| Objective:Diffuse gastric cancer has a poor prognosis and efficacy,which is still a lack of new targets related to immunotherapy.This study intends to screen out immune-related and specific high-expression targets in DGC,and a high-affinity and functional monoclonal antibody is prepared for this target.A new antibody immunotherapy is developed for DGC treatment by studying the efficacy and mechanism of the monoclonal antibody in the treatment of DGC.Methods:1.We used non-labeled quantitative protein histology（label free）and TCGA database to screen out the immune-related and differentially expressed genes in diffuse gastric cancer tissue and adjacent tissues.Then immunohistochemistry and western blot were used to verify its protein level expression in DGC cancer tissues and adjacent tissues,and the relationships were analyzed between the expression level of the target and the survival time or immune checkpoint PD-L1.2.Case 9 technology was used to knockout the target gene to verify the effect of the gene knockout on cell function and tumor growth.3.Monoclonal antibodies for this target were prepared by single-cell sequencing technology,and high-affinity monoclonal antibodies were screened by BIAcore technology and flow cytometry technology,and finally their functions were verified.4.Flow cytometry and western blot were used to verify the competitive effect of the monoclonal antibody on Pro-u PA and the blocking effect on ERK signaling pathway,respectively.cells with overexpression of this target were used to construct the CDX model,and the clinical tissue samples with high expression of this target were used to construct the PDX model,the intraperitoneal administration was used to studyαu PAR monoclonal antibody to enhance the efficacy of PD-1 m Ab in the treatment of diffuse gastric cancer and its related mechanism.Results:1.The differential expression analysis of cancer tissues and adjacent normal tissues by non-labeled quantitative protein histology technology and the TCGA database,it was found that u PAR and FCGR3A were immune-related and highly expressed genes in patients with diffuse gastric cancer.Furthermore,the expressions of m RNA were verified by q RT-PCR in diffuse gastric cancer tissues and paracancer tissues,and the results showed that the differential expression of u PAR in cancer tissues and paracancer tissues was statistically significant,and the expression of FCGR3A was not statistically significant.Finally,the protein level of u PAR was verified respectively in 8 pairs of diffuse gastric cancer tissues proteins and 202 pairs of diffuse gastric cancer tissue microarrays.The results found that the expression of u PAR in diffuse gastric cancer tissues was higher than that in paracancer tissues.In analyzing the relationship between u PAR expression level and survival time,the results showed that the survival time of diffuse gastric cancer patients with low u PAR expression were significantly higher than that of patients with high u PAR expression,and the differences were statistically significant.The analysis of the TISIDB database found that the m RNA expression level of u PAR was highly correlated with PD-L1.2.The functional experiments In vitro found that the proliferation and invasion abilities of SNU216 and AGS cells in the u PAR-/-group were significantly lower than those in the CTRL group.We constructed the mouse CDX model by MKN45 cells,the research showed that the tumor tissue volume and tumor weight of the u PAR-/-group were significantly lower than that of the CTRL group.At the same time,the u PAR gene was restored to the u PAR-/-MKN45 cells,and it was found that the tumor tissue volume and tumor weight of the u PAR-/-+u PAR+/+group were significantly close to those of the CTRL group.3.The affinity of E2 or A7 m Abs with 293T-u PAR+/+cells were verified by BIAcore technology,and it was found that the affinity of E2 antibody（Kd=1.95x10-9M）was significantly higher than that of A7 antibody（Kd=9.09x10-8M）.Finally,the ability of E2 antibodies binding to 293T-u PAR+/+cells or SNU216 cells were verified by immunofluorescence and immunoelectron microscopy respectively,the results showed that E2 monoclonal antibody had strong binding ability to u PAR antigen on cell membrane.In the antibody functional experiment,it was found thatαu PAR monoclonal antibody had obvious ADCC effect on SNU216 cells.The CDC effect induced byαu PAR was significantly higher than that of the homologous negative control group（Ig G）,the differences were statistically significant,which was same with the humanαHER2.4.The competitive binding experiment was verified by flow cytometry,and the result showed that the average fluorescence intensity of theαu PAR+Pro-u PA group was significantly lower than that of the Pro-u PA group alone,and the differences between the two groups were statistically significant.And the expression of activated p-ERK kinase in theαu PAR+Pro-u PA groups was significantly lower than those in the Ig G+Pro-u PA groups,and the expression of total T-ERK kinase did not change significantly.In the experiments of the CDX model and the PDX model,the results showed that the size and volume of tumors inαu PAR group,αPD-1 group andαu PAR+αPD-1 group were significantly lower than those in the CTRL group,and the survival time of the above three groups were also significantly higher than those of the CTRL group.Moreover,the size and volume of tumor inαu PAR+αPD-1 group were significantly lower than those in theαPD-1 group,and the survival time of theαu PAR+αPD-1 group were also significantly higher than those of theαPD-1 group,the differences were statistically significant.The analysis of Immunofluorescence,immunohistochemistry and flow cytometry showed that the infiltrating numbers of CD8+T cell（include activated CD8+T cell）in the tumor tissues of theαu PAR group,αPD-1 group andαu PAR+αPD-1 group were significantly higher than those in the CTRL group.And the infiltrating numbers of CD8+T cell（include activated CD8+T cell）in the tumor tissues of theαu PAR+αPD-1 group were significantly higher than those in theαPD-1group,the differences were statistically significant.Furthermore,the analysis of flow cytometry in the tumor tissues showed that the infiltrating numbers of M1 macrophages in the tumor tissues of theαu PAR group,αPD-1 group andαu PAR+αPD-1 group were significantly higher than those in the CTRL group.But the infiltrating numbers of M2 macrophages and Treg cells in the tumor tissues of the above three groups were significantly lower than those in the CTRL group.We also used q RT-PCR to verify the changes of T cell-related chemokine in the PDX model tumor tissue,it was found that the m RNA levels of CCL3,CCL4,and CXCL10 in theαu PAR group were significantly higher than those in the CTRL group.And the m RNA levels of CCL3,CCL4,CXCL9,and CXCL10 in theαu PAR+αPD-1 group were also significantly higher than those in the CTRL group,the difference was statistically significant.Conclusion:1.u PAR is a specific and highly expressed membrane protein in diffuse gastric cancer,and it has a potential new target to be combined with PD-L1 immunotherapy.2.The functional experiments in vivo and in vitro have found that the knockout of u PAR gene inhibits the proliferation and invasion abilities of diffuse gastric cancer.3.Theαu PAR monoclonal antibodies with high affinity and functionality are prepared,which have potential value for the targeted therapy of diffuse gastric cancer.4.Theαu PAR monoclonal antibodies can competitively bind to u PAR to affect the proliferation,invasion and adhesion of DGC cells By blocking the u PA-u PAR system and ERK signaling pathway5.αu PAR monoclonal antibody can not only independently improve the efficacy of DGC patients,but also significantly enhance the efficacy of PD-1 antibody in DGC patients6.αu PAR monoclonal antibodies have anti-tumor effects by increasing the infiltrating numbers of CD8+T cells and M1 macrophages,and reducing the infiltrating numbers of M2macrophages and Treg cells in tumor tissues.αu PAR monoclonal antibodies can increase the infiltrating numbers of T cell into tumor tissue by inducing the expression of T cell-related chemokines. |
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