys in each group were observed,and blood routine and blood biochemistry were monitored.ELISA was used to detect the changes in the levels of Th1:IL-2,IL-12,IFN-gamma;Th2:IL-4,IL-10;inflammatory cytokines:TNF-?,IL-1?,IL-6 and other cytokines in cynomolgus monkeys of each group,and tissues such as liver,lung,bone marrow,spleen,and thymus were taken for pathological analysis to observe their toxic reactions.[Results]PD-1 knockout T cells did not show an effect on their body weight,activity or other general conditions.Hematological examination indicators:white blood cell count and monocyte count did not show significant change compared with the control group;biochemical indicators such as total bilirubin,direct bilirubin,indirect bilirubin,glutamic-oxalotransferase,glutamic-pyruvic transferase,total protein,albumin,globulin,urea nitrogen,creatinine did not show significant change compared with the control group;histopathological analysis showed no damage related to infused PD-1 knockout T cells;flow cytometry monitored the in vivo persistence of PD-1 knockout T cells in peripheral blood till two months.[Conclusion]Our results demonstrate the utility of T cell immunotherapy in cynomolgus monkeys for assessing the safety of PD-1 knockout and provide a new strategy for T cell-based adoptive immunotherapy.