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[Human monoclonal antibody inhibiting reverse transcriptase activity of hepatitis B virus polymerase protein]. 2007

Sung Jae Park, and Sang Yong Seol, and Sam Ryong Jee, and Eun Taik Park, and Youn Jae Lee, and Sang Hyuk Lee, and Jung Myung Chung, and Hyun Dae Cho, and Young-Ju Jeong, and In Hak Choi, and Sae Gwang Park
Department of Internal Medicine, Inje University College of Medicine, Busanjin-Gu, Busan, Korea.

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.

UI MeSH Term Description Entries
D011993 Recombinant Fusion Proteins Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes. Fusion Proteins, Recombinant,Recombinant Chimeric Protein,Recombinant Fusion Protein,Recombinant Hybrid Protein,Chimeric Proteins, Recombinant,Hybrid Proteins, Recombinant,Recombinant Chimeric Proteins,Recombinant Hybrid Proteins,Chimeric Protein, Recombinant,Fusion Protein, Recombinant,Hybrid Protein, Recombinant,Protein, Recombinant Chimeric,Protein, Recombinant Fusion,Protein, Recombinant Hybrid,Proteins, Recombinant Chimeric,Proteins, Recombinant Fusion,Proteins, Recombinant Hybrid
D005822 Genetic Vectors DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition. Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D006515 Hepatitis B virus The type species of the genus ORTHOHEPADNAVIRUS which causes human HEPATITIS B and is also apparently a causal agent in human HEPATOCELLULAR CARCINOMA. The Dane particle is an intact hepatitis virion, named after its discoverer. Non-infectious spherical and tubular particles are also seen in the serum. Dane Particle,Hepatitis Virus, Homologous Serum,B virus, Hepatitis,Hepatitis B viruses,Particle, Dane,viruses, Hepatitis B
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000911 Antibodies, Monoclonal Antibodies produced by a single clone of cells. Monoclonal Antibodies,Monoclonal Antibody,Antibody, Monoclonal
D012194 RNA-Directed DNA Polymerase An enzyme that synthesizes DNA on an RNA template. It is encoded by the pol gene of retroviruses and by certain retrovirus-like elements. EC 2.7.7.49. DNA Polymerase, RNA-Directed,RNA-Dependent DNA Polymerase,Reverse Transcriptase,RNA Transcriptase,Revertase,DNA Polymerase, RNA Directed,DNA Polymerase, RNA-Dependent,RNA Dependent DNA Polymerase,RNA Directed DNA Polymerase
D015684 Gene Products, pol Retroviral proteins coded by the pol gene. They are usually synthesized as a protein precursor (POLYPROTEINS) and later cleaved into final products that include reverse transcriptase, endonuclease/integrase, and viral protease. Sometimes they are synthesized as a gag-pol fusion protein (FUSION PROTEINS, GAG-POL). Pol is short for polymerase, the enzyme class of reverse transcriptase. pol Gene Products,pol Polyproteins,pol Protein,Gene Product, pol,pol Polyprotein,Polyprotein, pol,Polyproteins, pol,Protein, pol,pol Gene Product
D018894 Reverse Transcriptase Inhibitors Inhibitors of reverse transcriptase (RNA-DIRECTED DNA POLYMERASE), an enzyme that synthesizes DNA on an RNA template. Reverse Transcriptase Inhibitor,Inhibitors, Reverse Transcriptase,Inhibitor, Reverse Transcriptase,Transcriptase Inhibitor, Reverse
D019151 Peptide Library A collection of cloned peptides, or chemically synthesized peptides, frequently consisting of all possible combinations of amino acids making up an n-amino acid peptide. Phage Display Peptide Library,Random Peptide Library,Peptide Phage Display Library,Phage Display Library, Peptide,Synthetic Peptide Combinatorial Library,Synthetic Peptide Library,Libraries, Peptide,Libraries, Random Peptide,Libraries, Synthetic Peptide,Library, Peptide,Library, Random Peptide,Library, Synthetic Peptide,Peptide Libraries,Peptide Libraries, Random,Peptide Libraries, Synthetic,Peptide Library, Random,Peptide Library, Synthetic,Random Peptide Libraries,Synthetic Peptide Libraries
D022801 Complementarity Determining Regions Three regions (CDR1; CDR2 and CDR3) of amino acid sequence in the IMMUNOGLOBULIN VARIABLE REGION that are highly divergent. Together the CDRs from the light and heavy immunoglobulin chains form a surface that is complementary to the antigen. These regions are also present in other members of the immunoglobulin superfamily, for example, T-cell receptors (RECEPTORS, ANTIGEN, T-CELL). Complementarity Determining Region,Complementarity Determining Region 1,Complementarity Determining Region 2,Complementarity Determining Region 3,Complementarity Determining Region I,Complementarity Determining Region II,Complementarity Determining Region III,Complementarity-Determining Region,Complementarity-Determining Region 3,Hypervariable Region, Immunoglobulin,Hypervariable Regions, Immunoglobulin,Third Complementarity-Determining Region,Complementarity-Determining Region 3s,Complementarity-Determining Region, Third,Complementarity-Determining Regions,Complementarity-Determining Regions, Third,Immunoglobulin Hypervariable Region,Immunoglobulin Hypervariable Regions,Region, Complementarity Determining,Region, Immunoglobulin Hypervariable,Regions, Complementarity Determining,Regions, Complementarity-Determining,Regions, Immunoglobulin Hypervariable,Third Complementarity Determining Region,Third Complementarity-Determining Regions

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