title: [Human monoclonal antibody inhibiting reverse transcriptase activity of hepatitis B virus polymerase protein].
abstract: OBJECTIVE
  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.affiliation: Department of Internal Medicine, Inje University College of Medicine, Busanjin-Gu, Busan, Korea.
authors.forename: Sung Jae
authors.initials: SJ
authors.lastname: Park
authors.forename: Sang Yong
authors.initials: SY
authors.lastname: Seol
authors.forename: Sam Ryong
authors.initials: SR
authors.lastname: Jee
authors.forename: Eun Taik
authors.initials: ET
authors.lastname: Park
authors.forename: Youn Jae
authors.initials: YJ
authors.lastname: Lee
authors.forename: Sang Hyuk
authors.initials: SH
authors.lastname: Lee
authors.forename: Jung Myung
authors.initials: JM
authors.lastname: Chung
authors.forename: Hyun Dae
authors.initials: HD
authors.lastname: Cho
authors.forename: Young-Ju
authors.initials: YJ
authors.lastname: Jeong
authors.forename: In Hak
authors.initials: IH
authors.lastname: Choi
authors.forename: Sae Gwang
authors.initials: SG
authors.lastname: Park
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D022801:Complementarity Determining Regions / Q000737:chemistry; D015684:Gene Products, pol / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology; D005822:Genetic Vectors; D006515:Hepatitis B virus / Q000201:enzymology / Q000235:genetics; D006801:Humans; D019151:Peptide Library; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D015684:Gene Products, pol; C066027:P protein, Hepatitis B virus; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D018894:Reverse Transcriptase Inhibitors; D012194:RNA-Directed DNA Polymerase