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WO1984004745A1 - Nouveaux polypeptides et leur utilisation - Google Patents

Nouveaux polypeptides et leur utilisation Download PDF

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Publication number
WO1984004745A1
WO1984004745A1 PCT/JP1983/000174 JP8300174W WO8404745A1 WO 1984004745 A1 WO1984004745 A1 WO 1984004745A1 JP 8300174 W JP8300174 W JP 8300174W WO 8404745 A1 WO8404745 A1 WO 8404745A1
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WO
WIPO (PCT)
Prior art keywords
antibody
arg
hours
human
ser
Prior art date
Application number
PCT/JP1983/000174
Other languages
English (en)
Japanese (ja)
Inventor
Kyozo Tsukamoto
Yuzo Ichimori
Mitshuhiro Wakimasu
Original Assignee
Takeda Chemical Industries Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takeda Chemical Industries Ltd filed Critical Takeda Chemical Industries Ltd
Priority to PCT/JP1983/000174 priority Critical patent/WO1984004745A1/fr
Priority to US06/534,091 priority patent/US4681848A/en
Priority to EP83109402A priority patent/EP0103898B1/fr
Priority to PH29569A priority patent/PH22559A/en
Priority to AT83109402T priority patent/ATE19088T1/de
Priority to DK430583A priority patent/DK430583A/da
Priority to IE2215/83A priority patent/IE56003B1/en
Priority to AU19345/83A priority patent/AU564620B2/en
Priority to NZ205666A priority patent/NZ205666A/en
Priority to IL69790A priority patent/IL69790A/xx
Priority to CA000437187A priority patent/CA1216807A/fr
Priority to DE8383109402T priority patent/DE3362936D1/de
Priority to KR1019830004438A priority patent/KR910001726B1/ko
Publication of WO1984004745A1 publication Critical patent/WO1984004745A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6866—Interferon
    • 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]
    • C07K14/57—IFN-gamma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249—Interferons

Definitions

  • the present invention relates to novel polypeptides and uses thereof.
  • IFN human interferon
  • ⁇ r-type interferon
  • I-IFN immune interferon
  • IFN-r properties include different antigenicity from type a interferon (IFN-a) and type 3 interferon (IFN- / 9), and different types of inducers. It is also known that the stability of foams, acid, and heat is poor (The Interferon System, Sp !; Nanger, New York, 1979).
  • IFN is defined as having an anti-viral effect produced by living organisms, but it has also been demonstrated that it has many other biological activities i) (Blood, Vol. 55, p. 71, 1980; Ibid., Vol. 56, p. 875, 1980).
  • Methods for suppressing tumor growth include a method of directly suppressing the growth of tumor cells and a method of suppressing the tumor indirectly through the host's immune response.
  • a method of directly suppressing the growth of tumor cells for example,
  • NK Bacha killer cells
  • Activation of macrophages or activation of killer T cells.
  • IFN has been proved to have various immunopotentiating activities in addition to its direct action (Biochemica et al., Vol. 516, p.231, 1978).
  • FN- r has much higher antitumor activity at INVIT and antitumor activity at in vivo than IF-a and IF! T- / 3 Its importance has been strongly pointed out (Cellular Immunology, Vol. 49, p. 390, 1980).
  • IFN-r induced in the mouth is generally low or appropriate. ⁇ ⁇ ⁇ ⁇ Most IFN-r-producing cell lines are difficult to purify due to poor heat and acid stability. Mass production and purification of IFN-T lags far behind IFN-a and IFN-i9 I have.
  • the present inventors performed cloning of the IFN- ⁇ gene based on the mBNA of IFiT-, which was derived from human peripheral blood lymphocytes, and clarified its nucleotide sequence.
  • a mammal was immunized with the polypeptide or the protein complex obtained here, and the extracted ⁇ ⁇ cells were fused with allogeneic or heterologous lymphoid cells by cell fusion.] The hybridoma was cloned. .
  • the obtained hybridoma was inoculated into a mammal to produce a monoclonal antibody, which was collected to produce a monoclonal antibody against the above-mentioned polypeptide.
  • a method for purifying human IFN- r from crude human IFN-I content using the nocloclonal antibody obtained here a radioimmunoassay (RIA) method, and a radiobiology enzyme immunoassay (EIA) method
  • the present invention provides a novel polypeptide (I), a protein complex thereof, a novel cloned hybridoma, a novel monoclonal antibody, a method for producing the same, and a use of the monoclonal antibody.
  • Y is Arg Arg Ala Ser Gin with respect to the polypeptide (I), and!
  • X is preferably Lys Arg.
  • polypeptide (I) is not a specific number of amino acid residues.
  • the peptide can be produced by conventional means of peptide synthesis. Either the solid phase synthesis method or the liquid phase synthesis method may be used, but the liquid phase synthesis method is advantageous in many cases.
  • the method for synthesizing such a peptide is described, for example, in “The Peptides-, Vol. 6 6), Schroder and Lbke, Academic Press, New York, USA, or "Peptide Synthesis", Izumiya et al., Maruzen Co., Ltd. (t975), al. , Pp. 207-400 "Haruaki Yajima, Tokyo Kagaku Dojin Co., Ltd. ⁇ 977.
  • J Examples include azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCc method, and activity.
  • Esthetic method method using wood reagent E, carbodimidazo method, redox method, DC CZ active (eg, HOWS, HOBt, HOSu) method.
  • the peptide has a reactive carboxy group corresponding to one of two types of fragments that are bisected at an arbitrary position in the peptide bond, and a reactive amino group corresponding to the other fragment.
  • Examples of the protecting group for the amino group of the raw material include canolepobenzoxy, t-butynoleoxycanoleboninole, t-aminole; , 2 black
  • the protecting group for the carboxyl group include, for example, akiester (eg, meth, eth, propyl, buty, t-butyl ester group), pendi est group, pout benzene V est group, "0— Methoxy pent diester group, one mouth; 1 pentyl 1 / ester / 1 group, penzhydri ester group, force; bopenzoxy hydrazide group, 1-butoxy carbazyl hydrazide group, triti / hydrazide group Can be
  • Protecting groups for the arginine guadino tomb include, for example, nitro, tol, p-methoxybenzenesulfonium, kabobenzoki ⁇ , isoboni, and adaman.
  • Tiloxycarb; / t, 4-Methoxy 2,6-dimethylbenzenesulfonol group, pentamethynolensens / U-hole group and the like.
  • the guadino group of ⁇ may be protected in the form of an acid (eg, venense; Hongjun, tonosulfonic acid, hydrochloric acid, sulfuric acid, etc.) salt.
  • the hydroxyl group of threonine can be protected, for example, by esterification or esterification.
  • Suitable groups for this esterification include, for example, groups derived from carbonic acid such as acetyl group, lower alkano group, penzoi group, alkaro group, benzyloxycarbo-nore group, ethynoleoxycanorebo-group and the like.
  • groups suitable for 3- or etherification, such as benzyl include benzyl, tetrahydroxy-, and 7-butyl groups.
  • Methione may be protected in the form of a sulfoxide.
  • Examples of the activated carboxyl group in the raw material include, for example, the corresponding chrysanthemic anhydride, azide, and hot-pottenol (pentachlorophenol, p--trophenol) Nore, IT-Hydroxysuccinimide, N-Hydroxydzintriazole, N-zdoxydoxy 5 -Nonolevo ⁇ nene-2, 3-Zikaboxymidido, etc.) can give.
  • the peptide bond formation reaction may be carried out in the presence of a dehydrating agent (eg, a carbodiimide reagent such as dicyclohexylcarbodimid or cabodimidazo).
  • the present peptide condensation reaction can be performed in the presence of a solvent.
  • the solvent can be appropriately selected from those known to be usable in peptide coupling reactions.
  • the reaction temperature is appropriately selected from a range known to be applicable to the peptide bond formation reaction, and is usually about -4 Ot to about 60, preferably about
  • -20 is appropriately selected from the range of about 0.
  • Such conventional methods include, for example, reductive methods (eg, hydrogenation using a catalyst such as palladium black, reduction with metal sodium in a liquid ammonia), and acid lysis (eg, triflic acid, fluoride).
  • reductive methods eg, hydrogenation using a catalyst such as palladium black, reduction with metal sodium in a liquid ammonia
  • acid lysis eg, triflic acid, fluoride.
  • sulfur-containing compound such as hydrogen, methanesulfonic acid, or thioazole
  • the peptide of the present invention produced as described above can be collected from the mixture after completion of the reaction by ordinary means for separating peptides, extraction, distribution, column chromatography, and the like.
  • the carrier' and the mixing ratio of carrier and hapten depend on the carrier. If an antibody can be efficiently produced against the hapten immunized by priming, any hapten may be coupled at any ratio.
  • any hapten may be coupled at any ratio.
  • bovine serum albumin, bovine thyroglobine, etc. may be used in a weight ratio. In this case, a method is used in which a force is applied to the hub ten at a rate of 0.1 to 20, preferably 1 to 5.
  • mammals to be immunized include experimental animals such as sheep, goats, rabbits, mo / remots, rats, and mice.
  • a rat mouse is preferable.
  • immunizing a mouse it is possible to immunize a mouse subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, etc.
  • inject Prefer to inject.
  • the immunity interval, immunity amount, etc. are also highly variable, and various methods are possible.
  • immunization is performed 2 to 6 times at 2 week intervals, and 1 to 5 days after the final immunization, preferably 2 to 4 times. A method using cells after days is often used.
  • the amount of immunization should be the amount of peptide at a time, per mouse]? 0.1 g or more, preferably! It is preferable to use 0 g to 300 g. Also, before ⁇ the ⁇ , partial blood collection line, after confirming the increase of antibody titer in blood, Shi desirable to perform fusion experiments with ⁇ cells 3
  • the above-mentioned saturating fusion between the ferocytic cells and the lymphoid cells can be performed, for example, by excising the extracted mouse cells from hypoxanthine-guanine-phosphoribosyltransferase deficiency (HGBBT ”) or thymidine kinase-deficient (K-).
  • HGBBT hypoxanthine-guanine-phosphoribosyltransferase deficiency
  • K- thymidine kinase-deficient
  • a fusion agent such as lietin glyco-nore (PEG) is used.
  • PEG lietin glyco-nore
  • PMSO dimethisoxide
  • the polymerization degree of PEG is usually 100 to 6000, the time is 0.5 to 30 minutes, and the concentration is 10 ⁇ -80.
  • the cells can be fused at a high impeachment rate by treating them for 4 to 10 minutes at ⁇ 55 to 6.
  • the fused cells can be grown in a hypoxanthine-aminopterin-thymidine medium (HAT medium; Nayya, Vol. 256, (P. 495, p. 1975) can be used to selectively proliferate.
  • HAT medium hypoxanthine-aminopterin-thymidine medium
  • the culture supernatant of the cultured cells can be screened for the production of the desired antibody, but the antibody titer can be screened as follows. I can do it. That is, in this case, the presence or absence of antibody production against the immunized peptide can be first examined as a first step by a method such as the BIA method or the EIA method, but various modifications of these methods are also possible. It is possible. As an example of a preferable measurement method, one method using EIA will be described.
  • a carrier such as cellulos beads is coupled with, for example, a persimmon anti-Macsimiminoglobulin antibody according to a conventional method, and the measured culture supernatant or mouse serum is added thereto, and the mixture is incubated at a constant temperature for a certain period of time. Less than
  • reaction product is thoroughly washed, and an enzyme-labeled peptide (purified after force-purifying the enzyme and the peptide according to a conventional method) is added, and the reaction is carried out at a constant temperature for a certain period of time.
  • enzyme substrate is added, and the reaction is allowed to proceed at a constant temperature for a certain period of time. Then, the resulting color product can be measured by absorbance or fluorescence.
  • -Capsule is added to a carrier such as beads according to a conventional method, and the hybridoma supernatant or mouse blood to be measured is added thereto and reacted at a constant temperature for a certain period of time. After that, the reaction mixture is added to the reaction solution in a constant amount.
  • a recombinant containing IFK-r derived from human peripheral blood lymphocytes with lectin and phorbo este etc.
  • ⁇ - IFH-r which can be used as a solution, react at constant temperature for a certain period of time, and then determine the activity of IFr contained in the reaction. In this way, the limb yield of IF-r activity of the target antibody can be measured.
  • Hybridomas cloned as described above can be used, for example, in a greedy medium, such as BPMI-164, medium supplemented with 0.1 to 40% bovine serum for 0 days, preferably 3 to 40%. 5 3 times, the culture solution
  • a greedy medium such as BPMI-164, medium supplemented with 0.1 to 40% bovine serum for 0 days, preferably 3 to 40%. 5 3 times, the culture solution
  • the monoclonal antibody can be obtained from the above, but in addition to the above, by inoculating intraperitoneally into a suitable mammal such as a mouse, and culturing the cell, and collecting ascites, the cell culture can be performed much more. It is possible to efficiently obtain a large amount of crab high titer antibody.
  • a suitable mammal such as a mouse
  • Also connexion inoculated before Minera old I and the like preferably 5's 1 0 5 - 2 I 0 6 Invite the hive 9-doma into the SC cavity, etc., and collect ascites narrow 7 to 20 days later, preferably 10 to 4 days later.
  • Antibodies produced and accumulated in ascites fluid are collected, for example, by Bowan's fraction, DEAE-cellose column chromatography, etc.! ;), The antibody can easily be isolated as pure immunoglobulin.
  • the Noclo-na antibody of the present invention has the following properties.
  • This monoclonal antibody can be safely manufactured, used and stored O
  • the obtained monoclonal antibody can be obtained by utilizing the EIA method or the B.IA method], and the amount of FN in the living body or in a test tube can be increased.
  • the production of antibody chromosomes has made natural or genetic
  • the resulting IFN-r can be used to purify very efficiently.
  • E PN - for detection of r as an example, the measured I 5 1 7 * previously including materials or standard IF N-r, e.g., radioactive ® -?
  • the monochrome - By the de or enzyme etc. j labeled the monochrome -After reacting with antibody and then precipitating an immune complex with protein A, for example, and reacting for a certain period of time, measure the radioactivity or Xe activity of the immunoprecipitate to easily reduce the amount of DiFir-r. Can be measured.
  • the competition method using an antigen for example, the polypeptide (I) is radioactively or enzymatically labeled, and when a certain amount of the unlabeled monoclonal antibody of the present invention is reacted, the measurement is performed.
  • IFN-r co-existing material containing IFN-r was used to precipitate the immune complex.] Or by binding to an anti-mouse antibody! ?, IF5-r can be quantified.
  • purified I Fir- r can be used instead of polypeptide (I).
  • a fixing method for example, I i ⁇ ⁇ -r or polybutide (I) is added to a phosphoric acid solution containing 0.1 ⁇ weight and nou to 0.,
  • the purified antibody is coated on a suitable carrier such as activated agarose beads according to a conventional method, and then applied to a column.
  • a suitable carrier such as activated agarose beads according to a conventional method
  • Material containing crude I-r such as culture supernatant or disrupted cells is applied to the column, washed after adsorption, and then washed with a pick-up reagent such as ⁇ sc ir or IFN-r
  • a pick-up reagent such as ⁇ sc ir or IFN-r
  • the antibody column can be prepared, for example, by force-purifying the monoclonal antibody of the present invention purified from ascites or the like inoculated with the hybridoma with an appropriate carrier], and the following methods. .
  • Any carrier can be used, as long as IFN-r is specifically and efficiently attached after cou- pling, and then can be appropriately eluted.
  • Activated agarose beads such as Affige-10 are conveniently used in the manner described below.
  • the reaction between Affige-10 and the antibody is carried out in a buffer solution of 0.001-1 M, preferably 0.1 M, such as biforce-bonate. Reaction conditions are 0 ⁇ ⁇ 20,! Various values of ⁇ ⁇ are possible from 0 minutes to 24 hours, but preferably, the conditions of 4 * C, 4 hours, and pH 3 to 10 are used.
  • the amount ratio of Abuigel-10 and antibody to be mixed is such that the amount of antibody
  • Antibodies of up to 50 W are more likely to be repelled as much as possible. Therefore, any antibody within this range may be used, but an antibody of 30 or less is preferably used in consideration of the r ratio.
  • the antibody-carrier conjugate thus obtained is thoroughly washed with the buffer used for the reaction, and then left for a few days or at a final concentration of 0.05 M.
  • O PI Use a method such as adding Tanoamine / hydrochloric acid and reacting with a rock for 1 hour. D. After blocking the remaining unreacted active groups, fill them with an appropriate solution], use as antibody column it can.
  • a sample containing human immunogenic protein is dissolved in a buffer solution near neutral, for example, phosphate buffer solution, tris-hydrochloric acid buffer solution, and adsorbed to the antibody column. .
  • a buffer solution near neutral for example, phosphate buffer solution, tris-hydrochloric acid buffer solution
  • IFN-r is eluted.
  • the solution include a weakly acidic solution such as an acetic acid solution, a solution containing polyethylene glycol, a solution containing ⁇ butide, which is more easily D-bound to the antibody than the sample, a high-purity salt solution, and a combination thereof. It is preferably used to promote the degradation of human IFN-IFN]).
  • the column eluate is neutralized with a buffer according to a conventional method. If necessary, the above-described purification operation using the antibody force column can be performed again.
  • the human I-IFIT protein solution obtained here is subjected to dialysis, and if necessary] this can be made into a powder by freeze-drying].
  • human immune Intafe port down protein shows a 1 0 7 UZ more specific activity in WE scan activity measurement by cytopathic effect deter test vesicular stomatitis Huy scan (VSV) on human amnion-derived WISH cells .
  • VSV vesicular stomatitis Huy scan
  • the method of producing U ZW (cut ⁇ ) as the activity of IFN was performed as follows.
  • the international standard IFN- ⁇ and the crude IFN-r derived from leukocytes were determined using the VSV cytopathic effect inhibition test on FL cell line derived from human amniotic membrane, and the titers were compared.
  • leukocyte-derived crude IFN-r were determined using the VSV cytopathic effect inhibition test on FL cell line derived from human amniotic membrane, and the titers were compared.
  • the efficiency IFN-r was always arranged, and the results were calculated using the WISH-VSV system described above, and the titer was calculated from the ratio.
  • the D-purified human immune interface protein can be obtained by conventional methods.
  • the human I-IFN protein of the present invention has anti-inoles, anti-tumor, cell growth inhibitory and immunopotentiating effects.
  • the human I-IFN protein of the present invention can be mixed with sterilized water, human serum albumin (HSA), physiological saline or other known physiologically acceptable carriers, and can be administered parenterally or topically. it can.
  • HSA human serum albumin
  • a normal person can receive 100,000 to 100 million ut, preferably 50 to 60 million ut, by intravenous injection or intramuscular injection.
  • the preparation containing the human I-IFN protein of the present invention may contain other physiologically acceptable activities such as salts, diluents, adjuvants, other carriers, buffers, binders, surfactants, and preservatives. Components may also be included. Preparations for parenteral administration may be sterile aqueous solutions or suspension ampoules in physiologically acceptable solvents, or sterile powders (usually I-I), which can be used after dilution in physiologically acceptable diluents. It is provided as an amplifier (obtained by freeze-drying FN solution).
  • the above-mentioned preparation containing the human immune interferon protein contains 1 to 99 other active ingredients relative to the substance of the present invention, such as IFN- ⁇ or IFN- (9 or interleukin 2 or any other lymphokine). May be.
  • a P Adenosine triphosphate
  • AeOEt firewood acid
  • Fig. 1 shows the elution pattern of the monoclonal antibody of the present invention on the DEAE cellose column
  • Fig. 2 shows the elution pattern of the monoclonal antibody of the present invention. 0 indicating the result
  • FIG. 3 shows the results of the electrophoresis of the human immunointerferon-feron protein obtained by the purification method of the present invention shown after Example 13, and FIG. 4 shows the molecular weight variation similarly shown after Example i3.
  • Figure 5 shows the primary structure (Tong group arrangement) of pHIT 3709 plasmid obtained in Reference Example II (V8)
  • Figure 6 shows the reference structure 2)
  • Figure 7 shows the reference example 2 (iii) pHIT trp 1101
  • Fig. 8 shows the construction of Reference Example 2 (iV) (pEIT cell rp 2101), and Fig.
  • FIGS. 10 and 11 show (a) Escherichia coli extract containing or not containing IFN-r and (b) enzyme against the Monica-Nanole antibody according to Examples 20- (i) and 20- (ii), respectively. 1 shows the competition with the paired peptide of the present invention which was recognized. --Best mode for carrying out the invention
  • thin-layer chromatography was performed using the following developing solvents, using Mek's Sili-Ige Plate 60 F £ 54 or Funakoshi Pharmaceutical's Cell-Port-Splate, Abysse SF. Was.
  • the black was reduced as a catalyst in a stream of hydrogen.
  • the catalyst was removed by filtration and the solvent was distilled off.
  • Z-A 1 a-S ⁇ rG 1 ⁇ -0 ⁇ u 43 ⁇ 46 was dissolved in methanol 300 W and reduced with palladium black as a catalyst in a hydrogen stream.
  • the catalyst was removed by filtration, and the residue obtained by removing the solvent was prepared from Z-Arg (Pme) -OH-CHA 4.549.
  • Z-Gl OBu 7 13.0 was converted to MeOH 500: and catalytically reduced in a stream of hydrogen using black as a catalyst. The solvent was separated off, the aqueous solution was decompressed and concentrated and the residue was dissolved in DMF200W.
  • Z-Arg (me) -Gly-OB ⁇ 1Q.0 was catalytically reduced in MeOH 500 W, and then transferred to DMF300 ⁇ J. This with Z-: Plie-OH 4.72,
  • Boc-Me 7-11 0H.DCEA 3.0 Add the prepared Boc Me Shichiichi OH, H0 1.39 ⁇ , cool on ice, add Dcc 1.59, stir for 15 hours, discriminate 3 folded DCs, concentrate, and concentrate BicQH- AcOE dissolved seven, 1 056 and washed with click E phosphate solution, then dried and concentrated in Na 2 S0 4, E -.. Tenore was ⁇ as crystals by adding yield 6.2 ⁇ (9 3.1%). mp. 1 9 2— ⁇ 95 3 C,
  • Z-Ser-iTHlTH-Boc3 was transferred to DMF50O after the thigh was placed in MeOH300. To this, add Z-Arg (Pme) -OH, ⁇ 1.5 prepared from Z-Arg (Pme) -OE-CHA S.29], cool on ice,
  • Z-Ar g (Pme) -Ser- ⁇ ⁇ - ⁇ oc 3.9 was subjected to catalytic reduction with MeOH 300 0 and then transferred to DMI “50”.
  • Example ⁇ The combination of holibeptide obtained in ( ⁇ ) and Cycloglobulin (hereinafter G-) was performed according to the method of Goodfriend et al. (Science,
  • the mixture was freeze-dried to obtain 4.7 W of the protein complex.
  • the antigen for EIA was prepared according to the method of Kitagawa et al. (Janab Biochemistry, Vol. 79, p. 233, 1976).
  • Antibody injection in serum or hybridoma supernatants immunized with the protein complex obtained in Example 2 was detected by the EIA method (Immunobu-Macologie, Vol. 1, p. 3, 19). 1978) to Wachi, serum some Haiburido - the Ma supernatant buffer - a (20 m M Na 2 HP0 4, 10 OmM -2-JTaCl, 0.1 Na3 ⁇ 4, 1 mM MgCl 2 , pH 7.0), diluted with 100 / ⁇ ])), the e obtained in Example 3. The mixture was thoroughly mixed with the peptide derivative 100, and was subjected to anti-JS at 24 for 24 hours.
  • mice Each of 6 6-8 week-old BALBZC female mice was well mixed with Freund's complete adjuvant as a protein, and inoculated subcutaneously (primary immunization). Two weeks after the first immunization, the same amount of antigen was mixed well with Freund's complete adjuvant, and subcutaneously inoculated (secondary immunization). after 6 days of immunization rows Natsuta 3 tertiary immune, blood was partially collected from the mice, was boss measure the anti ⁇ in serum by EIA method real 3 ⁇ 4 example 4 described. Of these, r-2 mice with high antibody titers were inoculated intravenously with 120 g of antigen dissolved in 5 ⁇ of drinking water to achieve final immunization. anti- ⁇ value of each mouse are shown in Table 1 3
  • Serum dilution is 1 ZS 300
  • Immunization was performed by the method described in Example 5, and 3 days after the final immunization, the spleen was excised from the ⁇ 2 mouse, squeezed with a stainless steel mesh, filtered, and placed in an e-Gluz Minimum 'Etsencia Medium (MEM). The cells were suspended to obtain a cell suspension.
  • a cell used for cell fusion BAL BZC mouse myeloma-ma cell P3-63.A 8. ⁇ ( ⁇ 31) was used (current topix-in-micro / kusaiguchi-g-and-immunology).
  • spleen cells and 3: 1 each contain serum and are washed three times with MEM and mixed so that the ratio of the number of stored cells to the number of P 3 LM is 5: 1.
  • HAT hypoxanthine ⁇ X 1 0 one 4 Micromax, Aminoputeri down 4 X 1 Cf '7 ⁇ , thymidine ⁇ .6 X ⁇ 0 "5) ⁇
  • PBMI 1 6 40 - 1 0 FCS medium HA 0? Medium
  • Each of three hybridomas (a2-! 1, 62, 100) showing positive antibody activity was cloned by limiting dilution. That is, two hybridomas were suspended in HPMI1S40-20FCS so as to reach 2 cells / cell, and the suspension was dispensed in a 96-well microphone plate (manufactured by Nunc Corporation) at a ratio of 0.1 per 1 kg. At the time of segregation, B mouse thymocytes were added as puper cells to make up to 5 x 10 "cells. Thus, after about two weeks, cell proliferation was observed. The supernatant was probed and the presence or absence of the antibody was examined by the EA method described in Example 4. As a result, in r2-11, 8 clones out of 19 clones, r2- In 62, 3 out of 54 clones, and in r2-100, 5 out of 47 clones showed anti-honey injection (Table 2).
  • the binding ability of the monoclonal antibody to IFH-r was examined by the following method. That is, a culture of 2 to 3 clones of r2-ll, r2-62 and r2--00 cells, respectively, was added to a three-sense solution 300 bound with a rabbit egan anti-mouse IgG antibody. Add 300 pieces of Kiyoshi at room temperature
  • the reaction was performed for 18 to 20 hours. After the reaction, the cells were mixed well with physiological saline, and 550 cells of the IFN-a obtained in the following manner were added, and the mixture was reacted for 3 to 4 hours. After the reaction, the supernatant was collected, and the IFIT-r activity in the supernatant was measured by the cell transfusion effect (c) reading method using a microplate. (Applied Microbiology, Vol. 16, pp. 1706, 1968). That is, 50 ⁇ m was put into all the 96-well microplates (manufactured by Nunc Corporation), and IFFT sample was added to the first layer at 5 ⁇ m, followed by serial 2-fold dilution.
  • the FN-r sample used was a human peripheral lymphocyte, which had a naphthalin A 40 / ul and a 12-0-tetradecanoinyl phorbo-1-13 acetate 1 2 hours after stimulation with 5 ng,
  • the culture medium contained human cultures (acid, unstable in ⁇ ⁇ processing) and 4400 cuts. -If an antibody capable of binding to ⁇ -r is present in the culture supernatant of the cloned cells, IFIT-r activity added later binds to the antibody on cellose. In addition, the activity in the supernatant is reduced:> Yugo shows relatively strong IN- ⁇ binding ability in the supernatant of r2-111 clone, and the added IP IT -r (550
  • BAL BZc mice that have been administered intraperitoneally with 1 x 10 ° clone cells in advance of mineral oil intraperitoneally, producing r 2-1 1.
  • Antibodies capable of binding to IF ir -r By inoculating intraperitoneally I made it.
  • the black - anti ⁇ in the culture supernatant of the emission cells are recognized to ⁇ 0 4-fold dilution, about 1 0 0 0 times antibody activity by to ascites rises.
  • OMPI The IgG subclass to which the ⁇ 2- ⁇ -nocchi-null antibody belongs was 10-fold diluted the fraction 17 purified by the method of Example 10 to obtain goat anti-mouse IgG ⁇ , G2a, G2b, Precipitation reaction in agar with G3 antibody (Miles) (Octa-orchid-method: immunologica method., ⁇ ⁇ ? R Ge fusion fusion technique, black black, oxford,
  • Supernatant 60 obtained in Reference Example 4 contains 1 mM EDTA and 0.15 M NaCl
  • Example 13 The protein obtained in Example 13 was treated with 2-butane ethanol.
  • the protein was identified as a single band when subjected to SDS-boriatari / u amide (17.5 *) electrophoresis (15 mV, 6 hours) and stained with Coomassie blue (FIG. 3). From the relationship between the migration distance of the molecular weight marker and the migration distance of the protein, which were simultaneously electrophoresed, the molecular weight of the protein was estimated to be 17,000 soil 1,000 (Fig. 4). On the other hand, another band was detected at a molecular weight of 33,000 ⁇ 2,000 for the protein that was not treated with 2-meadow ethanol. Since this value corresponds to about twice the molecular weight of IFN-r of 17,000 ⁇ 1,000, this band is considered to be derived from the IFN-r dimer.
  • Example 13 Transfer the protein obtained in Example 13 to a glass hydrolysis test tube], add 200 volumes (V / W) of constant boiling constant hydrochloric acid containing 4 thioglycolic acid, and seal the tube under reduced pressure. After that, the mixture was hydrolyzed for 24, 48, and 72 hours with 110 watts. After hydrolysis, open the tube, remove the salt under reduced pressure, dissolve the residue in 0.02 N acid, and use Hitachi 8335 high-speed amino acid analyzer to perform the amino acid analysis. Carried out.
  • Cystine and cystine were subjected to the formic acid oxidation of the above protein according to the method of Hirs [Methods EnzymoL 11, 197 (1967)], and then hydrolyzed for 24 hours in the same manner as described above to analyze amino acids. By all means]? Quantified as Sistine II. Amino acid analysis values were determined by averaging the values obtained after 24, 48 and 72 hours of hydrolysis. On the other hand, the values of serine, threon, tilidine and tributane were determined by extrapolating the hydrolysis time to 0 hours. Table 6 shows the results.
  • Example 5 The protein obtained in Example 5 was used according to the method of Hirs [Methods EnzymoL 11,
  • Example 6 Three days after the final immunization described in Example I-4, spleen was excised from the mouse, and cell fusion was performed and HAT selective culture was performed by the method described in Example 6. As a result, in March 43, hybridoma was found to be buried, and the presence or absence of antibodies was searched for by the EIA method described in Jeongseon-jo 4. The result was 2 ⁇ (r3-11, r3—19). In addition, strong antibody activity was observed.
  • the binding ability of the monoclonal antibody to IFH-r was examined by the method described in Example 8. However, the 15 ⁇ ⁇ -r sambu used in this example was obtained by combining human IFN-transducer with brassmid and working on E. coli.
  • ascites can be created by the method of *! ).
  • the ascites obtained was purified by the method described in Example I, Example 0, and the obtained subclass of Monoclonal-anti-Fyle was defeated. .
  • the results showed that a significant band was observed between goat anti-mouse Ig (Jl antibody) and that of both 7-3- -1.1 and r3-19-1.20 noclonal antibodies.
  • this 100 was dispensed to each tube of a 96-well microphone plate, and reacted at 4 for 24 hours. After the reaction, dissolve 100 ⁇ l / volume of phosphate buffer containing 2 cow beabumin in 100 / ⁇ each to block the excess binding site of the gel, process at 4 C for 24 hours, and use it for ELISA.
  • a mixture of the recombinant I PK— r prepared at 2 ⁇ 10 4 and 8 ⁇ 10 4 / W) was allowed to react at 37 ° C. for 3 hours, and reacted at room temperature for 3 hours.
  • the well was washed well with phosphate buffer, 100 g of a goat anti-mouse igG antibody labeled with horseradish oxidase was added, and the mixture was reacted at room temperature for 3 hours.
  • Example 20-1 Although the principle is the same as that of Example 20-1), a modified method under JK was carried out as a method for automatically and quickly measuring a large number of sambs using a multi-scan.
  • test sample or standard IFH- ⁇ is adjusted to an appropriate concentration with buffered narrow A (0.02 M phosphoric acid buffer at pH 7 containing 0.1 M HaCl, 1 mM MgCl 2 , 0.1 Si BSA, 0.1 Na). Dilute and inject 75 into each hole of the plate prepared in (2). Next, 5 ⁇ of the enzyme sample of Example 3 diluted 30 times with buffer A is injected into each well of the plate. The reaction was carried out for 3 hours with the addition of a bulk. The reaction was continued at room temperature for 3 hours. After removing the reaction ⁇ , the plate was washed four times with 0.01 M phosphate buffer ⁇ (pH 7.4) 75.
  • buffered narrow A 0.02 M phosphoric acid buffer at pH 7 containing 0.1 M HaCl, 1 mM MgCl 2 , 0.1 Si BSA, 0.1 Na.
  • each well of the plate was prepared with buffer ⁇ A in each of the wells of 1.5 4 trofu-1- ⁇ -1D-galactobino s / do (Wako fine, special grade)
  • Fig. 11 shows the standard curve of iFir-r.
  • Table 10 includes i FH-r
  • TSA 12-0-tetradecanoylhobo 13-acetate
  • concanavalin A 40 ⁇ gZ *
  • the cells were cultured in tfBPMl-l640 medium (including fetal bovine serum of ⁇ 0) for 3 hours to induce. 24 hours later, this induced one! 0 human lymphocytes were converted to a solution of guanidine (5 II guanidine, 5 liters of ethanol), 50 m ⁇ .
  • the BU precipitate was dissolved in a 0.25 K-lauroyza / u-sodium sodium cosinate solution and precipitated with ethanol to obtain 8.3 W of BIfA. this! ?
  • the NA was adsorbed on an oligo (dT) Senorelose column with a high salt solution [0.5 K NaC, 1 OmM Tris ⁇ EC £ pH 7.6, ImM EDTA, 0.3 SDS].
  • a solution of mRNA containing poly (A) in a low salt solution (1 OmM Tr-HCl-pH 7.
  • This mENA is further precipitated with ethanol, dissolved in a solution of ⁇ 2 ′′ (1 ⁇ Kris-HC1 pH 7. ⁇ , 2 mMED, 3% SDS), and treated with 65 for 2 minutes to obtain 1 Q to 35 % sucrose density gradient centrifugation (at 2 0 using a rotor of Beckman ST727, 25000rpm-C2 hour centrifugation) by the the child]? to obtain a fractionated by 2 2 fractions.
  • a reaction solution of 10 O 5 g of mfNA, 50 ori '(dT), 100 units of reverse transcriptase, 1 dATP, 1 dCTP, dGTP and iTTP, 8 mM MgCl 2l 50 mM PC 1, 1 Q mM dithionite, 50 mM Tris-HC 1 pH 8.3) 42 * C Incubate for 1 hour, then extract with phenol Then, the cells were treated with 0.1 N NaOH at 70'C for 20 minutes to decompose and remove RNA.
  • duplex 3 nuclease S 1 5 0 ⁇ a reaction solution NA (duplex]) NA 0.1 M Kokusan sodium ⁇ 4.5, 0.25 M NaCl, 1.521 ZnS0 4, 60 Interview - Tsu City of SI Nuclease) at room temperature for 30 minutes, deproteinize with phenol, precipitate DNA with ethanol, and add terminator transferase to 5 Q fig reaction mixture (double-stranded DMA, 0.14 M codylic acid).
  • 0.3MTris (base) pS 7.6, 2 Jichiosureito 'ImM C 0 C1 2, 0.15 mM dCTP, 3 0 Interview - Tsu Tota!
  • Bovine serum albumin 20 units of Pstl] was used for 3 hours at 37 t to excise one Ps11 recognition site in PBR322 DNA, deproteinized with phenol, and then terminated with terminal transferase. 5 0 / ⁇ ⁇ of reaction (:. DNA 1 0 a, 0.14M Kakoji acid strength Li, 0.3 M Tris (base) pH 7.6, 2 mM Jichiosurei toe, 1 mM C 0 C1 2 0.15mM dGTP, 3 0 Interview -Butardina transferase) for 3 minutes at 37 t! To extend about 8 deoxyguan chains at both ends of 3T of the above plasmid pBR322 DNA.
  • ptrp701 was partially digested with Clal to eliminate one of the two restriction enzyme Clal cleavage sites present in ptrp701 to obtain a fragment in which only one of the two Clal cleavage sites was cleaved. .
  • the resulting portion was filled in with DN JL volimase I page fragment and then religated with T4DNA ligase to obtain ptrp771 (FIG. 6).
  • pHITtrp2101 was constructed.
  • ptrp601 was treated with restriction enzymes Clal and Hpall to obtain a Clal-Hpa! I fragment 0.333 Kb containing a trp promoter. This fragment was cut with Clal and treated with ethanol phosphatase.
  • Trp promoter and combined using T 4 DNA ligase to obtain a pHITtr P 2101 entered the two series 1 ((2l ⁇ ).
  • Escherichia coli 294 was transformed with this plasmid ⁇ ⁇ trp2101 according to the method of Cohen et al.
  • the cells were cultured in a Meyer at 37 t, and when the growth reached KU220, 3 ⁇ -indolino-inorea-clinoleic acid (IAA) was added to 30 S 1 / ⁇ , and the cells were further cultured for 4 hours.
  • IAA 3 ⁇ -indolino-inorea-clinoleic acid
  • the culture solution 1.2 obtained in Reference Example 3 was centrifuged to collect bacterial cells, and the cells were suspended in 0.05 Tris-HC1 pH 7.6 containing 60i of 10% sucrose.
  • Add 2.4 « leave at 0 for 1 hour, treat with 3 7 for 5 minutes, and further apply ARTEK (USA) ultrasonic It was processed at 0'C for 30 seconds with the Xiao Xian.
  • the lysate was centrifuged at 105,000x for 1 hour to collect the supernatant 66.
  • the novel polypeptide of the present invention can be used to produce a monoclonal antibody that binds to a human interferon.
  • the monoclonal antibody is useful for detection and purification of human ⁇ -type interferon.

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Abstract

Nouveaux polypeptides représentés par la formule suivante:$(3,)$(où X représente une liaison ou un reste de peptide ou d'acide aminé comprenant de 1 à 16 acides aminés de la chaîne$(3,)$comptés depuis le côté de la terminaison C, et Y représente un reste de peptide ou d'acide aminé comprenant de 1 à 5 acides aminés de la chaîne$(3,)$comptés du côté de la terminaison N) et leur conjugués protéiques, les hybridomes et les anticorps monoclonaux induits à partir de ces polypeptides ou de ces conjugués protéiques, et procédé de détection et de purification de l'interféron gamma humain les utilisant.
PCT/JP1983/000174 1982-09-22 1983-05-31 Nouveaux polypeptides et leur utilisation WO1984004745A1 (fr)

Priority Applications (13)

Application Number Priority Date Filing Date Title
PCT/JP1983/000174 WO1984004745A1 (fr) 1983-05-31 1983-05-31 Nouveaux polypeptides et leur utilisation
US06/534,091 US4681848A (en) 1982-09-22 1983-09-20 Novel peptide and use thereof
EP83109402A EP0103898B1 (fr) 1982-09-22 1983-09-21 Peptide et son utilisation
PH29569A PH22559A (en) 1982-09-22 1983-09-21 Monoclonal antibody and method for purifying human gamma interferon
AT83109402T ATE19088T1 (de) 1982-09-22 1983-09-21 Peptid und dessen verwendung.
DK430583A DK430583A (da) 1982-09-22 1983-09-21 Polypeptider samt deres fremstilling og anvendelse
IE2215/83A IE56003B1 (en) 1982-09-22 1983-09-21 Novel peptide and use thereof
AU19345/83A AU564620B2 (en) 1982-09-22 1983-09-21 Peptides and protein-peptide complexes
NZ205666A NZ205666A (en) 1982-09-22 1983-09-21 Peptide derived from c-terminal region of human immune interferon,and conjugate with carrier protein;hybridomas and production of monoclonal antibodies against human immune interferon;purification or detection of human immune interferon
IL69790A IL69790A (en) 1982-09-22 1983-09-21 Peptides consisting of a sequence of human ifn-ypsilon and use thereof
CA000437187A CA1216807A (fr) 1982-09-22 1983-09-21 Peptide, et son emploi
DE8383109402T DE3362936D1 (en) 1982-09-22 1983-09-21 Novel peptide and use thereof
KR1019830004438A KR910001726B1 (ko) 1982-09-22 1983-09-22 펩티드의 제조방법

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7201897B2 (en) 1996-05-31 2007-04-10 Hoffmann-La Roche Inc. Interferon conjugates

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Miklos Bodanszky et al: "Peptide Sythesis (Second Edition)", (1976) John Wiley & Sons (New York) *
Nature, Vol. 295, "February 11, 1982" Go (11.02.82) p. 503-508 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7201897B2 (en) 1996-05-31 2007-04-10 Hoffmann-La Roche Inc. Interferon conjugates

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