WO2017018739A1 - Pharmaceutical composition for preventing cccdna formation of hepatitis b virus - Google Patents

Pharmaceutical composition for preventing cccdna formation of hepatitis b virus Download PDF

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WO2017018739A1
WO2017018739A1 PCT/KR2016/008039 KR2016008039W WO2017018739A1 WO 2017018739 A1 WO2017018739 A1 WO 2017018739A1 KR 2016008039 W KR2016008039 W KR 2016008039W WO 2017018739 A1 WO2017018739 A1 WO 2017018739A1
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hepatitis
virus
hbv
antibody
surface antigen
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Ki-Hwan Chang
Chun-Kyu KO
Wang-Shick Ryu
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Mogam Institute For Biomedical Research
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Priority to MYPI2018000085A priority patent/MY184876A/en
Priority to EA201890362A priority patent/EA201890362A1/en
Priority to BR112018001431A priority patent/BR112018001431A2/en
Publication of WO2017018739A1 publication Critical patent/WO2017018739A1/en

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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • G01N2500/00—Screening for compounds of potential therapeutic value
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Abstract

The present invention is based on a finding that a hepatitis B virus antibody inhibits binding of a surface antigen (HBsAg) of hepatitis B virus to heparan sulfate proteoglycan, thereby preventing cccDNA formation of the hepatitis B virus. It is expected that utilizing the pharmaceutical composition and the method of preventing cccDNA formation of hepatitis B virus of the present invention can fundamentally treat chronic hepatitis B and also prevent a recurrence of hepatitis in a hepatitis B patient after liver transplant surgery. Further, according to the present invention, a material for preventing or treating hepatitis B can be newly identified by confirming whether cccDNA formation is inhibited or not. In addition, according to the present invention, a treatment supplement capable of being administered in combination with the hepatitis B virus antibody can be newly identified.

Description

PHARMACEUTICAL COMPOSITION FOR PREVENTING cccDNA FORMATION OF HEPATITIS B VIRUS
The present invention relates to a pharmaceutical composition for preventing cccDNA formation of hepatitis B virus.
Hepatitis B virus (HBV) is a virus having DNA genome belonging to Hepadnaviridae family, and induces acute and chronic hepatitis. About 350 millions of the world populations are chronic HBV positive persons, and especially, Korea and China have about 5 to 8% of chronically infected persons. Such chronic HBV infection may induce hepatitis, hepatic cirrhosis and even liver cancer. The chronically infected persons show about 300-fold of higher incidence of liver cancer than the normal persons. According to a WHO research, 80% of the liver cancer is caused by chronic HBV.
Hepatitis B virus has been partially prevented due to a development of vaccines. However, once HBV invaded liver cells, there is no method to completely remove the HBV entered into the liver cells of a patient. Therefore, it is very difficult to completely cure hepatitis B at the present time. Further, when hepatitis B patients have a liver transplant, they show a high recurrence rate of hepatitis B. The reason is that HBV, which is not completely removed but remaining in the cells, invades the transplanted health liver cells.
If the HBV invades the liver cells, covalently closed circular (ccc) DNA is formed therein. Then, the formed cccDNA is used as a template for HBV genome DNA replication and continuously generates HBV until a lifespan of the corresponding liver cells is ended. Any treatment method currently used may inhibit HBV genome DNA replication to thus inhibit HBV formation in blood, however, cannot fundamentally block the replication of HBV.
Accordingly, there is still a strong requirement for developing a pharmaceutical composition for preventing cccDNA formation, which can fundamentally block the replication of HBV to enable principal treatment of chronic hepatitis B, and prevent a recurrence of hepatitis after liver transplant surgery to a hepatitis B patient, as well as a treatment method using the same.
[Prior Art Document]
[Patent Document]
Patent Document 1 Korean Patent Registration No. 0467706 (2005. 1. 13)
Patent Document 2 Korean Patent Registration No. 1072895 (2011. 10. 06.)
An object of the present invention is to provide a pharmaceutical composition for preventing cccDNA formation of hepatitis B virus.
Another object of the present invention is to provide a novel method of treating chronic hepatitis B using the pharmaceutical composition described above.
Further, another object of the present invention is to provide a method of preventing a recurrence of hepatitis in a hepatitis B patient after liver transplant surgery.
The above objects of the present invention will be achieved by the following characteristics:
(1) A pharmaceutical composition for preventing cccDNA formation of hepatitis B virus, including a hepatitis B virus antibody.
(2) The composition according to the above (1), wherein the hepatitis B virus antibody is adapted to inhibit binding of a surface antigen (HBsAg) of the hepatitis B virus to heparan sulfate proteoglycan.
(3) The composition according to the above (1), wherein the hepatitis B virus antibody includes: an amino acid sequence of SEQ ID NO: 1 encoding a human antibody H chain variable region to the surface antigen of the hepatitis B virus; and an amino acid sequence of SEQ ID NO: 2 encoding a human antibody L chain variable region to the surface antigen of the hepatitis B virus.
(4) The composition according to the above (1), wherein the hepatitis B virus antibody includes: an amino acid sequence of SEQ ID NO: 3 encoding a human antibody H chain variable region to the surface antigen of the hepatitis B virus; and any one of amino acid sequences of SEQ ID NOs: 4 to 6 encoding a human antibody L chain variable region to the surface antigen of the hepatitis B virus.
(5) The composition according to the above (1), wherein the composition is administered within 24 hours from a time of the hepatitis B virus infection.
(6) A method of preventing cccDNA formation of hepatitis B virus, including administrating a prophylactically- or therapeutically-effective amount of a hepatitis B virus antibody.
(7) The method according to the above (6), wherein the hepatitis B virus antibody is adapted to inhibit binding of a surface antigen (HBsAg) of the hepatitis B virus to heparan sulfate proteoglycan.
(8) The method according to the above (6), wherein the hepatitis B virus antibody includes: an amino acid sequence of SEQ ID NO: 1 encoding a human antibody H chain variable region to the surface antigen of the hepatitis B virus; and an amino acid sequence of SEQ ID NO: 2 encoding a human antibody L chain variable region to the surface antigen of the hepatitis B virus.
(9) The method according to the above (6), wherein the hepatitis B virus antibody includes: an amino acid sequence of SEQ ID NO: 3 encoding a human antibody H chain variable region to the surface antigen of the hepatitis B virus; and any one of amino acid sequences of SEQ ID NOs: 4 to 6 encoding a human antibody L chain variable region to the surface antigen of the hepatitis B virus.
(10) The method according to the above (6), wherein the composition is administered within 24 hours from a time of the hepatitis B virus infection.
(11) A method of treating chronic hepatitis, including administrating the pharmaceutical composition according to the above (1) to block genome DNA replication of hepatitis B virus.
(12) The method according to the above (11), wherein the chronic hepatitis is chronic hepatitis B.
(13) The method according to the above (11), wherein the hepatitis B virus antibody includes: an amino acid sequence of SEQ ID NO: 1 encoding a human antibody H chain variable region to the surface antigen of the hepatitis B virus; and an amino acid sequence of SEQ ID NO: 2 encoding a human antibody L chain variable region to the surface antigen of the hepatitis B virus.
(14) The method according to the above (11), wherein the hepatitis B virus antibody includes: an amino acid sequence of SEQ ID NO: 3 encoding a human antibody H chain variable region to the surface antigen of the hepatitis B virus; and any one of amino acid sequences of SEQ ID NOs: 4 to 6 encoding a human antibody L chain variable region to the surface antigen of the hepatitis B virus.
(15) A method of screening a material for preventing or treating hepatitis B, including: inoculating hepatitis B virus to liver cells; and after treating the liver cells with a subject material, confirming whether cccDNA formation is inhibited or not.
(16) The method according to the above (15), wherein the liver cells are HepG2-NTCP.
(17) The method according to the above (15), wherein the subject material is treated within 24 hours after the inoculation of hepatitis B virus.
(18) The method according to the above (15), wherein the confirmation of whether cccDNA formation is inhibited or not is performed by including: (a) extracting episome DNA from the liver cells after 2 to 10 days from a treatment time using the subject material; and (b) measuring an amount of cccDNA included in the episome DNA.
(19) A method of screening a hepatitis B treatment supplement, including: (a) contacting a hepatitis B virus antibody and a subject material to a biological sample including hepatitis B virus; and (b) confirming whether cccDNA formation is inhibited or not, and if the cccDNA formation is more reduced than a case in which the subject material does not contact to the sample, determining the subject material as a treatment supplement which is administered in combination with the hepatitis B virus.
According to the present invention, if the cccDNA formation of hepatitis B virus (HBV) is prevented, the HBV genome DNA replication does not proceed and a reproduction of the HBV is impossible, therefore, further propagation of the HBV into un-infected liver cells does not occur.
Meanwhile, in a case of the liver cell having cccDNA already formed therein, when a lifespan (about 5 months) of the liver cell is ended to reach cell death, cccDNA is also degraded and disappear. Accordingly, the pharmaceutical composition of the present invention may completely remove the HBV from the liver cells of a patient to make it possible to fundamentally and completely cure the chronic hepatitis B. Further, a recurrence of hepatitis in a hepatitis B patient after liver transplant surgery may also be fundamentally blocked.
The pharmaceutical composition of the present invention may be efficiently used for prevention of hepatic cirrhosis and liver cancer caused by chronic hepatitis B.
According to the present invention, the material for preventing or treating hepatitis B may be newly identified by confirming whether cccDNA formation is inhibited or not.
In addition, according to the present invention, the treatment supplement capable of being administered in combination with the HBV antibody may be newly identified.
FIG. 1 is diagrams illustrating results of assessment of HBV infection inhibitory efficacy by GC1102 through Southern blot analysis and HBsAg ELISA.
FIG. 2 is photographs illustrating results of assessment of HBV infection inhibitory efficacy by GC1102 according to immune-fluorescent technique.
FIG. 3 is a diagram illustrating results of assessment of HBV cccDNA formation inhibitory efficacy by GC1102.
FIG. 4 is diagrams illustrating analysis results of dose-response for HBV infection inhibition by GC1102.
FIG. 5 is a graph illustrating results of assessment of EC50 (effective concentration) by GC1102.
FIG. 6 is diagrams illustrating results of assessment of HBV infection inhibitory efficacy according to a time of treatment of GC1102 (anti-HBsAg).
The present invention is based on a finding that using a hepatitis B virus antibody may inhibit binding of Hepatitis B surface antigen (HBsAg) to heparan sulfate proteoglycan of a liver cell; block invasion of the liver cell by HBV; and prevent cccDNA formation of HBV.
Hereinafter, the present invention will be described in more detail.
One embodiment of the present invention provides a pharmaceutical composition for preventing cccDNA formation of HBV, which includes a HBV antibody.
Hepatitis B virus (HBV) is a virus belonging to Hepadnaviridae family, and invades into liver cells to induce acute and chronic hepatitis.
The HBV has a life history including: an invasion process of sticking to a surface of the liver cell and entering into the cell; a proliferation process of peeling off the skin of a cytoplasm, penetrating into a cell nucleus then replicating DNA; an assembly process of stacking a skin in the cytoplasm to recombine the same in a complete virus form; an emission process of escaping the liver cell; and a process of sticking to another liver cell and repeating the same proliferation processes as described above.
The HBV has a partially double-stranded DNA genome, and HBV genome DNA has a gap and protein (oval shape) or RNA (wavy line) linked by a covalent bond at both ends of the DNA. Since both ends thereof are not linked with no torsional stress, the above DNA is also referred to as relaxed circular DNA (RC DNA).
In order to proliferate HBV in the liver cell, DNA genome of HBV (RC DNA) should be injected into a cell nucleus of the liver cell, and then cccDNA (that is, covalently closed circular DNA) should be created from the same. Once cccDNA is created, the HBV may continuously proliferate until a lifespan of the cell is ended.
For cccDNA formation, first of all, invasion of liver cell by the HBV must precede.
An envelope of HBV includes the HBsAg and, in order for HBV to invade a liver cell, it is necessary for the HBsAg to be bound to heparan sulfate proteoglycan at the surface of the liver cell for allowing the HBV to be attached to the surface of the liver cell. When the HBV is attached to the surface of the liver cell, Pre-S1 domain in HBsAg is bound to a receptor existing on the surface of the liver cell, that is, sodium taurocholate contransporting polypeptide (NTCP) to allow the HBV to be entered into the liver cell.
A hepatitis B virus antibody (HBV antibody) may be a natural or recombinant antibody, and preferably, an antibody (HBIG-Gene) to a surface antigen of HBV produced from cell line BHAb-49 (KCLRFBP-00054).
The HBV antibody of the present invention may be a recombinant antibody which includes: an amino acid sequence of SEQ ID NO: 1 encoding a human antibody H chain variable region to the surface antigen of HBV; and an amino acid sequence of SEQ ID NO: 2 encoding a human antibody L chain variable region to the surface antigen of HBV.
Further, the HBV antibody of the present invention may be a recombinant antibody which includes: an amino acid sequence of SEQ ID NO: 3 encoding a human antibody H chain variable region to the surface antigen of HBV; and any one of amino acid sequences of SEQ ID NOs: 4 to 6 encoding a human antibody L chain variable region to the surface antigen of HBV.
The HBV antibody may prevent cccDNA formation of HBV. The HBV antibody may inhibit binding of the surface antigen (HBsAg) of the HBV to heparan sulfate proteoglycan. When the binding of HBsAg to heparan sulfate proteoglycan is inhibited, invasion of liver cells by the HBV is blocked, thus preventing the cccDNA formation.
The prevention of cccDNA formation of HBV is one of treatment methods directly acting the life history of the virus. This is distinguished from immunotherapy such as interferon treatment, cytokine treatment, cell immunity recovery, etc.
Further, the prevention of cccDNA formation of HBV is different from an extinction method of hepatitis B virus cccDNA (HBV cccDNA). The extinction method of HBV cccDNA has a problem of requiring a development of a suitable carrier in order to apply the same to clinical trials.
If the cccDNA formation of HBV is prevented, HBV genome DNA is not replicated and the HBV reproduction may become impossible, therefore, further propagation of HBV into un-infected liver cells does not occur. For the liver cells having cccDNA already formed therein, when a lifespan (about 5 months) of the liver cell is ended to reach cell death, cccDNA is also degraded and disappear.
Therefore, the pharmaceutical composition of the present invention may enable more comprehensive and fundamental treatment of hepatitis B. Further, the inventive composition may be more suitable for treating chronic hepatitis B requiring a long term treatment.
A period for administration of the pharmaceutical composition of the present invention is not particularly determined, however, in order to maximize the medical effects for preventing cccDNA formation of HBV, it is preferable that the composition is administered as soon as possible from a time of HBV infection. Within 72 hours, 48 hours or 24 hours from the time of HBV infection, the inventive composition may be administered.
According to any conventional method, the inventive composition may be prepared into a formulation. In preparing the formulations, HBV antibody may be mixed or diluted together with a carrier, or sealed into a carrier having a container form. When using the carrier as a diluent, the formulation may be a solid, semi-solid or liquid material which acts as a carrier, excipient or medium to the antibody.
The formulation may be in any form of: tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions and sterile powders, or the like.
Examples of suitable carriers, excipients and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolie, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.
The formation may further include fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The inventive composition may be formulated using methods well known in the related art to provide fast, sustained and delayed release of antibody after administering the composition to a mammal.
The pharmaceutical composition of the present invention may further include anti-HBV monoclonal antibody, anti-HBV polyclonal antibody, nucleoside analogue, DNA polymerase inhibitor, siRNA formulation or therapeutic vaccine as an antiviral agent.
In addition, one embodiment of the present invention provides a method of preventing cccDNA formation of HBV antibody, which includes administrating a prophylactically- or therapeutically-effective amount of a hepatitis B virus antibody.
The method of preventing cccDNA formation of HBV of the present invention may include administrating a prophylactically- or therapeutically-effective amount of the pharmaceutical composition described above. The above-mentioned HBV antibody may also be used in the method of preventing cccDNA formation of HBV.
Herein, the effective amount of HBV antibody means an amount sufficient to express medical effects for preventing cccDNA formation of HBV. For example, the HBV antibody may be included in any suitable formulation in a concentration of 0.1 to 50 mg/ml, preferably, 2 to 10 mg/ml, and more preferably, 3 to 7 mg/ml.
In order to maximize the medical effects for preventing cccDNA formation of HBV, it is preferable that the HBV antibody is administered as soon as possible from the time of HBV infection. For example, the HBV antibody may be administered within 72 hours, 48 hours or 24 hours from the time of HBV infection.
One embodiment of the present invention provides a method of treating hepatitis B, especially chronic hepatitis B, which includes administering the pharmaceutical composition described above to block genome DNA replication of HBV.
Further, the present invention provides a method of screening a material for preventing or treating hepatitis B, which includes: inoculating HBV to liver cells; and after treating the liver cells with a subject material, confirming whether cccDNA formation is inhibited or not.
According to one embodiment, the liver cell may be HepG2-NTCP.
The subject material may be used for treating within 24 hours, more preferably, within 12 hours and most preferably, within 6 hours after inoculating HBV.
The subject material may include a material for inhibiting invasion of HBV into the liver cell, or may include a material for inhibiting the binding of a surface antigen (HBsAg) of the HBV to heparan sulfate proteoglycan or sodium taurocholate cotransporting polypeptide (NTCP).
In addition, for example, the confirmation of whether cccDNA formation is inhibited or not may be performed by extracting episome DNA from liver cells after 2 to 10 days from a treatment point using the subject material, then measuring an amount of cccDNA included in the episome DNA.
The above processes of extracting episome DNA from the liver cells and measuring the amount of cccDNA included in the episome DNA may be executed by a variety of conventional methods used in the related art to which the present invention pertains.
Further, the present invention provides a method of screening a therapeutic supplement capable of being administered in combination with the HBV antibody. According to one embodiment, a biological sample including HBV is treated with HBV antibody and a subject material, followed by confirming whether cccDNA formation is inhibited or not. If the cccDNA formation is more reduced than a case in which the subject material does not contact with the sample, the subject material may be considered as a treatment supplement capable of being administered in combination with the HBV antibody described above. In the above screening method, the HBV antibody refers to an antibody having prevention and treatment effects of HBV, which includes the HBV antibody described above.
Hereinafter, the present invention will be described in more detail through examples. However, those skilled in the art will appreciate that such examples are provided for concretely describing the present invention and do not limit subject matters to be protected as disclosed in the detailed description and appended claims.
Example: Assessment of HBV infection inhibitory efficacy by human HBV antibody ( GC 1102 ) to HBsAg
<Material and method>
1) Anti-HBsAg antibody: The antibody (GC1102) used in the present experiment is a human antibody represented by: an amino acid sequence (HBAb-H4) of SEQ ID NO: 1 encoding a human antibody H chain variable region to a surface antigen of HBV; and an amino acid sequence (HBAb-L9) of SEQ ID NO: 2 encoding a human antibody L chain variable region to a surface antigen of HBV.
2) Control group: Both of a sample treated with IgG as a negative control and a sample treated with Myrcludex B (MyrB) as a positive control in progressing the experiment, were prepared to minimize an occurrence of experimental errors. MyrB used as the positive control is a lipo-peptide consisting of 47 amino acids in HBsAg pre-S1 domain and now is under clinical trials as a HBV entry inhibitor.
3) Cell culture: HepG2-NTCP cell line used in the present experiment is a cell line stably expressing human NTCP and has been established in the papers published by the present laboratory (Ko et al., 2014). Further, HepAD38 cell line is a cell line continuously producing HBV inoculum and has been used for obtaining HBV inoculum required in infection experiments.
4) Southern blot analysis: Southern blot analysis used in the present experiment is an analysis method of viral DNA separated from HBV capsids present in cells, and has been established by the present laboratory (Ko et al., 2014).
5) ELISA: ELISA is another method for determining HBV infection by directly measuring HBsAg or HBeAg secreted into a culture supernatant. More particularly, the supernatants at day 5 to 7 after infection were obtained and investigated by a diagnosis reagent for hepatitis B, "Genedia HBsAg Elisa 3.0." During performing the experiment, a standard curve was drawn using a HBV international standard and then compared the same with the measured values, so as to estimate an absolute amount of HBsAg secreted into the supernatant.
6) Immuno-fluorescence: Immuno-fluorescence has a significant advantage in that HBV infection can be observed directly using a fluorescent microscope without a cell lysis process. The infected cell was fixed with paraformaldehyde then subjected to a permeablization process using triton X-100. Next, it was confirmed with a HBcAg antibody whether there is HBcAg infection or not.
7) HBV infection: HBV infection was conducted in HepG2-NTCP cell line. More particularly, after seeding HepG2-NTCP cell in a culture dish, and after 24 hours, HBV inoculum was used for infecting HepG2-NTCP cell. In 16 to 24 hours after infection, the medium was changed into a new medium containing 2.5% DMSO. The medium was changed every second day, and in 8 to 10 days after infection, whether the infection was realized has been confirmed by utilizing various experimental techniques.
<2> Assessment of HBV infection inhibitory efficacy by GC1102
First, whether GC1102 can inhibit HBV infection or not has been investigated by Southern blot analysis and HBsAg ELISA measurement.
Results of the experiment are shown in FIG. 1. (A) Southern blot analysis: at a time after 9 days from HBV infection of HepG2-NTCP cells, cap seed DNA was measured by Southern blot. Cells were treated with MyrB (1 ㎍/ml), IgG (1 ㎍/ml) or GC1102 (1 ㎍/ml), simultaneously with the HBV infection. 3.2 kb HBV DNA was used as a size marker. RC: relaxed circular DNA; DL: double-stranded linear DNA; (B) HBsAg ELISA: after performing the HBV infection, a concentration of HBsAg present in a culture supernatant cultured for 2 days during 5 to 7 days after infection and 7 to 9 days after infection, respectively, was measured. Using a standard HBsAg product at a known concentration, an absolute amount of HBsAg (IU/ml) present in the culture supernatant was measured.
As a result of the experiment, it was confirmed that GC1102 (1 ㎍/ml) could efficiently inhibit production of HBV replication intermediates (RC and DL DNA). In addition, when quantifying HBsAg present in the culture supernatant, it was confirmed that the sample treated with GC1102 exhibits very low production of HBsAg. In the sample treated with MyrB as a positive control, the replication intermediates and HBsAg were not observed. Alternatively, in the sample treated with IgG as a negative control, the replication intermediates and HBsAg were observed similar to the sample subjected to only the HBV infection. From the above experimental results, it was observed that GC1102 could efficiently inhibit the HBV infection.
Further, HBV infection inhibitory effects by GC1102 were observed by immune-fluorescence. More particularly, at a time after 7 days from the infection of HepG2-NTCP cell line with HBV, HBV core protein was monitored by confocal microscopy. The cells were treated with 1ug/ml MyrB, 2ug/ml MyrB, 100ng/ml GC1102, or 200ng/ml GC1102, simultaneously with HBV infection. A nucleus of the cell was stained with DAPI (shown in blue or medium gray in FIG. 2) while staining a core protein with anti-HBcAg (shown in red or light gray in FIG. 2). Background where there is no fluorescence signal is shown in dark gray in FIG. 2.
Results of the experiment are shown in FIG. 2. Anti-HBcAg staining is shown in light gray; DAPI staining is shown in medium gray; and background is shown in dark gray. As shown in the figure, the cells were treated with 1ug/ml MyrB, 2ug/ml MyrB, 100ng/ml GC1102, or 200ng/ml GC1102, simultaneously with HBV infection. As a result of the treatment, an expression level of core protein (anti-HBcAg staining, shown in light gray) in the cell was considerably reduced in proportion to the concentration of GC1102. Further, it was observed that the experiment group treated with MyrB as the positive control also exhibits a decrease in an expression level of HBV core protein in proportion to the concentration of MyrB. Consequently, from the results shown in FIGS. 1 and 2, it could be interpreted that GC1102 inhibits HBV infection so as to efficiently prevent the expression of HBV protein as well as replication intermediate products.
<3> Assessment of HBV cccDNA formation inhibitory efficacy by GC1102
Based on the above results, in order to further investigate effects of GC1102 upon formation of covalently closed circular DNA (cccDNA), which is a determining factor of chronic HBV infection, an experiment was further executed.
Briefly, cccDNA of HBV refers to an episome DNA in which HBV genome DNA is converted into a plasmid form after HBV individuals entered into the liver cells of a target cell, which acts as a template for transcription. In other words, the process of converting the HBV genome DNA into cccDNA is necessarily required for genome replication of HBV.
On the ground of the above theory, an experiment was additionally executed to investigate effects of GC1102 upon the formation of covalently closed circular DNA (cccDNA), which is a determining factor of chronic HBV infection. More particularly, after treating HepG2-NTCP with GC1102 (100 ng/ml or 200 ng/ml) or MyrB (1 ㎍/ml), DNA was obtained by Hirt DNA extraction at a time after 3 days from the treatment, followed by measuring PF-RC DNA and cccDNA through Southern blot analysis. 3.2 kb, 2.1 kb, 1.9 kb and 1.4 kb HBV DNA were used as a size marker.
Results of the experiment are shown in FIG. 3. According to the experimental results, as compared to lane 2 in which only the HBV infection has proceeded, no cccDNA was found in the sample treated with MyrB used as the positive control and the sample treated with GC1102. This fact means that conversion of HBV genome DNA from the PF-RC DNA into the cccDNA was inhibited by the GC1102 treatment.
<4> Dose-response analysis of HBV infection inhibition by GC1102
Next, using various HBV studying techniques, HBV infection inhibitory effects for concentration of GC1102 were investigated to evaluate therapeutic efficacy thereof. While increasing the concentration of GC1102 in HepG2-NTCP cells from 6.25 to 200 ng/ml at a rate of 2 times, dose-response investigation was executed at 6 points. It was confirmed that at the concentration used in the previous experiment executed before the present experiment, non-cytotoxicity was determined and confirmed through cell viability assay (data not shown).
Results of the experiment are shown in FIG. 4. (A) Southern blot; (B) Western blot; (C) HBsAg ELSIA; and (D) HBeAg ELISA have been executed. Southern blot and ELISA were executed by the same procedure as shown in FIG. 1. Further, Western blot (B) is a result of measuring an amount of core protein in the cell using a part (6% input) of cell lysate obtained in order to conduct southern blot analysis. β-actin is a loading control demonstrating that the same amount of cell lysate was used.
According to the experimental result, an expression level of replication immediate products and virus core protein in the cell, and HBsAg and HBeAg secreted into a culture supernatant were reduced in a dose-dependent manner in proportion to an increased amount of GC1102. The above experimental results demonstrated that GC1102 is an effective HBV antiviral agent having no cytotoxicity and exhibiting dose-response according to the concentration.
<5> Assessment of EC50 (effective concentration) of GC1102
According to quantification of the results obtained from the experiment shown in FIG. 4, EC50 value was calculated. When performing the HBV infection, HBV infection extent obtained from a sample treated only with the IgG was set to be 100%. Then, values of samples treated with GC1102 at different concentrations were revised into relative values to the above extent, followed by drawing an inhibition curve (%).
Assayed results are shown in FIG. 5. When the cell was treated with GC1102 at a concentration of 14.97 ng/ml, HBV infection was inhibited by 50%. Such a treated concentration approximately corresponds to 100 pM when the concentration is converted into a molar concentration ('molarity') by applying a molecular weight of GC1102 (150 kDa). The above experimental results demonstrated that GC1102 could efficiently inhibit the HBV infection even at a very low concentration (EC50=100 pM).
<6> Assessment of HBV infection inhibitory efficacy according to a time of GC1102 (anti-HBsAg) treatment
Next, in order to investigate a time for treatment using GC1102 (anti-HBsAg) when HBV infection is most efficiently inhibited, an experiment was further executed.
Results of the experiment are shown in FIG. 6. (A) illustrating an experiment schedule to investigate a stage of acting GC1102 to HBV infection. HBV infection and GC1102 treatment schedule were indicated by solid lines. (B) Southern blot analysis: HepG2-NTCP cells were subjected to the same HBV infection and GC1102 treatment as described in the (A). Then, at a time after 9 days from the above treatment, cap seed DNA was measured by southern blot. 6% input is a result of measuring an amount of core protein in the cell by using a part of cell lysate obtained for executing the southern blot. β-actin is a loading control demonstrating that the same amount of cell lysate was used.
From the experimental results, it could be confirmed that, when GC1102 administration is conducted simultaneously with HBV infection, inhibition of the HBV infection by GC1102 is most effectively accomplished. This fact could be interpreted because GC1102 directly binds to a specific epitope of SORF in HBV during the HBV infection to inhibit attachment to hepatocyte in HBV, thereby effectively reducing the infection.
<7> Conclusion
Through the above experiments, it was demonstrated that human hepatitis B virus antibody (GC1102) of the present invention is a non-existing and very effective anti-HBV virus to fundamentally inhibit the HBV infection. GC1102 has EC50 value at a very low concentration, and has effectively inhibited not only the genome DNA replication of HBV but also cccDNA formation known as a transcription template of HBV genome. Accordingly, these results propose a new chance that GC1102 may very effectively inhibit the HBV infection in a liver transplant patient to thus very effectively prevent a recurrence of HBV after the liver transplant, in addition, a chronic hepatitis patient could be effectively treated by GC1102 administration alone or in combination with existing antiviral treatment agent.

Claims (19)

  1. A pharmaceutical composition for preventing cccDNA formation of hepatitis B virus, comprising a hepatitis B virus antibody.
  2. The composition according to claim 1, wherein the hepatitis B virus antibody is adapted to inhibit binding of a surface antigen (HBsAg) of the hepatitis B virus to heparan sulfate proteoglycan.
  3. The composition according to claim 1, wherein the hepatitis B virus antibody comprises: an amino acid sequence of SEQ ID NO: 1 encoding a human antibody H chain variable region to the surface antigen of the hepatitis B virus; and an amino acid sequence of SEQ ID NO: 2 encoding a human antibody L chain variable region to the surface antigen of the hepatitis B virus.
  4. The composition according to claim 1, wherein the hepatitis B virus antibody comprises: an amino acid sequence of SEQ ID NO: 3 encoding a human antibody H chain variable region to the surface antigen of the hepatitis B virus; and any one of amino acid sequences of SEQ ID NOs: 4 to 6 encoding a human antibody L chain variable region to the surface antigen of the hepatitis B virus.
  5. The composition according to claim 1, wherein the composition is administered within 24 hours from a time of the hepatitis B virus infection.
  6. A method of preventing cccDNA formation of hepatitis B virus, comprising administrating a prophylactically- or therapeutically-effective amount of a hepatitis B virus antibody.
  7. The method according to claim 6, wherein the hepatitis B virus antibody is adapted to inhibit binding of a surface antigen (HBsAg) of the hepatitis B virus to heparan sulfate proteoglycan.
  8. The method according to claim 6, wherein the hepatitis B virus antibody comprises: an amino acid sequence of SEQ ID NO: 1 encoding a human antibody H chain variable region to the surface antigen of the hepatitis B virus; and an amino acid sequence of SEQ ID NO: 2 encoding a human antibody L chain variable region to the surface antigen of the hepatitis B virus.
  9. The method according to claim 6, wherein the hepatitis B virus antibody comprises: an amino acid sequence of SEQ ID NO: 3 encoding a human antibody H chain variable region to the surface antigen of the hepatitis B virus; and any one of amino acid sequences of SEQ ID NOs: 4 to 6 encoding a human antibody L chain variable region to the surface antigen of the hepatitis B virus.
  10. The method according to claim 6, wherein the composition is administered within 24 hours from a time of the hepatitis B virus infection.
  11. A method of treating chronic hepatitis, comprising administrating the pharmaceutical composition according to claim 1 to block genome DNA replication of hepatitis B virus.
  12. The method according to claim 11, wherein the chronic hepatitis is chronic hepatitis B.
  13. The method according to claim 11, wherein the hepatitis B virus antibody comprises: an amino acid sequence of SEQ ID NO: 1 encoding a human antibody H chain variable region to the surface antigen of the hepatitis B virus; and an amino acid sequence of SEQ ID NO: 2 encoding a human antibody L chain variable region to the surface antigen of the hepatitis B virus.
  14. The method according to claim 11, wherein the hepatitis B virus antibody comprises: an amino acid sequence of SEQ ID NO: 3 encoding a human antibody H chain variable region to the surface antigen of the hepatitis B virus; and any one of amino acid sequences of SEQ ID NOs: 4 to 6 encoding a human antibody L chain variable region to the surface antigen of the hepatitis B virus.
  15. A method of screening a material for preventing or treating hepatitis B, comprising: inoculating hepatitis B virus to liver cells; and after treating the liver cells with a subject material, confirming whether cccDNA formation is inhibited or not.
  16. The method according to claim 15, wherein the liver cells are HepG2-NTCP.
  17. The method according to claim 15, wherein the subject material is treated within 24 hours after the inoculation of hepatitis B virus.
  18. The method according to claim 15, wherein the confirmation of whether cccDNA formation is inhibited or not is performed by comprising: (a) extracting episome DNA from the liver cells after 2 to 10 days from a treatment time using the subject material; and (b) measuring an amount of cccDNA included in the episome DNA.
  19. A method of screening a hepatitis B treatment supplement, comprising:
    (a) contacting a hepatitis B virus antibody and a subject material to a biological sample including hepatitis B virus; and
    (b) confirming whether cccDNA formation is inhibited or not, and if the cccDNA formation is more reduced than a case in which the subject material does not contact to the sample, determining the subject material as a treatment supplement which is administered in combination with the hepatitis B virus.
PCT/KR2016/008039 2015-07-24 2016-07-22 Pharmaceutical composition for preventing cccdna formation of hepatitis b virus WO2017018739A1 (en)

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MYPI2018000085A MY184876A (en) 2015-07-24 2016-07-22 Pharmaceutical composition for preventing cccdna formation of hepatitis b virus
EA201890362A EA201890362A1 (en) 2015-07-24 2016-07-22 PHARMACEUTICAL COMPOSITION FOR THE ADDITION OF EDUCATION OF THE HKVD VIRUS OF HEPATITIS B VIRUS
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