EP0300007A1 - Teilkationisierung von eiweissenthaltenden antigenen und deren verwendung zur immunisierung und desensibilisierung - Google Patents

Teilkationisierung von eiweissenthaltenden antigenen und deren verwendung zur immunisierung und desensibilisierung

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Publication number
EP0300007A1
EP0300007A1 EP88901145A EP88901145A EP0300007A1 EP 0300007 A1 EP0300007 A1 EP 0300007A1 EP 88901145 A EP88901145 A EP 88901145A EP 88901145 A EP88901145 A EP 88901145A EP 0300007 A1 EP0300007 A1 EP 0300007A1
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EP
European Patent Office
Prior art keywords
protein
cationized
containing substance
substance
antigen
Prior art date
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Withdrawn
Application number
EP88901145A
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English (en)
French (fr)
Inventor
Jacob Gabriel Michael
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Individual
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Individual
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]

Definitions

  • This invention relates to a method of preparing partially cationized protein-containing substance, su as an antiqen, having increased antigenicity, to t product of this method, and to a method of enhanci immune response to a native antigen in a mammal.
  • the basis of immunization is the exposure of th organism to be immunized to dead or weakened infectiou agents (viruses, bacteria, toxins, etc.) or extract thereof which contain a foreign, generally macromolecula substance which is capable of evoking an immune response
  • infectiou agents viruses, bacteria, toxins, etc.
  • allerqic reactions can be lessened b desensitization, wherein such a substance is used t suppress the normal allerqic response caused by foreig substances, referred to as allergens.
  • allergens Most antigens and allergens are either wholly o partially composed of orotein.
  • antiqens The action of antiqens is thouqht to be dependent i part on the antioen's affinity for certain binding site on cells of the livinq tissue affected by the antigen
  • tissue may be blood, internal organs, skin, eyes etc.
  • Interaction of the antigen to the binding sit stimulates the production of antibodies which in tur defend the organism against infectious agents containin the antigen.
  • antigens are foreign substances, they ca have adverse side affects on the organism sought to b immunized. This is most important in the treatment o humans and livestock. It is therefore desirable to b able to achieve an effective immunogenic response b utilizinq lower levels of the antigen. If antigenicit can be increased, it necessarily follows that a smalle dosage can be administered to achieve a given level o immunization.
  • An adjuvant is a substance which augments the immunogeni response by aiding the antigen's interaction with th living tissue.
  • adjuvants include alum salt for human use.
  • a problem which militates against the us of adjuvant ' s in _in_ vivo immunization is the toxicity an the side effects these compounds induce. Because o this, adjuvants are generally disfavored for use i humans or in any other organism where toxicity and/o side effects are a concern. The elimination of the nee for adjuvant cooperation is therefore desirable.
  • suc substances lack the threshold amount of bindin capability or strength to cause such a response. I would be advantageous to be able to convert suc substances from a non-antigenic form to an antigeni form. This would allow vaccines to be produced whic could elicit an immunogenic response to a substance wher this was not previously possible.
  • Immunization methods are normally carried out b subcutaneous or intramuscular injection of the vaccine
  • Oral intake of an antigen is not feasible in mos instances because it often causes suppression of rathe than increase in the immune response.
  • Exceptions to thi general rule include live attenuated bacterial and vira vaccines.
  • Oral vaccines offer advantages such as lowe expense and ease in administration and packaging Therefore, it would be better to be able to administe vaccines in oral form without loss in immunogenic effect.
  • protein-containing substance includes all proteins, as well as substances whos molecular makeup is in some part proteinacious, such a lipoproteins and proteosaccharides. These may b substances which do or do not have antigenic propertie in their native forms. More specific examples of sai substances include, without limitation, bovine seru albumin (BSA) , hen egg albumin (OVA) , bovin gammaglobulin (BGG), ferritin, bacterial endotoxin, vira proteins, diptheria toxin and tetanus toxoid, and kille microorganisms, including bacteria and viruses an protein-containinq products thereof.
  • BSA bovine seru albumin
  • OVA hen egg albumin
  • BGG bovin gammaglobulin
  • ferritin ferritin
  • bacterial endotoxin bacterial endotoxin
  • vira proteins diptheria toxin and tetanus toxoid
  • cationization means the conversio or substitution of functional groups on the protei portion of the protein-containing substance whereby th substance is rendered relatively more cationic.
  • Suc functional groups are normally anionic within physiologic pH range and are converted to or substitute for a cationic or nonionic moiety.
  • An example of such cationization is the reaction whereby anionic side chai carboxyl groups are substituted with polycationi aminoethylamide groups.
  • bovine serum albumin may be expresse as either “nBSA” as the native form, or "cBSA” as th cationized form.
  • the present invention provides a method of preparin an antigenic protein-containing substance which comprise reacting (1) a protein-containing substance, and (2) reagent capable of cationizing the protein-containin substance; and halting the -reaction between the protein containing substance and the reagent after a tim sufficient to result in partial cationization of th protein-containing substance.
  • a partially cationized antigenic protein-containin substance having increased antigenicity as compared t the same native protein-containing substance, th partially cationized substance having an isoelectri point ranging from about 6.5 to about 9.5, determined b isoelectric focusing as hereinafter described.
  • the invention also provides a method of immunizing mammal by administering to' the mammal an effective amoun of a partially cationized antigenic protein-containin substance having an isoelectric point in the range o about 6.5 to about 9.5 as determined by isoelectri focusing. Administration can be effected orally o parenterally, with or without an adjuvant.
  • a preferred method of partial cationization involve use of a reagent comprising at least one carbodiimide an at least one amine. Control of the degree of cationiza tion is effected by varying the pH, time parameters an concentration of- the reactants. The reaction may b halted or quenched readily at a desired degree of partia cationization, in a manner known in the art.
  • BSA reaction with a carbodiimide and amine mixture may be quenched after substitution of about 20 new amino groups in place of anionic carboxyl groups.
  • a fully cationized BSA would contain about 80 new amino groups. In general, an addition or substitution of fro about 20% to about 60% of the theoretical maximu possible number of amino groups will result in a isoelectric point ranging between about 6.5 and 9.5 a determined by isoelectric focusing.
  • the protein-containing substance can b cationized by several methods known in the art (15, 9) .
  • the preferred method is the reaction of ethylenediamine l-ethyl-3-( 3-dimethyl amino propyl)-carbodiimide (EDC) with the PCS.
  • EDC ethylenediamine l-ethyl-3-( 3-dimethyl amino propyl)-carbodiimide
  • This particular reaction involves th activation of the carboxyl groups of a protein wit carbodiimide and the subseguent reaction of the activate carboxyl with a nucleophile of the general type +R- H2 t obtain the primary amine type derivatives.
  • Considerabl versatility can be achieved since both the chemica nature of the modification, i.e. introduction of primary secondary or tertiary amine groups and the degree o modification of the protein carboxyls can be varied b proper choice of reagents, reaction time and pH of th coupling reaction.
  • the cationization methods suitable for use in th present invention are those which effect cationizatio under mild reaction conditions.
  • mil reaction conditions are those , under which the molecula character (e.g. 1°, 2° and 3° structure) of the subjec PCS is not substantially altered so as to adversel affect its antigenic pH immunogenic character.
  • Thes conditions can be most generally described as unde relatively low temperature and low ionic strength as wel as neither very acidic (e.g. below pH 4) nor very basi (e.g. above pH 8) nor presenting a very oxidative o reductive environment.
  • the pH of the EDC reaction is generally maintaine in the range of from about 4.5 to about 6.8. More rapi substitution of carboxyl groups in the EDC reactio occurs at the lower pH levels within this general range For example, BSA cationized at pH 6.0 for 30 minute migrated during electrophoresis the same distance as BS cationized at pH 4.75 for 15 minutes (i.e., 0.7cm) .
  • Reaction time is determined by the concentration o the reactants and by the degree of cationization desired
  • the preferred reaction time is in the range of from abou 5 minutes to no more than two hours.
  • the reaction is maintained within the general rang of from about 4° C to about 37° C and is 'generall maintained at about 25° C.
  • Each known method of cationization may be halted o quenched according to several methods known in the art
  • the EDC reaction is guenched with a buffer, preferably a acetate buffer, which halts the reaction. Concentratio of the acetate buffer is about 4M.
  • the protein-containing substance be cationized to an extent whereby it exhibits increased antigenic character such as an increas immunogenic character or an increased allergic respon suppressing character.
  • increased antigenic character such as an increas immunogenic character or an increased allergic respon suppressing character.
  • PCS types whi are modified to such an extent so as to increase the antigenic character will generally have from about 20% about 60% of the maximum possible number of those anion groups amenable to modification under "mild" conditio while that PCS is in its native state, so modified.
  • modified is intended to mean any type chemical modification which causes cationization of t PCS.
  • PCS types modified according the inventive method fall into the above-describ isoelectric point range, it is within the skill in t art to determine and adjust the degree of parti cationization which increases the antigenic character f all PCS types including those PCS types, in native cationized form, whose isoelectric points may fa outside this range.
  • Example 1 exhibit the inventive meth as practiced on several antigens. Variations of t parameters and methodology for optimization of the meth for any specific PCS is within the known art. Example 1
  • Bovine Serum albumin five tim crystallized, was cationized according to the gener procedure described by Border (9).
  • nBSA Five grams of nBSA was dissolved in distilled wat to a volume of 25 ml and admixed with a solution of 67 ethylene diamine in 500 ml distilled water. The pH this solution was adjusted to about 4.75 with 6N HCl. this was added 1.8 grams of 1-ethyl -3-(3 dimethyl ami propyl )-carbodi imide .
  • reaction was permitted to react for varyi periods of time with constant stirring, while t temperature was maintained at about 25° C and the pH w held constant. After quenching with 4M_ acetate buff mixture, the reaction was subjected to multiple dialys treatments against distilled water and lyophilized. was then passed through a column of Sephadex G-25 a lyophilized again before use.
  • Example 1 The method of Example 1 with the exception that t pH of the reaction was adjusted to about 6.0 with 6N H and the reaction was permitted to react for about 1 hour.
  • Example 4 The method of Example 1 with the exception that th PCS may be. native bacterial endotoxin instead of nBSA.
  • Example 4 The method of Example 1 with the exception that th PCS may be. native bacterial endotoxin instead of nBSA.
  • Example 1 The method of Example 1 with the exception that th PCS used was native tetanus toxoid instead of nBS (purchased from Lederle Laboratories, Pearl River, NY).
  • Example 2 The method of Example 2 with the exception that th PCS used was native hen egg albumin (OVA) instead o nBSA.
  • OVA native hen egg albumin
  • Example 1 The method of Example 1 with the exception that th PCS used was ferritin (purchased from Sigma Chemical Co. St. Louis, MO), samples of which were cationized for minutes, 15 minutes and 30 minutes.
  • ferritin purchased from Sigma Chemical Co. St. Louis, MO
  • Example 1 The method of Example 1 with the exception that th PCS used was heat killed E.coli bacteria.
  • Example 1 The method of Example 1 with the exception that th PCS used was bovine gamma globulin (BGG), purchased fro Sigma Chemical Co., St. Louis, MO.
  • BGG bovine gamma globulin
  • FITC is used in 0.5 mg concentration per mq protein. FITC is added to BSA solution and the pH adjusted to about 8.4 with borate buffer. conjugation is allowed to proceed for about 1 hour w continuous mechanical stirring at slow speed.
  • the suspension is dialyzed in a cold room w frequent changes of saline adjusted to a pH of about with borate buffer.
  • the dialysis reguires several d and is complete when the dialyzate is virtually free yellow-green color under ultraviolet light. conjugate is clarified by centrifugation.
  • hapte -carrier conjugations may require m complex chemical reactions to achieve covalent -bondi between the reacting molecules.
  • T products are readily soluble in water and show a sing band on gel electrophoresis in the presence of sodi dodecyl sulfate, with or without a reducing agent such mercaptoethanol .
  • the band migrates more rapidly towa the cathode than the native BSA monomer.
  • Passage throu Sephadex G 200 shows a single peak which appears slight before the nBSA peak.
  • Electrophoresis on agarose gel shows th the two hour reaction time produces a molecule whi appears to have attained a maximum positive charge in t case of cBSA since preparations which are permitted react for a longer period of time do not exhib increased migration toward the cathode.
  • the products were determined to have an isoelectri point greater than 6.5 and up to about 9.5. Th isoelectric point is used as one of the measures t determine the degree to which a given PCS has bee cationized.
  • the number of amino groups substituted in a protei molecule by cationization can be determined by means of ninhydrin reagent. Ninhydrin reagent, purchased fro Sigma Chemical Co. , was used to prepare a curve wit increasing concentrations of glycine, which served as a standard.
  • An experimental procedure with BSA was as follows :
  • degree to which a protein-containing substance has b cationized is useful in expressing degree to which a protein-containing substance has b cationized.
  • the degree of cationization may thus expressed as a percentage of those anionic groups am able to substitution under relatively mild conditi (i.e. the reaction conditions of the inventive cationi tion method disclosed herein) while the protein-c taining substance is in its native state, which substituted to effect cationization. This percenta though not all inclusive, is usually in the range of f about 20% to about 60%.
  • mice BDF ] _ and BALB/c mice. 6-8 weeks of age, w purchased from the Jackson Laboratory, Bar Harbor, ME.
  • Adjuvants Complete and incomplete Freund's Adjuva ( IFA) and bacterial lipopolysaccharide were purchas from Difco Laboratories, Detroit MI.
  • Aluminum hydroxi gel was prepared in our laboratory according to t method of Levine and Vaz (11) or was in the form commercial Maalox (Rorer Inc., Fort Washington, PA) .
  • Antibody Assays A quantitative ELISA technique was us as previously described (12). Standard curves were r each time an assay was performed, using known amounts antibody raised against either native or cationized BS The sera were then assayed on antigen-coated ELI plates. The coatings used on plates varied in some tes and these variations are described in the Tables belo where applicable.
  • T cell proliferation assays BDF mice were injected the hind footpads and tail base with 100 ug native cationized antigen emulsified in Incomplete Freud' Adjuvant. The inguinal and popliteal lymph nodes we removed 10 days later and the subsequent cell suspensi was passed over a nylon wool column as has been describ (13).
  • the nylon wool non-adherent cells were th resuspended in complete RPMI 1640 medium containing 1 horse serum, ImM n-onessential amino acids, 1 mM sodi pyruvate, 2 mM glutamine, 5 x 10 ⁇ 5 M 2-mereaptoethano 25 mM HEPES and 5 ug/ml gentamycin, and plated in 96 we flat bottom plates (Costar, Cambridge, MA) at 5 x 10 cells/well. Native or cationized antigen were added various concentrations in serum-free complete RPMI 16 to triplicate wells. Serum-free medium served as control.
  • Table A shows that cBSA is immunogenic when inject into mice.
  • Table B shows that the length of time of protei cationization controls its immunogenicity. Cationizatio optimizes immune responsiveness. However, if cationiza tion is excessive, immunogenicity is sharply reduced, i not totally eliminated. Administration wa intraperitoneal (i.p.) with alum adjuvant (1 mg/dose).
  • Table C shows that too extensive cationization o BSA creates a molecule incapable of stimulating BS primed T lymphocytes.
  • Cationization for a time up to 60 minutes greatl enhances the ability of BSA to stimulate lymphocyt proliferation, but excessive cationization creates non-immunogenic or poorly immunogenic molecule.
  • Table D shows that cBSA evoked greater immun response following initial pretreatment with cBSA wherea nBSA actually suppressed the production of anti BS antibodies .
  • Table E shows that cBSA evoked greater immun response following initial pretreatment with cBSA wherea nBSA actually suppressed the production of anti BS antibodies .
  • Table E shows results of a more detailed study o the immune response to BSA obtained with small quantitie (1-25 mg) of the cationized BSA and the resulting immun response, both in terms of increase in enhancement an the longevity of the increase.
  • Table F shows results of a more detailed study o the immune response to BSA obtained with small quantitie (1-25 mg) of the cationized BSA and the resulting immun response, both in terms of increase in enhancement an the longevity of the increase.
  • Feedisrig (100 ug) (ug/ml) (3 x 20 mg) in alum lOd 14d 24d
  • BSA cBSA 10 10 10 cBSA CBSA 20 20 20
  • Tables F and G show the effect of oral adminis tration of the cationized and native form of the antigen
  • Table F records the increase in anti-BSA IgG level resulting from cBSA immunization, while Table G show that cBSA suppresses anti-BSA IgE levels.
  • Table H shows that feeding with nOVA suppresses immune response to OVA.
  • feeding (oral administration) with cOVA enhances greatly the immune response to both nOVA and cOVA.
  • Antigen Administered ug/ml Anti-OVA Antibodies i.p. with 1 mg Alum 9 ⁇ 14d 21d
  • Table I shows cOVA is far more immunogenic than nO most notably at lower doses of antigenic challenge.
  • Cationized Ferritin, cF was produced as described in Example 6, and administered i.p. to BDF ] _ mice.
  • Antibody levels were determined from time-interval bleeds measured by ELISA on plates coated with native ferritin.
  • Table J shows a substantial increase in immunogenicity of partially cationized ferritin.
  • Heat killed E ⁇ coli bacteria were cationized as Example 7 and washed 3 times with saline and inject i.p. into mice, and the mice were later bled.
  • Antibo concentration was determined by ELISA in which plast plates were coated with untreated bacteria.
  • Antiba terial antibody titers in mice immunized with untreat bacteria served as controls and were compared wi antibody titers from mice immunized with partial cationized bacteria.
  • Table K shows a substantial increase i immunogenicity of partially cationized bacteria.
  • Table L shows a substantial increase i immunogenicity of partially cationized bacteria.
  • Tetanus Toxoid was cationized as described in Example 4.
  • Table L shows that partial cationization increases the immunogenicity of tetanus toxoid.
  • Anti- -BGG antibodies ug/ml
  • Bovine Gamma Globulin (BGG) was cationized as describe in Example 8.
  • Table M demonstrates that partial cationizatio greatly increases the immunogenicity of Bovine Gamm Globulin.
  • Table N demonstrates that partial cationizatio greatly increases the immunogenicity of Bovine Gamm Globulin.
  • FITC-BSA conjugates were prepared as described i Example 9.
  • Immune response was determined by measuring antibodies produced against FITC-nBSA or FITC-cBSA in mice on ELIS plates coated with FITC-KLH conjugate.
  • Table N shows that a small non-immunogenic molecule, a hapten such as FITC, becomes highly immunogenic whe conjugated to a partially cationized protein (cBSA) .
  • a hapten such as FITC
  • the method immunizing a mammal or suppressing allergic response in mammal in accordance with the invention includ administering an effective amount of a partial cationized antigenic protein having an isoelectric poi ranging from about 6.5 to about 9.5, the administrati being either oral or parenteral, and with or without adjuvant.
  • a method of enhancing immune response to native antigen in a mammal comprises administeri parenterally to a mammal an effective amount of th antigen which has been partially cationized such that i isoelectric point ranges from about 6.5 to about 9.5, a thereafter administering parenterally to the mammal amount of that native antigen sufficient to evoke immune response.
  • cationized proteins may be mo immunogenic for the following reasons:

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EP88901145A 1987-01-30 1988-01-25 Teilkationisierung von eiweissenthaltenden antigenen und deren verwendung zur immunisierung und desensibilisierung Withdrawn EP0300007A1 (de)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US923487A 1987-01-30 1987-01-30
US9234 1987-01-30
US14706388A 1988-01-22 1988-01-22
US147063 1988-01-22

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EP0300007A1 true EP0300007A1 (de) 1989-01-25

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EP (1) EP0300007A1 (de)
JP (1) JPH01502024A (de)
KR (1) KR890700355A (de)
AU (1) AU618946B2 (de)
CA (1) CA1338078C (de)
IL (1) IL85256A (de)
WO (1) WO1988005664A1 (de)

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US6764681B2 (en) 1991-10-07 2004-07-20 Biogen, Inc. Method of prophylaxis or treatment of antigen presenting cell driven skin conditions using inhibitors of the CD2/LFA-3 interaction
WO2003009740A2 (en) 2001-07-24 2003-02-06 Biogen Idec Ma Inc. Methods for treating or preventing sclerotic disorders using cd2-binding agents
CA2565259A1 (en) 2004-05-07 2005-12-08 Astellas Us Llc Soluble lfa-3 polypeptide for treating viral disorders
EP2233925A1 (de) 2009-03-26 2010-09-29 Corning Inc. Immobilisierungsverfahren für Protein mit niedrigem isoelektrischem Punkt

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US3825525A (en) * 1969-08-06 1974-07-23 Beecham Group Ltd Allergens reacted with carbodiimides
US3761585A (en) * 1971-04-05 1973-09-25 Beecham Group Ltd Vaccines containing modified allergenic material

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Title
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IL85256A (en) 1993-08-18
CA1338078C (en) 1996-02-20
WO1988005664A1 (en) 1988-08-11
IL85256A0 (en) 1988-07-31
AU618946B2 (en) 1992-01-16
AU1183688A (en) 1988-08-24
JPH01502024A (ja) 1989-07-13
KR890700355A (ko) 1989-04-24

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