Killing Effect Of CMet/Siglec15 CAR-T On Lung Adenocarcinoma And Its Mechanism On Tumor Microenvironment

Objective:LUAD is one of the most common forms of lung cancer.Immunotherapy has become a promising treatment for lung cancer.Because of the defects of low response rate and high toxicity of monoclonal antibody drugs developed by immune checkpoint inhibitors(ICIS),such as PD-1 and CTLA-4 inhibitors,we tried to use Siglec15,which is mutually exclusive with PD-1 expression,as a target to prepare monoclonal antibody,to explore its therapeutic effect on LUAD and verify the effectiveness of the target.On this basis,we prepared c Met CAR-T which can autocrine sigelc15sc Fv,and explored its killing effect on LUAD,further studied its mechanism of action on LUAD and its regulation on TME,so as to provide scientific basis for tumor immunotherapy and research and development of new medicine.Methods:1.Bioinformatics analysis was used to retrieve Siglec15gene,protein sequence and structure information from NCBI and IMgt human antibody databases,and the expression profile of Siglec15gene in tumor cells was retrieved from CCLE database to obtain the PPIs of the target,finally through KEGG database to access the target related pathways,to provide direction for future research.2.On the basis of previous studies,Sigelc15 antibody sequence was designed to prepare recombinant Siglec15gene protein;mice were immunized,and hybridoma cells secreting Siglec15 antibody stably were prepared by screening strains and antibody subtype detection.RNA of the cells was extracted and reverse transcripted to amplify antibody variable region gene PCR technology was used to recombine the expression plasmid,transform and clone it.After the sequence was confirmed to be consistent with the design,293F cells were used to express the antibody.Siglec15 VH and VL plasmids were cotransfected into 293F cells,and the supernatant was collected for protein purification.The purified antibody was purified by ELISA,SDS-PAGE and Western blot The specific binding ability of the antibody to the corresponding antigen and its correct expression in the corresponding molecular weight were identified by blot;the affinity of anti Siglec15 Ig G was detected by Blitz;the spatial and linear binding epitopes were identified by epitope mapping;the competitive binding ability of anti Siglec15 to s Tn protein and polypeptide was detected by ELISA;and the the proliferation of anti-Siglec15 antibody was detected by CCK8;the invasion and migration of tumor cells were detected by Transwell and scratch test;the differentiation,proliferation inhibition and migration of macrophages were detected by PMA induced differentiation of THP-1 cells;the nude mouse model of LUAD was established to evaluate the activity Objective to investigate the therapeutic effect of anti Siglec15 Ig G in nude mice.3.The c Met/Siglec15 CAR plasmid was designed and constructed by overlap PCR;the c Met/Siglec15 CAR lentiviral vector was constructed by infusion PCR;the lentiviral plasmid was transformed and cloned by competent technology,and the validity of the lentiviral vector was verified by sequencing;the c Met/Siglec15CAR lentiviral vector was constructed by in-fusion PCR:293T cells were transiently transfected with CAR lentivirus expression vector to prepare lentivirus and determine the titer of the virus;after PBMC were isolated from human peripheral blood,T cells were activated and cultured,and the prepared CAR lentivirus was used to infect T cells.The classification and transfection efficiency of c Met/Siglec15 CAR-T cells were detected by flow cytometry.4.LDH release assay was used to detect the killing effect of c Met/Siglec15 CAR-T cells on Siglec15 positive cells of different effect target ratios or fixed effect target ratios;ELISA was used to detect the secretion of IL-2 and IFN-γcytokines during the killing process of c Met/Siglec15 CAR-T cells;mouse c Met/Siglec15CAR-T was prepared as well as other four groups,after given these five gruops,detected the effects on body weight,tumor weight,blood biochemistry and organ index;the pathological changes,the expression of macrophage related markers and the infiltration of T cells were detected by immunohistochemistry.The expression of Siglec15,Tyrobp and Macrophage related genes and proteins were detected by RT-q PCR and Western blot,CD8~+,CD4~+T cells and M2subtypes were detected by FCM;RT-q PCR and Western blot were used to analyze the pathway genes expressions.Results:1.Using bioinformatics methods to find Siglec15has high express on the myeloid cells,immune cells and tumor cells,but low in normal tissues;there were 10proteins related to Siglec15,and 23 interacting pathways,which were mainly related to cell adhesion,immune regulation and osteoclast development.The structure of Siglec15consists of immunoglobulin(Ig)-like domain,transmembrane domain and short cytoplasmic tail.The immunoglobulin like domain consists of two extracellular Ig domains,including an N-terminal V-set domain containing sialic acid binding sites and a type 2 constant region.It interacts most closely with Tyrobp(DAP12).2.The anti-Siglec15 antibody was successfully purified and its identified as Ig G1.The results of denaturing SDS-PAGE showed that the size of anti body weight light chain was the same as expected,the heavy chain was about 55 k Da,the light chain was about 35 k Da,and the purity of antibody VH and VL was more than 95%.The Siglec15 Ig G can specifically bind to the corresponding antigen at drefferent 14 concentrations,and it can still specifically bind to the antigen at a lower concentration,indicating that the specific binding ability of the antibody meets the requirements of subsequent experiments.A375,U87MG,Raji,THP-1 and HCT-8 cells with Siglec15 positive expression could specifically bind to the purified anti Siglec15 antibody,while HLF-1 cells with Siglec15negative expression did not produce obvious bands.The KD values were all 5.719e(-8).When the peptide dilution ratio was 1:100,the highest OD is A9,indicating that the epitope had the strongest specific binding ability with the anti Sigelc15 Ig G.The competitive binding ability of Siglec15 antibody to glycosylated s Tn protein is strong,which is consistent with the literature description.In addition,the competitive ELISA test with the three sites with high OD value in epitope identification confirmed that it had good competitive binding ability to A9,B8 and B9 epitopes,and its competitive binding ability to A9 was better than the other two sites.3.Siglec15 antibody could inhibit the proliferation of A549,H1299,H460 and PC-9 cells,but had no significant effect on the proliferation of HBE cells.The antibody could inhibit the invasion of A549 cells,H1299cells and LLC cells,but had no significant effect on the invasion of H460 cells and HBE cells.It can significantly affect the morphology of macrophages induced by PMA.After adding anti Siglec15 antibody,the number of M1 adherent cells was more than that of the control group,while the number of M2 cells was significantly reduced.These results indicate that Siglec15 Ig G can increase the adhesion of M1 macrophage and inhibit M2macrophage.The order of cell proliferation rate was:M1+Siglec15 Mab>M1>M2>M2+Siglec15 Mab≈M0.The expression of M1 associated protein was significantly increased(P<0.01)and M2 associated protein was significantly decreased(P<0.01).4.Therapeutic effect of antibody:after modeling,the body weight of nude mice increased first and then decreased.The body weight of high-dose group and blank control group increased,but the body weight of low-dose group was lower than that of model group.After the second administration,the nude mice in the treatment group and the model group were accompanied with different degrees of death.The tumor weight of the model group was similar to that of the low-dose group,and the tumor weight of the medium dose and high-dose groups was significantly lighter than that of the model group(P<0.01).The tumor weight changed significantly among the groups.5.The recombinant plasmids were transformed into E.coli TOP10 cells,positive clones were selected,the plasmids were extracted,verified by double enzyme digestion and sequencing,the results were consistent with the theoretical sequence;Western blot showed that the expression of CD3ζwas detected in CAR plasmids,c Met/Siglec15 was transfected into IP products and there was a specific band about 35 k Da in the cell supernatant of CAR,which was close to the theoretical value,and the similarity was high after gel cutting and mass spectrometry analysis;the existence of HA tag in the swimming lane of c Met/Siglec15 CAR was detected by Western blot,which indicated that the c Met/Siglec15 CAR plasmid constructed in this experiment could secrete anti-Siglec15sc Fv in vitro;the titer of lentivirus was 7×10~8PFU/m L.The transfection efficiency of c Met/Siglec15 CAR,c Met CAR and CD19 CAR lentivirus was 45.8%,47.2%and 49.6%,respectively.6.LDH release assay showed that c Met/Siglec15 CAR-T had a certain killing effect on LUAD cells at different effect target ratio and fixed effect target ratio,the killing effect was:c Met/Siglec15 CAR-T>c Met CAR-T>CD19 CAR-T≈Activated T cells(P<0.01),with the decrease of the amount of target cells,the killing effect decreased,especially on A549and LLC.knockdown of Siglec15 and c Met could significantly affect the killing effect of effector cells on target cells;The levels of IL-2 and IFN-γwere higher than those of c Met CAR-T(P>0.05)and other T cells(P<0.0001);for HBE cells,the levels of IFN-γand IL-2secreted by c Met/Siglec15 CAR-T were not significantly different from those of other groups(P>0.05).There was no significant difference in the cytotoxic effect and cytokine secretion between the two groups(P>0.05).7.Animal experiment results:the weight of mice:c Met/Siglec15 CAR-T>c Met CAR-T+Siglec15 Mab>c Met CAR-T>CD19CAR-T>Activated T,the spleen and thymus index of mice were increased after treatment(P>0.05),suggesting that cell therapy may have no significant effect on the spleen and thymus function;tumor weight of mice:c Met/Siglec15 CAR-T<c Met CAR-T+Siglec15Mab<c Met CAR-T<CD19 CAR-T<Activated T.The results of serum biochemical test showed that glutamic pyruvic transaminase,glutamic oxaloacetic transaminase and urea nitrogen were significantly decreased(P<0.01),only c Met/Siglec15 CAR-T and c Met CAR-T+Siglec15 Mab could significantly reduce the serum creatinine level(P<0.05);pathological section showed that c Met/Siglec15 CAR-T could significantly reduce the serum creatinine level(P<0.05),the tumor cells were decreased,focal necrosis,interstitial fibrosis,infiltration of lymphocytes,histiocytes and multinucleated giant cells,and cholesterol crystal deposition could be seen.Compared with the other groups,the CAR-T groups had a good anti-tumor effect;IHC test results showed that c Met/Siglec15CAR-T and c Met CAR-T+Siglec15 Mab had a good anti-tumor effect.The results of IHC showed that the M2 markers of c Met/Siglec15 CAR-T and c Met CAR-T+Siglec15 Mab were significantly decreased(P<0.05).RT-q PCR showed that c Met/Siglec15 CAR-T could significantly downregulate DAP12,SYK,NF-κB,MUC-1,s Tn protein expression,downregulate M2 cytokines and up regulate M1 like cytokines(P<0.01).Conclusion:1.Siglec15 Mab has a certain therapeutic effect and kill effect on LUAD,which may affect the TME of LIAD by interfering macrophage polarization.2.The structure expression of c Met/Siglec15 CAR-T was complete and correct.Siglec15sc Fv could be test.3.c Met/Siglec15 CAR-T can significantly kill lung adenocarcinoma cells in vitro and in vivo.4.c Met/Siglec15 CAR-T can significantly inhibit LUAD,increase T cell infiltration in TME,target Siglec15,down regulate M2 macrophages,MUC-1 and s Tn,and inhibit the development of LUAD through DAP12/Syk/NF-κB pathway.