Screening and efficacy evaluation of anti-TMD5 monoclonal antibodies against Epstein-Barr virus-positive diffuse large B-cell lymphoma

Objective:To screen the anti-transmembrane domain 5 (TMD5) monoclonal antibodies of Epstein-Barr virus (EBV) latent membrane protein-1 and evaluate their inhibitory effect on monoclonal antibody against EBV positive diffuse large B-cell lymphoma(DLBCL).Methods:Synthesized TMD5 peptide fragment was used to immunize BALB/c mice.PEG method was used to prepare hybridomas.ELISA was used to screen the positive hybridoma cells.A limited dilution method was used to prepare monoclonal cells.Ascites was prepared by immunizing mice with positive monoclonal cells.Western blotting and ELISA were used to identify and titer determine the monoclonal antibodies in ascites, respectively.Cell counting kit-8 (CCK-8) method was used to measure the effect of ascites on the viability of myeloma Sp2/0 cells in vitro.Annexin V-FITC/PI double-stained flow cytometry was used to determine the effect of ascites on the apoptosis rate of Sp2/0 cells.The effect of ascites on the tumor growth in nude mice was determined in vivo.Results:Thirteen hybridomas were successfully formed, of which 3 were positive, namely 2B11, 5D07, and 6F04, with a positive rate of 23%.When the ascites prepared by 5D07 monoclonal cells were diluted at 1∶10⁵, the titer ratio of positive/negative monoclonal antibody against TMD5 was 6.763, and the titer ratio of positive/Sp2/0 was 5.952, which had the highest titer.Compared with the PBS group, the proliferative ability of DB cells at 48 h and 72 h in 2B11 and 5D07 groups decreased, the apoptosis rate increased, and the nude mice tumors were smaller (P< 0.05), while there was no significant difference was found in the parameters mentioned above in 6F04 group (P> 0.05).Compared with the 2B11 group, the proliferative ability of DB cells at 48 h and 72 h in the 5D07 group decreased, and the apoptosis rate increased, while the nude mice tumors were smaller(P< 0.05).Conclusion:The 5D07 monoclonal antibody screened by the hybridoma method has a high titer against TMD5 and has a good activity against EBV+DLBCL lymphoma in vitro and in vivo.