Korean J Gastroenterol.  2007 Feb;49(2):85-92.

Human Monoclonal Antibody Inhibiting Reverse Transcriptase Activity of Hepatitis B Virus Polymerase Protein

• Park SJ
• Seol SY
• Jee SR
• Park ET
• Lee YJ
• Lee SH
• Chung JM
• Cho HD
• Jeong YJ
• Choi IH
• Park SG

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 1.1x10(10) size, 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, 1microgram 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 8.15x10(-8) M to 1.75x10(-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.

Keyword

Hepatitis B virus; Reverse transcriptase; Phage display; Human monoclonal antibody

MeSH Terms

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