[Human monoclonal antibody inhibiting reverse transcriptase activity of hepatitis B virus polymerase protein].
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.
Park
Sung Jae
SJ
Department of Internal Medicine, Inje University College of Medicine, Busanjin-Gu, Busan, Korea.
Seol
Sang Yong
SY
Jee
Sam Ryong
SR
Park
Eun Taik
ET
Lee
Youn Jae
YJ
Lee
Sang Hyuk
SH
Chung
Jung Myung
JM
Cho
Hyun Dae
HD
Jeong
Young-Ju
YJ
Choi
In Hak
IH
Park
Sae Gwang
SG
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*
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