Three-dimensional epitope of hepatitis B surface antigen and antibody specifically binding thereto
阅读说明：本技术 乙型肝炎表面抗原的三维表位以及与其特异性结合的抗体 (Three-dimensional epitope of hepatitis B surface antigen and antibody specifically binding thereto ) 是由 金政焕 金佑眩 于 2020-01-14 设计创作，主要内容包括：本发明涉及乙型肝炎表面抗原的特异性三维表位以及与其结合的乙型肝炎中和抗体。本发明提供的表位具有特定的三维结构。另外,本申请的三维表位不包含在施用常规疫苗或HBIg时可产生逃逸突变的“a”决定簇。因此,能够与本申请的三维表位结合的抗体极不可能允许由常规疫苗引起的疫苗逃逸突变的出现,并因此可维持持续的作用。因此,这样的抗体或疫苗组合物可有效地应用于预防和治疗HBV中,具有很大的经济价值。(The present invention relates to a specific three-dimensional epitope of hepatitis b surface antigen and a hepatitis b neutralizing antibody binding thereto. The epitope provided by the invention has a specific three-dimensional structure. In addition, the three-dimensional epitopes of the present application do not comprise an "a" determinant that can generate escape mutations upon administration of conventional vaccines or HBIg. Therefore, antibodies capable of binding to the three-dimensional epitopes of the present application are highly unlikely to allow the emergence of vaccine escape mutations caused by conventional vaccines and thus can maintain sustained action. Therefore, such antibody or vaccine composition can be effectively applied to the prevention and treatment of HBV, and has great economic value.)
1. A conformational epitope of hepatitis b surface antigen (HBsAg) comprising:
a peptide represented by the general formula 1,
general formula 1
Thr-(X1)n1-A1-A2-(X2)n2-A3-(X3)n3-A4-(X4)n4-A5-(X5)n5-A6
In the general formula, A1Is Lys or Arg; a. the2Is Thr, Ala, Ile, Asn or Ser; a. the3Is Ser or Leu; a. the4Is Arg or His; a. the5Is Ser or Phe; and A is6Is Ser or Asn; and is
X1To X5Each independently a peptide molecule formed by n1 to n5 identical or different amino acids bonded to each other, wherein n1 is an integer from 4 to 8; n2 is an integer from 41 to 45; n3 is an integer from 0 to 2; n4 is an integer from 0 to 2; and n5 is an integer from 0 to 4.
2. The HBsAg conformational epitope of claim 1, wherein A1Is Lys or Arg; a. the2Is Thr; a. the3Is Ser; a. the4Is Arg; a. the5Is Ser; and A is6Is Ser or Asn.
3. The HBsAg conformational epitope of claim 2, wherein A1Is Lys, and A6Is Ser.
4. The HBsAg conformational epitope of claim 1, wherein n1 is 6; n2 is 43; n3 is 1; n4 is 1; and n5 is 2.
An HBV vaccine comprising as active ingredients:
the HBsAg conformational epitope of any one of claims 1 to 4.
6. The HBV vaccine of claim 5 wherein the HBsAg conformational epitope is native.
7. A hepatitis B virus neutralizing antibody or fragment thereof that specifically binds to the HBsAg conformational epitope as defined in any one of claims 1 to 4.
8. The hepatitis b virus neutralizing antibody or fragment thereof of claim 7 wherein said neutralizing antibody or fragment thereof has a therapeutic effect on infection with a vaccine escape mutant.
9. The hepatitis b virus-neutralizing antibody or fragment thereof of claim 7 wherein said neutralizing antibody or fragment thereof has a therapeutic effect on hepatitis b viruses that are resistant to drugs such as telbivudine, tenofovir, lamivudine, adefovir, cladribine, or entecavir.
10. The hepatitis B virus neutralizing antibody or fragment thereof of claim 7 wherein the amino acid sequence of the hepatitis B surface antigen (HBsAg) is SEQ ID NO 1.
11. The hepatitis B virus neutralizing antibody or fragment thereof of claim 7 wherein said antibody is produced in a cell line having accession number KCTC13760 BP.
12. A pharmaceutical composition for the treatment of HBV infection comprising as active ingredients:
the hepatitis b virus neutralizing antibody or fragment thereof of any one of claims 7 to 11.
13. Use of the hepatitis b virus neutralizing antibody or fragment thereof of any one of claims 7 to 11 for the manufacture of a pharmaceutical composition for the treatment of HBV infection.
14. A method for treating HBV infection comprising:
a step of administering to a subject an effective amount of the pharmaceutical composition of claim 12.
15. A method for producing the hepatitis b virus neutralizing antibody or fragment thereof of claim 7 comprising the steps of:
1) measuring the ability of an antibody specific for HBV to bind to a conformational epitope of HBsAg according to any of claims 1 to 4;
2) measuring the ability of said antibody specific for HBV to bind to a modified form of the conformational epitope of HBsAg by attaching a Thr, A selected from formula 1 to said conformational epitope of HBsAg1、A2、A3、A4、A5And A6Is obtained by replacing any one of the amino acid residues with another residue; and
3) selecting an antibody whose binding capacity to the HBsAg conformational epitope measured in step 1) is strong compared to its binding capacity to the modified form of the HBsAg conformational epitope measured in step 2).
16. The method of claim 15, wherein in the amino acid residue substitution performed in step 2),
A1may be substituted with an amino acid other than Lys or Arg;
A2can be replaced by an amino acid other than Thr, Ala, Ile, Asn or Ser；
A3May be substituted with an amino acid other than Ser or Leu;
A4may be substituted with an amino acid other than Arg or His;
A5may be substituted with an amino acid other than Ser or Phe; and
A6can be replaced by an amino acid other than Ser or Asn.
Technical Field
The present invention relates to conformational epitopes of hepatitis b surface antigen (hereinafter abbreviated as HBsAg) and antibodies that specifically bind thereto.
Background
Patients with chronic hepatitis b have no or very poor T cell response compared to spontaneously healing patients. It has been reported that this is because T cells specific to HBV disappear or fail to function properly due to continuous exposure to an excessive amount of viral antigen, such as hepatitis B surface antigen (HBsAg).
For chronic hepatitis B patients, the virus particles in blood reach 1010Individual particles/ml and HBsAg can even reach a level of 100 ug/ml. Such an excessive amount of virus particles or HBsAg suppresses immune functions of the human body and is thus considered to be an important cause of chronic hepatitis. In addition, it has been reported that HBV particles or HBsAg can enter dendritic cells to reduce activation of T cells, B cells and NK cells. In addition, in experiments using dendritic cells isolated from blood of healthy humans, it was observed that recombinant Hepatitis B surface antigen (rHBsAg) entered dendritic cells, and that the expression of activation marker protein of these dendritic cells was reduced. In addition, when these dendritic cells are co-cultured with NK cells and T cells, the ability of such dendritic cells to activate cells (e.g., NK cells and T cells) is also reduced (Woltman AM, PLos ONE, e15324, January 5,2011).
This phenomenon, in which loss of immune function or induction of immune tolerance occurs, is also observed in mice infected with Adeno-associated virus-hepatitis B virus (AAV-HBV). Interestingly, in the case where an anti-HBsAg monoclonal antibody was injected to reduce the HBsAg concentration in blood, it has been shown that the phenomenon of immune tolerance gradually decreases to restore the function of B cells and helper T cells.
Meanwhile, most antibodies produced by conventional hepatitis b vaccination are known to recognize an "a" determinant ('a' determinant), which is a site between amino acids 124 to 147 of HBsAg. In addition, it has been reported that, although the "a" determinant serves as a main neutralizing epitope of HBV, certain mutants having mutations within the "a" determinant, which appear in some patients, can escape antibodies produced by hepatitis b vaccination.
Therefore, there is an increasing need to develop new antibodies or vaccines for the prevention and treatment of hepatitis b, which can counteract such escape mutants produced against existing vaccines or hepatitis b immunoglobulin (HBIg). In particular, in order to generate and validate effective antibodies against HBsAg, the exact epitope of the antigen needs to be identified.
Disclosure of Invention
Technical problem
It is an object of the present invention to provide a conformational epitope of hepatitis b surface antigen, and an antibody or fragment thereof that specifically binds to the epitope and has excellent binding ability to various mutant hbsags.
Solution to the problem
To achieve the above objects, in one aspect of the present invention, a conformational epitope (conformational epitope) of hepatitis b surface antigen (HBsAg), and a hepatitis b virus-neutralizing antibody or a fragment thereof that specifically binds to the epitope are provided.
Advantageous effects
The conformational epitope of the hepatitis b surface antigen (HBsAg) provided herein comprises all critical residues that are important for specific binding to the hepatitis b virus neutralizing antibody and maintains an appropriate three-dimensional structure that allows the epitope to exhibit high affinity for the hepatitis b virus neutralizing antibody. Therefore, the epitope of the present invention can be used as an excellent HBV vaccine composition based on its high immunogenicity. In addition, the HBV neutralizing antibody produced by using the epitope of the present invention can effectively remove HBsAg present in blood and induce recovery of immunity in individuals infected with hepatitis b virus, thereby effectively treating hepatitis b. In addition, the antibody can effectively combine with a plurality of HBsAg variants, thereby effectively eliminating hepatitis B virus.
Drawings
Figure 1 shows a schematic diagram of the HBsAg structure.
FIG. 2 shows the results obtained by analyzing the characteristics of HBsAg virus-like particles (VLPs) on native agarose gel.
FIG. 3 shows an analysis method using denatured HBsAg VLP and HBsAg VLP-specific antibody (GC-100A).
FIG. 4 shows that denatured HBsAg VLPs did not bind to HBsAg VLP-specific antibodies (GC-100A). Here, N means natural conditions, and D means denaturing conditions.
Figure 5 shows a schematic of the binding sites on the conformational epitopes of HBsAg VLPs for binding to HBsAg VLP specific antibodies (GC-100A).
Figures 6 to 9 show the degree of binding between HBsAg VLP specific antibodies by natural immunoblotting (GC-100A) and 30 point mutations of HBsAg referred to as vaccine escape mutants (vacine escape mutants) involving 16 major amino acid residues.
Figure 10 shows the binding profile of GC-100A and DAKO antibodies to clinical ("a" determinant) variant HBsAg.
FIGS. 11 to 14 show the results obtained by ELISA of HBsAg in wild-type virus and vaccine escape mutant (G145R) in a mouse model of short term expression of HBV. In particular, fig. 11 shows the binding reaction with hIgG in mice infected with HBV. In addition, FIG. 12 shows the results of determining that an antibody specific to HBsAg VLP (GC-100A) binds well to HBsAg VLP produced in HBV-infected mice. In addition, fig. 13 shows the binding response to hIgG in wild-type mice infected with HBV vaccine escape mutant (G145R). In addition, fig. 14 shows the results of determining that HBsAg VLP-specific antibodies (GC-100A) bind well to HBsAg VLPs produced in wild-type mice infected with HBV vaccine escape mutant (G145R). Here, the Y-axis means the concentration of HBsAg in blood in IU/mL. The X axis means the number of days. On day 1, hydrodynamic injection (DNA injection) was performed; and on day 2 administration of IgG (which is a control) or GC-100A was performed. Each line in the same experiment represents each individual.
Fig. 15 to 18 show the results of determining the ability to eliminate wild-type virus and vaccine escape mutants (G145R) in a short-term HBV expression mouse model by quantification of HBV DNA. In particular, fig. 15 shows the results of determining that HBV was not eliminated by hIgG in mice infected with HBV. In addition, FIG. 16 shows the results of determining that HBV was effectively eliminated by GC-100A in mice infected with HBV. In addition, fig. 17 shows the results of determining that HBV was not eliminated by hIgG in wild-type mice infected with HBV vaccine escape mutant (G145R). In addition, fig. 18 shows the results of determining that HBV was effectively eliminated by GC-100A in a wild-type mouse infected with HBV vaccine escape mutant (G145R). Here, the Y-axis means the concentration of HBV DNA in blood in copy number/mL. The X axis means the number of days. On day 1, hydrodynamic injection (DNA injection) was performed; and on day 2 administration of IgG (which is a control) or GC-100A was performed. Each line in the same experiment represents each individual.
Figures 19 to 21 show the results obtained by natural immunoblotting of HBsAg for each genotype/serotype.
Figure 22 shows the binding profile of GC-100A and DAKO antibodies to HBsAg for each genotype/serotype.
FIG. 23 shows a graph representing the geographical distribution of each HBV genotype.
Figures 24 to 26 show the results obtained by natural immunoblotting of HBsAg mutants for each genotype/serotype.
Figure 27 shows the binding profile of GC-100A and DAKO antibodies to HBsAg for each genotype/serotype.
FIGS. 28 to 33 show the results of homology comparison analysis of various amino acid sequences of hepatitis B surface antigen. The horizontal axis represents the distribution of amino acids that may be present in the surface antigen. The vertical axis represents representative amino acids depending on the position of the surface antigen.
Detailed Description
Hereinafter, the present invention will be described in detail.
In one aspect of the present invention, there is provided a conformational epitope of hepatitis B surface antigen (HBsAg) comprising a peptide represented by formula 1,
general formula 1
Thr-(X1)n1-A1-A2-(X2)n2-A3-(X3)n3-A4-(X4)n4-A5-(X5)n5-A6
In the general formula, A1Is Lys or Arg; a. the2Is Thr, Ala, Ile, Asn or Ser; a. the3Is Ser or Leu; a. the4Is Arg or His; a. the5Is Ser or Phe; and A is6Is Ser or Asn; and is
X1To X5Each independently a peptide molecule formed by n1 to n5 identical or different amino acids bonded to each other, wherein n1 is an integer from 4 to 8; n2 is an integer from 41 to 45; n3 is an integer from 0 to 2; n4 is an integer from 0 to 2; and n5 is an integer from 0 to 4.
According to one embodiment, n1 may be an integer of 4, 5, 6, 7 or 8, and may preferably be an integer of 6. n2 may be an integer of 41, 42, 43, 44 or 45, and may preferably be an integer of 43. n3 may be an integer of 0, 1 or 2, and may preferably be 1. n4 may be an integer of 0, 1 or 2, and may preferably be 1. n5 may be 0, 1, 2, 3 or 4, and may preferably be 2.
Here, X1To X5May each independently consist of an amino acid selected from the group consisting of: ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.
The present inventors conducted studies to identify specific epitopes that exhibit excellent immunogenicity in HBsAg. As a result, the present inventors found that seven amino acid residues (Thr and A) represented by the general formula 1 in HBsAg1To A6) In thatPlays an important role in specific binding to a neutralizing antibody against hepatitis b virus, and a peptide molecule comprising 7 amino acid residues and maintaining an appropriate three-dimensional structure can be used as an excellent HBV vaccine composition, thereby completing the present invention.
The term "hepatitis B virus" as used herein refers to a DNA virus belonging to the family Hepodnaviridae (Hepadnaviridae) and having a nucleotide duplex structure of about 3.2kb in size. The hepatitis B virus has four genes, namely pre-core/core, pre-S/S, P and X. These genes encode HBeAg/HBcAg, HBsAg, DNA polymerase and HBx protein. The nucleotide sequence constituting HBV varies widely depending on the region and race. Depending on such nucleotide sequence changes, HBV serotypes are differentially expressed. For such serotypes, serotype adw is subdivided into subtypes adw, adw2, and adw 4; and serotypes adr or ayw are also further subdivided in a similar manner. HBV found in Korea is serotype adr.
The term "hepatitis B surface antigen" as used herein refers to a protein present on the hepatitis B virus coat and is also designated HBsAg. Here, the hepatitis b surface antigen (HBsAg) may be a polypeptide consisting of SEQ ID NO: 1. The antigen may contain a linear epitope having a one-dimensional structure and a conformational epitope having a three-dimensional structure. Typically, a linear epitope is composed of consecutive amino acids.
According to one embodiment of the present invention, in formula 1, A1Is Lys or Arg; a. the2Is Thr; a. the3Is Ser; a. the4Is Arg; a. the5Is Ser; and A is6Is Ser or Asn. More specifically, A1Is Lys, and A6Is Ser.
In such cases, the conformational epitope of HBsAg may have any of the structures of formulae 2 to 5:
general formula 2
Thr-(X1)n1-Lys-Thr-(X2)n2-Ser-(X3)n3-Arg-(X4)n4-Ser-(X5)n5-Ser
General formula 3
Thr-(X1)n1-Lys-Thr-(X2)n2-Ser-(X3)n3-Arg-(X4)n4-Ser-(X5)n5-Asn
General formula 4
Thr-(X1)n1-Arg-Thr-(X2)n2-Ser-(X3)n3-Arg-(X4)n4-Ser-(X5)n5-Ser
General formula 5
Thr-(X1)n1-Arg-Thr-(X2)n2-Ser-(X3)n3-Arg-(X4)n4-Ser-(X5)n5-Asn
According to a particular embodiment of the invention, n1 is 6; n2 is 43; n3 is 1; n4 is 1; and n5 is 2. Here, in particular, (X)1)6May have a sequence defined by SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. In addition, (X)2)43May have a sequence defined by SEQ ID NO: 3, and (b) an amino acid sequence represented by (3). In addition, (X)3)1May be Ser. In addition, (X)4)1May be Arg. In addition, (X)5)2Can be Trp-Leu.
According to one embodiment of the invention, the HBsAg conformational epitope of the invention may be at any position selected from HBsAg amino acid position 115, 122, 123, 167, 169, 171 and 174. In particular, an epitope may comprise all seven amino acids. In particular, the conformational epitope of hepatitis b surface antigen (HBsAg) may be an HBsAg conformational epitope consisting of a 7-mer-to 60-mer oligomer with residues at amino acids 115 to 174 of hepatitis b surface antigen (HBsAg), and the HBsAg conformational epitope may comprise amino acid residues at positions 115, 122, 123, 167, 169, 171 and 174. Here, the hepatitis b surface antigen (HBsAg) may have a sequence consisting of SEQ ID NO:1, or a pharmaceutically acceptable salt thereof.
In another aspect of the present invention, an HBV vaccine comprising an HBsAg conformational epitope as an active ingredient is provided. Herein, the conformational epitope of HBsAg contained in HBV vaccine as an active ingredient is characterized as being natural. Epitopes comprising amino acids present at the above positions may be used in combination with a carrier to maintain the three-dimensional structure of the epitope or to cause an increase in efficiency in a vaccine composition. Here, as for the carrier according to the present invention, any carrier may be used as long as it is biocompatible and can achieve the desired effect in the present invention. The carrier may be selected from, but is not limited to, serum albumin, peptides, immunoglobulins, hemocyanins, polysaccharides, and the like.
In another aspect of the invention, there is provided a hepatitis b virus neutralizing antibody or fragment thereof that specifically binds to a conformational epitope of the hepatitis b surface antigen (HBsAg) of the invention as described above. In particular, the hepatitis b virus neutralizing antibody or fragment thereof specifically binds to the above-described HBsAg conformational epitope at amino acid positions 115, 122, 123, 167, 169, 171, and 174 of HBsAg.
Here, neutralizing antibodies or fragments thereof may have a therapeutic effect on infection under vaccine escape mutants (see fig. 10).
Here, the antibody may be a monoclonal antibody. Alternatively, the antibody may be produced in a cell line having accession number KCTC13760 BP. In addition, in the case where the antibody has the same epitope (complementary-determining region, CDR) as the antibody produced in the above-described deposited strain, such an antibody can be determined to belong to the antibody disclosed in the present invention. Alternatively, the antibody fragment may be selected from Fab, sFv and F (ab')2And may be a diabody (diabody) or a chimeric antibody. However, the fragment is not limited thereto.
The antibody or fragment thereof according to the present invention can bind to hepatitis b virus having a genotype of A, B, C, D, E, F, G or H and thus has neutralizing activity against these. In addition, the antibody or a fragment thereof may bind to any one or more selected from among hepatitis b surface antigen (HBsAg) subtypes adw, adr, ayw, and ayr, and thus have neutralizing activity against hepatitis b virus (experimental example 3.3 and fig. 19 to 22).
In addition, the antibody or a fragment thereof according to the present invention may be combined with hepatitis b virus resistant to therapeutic drugs against hepatitis b virus, such as telbivudine (telbivudine), tenofovir (tenofovir), lamivudine (lamivudine), adefovir (adefovir), clevudine (clevudine), or entecavir (entecavir), on the market or under development, and thus have neutralizing activity against the virus (experimental example 4 and fig. 24 to 27).
In addition, the antibody or a fragment thereof according to the present invention may bind to HBsAg having a mutation at amino acid positions 80, 101, 112, 126, 129, 133, 143, 172, 173, 175, 181, 184, 185, 195, 196, 204 or 236, and thus have neutralizing activity against mutant hepatitis b virus. In one embodiment of the invention, the mutant HBsAg may be an antigen having a mutation at position 80, 171, 172, 173, 195, 196, 204 or 236 of the HBsAg. However, the antigen is not limited thereto.
In still another aspect of the present invention, there is provided a pharmaceutical composition for treating HBV infection, comprising a hepatitis b virus-neutralizing antibody or a fragment thereof as an active ingredient.
Here, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. For a pharmaceutically acceptable carrier, any carrier can be used so long as it is a non-toxic material suitable for delivery to a patient. Some examples of carriers may include distilled water, alcohols, fats, waxes, and inert solids. Pharmaceutically acceptable adjuvants (buffers or dispersants) may also be included in the pharmaceutical composition.
In particular, the pharmaceutical compositions of the present application may contain, in addition to the active ingredient, a pharmaceutically acceptable carrier and thus be prepared as parenteral formulations, depending on the route of administration, using any conventional method known in the art. As used herein, the term "pharmaceutically acceptable" refers to a substance that does not inhibit the activity of the active ingredient and has a toxicity that does not exceed the toxicity to which the subject receiving it is adapted.
In the case where the pharmaceutical composition of the present application is prepared as a parenteral formulation, it may be formulated in the form of an injection, a drug for transdermal delivery, a drug for nasal inhalation, or a suppository, together with a suitable carrier according to any method known in the art. In the case where the pharmaceutical composition is formulated as an injection, a suitable carrier to be used may include sterile water, ethanol, a polyhydric alcohol (e.g., glycerol or propylene glycol) or any mixture thereof, and may preferably include Ringer's solution, sterile water for injection or Phosphate Buffered Saline (PBS) containing triethanolamine, an isotonic solution such as 5% dextrose, or the like. Methods related to the formulation of pharmaceutical compositions are known in the art.
The preferred dose of the pharmaceutical composition of the present application may be 0.01ug/kg to 10g/kg per day, or 0.01mg/kg to 1g/kg per day, depending on the condition, body weight, sex, age, disease severity and administration route of the patient. Administration may be performed once daily or divided into several administrations. Such dosages should not be construed as limiting the scope of the invention in any way.
Subjects to which the compositions of the present application may be applied include mammals and humans, with humans being particularly preferred. In addition to the active ingredients, the pharmaceutical compositions of the present application may also comprise any compound or natural extract that has been validated for safety and is known to have a therapeutic effect on infectious diseases, to enhance or enhance its therapeutic activity.
In yet another aspect of the present invention, there is provided a use of a hepatitis b virus neutralizing antibody or fragment thereof for the manufacture of a pharmaceutical composition for the treatment of HBV infection.
In yet another aspect of the present invention, a method for treating HBV infection is provided, comprising the step of administering to an individual an effective amount of a pharmaceutical composition.
In yet another aspect of the present invention, there is provided a method for producing a hepatitis b virus neutralizing antibody or fragment thereof that specifically binds to the above-described HBsAg conformational epitope, comprising the steps of: 1) measuring the binding capacity of an antibody specific for HBV to the conformational epitope of HBsAg; 2) measuring the binding capacity of an antibody specific for HBV to a modified form of the conformational epitope of HBsAg as defined aboveThe form is formed by selecting Thr and A in the HBsAg conformational epitope from general formula 11、A2、A3、A4、A5And A6Is obtained by replacing any one of the amino acid residues with another residue; and 3) selecting an antibody whose binding capacity to the HBsAg conformational epitope measured in step 1) is strong compared to its binding capacity to the modified form of the HBsAg conformational epitope measured in step 2).
Here, in the amino acid residue substitution mentioned in step 2), A1May be substituted with an amino acid other than Lys or Arg. In addition, A2May be substituted with an amino acid other than Thr, Ala, Ile, Asn or Ser. In addition, A3May be substituted with an amino acid other than Ser or Leu. In addition, A4May be substituted by an amino acid other than Arg or His, and A5May be substituted with an amino acid other than Ser or Phe. In addition, A6Can be replaced by an amino acid other than Ser or Asn.
MODE OF THE INVENTION
Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.
Preparation example 1 preparation of antibody specifically binding to HBsAg
In the present invention, for the antibody specifically binding to HBsAg, a monoclonal antibody produced by a strain with accession number KCTC13760BP was used. In addition, the monoclonal antibody produced by this strain was designated GC-100A for convenience.
Experimental method 1 Natural Agarose Gel Electrophoresis (NAGE)
Huh-7 cells were transfected with empty (mock) vector or Flag-small HBsAg expression plasmid using Lipofectamine 3000. After 2 days, cells were lysed using RIPA buffer (Thermo Fisher, 89901). Centrifugation was carried out at 4 ℃ and 12,000rpm for 15 minutes, and then the supernatant without precipitate was transferred to a new Eppendorf tube. To this was added 6X dye loaded agarose (50% glycerol, 0.1% BPB) to a final concentration of 1X and mixed well. A1.2% TBE agarose gel was prepared and loaded with a sample mixed with dye-loaded agarose. Then, gel electrophoresis was performed at 50V for 1 hour.
After completion of gel electrophoresis, upward capillary transfer was performed on a polyvinylidene fluoride membrane (PVDF membrane) using 20X SSC buffer (3M NaCl, 0.3M sodium citrate, pH 7.2). The membrane on which the transfer of the substance was completed was blocked with a 5% skim milk solution for 30 minutes and washed twice with 1XTBST buffer (50mM Tris, 150mM NaCl, 0.1% Tween 20). Subsequently, GC-100A or anti-Flag antibody (5ug) was diluted in 10ml TBS blocking buffer (Thermo Fisher, 37571) and then incubated with the membrane on a shaker for 2 hours. After the incubation was completed, washing with 1X TBST buffer was performed on a shaker for 5 minutes. This process was repeated 4 times.
Anti-human or anti-mouse antibodies were diluted in 10ml TBS blocking buffer and subsequently incubated with the membrane on a shaker for 1 hour. Finally, the wash with 1X TBST buffer was performed on a shaker for 5 minutes as described above. This process was repeated 4 times. The membrane on which the reaction with the primary and secondary antibodies was completed was reacted with ECL (GE Healthcare, RPN 2235). Then, images were obtained using a ChemiDoc MP system (Bio-rad, 1708280).
Experimental method 2 denatured immunoprecipitation experiment
HEK293T cells were transfected with either empty vector or Flag-small HBsAg expression plasmid using Lipofectamine 3000. After 2 days, cells were lysed using NP40 cell lysis buffer (150mM NaCl, 50mM Tris-HCl, pH 7.5, 1% NP-40). Centrifugation was carried out at 4 ℃ and 12,000rpm for 15 minutes, and then the supernatant without precipitate was transferred to a new Eppendorf tube.
Dividing the cell lysate into 4 samples; and then 2 of them were subjected to native IP, and the other 2 were denatured (heating at 100 ℃ for 10 minutes, final concentration of 1% SDS, 1% β -mercaptoethanol). Each of the 4 samples was transferred to a 15ml conical tube and subsequently diluted 20-fold with NP40 cell lysis buffer. To match the conditions under which antibody binding occurs in the native and denatured samples, SDS at a final concentration of 0.05% and 0.05% beta-mercaptoethanol were added to 2 native samples. To each of the native and denatured samples was added 5ug of anti-Flag antibody, and to each of the remaining non-denatured and denatured samples was added 5ug of GC-100A. Then, incubation was performed overnight at 4 ℃ in a cold room with shaking.
The following day, 100ul of each of protein A agarose beads (GE Healthcare, 17-0780-01) was added thereto, and it was incubated again for 5 hours in a cold room with shaking. For each sample, after completion of the reaction, centrifugation was performed at 4 ℃ and 2,000rpm for 3 minutes. The supernatant was discarded and the remaining beads were transferred to a new Eppendorf tube using NP40 cell lysis buffer. Centrifugation was again carried out at 4 ℃ and 4,000rpm for 3 minutes. The supernatant was discarded and washed again with 500ul NP40 cell lysis buffer. This process was repeated 4 times. The supernatant was discarded completely. To this was added 50ul of 1.5 Xsample buffer (obtained by adding 4% final concentration of. beta. -mercaptoethanol to NP0007(Thermo Fisher) and diluting at 1.5X) and boiled at 100 ℃. Subsequently, centrifugation was performed at 4 ℃ and 12,000rpm for 3 minutes, and then the supernatant was transferred to a new Eppendorf tube.
Each of the above samples was loaded on a 12% Bis-Tris protein gel (Thermo Fisher, NP0342BOX) and subjected to gel electrophoresis at 110V for 2 hours (until the dye was electrophoresed to the end of the gel). After completion of gel electrophoresis, the material was transferred to a PVDF membrane. The membrane on which the transfer of the substance was completed was blocked with a 5% skim milk solution for 30 minutes, and washed twice with 1 XTSST buffer (50mM Tris, 150mM NaCl, 0.1% Tween 20). Subsequently, GC-100A or anti-Flag antibody (5ug) was diluted in 10ml TBS blocking buffer (Thermo Fisher, 37571) and then incubated with the membrane on a shaker for 2 hours.
After the incubation was completed, washing with 1X TBST buffer was performed on a shaker for 5 minutes. This process was repeated 4 times. Anti-human or anti-mouse antibodies were diluted in 10ml TBS blocking buffer and subsequently incubated with the membrane on a shaker for 1 hour. Finally, as described above, on the shaker for 5 minutes with 1 TBST buffer washing. This process was repeated 4 times. The membrane on which the reaction with the primary and secondary antibodies was completed was reacted with ECL (GE Healthcare, RPN 2235). Then, images were obtained using a ChemiDoc MP system (Bio-rad, 1708280).
Experimental method 3 epitope mapping between HBsAg and GC-100A
To form the immune complex, HBsAg VLPs were mixed with GC-100A Fab as shown in table 1.
[ Table 1]
1mg of d0 crosslinker was mixed with 1mg of d12 crosslinker. Again, the mixture was mixed with 1ml of DMF to obtain a 2mg/ml solution of DSS d0/d 12. 10ul of the previously prepared antibody/antigen mixture was mixed with 1ul of a solution of the cross-linking agent d0/d 12. For the crosslinking reaction, the solution was incubated at room temperature for 180 minutes.
Experimental example 1 identification of HBsAg epitope of GC-100A antibody
Experimental example 1.1 identification of possibility of detecting HBsAg VLP Using GC-100A
Up to 2016, ELISA was the only test that made it possible to detect HBsAg with GC-100A. In addition, in Western blot analysis using SDS-PAGE, it was impossible to detect HBsAg with GC-100A, which is thought to have a conformational epitope. Therefore, a technique for detecting HBV capsid in natural state was designed and attempted to detect HBsAg VLP (consisting of 100 single HBsAg molecules) using NAGE disclosed in Experimental method 1. Since NAGE is a method for identifying macromolecules without causing protein denaturation, this method makes it possible to detect HBsAg VLPs with anti-Flag antibodies with linear epitopes and GC-100A with conformational epitopes (fig. 2).
There was a difference between the HBsAg VLP patterns detected by GC-100A and anti-Flag antibodies. In each upper band, the VLP can be said to be in the intact state desired herein. On the other hand, in the lower band of the HBsAg VLP detected by the anti-Flag antibody, the VLP is expected to be in a denatured state. The reason for this is as follows. In HBsAg VLPs, the lipid component comprises about 30% or more; and therefore, it is expected that in the step of obtaining cell lysates in NAGE, the VLP lipids will be disrupted by Triton X-100 as a detergent in RIPA buffer to produce complexes.
Thus, since GC-100A with conformational epitopes recognizes the VLP structure, only the upper band, where the VLP is in the intact state, can be detected. However, in the case of anti-Flag antibodies with linear epitopes, VLPs in a slightly disrupted state can be detected. In conclusion, in the case of using NAGE without causing protein denaturation in HBsAg VLPs, VLPs can be detected by GC-100A (fig. 2).
EXAMPLE 1.2 identification of whether the binding site for GC-100A is a conformational epitope
To identify whether the binding site of GC-100A has a conformational epitope, denatured IP was performed. According to the results of Experimental example 1.1, native HBsAg VLPs can be detected using both GC-100A and anti-Flag antibodies. Thus, GC-100A binds to native HBsAg VLPs; however, in cases where HBsAg VLPs are denatured, GC-100A with conformational epitopes cannot bind to it. In addition, GC-100A failed to detect HBsAg in SDS-PAGE. Therefore, the last detection was intended to be performed using an anti-Flag antibody. In other words, a method in which binding to natural or denatured HBsAg VLPs was performed with GC-100A (IP) and detection of HBsAg VLPs bound to GC-100A was performed with anti-Flag antibody (IB) was selected. An overview of this experiment is shown in figure 3.
GC-100A binds to native HBsAg VLPs when Immunoprecipitation (IP) is performed with GC-100A. However, denatured HBsAg VLPs did not bind to GC-100A (FIG. 4). anti-Flag antibodies with linear epitopes bind to denatured HBsAg VLPs as well as native HBsAg VLPs. The results obtained by denaturing IP with anti-Flag antibody indicate that conditions were shown to be sufficient for the control of antibody binding to antigen. However, GC-100A did not bind to denatured HBsAg VLPs, and this result supports GC-100A with conformational epitopes.
In addition, unlike the case where Immunoblotting (IB) was performed with an anti-Flag antibody, no band was observed in the case where IB was performed with GC-100A; and this becomes the supporting basis independent of the results as described above. Thus, from the results obtained by performing the denaturing IP experiment, it was found that GC-100A binds to a conformational epitope.
Experimental example 2 identification of epitope position of antibody of the present invention Using protease
The experiment was performed as follows. The two proteins are allowed to react naturally and then cross-linked at sites close to each other using a cross-linking agent. The protein is then cleaved into peptide fragments using a protease in order to find the crosslinking site.
In particular, 1mg of d0 crosslinker was mixed with 1mg of d12 crosslinker, and the mixture was subsequently mixed with 1ml of DMF to obtain a 2mg/ml DSS d0/d12 solution. 1ul of the solution was mixed with 10ul of GC-100A/HBsAg VLP complex and incubated for 180 minutes at room temperature.
Then, the denatured GC-100A/HBsAg VLP complex crosslinked with d0/d12 was digested with trypsin (Roche Diagnostic) that cleaves lysine or arginine residues, and subsequently 9 crosslinked peptides between Fab and HBsAg VLPs were identified as fragments of GC-100A by nLC-orbitrap MS/MS analysis. These cross-linked peptide moieties were detected by both Xquest and Stavrox software (Table 2).
[ Table 2]
Denatured GC-100A/HBsAg VLP complexes crosslinked with d0/d12, prepared by the same preparation method as described above, were digested with chymotrypsin (Roche Diagnostic) cleaving tryptophan, tyrosine, phenylalanine, leucine and methionine residues. Then, a cross-linking peptide between Fab and HBsAg VLP was detected by nLC-orbitrap MS/MS analysis. These cross-linked peptide moieties were detected by both Xquest and Stavrox software (Table 3).
[ Table 3]
The denatured GC-100A/HBsAg VLP complex crosslinked with d0/d12, prepared by the same preparation method as described above, was digested with ASP-N enzyme (Roche Diagnostic) that cleaves aspartic acid and glutamic acid. Then, nLC-orbitrap MS/MS analysis is carried out; however, no cross-linking peptide between Fab and HBsAg VLPs was detected. The GC-100A/HBsAg VLP complex was digested with serine cleaving elastase (Roche Diagnostic) using the same method as described above, and then 5 cross-linking peptides between Fab and HBsAg VLP were detected by nLC-orbitrap MS/MS analysis. These cross-linked peptide moieties were detected by both Xquest and Stavrox software (Table 4).
[ Table 4]
The GC-100A/HBsAg VLP complex was digested with thermolysin (Roche Diagnostic) which cleaves hydrophobic amino acid residues using the same method as described above, and then a cross-linking peptide between Fab and HBsAg VLP was detected by nLC-orbistrap MS/MS analysis. These cross-linked peptide moieties were detected by both Xquest and Stavrox software (Table 5).
[ Table 5]
In particular, protein 1 is the heavy chain and protein 2 is the VLP; and sequence proteins 1 and 2 are peptide moieties bound thereto. The terminal nAA1 and nAA2 are moieties linked by a crosslinker. The data obtained so far were used to infer the epitope for GC-100A. As a result, the epitope was identified to comprise two parts, one part being located at amino acid residues 115, 122 and 123 of HBsAg, and the other part being located at amino acid residues 167, 169, 171 and 174 of HBsAg (fig. 5).
Experimental example 3 examination of binding ability of GC-100A to HBsAg mutant
GC-100A binds to a conformational epitope of HBsAg and may therefore have different binding capacities for HBsAg, depending on the type of HBsAg and the mutations therein. Thus, several types of HBsAg mutants were constructed and examined for the ability of GC-100A to bind to them.
Experimental example 3.1 examination of the binding Capacity of GC-100A to clinical ("a" determinant) variants
In the Clinical variants reported so far (Alavian SM, J Clinical virol.57: 201, 2013), 30-point mutants of 16 major amino acid positions (e.g.G 145R, known as vaccine escape mutants) were selected as the primary analytical target in view of the membrane distribution structure of HBsAg (Rezaee R.et al, Heapat Mon.16: e39097, 2016), with emphasis placed on the "a" determinant region (amino acids positions 120 to 150).
For the data obtained by NAGE in experimental method 1, the band intensity was measured with ImageJ software to determine the extent of binding of each anti-HBsAg antibody relative to the binding capacity of anti-HA. As a result, a significant difference was identified in antigen binding properties between the antibodies GC-100A and DAKO (anti-HBsAg polyclonal antibody) (fig. 6 to 9). The clinical mutants of HBsAg are listed in Table 6 below.
[ Table 6]
Amino acid position
Wild type (ayw)
Mutants
117
S
R/T
120
P
E/S/T
123
T
N
124
C
R/Y
126
T
I/A/N/S
129
Q
H/L
130
G
D/R
133
M
L
134
Y
N/R
141
K
E/I
142
P
S/L
144
D
A/E
145
G
R/K
146
N
S
148
T
I
149
C
R
As illustrated in fig. 6 to 9, the bands of the HBsAg mutant have different positions in NAGE. In this phenomenon, proteins with substitutions by basic amino acids (e.g., S117R and G130R) migrate less. In contrast, clones with substitutions by acidic amino acids (e.g., P120E and G130D) migrated farther. From these results, it was found that in the above NAGE, the charge of the protein is an important transfer factor. Some clones (e.g., S117R and G130R) did not exhibit or exhibited significantly lower levels of antigenic protein compared to WT. In view of the fact that: such clones exhibited intracellular protein expression levels that were not significantly lower than those of other clones when examined with anti-HA antibodies as described above, confirming that these mutants have low efficiency in secreting proteins to the outside of cells, or that proteins secreted therefrom have low stability. GC-100A showed approximately similar binding capacity to 30 clinical ("a" determinant) variants. On the other hand, the DAKO antibody produced against the blood-derived HBsAg antigen of the patient showed very low binding ability to all 10 mutants of K141E to T148I in terms of antigen binding; and the results are significantly different from those of GC-100A.
The portion consisting of about 10 amino acids is at the position corresponding to the second loop of the "a" determinant and appears to be important for binding of the DAKO antibody. On the other hand, unlike most "a" determinant-dependent anti-HBsAg antibodies, it was determined that GC-100A showed reasonably consistent binding to various clinical variants, including vaccine escape mutants such as G145K or G145R (fig. 10). In particular, it appears that binding of the DAKO antibody is attenuated at positions 141 to 149. This may represent a vaccine escape mutant. In contrast, GC-100A showed no difference in binding capacity at the same location, thereby predicting that GC-100A would be effective against vaccine escape mutants. Here, the vaccine escape mutant means a case where HBV infection is caused again in a subject who has received HBV vaccination and produced antibodies.
Experimental example 3.2 identification of the potency of GC-100A against the vaccine escape mutant G145R
The elimination capacity against the vaccine escape mutant G145R was examined in mice. For the short-term expression mouse model of wild-type HBV or vaccine escape mutant G145R, HBsAg of wild-type virus or vaccine avoidance mutant (G145R) was identified to be maintained in blood until about day 6 in the group treated with hIgG as a negative control (fig. 11 and 13). In contrast, in the group treated with GC-100A, HBsAg of the wild-type virus or vaccine avoidance mutant (G145R) was identified to decrease dramatically on day 1 after treatment (fig. 12 and 14).
qPCR was performed to quantitatively analyze infectious virus particles (virions) other than viral HBsAg. As a result, in the group treated with GC-100A, DNA of the wild-type virus or the vaccine avoidance mutant (G145R) was identified to be drastically reduced in blood on day 1 after the treatment. However, in the group treated with hIgG, which was a negative control, DNA of the wild-type virus or vaccine avoidance mutant (G145R) was identified to be maintained in blood until day 7 to day 10 (fig. 15 to 18). Taken together, the above results identified that GC-100A eliminated HBsAg and infectious viral particles (virions) present in the blood of mice, and this effect was observed in the vaccine escape mutant (G145R) as well as in the wild-type virus.
Experimental example 3.3 examination of binding Capacity of GC-100A against genotype/serotype
HBV consists of 10 genotypes (A, B, C, D, E, F, G, H, I and J) and 4 serotypes (ayw, ayr, adw and adr). The binding ability of GC-100A to various genotypes and serotypes of HBV was examined.
For amino acid sequences representing the respective genotypes, several sequences derived from patient samples are compared and a consensus sequence is selected. The number of consensus sequences selected for each type is as follows: 2 of A type; 2 of type B; 2 of C type; 2 in D type; 2 of E type; 3 of F type; 2 of G type; and 3 in H type. Since type I and J are the most recently isolated genotypes, there is not much sequence information. Therefore, the sequences possessed by the present inventors were selected as representative sequences. In addition, since serotypes adw, adr, and ayw exist in 21 representative sequences of respective genotypes, sequences of serotype ayr possessed by the present inventors are added. Thus, a total of 22 sequences were used to examine the binding capacity of GC-100A.
As a result of the identification by the above method, it was shown that GC-100A responded to genotype C somewhat strongly and genotypes F and H somewhat weakly as compared with the DAKO antibody (FIGS. 19 to 22). These results indicate that GC-100A is able to bind multiple hbsags of all genotypes/serotypes. In addition, most patients have genotypes A, B, C and D; and genotypes F and H are representative genotypes only in south america in local areas (fig. 23).
Experimental example 4 examination of binding Capacity of GC-100A to drug resistant variants
Currently, there are two types of FDA approved antiviral drugs against chronic hepatitis b virus (CHB), namely nucleotide/nucleoside(s) ide based and interferon-a related types. Among them, nucleotide/nucleoside drugs include lamivudine, telbivudine, adefovir, tenofovir and entecavir, and have a mechanism acting on HBV polymerase to prevent viral replication. However, long-term administration of nucleotide/nucleoside drugs results in resistant mutants against them, and thus the viral titer is restored again despite the administration of the drug. Thus, combination therapy using nucleotide/nucleoside drugs has been performed. However, such treatment is a big problem because it causes multidrug resistance.
The relationship between the drugs acting on the polymerase and GC-100A bound to HBsAg is as follows. HBV has a small genome of 3.2kb and thus the polymerase ORF and HBsAg ORF overlap therein. Thus, in the case where a mutation occurs in the polymerase, HBsAg also undergoes a mutation that can affect GC-100A binding. Therefore, the inventors aimed to examine the binding ability of GC-100A to these mutants.
Mutations in the polymerase and resulting HBsAg caused by lamivudine, telbivudine, adefovir, tenofovir, entecavir, and multidrug were examined (Table 7).
[ Table 7]
The sites of mutation in the polymerase caused by the 5 RT inhibitors were 180L, 181A, 204M and 236N in the polymerase, and the resulting mutation in HBsAg occurred at position 172, 173, 195 or 196. The polymerase mutant becomes more resistant in the case where a double mutation occurs therein; however, they become more resistant even in cases where a single mutation occurs in one of the two sequences. Therefore, in consideration of the combinations thereof, 14 combinations are created. Drug resistant strains are listed in table 8 below.
[ Table 8]
The binding ability of GC-100A to the mutants was examined using NAGE. For all 14 constructs, GC-100A showed similar binding capacity to it as to WT (FIGS. 24 to 26). Thus, GC-100A showed good binding regardless of the nucleotide/nucleoside resistant mutant, and thus it could be applied to patients who could not be cured due to the generation of resistant mutant.
The antibody was deposited at 12/5 in 2018 at the institute of bioscience and biotechnology under accession number KCTC13760 BP.
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