Establishment And Characterization Of Monoclonal Fab Antibodies Specific For Epstein-Barr Virus Latent Membrane Protein 1

The Epstein-Barr virus(EBV)is related to multiple malignancies,including B Hodgkin lymphoma and nasopharyngeal carcinoma.Latent membrane protein 1(LMP1)is a membrane protein encoded by EBV.It critically contributes to pathogenesis and disease phenotypes,and is an ideal target in development for cancer immunotherapy.The peptides containing extramembrane part(96-107 aa)of LMP1 was sythesised and immunized mice for 3 rounds.The serum obtained after three immunizations showed high titer in ELISA assay.However,the binding capacity towards full length and natural LMP1 protein was further evaluated by Western blot and flow cytometry on EBV-BJAB,EBV+ B95-8 and overexpressed 293 T cell lines.The data showed that the serum had no specific recognition to full length and natural LMP1 protein.Therefore,the attempt to generate specific monoclonal antibody from mice origin failed.Subsequently,we constructed a eukaryotic expressing vector pSegTag-hFc containing DNA fragment encoded LMP1 gene extracellular domain segments.LMP1-Fc protein was expressed in 293 T cells,purified and labeled with biotin as bait for large phage-display na?ve human antigen-binding fragment(Fab)library panning.The library was panned with reduced antigen concentration for four rounds and polyclonal ELISA data confirmed that the Fab clones against LMP1 extracellular domain was effectively enriched.384 monoclones were randomly selected to perform monoclone ELISA.ELISA data showed that 14 human Fab clones could bind to the extracellular segment of LMP1 2 folds higher than Fc antigen.Sequencing and translation of the 14 clones into peptide sequences showed that all of them were valid Fab clones,and two of them were identical with same VH and VL insertions.Antigen dilution binding assay showed that the binding of these Fab clones to LMP1-Fc varied linearly with the concentration,and clones 10-B2 and 15-H10 had strong antigen binding ability.Western blot results confirmed that except 1-A11 was a binding Fab clone at Fc end,all the other clones were LMP1 binding clones.Western Blot results showed that endogenous LMP1 overexpressed in 293 T or B95-8 cells could be effectively detected by 6-C6,7-G9,10-B2 and 15-H10.Flow cytometry showed that clones 1A11,6-C6,7-G9,10-B2 and 15-H10 could stain EBV+ cell line B95-8.Clones 10-B2 and 15-H10 could stain EBV+ cell line LCL,while control EBV-cell line BJAB had no specific staining.Immunofluorescence staining and confocal microscopy showed that clone 15-H10 could specifically stain B95-8.In conclusion,LPM1-specific Fab clones 6-C6,7-G9,10-B2 and 15-H10 have been isolated in this study.These clones may provide materials in the diagnosis of EB virus and adoptive immunotherapy targeting EB virus-related tumors.