Antitumor Efficacy Of NKG2D CAR-T Cells Against Human Colorectal Cancer Cells In Vitro And In Vivo

Colon cancer is one of the most common malignant tumors of digestive tract.It has the characteristics of difficult treatment,high morbidity and high mortality.The incidence of colon cancer ranks second in digestive tract tumors,and it is increasing worldwide.For patients with early or local metastasis,the prognosis is generally better if they can get the chance of radical surgery.For patients with advanced stage,they often lose the chance of radical surgery,and have to use treatment such as radiotherapy,chemotherapy,symptomatic supportive treatment,and the prognosis is generally poor.Therefore,a safe and effective treatment strategy for colon cancer treatment is needed.Chimeric Antigen Receptor T-Cell（CAR-T）Immunotherapy is one of the most important methods in cancer immunotherapy.Chimeric Antigen Receptor T-Cell is a kind of immune cells that can specifically kill tumor cells by gene modification of chimeric antigen receptor.Chimeric Antigen Receptor（CAR）can specifically recognize tumor-associated antigen（TAA）or tumor-specific antigen（TSA）,and then transmit signals to T cells,causing T cell activation and proliferation,thus effectively killing tumor cells.In 2017,the Food and Drug Administration（FDA）of the United States approved the Kymriah（a CAR-T drug from Novartis Pharmaceutical Company）and Yescarta（a CAR-T drug from Kite Pharmaceutical Company）to be used for the treatment of hematological diseases,and achieved amazing results.Recent studies have found that CAR-T immunotherapy has a certain therapeutic effect on solid tumors.NKG2D（natural killer cell group 2D）is an important activated receptor encoded by Klrk1 gene of NK cell lectin-like receptor family.It mainly exists in NK cells of mice and humans,and is constitutively expressed in CD8⁺T cells of humans,but only expressed in activated T cells of mice.Human NKG2D receptors recognize a variety of highly diverse ligands,including MICA,MICB,ULBP1,ULBP-2/5/6,ULBP-3 and NKG2DR6.Normally,NKG2D ligands do not exist in normal cells,but can be induced by various stimuli.In cancer cells,NKG2D ligands bind to NKG2D,then activate NK cells and participate in immune cell mediated antitumor efficacy.NKG2D ligand can be used as an effective target for the CAR-T cells immunotherapy.In this study,we will construct NKG2D CAR using non-viral minicircle DNA vector and electroporate the vector into T cells to obtain the third generation NKG2D CAR-T cells,and explore the killing effect of NKG2D CAR-T cells on human colon cancer cells in vitro and in vivo.Non-viral vectors can avoid the risk of virus replication and bacterial replication,and have good security.Therefore,this study will provide new targets and ideas for the treatment of colon cancer and other solid tumors,and has important clinical application value.Part one Preparation and functional detection of NKG2D chimeric antigen receptor T-cellObjectives:To construct NKG2D CAR minicircle DNA vector,generate and verify NKG2D CAR-T cell.Methods:According to the references and Genebank,the amino acid sequence of NKG2D CAR was determined.The structure of CAR was determined according to the sequence of CD8αsignal peptide,human NKG2D extracellular domain,human CD8αhinge region,human CD28transmembrane domain and intracellular domain,human 4-1BB intracellular signal sequence and human CD3 intracellular signal sequence.In addition,two restriction sites,EcoRI and BamHI,were placed at the front and back ends of the whole NKG2D CAR gene sequence to form the whole NKG2D CAR gene sequence,respectively.The whole gene sequence of NKG2D CAR was synthesized and linked to the gene of E.coli cloning plasmid pUC57,named NKG2D CAR-pUC57 plasmid.NKG2D CAR-pUC57 plasmid were digested by EcoRI and BamHI.Agarose gel electrophoresis was used to detect the results of enzyme digestion,and NKG2D CAR fragments were recovered.NKG2D CAR fragments were ligated with the parental minicircle DNA,and transformed into ZYCY10P3S2T E.coli,which was cultured in LB solid medium containing kanamycin.Two monoclonal colonies were selected to extract NKG2D CAR minicircle DNA plasmid.The plasmid was analyzed by double enzyme digestion and agarose gel electrophoresis.T lymphocyte was isolated from human peripheral blood by degree gradient centrifugation.NKG2D CAR minicircle DNA plasmid was transfected into T lymphocyte by electroporation to obtain NKG2D CAR-T cells.Cell counts were performed before medium exchange.Cell viability was calculated by trypan blue staining.GFP expression was observed by fluorescence microscopy.NKG2D and GFP expression and cell localization were observed by confocal microscopy.NKG2D and GFP expression were observed by flow cytometry.CD3ζexpression was detected by Western blotting.The proportion of CD4⁺and CD8⁺T cell subgroups were detected by flow cytometry.Result:1.Agarose gel electrophoresis showed that a band of about 1200bp size was visible in the gel.The size of band was consistent with that of designed NKG2D CAR.This is NKG2D CAR fragment,suggesting that the NKG2D CAR-pUC57 plasmid was successfully constructed.2.After overnight inversion culture,monoclonal colonies were observed in LB solid medium,suggesting that NKG2D CAR parental minicircle DNA plasmids were successfully transformed into competent bacteria and obtained kanamycin resistance.3.Two monoclonal colony plasmids were extracted.Double enzyme digestion and agarose gel electrophoresis showed that the second plasmid has a band of about 1200bp size.The size of band was consistent with that of designed NKG2D CAR.It was suggested that the second plasmid containing the NKG2D CAR fragment,and NKG2D CAR minicircle DNA plasmid was successfully constructed.The results of gene sequencing showed that the NKG2D CAR minicircle DNA plasmid did not undergo gene mutation and included complete gene sequence of NKG2D CAR.4.After management with FICOLL separation solution,each person’s peripheral blood leukocyte removal filter can obtain 3×10⁶ to 5×10⁶lymphocytes.Under the inverted microscope,lymphocyte showed suspended growth,round or elliptic.A part of large and irregular cells showed adherent growth.After 12 hours of cell culture,lymphocytes began to aggregate and form proliferative colonies.Meanwhile,the number of large and irregular cells decreased significantly.5.After electroporation,trypan blue staining showed that the survival rate of normal T cells was 97%,and that of NKG2D CAR-T cells was 76%.It was concluded that electroporation can decrease the survival rate of cells.6.The results of cell counting showed that normal T cells and NKG2D CAR-T cells had similar amplification rates after 12 days of culture,with the amplification ratio reaching 5-6 times.In addition,the number of NKG2D CAR-T cells decreased significantly on day 2-4,which may be due to cell damage caused by electroporation.7.The results of fluorescence microscopy showed that GFP began to express in NKG2D CAR-T cells 24 hours after electroporation,peaked at 48hours,and lasted for at least 72 hours.8.The results of confocal microscopy showed that the expression of NKG2D and GFP in CAR-T cells was significantly stronger than that in normal control T cells.NKG2D protein was mainly localized in cell membrane and cytoplasm,suggesting that NKG2D CAR could fold smoothly and bind specifically to T cell membrane.9.The results of western blotting showed that both NKG2D CAR-T cells and normal control T cells had endogenous CD3 expression.NKG2D CAR-T cells also had exogenous CD3 expression,while normal control T cells had no exogenous CD3 expression.10.The results of flow cytometry showed that,compared with the normal control group,the percentage of NKG2D CAR-T cells with CD4⁺increased slightly,and that of NKG2D CAR-T cells with CD8⁺decreased slightly.Summary:1.NKG2D CAR minicircle DNA plasmid was successfully constructed by gene recombination technology.2.NKG2D CAR minicircle DNA plasmid was successfully electroporated into human peripheral blood lymphocytes,and cultured and amplified in vitro.NKG2D CAR-T cells were successfully obtained.3.Human colon cancer cells LS174T expressed high levels of MICA and ULBP-3.HCT-116 cells expressed high levels of MICA,MICB,ULBP-2/5/6and ULBP-3.Part two Killing effect of NKG2D chimeric antigen receptor T cells on human colon cancer cells in vitroObjectives:To observe the function of CAR-T cells and their killing effect on human colon cancer cell line in vitro.Methods:Human colon cancer cell LS174T and HCT-116 were resuscitated and subsequently cultured and expanded in RPMI 1640supplemented with 1%penicillin-streptomycin double antibody and 10%fetal bovine serum in a 95%air,5%CO₂ atmosphere at 37℃.The morphology of cells was observed under inverted microscopy.The culture medium was replaced every two days.When cell adherence grew to 90%fusion,cell passage was carried out by trypsin EDTA digestion method according to1:3-1:4 multiplication.The expression of NKG2D ligands such as MICA,MICB,ULBP-1,ULBP-2/5/6 and ULBP-3 in human colon cancer cell lines LS174T and HCT-116 was detected by flow cytometry.T cells or NKG2D CAR-T cells were co-cultured respectively with LS174T or HCT-116 at 5:1,10:1 and 20:1 effector cells:target cell ratio（E:T）for 24 hours.The cytotoxicity of NKG2D CAR-T cells to LS174T and HCT-116 was detected by LDH Cytotoxicity Assay Kit.Enzyme-linked immunosorbent assay（ELISA）was used to detect the release of cytokines such as IL-2 and IFN-γfrom NKG2D CAR-T co-cultured with LS174T or HCT-116 colon cancer.Result:1.The results of flow cytometry showed that MICA and ULBP-3 were highly expressed on the surface of human colon cancer cells LS174T,but MICB,ULBP 1 and ULBP-2/5/6 were low.2.The results of flow cytometry showed that the expressions of MICA,MICB,ULBP-2/5/6 and ULBP-3 on the surface of HCT-116 cells were high,but ULBP1 was low.3.The cytotoxicity test of lactate dehydrogenase showed that NKG2D CAR-T cells had stronger killing effect on LS174T cells than normal control T cells in all effective target ratios（E:T）.When the effective target ratio（E:T）was 20:1,NKG2D CAR-T cells had the best killing effect on LS174T,and the killing rate was 47%.4.The cytotoxicity test of lactate dehydrogenase showed that NKG2D CAR-T cells had stronger killing effect on HCT-116 cells than normal control T cells in all effective target ratios（E:T）.When the target-to-effect ratio（E:T）was 20:1,NKG2D CAR-T cells had the best killing effect on HCT-116,with the killing rate reaching 52%.5.ELISA results showed that NKG2D CAR-T cells could release a large amount of IL-2,compared with normal control T cells.When NKG2D CAR-T cells and LS174T cells were co-cultured for 24 hours,the concentration of IL-2 in the medium reached 3265.83±840.45pg/mL.When NKG2D CAR-T cells and HCT-116 cells were co-cultured for 24 hours,the concentration of IL-2 in the medium reached 4855.71±1166.61pg/mL.6.ELISA results showed that NKG2D CAR-T cells could release a large amount of IFN-γcompared with normal control T cells.When NKG2D CAR-T cells and LS174T cells were co-cultured for 24 hours,the concentration of IFN-γin the medium reached 8621.43±1594.91pg/mL.When NKG2D CAR-T cells and HCT-116 cells were co-cultured for 24 hours,the concentration of IFN-γin the medium reached 10757.14±2163.74pg/mL.Summary:1.In vitro studies showed that NKG2D CAR-T cells had good killing effects on human colon cancer cells LS174T and HCT-116,and the killing efficiency was positively correlated with the target-to-effect ratio.2.NKG2D CAR-T cells could secrete a large number of cytokines such as IL-2 and IFN-γ.Part three Killing effect of NKG2D chimeric antigen receptor T cells on human colon cancer cells in vivoObjectives:To establish the xenotransplantation NOD/SCID model of HCT-116 cells,and observe the killing effect of NKG2D CAR-T cells on human colon cancer cells in vivo.Methods:Human colorectal cancer cell line HCT-116-Luc was resuscitated,and subsequently cultured and expanded in RPMI 1640supplemented with 1%penicillin-streptomycin double antibody and 10%fetal bovine serum in a 95%air,5%CO₂ atmosphere at 37℃.The morphology of was observed under inverted microscopy.The culture medium was replaced every two days.When cell adherence grew to 90%fusion,cell passage was carried out by trypsin EDTA digestion method according to 1:3-1:4multiplication.Hygromycin B solution was added to the medium of newly passaged human colon cancer cell line HCT-116-Luc.HCT-116-Luc cells with hygromycin B resistance were obtained after 3-4 passages.Male NOD/SCID（non-obese diabetes mellitus/severe combined immunodeficiency）mice were fed in SPF-grade animal laboratory.HCT-116-Luc tumor cell suspension（1×10⁶ cells per mice）was injected subcutaneously into the right shoulder of mice to establish a subcutaneous transplanted model of colon cancer.When the tumor load reached 150-250mm³,the mice were randomly divided into three groups according to the tumor size,body weight and experimental design:（1）Control group:five NOD/SCID mice were fed normally and injected PBS on day 0 and 7,respectively.（2）T cells group:five NOD/SCID mice were given normal T cells treatment on day 0 and 7,respectively.（3）NKG2D CAR-T cells group:five NOD/SCID mice were given NKG2D CAR-T cells treatment on day 0 and 7,respectively.During the treatment,the mental state,hair condition,diarrhea and allergic reaction were observed,and the survival time of the mice was recorded.The body weight and tumor volume of mice were recorded every 5 days,and the weight curve and tumor growth curve were drawn.On the 20th day after treatment,the growth of the tumors in mice was observed by using in vivo imaging system.On the 25th day of treatment,mice were executed.Tumors,heart,liver,lung,spleen,kidney and other important organs and tissues were weighed and placed in 4%polyformaldehyde universal tissue stationary solution for immunohistochemical stain and hematoxylin and eosin（HE）stain.Result:1.Human colon cancer cell line HCT-116-Luc was subcutaneously injected into the right shoulder and back of NOD/SCID mice.The transplanted colon cancer model of mice was successfully constructed and all tumors were formed in 12 days.Mice were divided into normal control group,T cell treatment group and NKG2D CAR-T cell treatment group.2.Weight measurements showed that,during the treatment,the weight of mice in the normal control group increased slightly,while the weight of mice in the T cell treatment group did not change significantly,but the weight of mice in the NKG2D CAR-T cell treatment group decreased slightly.3.In vivo imaging results showed that,compared with normal control group and T cell treatment group,NKG2D CAR-T cell treatment group significantly inhibited the growth of tumors in mice.4.Vernier caliper measurements showed that the tumor volume of mice in normal control group and T cell treatment group increased rapidly during the treatment period,but that of mice in NKG2D CAR-T cell treatment group increased slowly.5.The survival time of mice in NKG2D CAR-T cell treatment group was longer than that of normal control group and T cell treatment group.6.Immunohistochemical stain results showed that NKG2D CAR-T cell treatment group had a large number of lymphocyte infiltration around the tumor tissue compared with the normal control group and T cell treatment group.7.HE stain results showed that no obvious morphological changes were found in the sections of heart,liver,spleen,lung and kidney of mice treated with NKG2D CAR-T cells.NKG2D CAR-T cell treatment was safe.Summary:1.In vivo results showed that the transplanted colon cancer model was successfully constructed by subcutaneous injection of human colon cancer cell HCT-116-Luc into the right shoulder and back of NOD/SCID mice.2.NKG2D CAR-T cell therapy resulted in weight loss,prolonged survival time and inhibited tumor growth in mice.3.NKG2D CAR-T cells can infiltrate around the tumor tissues of mice,without causing morphological changes of the heart,liver,spleen,lung and kidney.NKG2D CAR-T cell treatment was safe.Conclusion:1.NKG2D CAR microcyclic DNA plasmid was successfully prepared by gene recombination technology.NKG2D CAR minicircle DNA plasmid was successfully transferred to human peripheral blood lymphocyte by electroporation and cultured and amplified in vitro.NKG2D CAR-T cells were successfully obtained.2.LS174T cells expressed high levels of MICA and ULBP-3,HCT-116cells expressed high levels of MICA,MICB,ULBP-2/5/6 and ULBP-3.3.In vitro studies showed that NKG2D CAR-T cells had good killing effects on LS174T and HCT-116 cells,and the killing efficiency was positively correlated with the target-to-effect ratio.NKG2D CAR-T cells could secrete a large number of cytokines such as IL-2 and IFN-γ.4.In vivo results showed that the transplanted colon cancer model of NOD/SCID mice was successfully constructed by subcutaneous injection of HCT-116-Luc cells into the right shoulder and back of NOD/SCID mice.NKG2D CAR-T cell therapy resulted in weight loss,prolonged survival time and inhibited tumor growth in mice.NKG2D CAR-T cells can infiltrate around the tumor tissue of mice,without causing morphological changes of the heart,liver,spleen,lung and kidney,and have good safety.