WO1998007863A1 - VARIANTS DE L'INTERFERON τ HUMAIN - Google Patents

VARIANTS DE L'INTERFERON τ HUMAIN Download PDF

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Publication number
WO1998007863A1
WO1998007863A1 PCT/JP1997/002879 JP9702879W WO9807863A1 WO 1998007863 A1 WO1998007863 A1 WO 1998007863A1 JP 9702879 W JP9702879 W JP 9702879W WO 9807863 A1 WO9807863 A1 WO 9807863A1
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Prior art keywords
sequence
amino acid
polypeptide
leu
dna
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PCT/JP1997/002879
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English (en)
Japanese (ja)
Inventor
Masako Ishimura
Takashi Nishigaki
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Sankyo Company, Limited
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Publication date
Application filed by Sankyo Company, Limited filed Critical Sankyo Company, Limited
Priority to AU38667/97A priority Critical patent/AU3866797A/en
Publication of WO1998007863A1 publication Critical patent/WO1998007863A1/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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a virus !; an interferin mutant with low cytotoxicity, which is effective in treating infectious diseases, thigh ulcers and autoimmune diseases, and a method for producing the same by genetic manipulation. ,:.
  • Interferon produces various cells such as lymphocytes and fibroblasts in vivo.
  • Active bioactive protein has anti-virus activity /]] ⁇ anti-cancer activity ffj:
  • Interferon has various subtypes: fi: power 5 ', of which type I interface Interferon ", interferon, and interferon ⁇ are well-known as being classified as anti-viral.
  • interferon and are used as antiviral agents, and are used for hepatitis C.
  • Efficacy has been shown in 20 to 40% of patients with chronic hepatitis due to the virus (HCV) by high-dose administration (10 fi IU / i body, 3 times a week, 6 times) [Davis, GL, et.
  • HCV virus
  • interferon r is a protein originally secreted by the ovine conceptus (conceptus) from 10 to 21 days after pregnancy.
  • 0 TP-1 has the effect of inhibiting the secretion of prostaglandin F2a, which has ovulatory action, into the uterus.
  • et al. (1986) Biology of Reproduction 76, 841] which is thought to have a function involved in the establishment of pregnancy.
  • the present invention is a.
  • amino acid sequence represented by SEQ ID NO: S in the Sequence Listing at least Arg of amino acid No. 86 is substituted with Cys, and amino acid No. 85 is substituted with an amino acid other than Cys.
  • Consisting of amino acid sequences A polypeptide characterized by having a long-term activity (however, I: ⁇ ).
  • the polypeptide is preferably
  • polypeptide according to any one of (1) to (3), comprising: a) an amino ft sequence which is not replaced by an amino acid of SEQ ID NO: 4 of SEQ ID NO: 1 to 172.
  • polypeptide according to any one of (1) to (3) which comprises an amino acid sequence represented by amino numbers 1 to 172 of SEQ ID NO: 6 in the sequence list.
  • polypeptide according to any one of (1) to (3) which consists essentially of an amino acid sequence represented by amino acid numbers 1 to 172 of SEQ ID NO: 2 in the sequence listing.
  • polypeptide according to any one of (1) to (3), which essentially consists of the amino acid sequence represented by amino acid numbers 1 to 172 of SEQ ID NO: 6 in the sequence listing. And most preferably the polypeptide described in (7) to (9).
  • the present invention also provides
  • the DNA is preferably
  • E. c01ipT7 IFN rd3 (FERMBP-5604) can be obtained from a recombinant DNA vector, (10) ⁇ listed DNA,
  • a DNA comprising a nucleotide sequence that hybridizes with the DNA according to any one of (1)) to (16) and encodes a polypeptide having an interferon activity.
  • a polypeptide having an interferon activity which is hybridized with the DNA described in any one of (11) to (16) under the conditions of 6 XSSC and 55 ° C.
  • DNA comprising a nucleotide sequence
  • the vector is preferably
  • E. coli transformant E. c 0 1 i P T 7 IFN rd 2 (FE RM BP - 5 6 0 3) is held in, (1 9), wherein the recombinant DNA vector of all, (22) The recombinant DNA vector described in (19), which is retained in transformed E. colip T7 I FN rd 3 (FERMBP-56004).
  • the present invention
  • the cell is preferably
  • the present invention provides:
  • the host cell according to any one of (23) to (27) is cultured under conditions capable of producing a polypeptide having interferon activity, and then the culture is performed.
  • a method for producing a polypeptide having interferon activity comprising recovering a polypeptide having interferon activity from a substance.
  • the present invention also provides:
  • a pharmaceutical composition for preventing or treating autoimmunity comprising the polypeptide according to any one of (1) to (9) in a therapeutically effective view, and
  • the “h I FN r ⁇ i form” refers to the h I FN stimulated in the literature of Imagawa et al. [Imakawa, K., et al. (1994) J. Biol. Chem. 269. 10864].
  • Consists of an amino acid sequence that has been substituted, deleted or deleted and has interferon activity The degree of “addition, substitution, deletion, or deletion” in the definition above is not particularly limited, but administration of the IFN r mutant as a drug into human organisms is not particularly limited.
  • Ming's polypeptide should have at least the amino acid of sequence number S in column K and the Cys force of ⁇ £ 3 ⁇ 4S5?
  • An amino acid other than Cys which is an amino acid having the Arg force of amino acid ⁇ 86, which has been subjected to iS exchange with Cys, and preferably at least one amino acid in the S column of the sequence list.
  • Cys of 5-S5 is Leu
  • the amino acid number 86 is a polypeptide that has been replaced by an Arg force ⁇ ys, respectively. More preferably, at least the amino acid ⁇ of sequence No.
  • DN comprising the nucleotide sequence encoding the polypeptide of the present invention has an amino acid sequence known as hIFNr [Iraakawa, , Et al.
  • hIFN r sequence amino acid numbers 1 to 172 of SEQ ID NO: 8 in the sequence listing: hereinafter referred to as “hIFN r sequence”
  • nucleotides that encode the sequence at hIF such that one or more amino acid residues at two or more sites encode an added, replaced, deleted or inserted amino acid sequence.
  • hIFN r sequence amino acid numbers 1 to 172 of SEQ ID NO: 8 in the sequence listing: hereinafter referred to as “hIFN r sequence”
  • nucleotides that encode the sequence at hIF such that one or more amino acid residues at two or more sites encode an added, replaced, deleted or inserted amino acid sequence.
  • DNA encoding the hIFN r sequence can be obtained by cloning from a cDNA library made from a human tissue or cell line producing IFN according to methods described in the literature. [See Imakawa, K., et al. (1994) J. Biol. Chem. 269, 10864] .: Also, for example, the recombinant protein S is found in Escherichia coli. It is also possible to artificially prepare a DNA consisting of a nucleotide sequence encoding the hIFNrL sequence with a high codon.
  • PCR polymerase chain reaction
  • colip T 7 I FN rd 3 were released on July 23, 1996 (Heisei S (1996)) by FERMBP-5602, FERM BP-5603 and FERM BP- It is marked with 564: By separating the plasmid from these ffi strains, it is possible to prepare a suitable DNA of the present invention. You can also synthesize the DNA to encode
  • the sense and antisense subsequences are designed so that both ends overlap, and DNA polymerases such as PCR All the subsequences are linked to 0 [!
  • the sequence of eukaryotes may show many ⁇ (po lynio ⁇ hi sm) [for example, Ni shi, T., et al. (1985) J. Biochem.
  • h One or more amino acid residues are missing at one or more sites in the amino acid sequence of IFN r. Loss, iip, or even substituted polypeptides may exhibit interferon activity: these polypeptides may be substituted with the equivalents of hIFN r polypeptide in the present invention. Call.
  • a protein obtained by converting the nucleotide ⁇ sequence corresponding to the interleukin 2 (IL-2) gene cysteine into a nucleotide sequence corresponding to serine may retain IL-12 activity.
  • IL-2 interleukin 2
  • the codon for the desired amino acid is known per se and may be selected arbitrarily.For example, it can be determined according to a conventional method in consideration of the frequency of the codon usage ffl of the host to be used [Grantham, R., et al. (1981) Nucleic Acids Res. 9, r43-r74] Further, the codons in these nucleotide sequences may be partially modified according to the site-directed mutagenesis method described above.
  • Whether or not a certain DNA hybridizes with DNA having a nucleotide sequence represented by nucleotides 4 to 5 of nucleotides 1, 3, or 5 in the sequence listing can be determined as follows. .: First, the DNA of the test sample was subjected to agarose gel electrophoresis as necessary, and then blotted to the crotch of nitrocellulose or nylon. ⁇ ⁇ ⁇ ⁇ D.. D. D. D... D D D D D. D D D D D D D D D D D D D D D D D D D [Feinberg, AP, et al. (1983) Anal. Biochem., 132, 6-13], Nick translation method [Maniatis, T., et al.
  • SSC serumd-sodium citrate: “Sodium citrate mono-saline solution” contained in the hybridization solution; 155 contains 0.15 M sodium chloride and 15 mM sodium citrate Is preferably 4 to 8 XSSC, more preferably 6 XSSC.:, And the incubation degree is preferably 30 to 7 (TC, more preferably 60 nm).
  • Prokaryotic or eukaryotic ⁇ The main cells may be transformed.
  • i ⁇ human sequences related to suitable promoter and transformed into these vectors one, you to each host fine
  • large ij3 ⁇ 4L3 ⁇ 4 Escherichia coli
  • B. subtilis can be used as a host for nuclear cells that can express genes.
  • the ffi main cell can be transformed with a plasmid vector containing the sequence .
  • the vector should also have a single line that will allow the transformed cells to exhibit the 3 ⁇ 43 ⁇ 4 form selectivity. Desirable ..
  • E. coli K12 ⁇ is often used as the large intestine, and as a vector, the ability to insert pBR322 and pUC-based plasmids is generally fU.
  • the present invention is not limited thereto, and any of various known strains and vectors can be used.
  • promoters such as tributofan (trp) promoter, lactose (lac) promoter and tributofan are used.
  • trp tributofan
  • lactose lac
  • tributofan lactose
  • tc Latency Promos Examples include the popprotein (lpp) promoter, a lambda (s) PL promoter derived from nocteriophage, a polypeptide chain elongator Tu (tfB) promoter, and the like. Any promoter can be used to produce the polypeptide of the present invention.
  • Bacillus subtilis for example, 2007-25 ⁇ is preferable, and as a vector, pTUB22S [Ohmura, K., et al. (1984) J. Biochem. 95, 87- 93] is used, but is not limited to this .:
  • the B. subtilis ffl promoter the regulatory sequence of the "-amylase gene of B. subtilis is often used. — By linking the DNA sequence encoding the amylase signal peptide sequence, extracellular secretory expression is possible.
  • the expression vector has a pBR322 replication origin and is capable of autonomous propagation in Escherichia coli.
  • the vector containing the promoter and translational fj initiation signal can be used.
  • the expression vector can be obtained by the calcium chloride method [see Mandel,. And Higa, A. (1970) J. Mol. Biol. 53, 154], Hanahan's / j-method iHanahan. D. (1983) J. Mol. Biol. 166, 557-580] and pulsed air perforation [Neumann, E., et al. (1982) EMBO J.
  • ⁇ ⁇ ⁇ ⁇ cells are rare, and as insert cells, for example, C0S cells [see Gluzman, Y. (1981) Cell 23, 175-182] and Chinese cells A strain lacking dihydrophosphate reductase from hamster egg cell (CH0) [Urlaub, G. and Chasm, LA (1980) Pro Natl. Acad. Sci. USA 77-4216-4220 ! ': Cell, hamster BHK cells are often found but not included.
  • vertebrate cell expression vectors are those that are to be expressed; promoters located upstream of offspring, splice sites for RNA, and polyadenylation. A site having a site and a transcription termination sequence may be used, and this may be further provided with an origin of replication if necessary .
  • An example of the expression vector is an initial promoter of SV40. one coater to ⁇ to that p SV 2 dhf 1- [Subramani, S., et al. (1981) Mol. Cell. Biol. ⁇ , 854-864 Browse not 13 ⁇ 4 constant to force 5 capable example ⁇ and the like.
  • Yeasts are generally used as eukaryotic microorganisms.
  • yeasts of the genus Saccharomyces for example, Saccharomyces cerevisiae are preferable.
  • yeast examples include, for example, the promoter of the alcohol dehydrogenase gene [see Bennetzen, J. Shi and Hall, BD (1982) J. Biol. Chem. 257, 3018-3025. And a promoter of the acid phosphatase gene [see Miyanohai-a, A., et al. (1983) Proc. Natl. Acad. Sci. USA 80,] -5].
  • the expression vector has an SV40 replication origin and is capable of autonomous growth in COS cells.
  • transcriptional promoters, transcriptional signals and RNA splice sites can be used.
  • the vector can be obtained by the DEAE-dextran method.
  • the desired transformant obtained by the above-described method can be cultured according to a conventional method, and the culture of the present invention can be carried out inside or outside the cell by the culture.
  • the medium used for the culture in which the peptide is produced can be appropriately selected from various conventional ones depending on the host cells collected.
  • a tributon-yeast medium Pak Tribton 1. 6%, yeast extract 1.0 ", sodium chloride 0.5% ( ⁇ ⁇ 7.0)
  • peptone medium Difco
  • a medium such as RII-1640 medium or Dulbecco's modified Eagle medium (DMEM) to which serum components such as fetal bovine serum (FBS) are added as necessary.
  • E. coli strain E. c01ipT7I FN rdl E. coli transformed with a DNA encoding a suitable polypeptide of the present invention. . colip T 7 I FN: ⁇ d 2 and E. col ⁇ p T 7 I FN rd 3
  • polypeptide of the present & HJJ can be produced by the above method.
  • the polypeptide of the present invention which is intracellularly or extracellularly isolated, can be separated and purified by the known separation method described in [II], which takes advantage of its physical properties and chemical properties. Examples of such methods include, but are not limited to, preparative protein precipitation, ultrafiltration, molecular sieving chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography.
  • Takachi liquid chroma examples include various types of chromatography, such as chromatography (HPLC), dialysis, and combinations thereof.: If foreign genes are introduced into Escherichia coli or the like and expressed in large amounts, production: produced polypeptides ', May form water-insoluble aggregates called inclusion bodies.c. In such cases, denaturing the polypeptide by applying a powerful modifying agent, such as guanidine isothiocynate. Can solubilize the polypeptide, but the denatured polypeptide often has no activity because it does not form an original higher-order structure. Even in such cases, the polypeptide produced may be obtained as an active form by leaving it without forming the correct higher-order structure or by denaturing during production.
  • HPLC chromatography
  • polypeptide of the present invention obtained as described above has interferon activity. Can be determined by, for example, the method described below.
  • Antiviral activity [See Pestka, S. (1981) Methods in Enzymology vol. 78, 386! I]: A 96-well cell culture plate containing a strain of MDBK (for example, The American 'Type' Culture 'Collection (hereinafter referred to as “ATCC”) No. CCL22) was cultured in a medium containing the test sample, and vesicular stomatitis virus.
  • ATCC The American 'Type' Culture 'Collection
  • the polypeptides of the present invention are useful for viral infections, sickness, autoimmune diseases and In the treatment of various concomitant diseases, it can be administered by fif-ffl administration with ⁇ ⁇ ⁇ 1 ⁇ or other therapeutic agents.
  • Effective Consisting of the present invention and a polypeptide, the present composition can be administered in various forms, such as tablets, tablets, and tablets. 1 administration via granules, powders, syrups, or non-transfusions via injections, infusions, medicines, etc.! Administration: can be administered as an injection or infusion.
  • one dose is about 11 g to 11 O mg to 11: 1 for an adult, and these can be administered once or in several divided doses.
  • about 1 ng to 100 mg can be administered at one time by injection, intramuscular injection, or intravenous dragon.
  • the composition for preventing or treating an immune disease can be used in combination with other suitable agents for preventing or treating viral infections, femoral or autoimmune diseases.
  • the prophylactic or therapeutic agent for other viral infections, tumors or autoimmune diseases may be used as a component of the prophylactic or therapeutic ffi composition for viral infections, thighs or autoimmune diseases of the present invention.
  • the river can be if administered as a separate composition to the same patient,:.
  • A can promote the prevention or cure of autoimmune diseases by itself or the activity or efficacy of self-treatment.
  • the virus infection of the present invention, Preventive or therapeutic ffl compositions include '.; £ h IFN ⁇ mutant as a component.
  • general medical additives are selected: the purpose of the present invention is achieved without additives, but additives are generally added mainly for stabilization.
  • the pharmaceutical is selected from the proteins or saccharides described in the Pharmacopoeia that can be used as pharmaceuticals]. Particularly preferred is human ifti-albumi. , Gelatin, mannitol, sorbitol, lactose, etc.
  • LAPCR kit version 2 (Takara Shuzo Co., Ltd. tt) was used. As described in the following:
  • the complementary strands are annealed first, and then the remaining strands are annealed in PCR in which the 3′-terminal side has mutually opposite sequences.
  • a complementary strand is generated in the main strand by polymerase chain reaction, and as a result, two strands of DNA are obtained with the total length of the original nucleotides excluding the overlapped portion.
  • the product is subjected to polyacrylamide gel electrophoresis, the gel corresponding to the target strand is cut out, and the DNA fragments (A- (1 + 2)) and A- (3 + 4) ”,“ A— (5 + 6) ”,“ A—
  • the PCR was in each case 30 cycles at 9 S ° C, 1 minute at 54 ° C, and 7 2 X: per cycle. After 20 cycles under the conditions of 1 min: each of the 2 types in the liquid has a complementary sequence at the 3 ′ end, and was ligated with these complementary acs by PCR.
  • Each reaction product from which DNA is generated is subjected to polyacrylamide gel electrophoresis, bands corresponding to the H-like chain length are cut out, and the DNA fragments amplified by each PCR (each of “A (1 , ⁇ ! J, "A (5, S)" and IA (9-12) ”) were purified.
  • A— (1 to 4) and A— (5, S) are mixed to form a gun, and A—1Pr and A—8P1- are primers.
  • S. C for 1 minute, 54 ° C for 2 minutes, 72 ° C for 2 minutes; 20 cycles) to connect A— (1-4) and A— (5-8) DN A (“A— (1 to 8)”) was prepared, and further, A (1 to S) and A (9-12) were combined to form a type A 1 P PCR was performed using ⁇ ⁇ and A—12P ⁇ ⁇ as primers to obtain a DNA containing the nucleotide sequence shown in SEQ ID NO: 7 of sequence 3 ⁇ 4. A translational methionine codon is added to the 5 'end of the DNA encoding the amino acid sequence.
  • the pAR3040 vector [see Rosenberg, A. II .. et al. (1987) Gene 56,] 25-135] was used as type III, and the above S 1 Pr and S 2 PCR using P i- as primer (1 min at 9 S per cycle, 2 min at 54, 4 min at 72 "C;
  • the vector containing the T7 promoter present in the vector ('((Sa1I cleavage site to NdeI cleavage site i)) and hI obtained in 1) above . '1 8 nucleotides force end;' 5 of the FN gamma code sul DNA ⁇ binding the DNA: was ⁇ the (column table arrangement 2 9 hereinafter referred to as "T 7 promoter one ⁇ Yes fragment").
  • the DNA coding for hIFN r obtained in . ⁇ -1) is digested with the restriction elements NdeI and BamHI, and the digestion is performed so as to obtain 0.1 to 0.3 pmol.
  • Ethylenediaminetetraacetic acid (hereinafter referred to as “EDTAJ”) dissolved in 1 OmM Tris-HCl buffer (pH 8.0; hereinafter referred to as ⁇ buffer).
  • EDTAJ Ethylenediaminetetraacetic acid
  • ⁇ buffer 1 OmM Tris-HCl buffer
  • S1Pr and A—12P1- primers (1 minute at 98 ° C per cycle, 2 minutes at 54 ° C, 72 ° C).
  • a cloning kit pCR—Script SK (+) cloning kit: Stratagene 1 ⁇
  • the plasmid was introduced into the plasmid vector pCR—Script SK (+) by using the method described in the written instructions, and the hIFN obtained in 1) above was coded. That the nucleotide sequence of DNA Jidokishi method [Sanger, F. (1981) Science 214, 1205 Reference was confirmed by DNA sequencing by.:.
  • the ampicillin-resistant colonies that emerged were inoculated into the I 9 CA site after being isolated from the iji, and the absorbance of the solution at 600 ⁇ 11 (006 ( , 1, ,,, notedincreased from 0.-J to 0.S) After culturing with shaking at 24 to 6; adding L: ground and isopropyl-D-thiogalacto viranoside (final concentration of dumplings: 1 mM), and culturing with shaking at 37 for another 1 hour.
  • the composition of the medium used was as follows: I was sick:
  • the hIFN obtained in 4) above was applied to a DEAE-Sepharose column (High Trap 'Fast Flow, 5 nil; manufactured by Pharmacia) equilibrated with 0.02 mM Tris-hydrochloric acid squirting solution (pHS.0). r, Inject the charge, 0 force, etc. 0.5M NaCl It was released at a flow rate of 2 m 1 / min with a linear thorium concentration gradient K .: The fraction eluted with about 0.2 M sodium chloride was used as h I FN r ⁇ ⁇ .n.
  • the sequence K (fid sequence 3 ⁇ 4 sequence-number 30) was created .: This sequence is the sequence of the sequence ⁇ the amino acid sequence of S ⁇ ⁇ Therefore, the methine nin added at the ⁇ end for the first time is lost in any of the processes from 5 to i, and consequently the mature mature h I FN r It was determined that a recombinant having the same N-terminal amino acid sequence was found.
  • Example 1 Preparation and expression of DNA encoding mutant (hlFN rdl) Nucleotides described below:
  • the hIFNr expression vector DNA prepared in 1) of the above reference example was digested with restriction enzymes Kp ⁇ I and SacI, and after agarose gel electrophoresis, a DNA fragment corresponding to about 1 kbp was cut out.
  • PCR was carried out (per cycle) using A-1Pr and rd1-1Pr, and rd1_2P1- and A-12P1- as primers. (1 minute at 94 ° C, 1 minute at 37 ° C, 3 minutes at 72 ° C: 25 cycles), and recovered DNA fragments were recovered. The two obtained DNA fragments were mixed.
  • Plasmid 1 which allows a DNA containing a nucleotide sequence (sequence number 2 in the sequence listing) coding for N rd 1 (sequence No.
  • the h I FN ⁇ 3 ⁇ 4 expression vector DNA prepared in 1) of the above reference example was digested with restriction enzymes K pn I and S ac I, and after agarose gel, a DNA fragment corresponding to about 1 kbp was cut out. ...
  • r d2 1 1 P i- and rd 1 — 1 P l-, or: ⁇ d 1 — 2 P ⁇ ⁇ and A— 12 P ⁇ ⁇ PCR (1 minute at 9 S ° C for 1 cycle, 54. C for 2 minutes, 7: TC for 2 minutes; 20 cycles) was performed.
  • Escherichia coli E.c01ipT7IFNrd2 which had been transformed with pT7IFNrd2, was 99 6) Deposited on July 23, 1980 at Iwaki Technical Research Institute, Ibaraki ⁇ Tsukuba ⁇ 1 RM BP 5603 was attached.
  • DNA encoding IFN rdl produced in Example 1 was digested with the restriction enzymes Kp ⁇ I and Sac I, and after agarose gel electrophoresis, equivalent to about 1 kbp.
  • the DNA fragment to be excised is: ⁇ .
  • This DNA fragment is made into ⁇ -type, and A—1P1- and rd1-1—Pr, and rd3-1—P1- and A—12Pr
  • As a first step perform 20 cycles of PCR (1 minute at 9 S ° C, 2 minutes at 54 ° C, 2 minutes at 72 ° C), and collect the two amplified DNA fragments.
  • VSV vesicular Stomatitis virus
  • MDBK cells were suspended in Dulbecco's modified Eagle's medium (hereinafter referred to as “DMEM”) containing 10% ⁇ ⁇ 3 ⁇ 4 blood so that they became 3 ⁇ 10 ' ⁇ fine iJ Zm 1. Then, this cell suspension was dispensed in a quantity of 101 to each cell of a 96-well thin plate]]; ⁇ ffiffl plate, and the cells were removed at 37 ° C and 5% carbon dioxide f. After passing through the bottom of the gel, 100 ml of serial dilutions of the ⁇ obtained in Reference Example and Example 1-3 with DMEM were added, and the mixture was added at 37 ° C, 5% CO 2 gas.
  • DMEM Dulbecco's modified Eagle's medium
  • DMEM containing VSV with an infectious titer of 1.25 ⁇ 10 5 plaque forming unit (PFU) / m 1 was added in 501 wells, followed by addition of 37 The culture was allowed to stand for 20 hours at 5 ° C. and 5% carbon dioxide.
  • This 3 ⁇ 4 Ming Inta -. Fuweron gamma 3 ⁇ 4 ⁇ body is subjected to use if] ampoules of water or non-Minami solution or suspension 3 ⁇ 4 solution prepared by dissolving any other drugs ⁇ manner acceptable solution also Sterile powder preparations (preferably drying the interferon variant) are filled with ampoules and diluted with a pharmaceutically acceptable solution at the time of use.
  • the polypeptide of the present invention has high antiviral activity and low cytotoxicity, it can be used as an excellent prophylactic or therapeutic agent against viral infections, tumors or S autoimmune diseases.
  • Val Arg Leu Glu lie Met Arg Ser Phe Ser Ser Leu He Ser Leu Gin
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence species Si other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid Number of chains: single strand
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence family other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence species 3 ⁇ 4 ⁇ Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Type of row K other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Fragment type ⁇ terminal fragment
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • sequence is ⁇ !: Other nucleic acid (synthetic DNA)

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Abstract

Cette invention se rapporte à des variants d'interféron τ humain hautement actifs qui sont utiles comme agents de prévention ou comme remèdes contre les infections virales, les tumeurs ou les maladies auto-immunes. Plus particulièrement, ces variants sont des polypeptides se caractérisant en ce qu'ils contiennent la séquence d'acides aminés représentée par SEQ ID NO:8 dans la liste des séquences, mais comportant au moins une substitution d'Arg de l'acide aminé No. 86 par Cys et une autre substitution de Cys de l'acide aminé No. 85 par un acide aminé autre que Cys et ayant des activités d'interféron. L'administration de ces variants permet de prévenir ou de traiter les maladies mentionnées ci-dessus.
PCT/JP1997/002879 1996-08-21 1997-08-20 VARIANTS DE L'INTERFERON τ HUMAIN WO1998007863A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6833256B1 (en) 1999-06-22 2004-12-21 University Of Maryland Interferon tau mutants and methods for making them

Non-Patent Citations (4)

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US6833256B1 (en) 1999-06-22 2004-12-21 University Of Maryland Interferon tau mutants and methods for making them

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