[Human monoclonal antibody inhibiting reverse transcriptase activity of hepatitis B virus polymerase protein].

Abstract

BACKGROUND/AIMS:

To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy.

METHODS:

Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1 x 10(10), human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing.

RESULTS:

pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1 micro g of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15 x 10(-8) M to 1.75 x 10(-6) M.

CONCLUSIONS:

Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.

• Authors: Sung Jae Park, Sang Yong Seol, Sam Ryong Jee, Eun Taik Park, Youn Jae Lee, Sang Hyuk Lee, Jung Myung Chung, Hyun Dae Cho, Young-Ju Jeong, In Hak Choi, Sae Gwang Park
• Journal: The Korean journal of gastroenterology = Taehan Sohwagi Hakhoe chi (Korean J Gastroenterol) Vol. 49 Issue 2 Pg. 85-92 (Feb 2007) ISSN: 1598-9992 [Print] Korea (South)
• PMID: 17322787 (Publication Type: English Abstract, Journal Article)

Chemical References

• Antibodies, Monoclonal
• Complementarity Determining Regions
• Gene Products, pol
• P protein, Hepatitis B virus
• Peptide Library
• Recombinant Fusion Proteins
• Reverse Transcriptase Inhibitors
• RNA-Directed DNA Polymerase

Topics

• Antibodies, Monoclonal (biosynthesis, genetics, pharmacology)
• Complementarity Determining Regions (chemistry)
• Gene Products, pol (antagonists & inhibitors, genetics, immunology)
• Genetic Vectors
• Hepatitis B virus (enzymology, genetics)
• Humans
• Peptide Library
• RNA-Directed DNA Polymerase (genetics, immunology)
• Recombinant Fusion Proteins (biosynthesis, genetics)
• Reverse Transcriptase Inhibitors (chemistry, metabolism, pharmacology)