Patent Application
20100291545
The present invention relates to an antibody having inhibitory activity on infection with hepatitis C virus (HCV) and use thereof.
Hepatitis C viruses (HCV) were found and identified as major causative viruses of non-A and non-B hepatitis (Non-patent Document 1). Further, highly sensitive methods for detecting HCV had been established recent years, and the number of new HCV patients because of blood transfusion dramatically decreased. However, the number of virus carriers in Japan is deduced to be over 2,000,000, and the world wide figure is over 170,000,000, including so-called virus carriers who have not yet developed hepatitis symptoms. This is mainly because the rate of chronicity of hepatitis due to HCV infection is as high as 70% to 80% and there are no effective antivirus agents other than interferons at present. Further, hepatitis C is a serious infection with a poor prognosis since approximately half of the patients with chronic hepatitis C would unexceptionally experience worsening of symptoms from hepatitis C to cirrhosis and hepatic cancer. Accordingly, development of antivirus agents or vaccines aimed at preventing virus carriers from developing the disease or eliminating viruses has been awaited.
When a virus having an envelope such as HCV infects a cell, a virus first needs to bind to a specific protein (i.e., a virus receptor) on the cell surface. Thereafter, the virus membrane is fused with a cell membrane and the virus gene is injected into the cell. Treatment and prevention of HCV require the development of an antibody that can inhibit cellular infection. In particular, an antibody that can prevent HCV from binding to a virus receptor on the cell surface and invading into the cell is important.
An HCV envelope protein is considered to play a key role in binding of HCV to a cell surface. Thus, research has been made regarding detection of an antibody reacting with an envelope protein in the blood serum of the patient. The percentage of patients exhibiting positive reactions with either the C100 antibody (the NS4-NS5 antibody) or the core antibody and exhibiting positive reaction with an envelope protein antibody was found to be approximately 10%. Since only 3 of 31 patients exhibiting positive reactions with the envelope protein antibody are naturally cured, the development of neutralizing antibodies in these 3 patients was considered (Non-patent Document 2). The percentage of patients who experienced the development of neutralizing antibodies was as low as 1% of all patients.
In the case of acute hepatitis B, however, development of antibodies against envelope proteins of the hepatitis B virus is 100%, and viruses are destroyed. In the case of HIV, antibodies against HIV envelope proteins are detected with high frequency. The fact that antibodies against HCV envelope proteins are detected in only 10% of the patients is deduced to result from the presence of a mechanism that may complicate development of antibodies against HCV envelope proteins (Non-patent Document 3).
Meanwhile, it was demonstrated that HCV E2 proteins expressed in mammalian animal cells would specifically bind to CD81, which is present on the human cell surface (Non-patent Document 4). With the use of such experimentation system, development of an antibody exhibiting activity of neutralizing binding (i.e., neutralization of binding, referred to as “NOB”) of E2 proteins and CD81 from hepatitis C patients has been attempted.
A representative example is an antibody exhibiting NOB activity that is obtained by preparing an antibody gene library from marrow lymphocytes of patients of chronic hepatitis C of genotype 1a and obtaining an antibody of interest via phage display (Patent Document 1). Another research group prepared hybridomas from peripheral B cells of patients of hepatitis C of genotype 1b and obtained an antibody exhibiting NOB activity (Non-patent Document 5, Patent Document 2).
According to the abovementioned method of obtaining monoclonal antibodies from HCV patients, however, the number of patients is limited, and the source is limited to patients in which antibodies inhibiting infection with HCV are present. Thus, it is difficult to increase the repertoires of antibodies inhibiting infection and to discover antibodies useful as anti-HCV agents.
Further, a method in which recombinant envelope proteins are administered to a mouse to induce antibodies (Patent Document 3) and a method in which lymphocytes are fused to myeloma cells to prepare antibody-producing hybridomas to obtain antibodies against envelope proteins (Patent Document 4, Non-patent Document 6) have been attempted. However, antibodies that inhibit HCV infection have not yet been demonstrated.
As a reason why an antibody that neutralizes HCV infection via immunization of an animal with an envelope protein has not yet been prepared, it is suggested that a recombinant envelope protein used for immunization has a structure different from that of the envelope protein inherent to the virus. It has already been reported that a recombinant envelope protein is likely to undergo aggregation and is not capable of forming a native conformation (Non-patent Document 7).
It has also been demonstrated that an antibody exhibiting NOB activity would not always inhibit infection (Non-patent Document 8). Thus, development of a method for effectively inducing an antibody against an envelope protein that is able to inhibit virus infection and an antibody that inhibits virus infection is necessary, from the viewpoint of treatment and prevention of HCV with the use of an antibody.
Under the above circumstances, a technique for preparing infectious HCV particles in a cell culture system has been developed recent years (Patent Document 5, Patent Document 6 and Patent Document 7). Compared with the case in which an envelope protein is expressed via a gene recombination technique and the resultant is used as an antigen, the HCV particles exhibit infectivity, and the conformation of the HCV antigen is thus considered to be maintained. Thus, such HCV particles can be suitable as antigens for producing antibodies for inhibiting HCV infection.
[Patent Document 1] JP Patent Publication (kohyo) No. 2005-531286 A
[Patent Document 2] JP Patent Publication (kohyo) No. 2006-504645 A
[Patent Document 3] JP Patent Publication (kohyo) No. 2004-500366 A
[Patent Document 4] JP Patent Publication (kohyo) No. H-06-505389 A
[Non-patent Document 7] Op de Beeck et al., J. Gen. Virol., 2001, vol. 82, pp. 2589-2595
The object of the present invention is to provide an antibody that inhibits infection with hepatitis C virus (HCV).
The present inventors administered hepatitis C virus (HCV) particles of the JFH-1 strain or a chimera virus of the J6CF strain and the JFH-1 strain as an antigen to mice and measured inhibitory activity on infection with HCV in the blood serum of the mice to which HCV had been administered. As a result, they discovered that such HCV particles had inhibitory activity on infection with HCV.
Further, spleen cells were prepared from mice to which HCV particles had been administered, the spleen cells were fused to mouse myeloma cell lines to prepare antibody-producing hybridomas, inhibitory activity on infection with HCV of antibodies produced by the hybridomas was evaluated, and monoclonal antibodies inhibiting HCV infection were obtained. This has led to the completion of the present invention. Specifically, the present invention includes the following.
[1] An antibody that recognizes a hepatitis C virus (HCV) particle obtained from the genome of a hepatitis C virus (HCV) comprising the following (i) and (ii) ligated to each other as an antigen and has inhibitory activity on infection with hepatitis C virus (HCV):
(i) (a) the 5′ untranslated region, the core protein-encoding sequence, the E1 protein-encoding sequence, the E2 protein-encoding sequence, and the p7 protein-encoding sequence of the JFH-1 strain of the hepatitis C virus (HCV) or (b) the 5′ untranslated region, the core protein-encoding sequence, the E1 protein-encoding sequence, the E2 protein-encoding sequence, and the p7 protein-encoding sequence of the J6CF strain of the hepatitis C virus (HCV); and
(ii) the NS2 protein-encoding sequence, the NS3 protein-encoding sequence, the NS4A protein-encoding sequence, the NS4B protein-encoding sequence, the NS5A protein-encoding sequence, the NS5B protein-encoding sequence, and the 3′ untranslated region of the JFH-1 strain.
[2] The antibody according to [1] above, wherein the hepatitis C virus (HCV) genome is a nucleic acid comprising the nucleotide sequence as shown in SEQ ID NO: 1 or 2 of the Sequence Listing (provided that nucleotide “T” in the nucleotide sequence is read as “U” when the nucleic acid is RNA).
[3] The antibody according to [1] or [2] above, wherein the antibody class is IgM.
[4] The antibody according to any of [1] to [3] above, which recognizes at least 10 continuous amino acids in the amino acid sequence as shown in SEQ ID NO: 24 of the Sequence Listing.
[5] The antibody according to [4] above, which has a heavy chain variable region containing the amino acid sequence as shown in SEQ ID NO: 52 of the Sequence Listing.
[6] The antibody according to [4] or [5] above, which has a light chain variable region containing the amino acid sequence as shown in SEQ ID NO: 54 of the Sequence Listing.
[7] The antibody according to any of [4] to [6] above, which is produced by the hybridoma cell line of Accession Number FERM BP-10982.
[8] The antibody according to any of [1] to [3] above, which recognizes at least 10 continuous amino acids in the amino acid sequence as shown in SEQ ID NO: 44 or 45 of the Sequence Listing.
[9] The antibody according to [8] above, which is produced by the hybridoma cell line of Accession Number FERM BP-10980.
[10] The antibody according to any of [1] to [3] above, which recognizes at least 10 continuous amino acids in the amino acid sequence as shown in SEQ ID NO: 36, 45, 46, 47, or 48 of the Sequence Listing.
[11] The antibody according to [10] above, which is produced by the hybridoma cell line of Accession Number FERM BP-10981.
[12] An inhibitory agent for infection with hepatitis C virus (HCV) comprising, as an active ingredient, the antibody according to any of [1] to [11] above.
[13] A medicament comprising, as an active ingredient, the antibody according to any of [1] to [11] above.
[14] A therapeutic or preventive agent for hepatitis C comprising, as an active ingredient, the antibody according to any of [1] to [11] above.
[15] A method for detecting hepatitis C virus (HCV) comprising detecting hepatitis C virus (HCV) particles using the antibody according to any of [1] to [11] above.
The antibody having inhibitory activity on infection with HCV of the present invention and the use thereof can be used for treatment or prevention of hepatitis C and research for elucidation of the mechanism of HCV infection.
The present invention relates to an antibody that can be induced with the use of an infectious HCV particle produced from the specific HCV genome as the antigen and that can inhibit HCV infection.
The present invention can be implemented via conventional molecular biological and immunological techniques within the technical scope in the art. Such techniques are thoroughly described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (vol. 3, 2001) or Ed Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
This description includes part or all of the contents as disclosed in Japanese Patent Application No. 2007-193413, to which the present application claims priority.
Infectious HCV particles that can be used as antigens in the present invention can be produced in a cell culture system. Fundamental techniques for doing so are described in WO 04104198 A1, WO 06022422 A1, WO 06096459 A2, Wakita, T. et al., Nat. Med. 11: 791-796, 2005, Lindenbach, B. D. et al., Science 309:623-626, 2005, and Pietschmann, T. et al., Proc. Natl. Acad. Sci. U.S.A., 103: 7408-7413, 2006.
A specific example of the HCV genome that can be used for generating infectious HCV particles used for producing the antibody having inhibitory activity on infection with HCV of the present invention is virus genome RNA of the JFH-1 strain of genotype 2a, which comprises, sequentially from the 5′ end to the 3′ end, the 5′ untranslated region, the core protein-encoding sequence, the E1 protein-encoding sequence, the E2 protein-encoding sequence, the p7 protein-encoding sequence, the NS2 protein-encoding sequence, the NS3 protein-encoding sequence, the NS4A protein-encoding sequence, the NS4B protein-encoding sequence, the NS5A protein-encoding sequence, the NS5B protein-encoding sequence, and the 3′ untranslated region. Alternatively, such HCV genome can be composed of virus genome RNAs of two or more types of HCV strains, comprising, sequentially from the 5′ end to the 3′ end, the 5 untranslated region, the core protein-encoding sequence, the E1 protein-encoding sequence, the E2 protein-encoding sequence, the p7 protein-encoding sequence, and NS2 protein-encoding sequence of an HCV strain other than the JFH-1 strain and the NS3 protein-encoding sequence, the NS4A protein-encoding sequence, the NS4B protein-encoding sequence, the NS5A protein-encoding sequence, the NS5B protein-encoding sequence, and the 3′ untranslated region of the JFH-1 strain. Further, the HCV genome can be composed of virus genome RNAs of two or more types of HCV strains, comprising, sequentially from the 5′ end to the 3′ end, the 5′ untranslated region, the core protein-encoding sequence, the E1 protein-encoding sequence, the E2 protein-encoding sequence, and the p7 protein-encoding sequence of an HCV strain other than the JFH-1 strain and the NS2 protein-encoding sequence, the NS3 protein-encoding sequence, the NS4A protein-encoding sequence, the NS4B protein-encoding sequence, the NS5A protein-encoding sequence, the NS5B protein-encoding sequence, and the 3′ untranslated region of the JFH-1 strain.
According to a preferable embodiment of the present invention, the HCV genome is a chimeric genome comprising, sequentially from the 5′ end to the 3′ end, the full-length genome derived from the JFH-1 strain of genotype 2a (e.g., the genome comprising the nucleotide sequence as shown in SEQ ID NO: 1) and the 5′ untranslated region, the core protein-encoding region, the E1 protein-encoding region, the E2 protein-encoding region, the p7 protein-encoding region, a region up to amino acid 16 at the N-terminus of the NS2 protein-encoding region, a region from amino acid 17 at the N terminus to the C terminus of the NS2 protein-encoding region of the J6CF strain, the NS3 protein-encoding region, the NS4A protein-encoding region, the NS4B protein-encoding region, the NS5A protein-encoding region, the NS5B protein-encoding region, and the 3′ untranslated region derived from the JFH-1 strain of genotype 2a. A particularly preferable example thereof is a nucleic acid cloned into J6/JFH-1 comprising the nucleotide sequence as shown in SEQ ID NO: 2.
According to a particularly preferable embodiment of the present invention, the HCV genome is a nucleic acid comprising the nucleotide sequence as shown in SEQ ID NO: 1 or 2, provided that nucleotide “T” in the nucleotide sequence is read as “U” when the nucleic acid is RNA. The infectious HCV particles of the present invention can be produced with the use of HCV genomic RNA or HCV genomic DNA.
Specifically, the present invention relates to an antibody having activity of inhibiting infection with HCV, which reacts with, as an antigen, the HCV particle obtained from (a) the HCV genome comprising, sequentially from the 5′ end to the 3′ end, the 5′ untranslated region, the core protein-encoding sequence, the E1 protein-encoding sequence, the E2 protein-encoding sequence, and the p7 protein-encoding sequence of the JFH-1 strain, the NS2 protein-encoding sequence, the NS3 protein-encoding sequence, the NS4A protein-encoding sequence, the NS4B protein-encoding sequence, the NS5A protein-encoding sequence, the NS5B protein-encoding sequence, and the 3′ untranslated region of the JFH-1 strain or (b) the HCV genome comprising, sequentially from the 5′ end to the 3′ end, the 5′ untranslated region, the core protein-encoding sequence, the E1 protein-encoding sequence, the E2 protein-encoding sequence, and the p7 protein-encoding sequence of the J6CF strain of HCV and the NS2 protein-encoding sequence, the NS3 protein-encoding sequence, the NS4A protein-encoding sequence, the NS4B protein-encoding sequence, the NS5A protein-encoding sequence, the NS5B protein-encoding sequence, and the 3′ untranslated region of the JFH-1 strain (preferably the HCV genome comprising, sequentially from the 5′ end to the 3′ end, the 5′ untranslated region, the core protein-encoding sequence, the E1 protein-encoding sequence, the E2 protein-encoding sequence, the p7 protein-encoding sequence, and a sequence encoding a region up to amino acid 16 at the N terminus of the NS2 protein-encoding region of the J6CF strain and a sequence encoding a region from amino acid 17 at the N terminus to the C terminus of the NS2 protein-encoding region, the NS3 protein-encoding sequence, the NS4A protein-encoding sequence, the NS4B protein-encoding sequence, the NS5A protein-encoding sequence, the NS5B protein-encoding sequence, and the 3′ untranslated region of the JFH-1 strain).
When activity of the antibody of the present invention for inhibiting infection is evaluated, in addition to the HCV particles (a) or (b) above, HCV particles obtained from the HCV genome (c) comprising the 5′ untranslated region of the JFH-1 strain, the core protein-encoding sequence, the E1 protein-encoding sequence, the E2 protein-encoding sequence, and the p7 protein-encoding sequence of the TH strain of genotype 1b (Wakita, T. et al., J. Biol. Chem., 269, 14205-14210, 1994, JP Patent Publication (kokai) No. 2004-179 A), the NS2 protein-encoding sequence, the NS3 protein-encoding sequence, the NS4A protein-encoding sequence, the NS4B protein-encoding sequence, the NS5A protein-encoding sequence, the NS5B protein-encoding sequence, and the 3′ untranslated region of the JFH-1 strain ligated in that order (preferably the HCV genome comprising the 5′ untranslated region derived from the JFH-1 strain, the core protein-encoding sequence, the E1 protein-encoding sequence, the E2 protein-encoding sequence, the p7 protein-encoding sequence, and a sequence encoding a region up to amino acid 33 at the N terminus of the NS2 protein-encoding region of the TH strain, a sequence encoding a region from amino acid 34 at the N terminus to the C terminus of NS2 protein-encoding region, the NS3 protein-encoding sequence, the NS4A protein-encoding sequence, the NS4B protein-encoding sequence, the NS5A protein-encoding sequence, the NS5B protein-encoding sequence, and the 3′ untranslated region of the JFH-1 strain ligated in that order) can be used.
The above infectious HCV particles can be prepared by synthesizing RNA from a vector comprising cDNA of full-length HCV genomic RNA according to any of (a) to (c) above cloned into a site downstream of the transcription promoter (e.g., a vector comprising HCV genomic RNA cloned under the control of the T7 promoter) and introducing the RNA into cells. RNA can be synthesized in vitro with the use of a nucleic acid comprising HCV cDNA cloned under the control of the T7 promoter as a template using a kit, such as the MEGAscript T7 kit (Ambion). RNA may be introduced into any cells, provided that such cells permit generation of HCV particles. Examples thereof include cultured cells of Huh7 cells, HepG2 cells, IMY-N9 cells, HeLa cells, and 293 cells. A more preferable example is liver-derived cultured cells such as Huh7 cells. Further preferable examples include Huh7 cells and cells derived from Huh7 cells (i.e., Huh7.5 cells and Huh7.5.1 cells). Further, cells obtained by expressing CD81 and/or Claudin1 genes in Huh7 cells, HepG2 cells, IMY-N9 cells, HeLa cells, or 293 cells can be used. Huh7 cells or derivative strain cells from Huh7 cells are particularly preferable. In the present invention, the term “derivative strain” refers to cell lines derived from the relevant cells.
RNA can be introduced into cells by any known methods. Examples of such methods include calcium phosphate coprecipitation, the DEAE-dextran method, lipofection, microinjection, and electroporation. Lipofection and electroporation are preferable and electroporation is more preferable.
The capacity of the cells for virus particle production can be evaluated with the use of an antibody reacting with a factor that constitutes HCV particles released in the culture solution, such as the core protein, the E1 protein, or the E2 protein. Also, HCV genomic RNA contained in the HCV particles in the culture solution may be amplified via RT-PCR using specific primers to indirectly detect the presence of HCV particles.
Whether or not the prepared viruses are infectious can be evaluated by culturing cells into which HCV RNA has been introduced in the aforementioned manner, applying (adding) the resulting supernatant to HCV permissive cells (e.g., Huh7 cells), and immunologically staining the cells with an anti-core antibody 48 hours later to count the number of infected cells. Alternatively, evaluation can be carried out by subjecting the cell extract to electrophoresis on SDS-polyacrylamide gel and detecting core proteins via Western blotting.
The virus solution containing the infectious HCV particles obtained in (1) above is subjected to, for example, centrifugation and/or filtration through a filter to remove cells and cell residues. A solution from which residues have been removed can be concentrated approximately 10- to 100-fold using a ultrafiltration membrane with a molecular weight cut off of 100,000 to 500,000. A solution containing HCV from which residues have been removed can be purified via chromatography and density-gradient centrifugation in arbitrary combinations or alone. Hereafter, representative chromatography or density-gradient centrifugation techniques are described, although the techniques are not limited thereto.
Gel filtration chromatography can be carried out preferably using a chromatography support comprising a cross-linked polymer of allyl dextran and N,N′-methylenebisacrylamide as the gel matrix, and more preferably by using Sephacryl® S-300, 5-400, and S-500 chromatography to purify infectious HCV particles.
Ion-exchange chromatography can be carried out using, preferably, Q-Sepharose® as an anion exchange resin and SP Sepharose® as a cation exchange resin to purify HCV particles.
Affinity chromatography can be carried out preferably using, as a support, resin comprising a substrate selected from among heparin, sulfated cellulofine, lectin, and various dyes bound thereto as a ligand to purify HCV particles. Further preferably, HCV particles can be purified with the use of a support comprising HiTrap Heparin HP®, HiTrap Blue HP®, HiTrap Benzamidine FF®, sulfated cellulofine, LCA, ConA, RCA-120, and WGA bound thereto. The most preferable method is purification of HCV particles with the use of sulfated cellulofine as a support. HCV particles can be purified 30-fold or more in terms of the ratio of the HCV RNA copy number to the total protein amount in the solution.
Purification via density-gradient centrifugation can be preferably carried out with the use of a sugar polymer, such as cesium chloride, sucrose, Nycodenz®, Ficoll®, or Percoll®, as a solute that makes density gradient. Sucrose can be further preferably used. Preferably, water or a buffer, such as phosphate, Tris, acetate, or glycine buffer, can be used. The centrifugal force that is employed at the time of purification via density-gradient centrifugation is preferably 1×104 to 1×109 g, more preferably 5×104 to 1×107 g, and most preferably 5×104 to 5×105 g.
Purification is carried out preferably at 0° C. to 40° C., more preferably 0° C. to 25° C., and most preferably 0° C. to 10° C.
When purification is carried out via density-gradient centrifugation in combination with column chromatography, such techniques may be carried out in any order. Preferably, HCV particles are first purified through a plurality of chromatography columns followed by density-gradient centrifugation. More preferably, a fraction containing HCV particles obtained via anion exchange column chromatography followed by affinity chromatography is subjected to purification via density-gradient centrifugation. Most preferably, a fraction containing HCV particles obtained with the use of a Q-Sepharose® column is further purified with the use of a sulfated cellulofine-based column, and the resulting fraction containing HCV particles is then purified via density-gradient centrifugation. During the steps of column chromatography and density-gradient centrifugation, dialysis or ultrafiltration may be carried out to substitute a solute of a solution containing HCV particles and/or to concentrate HCV particles.
The antibody of the present invention reacts with the HCV particle as an antigen. When producing the antibody of the present invention, infectivity of the HCV particles is not correlated with antigenicity, although use of inactivated HCV particles is preferable. Infectious HCV particles can be inactivated by adding and mixing an inactivator such as formalin, β-propiolactone, or glutardialdehyde in, for example, a virus suspension and allowing the inactivator to react with viruses (Appaiahgari et al., Vaccine, 22: 3669-3675, 2004). Further, infectious HCV particles may be irradiated with ultraviolet rays to eliminate infectivity of viruses, and viruses can be immediately inactivated. Irradiation with ultraviolet rays realizes virus inactivation with little influence on proteins that constitute viruses. A source of ultraviolet rays used for inactivation can be a commercially available germicidal lamp. In particular, a 15 w germicidal lamp can be used, although the source is not limited thereto. In the present invention, HCV particles that are purified by the method described above are used, and methods of inactivation are not limited by a purified or unpurified state. Preferably, a solution containing infectious HCV particles may be irradiated with ultraviolet rays at 20 mW/cm2 at room temperature for at least 5 minutes to inactivate infectious HCV particles.
The antibody of the present invention can be obtained by administering HCV particles to animals to induce the antibody via immune reaction. Any non-human animals (non-human mammalian animals), such as mice, rats, hamsters, or rabbits, which can produce spleen cells capable of producing hybridomas, may be used for immunization. In the present invention, mice are preferable, and examples involving the use of mice are described below.
In general, 4- to 10-week-old mice may be immunized with the HCV particle antigen obtained in the steps (1) to (3) above. According to circumstances, the step of purification may be altered or omitted, and virus inactivation may be omitted. In general, immunization is carried out by hypodermically or intraperitoneally administering an antigen with an adjuvant several times. Examples of adjuvants include, but are not limited to, the Freund's complete adjuvants, the Freund's incomplete adjuvant, aluminum hydroxide gel with pertussis vaccine, Titer Max Gold (Vaxel), and the GERBU adjuvant (GERBU Biotechnik). Final immunization is carried out without the use of an adjuvant, the antigen is intravenously or intraperitoneally administered, and polyclonal antibodies are obtained from the blood serum of the immunized mice 3 to 10 days, and preferably 4 days, after the final administration of HCV particles in accordance with a known method (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). Whether or not the immunized animal produces the antibody of the present invention can be inspected by sampling the blood from the venous plexus of the ocular fundus or caudal vein of the immunized animal before the final immunization and measuring the activity of inhibiting infection with HCV used as the antigen.
The antibody according to the present invention may be a monoclonal or polyclonal antibody, and preferably, a monoclonal antibody. Also, the antibody of the present invention may be derived from any organism, preferably a mammalian animal, and more preferably a human. The antibody of the present invention may be a chimeric antibody (e.g., a chimeric antibody of a human antibody and an antibody derived from another mammalian animal). An example of particularly preferable chimeric antibody is a humanized antibody obtained by transplanting a sequence at the hypervariable region of a mouse antibody into a human antibody. The antibody of the present invention may be a chimeric antibody having a framework region of a human antibody and a heavy chain variable region and/or a light chain variable region including a hypervariable region of an antibody derived from another mammalian animal.
(5) Preparation of Hybridoma Cell that Produces Monoclonal Antibody
When the antibody of the present invention, and in particular, when the monoclonal antibody, is prepared, the spleen is removed from the immunized animal, and the spleen cells are fused to the myeloma cells to prepare hybridoma cells. Any myeloma cells that can be multiplied in vitro can be used for cell fusion. Examples thereof include mouse-derived established cells, such as 8-azaguanine-resistant mouse (BALB/c-derived) myeloma cell lines P3-X63Ag8-U1 (P3-U1), SP2/0-Ag14 (SP2/0), P3-X63-Ag8653 (653), P3-X63-Ag 8 (X63), and P3/NS1/1-Ag4-1 (NS1). These cell lines are available from RIKEN BioResource Center, ATCC (American Type Culture Collection), or ECACC (European Collection of Cell Cultures). Culture and subculture are carried out in accordance with a conventional technique (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, Selected Methods in Cellular Immunology W.H. Freeman and Company, 1980).
The spleen cells and the myeloma cells obtained above are washed, the myeloma cells are mixed with the spleen cells at a proportion of 1:1 to 10, and polyethylene glycol is added thereto for cell fusion. Polyethylene glycol is capable of fusing cells, polyethylene glycol with less cytotoxicity is preferable, and, for example, polyethylene glycol-1500 (PEG-1500) can be preferably used. After cell fusion, cells are suspended and washed in a medium. Cells are washed using a medium that is used for myeloma cell culture, such as Dulbecco's modified MEN medium or RPMI-1640. Culture medium for fused cells is selected so as to selectively obtain fused cells of interest, and an example of such medium is a medium prepared by adding hypoxanthine (10−4 M), thymidine (1.5×10−5M), and aminopterin (4×10−7M) to HAT medium (a medium prepared by adding 2-mercaptoethanol (5×10−5M), penicillin (100 units/ml), streptomycin (100 μg/ml), and fetal calf serum (FCS)(10%, Invitrogen) to a medium used for myeloma cell culture, such as Dulbecco's modified MEN medium).
After culture, part of the culture supernatant is separated, and cells producing antibodies that inhibit infection with HCV are selected via an HCV infection test. Alternatively, cells producing antibodies that react with HCV proteins can be selected via enzyme immunoassay. Subsequently, cloning is carried out via limiting dilution or colony formation in a methylcellulose medium, and cells that have been proven to produce antibodies reacting with HCV proteins and antibodies having inhibitory activity on infection with HCV are selected as a monoclonal antibody-producing hybridoma cell line.
(6) Selection of Anti-HCV Monoclonal Antibody Having Inhibitory Activity on Infection with HCV
Hybridomas that produce anti-HCV monoclonal antibodies having inhibitory activity on infection with HCV can be selected via a method for assaying inhibitory activity on infection with HCV using infectious HCV particles as follows or described in the Examples below.
At the outset, infectious HCV particles (the method for producing the same was described above) are mixed with the antibody sample, and the reaction is allowed to proceed at 37° C. for 1 hour. The sample (50 μl) is added to the Huh7 cells, which had been cultured on the previous day at 5×103 cells/well on a 96-well plate, and culture is conducted at 37° C. for 2.5 hours. After culture, the sample is removed, cells are washed with PBS, a fresh medium is added, and culture is continued. The culture supernatant is removed 48 hours later, cells are washed once with PBS, 100 μl of ISOGEN (Nippon Gene) is added, RNA is prepared from the cells, RNA is quantified, and the amount of HCV genomic RNA is then measured. HCV RNA is detected via quantitative RT-PCR by detecting RNA in the 5′ end untranslated region of HCV RNA according to the method of Takeuchi et al. (Takeuchi T. et al., Gastroenterology, 116: 636-642, 1999).
Alternatively, inhibitory activity on infection with HCV can be evaluated by the following method. At the outset, the antibody sample is mixed with infectious HCV particles, and the resulting mixture is subjected to a reaction at 37° C. for 1 hour. Subsequently, the sample (50 μl) is added to the Huh7 cells, which had been cultured on the previous day at 1×104 cells/well on a 96-well plate, and culture is conducted at 37° C. for 2.5 hours. After culture, the sample is removed, cells are washed with PBS, a fresh medium is added, and culture is continued. The culture supernatant is removed 72 hours later, the plate is introduced into ice-cooled methanol, and cells are fixed. Thereafter, methanol is removed via air drying, and cells are permeabilized with the use of Block Ace® (Dainippon Pharmaceutical Co., Ltd.) containing 0.3% Triton®-X 100 (GE Healthcare). The number of HCV-infected cells is counted under a fluorescent microscope (IX-70; Olympus) using a clone 2H9 anti-HCV-core antibody (Nat. Med., 2005, 11: pp. 791-6) and goat anti-mouse IgG-Alexa488 (Molecular Probes), and the samples in the wells in which HCV infection is inhibited are designated as positive clones. Thus, target hybridomas can be selected. A monoclonal antibody produced by the hybridoma thus selected is an antibody according to a preferable embodiment of the present invention.
Hybridomas that produce the antibodies of the present invention are not particularly limited, provided that such hybridomas are selected in the above manner. Hybridoma cell lines of Accession Number FERM BP-10980, Accession Number FERM BP-10981, and Accession Number FERM BP-10982 are preferable. Hybridoma cell lines J6/1G11-25 (Accession Number: FERM BP-10980; transferred from Accession Number: FERM P-21318 deposited on Jul. 19, 2007 (Receipt Number: FERM AP-21318) to international deposit), J6/4D4-2 (Accession Number: FERM BP-10981; transferred from Accession Number: FERM P-21319 deposited on Jul. 19, 2007 (Receipt Number: FERM AP-21319) to international deposit), and JF/M1-4 (Accession Number: FERM BP-10982; transferred from Accession Number: FERM P-21320 deposited on Jul. 19, 2007 (Receipt Number: FERM AP-21320) to international deposit) were deposited at the International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan) as of Jul. 19, 2007. These hybridoma cell lines can be preferably cultured in a medium prepared by adding 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, and 10% fetal calf serum to Dulbecco's modified Eagle medium (high glucose) at 37° C.
In order to analyze an epitope of the antibody of the present invention produced by the hybridoma cell line selected in the above-described manner, HCV protein-based enzyme immunoassay (EIA), Western blotting, dot blotting, or other means can be employed. By performing such epitope analysis for the primary screening of the anti-HCV monoclonal antibody, the anti-HCV monoclonal antibody that targets a given HCV protein can be effectively screened.
A more preferable antibody having inhibitory activity on infection with HCV according to the present invention recognizes the amino acid sequence WLTPKCLVHYPYRLWHYPC (SEQ ID NO: 24) or an amino acid sequence comprising at least 10 continuous amino acids of WLTPKCLVHYPYRLWHYPC (SEQ ID NO: 24) of the E2 protein of HCV (and in particular, the JFH-1 strain of HCV) as an epitope. An amino acid sequence comprising at least 10 continuous amino acids in WLTPKCLVHYPYRLWHYPC is preferably LVHYPYRLWH (SEQ ID NO: 18), WLTPKCLVHY (SEQ ID NO: 19), PKCLVHYPYR (SEQ ID NO: 20), or YPYRLWHYPC (SEQ ID NO: 21), with LVHYPYRLWH (SEQ ID NO: 18) being more preferable. An antibody that recognizes NFTIFKIRMY (SEQ ID NO: 22) or IFKIRMYVCG (SEQ ID NO: 23), which is an amino acid sequence of the HCV E2 protein, is a more preferable antibody having inhibitory activity on infection with HCV according to the present invention. In the present description, for example, the term “amino acids 1 to 162 of the E1 protein” refers to a region from amino acids 1 to 162 in the amino acid sequence of the E1 protein. Also, a similar expression representing part of a protein or nucleic acid is to be similarly understood.
An example of the antibody according to a preferable embodiment of the present invention is an antibody having a heavy chain variable region containing the amino acid sequence as shown in SEQ ID NO: 52. Further, another example of the antibody according to a preferable embodiment of the present invention is an antibody having a light chain variable region containing the amino acid sequence as shown in SEQ ID NO: 54. An antibody having a heavy chain variable region containing the amino acid sequence as shown in SEQ ID NO: 52 and a light chain variable region containing the amino acid sequence as shown in SEQ ID NO: 54 (preferably a monoclonal antibody) is more preferable. An example of such monoclonal antibody is described as the JF/M1-4 monoclonal antibody in the Examples below. An antibody having a heavy chain variable region containing the amino acid sequence as shown in SEQ ID NO: 52 and/or a light chain variable region containing the amino acid sequence as shown in SEQ ID NO: 54 may be an antibody derived from any organism, preferably an antibody derived from a mammalian animal, and more preferably a human antibody or a chimeric antibody. A chimeric antibody obtained by transplanting a heavy chain and/or light chain variable region derived from an arbitrary organism (e.g., a mouse that can be easily genetically engineered) or a complementary determining region (a hypervariable region) thereof into an equivalent site of an antibody (preferably a monoclonal antibody) derived from an organism (e.g., a human) to which the antibody of interest is to be administered can be used. A particularly preferable example of a chimeric antibody is a humanized antibody obtained by transplanting a sequence of a hypervariable region (also referred to as the “complementary determining region (CDR)”) of a mouse antibody into the relevant site of a human antibody.
The antibody of the present invention can comprise, as the complementary determining regions (CDRs) of the H chain V region (i.e., the heavy chain variable region), CDR1, CDR2, and CDR3 having the amino acid sequences as shown in SEQ ID NOs: 56, 58, and 60 derived from the JF/M1-4 monoclonal antibody, respectively. Also, the antibody of the present invention can comprise, as the complementary determining regions (CDRs) of the L chain V region (i.e., the light chain variable region), CDR1, CDR2, and CDR3 having the amino acid sequences as shown in SEQ ID NOs: 63, 65, and 67 derived from the JF/M1-4 monoclonal antibody, respectively. According to a further preferable embodiment, the antibody of the present invention can comprise, as the complementary determining regions (CDRs) of the H chain V region (i.e., the heavy chain variable region), CDR1, CDR2, and CDR3 having the amino acid sequences as shown in SEQ ID NOs: 56, 58, and 60, respectively, and, as the complementary determining regions (CDRs) of the L chain V region (i.e., the light chain variable region), CDR1, CDR2, and CDR3 having the amino acid sequences as shown in SEQ ID NOs: 63, 65, and 67, respectively. Such antibody can comprise any framework 1 to 4 regions in the heavy chain variable region and the light chain variable region. Such antibody can also comprise framework 1 to 4 regions of an any antibody class derived from an organism other than a mouse (e.g., human-derived framework 1 to 4 regions). Such antibodies having CDR1 to CDR3 of the H chain V region and/or CDR1 to CDR3 of the L chain V region derived from the JF/M1-4 monoclonal antibody show activities of inhibiting infection with HCV particles of genotype 2a.
According to another embodiment of the present invention, a more preferable antibody having inhibitory activity on infection with HCV recognizes an amino acid sequence comprising at least 10 continuous amino acid residues in the amino acid sequence NVTNPEDMPRPYCW (SEQ ID NO: 44) or NYTIFKIRMYVGG (SEQ ID NO: 45) of the E2 protein of HCV (and in particular, HCV of the J6CF strain) as an epitope. Examples of amino acid sequences comprising at least 10 continuous amino acids in the amino acid sequence NVTNPEDMPRPYCW (SEQ ID NO: 44) or NYTIFKIRMYVGG (SEQ ID NO: 45) include NVTNPEDMRP (SEQ ID NO: 29), NPEDMRPYCW (SEQ ID NO: 30), NYTIFKIRMY (SEQ ID NO: 31), and IFKIRMYVGG (SEQ ID NO: 32).
According to a further embodiment of the present invention, a more preferable antibody having inhibitory activity on infection with HCV recognizes an amino acid sequence comprising at least 10 continuous amino acids in the amino acid sequence FIVSPQHHWFVQDCNC (SEQ ID NO: 46) or MAWDMMMNWS (SEQ ID NO: 36) of the E1 protein of HCV (and in particular, HCV of the J6CF strain) or the amino acid sequence LIDYPYRLWHYPC (SEQ ID NO: 47), NPEDMRPYCWHYPPRQ (SEQ ID NO: 48), or NYTIFKIRMYVGG (SEQ ID NO: 45) of the E2 protein as the epitope. Examples of amino acid sequences each comprising at least 10 continuous amino acids of the amino acid sequences in FIVSPQHHWFVQDCNC (SEQ ID NO: 46), MAWDMMMNWS (SEQ ID NO: 36), LIDYPYRLWHYPC (SEQ ID NO: 47), NPEDMRPYCWHYPPRQ (SEQ ID NO: 48), or NYTIFKIRMYVGG (SEQ ID NO: 45) of the E2 protein include FIVSPQHHWF (SEQ ID NO: 33), SPQHHWFVQD (SEQ ID NO: 34), HHWFVQDCNC (SEQ ID NO: 35), and MAWDMMMNWS (SEQ ID NO: 36) of E1-derived epitopes. Examples of E2-derived epitopes include LIDYPYRLWH (SEQ ID NO: 37), YPYRLWHYPC (SEQ ID NO: 38), DMRPYCWHYP (SEQ ID NO: 39), NPEDMRPYCW (SEQ ID NO: 40), and PYCWHYPPRQ (SEQ ID NO: 41).
In order to select the antibody of the present invention, more specifically, HCV proteins are immobilized on a support (i.e., solid-phased), the antibody sample is then added thereto, and the reaction is then allowed to proceed for a certain period of time under conditions that are sufficient for forming an antibody/antigen complex. Subsequently, the thus-formed complex is brought into contact with an antibody (i.e., a secondary antibody) that recognizes the antibody sample to which a signal enzyme, dye, or radioisotope has been bound to form the second mixture. The second mixture is subjected to the reaction for a certain period of time under the conditions that are sufficient for forming an antibody/antigen complex. The presence of an antibody that recognizes the HCV protein is detected with the aid of a signal of an enzyme, dye, or radioisotope.
As HCV proteins to be immobilized on a support, HCV particles may be used. Alternatively, proteins expressed in E. Coli, yeast, mammalian animal cells, insect cells, or the like with the use of cDNA comprising the core protein-encoding sequence, the E1 protein-encoding sequence, the E2 protein-encoding sequence, the p7 protein-encoding sequence, the NS2 protein-encoding sequence, the NS3 protein-encoding sequence, the NS4A protein-encoding sequence, the NS4B protein-encoding sequence, the NS5A protein-encoding sequence, or the NS5B protein-encoding sequence of the HCV genome may be used. Further, such proteins may be chemically synthesized and used. The length of an amino acid sequence of a protein to be used for immobilization is not limited, and such length is 3 or amino acids and more preferably 8 amino acids.
Examples in which the E1 protein and the E2 protein, which are envelope proteins of the JFH-1 strain or the J6CF strain, are expressed in mammalian animal cells are described below. The E1 protein of the JFH-1 strain or the J6CF strain starts from amino acid 192 and ends at amino acid 383, when amino acid 1 starts at the initiator methionine of the amino acid sequence of the full-length JFH-1 protein (NCBI Protein Accession Number BAB32872). A region from amino acid 353 to amino acid 383 of the E1 protein is considered to be a transmembrane domain (also referred to as a “C-terminal hydrophobic domain”) (Cocquerel, L. et al., J. Virol. 74:3623-3633, 2000).
The E2 protein of the JFH-1 strain or the J6CF strain starts from amino acid 384 and ends at amino acid 750 of the above amino acid sequence. A region from amino acid 722 to amino acid 750 of the E2 protein is considered to be a transmembrane domain (Cocquerel, L. et al., J. Virol. 74: 3623-3633, 2000).
When proteins are secreted and expressed in the culture supernatant in mammalian animal cells, it is necessary for such proteins to have signal peptides, but it is not necessary for them to have transmembrane domains.
Accordingly, a protein that does not have the transmembrane domain of the E1 or E2 protein comprises a region from amino acid 192 to amino acid 352 or a region from amino acid 384 to amino acid 721. Shift of an amino acid location is not problematic, provided that the amino acid is qualitatively equivalent. In the present invention, a sequence from amino acid 192 to amino acid 352 of the E1 protein, a sequence from amino acid 384 to amino acid 720 (and preferably a sequence from amino acid 384 to amino acid 714) of the E2 protein of the JFH-1 strain, and a sequence from amino acid 192 to amino acid 352 of the E1 protein, and a sequence from amino acid 384 to amino acid 720 of the E2 protein of the J6CF strain can be used as proteins that do not comprise a transmembrane domain. When such amino acid numbers are applied to other sequences, sequences may be designated with the amino acid numbers corresponding in the alignment with the amino acid sequences of the full-length JFH-1 proteins.
A nucleic acid that encodes a protein not containing any transmembrane domain of the E1 or E2 proteins can be synthesized via PCR using cDNA of JFH-1 as a template based on the nucleic acid sequence of JFH-1 given in GenBank, or such nucleic acid can be fully synthesized.
The corresponding E1 and E2 protein regions of HCV strains other than JFH-1 can be easily determined by aligning the sequences (making alignment), so that the length of aligned sequences become the longest in comparison with the JFH-1 sequence, while taking substitution or deletion of the sequence of the HCV strain into consideration. Such analysis can be carried out using genetic information processing software (e.g., GENETYX, Software Development Co., Ltd.).
When a protein that does not contain a transmembrane domain of the E1 or E2 protein is secreted and expressed in a mammalian animal cell, a nucleic acid encoding such protein is ligated to a site downstream of a nucleic acid that encodes a signal peptide in such a manner that frames of codons are correctly aligned (i.e., in-frame), a stop codon is added to the 3′ terminus, and the resultant is then inserted into an expression vector. A signal peptide is mainly composed of hydrophobic amino acids comprising 15 to 30 amino acid residues located at the N terminus of the secretory protein and a signal peptide is associated with the mechanisms of protein transport through the cell membrane.
A signal peptide that can be used for protein secretion and expression in a mammalian animal cell may be a signal peptide of a secretory protein. Examples of vectors having signal peptides include a vector having a signal peptide sequence of mouse GM-CSF (JP Patent Publication (kokai) No. S63-276490 A (1988)), a pSecTag/FRT/V5-His vector having a signal peptide sequence of the IgG κ strand (Invitrogen), a p3×FLAG-CMV13 vector having a signal peptide sequence of preprotrypsin (Sigma), a pFUSE-Fc2 vector having a signal peptide sequence of IL-2 (InvivoGen), and a pTriEx-7 vector having a signal peptide sequence of IgM (Novagen).
When proteins are expressed, such proteins are expressed as fusion proteins of target proteins and label proteins, and fusion proteins can be detected or purified with the use of an antibody reacting with the label protein or a molecule that specifically binds thereto. Such label proteins are also referred to as “tags.” Label proteins are not limited, and examples thereof include FLAG peptide (also referred to as flag peptide or Flag peptide), 3×FLAG peptide (also referred to as 3× FLAG peptide, 3× Flag peptide, or 3× flag peptide), HA peptide, 3× HA peptide, myc peptide, 6× His peptide, GST polypeptide, MBP polypeptide, PDZ domain polypeptide, alkaline phosphatase, immunoglobulin, and avidin. Such peptides or polypeptides are generally fused to the N- or C-terminus of the target proteins, but such peptides or polypeptides can be inserted into the target proteins according to need. A vector having a fusion polypeptide of preprotrypsin signal peptide and 3× FLAG peptide is available as the p3×FLAG-CMV-9 vector from Sigma.
The hybridomas selected in (6) above are conditioned to serum-free medium, such as Hybridoma-SFM (Invitrogen), and the supernatant cultured in serum-free medium can be designated as a monoclonal antibody sample. Culture can be conducted with the use of a flask, petri dish, spinner culture bottle, roller bottle, or high-density culture flask (CELLine, Becton, Dickinson and Company).
When monoclonal antibodies are prepared from animals, the anti-HCV monoclonal antibody-producing hybridoma cells obtained above are intraperitoneally injected into pristane-treated 8- to 10-week-old mice, nude mice, or SCID mice (0.5 ml of 2,6,10,14-tetramethylpentadecane (pristane) was administered intraperitoneally and grown for 2 weeks) at 2×107 to 5×106 cells/mouse. Hybridomas experience ascites tumor formation within 10 to 21 days. Ascites fluid is sampled from the mice or nude mice to prepare monoclonal antibody samples. The obtained antibody samples are centrifuged to remove cells or fractured cells, the samples are subjected to salting-out with 40% to 50% saturated ammonium sulfate, caprylic acid precipitation, DEAE-Sepharose column, Protein-A column, Protein-G column, HiTrap IgM Purification HP-column (GE Healthcare), mannan binding protein-column (Pierce), or gel filtration column, and such techniques are carried out alone or in adequate combination to recover IgG or IgM fractions. Thus, purified monoclonal antibodies are obtained. The purified monoclonal antibody subclasses are determined with the use of, for example a mouse monoclonal antibody typing kit (Pierce). The classes of the antibodies having inhibitory activity on infection with HCV of the present invention obtained in (4) to (7) above are not particularly limited. In the present invention, IgG or IgM is preferable, with IgM antibodies being more preferable.
Humanized antibodies are also referred to as reshaped human antibodies, such reshaped antibodies are obtained by transplanting complementary determining regions (CDRs) of mammalian animals other than humans (e.g., mouse antibodies) into the complementary determining regions of human antibodies, and general gene recombination techniques are known (EP Patent Publication No. EP 125023). Specifically, a DNA sequence that is designed to ligate CDR of a mouse antibody to a framework region (FR) of a human antibody is synthesized via PCR using several oligonucleotide primers prepared so as to have regions overlapping at the terminal regions of CDR and FR.
Framework regions of human antibodies ligated via complementary determining regions (CDRs) in which CDRs form good antigen-binding sites are selected. According to need, amino acids in the framework regions in the antibody variable region may be substituted, so that the CDRs of the reshaped human antibodies form adequate antigen-binding sites (Sato, K., et al., Cancer Res. 53:851-856, 1993).
When humanized anti-HCV monoclonal antibodies having inhibitory activity on infection with HCV of the present invention are prepared, for example, cDNA encoding the H chain V region (VH) and the L chain V region (VL) of the anti-HCV monoclonal antibody is first obtained in the following manner. mRNA is extracted from hybridomas that produce anti-HCV monoclonal antibodies to synthesize cDNA. The synthesized cDNA is inserted into a phage or plasmid vector to prepare a cDNA library. Recombinant phages or plasmids having cDNA encoding VH and recombinant phages or plasmids having cDNA encoding VL are isolated from the resulting library with the use of the C or V region of the mouse antibody as a probe. The whole nucleotide sequences of VH and VL of the target antibody on the recombinant phage or plasmid are determined, and the whole amino acid sequences of V11 and VL are deduced based on the nucleotide sequences.
Alternatively, cDNA encoding VH and VL can be cloned via PCR. cDNAs of hybridomas prepared in the above-described manner are used as templates, the templates are amplified using a plurality of primers designed based on the amino acid sequences conserved in the relevant genes, and the cDNA fragments are cloned into cloning vectors. Thus, cDNA encoding V11 and VL can be obtained.
cDNA encoding VH and VL of the anti-HCV antibody may be inserted into a region upstream of the gene encoding the H chain C region (CH) and the L chain C region (CL) of the human antibody to construct cDNA encoding a humanized anti-HCV monoclonal antibody. In CH, Cγ1, Cyγ2, Cyγ3, and Cyγ4 can be used, and in CL, Cκ and Cλ can be used. In order to improve stability of the antibody or production thereof, the C region may be modified.
In the case of mammalian animals, a promoter that can express the gene of interest in mammalian animals, the antibody gene to be expressed, and poly A signal is operably linked to a site downstream of the 3′ terminus. The gene of interest can be expressed using it. Examples of promoters that can be used include virus promoters/enhancers, such as human cytomegalovirus, retrovirus, polyoma virus, adenovirus, and simian virus 40 (SV40) and promoters/enhancers derived from mammalian cells, such as the elongation factor 1α (EF1α).
In the case of E. coli, a promoter that can express the gene of interest in E. coli cells, a signal sequence for antibody secretion, and the antibody gene to be expressed may be operably linked to express the gene of interest. Examples of promoters include lacz promoter, araB promoter, Trp promoter, and T7 promoter.
In the case of insect cells, a promoter that can express the gene of interest in insect cells, the antibody gene to be expressed, and poly A signal may be operably linked to a site downstream of the 3′ terminus to express the gene of interest. Examples of promoters that can be used include polyhedrin promoter and baculovirus OpNMPV-derived immediate-early OpIE2 promoter.
The antibody having a heavy chain variable region containing the amino acid sequence as shown in SEQ ID NO: 52 and/or a light chain variable region containing the amino acid sequence as shown in SEQ ID NO: 54 according to the present invention can be prepared in the similar manner to the above-mentioned method for preparing the humanized monoclonal antibody.
When the antibody having inhibitory activity on infection with HCV according to the present invention is used as an inhibiting agent of HCV infection, the antibody is used as a research tool for elucidating the HCV infection mechanism. Further, it is preferably used as a medicament (e.g., a pharmaceutical composition) and it is more preferably used as a therapeutic or preventive agent against hepatitis C. When the antibody according to the present invention is used as a therapeutic or preventive agent against hepatitis C, it is used for preventing HCV infection caused by organ transplantation from a donor organ. Further, it can be used in combination with existing antiviral agents, such as interferon and ribavirin.
The antibody having inhibitory activity on infection with HCV according to the present invention can be effective on arbitrary forms of hepatitis C. For example, it is effective on chronic hepatitis or fulminant hepatitis, and it is particularly effective on hepatitis C induced by HCV of genotype 2a or 1b.
When the therapeutic or preventive agent against hepatitis C of the present invention is administered to a patient, an effective dose of the antibody of the present invention as an active ingredient is between 0.001 mg and 1,000 mg per kg of the body weight. Alternatively, an dose can be between 0.01 to 100,000 mg/body of a patient, although the dose is not limited thereto. Also, the therapeutic or preventive agent can be administered before or after the patient develops clinical symptoms of the disease.
The therapeutic or preventive agent against hepatitis C of the present invention is prepared in accordance with a conventional technique (Remington's Pharmaceutical Science, the latest edition, Mark Publishing Company, Easton, U.S.A.), and such agent may comprise pharmaceutically acceptable carriers or additives.
Examples of such pharmaceutically acceptable carriers and additives include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, carboxymethylcellulose sodium, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methylcellulose, ethylcellulose, xanthan gum, gum Arabic, casein, agar, polyethylene glycol, diglycerine, glycerine, propylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, and a surfactant, which is acceptable as a pharmaceutical additive.
Actual additives are selected from among the above-mentioned additives in accordance with a dosage form of the therapeutic or preventive agent against hepatitis C according to the present invention, and such additive can be used alone or in adequate combinations, although additives are not limited to the above-mentioned additives. When an additive is used for an injection preparation, for example, the purified anti-HCV antibody is dissolved in a solvent, such as physiological saline, buffer, or a glucose solution, an adsorption inhibitor, such as Tween 80, Tween 20, gelatin, human serum albumin, or the like is added thereto, and the resulting mixture can be used. Alternatively, an additive may be lyophilized so as to be in a dosage form that allows it to be dissolved and reconstructed before use. Examples of excipients that can be used for lyophilization include sugar alcohols, such as mannitol or dextrose, and sugars.
The antibody of the present invention can be applied to a diagnostic composition for HCV detection.
For example, a carrier or plate upon which the antibody of the present invention has been immobilized is brought into contact with a test sample, which may contain the HCV antigen, to produce a mixture. The resulting mixture is subjected to a reaction for a period of time under conditions that are sufficient for forming an antibody/antigen complex. Subsequently, the thus-generated complex is brought into contact with an antibody that recognizes the HCV antigen to which a signal enzyme, dye, or radioisotope has been bound to generate a second mixture. The second mixture is subjected to the reaction for a period of time under conditions that are sufficient for forming an antibody/antigen complex. The presence of the HCV antigen is detected with the aid of a signal of an enzyme, dye, or radioisotope.
Alternatively, a test sample, which may contain the HCV antigen, is spotted on a membrane on which proteins can be immobilized (e.g., a nitrocellulose membrane or PVDF membrane) to immobilize proteins contained in a test sample. Subsequently, this membrane is soaked in a 5% skimmed milk or 1% BSA solution to block the membrane. After the membrane has been washed, the membrane is soaked in a solution of the antibody of the present invention to which an enzyme, dye, or radioisotope has been bound, and the reaction is allowed to proceed for a period of time under the conditions that are sufficient for the antigen immobilized on the membrane and the antibody of the present invention to form a complex. After the membrane has been washed, the presence of the HCV antigen immobilized on the membrane is then detected with the aid of a signal of an enzyme, dye, or radioisotope.
As another use of the present invention, an anti-HCV antibody that recognizes the same epitope as the antibody of the present invention can be detected. The present invention can be used as a competitive probe against the antibody of the present invention reacting with the HCV antigen and a test antibody. For example, the HCV antigen is immobilized upon a plate or membrane, the antibody of the present invention to which an enzyme, dye, or radioisotope has been bound and the test antibody are added, and the reaction is allowed to proceed for a certain period of time under conditions that are sufficient for the HCV antigen and the antibody to form a complex. By assaying a decrease in binding of the enzyme, dye, or radioisotope signal to a solid phase, whether or not the test antibody detects the same epitope that is recognized by the antibody of the present invention can be detected.
Hereafter, the present invention is described in greater detail with reference to the examples. It should be noted that these examples are provided for illustrative purposes and the technical scope of the present invention is not limited to these examples.
pJFH-1, which is plasmid DNA obtained by cloning cDNA (genome-length cDNA) obtained via reverse transcription of the entire genome RNA region of the JFH-1 strain (genotype 2a) of the hepatitis C virus (HCV) isolated from a fulminant hepatitis patient downstream of the T7 RNA promoter sequence of the pUC19 plasmid, was prepared as described in Wakita, T. et al., Nat. Med., 11, 2005, pp. 791-796 and International Publication WO 2004/104198. The nucleotide sequence of the genome-length cDNA derived from the JFH-1 strain inserted into pJFH-1 is as shown in SEQ ID NO: 1. pJFH-1 was digested with EcoRI and then partially digested with Bell to prepare a plasmid DNA fragment, which lacks a fragment of approximately 2,840 bp from the EcoRI site to the first Bell site, and the resulting fragment was purified.
pJ6CF, which is obtained by cloning genome-length cDNA derived from the HCV J6CF strain (GenBank Accession Number AF177036, Yanagi, M., et al., Virology 262: 250-263, 1999) downstream of the T7 RNA promoter sequence of the pUC19 plasmid, was prepared as described in International Publication WO 2006/022422. pJ6CF was partially digested with EcoRI and Bell, the resulting fragment of approximately 2,840 bp was purified, and the resultant was ligated to the pJFH-1 fragment lacking EcoRI-BclI as described above to prepare plasmid DNA pJ6/JFH-1. DNA (SEQ ID NO: 2) cloned into pJ6/JFH-1 is a chimeric viral genome-length cDNA comprising a 5′ untranslated region, the sequence encoding core, E1, E2, and p7 proteins, and the sequence encoding a region from the N terminus to amino acid residue 16 of the NS2 protein of the genome-length cDNA of the J6CF strain; and the sequence encoding a region from amino acid residue 17 to the C terminus of the NS2 protein, the sequence sequentially encoding the NS3, NS4A, NS4B, NS5A, and NS5B proteins, and the 3′ untranslated region of the genome-length cDNA of the JFH-1 strain ligated thereto.
Subsequently, plasmid DNA pTH/JFH-1 was prepared in the following manner. At the outset, 10 μl of 10× buffer supplied with the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), 4 μl of 2 mM dNTP mix, 1 μl each of 10 μM primers JFH-1-A (SEQ ID NO: 4: TGTAAAACGACGGCCAGT) and JFH-1-B (SEQ ID NO: 5: GGTTTAGGATTCGTGCTCATGGTGCACGGTCTACGAGACC) were added to the genome-length cDNA in pJFH-1 as a template, and deionized water was added to bring the total amount to 49.5 μl in the end. Then, 0.5 μl of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out to amplify a DNA fragment comprising a part of the core protein-encoding sequence of the HCV TH strain contained in the JFH-1-B primer sequence, in addition to the 5′ untranslated region of the JFH-1 strain. PCR was carried out with 30 cycles of 98° C. for 10 seconds, 55° C. for 15 seconds, and 72° C. for 30 seconds. The resulting PCR product was designated as PCR product No. 1. Subsequently, 10 pd of 10× buffer supplied with the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), 4 μl of 2 mM dNTP mix, and 1 μl each of 10 μM primers TH-C (SEQ ID NO: 6: GGTCTCGTAGACCGTGCACCATGAGCACGAATCCTAAACC) and TH-D (SEQ ID NO: 7: AGATAGCACAACCACCACAGGAGCTTGGCGAGGAATGCCT) were added to the pTH plasmid containing genome-length cDNA derived from the HCV TH strain (Wakita et al., J. Biol. Chem., 269: 14205-14210, 1994; and Moradpour et al., Biochem. Biophys. Res. Commun., 246: 920-924, 1998) as a template, and then deionized water was added to bring the total amount to 49.5 μl in the end. 0.5 μl of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out to amplify a DNA fragment comprising a part of the 5′ untranslated region of the JFH-1 strain contained in the TH-C primer sequence and a part of the NS2 protein-encoding sequence of the JFH-1 strain contained in the YH-D primer sequence, in addition to a sequence comprising a region from a major part of the core protein-encoding sequence to a part of the NS2 protein-encoding sequence of the TH strain. PCR was carried out with 30 cycles of 98° C. for 10 seconds, 55° C. for 15 seconds, and 72° C. for 30 seconds. The resulting PCR product was designated as PCR product No. 2. Further, 10 μl of 10× buffer supplied with the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), 4 μl of 2 mM dNTP mix, and 1 μl each of 10 μM primers JFH-1-E (SEQ ID NO: 8: AGGCATTCCTCGCCAAGCTCCTGTGGTGGTTGTGCTATCT) and JFH-1-F (SEQ ID NO: 9: CAGCTACCGAGGGGTTAAGC) were added to the above-mentioned pJFH-1 as a template, and then deionized water was added to bring the total amount to 49.5 μA in the end. 0.5 μl of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out to amplify a DNA fragment comprising a part of the NS2 protein-encoding sequence of the TH strain contained in the JFH-1-E primer sequence, in addition to a part of the NS2 and NS3 protein-encoding sequence of the JFH-1 strain. PCR was carried out with 30 cycles of 98° C. for 10 seconds, 55° C. for 15 seconds, and 72° C. for 30 seconds. The resulting PCR product was designated as PCR product No. 3.
Then, PCR was carried out using the PCR products No. 1, No. 2, and No. 3 prepared as described above, with the JFH-1-A primer (SEQ ID NO: 4) and the JFH-1-E primer (SEQ ID NO: 8). Thus, a fragment comprising the 5′ untranslated region of the JFH-1 strain, a region from the core protein-encoding sequence to a part of the NS2 protein-encoding sequence of the TH strain, and a region from a part of the NS2 protein-encoding sequence to a part of the NS3 protein-encoding sequence of the JFH-1 strain ligated to each other was obtained (PCR product No. 4).
Subsequently, pJFH-1 and PCR product No. 4 were treated with the EcoRI and SpeI restriction enzymes, and the resultants were ligated to obtain plasmid DNA pTH/JFH-1. The nucleotide sequence of the chimeric viral genome-length cDNA cloned into the resultant pTH/JFH-1 is shown in SEQ ID NO: 3.
pJFH-1, pJ6/JFH-1, and pTH/JFH-1 were cleaved with XbaI, and then subjected to phenol/chloroform extraction and ethanol precipitation. The cleaved plasmids each were used as a template and RNAs were synthesized using the MEGAscript T7 kit (Ambion) (see WO 2006/022422). The thus-synthesized HCV RNAs each were used for introduction into cells as described below.
The Huh-7 cells (3×106 cells) and 5 μg of the HCV RNA were suspended in 400 μl of the Cytomix solution (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4, 25 mM Hepes, 2 mM EGTA, 5 mM MgCl2, 20 mM ATP, and 50 mM glutathione), the suspension was transferred to a 4-mm cuvette, and subjected to electroporation of HCV RNA into Huh-7 cells using the Gene Pulser (BioRad) at 260 V and 950 μF. Thereafter, the cells into which HCV RNA had been introduced were seeded on a 10 cm2 dish and then subcultured. At the time of subculture, the HCV core proteins contained in the culture supernatant were quantified using the HCV antigen ELISA test kit (ORTHO) to confirm the production of HCV particles. The culture supernatant comprising large quantities of core proteins and having high HCV particle production activity was selected and stored as a virus stock solution.
To the Huh-7 cells that had been cultured in 10% FCS-DMEM medium (1% MEM Non-essential Amino Acid Solution (Invitrogen), 10 mM HEPES-Tris (pH 7.3), and 1 mM sodium pyruvate) in a 10-cm dish, approximately 100 μl of the JFH-1 or J6/JFH-1 virus stock solution obtained above (4×104 ffu/ml) was added to infect the Huh-7 cells with the HCV viruses.
The cells were adequately subcultured while avoiding cell culture becoming confluent, and culture expansion was carried out in 225 cm2 flasks from one flask to 4 flasks and then 12 flasks. Subsequently, cells were detached from 8 of such 225-cm2 flasks, and seeded in two 5-layer Cellstacks™ (Corning), and a medium was added thereto in an amount of 650 ml/cellstack. The cells obtained from the other 4 flasks were seeded in 12 flasks and virus production was effectively continued.
On the day following subculture, the media were discarded and 650 ml of 2% FCS-DMEM medium (1% MEM Non-essential amino acid solution (Invitrogen), 10 mM HEPES-Tris (pH 7.3), 1 mM sodium pyruvate) was added. The media were recovered 3 days after medium exchange, passed through a 0.45-μm filter, and the resultants were stored in a deep freezer. Also, 650 ml of 2% FCS-DMEM medium (1% MEM Non-essential amino acid solution, 10 mM HEPES-Tris (pH 7.3), and 1 mM sodium pyruvate) was added to the Cellstacks after the culture supernatants were recovered, and culture was continued. A similar procedure was repeated 2 days after the medium exchange and the culture supernatants were recovered. A similar procedure was then repeated one more time. The culture supernatants recovered herein were used in the Examples below.
Thus, it was demonstrated that infectious HCV particles were produced in the culture supernatants of cells into which RNAs synthesized from pJFH-1, pJ6/JFH-1, and pTH/JFH-1 each had been introduced.
The virus particles produced in Example 1 were purified via the following 4 steps.
With the use of Pellicon 2 Mini Ultrafiltration Module PLCMK V 0.1 m2 (300 kDa cut-off, Catalog No. P2C300V01, Millipore, hereafter referred to as “pellicon 2 mini”), the culture supernatant containing the HCV particles obtained in the Example above was concentrated to approximately 30- to 50-fold. The concentrate was filtered through a 0.45-μm filter and then stored at −80° C.
To the Ultra-clear 25×89 mm centrifuge tube (Catalog No. 344058, Beckman Coulter), 3 ml of TNE buffer containing cold 60% sucrose (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA) was added, and 7 ml of TNE buffer containing 20% sucrose was overlaid thereon. Further, 25 ml of the sample was overlaid onto the TNE buffer containing 20% sucrose. Ultracentrifugation was carried out using a SW-28 (Beckman Coulter) at 28,000 rpm for 4 hours at 4° C.
The bottom of the tube was perforated using the 25 G injection needle (Terumo) and 2.5 ml, 3 ml, 4.5 ml, and 25 ml fractions were successively obtained.
A 5-ml HiTrap Heparin HP column was equilibrated with 20 mM phosphate buffer (pH 7). The diluted fraction (not more than 6 columns) was applied to HiTrap Heparin at a flow rate of 5 ml/min or slower. The column was washed by passing 50 ml of 20 mM phosphate buffer (pH 7). The elution fraction was obtained by applying 25 ml of 20 mM phosphate buffer (pH 7) containing 500 mM NaCl thereto. Further, the column was washed by passing 50 ml of 20 mM phosphate buffer (pH 7) containing 3.8 M NaCl, the column was then equilibrated with 20 mM phosphate buffer (pH 7), and the column was stored.
The elution fraction was subjected to buffer exchange and concentration using Amicon Ultra-15 Centrifugal Filter Units (Millipore) and TNE buffer. The thus-obtained concentrate was used as a virus solution containing infectious virus particles in the immunization step described below.
The concentrated hepatitis C viruses obtained by the steps 1) to 4) in Example 2 were inactivated via ultraviolet irradiation. As a source of ultraviolet rays, GL-15 (Toshiba) was used. The solution containing purified hepatitis C virus particles (the JFH-1 strain) having an infectious titer of 1×106 ffu/ml was introduced into a silicon-coated polyethylene Eppendorf tube (Assist Co., Ltd,), the tube was placed at a distance from the source of ultraviolet rays, so that the ultraviolet rays would be applied at the intensity of 20 mW/cm2, and UV-C was applied for 5 minutes.
After ultraviolet irradiation, hepatitis C virus particles were serially diluted 50-fold, 250-fold, 1,250-fold, 6,250-fold, 31,250-fold, 156,250-fold, and 781,250-fold in Dulbecco's modified Eagle medium (DMEM).
On the previous day, the Huh-7 cells were seeded on a 96-well poly-L-lysine-coated plate (Corning 96 Well Clear Flat Bottom Poly-L-Lysine Coated Microplate, Corning) at 1×104 cells/well, the serially-diluted virus particles were seeded thereonto, and culture was conducted at 37° C. for 72 hours.
After the culture supernatant was removed, the plate was soaked in ice-cold methanol to fix the cells. Thereafter, methanol was removed via air drying, and cells were permeabilized with the use of Block Ace® (Dainippon Pharmaceutical Co., Ltd.) containing 0.3% Triton®-X 100 (GE Healthcare). HCV-infected cells were detected using the clone 2H9 anti-HCV-core antibody (Wakita, T. et al., Nat. Med. 11:791-796, 2005) and goat anti-mouse IgG-Alexa488 (Molecular Probes), and the number of HCV-infected cells was counted under a fluorescent microscope (IX-70; Olympus). The infectious titer of the ultraviolet-irradiated hepatitis C viruses was found to be below detection limit. HCV particles, which were demonstrated to have completely lost infectivity, were used for administration to mice in the Examples below.
Adjuvant was first prepared. To 100 μl of a solution containing J6/JFH-1-HCV particles (equivalent to 1.4 μg of HCV core proteins; equivalent to 10 μg of total HCV proteins), which had been inactivated as described in Example 3, the equivalent amount of Freund's complete adjuvant (Difco) was added to generate an emulsion. Generation of an emulsion was confirmed by preparing an adequate amount of water in a beaker, dropping a drop of the relevant mixture on the liquid surface, and observing that the liquid would not disperse. Balb/c mice (7-week-old, females) were etherized, and the emulsion containing inactivated J6/JFH-1-HCV particles thus prepared was intraperitoneally administered thereto to immunize the mice.
Two weeks later, to 100 μl of a solution containing J6/JFH-1-HCV particles (equivalent to 1.4 μg of HCV core proteins; equivalent to 10 μg of total HCV proteins), the equivalent amount of Freund's incomplete adjuvant (Difco) was added to generate an emulsion, and then mice were further immunized with the emulsion via intraperitoneal administration as described above. The emulsion was further administered intraperitoneally to mice 3 weeks later for further immunization.
Also, mice were immunized with the use of the JFH-1-HCV particles inactivated in Example 3 according to the above immunization schedule in the same manner.
The blood serum samples that were obtained via partial blood sampling from mice immunized with inactivated HCV particles (J6/JFH-1-HCV particles or JFH-1-HCV particles) having structural proteins of genotype 2a among the mice that had been immunized in Example 4 were subjected to measurement of inhibitory activity on infection with HCV.
Huh7 cells were seeded on a 96-well plate at 5×103 cells/well, and the cells were cultured overnight. The serum and heparin (10 units/ml) were added to the concentrated J6/JFH-1-HCV virus solution obtained in step 1) of Example 2 and the resultant was incubated at 37° C. for 1 hour. The serum was diluted 20-fold and 100-fold using medium (DMEM containing 10% FCS (Invitrogen), 1% MEM Non-essential amino acid solution (Invitrogen), 10 mM HEPES-Tris (pH 7.3), and 1 mM sodium pyruvate) before using. After the medium was discarded, the virus solution was added at 50 μl/well and incubation was then carried out at 37° C. for 3 hours. After the medium was discarded, the plate was washed once with PBS, medium was added at 100 μl/well and incubation was then carried out at 37° C. for 48 hours. After the medium was discarded, the plate was washed once with PBS, Isogen (Wako Pure Chemical Industries, Ltd.) was added at 100 μl/well, and total RNA was extracted according to accompanying instruction. Total RNA was quantified via quantitative RT-PCR, and the RNA copy number per 1 μg of total RNA was determined.
Further, whether or not the serum derived from mice immunized with J6/JFH-1-HCV particles having structural proteins of genotype 2a has activity of inhibiting infection with HCV having structural proteins of genotype 1b was examined in the following manner. To this end, chimeric HCV particles (TH/JFH-1-HCV) having structural proteins of the TH strain of genotype 1b and chimeric J6/JFH-1-HCV particles having structural proteins of genotype 2a prepared in Example 1 and in Example 2 were used as infectious agents to measure activity of the serum for inhibiting the infection in the same manner as described above. A control experiment was carried out in the same manner, except for the use of normal mouse serum sampled before administration of HCV particles.
As a result, the 20-fold diluted serum obtained from mice 62 days after immunization thereof with J6/JFH-1-HCV particles was found to have an infection level of J6/JFH-1-HCV lowered to 15% compared with the control experiment (100%), and thus the blood serum was found to have activity of inhibiting infection with HCV particles having structural proteins of genotype 2a (
Even when the sera from two mice obtained 42 days after immunization with JFH-1-HCV particles had been diluted 100-fold for use, the infection levels of JFH-1-HCV of genotype 2a were decreased, compared with the blood serum collected before HCV particle administration (100%) in the control experiment, and thus it was confirmed that the sera have activity of for inhibiting the infection (
Subsequently, 200 μl of the blood sera from a mouse to which 10J6/JFH-1-HCV particles had been administered and 200 μl of the serum collected from a normal mouse before HCV particle administration each were applied to 1-ml Protein G Sepharose columns (GE Healthcare), and the samples were eluted with 0.1M glycine buffer (pH 3.0) in amounts of 1 ml/fraction, following column washing. The eluted protein peak fraction was designated as the IgG fraction, and the IgG fraction and the non-adsorbed fraction (the IgM fraction) were separately prepared as purified fractions. Their inhibitory activities on infection with HCV thereof were measured in a similar manner as described above, as follows.
Instead of the blood serum samples used in the above-mentioned measurement, purified IgG obtained from the blood serum of mice to which HCV particles had been administered was used at 100 μg/ml or 10 μg/ml, or purified IgG obtained from normal mouse serum was used at 100 μg/ml. HCV particles (in the virus solution) were mixed with IgG and the like so as to achieve 107 copies/ml, and allowed to stand for 1 hour at room temperature. Subsequently, the medium used for the Huh-7 cells that had been seeded at 4×104 cells/well on a 24-well plate on the previous day and cultured therein was discarded, and the mixture of virus particles and IgG antibody as described above was added to the cells at 200 μl/well. The cells were incubated for 3 hours at 37° C., the solution containing virus particles was discarded, and the wells were washed once with PBS. A fresh medium was added thereto, culture was conducted for 2 days, the medium was discarded, the wells were washed once with PBS, and then RNA was extracted from the cells using the Trizol reagent (Invitrogen). The copy number of HCV RNA contained in the extracted RNA was determined. The determination of the RNA copy number was done according to the method of Takeuchi et al. (Gastroenterology, 1999, 116, pp. 636-42.).
Subsequently, the activity of inhibiting infection of the fraction that did not adsorb to Protein G Sepharose (i.e., the IgM fraction) was measured in the same manner. As a positive control, a blood serum (unpurified) sample obtained from a mouse to which J6/JFH-1-HCV particles had been administered was used. Protein content in the blood serum sample obtained from a mouse to which J6/JFH-1-HCV particles had been administered and protein content in the IgM fraction were measured, and then the experiment for measurement of the activity of inhibiting infection was carried out while adding the same amounts of proteins to the samples. Specifically, the blood serum obtained from a mouse to which J6/JFH-1-HCV particles had been administered was mixed so as to become 5% and HCV particles were mixed so as to become 107 copies/ml in the mixture. The IgM fraction was mixed in an amount so as to contain the same amount of proteins as in the aforementioned 5% mouse serum, and HCV particles were mixed so as to become 107 copies/ml. The mixtures were allowed to stand for 1 hour at room temperature. The medium used for the Huh-7 cells that had been seeded at 4×104 cells/well on a 24-well plate on the previous day and cultured therein was discarded, and the mixture of virus particles and serum or IgM was added thereto at 200 μl/well. The cells were incubated for 3 hours at 37° C., a solution containing virus particles was discarded, and the wells were washed once with PBS. A fresh medium was added, culture was conducted for 2 days, the medium was discarded, the wells were washed once with PBS, and RNA was extracted from the cells using the Trizol reagent (Invitrogen). The copy number of HCV RNA contained in the extracted RNA was determined. The determination of the RNA copy number was done according to the method of Takeuchi et al. (Gastroenterology, 1999, 116, pp. 636-42.).
As a result, the IgM fraction derived from the blood serum obtained from a mouse to which J6/JFH-1-HCV particles had been administered was found to have inhibitory activity on infection with HCV, as shown in
The mouse myeloma cell line SP2/0 cells (obtained from ECACC) were cultured in Dulbecco's modified MEM medium (Invitrogen) containing 5×10−5 M 2-mercaptoethanol, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum (FCS, Invitrogen), and SP2/0 cells were obtained in the logarithmic growth phase. The cells were washed three times with serum-free Dulbecco's modified MEM.
Subsequently, spleen cells were prepared from mice to which HCV particles of Example 4 (JFH-1 particles or J6/JFH-1 particles) had been administered, and washed three times with serum-free Dulbecco's modified MEM. The SP2/0 cells and the mouse spleen cells were introduced at a ratio of 1:5 into a 50-ml centrifuge tube and the cells were centrifuged at 1,000 rpm for 10 minutes. The supernatant was completely aspirated off, and the bottom of the tube was tapped with fingers to loosen the cell pellet. 1 ml of 50% polyethylene glycol-1500 solution heated at 37° C. (Roche) was added to the cells for 1 minute, and the reaction was allowed to continue at 37° C. for 1 minute. Subsequently, 1 ml of serum-free Dulbecco's modified MEM was gradually added for 1 minute, and 1 ml of serum-free Dulbecco's modified MEM was gradually added again for 1 minute. In the end, 7 ml of serum-free Dulbecco's modified MEM was added for 3 minutes to dilute the ethylene glycol solution. The cells in the diluent were centrifuged at 1,000 rpm for 10 minutes, 50 ml of HT medium (Dulbecco's modified MEM medium containing 5×10−5M 2-mercaptoethanol, 100 units/ml penicillin, 100 μg/ml streptomycin, 10% FCS, 10−4M hypoxanthine, and 1.5×10−5M thymidine) was added thereto, and the cell pellet was loosened via pipetting. The cells were transferred to two 75-cm2 flasks and cultured at 37° C. in a 5% CO2 incubator overnight.
The cells were centrifuged at 1,000 rpm for 10 minutes and recovered. The cell pellet was loosened via tapping and suspended in 10 ml of Dulbecco's modified MEM. The cell suspension was added to and thoroughly mixed in 90 ml of methylcellulose HAT selection medium (Stem Cell Technology), the resultant was added to 10-cm dishes in amounts of 10 ml per dish, and culture was conducted at 37° C. in a 5% CO2 incubator.
After the culture for 10 to 14 days, hybridoma colonies, each of which was considered to have grown from a single cell, were suctioned with a pipette chip, and each introduced into wells of a 96-well plate to which 200 μl each HT medium containing 10% hybridoma growth factors (Bio Veris) had been added, and culture was then conducted.
When the hybridomas prepared in Example 6 had sufficiently proliferated, the culture supernatants were recovered, and screening thereto was carried out as follows.
Screening was carried out by immobilizing E1 proteins and E2 proteins on a plate, evaluating whether or not the antibodies in the hybridoma supernatant would bind to the proteins immobilized on the plate via EIA, and evaluating whether or not the antibodies in the hybridoma supernatant would be able to inhibit HCV infection.
1) Preparation of E1 and E2 Proteins Derived from J6CF Strain
E1 and E2 proteins of the J6CF strain were prepared as follows. With the use of genome-length cDNA derived from the J6CF strain of genotype 2a (GenBank Accession Number AF177036) as a template, a gene encoding an E1 protein lacking a transmembrane region, which is equivalent to a region from amino acids 192 to 352 when amino acid 1 starts at the initiator methionine at the N terminus of the full-length protein sequence of J6CF (i.e., the continuous protein sequence encoded by the genome sequence of the J6CF strain: GenBank Accession Number AF177036), was amplified via PCR using the Advantage GC2 PCR kit (Takara Bio) with J6E1dTM-s (SEQ ID NO: 10: CACAAGCTTGCCGAAGTGAAGAACATCAGT) and J6E1dTM-as (SEQ ID NO: 11: GCTCTAGATTAATGAGCCCCGCTAATGATGTC). The amplified DNA fragment was cloned into pCR-TOPO (Invitrogen), and 3 clones were subjected to nucleotide sequence analysis. A clone containing an insert having a correct nucleotide sequence was designated as pTOP0-J6E1dTM.
pTOPO-J6E1dTM was digested with HindIII and XbaI and a gel containing the resultant DNA fragment of approximately 500 bp (the E1 fragment) was excised. The DNA fragment was purified from the gel using GeneElute (SIGMA). Similarly, p3×FLAG-CMV-9 (SIGMA) was digested with HindIII and XbaI, the resultant was electrophoresed on 1% agarose gel, a gel containing a fragment of approximately 6,400 bp was excised, and the DNA fragment was purified from the gel using GeneElute (SIGMA). The purified DNA fragments were ligated to each other using T4 ligase (Takara Bio), thereby obtaining an animal cell expression vector CMV-3×FLAGJ6E1dTM into which the J6CF E1 fragment had been incorporated.
Subsequently, a gene encoding the E2 protein lacking a transmembrane region, which is equivalent to a region from amino acids 384 to 720 when amino acid 1 starts at the initiator methionine at the N terminus of the full-length protein sequence of the J6CF strain, was amplified via PCR using the Advantage GC2 PCR kit (Takara Bio) with J6E2dTM-s (SEQ ID NO: 12: CACAAGCTTCGCACCCATACTGTTGGGG) and J6E2dTM-as (SEQ ID NO: 13: GCTCTAGATTACCATCGGACGATGTATTTTGT). The amplified DNA fragment was cloned into pCR-TOPO (Invitrogen), and 3 clones were subjected to nucleotide sequence analysis. A clone containing an insert having a correct nucleotide sequence was designated as pTOP0-J6E2dTM.
Subsequently, DNA obtained by digesting p3×FLAG-CMV-9 (SIGMA) with HindIII and XbaI was ligated with the aid of T4 DNA ligase to the DNA fragment of approximately 1,000 bp excised from pTOP0-J6E2dTM with HindIII and XbaI for cyclization. The resulting vector was designated as CMV-3×FLAGJ6E2dTM.
CMV-3×FLAGJ6E1dTM and CMV-3×FLAGJ6E2dTM were introduced into the COS1 cells derived from monkey kidney cells (Accession Number RCB0143, obtained from Riken Cell Bank) to express proteins therein as described below.
The COS1 cells were cultured in Dulbecco's MEM (D-MEM: Invitrogen) containing 10% fetal calf serum (Invitrogen), 100 U/ml and 100 μg/ml streptomycin sulfate. The COS1 cells were seeded in a 150-cm2 culture flask (Corning Coaster) at a split ratio of 1:2 on the previous day of gene introduction, and the cells were cultured at 37° C. in a 5% CO2 incubator overnight.
Separately, DEAE dextran (Pharmacia) and chloroquine (SIGMA) were added to D-MEM medium (Invitrogen) to result in final concentrations of 400 μg/ml and 100 μM, respectively, 50 μg of the expression vector (CMV-3×FLAGJ6E1dTM or CMV-3×FLAGJ6E2dTM) was added at 0.1 μg/μl per 13 ml of the solution, and culture was then conducted. Subsequently, the supernatant of the cultured COS1 cells was aspirated off, and 10 ml of PBS(-) (Nissui) was added thereto and the cells were washed once. After PBS(-) was aspirated off, 13 ml of the DEAE dextran-DNA mixture was added thereto per 150-cm2 flask, and incubation was then carried out at 37° C. in the presence of 5% CO2 for 4 hours.
4 hours later, the DEAE dextran-DNA mixture was aspirated off, and the cells were washed once with 10 ml of PBS. CHO-SFM medium (Invitrogen) was added thereto in amounts of 50 ml per flask, and culture was conducted at 37° C. in the presence of 5% CO2. The culture supernatant was collected in a 50-ml centrifuge tube (Corning Coaster) 4 days later. The collected supernatant was centrifuged at 6,000 rpm (with the use of a HITACHI RPR9-2 rotor) for 30 minutes at 4° C. and filtered through a 0.2-μm filter (Whatman).
The culture supernatant was purified with the use of anti-FLAG M2 agarose (SIGMA) as follows. To 500 ml of the culture supernatant, 1 ml of anti-FLAG M2 agarose was added, and the reaction was allowed to proceed at 4° C. (in a low-temperature chamber) for 2 hours while undergoing agitation in a spinner bottle. A mixture of the supernatant and anti-FLAG M2 agarose was transferred to the Econo-Column (BIO-RAD) 2 hours later, and flow-through fractions were collected. Subsequently, the column was washed twice with 10 ml of TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Six fractions (1 ml of each fraction) were eluted with the use of 0.1M glycine-HCl (pH 3.5). Immediately after elution, 1M Tris-HCl (pH 9.5) was added for neutralization. The fractions (20 μl each) were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions and stained with Coomassie brilliant blue. As a result, E1 proteins or E2 proteins derived from the J6CF strain were found to be purified.
2) Preparation of E1 and E2 Proteins Derived from JFH-1 Strain
E1 and E2 proteins of the JFH-1 strain were prepared as follows. With the use of genome-length cDNA derived from the JFH-1 strain of genotype 2a as a template, a gene encoding the E1 protein, which is equivalent to a region from amino acids 192 to 352 when amino acid 1 starts at the initiator methionine at the N terminus of the full-length protein sequence (i.e., the continuous protein sequence encoded by the genome sequence of the JFH-1 strain: the amino acid sequence disclosed in GenBank Accession Number AB047639; Kato, T. et al., Gastroenterology, 125, 2003, pp. 1808-1817), was amplified via PCR using the Advantage GC2 PCR kit (Takara Bio) with JFHE1dTM-s (SEQ ID NO: 14: CACAAGCTTGCCCAGGTGAAGAATACCAGT), and JFHE1dTM-as (SEQ ID NO: 15: GCTCTAGATTAGTGAGCCCCGCTAACGATGTC). The amplified DNA fragment was cloned into pCR-TOPO. Three clones were subjected to nucleotide sequence analysis, and a clone containing an insert having a correct nucleotide sequence was designated as pTOPO-JFHE1dTM.
pTOPO-JFHE1dTM was digested with HindIII and XbaI and a gel containing the resultant DNA fragment of approximately 500 bp (the E1 fragment) was excised. The DNA fragment was purified from the gel using GeneElute (SIGMA). Similarly, p3×FLAG-CMV-9 (SIGMA) was digested with HindIII and XbaI, the resultant was electrophoresed on 1% agarose gel, a gel containing a fragment of approximately 6,400 bp was excised, and the DNA fragment was purified from the gel using GeneElute (SIGMA). The purified DNA fragments were ligated to each other using T4 ligase (Takara Bio), thereby obtaining an animal cell expression vector CMV-3×FLAGJFHE1dTM into which the JFH-1 E1 fragment had been incorporated.
Subsequently, a gene encoding the E2 protein lacking a transmembrane region, which is equivalent to a region from amino acids 384 to 714 when amino acid 1 starts at the initiator methionine at the N terminus of the full-length protein sequence of the JFH-1 strain, was amplified via PCR using the Advantage GC2 PCR kit (Takara Bio) with JFE2dTM-s (SEQ ID NO: 16: CACAAGCTTGGCACCACCACCGTTGGAG) and JFE2dTM-as (SEQ ID NO: 17: GCTCTAGATTATGTGATAGCAGGTGAGAGGCC). The amplified DNA fragment was cloned into pCR-TOPO (Invitrogen), and 3 clones were subjected to nucleotide sequence analysis. A clone containing an insert having a correct nucleotide sequence was designated as pTOPO-JFHE2dTM.
pTOPO-JFHE2dTM was digested with HindIII and XbaI and a gel containing the resultant E2 fragment of approximately 1,000 bp was excised. The DNA fragment was purified from the gel using GeneElute (SIGMA). Similarly, the p3×FLAG-CMV-9 vector (SIGMA) was digested with HindIII and XbaI, the resultant was electrophoresed on 1% agarose gel, a gel containing a fragment of approximately 6,400 bp was excised. The DNA fragment was purified from the gel using GeneElute (SIGMA). The purified DNA fragments were ligated to each other using T4 ligase (Takara Bio), thereby obtaining an animal cell expression vector CMV-3×FLAGJFHE2dTM into which the JFH-1 E2 fragment had been incorporated.
CMV-3×FLAGJFHE1dTM and CMV-3×FLAGJFHE2dTM were introduced into the COS1 cells derived from monkey kidney cells (Accession Number RCB0143, obtained from Riken Cell Bank) to express proteins therein as described below.
The COS1 cells were cultured in Dulbecco's MEM (D-MEM: Invitrogen) containing 10% fetal calf serum (Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate. The COS1 cells were seeded in a 150-cm2 culture flask (Corning Coaster) at a split ratio of 1:2 on the previous day of gene introduction, and the cells were cultured at 37° C. in a 5% CO2 incubator overnight.
Separately, DEAE dextran (Pharmacia) and chloroquine (SIGMA) were added to D-MEM medium (Invitrogen) to result in final concentrations of 400 μg/ml and 100 μM, respectively, 50 μg of the expression vector (CMV-3×FLAGJFHE1dTM or CMV-3×FLAGJFHE2dTM) was added at 0.1 μg/μl per 13 ml of the solution, and culture was then conducted. Subsequently, the supernatant of the cultured COS1 cells was aspirated off, 10 ml of PBS(-) (Nissui) was added thereto and the cells were washed once. After PBS(-) was aspirated off, 13 ml of a DEAE dextran-DNA mixture was added thereto per 150-cm2 flask, and incubation was then carried out at 37° C. in the presence of 5% CO2 for 4 hours.
4 hours later, the DEAE dextran-DNA mixture was aspirated off, and the cells were washed once with 10 ml of PBS. CHO-SFM medium (Invitrogen) was added thereto in amounts of 50 ml per flask, and culture was conducted at 37° C. in the presence of 5% CO2. The culture supernatant was collected in a 50-ml centrifuge tube (Corning Coaster) 4 days later. The collected supernatant was centrifuged at 6,000 rpm (with the use of a HITACHI RPR9-2 rotor) for 30 minutes at 4° C. and filtered through a 0.2-1 μm filter (Whatman).
The culture supernatant was purified with the use of anti-FLAG M2 agarose (SIGMA) as follows. To 500 ml of the culture supernatant, anti-FLAG M2 agarose was added, and the reaction was allowed to proceed at 4° C. (in a low-temperature chamber) for 2 hours while undergoing agitation in a spinner bottle. A mixture of the supernatant and anti-FLAG M2 agarose was transferred to the Econo-Column (BIO-RAD) 2 hours later, and flow-through fractions were collected. Subsequently, the column was washed twice with 10 ml of TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Six fractions (1 ml of each fraction) were eluted with the use of 0.1M glycine-HCl (pH 3.5). Immediately after elution, 1M Tris-HCl (pH 9.5) was added for neutralization. The fractions (20 μl each) were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions and stained with Coomassie brilliant blue. As a result, E1 proteins or E2 proteins derived from the JFH-1 strain were found to be purified.
The E1 and E2 proteins derived from the J6CF strain were diluted with PBS to become 1 μg of each of the two proteins/ml. The solution of E1 and E2 protein mixture (50 μl) was added to wells of the immunoplate (NUNC), the plate was allowed to stand at 4° C. overnight, and the proteins were immobilized on the plate. The protein solution was removed, 150 μl of Block Ace (Dainippon Pharmaceutical Co., Ltd.) was added to each well, and the plate was subjected to blocking at room temperature for 4 hours.
E1 and E2 proteins derived from the JFH-1 were also diluted with PBS to become 1 μg of each of the two proteins/ml. The solution of E1 and E2 protein mixture (50 μl) was added to wells of the immunoplate (NUNC), the plate was allowed to stand at 4° C. overnight, and the proteins were immobilized on the plate. The protein solution was removed, 150 μl of Block Ace (Dainippon Pharmaceutical Co., Ltd.) was added to each well, and the plate was subjected to blocking at room temperature for 4 hours.
These plates were used for screening for the anti-HCV antibody in the culture supernatant of hybridomas as described below.
The plate prepared in 3) above upon which E1 and E2 proteins derived from the J6CF strain have been immobilized was washed 4 times with PBS containing 0.1% Tween 20 (SIGMA), 50 μl of the hybridoma supernatant samples obtained in Example 6 each was added to the wells, and the reaction was allowed to proceed at room temperature for 1 hour while the plate was shaken with a plate mixer. After the reaction, the plate was washed 4 times with PBS containing 0.1% Tween 20 (SIGMA), 50 μl of the HRP-labeled anti-mouse IgG antibody (Amersham Biosciences), which had been diluted 5,000-fold with PBS containing 0.1% Tween 20, was added to each well, and the reaction was allowed to proceed at room temperature for 1 hour while the plate was shaken. After the reaction, the plate was washed 4 times with PBS containing 0.1% Tween 20 (SIGMA), color was developed using a color development kit for peroxidase (Sumitomo Bakelite Co., Ltd.), the absorbance at 450 nm was measured using Multi-Scan (Titer-Tech), and positive clones were selected.
Subsequently, inhibitory activity on infection with HCV of the culture supernatants of the positive clones was evaluated using J6/JFH-1-HCV particles as infectious agents. Specifically, a solution containing J6/JFH1-HCV particles having the infectious titer of 1×106 ffu/ml was mixed with the equivalent amount of the hybridoma supernatant, and the mixture was cultured at 37° C. for 1 hour. Thereafter, the mixture sample of J6/JFH-1-HCV particles and hybridoma culture supernatant was added to a 96-well poly-L-lysine-coated plate (Corning 96 Well Clear Flat Bottom Poly-L-Lysine Coated Microplate, Corning) to which the Huh-7 cells had been seeded at 1×104 cells/well on the previous day, and then culture was conducted at 37° C. for 72 hours.
After the sample was removed, the plate was soaked in ice-cold methanol to fix the cells. Thereafter, methanol was removed via air drying, and cells were permeabilized with the use of Block Ace® (Dainippon Pharmaceutical Co., Ltd.) containing 0.3% Triton®-X 100 (GE Healthcare). HCV-infected cells were detected using the clone 2H9 anti-HCV-core antibody (Wakita, T. et al., Nat. Med. 11:791-796, 2005) and goat anti-mouse IgG-Alexa488 (Molecular Probes), and the number of HCV-infected cells was counted under a fluorescent microscope (IX-70; Olympus). Two samples each exhibiting a smaller number of infected cells were selected as hybridoma cell lines that produce monoclonal antibodies having inhibitory activity on infection with HCV. These cell lines were cloned via limiting dilution and clones J6/1G11-25 (Accession Number: FERM BP-10980) and J6/4D4-2 (Accession Number: FERM BP-10981) were obtained.
The plate prepared in 3) above upon which E1 and E2 proteins derived from the JFH-1 strain had been immobilized was washed 4 times with PBS containing 0.1% Tween 20 (SIGMA), 50 μl of the hybridoma supernatant samples obtained in Example 6 each was added to the wells, and the reaction was allowed to proceed at room temperature for 1 hour while shaking the plate with a plate mixer. After the reaction, the plate was washed 4 times with PBS containing 0.1% Tween 20 (SIGMA), 50 μl of the HRP-labeled anti-mouse IgG antibody (Amersham Biosciences), which had been diluted 5,000-fold with PBS containing 0.1% Tween 20, was added to each well, and the reaction was allowed to proceed at room temperature for 1 hour while the plate was shaken. After the reaction, the plate was washed 4 times with PBS containing 0.1% Tween 20 (SIGMA), color was developed using a color development kit for peroxidase (Sumitomo Bakelite Co., Ltd.), and the absorbance at 450 nm was measured using Multi-Scan (Titer-Tech). Four clones were selected from among the positive clones as hybridoma cell lines that produce monoclonal antibodies recognizing the E1 and/or E2 proteins of the JFH-1 strain. Subsequently, inhibitory activity on infection with HCV of the culture supernatant of positive clones was evaluated in the same manner as in 4) using the J6/JFH-1-HCV particles as infectious agents. As a result, a positive clone JF/M1-4 (Accession Number: FERM BP-10982) was obtained. The positive clone JF/M1-4 is a hybridoma cell line that produces a monoclonal antibody recognizing the E1 and/or E2 protein of the JFH-1 strain and produces an antibody inhibiting infection with J6/JFH-1-HCV.
The three hybridoma cell lines selected in example 7: J6/1G11-25 (Accession Number: FERM BP-10980); J6/4D4-2 (Accession Number: FERM BP-10981); and JF/M1-4 (Accession Number: FERM BP-10982) were deposited at the International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan) as of Jul. 19, 2007.
Properties of monoclonal antibodies inhibiting infection with HCV produced from these hybridoma cell lines were analyzed as follows.
Mouse antibody subclass was determined by analyzing the culture supernatant of the positive clones using the ImmunoPure Monoclonal Antibody Isotyping Kit (Pierce). As a result of isotype analysis of the clones, all clones were found to have μ-type H chains and κ-type L chains. Thus, these clones were found to be of the IgM subclass.
The hybridoma was cultured in serum-free medium, and the culture supernatant was subjected to salting-out with 50% ammonium sulfate, followed by dialysis against PBS. Thereafter, the resultant was subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions and stained with Coomassie brilliant blue, and the molecular weights of the H chains and the L chains of the antibodies were estimated. As a result, antibodies from all clones were found to have H chains with a molecular weight of 70 kDa and L chains with a molecular weight of 23 kDa.
3) Evaluation of Inhibitory Activity on Infection with HCV of Monoclonal Antibodies
Monoclonal antibodies were purified from each of culture supernatants of the hybridoma cell lines. The purification of the antibodies was done by subjecting the supernatant of hybridomas conditioned to and cultured in serum-free medium Hybridoma-SFM to 50% ammonium sulfate precipitation and dialysis against PBS. The concentration of the purified antibody sample was determined by performing SDS-PAGE using purified mouse IgMκ (Sigma) as the standard and Coomassie staining.
Inhibitory activity on infection with HCV of the thus-prepared purified monoclonal antibodies derived from the hybridoma cell lines was evaluated in the same manner as described above. That is, the antibodies were added with infectious HCV particles to cells, incubation was carried out, the HCV RNA concentration in the infected cells was measured, and the HCV RNA amount in the cells was analyzed. Since HCV RNA in the cells are replicated from infectious HCV particles, the amount of HCV RNA in the cell should decrease if the infection is inhibited by the monoclonal antibodies.
Specifically, purified monoclonal antibodies each was mixed with infectious HCV particles (J6/JFH-1-HCV particles) such that the monoclonal antibodies became 10 μg/ml and the infectious HCV particles achieved 107 copies/ml in the mixture, and the resultant was allowed to stand for 1 hour at room temperature. The medium used for the Huh-7 cells that had been seeded at 4×104 cells/well on a 24-well plate on the previous day and cultured therein was discarded, and the mixture of J6/JFH-1-HCV particles and purified monoclonal antibodies was added thereto at 200 μl/well. The cells were incubated for 3 hours at 37° C., the solution containing virus particles was discarded, and the wells were washed once with PBS. A fresh medium was added, culture was conducted for 2 days, the medium was discarded, the wells were washed once with PBS, and RNA was extracted using the Trizol reagent (Invitrogen). The copy number of HCV RNA contained in the extracted RNA was determined. The determination of the RNA copy number was done according to the method of Takeuchi et al. (Gastroenterology, 1999, 116, pp. 636-42.).
Regarding amino acids 1 to 162 of the E1 protein of the JFH-1 strain (SEQ ID NO: 25) and amino acids 1 to 337 of the E2 protein of the JFH-1 strain (SEQ ID NO: 26), peptides comprising amino acid sequences having 10 continuous amino acids which were designed to each be shifted by 3 amino acids from the N terminuses were synthesized. The N terminuses of the peptides were biotinylated, and the C terminuses were glycine amide (the peptides were synthesized by JPT on consignment).
The synthesized peptides were dissolved in DMSO and then dissolved in PBS at a concentration of 0.01 nmol/μl. The peptide solution (50 μl) was added to wells of the streptavidin-coated plate (Nunc) and the reaction was allowed to proceed at room temperature for 1 hour. Subsequently, the peptide solution was discarded, Blocking One (Nacalai Tesque) was added at 200 μl/well, and the plate was allowed to stand at room temperature for 5 hours. The Blocking One solution was discarded, the plate was washed 4 times with 150 μl/well of PBS (pH 7.2) containing 0.05% Tween 20, the JF/M1-4 monoclonal antibody diluted 1 μg/ml with PBS (pH 7.2) containing 0.05% Tween 20 was added at 50 μl/well, and the reaction was allowed to proceed at room temperature for 1 hour. Subsequently, the antibody solution was discarded, the plate was washed 5 times with 150 μl/well of PBS (pH 7.2) containing 0.05% Tween 20, 50 μl/well of the HRP-labeled anti-mouse IgG goat antibody (GE Healthcare) diluted 5,000-fold with PBS containing 0.05% Tween 20 was added, and the reaction was allowed to proceed at room temperature for 1 hour. After the reaction, the antibody solution was discarded, and the plate was washed 5 times with 150 μl/well of PBS (pH 7.2) containing 0.05% Tween 20. After washing, antibodies bound to peptides were detected using a color development kit for HPR (Sumitomo Bakelite Co., Ltd.).
As a result, the JF/M1-4 monoclonal antibody did not recognize any peptides derived from the E1 protein of the JFH-1 strain (
Thus, the JF/M1-4 monoclonal antibody was found to be capable of recognizing, as the epitope, WLTPKCLVHYPYRLWHYPC (SEQ ID NO: 24) within the E2 protein of the JFH-1 strain, which contains a strong epitope.
Regarding amino acids 1 to 162 of the E1 protein of the J6CF strain (SEQ ID NO: 27) and amino acids 1 to 337 of the E2 protein of the J6CF strain (SEQ ID NO: 28), peptides comprising amino acid sequences having 10 continuous amino acids which were designed to each be shifted by 3 amino acids from the N terminuses were synthesized. The N terminuses of the peptides were biotinylated, and the C terminuses were glycine amide (the peptides were synthesized by JPT on consignment).
The synthesized peptides were dissolved in DMSO and then dissolved in PBS at a concentration of 0.01 nmol/μl. The peptide solution (50 μl) was added to wells of the streptavidin-coated plate (Nunc) and the reaction was allowed to proceed at room temperature for 1 hour. Subsequently, the peptide solution was discarded, Blocking One (Nacalai Tesque) was added at 200 μl/well, and the plate was allowed to stand at room temperature for 5 hours. The Blocking One solution was discarded, each well of the plate was washed 4 times with 150 μl of PBS (pH 7.2) containing 0.05% Tween 20, the J6/1G11-25 or J6/4D4-2 monoclonal antibody diluted 1 μg/ml with PBS (pH 7.2) containing 0.05% Tween 20 was added at 50 μl/well, and the reaction was allowed to proceed at room temperature for 1 hour. Subsequently, the antibody solution was discarded, the plate was washed 5 times with 150 μl/well of PBS (pH 7.2) containing 0.05% Tween 20, 50 μl/well of the HRP-labeled anti-mouse IgG goat antibody (GE Healthcare) diluted 5,000-fold with PBS containing 0.05% Tween 20 was added, and the reaction was allowed to proceed at room temperature for 1 hour. After the reaction, the antibody solution was discarded, and the plate was washed 5 times with 150 μl/well of PBS (pH 7.2) containing 0.05% Tween 20. Subsequently, antibodies bound to peptides were detected using a spectrophotometer (at OD 450 nm) with the aid of a color development kit for HPR (Sumitomo Bakelite Co., Ltd.).
As a result, the J6/1G11-25 monoclonal antibody did not recognize any peptides derived from the E1 protein of the J6CF strain (
As a result, the J6/4D4-2 monoclonal antibody recognized some peptides derived from the E1 protein of the J6CF strain and some peptides derived from the E2 protein of the J6CF strain, as shown in
E2-derived strong epitopes were LIDYPYRLWH (SEQ ID NO: 37; peptide no. 77 in
A relatively strong epitope was NYTIFKIRMY (SEQ ID NO: 31; peptide no. 82 in
Further, STRPPLGSWF (SEQ ID NO: 42; peptide no. 54 in
Thus, the J6/1G11-25 monoclonal antibody was found to recognize the amino acid sequences within the E2 protein of the J6CF strain, NVTNPEDMPRPYCW (SEQ ID NO: 44) and NYTIFKIRMYVGG (SEQ ID NO: 45), as epitopes.
Also, the J6/4D4-2 monoclonal antibody was found to recognize the amino acid sequences within the E1 protein of the J6CF strain, FIVSPQHHWFVQDCNC (SEQ ID NO: 46) and MAWDMMMNWS (SEQ ID NO: 36), as epitopes as well as the amino acid sequences within the E2 protein of the J6CF strain, LIDYPYRLWHYPC (SEQ ID NO: 47), NPEDMRPYCWHYPPRQ (SEQ ID NO: 48), and NYTIFKIRMYVGG (SEQ ID NO: 45), as epitopes.
DNA encoding a variable region of the JF/M1-4 mouse monoclonal antibody against HCV particles was cloned as follows.
Total RNA was prepared from the JF/M1-4 hybridoma cell line using PureLink Micro-to-Midi (Invitrogen) in accordance with the method described in the accompanying manual. Specifically, 2×108 hybridoma line JF/M1-4 cells were suspended in 2.4 ml of RNA Lysis solution, and the cells were passed through a syringe with a 18 gauge needle several times for homogenization. The resulting homogenate was centrifuged, 70% ethanol was added to the same amount of the resulting supernatant, and the mixture was applied on an RNA spin cartridge. The cartridge was thoroughly washed with the addition of a washing solution and RNA was eluted with RNase-free water.
2) Synthesis of Single-Stranded cDNA
With the use of μ-type H chain- and κ-type L chain-specific primers, single-stranded cDNA was synthesized from the total RNA prepared in 1) above as follows.
i) Synthesis of cDNA Encoding Pt-Type H Chain of JF/M1-4 Monoclonal Antibody
To 5 μg of RNA, 50 pmol of the μ-type H chain-specific primer mCHm-as (SEQ ID NO: 49; 5′-AGGCTTACTAGTAGCTGGAATGGGCACATGCAGATC-3′) was added for annealing, and 5 μl of 0.1M DTT, 1.25 μl of 20 mM dNTPs solution, and 10 μl of 5× RT Buffer supplied with SuperScriptII were added thereto, and distilled water (DW) was added thereto to bring the final volume to 49 μl.
Subsequently, 1 μl of a reverse transcriptase SuperScriptII (Invitrogen) was added, the reaction was carried out at 42° C. for 30 minutes and at 50° C. for 30 minutes, and the reaction solution was directly used for the next step, i.e., polymerase chain reaction (PCR).
ii) Synthesis of cDNA Encoding κ-Type L Chain of JF/M1-4 Monoclonal Antibody
To 5 μg of RNA, 50 pmol of the κ-type L chain-specific primer mCK1-as (SEQ ID NO: 50; 5′-CATGTCTAGAACACTCATTCCTGTTGAAGCTCTTG-3′) was added for annealing, and 5 μl of 0.1M DTT, 1.25 μl of 20 mM dNTPs solution, and 10 μl of 5× RT Buffer supplied with SuperScriptII were added thereto, and distilled water (DW) was added thereto to bring the final volume to 49 μl. Subsequently, 1 μl of a reverse transcriptase SuperScriptII (Invitrogen) was added, the reaction was carried out at 42° C. for 30 minutes and at 50° C. for 30 minutes, and the reaction product was directly used for the next step, i.e., polymerase chain reaction (PCR).
PCR was carried out with the GeneAmp® PCR System 9700 (Applied Biosystems) using the Advantage GC2 DNA polymerase kit (TAKARA).
i) Amplification of cDNA Encoding Mouse H Chain V Region
PCR was carried out using the Mouse Ig-Primer Set (69831-3, Novagen) in accordance with the conditions described in the accompanying manual.
Specifically, PCR was carried out using the MuIgVH5′-A to MuIgVH5′-F primers (that hybridize to the leader sequence of the mouse H chain) and the MuIgMVH3′-1 primer (that hybridizes to the mouse μ-type H chain C region) supplied with the kit.
To 5 μl of the reaction mixture resulting from synthesis of single-stranded cDNA encoding the μ-type H chain prepared above, 10 μl of 5× PCR buffer supplied with the kit, 10 pd of GC Melt, 4 μl of 2.5 mM dNTPs solution, 1.25 units of Advantage GC2 DNA polymerase mix (TAKARA), 1 μl of 10 pmol/μl MuIgVH5′ primer, and 1 μl of 5 pmol/μl MuIgMVH3′-1 primer were added. Distilled water (DW) was further added to bring the final volume to 50 μl. The PCR solution (50 μl) was incubated at 94° C. for 4 minutes and then heated at 94° C. for 1 minute, 50° C. (in the case of MuIgVH5′-A and MuIgVH5′-B) or 60° C. (in the case of MuIgVH5′-C to MuIgVH5′-F) for 1 minute, and 72° C. for 2 minutes, in that order. This thermal cycle was repeated 40 times and the reaction mixture was subsequently further incubated at 72° C. for 6 minutes.
PCR was carried out using the Mouse Ig-Primer Set (69831-3, Novagen) in accordance with the conditions described in the accompanying manual.
Specifically, PCR was carried out using the MuIgκVL5′-A to MuIgκVL5′-F primers (that hybridize to the leader sequence of the mouse κ-type L chain) and the MuIgκVL3′-1 primer (that hybridizes to the mouse κ-type L chain C region) supplied with the kit.
To 5 μl of the reaction mixture resulting from synthesis of single-stranded cDNA encoding the κ-type L chain prepared above, 10 μl of 5× PCR buffer supplied with the kit, 10 μl of GC Melt, 4 μl of 2.5 mM dNTPs solution, 1.25 units of Advantage GC2 DNA polymerase mix (TAKARA), 1 μl of 10 pmol/μl MuIgκVL 5′ primer, and 1 μl of 5 pmol/μl, MuIgκVL3′-1 primer were added. Distilled water (DW) was further added to bring the final volume to 50 μl. The PCR solution (50 μl) was incubated at 94° C. for 4 minutes and then heated at 94° C. for 1 minute, 50° C. (in the case of MuIgVL5′-A and MuIgVL5′-B) or 60° C. (in the case of MuIgVL5′-C to MuIgVL5′-F) for 1 minute, and 72° C. for 2 minutes, in that order. This thermal cycle was repeated 40 times and the reaction mixture was subsequently further incubated at 72° C. for 6 minutes.
4) Purification of PCR Product and Cloning Thereof into Plasmid Vector
The DNA fragments PCR amplified as described above were separated via agarose gel electrophoresis. A DNA fragment of approximately 450 bp in length was purified using GeneElute (Sigma). A DNA fragment was cloned using the TOPO TA Cloning kit (Invitrogen). The pCR-TOPO vector supplied with the kit, the DNA fragment, and a salt solution supplied with the kit were added, the resultant was allowed to stand at room temperature for 5 minutes, and a part of the reaction solution was added to competent cells of E. Coli Mach1 (Invitrogen). The E. Coli competent cells were placed on ice for 30 minutes, subsequently heated at 42° C. for 30 seconds, and placed again on ice for 1 minute. SOC medium (room temperature) was added thereto, the cells was cultured at 37° C. for 1 hour and seeded on an agar medium, and then cultured at 37° C. overnight. Plasmid DNA was prepared from the resulting transformant, and the nucleotide sequence of the cloned DNA was determined in accordance with a conventional technique.
5) Analysis of Nucleic Acid Sequence of cDNA Encoding V Region of JF/M1-4 Mouse Monoclonal Antibody
DNAs cloned in the plasmids, the nucleotide sequences of which were determined in 4) above (which are cDNAs encoding the H chain V region and the L chain V region of the JF/M1-4 mouse monoclonal antibody), were analyzed as follows.
i) Analysis of Nucleic Acid Sequence of cDNA Encoding Mouse H Chain V Region
Nucleic acid sequences of the H chain V regions cloned in many resultant clones were found to be identical except for the regions of the mouse H chain leader sequences. This indicates that a plurality of clones tested are derived from independent isolates obtained via PCR utilizing a mixture of various primers encoding the mouse H chain leader sequences. Further, by searching a database for encoded amino acid sequences deduced from their nucleic acid sequences, sequences of regions from the framework 1 to the J segment (framework 4) of the H chain V region of the JF/M1-4 mouse monoclonal antibody were deduced (SEQ ID NOs: 55 to 61). The nucleic acid sequence and the amino acid sequence thereby determined are shown in SEQ ID NOs: 51 and 52 of the Sequence Listing, respectively. Further, CDR1 to CDR3 deduced from the amino acid sequences are shown in
ii) Analysis of Nucleic Acid Sequence of cDNA Encoding Mouse L Chain V Region
Nucleic acid sequences of the L chain V regions cloned in many resultant clones were found to be identical except for the regions of the mouse L chain leader sequences. This indicates that a plurality of clones tested are derived from independent isolates obtained via PCR utilizing a mixture of various primers encoding the mouse L chain leader sequences. Further, by searching a database for encoded amino acid sequences deduced from their nucleic acid sequences, sequences of regions from the framework 1 to the J segment (framework 4) of the L chain V region of the JF/M1-4 mouse monoclonal antibody were deduced (SEQ ID NOs: 62 to 68). The nucleic acid sequence and the amino acid sequence thereby determined are shown in SEQ ID NOs: 53 and 54. Further, CDR1 to CDR3 deduced from the amino acid sequences are shown in
The antibody according to the present invention which has inhibitory activity on infection with HCV can be used as a therapeutic or preventive medicament for HCV infection.
SEQ ID NO: 1 discloses the HCV genome-length cDNA cloned into pJFH-1.
SEQ ID NO: 2 discloses the chimeric HCV genome-length cDNA sequence cloned into pJ6/JFH-1.
SEQ ID NO: 3 discloses the HCV genome-length cDNA sequence cloned into pTH/JFH-1.
SEQ ID NO: 4 discloses the sequence of the primer (JFH-1-A).
SEQ ID NO: 5 discloses the sequence of the primer (JFH-1-B).
SEQ ID NO: 6 discloses the sequence of the primer (TH-C).
SEQ ID NO: 7 discloses the sequence of the primer (TH-D).
SEQ ID NO: 8 discloses the sequence of the primer (JFH-1-E).
SEQ ID NO: 9 discloses the sequence of the primer (JFH-1-F).
SEQ ID NO: 10 discloses the sequence of the primer (J6E1dTM-s).
SEQ ID NO: 11 discloses the sequence of the primer (J6E1dTM-as).
SEQ ID NO: 12 discloses the sequence of the primer (J6E2dTM-s).
SEQ ID NO: 13 discloses the sequence of the primer (J6E2dTM-as).
SEQ ID NO: 14 discloses the sequence of the primer (JFHE1dTM-s).
SEQ ID NO: 15 discloses the sequence of the primer (JFHE1dTM-as).
SEQ ID NO: 16 discloses the sequence of the primer (JFHE2dTM-s).
SEQ ID NO: 17 discloses the sequence of the primer (JFHE2dTM-as).
SEQ ID NOs: 18 to 24 disclose the sequences of synthetic peptides.
SEQ ID NO: 25 discloses the amino acid sequence comprising amino acids 1 to 162 of E1 protein of the JFH-1 strain.
SEQ ID NO: 26 discloses the amino acid sequence comprising amino acids 1 to 337 of the E2 protein of the JFH-1 strain.
SEQ ID NO: 27 discloses the amino acid sequence comprising amino acids 1 to 162 of the E1 protein of the J6CF strain.
SEQ ID NO: 28 discloses the amino acid sequence comprising amino acids 1 to 337 of the E2 protein of the J6CF strain.
SEQ ID NOs: 29 to 48 disclose the sequences of synthetic peptides.
SEQ ID NO: 49 discloses the sequence of the primer (mCHm-as).
SEQ ID NO: 50 discloses the sequence of the primer (mCK1-as).
SEQ ID NO: 51 discloses the nucleotide sequence of the gene encoding the H chain V region (i.e., the heavy chain variable region) of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 52 discloses the amino acid sequence of the H chain V region (i.e., the heavy chain variable region) of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 53 discloses the nucleotide sequence of the gene encoding the L chain V region (i.e., the light chain variable region) of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 54 discloses the amino acid sequence of the L chain V region (i.e., the light chain variable region) of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 55 discloses the amino acid sequence of the framework 1 of the H chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 56 discloses the amino acid sequence of CDR1 of the H chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 57 discloses the amino acid sequence of the framework 2 of the H chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 58 discloses the amino acid sequence of CDR2 of the H chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 59 discloses the amino acid sequence of the framework 3 of the H chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 60 discloses the amino acid sequence of CDR3 of the H chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 61 discloses the amino acid sequence of the framework 4 (i.e., the J segment) of the H chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 62 discloses the amino acid sequence of the framework 1 of the L chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 63 discloses the amino acid sequence of CDR1 of the L chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 64 discloses the amino acid sequence of the framework 2 of the L chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 65 discloses the amino acid sequence of CDR2 of the L chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 66 discloses the amino acid sequence of the framework 3 of the L chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 67 discloses the amino acid sequence of CDR3 of the L chain V region of the JF/M1-4 monoclonal antibody.
SEQ ID NO: 68 discloses the amino acid sequence of the framework 4 (i.e., the J segment) of the L chain V region of the JF/M1-4 monoclonal antibody.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-193413 | Jul 2007 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2008/063424 | 7/25/2008 | WO | 00 | 1/22/2010 |
