Generation and characterization of a novel recombinant antibody against LMP1-TES1 of Epstein-Barr virus isolated by phage display

Abstract

Latent Membrane Protein 1 (LMP1) is a primary target for controlling tumorigenesis in Epstein-Barr virus related malignancies; in this study, we aimed to develop a specific antibody against the TES1 domain of the oncogenic LMP1. We screened a full human naïve Fab phage library against TES1 peptide, which consisted of C terminal-activating regions proximal 44 amino acids. After three rounds of panning, enrichment and testing by phage ELISA and further analyzed by DNA sequencing, we selected a phage clone with the highest affinity to LMP1-TES1 and designated it as htesFab. The positive clone was expressed in Escherichia coli and the purified htesFab was characterized for its binding specificity and affinity to LMP1. ELISA, immunofluorescence and FACS analysis confirmed that htesFab could recognize LMP1 TES1 both in vitro and in LMP1 expressing HNE2-LMP1 cells. Furthermore, MTT assay showed that htesFab inhibited the proliferation of HNE2-LMP1 cells in a dose-dependent manner. In summary, this study reported the isolation and characterization of human Fab, which specifically targets the C terminal region/TES1 of LMP1, and has potential to be developed as novel tool for the diagnosis and therapy of Epstein-Barr virus related carcinoma.

Figure 1

ELISA results of 40 individual phage clones randomly picked up from the eluted phage pool after the 3rd round of bio-panning. Purified human pLMP1-TES1 was coated at 400 ng per well, and 50 μL supernatant of each phage was added to each well for ELISA.

Figure 2

Western blotting characterization of htesFab fragment expressed in E. coli. Lane 1: Top10F' cell lysate control; lane 2: 25 mL of cell lysates after sonication. Mouse antihuman Fab HRP conjugate was used at 1:1,000 dilution (A). Purified htesFab fragment was separated on a 10% SDS-PAGE gel and stained with Coomassie blue. The double bands were heavy chain Fd (top) and light chain k (bottom) (B).

Figure 3

Characterization of Fab binding with LMP1-TES1 domain. (A) ELISA showed that htesFab bound LMP1-TES domain in native confirmation (p < 0.05); (B) Immunoprecipitation analysis for the detection of LMP1 protein. LMP1 was 53 kDa; Line 1: HNE2 cells; line 2: HNE2-LMP1 cells; line 3: unrelated Fab fragment (C); Immunofluorescence analysis showed that htesFab labeled LMP1 in the intracellular and plasma membranes in HNE2-LMP1 cells (green), cell nuclei were stained with DAPI (blue) (×200, Olympus digital camera); (D) FACS analysis showed the binding of htesFab to HNE2-LMP1(40.35%) and HNE2 cells(4.15%) (blue dots). Background staining was obtained by PBS.

Figure 4

htesFab inhibits the proliferation of HNE2-LMP1 cells in vitro. The cells were treated as indicated and harvested 48 h later. The proliferation of cells was assessed by MTT assay to calculate the proliferation inhibition rate (%).