Screening And Identification Of Monoclonal Antibodies Against Human Pancreatic Cancer Cell SW1990 And Evaluation Of Their Antitumor Effects

Pancreatic cancer is a gastrointestinal tumor with a high degree of malignancy.Because of its difficulties in early diagnosis,complicated tumor microenvironment,easy metastasis and recurrence,the 5-year survival rate of patients with cumulative cancer is often less than 10%.With the increase of the aging population and changes in the way people live and eat,the incidence of pancreatic cancer has also increased year by year.At present,the treatment of pancreatic cancer is mainly surgical resection,but because most patients have metastasized at the time of diagnosis,only 20%of patients have the opportunity to undergo surgical resection.In recent years,immunotherapy and targeted therapy for tumors have shown good application prospects,but because the current understanding of tumor-specific diagnosis and treatment targets for pancreatic cancer is still relatively limited,research on immunotherapy and targeted therapy for pancreatic cancer no significant progress has been made achieved.Therefore,the exploration and discovery of potential targets for specific diagnosis and treatment of pancreatic cancer are of great significance for the development of new methods of diagnosis and treatment of pancreatic cancer.This study intends to obtain specific monoclonal antibodies against human pancreatic cancer cells through screening,and then carry out research on the discovery of potential targets for pancreatic cancer based on specific or functional antibodies.In this study,human pancreatic cancer cell SW1990 was used as the immunogen,and multiple immune strategies were combined with differential screening to screen antihuman pancreatic cancer cell SW1990 monoclonal antibody.After immune screening,a total of 9 monoclonal antibodies specifically binding to human pancreatic cancer cell SW1990 were obtained in this study,of which 5 monoclonal antibodies were of IgG1 subtype and 4 were of IgM subtype.Using flow cytometry and immunospot method,this study evaluated the reactivity and specificity of 5 monoclonal antibodies against IgG subtypes.In order to evaluate whether the obtained monoclonal antibodies of the five IgG subtypes have anti-tumor activity,this study applied in vivo and in vitro models to verify the function of the monoclonal antibodies.In the in vitro cell model,the analysis results of the CCK8 experiment showed that SW1990 cells showed no significant changes in cell activity after treatment with 5 antibody strains for 6 h,12 h,24 h,and 48 h,However,in the clone formation experiment,the antibodies 2D8 and 7C3 showed significant ability to inhibit the colony formation of SW1990 cells,and the ability of the above antibodies to inhibit colony formation of SW1990 cells was dose-dependent.In this study,the Transwell and wound healing assay were used to investigate whether the antibodies 2D8 and 7C3 have the function of inhibiting the migration of SW1990 cells in vitro.Transwell analysis results show that both 2D8 and 7C3 antibodies have the ability to inhibit the migration of SW1990 cells in vitro.The Transwell assay results showed that both monoclonal antibodies had the ability to inhibit the migration of SW1990 cells in vitro.The results of the wound healing assay also confirmed this conclusion,while the antibody 2D8 and 7C3 inhibited the migration of SW1990 cells in vitro in a dose-dependent manner.In addition,this study further evaluated the effects of antibodies 2D8 and 7C3 on the clonal formation and migration inhibition of human pancreatic cancer cell line HPAF-Ⅱ and human colorectal cancer cell line HT29 by clone formation and wound healing assay in vitro.The results show that both antibodies 2D8 and 7C3 can inhibit the colony formation of pancreatic cancer cell HPAF-Ⅱ and colorectal cancer cell HT29.The results of the wound healing assay also show that for colorectal cancer cell HT29,antibodies 2D8 and 7C3 can inhibit cell migration,but for pancreatic cancer cell HPAF-Ⅱ,only antibody 7C3 can significantly inhibit cell migration in vitro,antibody 2D8 There is no significant difference between the effect and the control.In the in vivo model evaluation,a nude mouse model ectopically inoculated with SW1990 cells was selected for evaluation in this study.The experimental results show that both antibodies 2D8 and 7C3 have the effect of inhibiting tumor growth.This study further carried out preliminary studies on the potential targets of antibodies 2D8 and 7C3.In this study,Western blot,immunofluorescence,and immunoprecipitation combined with mass spectrometry were used to analyze the results.The results showed that both antibodies 2D8 and 7C3 could bind to cell membrane proteins,and antibodies 2D8 and 7C3 might bind to the same target.The experimental results show that both antibodies 2D8 and 7C3 can bind to cell membrane proteins,and antibody 2D8 And 7C3 may bind the same target.However,Western blot and immunofluorescence results showed that antibodies 2D8 and 7C3 did not react with the three suspicious targets CD 166,CD6 and CDCP1.The targets of antibodies 2D8 and 7C3 need to be further analyzed.In summary,this study screened and obtained 9 specific monoclonal antibodies against human pancreatic cancer cell SW1990,of which antibodies 2D8 and 7C3 showed significant antitumor activity in both in vivo and in vitro models.In the preliminary exploration of the targets of antibodies 2D8 and 7C3,this study found that antibodies 2D8 and 7C3 can bind to cell membrane proteins,but the targets of antibodies 2D8 and 7C3 still need to be confirmed.The work of this study can provide support for the study of therapeutic antibody drugs for pancreatic cancer,and provide a basis for further research on finding tumor targets based on functional antibodies.