WO2002077239A1 - Polypeptides se liant au virus hcv - Google Patents

Polypeptides se liant au virus hcv Download PDF

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Publication number
WO2002077239A1
WO2002077239A1 PCT/JP2002/002954 JP0202954W WO02077239A1 WO 2002077239 A1 WO2002077239 A1 WO 2002077239A1 JP 0202954 W JP0202954 W JP 0202954W WO 02077239 A1 WO02077239 A1 WO 02077239A1
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seq
amino acid
acid sequence
positions
polypeptide
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PCT/JP2002/002954
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English (en)
Japanese (ja)
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Nobuyuki Kato
Akito Nozaki
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Sumitomo Pharmaceuticals Company, Limited
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Priority to JP2002576681A priority Critical patent/JPWO2002077239A1/ja
Publication of WO2002077239A1 publication Critical patent/WO2002077239A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the present invention relates to a technology capable of inhibiting, suppressing or reducing hepatitis C virus (HCV) infection.
  • HCV hepatitis C virus
  • the present invention provides an HCV-binding polypeptide having an activity of binding to HCV, in particular, an activity of binding to E2 (envelope 2) protein of HCV; it is capable of inhibiting, suppressing or reducing HCV infection.
  • the present inventors have recently shown that in an HCV-infected system using HCV and human hepatocyte-derived cultured cells, lactofurin, which is abundantly contained in HCV colostrum and is a member of the iron transport family, is known. Has been found to protect HCV infection of human hepatocyte-derived cultured cells [Ikeda et al, Biochem Biophys Res Commun., 245 (2), 549-553 (1998); Ikeda et al, Virus Res., 66 (1), 51-63 (2000)].
  • the present inventors have reported a case in which percutaneous administration of ⁇ cilactoferin was effective in chronic hepatitis C patients [Tanaka et al, Jpn. J. Cancer Res., 90 (4): 367-371 (1999)].
  • the present inventors have studied the use of the HCV infection system to absorb HCV into cells.
  • the temporal relationship between the interaction of lactofurin with garment was analyzed.
  • the present inventors have found that even when lactoferrin and HCV are mixed and immediately added to cells, HCV infection is completely inhibited, whereby the interaction between HCV and lactoferrin is reduced by HCV. It has been clarified that it occurs faster than adsorption to cells [The 58th Annual Meeting of the Cancer Society of Japan, 145, No.223, (1999)].
  • lactoferrin is HC2 E2 tanno. It has been shown that it directly binds to proteins [The 58th Annual Meeting of the Cancer Society of Japan, 145 pages, No.223, (1999)].
  • lactofurin As described above, the interaction between lactofurin and the E2 protein of HCV has been shown to be important for the protection of HCV infection. However, at present, it has not been sufficiently clarified which limited region in lactofurin consisting of about 700 amino acids is a region essential for binding to HC2 E2 protein. Disclosure of the invention
  • An object of the present invention is to provide a means for inhibiting, suppressing or reducing HCV infection in a living body.
  • an object of the present invention is to provide an HCV-binding polypeptide having an activity of binding to HCV and an application thereof. More specifically, the present invention relates to an HCV binding agent capable of inhibiting, suppressing or reducing HCV infection, and having a binding activity with HC2 E2 protein, which is a size more suitable for incorporation into a living body. It is intended to provide a polypeptide.
  • Another object of the present invention is to provide an HCV infection protective agent capable of inhibiting, suppressing or reducing HCV infection.
  • a portion of positions 1 to 33 in the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, or positions 11 to 53 in the amino acid sequence A polypeptide having an activity of binding to an HCV E2 protein, comprising at least a position moiety;
  • (II) a sequence consisting of a part of the amino acid sequence according to (I), wherein the sequence is a portion of positions 1 to 33 in the amino acid sequence or a position of positions 11 to 33 in the amino acid sequence.
  • polypeptide according to (1) or (2) comprising an amino acid sequence selected from the group consisting of:
  • [6] consists of an amino acid sequence having at least 71% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, and (i) and ( ii):
  • amino acid substituted at the substitution position is selected from the amino acids present at the substitution position on the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.
  • amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.
  • the therapeutic or prophylactic agent for chronic hepatitis C comprising the polypeptide according to any one of (1) to (12) as an active ingredient.
  • FIG. 1 is a diagram showing the results of furestane blotting for examining the binding between the carboxyl-terminal 93amino acid moiety of human, pacific, and pmalactoferrin with the E2 protein of HCV.
  • lane 1 shows the results of thioredoxin tag (22 kDa) obtained by expressing empty vector pET32a
  • lane 2 shows the results of human lactofurin
  • lane 3 shows the results of human transferrin
  • Lane 4 shows the results of the 93 carboxyl-terminal amino acids of human lactoferrin
  • lane 5 shows the results of the 93 carboxyl-terminal amino acids of ⁇ lactoferrin
  • lane 6 shows the results of the carboxyl-terminal 93 amino acids of ⁇ malactoferrin.
  • Lane 7 shows the results of amino acids 113 to 201 of human CD81 (Genebank accession number: XM-006475).
  • FIG. 2 shows the amino acid identity of the amino acid sequence at the carboxyl terminus of human, human and human malaria tofuerin.
  • the amino acid indicated by * indicates that it is the same amino acid as human lactoferrin.
  • FIG. 3 is a diagram showing the results of far-western plotting for examining the binding between various partial polypeptides of human lactofurin and the E2 protein of HCV.
  • lane 1 shows the result of full-length human lactoferrin
  • lane 2 shows the result of full-length human transfusin
  • lane 3 shows the result of human lactoferrin (600-692)
  • lane 4 shows the result of human lactoferrin.
  • lane 5 shows the results of human lactoferrin (610-692)
  • lane 6 shows the results of human lactoferrin (624-692)
  • lane 7 shows the results of human lactoferrin.
  • Lane 8 shows the results for phosphorus (640-692)
  • lane 8 shows the results for human lactofurin (653-692).
  • FIG. 4 is a diagram summarizing the results shown in FIGS. 2 and 3. Number in figure The values indicate the amino acid numbers of human lactoferrin and human CD81.
  • FIG. 5 is a diagram showing the results of FW Wesson blotting in which the binding between various partial polypeptides of human lactofurin and the E2 protein of HCV was examined.
  • lane 1 shows the results for full-length human lactoferrin
  • lane 2 shows the results for full-length human transferrin
  • lane 3 shows the results for human lactoferrin (600-692)
  • lane 4 shows the results for human lactoferrin.
  • lane 5 are the results of human fertographin (610-652)
  • lane 6 are the results of human lactofurin (610-652) (however, the 635th phenylalanine was replaced with leucine).
  • Lane 7 shows the results for human lactofurin (610-642), lane 8 shows the results for human lactofurin (620-652), and lane 9 shows the results for human lactofurin (620-642).
  • Lane 10 shows the result of the thioredoxin tag (22 kDa) obtained by expressing the empty vector.
  • FIG. 6 is a diagram showing the results of far-western blotting for examining the binding between various partial polypeptides of human lactofurin and the E2 protein of HCV.
  • lane 1 shows the results for human lactoferrin (600-622)
  • lane 2 shows the results for human lactofurin (600-627)
  • lane 3 shows the results for human lactoferrin (600-632).
  • Lane 4 shows the results for human lactofurin (600-637)
  • Lane 5 shows the results for human lactofurin (600-642)
  • Lane 6 shows the results for human lactoferrin (600-647)
  • Lane 7 shows the results for human lactoferrin (600-647). Show results for lactofurin (600-652) o
  • FIG. 7 is a graph showing the results of examining the effects of each of human tractoferrin (600-632) and peractyltoferrin (599-631) on the infection of cultured hepatocytes with HCV.
  • panel A shows the results for human phractoferin (600-632)
  • panel B shows the results for ⁇ -lactofurin (599-631).
  • BEST MODE FOR CARRYING OUT THE INVENTION One embodiment of the present invention relates to a binding activity of HCV to E2 protein, which is capable of inhibiting, suppressing or reducing the adsorption of HCV to cells, and having a size more suitable for incorporation into a living body.
  • polypeptide of the present invention is a portion of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6 at positions 1 to 33 or the amino
  • One major feature is that it contains at least the positions 1 to 53 in the acid sequence.
  • the polypeptide of the present invention expresses an excellent property of exhibiting a binding activity to HCVE2 protein.
  • polypeptide of the present invention exhibits an excellent property that it can actually inhibit, suppress or reduce HCV infection despite having a shorter length than full-length lactoferrin.
  • polypeptide of the present invention exhibits the binding activity to HCV disclosed in each of WO 01/73222 and WO 01/47545 pamphlet.
  • lactoferrin refers to the amino acid sequence of the mature lactoferrin protein in which the signal sequence has been processed.
  • the amino acid sequence of lactoferrin is expressed as “610th to 652nd”, the amino acid sequence is the number of amino acids at the N-terminal amino acid of the mature protein of lactoferrin as the first amino acid. Is shown.
  • human lactoferrin (XY) refers to the Xth to Yth positions in the amino acid sequence of human lactofurin described in Genebank Accession No .: X52941, unless otherwise specified.
  • ⁇ -lactofurin (XY) J is referred to as" X-lactofurin (XY) J, unless otherwise specified, from the X-th position in the amino acid sequence of ⁇ -lactofurin described in Genebank Accession No .: M63502. " It shows a polypeptide consisting of the amino acid residue at position Y. Further, in this specification, unless otherwise specified, the expression “ ⁇ malactoferin (XY)” is described in Genebank Accession Number: AJ010930. 2 shows a polypeptide consisting of amino acid residues at positions X to Y in the amino acid sequence of sylactofurin.
  • lactoferrin has recently been shown to be effective in protecting against HCV infection [Ikeda et al, Biochem Biophys Res Com Mardan. '245 (2), 549-553 (1998); Ikeda et al, Virus Res., 66 (1), 51-63 (2000); Tanaka et al, Jpn. J. Cancer Res. 90 (4), 367-371 (1999)].
  • lactofurin has been shown to bind to the HCV E2 protein (58th Annual Meeting of the Japanese Cancer Society, 145 pages 145 ⁇ .223, 1999). However, it has not been clarified which restricted region in lactofurin is essential for the binding of HCV to the E2 protein.
  • the present inventors have conducted intensive studies on which region in lactofurin has a binding activity to the E2 protein of HCV and the strength of the binding.
  • the present invention relates to a polypeptide comprising the carboxyl terminal 93 amino acid portion of each of human, human, and malactoferrin, specifically, the following three sequences:
  • the 93 amino acid portion contains a binding region to HC2 E2 protein. Furthermore, according to these polypeptides, the binding strength is expected to be about twice as strong as the binding activity of HCV to CD81 large trajectory loop, which is the original receptor for HCV. Demonstrate the outside effect.
  • the present invention relates to the 93 amino acid portion (SEQ ID NO: 2) of which limited region of the 93 carboxyl terminal amino acids of human lactoferrin is essential for binding to the E2 protein of HCV.
  • SEQ ID NO: 2 the positions 600-632 of human lactoferrin (positions 1 to 3 of SEQ ID NO: 2) If it has the 33 amino acid portion of the 3 amino acid portion or the 43 amino acid portion of the human lactofurin at positions 610 to 652 (positions 1 to 53 of SEQ ID NO: 2), Based on the surprising findings of the present inventors that they have a binding activity to E2 protein. Further, the present invention is based on the inventor's opinion that the retention of 635-position of human lactoferrin (position 36 of SEQ ID NO: 2) is important for binding to HCV0E2 protein. Based on their surprising findings.
  • (II) A sequence consisting of a part of the amino acid sequence according to (I), wherein the sequence is a portion of the amino acid sequence at positions 1 to 33 or the first amino acid sequence in the amino acid sequence. A sequence comprising a portion from position 1 to position 53,
  • HCVE2 protein comprising at least:
  • the length of the polypeptide of the present invention when containing the above (I), is at least 93 amino acid residues, preferably 93 to 500 residues, more preferably 93 to 3 residues. 0 residues, more preferably 93 to 200 residues, even more preferably 93 to 100 residues, or SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.
  • the full length of the amino acid sequence selected from the group consisting of
  • the length of the polypeptide of the present invention is at least 33 amino acids. Residues, preferably 33-92 residues, more preferably 33-80 residues, even more preferably 33-60 residues, even more preferably 33- It is desirable that the length of the polypeptide of the present invention is at least 43 amino acid residues, preferably 43 to 50 residues. It is desirable that the number of residues is 92, more preferably 43 to 80, still more preferably 43 to 60, and still more preferably 43 to 50.
  • the compound containing both the first to third position in the amino acid sequence and the first to fifth position in the amino acid sequence Those containing the positions -53 are also included.
  • the polypeptide comprises the full length of the amino acid sequence shown in SEQ ID NO: 2 or a part thereof. And a polypeptide comprising at least the 1st to 33rd positions of the amino acid sequence, or a polypeptide consisting of the full length of the amino acid sequence shown in SEQ ID NO: 2 or a part thereof. And a polypeptide containing at least the 11th to 53rd positions of the amino acid sequence. More specifically,
  • polypeptide consisting of a part of the amino acid sequence shown in SEQ ID NO: 2, and containing at least the 1st to 53rd positions of the amino acid sequence;
  • polypeptide that includes the portion and extends in the N-terminal direction and / or the C-terminal direction on the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 from the position in parentheses, A polypeptide in which an amino acid residue is added to the N-terminus and the C- or C-terminal of the amino acid sequence
  • the number of amino acid residues added at the N-terminus and the amino or C-terminus of the amino acid sequence is such that the polypeptide exhibits the binding activity and inhibits, suppresses or reduces HCV infection. Any range is acceptable as long as the ability is exhibited.
  • the amino acid residue to be added may be a natural amino acid sequence, specifically, the same amino acid residue as the corresponding portion in the amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4 and 6. Any amino acid having physicochemical properties suitable for exerting the binding activity of the polypeptide and the ability to inhibit, suppress or reduce HCV infection. Further, such amino acid residues may be various modified residues capable of imparting affinity to a living body.
  • polypeptides of the present invention include, for example,
  • polypeptides can be made by either chemical synthesis or genetic engineering techniques. That is, if the polypeptide can be synthesized by a conventional chemical synthesis method using a peptide synthesizer or the like, by expressing a DNA encoding the polypeptide by a conventional genetic engineering technique, Polypeptides can also be prepared.
  • the peptide can be synthesized according to a method used in ordinary peptide chemistry.
  • the known method is described in the literature [Peptide Synthesis, Interscience, New York, (1966); (The Proteins), Vol 2, Academic Press Inc., New York, (1976); Peptide Synthesis, Maruzen Co., Ltd., (1975); Peptide Synthesis Fundamentals and Experiments, Maruzen Co., Ltd., (1985) ); Pharmaceutical development continued Volume 14 ⁇ Peptide synthesis, Hirokawa Shoten, (1991)].
  • the DNA encoding the above-mentioned various polypeptides is human lactoferrin (Genebank cession number: X52941), ⁇ -silactoferin (Geneban quaxion number: M63502), or ⁇ malactoferin (Genebank). Accession number: AJ010930) can be isolated by amplifying the desired portion on the cDNA sequence by PCR.
  • Examples of the nucleotide sequences of the DNAs encoding the polypeptides described in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 are SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, respectively.
  • the base sequences described can be exemplified. In addition, those skilled in the art will understand that such base sequences may be different sequences through degeneracy or nucleic acid polymorphism.
  • the expression vector into which the DNA is inserted can be appropriately selected depending on the host used, the purpose, and the like, and examples thereof include plasmids, phage vectors, and virus vectors.
  • examples of the vector include a plasmid vector such as pUC118, pUC119, pBR322, and PCR3, and a phage vector such as ⁇ ; igtll.
  • examples of vector one, pYES2, etc. P YBUra3 is like et be.
  • examples include pAcSGHisNT-A.
  • the host is an animal In the case of cells, PKCR, pCD8, pG2, pcD secretion, pRc / RSV, pRc / CMV and the like are listed.
  • the vector may appropriately have factors such as a promoter capable of inducing expression, a marker gene for selection, and a terminator.
  • a sequence expressed as a fusion protein with thioredoxin, His tag, or GST may be added.
  • a GST fusion protein eg, PGEX4T
  • an appropriate promoter eg, lac, tac, trc, trp, CMV, SV40 early promoter
  • Myc His vector one having the sequence (pcDNA3. 1 / Myc-His, etc.)
  • P ET32a a sequence expressed as a fusion protein with thioredoxin, His tag, or GST (glutathion S-transferase)
  • the host used herein includes Escherichia coli, yeast, insect cells, animal cells, and the like.
  • Escherichia coli include the E. coli K-12 strain HB101 strain, C600 strain, JM109 strain, DH5 strain, and AD494 (DE3) strain.
  • yeast include Saccharomyces-cellubiye.
  • Animal cells include L929 cells, BALB / C3T3 cells, C127 cells, CH0 cells, COS cells, Vero cells, Hela cells, and the like. Insect cells include Sf9.
  • a normal introduction method suitable for the host cell may be used. Specific examples thereof include a calcium phosphate method, a DEAE-dextran method, an electroporation method, and a method using a lipid for gene introduction (Lipofectamine, Lipofect in; Gibco-BRL).
  • the cells are cultured in a normal medium containing a selection marker to select transformed cells or transduced cells in which the expression vector has been introduced into host cells.
  • the polypeptide of the present invention By continuously culturing the transformed cells or transduced cells obtained as described above under conditions suitable for expression of the polypeptide, the polypeptide of the present invention can be obtained. Can be obtained.
  • the obtained polypeptide can be further isolated and purified by general biochemical purification means.
  • the purification means include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography and the like.
  • the polypeptide of the present invention when expressed as a fusion protein with the aforementioned thioredoxin, His tag, GST, etc., it can be isolated and purified by a purification method utilizing the properties of these fusion proteins and tags. .
  • the fact that the thus prepared polypeptide of the present invention has an activity of binding to HC2 E2 protein can be carried out, for example, by a conventional immunological technique, for example, phage stamp lotting or a physical technique, For example, it can be analyzed by surface plasmon analysis.
  • the polypeptide of the present invention is subjected to SDS-PAGE, and this is transferred to a membrane (PVDF membrane).
  • the membrane is reacted with the HC2 E2 protein [Vaccine, 14 (17/18), 1590-1596 (1996)], and then reacted with an anti-HCVE2CHCV E2 protein) antibody.
  • the anti-HCVE2 antibody used here includes, for example, rat anti-HCVE2 monoclonal antibody Mo-12 [Microbiol I bandage unol., 42 (12), 875-877 (1998)]. Thereafter, the labeled secondary antibody is further reacted.
  • an HRP-labeled anti-rat IgG secondary antibody for example, manufactured by Amersham Pharmacia, catalog number: NA935 etc.
  • the binding activity between the polypeptide of the present invention and the E2 protein of HCV can be measured by detecting the label by a technique such as chemiluminescence and exposing the label to an X-ray film. .
  • the detection means include optical detection means such as fluorescence intensity and fluorescence polarization degree; matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer (MALDI-TOF MS), electrospray ionization mass spectrometer (ESI-MS) and other detection means in combination with a mass spectrometer; detection means based on changes in the natural frequency of a substance such as quartz.
  • the binding is represented by one gram of a sensor showing the formation of a complex between the polypeptide of the present invention and the E2 protein of HCV, for example, one gram of an optical sensor fluctuated by the introduction of protein by liquid transfer or The mass can be measured using the sensorgram as an index.
  • the affinity of the polypeptide of the present invention for the E2 protein of HCV can be estimated from the binding rate.
  • methods for examining that the polypeptide of the present invention protects HCV from infecting hepatocyte-derived cells include, for example, Biochem Biophys Res Com Band., 245 (2), 549-553 (1998) and Virus Res., 66 (1): 51-63 (2000). An example is shown below.
  • a human hepatocyte cell line PH5CH8 was added to a hepatocyte culture medium [composition: 500 ml F12 medium, 500 ml D-ME, 1% fetal serum, 100 ng / ml BGF, 200 g / ml kanamycin, 10 zg / ml gentamicin ) And culture at 37 ° C for about 2 days.
  • HCV virus for example, Serum IB-2 Genotype lb
  • the polynucleotide of the present invention Mix with the peptide and react at 4 ° C for about 60 minutes. Add this reaction to the previous PH5CH8 cells and incubate at 37 ° C for 90 minutes.
  • RNA is recovered from the obtained cells by a conventional method.
  • a part of the HCV genome is amplified by performing RT-nested PCR on the RNA, and the obtained product is subjected to electrophoresis to detect an amplified fragment.
  • the fact that the amplified fragment is not detected or, if detected, is extremely weak confirms that the polypeptide of the present invention has a protective effect against HCV infection.
  • the amount of the amplified fragment can be evaluated by staining the nucleic acid on the gel after electrophoresis by a conventional means, and measuring the intensity of the band by densitometry, etc.
  • polypeptide of the present invention a structure similar to the sequence of lactofurin having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 (sequence ), And a polypeptide having a binding activity to HC2 E2 protein.
  • the present inventors have selected HCV from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, although it is known that HCV infects only humans and chimpanzees.
  • the fact that the polypeptide consisting of the amino acid sequence binds to the HCV and the sequence identity between the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 are as follows: 74.2%, and 71% between humans and dogs. That is, the present inventors have thus obtained not only human lactoferrin, but also a human lactoferrin-derived polypeptide, and an amino acid residue that retains the above-mentioned identity with human lactoferrin. It was clarified that HCV retains the activity of binding to E2 protein even with a substituted polypeptide.
  • the present invention relates to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. Amino acid sequence having a sequence identity of at least No acid sequence ”) or a portion thereof, and
  • a polypeptide consisting of a portion corresponding to positions 1 to 38; a polypeptide consisting of a portion corresponding to positions 1 to 43; corresponding to positions 1 to 48 A polypeptide consisting of a part corresponding to positions 1 to 53, a polypeptide consisting of a part corresponding to positions 1 to 53, and a polypeptide consisting of a part corresponding to positions 1 to 53
  • a polypeptide consisting of a portion corresponding to position 93 or a polypeptide consisting of a portion corresponding to positions 1 to 93 and having the above-mentioned activity is exemplified.
  • the “corresponding portion” of a specific portion of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 is SEQ ID NO: 2, A member selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6
  • SEQ ID NO: 2 A member selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6
  • an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 Refers to the part of the sequence that matches the above part.
  • sequence identity of an amino acid sequence refers to residue identity and homology between two polypeptides. That is, the sequence identity is determined not only when the amino acid residues are completely identical (identity) between the two sequences to be compared, but also when the similarity (homology) of the properties of the amino acid residues is taken into account. It may be done.
  • sequence identity is determined by comparing two optimally aligned sequences over the region of the sequence to be compared.
  • the polypeptide to be compared may have an addition or deletion (for example, a gap or the like) in the optimal alignment of the two sequences.
  • amino acid sequence having at least 71%, preferably at least 75%, more preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity is mentioned.
  • amino acid substitution position and the type of amino acid the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6 is substituted with the 3rd-position of furanalanine. It is preferable that the modified polypeptide is retained without any modification. For other points, see the section of the modified polypeptide of the present invention described later.
  • homologous polypeptides of the present invention include camel lactofu X phosphorus (Gene Bank Accession No .: AJ131674), bushu lactoferrin (Gene Bank Accession No .: M92089), and jagi lactoferrin ( Gene bank accession number: X78902) or the full-length or a part of the C-terminal 93 amino acids of mouse lactoferrin (Gene bank accession number: NM-008522) (1st to 33rd positions, Or including the 11th to 53rd positions).
  • the results of calculating the sequence identity between the C-terminal 93 amino acid portion of these other lactoferrins and the amino acid sequence shown in SEQ ID NO: 2 using Vector NTI are shown below. All retain 71% or more sequence identity in this region. table 1
  • the present invention relates to a polypeptide other than funnilaranine at position 36 in the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.
  • At least one amino acid sequence having a substitution, deletion, addition or insertion hereinafter referred to as a modified amino acid sequence or at least a part thereof, and
  • polypeptide comprising a part of the modified amino acid sequence is “polypeptide comprising a part of the modified amino acid sequence”.
  • a portion corresponding to positions 1 to 33 of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, or 11 A polypeptide consisting of a portion corresponding to positions 3 to 53, or
  • modified polypeptide include at least one residue other than phenylalanine at position 36 in the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6.
  • a portion corresponding to positions 1 to 33 of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 A polypeptide consisting of portions corresponding to positions 1 to 38; a polypeptide consisting of portions corresponding to positions 1 to 43; a portion corresponding to positions 1 to 48 A polypeptide consisting of portions corresponding to positions 1 to 53, a polypeptide consisting of portions corresponding to positions 1 to 53, and positions 1 to 93
  • the polypeptide include a polypeptide corresponding to the position or a polypeptide corresponding to the positions 1 to 93 and having the above-mentioned activity.
  • a specific portion of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 As a result of performing the same alignment as in the above, it indicates a sequence portion that matches the corresponding portion on the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.
  • the polypeptide relating to the modification of the amino acid sequence can be obtained by subjecting the DNA encoding the polypeptide to conventional site-specific mutagenesis and then expressing the DNA by a conventional method.
  • the site-directed mutagenesis method include a method using an amber mutation (gapped, duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), and PCR using a mutagenesis primer. And the like.
  • the number of amino acids modified as described above is at least one residue, specifically, one or several, or more. The number of such modifications may be, for example, in a range where the activity can be found by measuring the activity according to the method for measuring the binding activity of HCV to E2.
  • amino acid sequence selected from the range described in the above section “Homologous polypeptide”, ie, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6 and at least 71% It is desirable that the modification be performed within the range having the sequence identity of the above.
  • the localization position of such a substitution is a position where amino acid residues in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 are not identical (see FIG. 2). ) Is preferred. That is, specifically, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, amino acid sequence 4, position 5, position 7, position 8, position 9 of the amino acid sequence selected from the group consisting of , 10th, 11th, 13th, 16th, 17th, 20th, 21st, 24th, 27th, 28th , 31st, 37th, 54th, 56th, 58th, 59th, 63rd, 67th, 68th, 71st , No. 72, No. 74, No. 77, No. 78, No. 81, No. 89, No. 92, No. 93.
  • it is desirable that the phenylalanine in the 36th position is not substituted.
  • the type of amino acid substituted at the substitution position may be the amino acid residue at the substitution position on the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. It is preferable to be selected from among them. That is, for example, substitution of arginine at position 4 of the amino acid sequence shown in SEQ ID NO: 2 with glutamine (human ⁇ puma type) or lysine at position 7 to arginine (human-p And ⁇ type).
  • substitutions may have similarities in hydrophobicity, charge, pK, steric features, etc. And substitution with amino acids having the above properties.
  • substitutions include, for example, 1 glycine, alanine; 2 valine, isoleucine, leucine; 3 aspartic acid, glutamic acid, asparagine, glutamine, 4 serine, threonine; 5 lysine, arginine; Substitution within the group of lualanin and tyrosine is mentioned.
  • modified polypeptide of the present invention includes camelid lactoferrin (Genebank cession number: AJ131674), butalactoferrin (Genebank cession number: M92089), and jagiract toferin (Genebank cession number). : X78902) or the entire C-terminal 93 amino acids of mouse lactoferrin (Genebank Accession Number: NM-008522) or a part thereof (positions 1 to 33, or positions 11 to 53) (Including the position part).
  • the method for producing the modified polypeptide described above and the method for measuring the activity are the same as those for the above-described polypeptide of the present invention.
  • lactoferrin has an increased anti-HIV and anti-HCV activity by acylating or aminating a specific amino acid residue of lactoferrin [J. pept. Sci., 5 ( 12), 563-576 (1999)], and it is expected that the activity of the polypeptide of the present invention is also increased by the same acylation and amination.
  • Polypeptides (including homologous polypeptides and modified polypeptides). That is, since the above-mentioned polypeptide has a binding activity to HC2 E2 protein, it is clear that a polypeptide containing the polypeptide also has the same binding activity.
  • human lactoferrin (gene bank accession number: X52941), ⁇ lactoferrin (gene bank accession number: M63502), and A polypeptide extending from the C-terminal 93 amino acid portion, which is the corresponding position on maltoferrin (GeneBank Accession No .: AJ010930), toward the N-terminus.
  • the polypeptide consisting of the amino acid sequences shown in Gene Bank Accession Nos .: X52941, M63502 and AJ01093 It is excluded from the scope of the present invention.
  • positions 1 to 3 of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 positions 610 to 632 of human lactofurin
  • polypeptide comprising the full length or a part of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, and the amino acid sequence thereof.
  • a polypeptide having at least a portion of positions 1 to 33 of the HCV and having an activity of binding to HC2 E2 protein can also be provided.
  • All of the polypeptides of the present invention described above have an activity of binding HCV to the E2 protein, and furthermore, have the property of inhibiting, suppressing or reducing HCV infection, and thus protect HCV infection. It can be used effectively as an agent. That is, the present invention provides a protective agent against HCV infection, comprising the polypeptide of the present invention as an active ingredient.
  • HCV infection protective agents of the present invention are those containing a shorter polypeptide having a sequence portion important for binding to HCV as an active ingredient. That is, the above-mentioned polypeptide of the present invention, in particular, a polypeptide consisting of positions 1 to 33 of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; , SEQ ID NO: 2, selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6.
  • a polypeptide consisting of positions 11 to 53 of the amino acid sequence obtained is exemplified as a preferable active ingredient of the HCV infection protective agent of the present invention.
  • Such a polypeptide has the effect of being able to reduce the dose as compared to the case of administering lactoferrin itself, decomposing proteins during absorption during oral administration, and reducing side effects during intravenous injection. Such an effect can be obtained.
  • the polypeptide of the present invention which is an active ingredient, can be administered as it is, or can be administered in a particulate dosage form.
  • the dosage form can be a liposome formulation, a particulate formulation bound to beads having a diameter of m; m, a formulation bound to lipid, and the like.
  • the dosage can be adjusted as appropriate depending on the severity of the symptoms, the age of the patient, body weight, etc., but is usually 0.001 mg / kg / time to 1000 mg / kg / time. Thereafter, it is preferably administered once every several days to several months depending on the amount of virus in the cells.
  • oral administration arterial injection, intravenous injection, intramuscular injection, subcutaneous administration, intradermal administration, local injection to the liver, and the like are possible.
  • polypeptide of the present invention which is an active ingredient, is preferably stable in blood.
  • the polypeptide may be modified.
  • polypeptides that are stable in blood can be produced by modifying the polypeptide at the N-terminus or substituting it with D-amino acids.
  • the HCV infection protective agent of the present invention is specifically used as a therapeutic or preventive agent for hepatitis C. Therefore, the present invention provides a therapeutic or preventive agent for chronic hepatitis C. More specifically, it is used for chronic hepatitis C and acute hepatitis C. In particular, it is effectively used in the treatment or prevention of chronic hepatitis C showing resistance to existing insulin therapy.
  • the HCV infection protective agent of the present invention can be used in combination with interferon.
  • Polypeptides of the invention directly inhibit the binding of HCV to target cells Therefore, it is possible to further reduce the relapse of the interface-resistant virus at the end of administration of interferon, for example.
  • a method for treating or preventing chronic hepatitis C is characterized in that the polypeptide of the present invention is administered to a subject to be administered in a therapeutically effective amount sufficient to prevent the binding of HCV to cells.
  • a therapeutically effective amount of the agent for preventing HCV infection of the present invention or the agent for treating or preventing chronic hepatitis C of the present invention may be administered.
  • interferon may be further administered in combination. Examples of the administration target include an individual who has chronic hepatitis C, an individual who may have chronic hepatitis C, and the like.
  • the present invention also provides a polynucleotide encoding the polypeptide of the present invention.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • These polynucleotides of the present invention can be obtained by combining the cDNA sequence of human lactoferrin (Genebank Accession No .: X52941), ⁇ -syruptoprin (Genebank Question No .: M63502), or ⁇ -malactoferin (Genebank Accession No .: AJ010930). Based on this, a person skilled in the art can easily produce it. For details, refer to the section of the polypeptide of the present invention.
  • polynucleotide of the present invention examples include, for example, a polynucleotide encoding any of the aforementioned polypeptides of the present invention, SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5.
  • the polynucleotide of the present invention includes a base sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, etc., from a sequence that differs through degeneracy or nucleic acid polymorphism.
  • polynucleotide comprising a part of the nucleotide sequence.
  • the polynucleotide of the present invention has a length of at least 279 nucleotides, preferably 279 to 150 nucleotides, more preferably 27.9 nucleotides in the case of a polypeptide containing the entire amino acid sequence.
  • 79-900 nucleotides more preferably, 279-600 nucleotides, more preferably, 279-300 nucleotides.
  • Such a polynucleotide can be used for production of the polypeptide of the present invention, application to the therapeutic or preventive agent, and the like.
  • the “part of the base sequence” includes the first to third positions in the amino acid sequence when used for production of the polypeptide of the present invention, application to the therapeutic or preventive agent, and the like.
  • At least 99 nucleotides, preferably 99-276 nucleotides, more preferably 99-240 nucleotides, even more preferably 99-180 nucleotides, corresponding to the polypeptide More preferably, it is preferably from 99 to 150 nucleotides, and at least 12 9 nucleotides, preferably 12 9 nucleotides, corresponding to the polypeptide containing the 11th to 53rd positions.
  • To 276 nucleotides more preferably, 129 to 240 nucleotides, even more preferably, 129 to 180 nucleotides.
  • the ⁇ part of the base sequence '' may be, when producing the polypeptide of the present invention using a polynucleotide as a probe or a primer, at least 12 nucleotides, preferably 15 to 200 nucleotides, More preferably, it is 15 to 100 nucleotides, and still more preferably, it is 15 to 50 nucleotides.
  • the polynucleotide of the present invention can be effectively used when producing the polypeptide of the present invention, and can make it possible to efficiently supply the polypeptide.
  • the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.
  • position 270 of the nucleotide sequence described in SEQ ID NO: 1 is c in Genebank Accession Number: X52941, but is t in the actually obtained cDNA. This is thought to be due to genetic polymorphism.
  • the regions to be expressed ((1) to (4) below) were amplified by PCR using a set of primers for amplification and KOD DNA polymerase.
  • the amplification product was incorporated into BamHI-Hindl lsite of pET32a [manufactured by Novagen], which can be expressed as a fusion protein with the reduced protein thioredoxin and His tag by IPTG induction.
  • amino acid sequence SEQ ID NO: 2, base sequence; SEQ ID NO: 1
  • a reverse transcription reaction is performed using RNA extracted from peripheral blood lymphocytes in the same manner as in 1) above, and the cDNA of malactofurin (Genebank Accession No .: AJ010930) is amplified by KOD DNA polymerase. did.
  • the region encoding the carboxyl terminal 93 amino acids was further amplified and assembled into the BamHI-Hindlll site of pET32a. I was crowded.
  • Reverse transcription was also performed using RNA extracted from mammary gland tissue, and cDNA of lactoferrin (Genebank Accession No .: M63502) was amplified.
  • the region (amino acid sequence; SEQ ID NO: 4, base sequence; SEQ ID NO: 3) encoding the carboxyl terminal 93 amino acids was further amplified using the obtained amplification product as type II, and the BamHI-Hindl 11 si of pET32a was further amplified. Built in te.
  • RNA was extracted from PH5CH8 cells, which are human hepatocytes immortalized with 40 large T antigen. Using the obtained RNA as type III, a reverse transcription reaction was performed using an oligo dT primer, and a cDNA containing human CD81 (GeneBank Accession No .: XM-006475) was amplified using K0D DNA polymerase.
  • a region containing amino acids 113 to 201 is further amplified using KOD DNA polymerase, and the amplified product is incorporated into the BamHI-Hindlll site of pET32a, resulting in a human CD81 large exclusion loop.
  • KOD DNA polymerase KOD DNA polymerase
  • the expression vector obtained in the above 1)) to 3) was transformed into host E. coli AD494 (DE3) (Novagen). Then, the cells cultured in 10 ml of LB medium at 37 ° C. for 16 hours were added to 200 ml of LB medium supplemented with 50 / zg / ml of ampicillin and 15 ⁇ g / ml of kanamycin, and cultured at 37 ° C. for 4 hours. Thereafter, ImM IPTG was added to the culture, which was further cultured for 4 to 5 hours. The resulting culture was centrifuged at 3000 ⁇ g for 10 minutes. Next, the pellet was frozen at ⁇ 80 ° C.
  • the pellet was dissolved again with 10 ml of B-PER® containing 200 / xg / ml lysozyme (Sigma), and the mixture was stirred at room temperature. The resulting mixture was then centrifuged at 20000 xg for 30 minutes. The pellet was then dissolved in 20 ml of 10-fold diluted B-PER® and centrifuged at 20,000 xg for 30 min.Three times, the resulting pellet was reconstituted in Binding Buffer containing 6M urea containing urea. Novagene (Novagen)] and used for purification.
  • Example 1 Whether the purified human lactofurin fragment protein obtained in Example 1 binds to E2 protein, which is an HCV envelope protein, was examined by far-stamping. As a probe, E2 protein [Inudoh et al, Vaccine, 14 (17/18), 1590-1596 (1996)] secreted into the culture solution by CH0 cells was used.o
  • the purified protein obtained in Example 1 was subjected to 12% SDS-PAGE using 2 jag each, and was transferred to a PVDF membrane. Then, blocking was performed for 1 hour with N-buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Triton-X, 0.25% gelatin] to which 2 BSA (manufactured by Sigma) was added. did.
  • the culture supernatant of the CH0 cells containing E2 protein (total protein amount 250 g) was diluted with 5 ml of N-buer containing the same BSA. The resulting solution was brought into contact with the PDVF membrane, and a binding reaction was performed at room temperature for 60 minutes. The PDVF membrane was washed three times with N-buffer for 10 minutes ⁇ 3 times, and then blocked again with N-buffer supplemented with 2% BSA (Sigma) for 1 hour.
  • the membrane was contacted, and the reaction was performed for 60 minutes.
  • the PVDF membrane was washed three times with 0.1% Tris-line solution [10 mM Tris (pH 7.5), 150 mM NaCl, 0.1 Tween 20] for 5 minutes and then diluted 1000-fold with HRP-labeled anti-rat cells.
  • the antibody was brought into contact with 0.1% Tris-saline solution to which a secondary antibody (Amersham Pharmacia cat.
  • Fig. 1 shows the results of Western plotting of the 93 amino acid portions at the carboxyl terminus of human, male and female malachtoferrins. Lanes 1 to 7 in FIG. 1 show the following results.
  • Lane 1 Thioredoxin tag (22 kDa) obtained by expressing empty vector pET32a
  • Lane 2 Human lactofurin (Sigma, L3770)
  • Lane 3 human transpurin (Sigma, T6549, 80kDa)
  • Lane 4 Carboxyl terminal 93 amino acids of human lactoferrin
  • Lane 5 93 amino acids at the carboxyl terminus of ⁇ -silactoferin
  • FIG. 2 shows the amino acid identity of the 93 amino acid sequence at the carboxyl terminus of human, human and maleactoferrin.
  • the amino acid indicated by * indicates that it is the same amino acid as human lactoferrin.
  • the sequence identity to human lactoferrin (100%) was 74.2% for peractylfurin and 71.0% for malactoferin. This value was higher than the sequence identity to human transfulin (59.1) and the sequence identity to dystransulin (51.6%).
  • FIG. 3 shows the results of far western plotting performed in the same manner as in Example 2 when various partial polypeptides of human lactoferrin (see (1) to (4) in Example 1-1). Lanes 1 to 8 in FIG. 3 show the following results.
  • Figure 4 summarizes the results. Human lactoferrin and HC2 E2 protein bind at least between amino acids 610 and 652 of human lactoferrin (positions 11-53 of SEQ ID NO: 2). It was expected that there would be important areas.
  • FIG. 5 shows the results of Far Western plotting performed in the same manner as in Example 3 for the amino acid sequences at positions 610 to 652 of human lactofurin and the sequences before and after it. Lanes 1 to 10 in FIG. 5 show the following results.
  • Lane 1 human lactofurin (Sigma, L3770) Lane 2 human transferrin (Sigma, T6549, 80kDa)
  • Lane 7 Human lactofurin (610-642)
  • Lane 8 human lactoferrin (620-652)
  • Lane 10 Thioredoxin tag (22kDa) obtained by expressing empty vector
  • FIG. 6 shows the results of the same amino acid sequence at positions 600 to 652 of human lactofurin and a further reduced version of the amino acid sequence, which was subjected to the same Farwesian blotting as in Examples 2 to 4. Lanes 1 to 7 in FIG. 6 show the following results.
  • Lane 1 Human lactoferrin (600-622)
  • Example 5 human Ractophilin (600-632) in which binding of HCV to the E2 protein was observed:
  • Psylactoferrin (599-631), which is a sequence portion corresponding to the polypeptide:
  • VVSRSDRAAH VKQVLLHQQA LFG NGKNCP DKF (SEQ ID NO: 8)
  • the human hepatocyte cell line PH5CH8 was used for hepatocyte culture medium [Composition: F12 medium 500 ml, D-MEM 500 0 ml, 13 ⁇ 4 fetal serum, 100 ng / ml EGF, 200 zg / ml kanamycin, 10 g / ml gentamicin] 100 ⁇ ⁇ ⁇ ⁇ 5 xlO cells were seeded on a 96-well plate treated with collagen containing 1 and cultured at 37 ° C for 2 days.
  • HCV virus-positive human serum (containing IB-2 / genotype lb: 2xlO 4 copies of HCV RNA) is mixed with the above-mentioned polypeptide derived from human lactoferrin or dilactoferrin. For 60 minutes. The resulting mixture was added to PH5CH8 cells and incubated at 37 ° C for 90 minutes. Next, the PH5CH8 cells were washed three times with the above-mentioned medium for culturing hepatocytes, and further cultured at 32 ° C for 24 hours.
  • RNA was recovered from the cells by a conventional method, and HCV RNA contained in 0.5 g RNA was analyzed using LightCycler (Roche Diagnostics) in the following literature (Nozaki A. and Kato N., Quantitative Method of Intracellular Hepatitis C Virus RNA using LightCycler PCR. Acta Medica Okayama, 56 (2), 107-110 (2002)).
  • an HCV binding polypeptide having an activity of binding to the HC2 E2 protein is provided.
  • the binding of HCV to the E2 protein prevents the binding of HCV to hepatocytes, and further prevents the infection, thereby treating chronic hepatitis C or the like. It becomes possible to prevent. Accordingly, a therapeutic or preventive agent for chronic hepatitis C and a method for treating or preventing chronic hepatitis C are provided.

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Abstract

L'invention concerne des moyens d'inhibition, de régulation ou de diminution de l'infection corporelle par le virus HCV. Elle concerne des polypeptides, ou des dérivés de ces polypeptides, possédant une activité de liaison à la protéine E2 du virus HCV portant au moins les acides aminés aux positions 1 à 33 dans la séquence représentée par les séquences SEQ ID NO:2, SEQ ID NO:4 et SEQ ID NO:6 ou les acides aminés aux positions 11 à 53 de ces séquences. Elle concerne encore des polypeptides contenant ces polypeptides, des préventifs de l'infection par le virus HCV contenant ces polypeptides en tant que principe actif, des préventifs ou des remèdes contre l'hépatite C chronique contenant ces polypeptides comme principe actif, des polynucléotides codant pour ces polypeptides, un procédé de traitement ou de prévention de l'hépatite C chronique consistant à administrer une dose, efficace sur le plan thérapeutique, d'un tel polypeptide à un sujet afin d'inhiber la liaison du virus HCV aux cellules, et l'utilisation de ces polypeptides afin de produire les préventifs contre l'infection par le virus HCV, ainsi que les remèdes et les préventifs décrits ci-dessus.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050116A1 (fr) * 2002-11-29 2004-06-17 Morinaga Milk Industry Co., Ltd. Inhibiteur de protease
FR2875501A1 (fr) * 2004-09-21 2006-03-24 Univ Paris Descartes Nouveaux derives peptidiques, compositions et utilisations dans un traitement therapeutique de l'infection par un herpesvirus

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WO2001047545A1 (fr) * 1999-12-28 2001-07-05 Sumitomo Pharmaceuticals Company, Limited Produits therapeutiques et prophylactiques pour l'hepatique chronique
WO2001072322A2 (fr) * 2000-03-27 2001-10-04 Pharming Intellectual Property B.V. Administration parenterale de fortes doses de lactoferrine

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Publication number Priority date Publication date Assignee Title
WO2001047545A1 (fr) * 1999-12-28 2001-07-05 Sumitomo Pharmaceuticals Company, Limited Produits therapeutiques et prophylactiques pour l'hepatique chronique
WO2001072322A2 (fr) * 2000-03-27 2001-10-04 Pharming Intellectual Property B.V. Administration parenterale de fortes doses de lactoferrine

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IKEDA M. ET AL.: "Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultured cells", VIRUS RES., vol. 66, no. 1, January 2000 (2000-01-01), pages 51 - 63, XP002952964 *
IKEDA W. ET AL.: "Lactoferrin markedly inhibits hepatitis C virus infection in cultured human hepatocytes", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 245, no. 2, 17 April 1998 (1998-04-17), pages 549 - 553, XP002952966 *
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TANAKA K. ET AL.: "Lactoferrin inhibits hepatitis C virus veremia in patients with chronic hepatitis C: a pilot study", JPN. J. CANCER RES., vol. 90, no. 4, April 1999 (1999-04-01), pages 367 - 371, XP002952965 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050116A1 (fr) * 2002-11-29 2004-06-17 Morinaga Milk Industry Co., Ltd. Inhibiteur de protease
EP1576964A4 (fr) * 2002-11-29 2008-06-11 Morinaga Milk Industry Co Ltd Inhibiteur de protease
FR2875501A1 (fr) * 2004-09-21 2006-03-24 Univ Paris Descartes Nouveaux derives peptidiques, compositions et utilisations dans un traitement therapeutique de l'infection par un herpesvirus
WO2006032781A2 (fr) * 2004-09-21 2006-03-30 Universite Rene Descartes - Paris 5 Derives peptidiques , compositions et utilisations dans un traitement therapeutique de l’infection par un herpevirus
WO2006032781A3 (fr) * 2004-09-21 2006-08-31 Univ Paris Descartes Derives peptidiques , compositions et utilisations dans un traitement therapeutique de l’infection par un herpevirus

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