The Antitumor Effects Of CAR-T Cells Targeting EpCAM Antigen On Colorectal Cancer

Objective:In this study,the scFv,the anti-human epithelial cell adhesion molecule(EpCAM)monoclonal antibody,is an extracellular single-chain variable region and its sequence includes Kozak sequence and CD8αsignal peptide sequence、the scFv region,CD8hinge region,CD28 transmembrane region,CD28 signal area,CD137 signal area and CD3ξsignal area.Using LV5 as lentiviral vector,we want to construct a third generation chimeric antigen receptor(CAR)targeting colorectal cancer antigen EpCAM for transfecting T cells to construct CAR-T cells targeting EpCAM antigen(Anti-EpCAM-CAR-T).Asthesametime,westudiedtheexpressionof anti-EpCAM-CAR on T cells、investigated the killing effect of anti-EpCAM-CAR-T on EpCAM antigen positive tumor cells and EpCAM antigen negative tumor cells in vitro,and observed the secretion of T cell killer cytokines.And we investigaed the therapeutic effect of anti-EpCAM-CAR-T cells on bearing colorectal cancer in the vivo of tumor-bearing nude mice,and observed the secretion of killer cytokines in the serum of nude mice,and preliminarily analyzed the side effects that were related after anti-EpCAM-CAR-T treatment.Methods:(1)The scFv sequences and related sequence information of anti-human EpCAM mAbs were determined and all sequences were synthesized as a whole sequence by total gene synthesis:CD8αsignal peptide-anti-EpCAM-scfv-CD8 hinge region-CD28transmembrane region-CD28 signal region-CD137 signal area-CD3 signal area.Then,EpCAM-CAR was cloned into the LV5 lentiviral vector and transfected into the 293T cells using the three-plasmid system to prepare the lentivirus.The sequence of DNA sequencing was correct.(2)The T cells were expanded and cultured.Peripheral blood mononuclear cells were extracted,activated with CD28 and CD3,and cultured in GT-T551 complete medium containing interleukin-2(IL-2).And then the T cell ratios of CD8+and CD4+were detected by flow cytometry.(3)whether EpCAM-CAR was expressed in transfected EpCAM-CAR-T cells was detected by Flow cytometry.In vitro killing experiments were performed after the construction of EpCAM-CAR-T was completed.(4)The EpCAM-CAR-T cells were co-cultured with EpCAM positive and negative cells.The killing efficiency was then measured by MTT assay.And then The secretion of the killer cytokines tumor necrosis factor-alpha(TNF-a)and interference-gamma(IFN-γ)were detected by ELISA.(5)BALB/C nude mice were divided into four groups with 6 mice in each group.Tumor models of BALB/C nude mice were implanted inHT-29 colorectal cancer cells were implanted into the peritoneal cavity of the tumor models of BALB/C nude mice to study the inhibitory effect of EpCAM-CAR-T on colorectal cancer in vivo.The mortality and tumor size of the nude mice were recorded.And then Pathological changes were observed in pathological sections of the tumor tissues and the secretion of serum killing factors in nude mice was detected.(6)At the same time,the liver and kidney of the tumor-bearing mice were pathologically sectioned to observe whether it had side effects that were related.Results:(1)The chimeric antigen receptor LV5-EpCAM-CAR targeting EpCAM antigen was successfully constructed and packaged with lentivirus.DNA sequencing was consistent with the expected results.(2)After the extracted peripheral blood mononuclear cells were activated and cultured,T cells were greatly expanded,and the ratio of CD3~+and CD8~+and CD4~+T cells was more than 70%,respectively.(3)After transfection,the expression rate of EpCAM-CAR on T cell membrane that was detected by flow cytometry was19.7%.(4)MTT results showed that the tumor suppressing rate of EpCAM-positive group was higher than that of negative group(P<0.05).ELISA results suggested that the cytokines of TNF-αand IFN-γof the positive group were higher than that of negative group(P<0.05).(5)After Seven days in tumorigenesis in nude mice,nodules appeared subcutaneously,and the abdomen slightly swelled.Pressing the abdomen affected masses of varying sizes and sizes,and pathological sections confirmed them as colorectal cancers.We found that in the blank group mice began to die from the 16th day and mice died with swollen abdomen on the 18th day in the control group.However,in the CAR-T group micebegan to die on the 26th day and there was almost no swelling in the abdomen,and the tumor volume was also smaller than that of the blank group and the control group(P<0.05).There was a significant difference in mortality between the blank group and the experimental group(P<0.05),but the survival rate was the same on the28th day in the experimental group and the control group.The serum levels of TNF-αand IFN-γin tumor-bearing nude mice in the experimental group were higher than those in the control group and blank control group(P<0.05).(6)The pathological sections of liver and kidney of nude mice showed that there were metastases in the liver and kidney in all group,but the pathological sections of the dead nude mice in the experimental group showed many changes in cachexia,such as pigmentation and cell atrophy,indicating that the treatment may produce off-target effects to cause a cytokine storm.The experimental group nude mice may die from the side effects.Conclusion:A third-generation chimeric antigen receptor structure,EpCAM-CAR,was successfully constructed.T cells transfected with EpCAM-CAR can be targeted to recognize EpCAM-positive tumor cells,secrete a large number of killer cytokines,TNF-αand IFN-γ,inhibit the growth and metastasis of colorectal cancer in vivo,and improve the quality of tumor-bearing nude mice.EpCAM-CAR also causes off-target effects to ressult in cytokine storms.Therefore,in treatment,it is necessary to combine immunosuppressive agents and gamma globulin to reduce side effects.