WO1994012531A1 - Antagonistes de l'interferon gamma humain - Google Patents

Antagonistes de l'interferon gamma humain Download PDF

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WO1994012531A1
WO1994012531A1 PCT/US1993/011110 US9311110W WO9412531A1 WO 1994012531 A1 WO1994012531 A1 WO 1994012531A1 US 9311110 W US9311110 W US 9311110W WO 9412531 A1 WO9412531 A1 WO 9412531A1
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amino acid
seq
polypeptide
ifn
ser
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PCT/US1993/011110
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English (en)
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Gail F. Seelig
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Schering Corporation
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Priority to AU56696/94A priority Critical patent/AU5669694A/en
Priority to JP6513217A priority patent/JPH07508763A/ja
Priority to EP94902267A priority patent/EP0668873A1/fr
Publication of WO1994012531A1 publication Critical patent/WO1994012531A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to antagonists of human gamma interferon that are based upon a critical region of the human gamma interferon receptor.
  • IFN- ⁇ Gamma interferon
  • MHC Major Histo- compatibility Complex
  • IFN- ⁇ is also known to upregulate the expression of class II antigens in cells that are not primary antigen-presenting cells, such as epithelial cells, fibroblasts, astrocytes, endothelial and smooth muscle cells.
  • the upregulation of class II antigens in these cell types is often correlated with the development of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.
  • IFN- ⁇ binds to specific cellular receptors [Langer et al., Immunology Today
  • IFN- ⁇ acts at specific cellular receptors and is implicated in autoimmune diseases such as rheumatoid arthritis and multiple sclerosis, there is a need for agents that inhibit the binding of such interferon to cellular receptors.
  • the present invention fills this need by providing IFN- ⁇ antagonists, compositions and methods for inhibiting the biological activity of human IFN- ⁇ .
  • this invention provides antagonists of human IFN- ⁇ that mimic, comprise or specifically bind to an amino acid sequence of a region of the human IFN- ⁇ receptor, which region has an amino acid sequence defined by the sequence of SEQ ID NO: 2.
  • This invention further provides methods for inhibiting the biological activity of human IFN- ⁇ comprising contacting human IFN- ⁇ or cells bearing receptors for human IFN- ⁇ with an antagonist of human IFN- ⁇ that mimics, comprises or specifically binds to an amino acid sequence of a region of the human IFN- ⁇ receptor, which region has an amino acid sequence defined by the sequence of SEQ ID NO: 2.
  • the antagonists are polypeptides which contain a core sequence defined by SEQ ID NO: 3 and comprise from about 22 to 48 amino acid residues of the amino acid sequence defined by SEQ ID NO: 4, wherein in both sequences residues represented as Xaa at positions 2 and 3 can be Tyr or Val and Ser or Cys, respectively, and the sulfhydryl groups of Cys residues in the polypeptides can be free or blocked by a sulfhydryl blocking group.
  • the antagonists are antibodies or fragments thereof that specifically bind to an epitope of a polypeptide having an amino acid sequence defined by part or all of the sequence of SEQ ID NO: 2, and to the human IFN- ⁇ receptor.
  • the antagonists are anti-idiotypic antibodies or fragments thereof produced against an antibody or a fragment thereof that specifically binds to an epitope of a polypeptide having an amino acid sequence defined by part or all of the sequence of SEQ ID NO: 2, and to the human IFN- ⁇ receptor.
  • Fig. 1 is a graphical representation of the inhibition of IFN- ⁇ -induced expression of HLA DR antigen on Colo 205 cells by a polypeptide antagonist having an amino acid sequence defined by SEQ ID NO: 5, wherein the sulfhydryl group of the cysteine residue at position 3 was blocked by an acetamidomethyl group.
  • Fig. 2 is a graphical representation of the binding to human IFN- ⁇ of a polypeptide antagonist having an amino acid sequence defined by SEQ ID NO: 5, wherein the sulfhydryl group of the cysteine residue at position 3 was blocked by an acetamidomethyl group.
  • the amount of IFN- ⁇ bound to the polypeptide coated onto the wells of a microtiter plate is shown as a function of absorbance at 405 nm.
  • the human "IFN- ⁇ receptor” means a protein which (a) has an amino acid sequence substantially as defined in the Sequence Listing by SEQ ID NO: 1 and (b) has biological activity that is common to the native IFN- ⁇ receptor and which binds to human IFN- ⁇ .
  • the antagonists of this invention can potentially be used to treat any medical condition caused by IFN- ⁇ , such as autoimmune disease. They can also be used to elucidate the mechanism of action of IFN- ⁇ and can be used as part of a screening system to identify agonists and/or other antagonists of IFN- ⁇ .
  • the term "antagonist” is defined as a substance that blocks or inhibits the binding of human IFN- ⁇ to cellular receptors and thereby inhibits one or more of the known biological activities of IFN- ⁇ . Depending upon the particular antagonist, such inhibition may involve binding of an antagonist to IFN- ⁇ or to the IFN- ⁇ receptor.
  • the critical region of the human IFN- ⁇ receptor has an amino acid sequence defined by the sequence of residues 120 to 167 of SEQ ID NO: 1.
  • polypeptides containing a core sequence based upon the sequence of residues 120 to 141 of SEQ ID NO: 1 are effective antagonists of IFN- ⁇ .
  • the present invention provides such polypeptides, as well as compounds that can mimic such polypeptides.
  • any polypeptide comprising the core sequence defined by the sequence of residues 120 to 141 of SEQ ID NO: 1 will inhibit the binding of IFN- ⁇ to cellular receptors and, hence, biological activity.
  • this invention encompasses not only the above- mentioned polypeptides, but also others that are intermediate in length (i.e., those which contain in addition to the 22-residue core sequence of SEQ ID NO: 3, one or more of the other amino acid residues shown in SEQ ID NO: 4) and inhibit the binding and biological activity of IFN- ⁇ .
  • Residues - represented as Xaa at positions 2 and 3 in both sequences can be Tyr or Val and Ser or Cys, respectively. Any or all of the sulfhydryl groups of the cysteine residues in the polypeptides can be free or covalently blocked by any of the known sulfhydryl blocking groups, such as the acetamidomethyl group.
  • reagents that can be used to block sulfhydryl groups include, e.g., alkylating agents, such as iodoacetate or iodoacetamide; anhydrides such as maleic or succinic anhydride; and DTNB [5,5'-dithiobis(2-nitrobenzoic acid)].
  • alkylating agents such as iodoacetate or iodoacetamide
  • anhydrides such as maleic or succinic anhydride
  • DTNB 5,5'-dithiobis(2-nitrobenzoic acid)
  • the antagonists of this invention will inhibit the binding of IFN- ⁇ to any cells bearing IFN- ⁇ receptors, such as B cells, T cells, eosinophils, smooth muscle cells, promyelocytes, macrophages, erythroid cells, monocytes and granulocytes.
  • IFN- ⁇ receptors such as B cells, T cells, eosinophils, smooth muscle cells, promyelocytes, macrophages, erythroid cells, monocytes and granulocytes.
  • Daudi cells a well-characterized B lymphoblast cell line derived from a Burkitt lymphoma patient which are available from the American Type Culture Collection under Accession No. CCL 213, can also be used. Effects of the antagonists can be observed by measuring inhibition of the binding of 125 I-labeled IFN- ⁇ to cellular receptors on such cells.
  • Other cell lines can also be used for this purpose, such as U-937 human lymphoma line (ATCC CRL 1593).
  • polypeptide antagonists of the invention can be synthesized by a suitable method such as by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis.
  • the polypeptides are preferably prepared by solid phase peptide synthesis as described, e.g., by Merrifield [J. Am. Chem. Soc. 55:2149 (1963); Science 232:341 (1986)] and Atherton et al. (Solid Phase Peptide Synthesis: A Practical Approach, 1989, IRL
  • the synthesis is carried out with amino acids that are protected at the alpha-amino terminus. Trifunctional amino acids with labile side-chains are also protected with suitable groups to prevent undesired chemical reactions from occurring during the assembly of the polypeptides.
  • the alpha-amino protecting group is selectively removed to allow subsequent reaction to take place at the amino-terminus. The conditions for the removal of the alpha-amino protecting group do not remove the side-chain protecting groups.
  • the alpha-amino protecting groups are those known to be useful in the art of stepwise polypeptide synthesis. Included are acyl type protecting groups (e.g., formyl, trifluoroacetyl, acetyl), aromatic urethane type protecting groups [e.g., benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and 9-fluorenylmethyloxycarbonyl (Fmoc)], aliphatic urethane protecting groups (e.g., t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl type protecting groups (e.g., benzyl, triphenylmethyl).
  • acyl type protecting groups e.g., formyl, trifluoroacetyl, acetyl
  • aromatic urethane type protecting groups e.g., benzyloxycarbonyl (Cb
  • the preferred protecting group is Boc.
  • the side-chain protecting groups for Tyr include tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, 4-Br-Cbz and 2,6-dichlorobenzyl.
  • the preferred side-chain protecting group for Tyr is 2,6-dichlorobenzyl.
  • the side-chain protecting groups for Asp include benzyl, 2,6-dichlorobenzyl, methyl, ethyl and cyclohexyl.
  • the preferred side-chain protecting group for Asp is cyclohexyl.
  • the side-chain protecting groups for Thr and Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl and Cbz.
  • the preferred protecting group for Thr and Ser is benzyl.
  • the side-chain protecting groups for Arg include nitro, Tos, Cbz, adamantyloxycarbonyl and Boc.
  • the preferred protecting group for Arg is Tos.
  • the side-chain amino group of Lys may be protected with Cbz, 2-Cl-Cbz, Tos or Boc.
  • the 2-Cl-Cbz group is the preferred protecting group for Lys.
  • the side-chain protecting groups selected should remain intact during coupling and not be removed during the deprotection of the amino-terminus protecting group or during coupling conditions.
  • the side-chain protecting groups should also be removable upon the completion of synthesis, using reaction conditions that will not alter the finished polypeptide.
  • Solid phase synthesis is usually carried out from the carboxy-terminus by coupling the alpha-amino protected (side-chain protected) amino acid to a suitable solid support.
  • An ester linkage is formed when the attachment is made to a chloromethyl or hydroxymethyl resin, and the resulting polypeptide will have a free carboxyl group at the C-terminus.
  • a benzhydrylamine or p-methylbenz- hydrylamine resin is used, an amide bond is formed and the resulting polypeptide will have a carboxamide group at the C-terminus.
  • These resins are commercially available, and their preparation has described by Stewart et al. , Solid Phase Peptide Synthesis (2nd Edition), Pierce Chemical Co., Rockford, IL., 1984.
  • DCC dicyclohexylcarbodiimide
  • N,N'- diisopropylcarbodiimide N,N'- diisopropylcarbodiimide
  • carbonyldiimidazole carbonyldiimidazole
  • Dimethylsulfide is added to the TFA after the introduction of methionine (Met) to suppress possible S-alkylation. After removal of the alpha-amino protecting group, the remaining protected amino acids are coupled stepwise in the required order to obtain the desired sequence.
  • Various activating agents can be used for the coupling reactions including DCC, N,N'-diisopropyl- carbodiimide, benzotriazol-l -yl-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate (BOP) and DCC- hydroxybenzotriazole (HOBt).
  • BOP benzotriazol-l -yl-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate
  • HOBt DCC- hydroxybenzotriazole
  • the polypeptide-resin is cleaved with a reagent such as liquid HF for 1-2 hours at 0°C, which cleaves the polypeptide from the resin and removes all side-chain protecting groups.
  • a scavenger such as anisole is usually used with the liquid HF to prevent cations formed during the cleavage fom alkylating the amino acid residues present in the polypeptide.
  • the polypeptide-resin may be deprotected with TFA/dithioethane prior to cleavage if desired.
  • Recombinant DNA methodology can also be used to prepare polypeptide antagonists. See, e.g., Sambrook et al. , Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Press, Cold Spring Harbor, New York.
  • the known genetic code tailored if desired for more efficient expression in a given host organism, can be used to synthesize oligonucleotides encoding the desired amino acid sequences.
  • the phosphoramidite solid support method of Matteucci et al. [J. Am. Chem. Soc. 705:3185 (1981)], the method of Too et al. [J. Biol. Chem. 764:17078 (1989)], or other well known methods can be used for such synthesis.
  • oligonucleotides can be inserted into an appropriate vector and expressed in a compatible host organism.
  • standard molecular biology techniques can be used to permit engineering of an appropriate gene for efficient expression, including tandemly repeated segments having convenient protease sites for later cleavage and processing.
  • polypeptides can be purified using HPLC, gel filtration, ion exchange and partition chromatography, countercurrent distribution or other known methods.
  • the present invention also encompasses polypeptide analogs and mimetics, as well as other polypeptides comprising amino acid sequences which differ slightly from the sequences defined above.
  • this invention also includes modifications of the polypeptide antagonists which have undergone conservative amino acid substitution, deletion and or addition, as long as the modified polypeptides retain the ability to bind to and thereby inhibit the biological activity of IFN- ⁇ .
  • Polypeptide antagonists produced in prokaryotic expression systems may also contain an additional N-terminal methionine residue, as is well known in the art.
  • the terms “mimetic” and “analog” include polypeptides, organic compounds or peptidomimetics which adopt the same characteristics as the polypeptide antagonists. Included are molecules which adopt a portion of the same physical structure, contain a portion of the same epitope, or adopt a secondary structure and binding conformation similar to those of a polypeptide antagonist.
  • the mimetics and analogs include organic gamma and beta turn mimetics [Sato et al., Biochem. Biophys. Res. Commun. 187:999 (1992); Kahn et al., Tetrahedron Letters 30:2317 (1989)], alpha helix and beta sheet mimetics [Regan et al., Science 241 :974 (1988)] , and conformationally- restricted analogs [Kessler et al., Intl. J. Pep. Protein Res. 32:183 (1988); Dutta et al., Biochem. Biophys. Res. Commun.
  • the antagonists of this invention should preferably produce at least about 25% inhibition of a biological activity of IFN- ⁇ in cells bearing IFN- ⁇ receptors. More preferably, the degree of inhibition will be at least about 75% and, most preferably, at least about 95%.
  • the IFN- ⁇ antagonists of this invention also include antibodies or fragments thereof which specifically bind to the polypeptides and to the human IFN- ⁇ receptor. By binding to the receptor, these antibodies and antibody fragments also inhibit the binding, and hence the biological activity, of human IFN- ⁇ .
  • polypeptide antagonists which can be used as antigens to produce such antibodies and fragments, comprise one or more antigenic determinants (epitopes) against which the production of antibodies can be elicited.
  • epitopes generally contain at least about 5 amino acid residues [Ohno et al., Proc. Natl. Acad. Sci. USA 52:2945 (1985)].
  • Antibodies produced using the polypeptide antagonists as antigens will specifically bind to an epitope on the polypeptides and to the human IFN- ⁇ receptor as well.
  • Polyclonal antibodies can be produced by immunizing a host animal such as a rabbit, rat, goat, sheep, mouse, etc. with one of the polypeptides. Preferably, one or more booster injections are given after the initial injection, to increase the antibody titer. Blood is then drawn from the animal and serum is prepared and screened by standard methods such as enzyme-linked immunosorbent assay (ELISA) using the polypeptides as the antigen.
  • ELISA enzyme-linked immunosorbent assay
  • Hybridomas and monoclonal antibodies can be produced by standard methods [Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976)], using one of the polypeptide antagonists as the antigen.
  • the immunogenicity of the polypeptides is increased by combination with an adjuvant and/or by conversion to a larger form prior to immunization of a suitable host animal.
  • the immunogenicity of the polypeptides can also be enhanced by using standard methods to cross-link the polypeptides or to couple them to an immunogenic carrier molecule such as keyhole limpet hemocyanin or a mammalian serum protein such as human or bovine gammaglobulin, or human, bovine or rabbit serum albumin.
  • an immunogenic carrier molecule such as keyhole limpet hemocyanin or a mammalian serum protein such as human or bovine gammaglobulin, or human, bovine or rabbit serum albumin.
  • the protein carrier will be foreign to the host animal in which antibodies against the polypeptides are to be elicited.
  • This invention also provides anti-idiotypic antibodies or fragments thereof which are directed against the above-mentioned antibodies or antibody fragments.
  • anti-idiotypic antibodies mimic or act like the original polypeptide antagonist antigen (see, e.g., U.S. Patent No. 4,731,237 to Regan et al.).
  • these antibodies are presumed to bind specifically and directly to IFN- ⁇ .
  • Such anti-idiotypic antibodies are prepared by vaccinating an animal with an antibody (polyclonal or monoclonal) against a polypeptide of the present invention. They may be recovered as a whole polyclonal antiserum or as an IgG or other fraction thereof, or as monoclonal antibodies produced by cloned hybridomas.
  • DNA encoding the antibody can be cloned and sequenced, and techniques can be used to produce interspecific monoclonal antibodies wherein the binding region of one species is combined with a non-binding region of the antibody of another species [Liu et al., Proc. Natl. Acad. Sci. USA 54:3439 (1987)].
  • the CDRs from a rodent monoclonal antibody can be grafted onto a human antibody, thereby "humanizing" the rodent antibody [Riechmann et al. , Nature 332:323 (1988)]. More particularly, the CDRs can be grafted into a human antibody variable region with or without human constant regions.
  • Such methodology has been used, e.g., to humanize a mouse monoclonal antibody against the p55 (Tac) subunit of the human interleukin-2 receptor [Queen et al, Proc. Natl. Acad. Sci. USA 56:10029 (1989)]. Fragments of such humanized antibodies can also be made.
  • CDR sequence information can be used to design non-peptide mimetic compounds which mimic the functional properties of the antibody. Methods for producing such mimetic compounds have been described, e.g., by Saragovi et al. [Science 253: 792 (1991)]. CDR sequence information can also be used to produce single-chain binding proteins comprising linked CDRs from the light and/or heavy chain variable regions, as described by Bird et al. [Science 242:423 (1988)], or biosynthetic antibody binding sites (BABS), as described by Huston et al. [Proc. Natl. Acad. Sci. USA 55:5879 (1988)]. Single-domain antibodies comprising isolated heavy-chain variable domains [Ward et al., Nature 347 :544 (1989)] can also be prepared using the sequence information.
  • the antibody-based IFN- ⁇ antagonists used in this invention are preferably antibody fragments, BABS, mimetic compounds or single-domain antibodies.
  • the use of humanized antibody sequences is also preferred.
  • compositions can be prepared using one or more of the IFN- ⁇ antagonists.
  • Such compositions which can be used to treat any IFN- ⁇ -related disease, can be prepared by admixing an effective amount of one or more of the antagonists and a physiologically acceptable carrier.
  • Useful pharmaceutical carriers can be any compatible, non-toxic substance suitable for delivering the compositions of the invention to a patient. Sterile water, alcohol, fats, waxes, and inert solids may be included in a carrier.
  • Pharmaceutically acceptable adjuvants (buffering agents, dispersing agents) may also be incorporated into the pharmaceutical composition.
  • compositions useful for parenteral administration of such drugs are well known; e.g. Remington 's Pharmaceutical Science, 15th Ed. (Mack Publishing Company, Easton, PA, 1980). Single-dose packaging will often be preferred, e.g., in sterile form.
  • compositions of the invention may be introduced into a patient's body by implantable drug delivery systems [Urquhart et al., Ann. Rev. Pharmacol. Toxicol. 24:199 (1984)].
  • implantable drug delivery systems include implantable drug delivery systems
  • Such carriers are well known to those skilled in the art.
  • the antagonists can also be incorporated into liposomes, or delivered by standard gene therapy techniques, including, e.g., direct DNA injection into tissues, the use of recombinant viral vectors and implantation of transfected cells. See, e.g., Rosenberg, J. Clin. Oncol. 70: 180
  • Determination of the appropriate dosage of an antagonist for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages that are less than optimum. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.
  • the amount and frequency of administration of the antagonists and the pharmaceutically acceptable salts thereof will be regulated according to the judgment of the attending clinician, taking into account such factors as age, condition and size of the patient and severity of the symptom(s) being treated .
  • the present invention can be illustrated by the following, non-limiting example. Unless otherwise specified, percentages given below for solids in solid mixtures, liquids in liquids, and solids in liquids are on a wt/wt, vol/vol and wt/vol basis, respectively.
  • Recombinant human IFN- ⁇ s A and D [specific activity about 5 x 10 ⁇ units/mg; Seelig et al., Biochemistry 27: 1981 (1988)] were prepared and purified from transformed E. coli, essentially as described in U.S. Patent No. 4,751 ,078.
  • COLO-205 cells (ATCC CLL 222) were used to measure the induction by the interferon of class II major histocompatibility antigens (HLA-DR).
  • HLA-DR major histocompatibility antigens
  • ELISA Enzyme-Linked Immunosorbent Assay
  • Protein determinations were carried out by the method of Lowry et al [J. Biol. Chem. 793:265 (1951 )] using bovine serum albumin as a standard. Polypeptide concentrations were determined by amino acid analysis using gas phase HC1 and 1 hour incubation at 150°C.
  • TBS tris-buffered saline
  • TWEEN 20 polyoxethylenesorbitan monolaurate
  • the plate was subsequently blocked with 1 % bovine serum albumin (BSA) for 1 hour, washed 5 times with TBS, and coated with 2.5 ng of horseradish peroxidase-conjugate goat anti-rabbit IgG, or goat anti-mouse IgG.
  • BSA bovine serum albumin
  • Immunosorbent assays were carried out on polypeptides immobilized on pins as follows. The pins were blocked for 1 hour by inverting the pins onto a standard
  • An anti-idiotypic antibody was used to carry out analyses to identify the critical region of the human IFN- ⁇ receptor.
  • This antibody which was prepared against an IgG antibody fraction specific for a polypeptide having an amino acid sequence corresponding to that of a region of human IFN- ⁇ , mimics IFN- ⁇ itself and thereby specifically binds to the IFN- ⁇ receptor.
  • a complete description of the anti-idiotypic antibody can be found in International Application Publication No. WO 92/06115.
  • the analysis was carried out by first synthesizing polypeptide octamers corresponding to continuously overlapping regions of the human IFN- ⁇ receptor, and then through a standard ELISA determining which of the octamers bound to the anti-idiotypic antibody.
  • Overlapping octamer polypeptides were synthesized on polyethylene pins in a 96-pin format using the method of Geysen et al [Proc. Natl. Acad. Sci. USA 57:3998 (1984); Proc. Natl. Acad. Sci. USA 52:178 (19845)].
  • the polypeptides were synthesized using Fmoc/t-butyl protecting groups and the amino acids being coupled were highly activated pentafluorophenyl and oxo-benzotriazine esters. Approximately 20 to 50 pmoles of peptide were estimated to be synthesized on each pin.
  • Polypeptides having amino acid sequences defined by SEQ ID NOs: 5-8 were synthesized using the solid-phase method of Merrifield [J. Am. Chem. Soc. 55:2149 (1963)] .
  • An Applied Biosystems (Foster City, CA) Model 430A solid-phase peptide synthesizer was used with t-butyloxycarbonyl chemistry, and the polypeptides were built upon a PAM resin.
  • Hydrogen fluoride was used to cleave the polypeptides from the resin, after which the polypeptides were purified on a PHARMACIA FPLC using a 20 ml Pep/RPC column with a reverse phase chromatography solvent system of TFA/acetonitrile.
  • the cysteine group of some of the polypeptide defined by SEQ ID NO: 5 was modified by standard methods with acetamidomethyl protecting groups, which were not removed. Some of the data described below were produced with this sulfhydryl-blocked polypeptide.
  • Antibodies against the polypeptides having sequences defined by SEQ ID NOs: 5 (with and without sulfhydryl block) and 6-8 were produced in New Zealand White rabbits (Hazelton Labs) by intradermal immunization with 500 ⁇ l volumes (0.1 ml per injected site) of aqueous pH 7.1 solutions containing 0.5 to 1.0 mg of the various polypeptides emulsified with equal volumes of Freund's complete adjuvant.
  • Booster injections containing about 0.25 to 0.5 mg of polypeptide in Freund's incomplete adjuvant were administered at approximately 4-week intervals as required, as judged by ELISA responses to the polypeptides and to the human IFN- ⁇ receptor.
  • ELISA of the antisera thus produced showed that all of the antibodies tested were reactive against the polypeptide antigens used to elicit production of the antibodies.
  • the antibodies against the polypeptides having sequences defined by SEQ ID NOs: 5 (with blocked sulfhydryl group) and 8 also bound to the IFN- ⁇ receptor. Presumably, the antibodies against the other polypeptides would also have bound to the receptor, although this was not determined experimentally.
  • IFN- ⁇ was quantified essentially as described by Gibson et al. [J. Immunol. Meth. 725:103 (1989)]. Briefly, control culture medium and various dilutions in culture medium of the polypeptide defined by SEQ ID NO:5 (blocked at the Cys sulfhydryl group) were incubated in the presence of a fixed concentration (150 pM) of the interferon in 0.1 ml volumes in microtiter plate wells for one hour at 37°C.
  • the wells were washed with 0.2 ml of phosphate buffered saline (PBS; 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4) and then fixed for two minutes with ice-cold anhydrous ethyl alcohol. The alcohol was removed, and the wells were washed once with 0.2 ml of PBS. Fifty microliters of a 1 :50 dilution of the mouse monoclonal anti-HLA-DR antibody in PBS containing 0.5% bovine serum albumin were then added to each well, and the plates were incubated for one hour at room temperature.
  • PBS phosphate buffered saline
  • Excess reagent was removed by washing the wells three times with 0.2 ml of PBS, after which 0.1 ml of a 1 :5,000 dilution of peroxidase-labeled goat anti-mouse IgG was added to each well. The plates were incubated for one hour at room temperature. After washing each well three times with PBS as before, color was developed by the addition of ABTS for 5-10 minutes at room temperature. Absorbance was measured at 405 nm using an ELISA plate reader.
  • SEQ ID NO: 5 (blocked at the Cys sulfhydryl group) are shown in Fig. 1 , where it can be seen that increasing concentrations of the polypeptide antagonist of from about 10 to 100 ⁇ M produced progressively increasing inhibition of HLA-DR antigen expression. At the higher concentrations, the antagonist produced essentially complete inhibition.
  • Fig. 1 To determine whether the inhibition observed in Fig. 1 was the result of binding of the polypeptide antagonist to the IFN- ⁇ , 0.1 ml aliquots of a 100 pM solution of the polypeptide were coated onto the wells of a microtiter plate and the plate was blocked with 1% BSA. Varying amounts of IFN- ⁇ were then added to the wells and the plates were incubated and analyzed by ELIZA as described above. Specifically bound IFN- ⁇ was detected colorimetrically at
  • NMR NMR spectroscopy. NMR spectra of the free polypeptide collected in 20 mM phosphate, pH 7.0, at 5°C with a polypeptide concentration of 7.0 mg/ml (2.66 mM) showed that the polypeptide alone had very little secondary structure. In contrast, NMR analysis of the polypeptide (1.0 mg/ml; 0.38 mM) in the presence of recombinant human IFN- ⁇ E (6.7 mg/ml; 0.20 mM) in the same buffer at 5°C produced a Nuclear Overhauser Effect spectrum indicative of specific binding of the polypeptide to the IFN- ⁇ .

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  • Cell Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des antagonistes de IFN-η humain basant sur une région critique du récepteur de l'IFN-η chez l'homme. On peut citer à titre d'exemples de ces antagonistes imitant, comprenant la région critique ou le récepteur, ou se fixant spécifiquement à ceux-ci, des polypeptides et des anticorps ou des fragments de ces derniers. L'invention concerne également des compositions et des procédés d'inhibition de l'activité biologique de l'IFN-η humain.
PCT/US1993/011110 1992-11-20 1993-11-19 Antagonistes de l'interferon gamma humain WO1994012531A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU56696/94A AU5669694A (en) 1992-11-20 1993-11-19 Antagonists of human gamma interferon
JP6513217A JPH07508763A (ja) 1992-11-20 1993-11-19 ヒトγインターフェロンのアンタゴニスト
EP94902267A EP0668873A1 (fr) 1992-11-20 1993-11-19 Antagonistes contre le gamma-interferon humain

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US98052792A 1992-11-20 1992-11-20
US07/980,527 1992-11-20

Publications (1)

Publication Number Publication Date
WO1994012531A1 true WO1994012531A1 (fr) 1994-06-09

Family

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Family Applications (1)

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PCT/US1993/011110 WO1994012531A1 (fr) 1992-11-20 1993-11-19 Antagonistes de l'interferon gamma humain

Country Status (6)

Country Link
EP (1) EP0668873A1 (fr)
JP (1) JPH07508763A (fr)
AU (1) AU5669694A (fr)
CA (1) CA2149785A1 (fr)
EE (1) EE9400365A (fr)
WO (1) WO1994012531A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2304342A (en) * 1995-08-18 1997-03-19 Univ Manchester Pharmaceutical comprising either an inhibitor or a stimulator of interferon gamma
WO2006099701A1 (fr) * 2005-03-21 2006-09-28 Ivan Ivanov Inhibiteur de l’interferon-gamma humain endogene
US7220413B2 (en) 1995-08-18 2007-05-22 Renovo Limited Pharmaceutical composition containing inhibitors of interferon-γ
EP2284192A3 (fr) * 2002-11-08 2011-07-20 Ablynx N.V. Anticorps de Camelidae pour administration sublinguale
US9320792B2 (en) 2002-11-08 2016-04-26 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
US9371381B2 (en) 2002-11-08 2016-06-21 Ablynx, N.V. Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0393502A1 (fr) * 1989-04-19 1990-10-24 F. Hoffmann-La Roche Ag Récepteurs solubles de l'interféron gamma ainsi que leurs méthodes de production
EP0416652A2 (fr) * 1989-09-07 1991-03-13 Yeda Research And Development Company Limited Fragment du récepteur de l'interféron-gamma et sa production
WO1991016431A1 (fr) * 1990-04-24 1991-10-31 Schering Corporation RECEPTEURS DE η-INTERFERONS SOLUBLES TRONQUES
WO1992006115A1 (fr) * 1990-09-27 1992-04-16 Schering Corporation Antagonistes de l'interferon gamma humain

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0393502A1 (fr) * 1989-04-19 1990-10-24 F. Hoffmann-La Roche Ag Récepteurs solubles de l'interféron gamma ainsi que leurs méthodes de production
EP0416652A2 (fr) * 1989-09-07 1991-03-13 Yeda Research And Development Company Limited Fragment du récepteur de l'interféron-gamma et sa production
WO1991016431A1 (fr) * 1990-04-24 1991-10-31 Schering Corporation RECEPTEURS DE η-INTERFERONS SOLUBLES TRONQUES
WO1992006115A1 (fr) * 1990-09-27 1992-04-16 Schering Corporation Antagonistes de l'interferon gamma humain

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J.BIOL.CHEM. vol. 265, no. 12, 25 April 1990, pages 6908 - 6915 GAROTTA, G. ET AL. 'Human interferon-gamma receptor' *
PHARMACOLOGICAL RESEARCH vol. 21, no. 2, 1989, pages 5 - 17 GAROTTA, G. ET AL. 'Development of interferon-gamma antagonists ...' *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2304342A (en) * 1995-08-18 1997-03-19 Univ Manchester Pharmaceutical comprising either an inhibitor or a stimulator of interferon gamma
US7220413B2 (en) 1995-08-18 2007-05-22 Renovo Limited Pharmaceutical composition containing inhibitors of interferon-γ
EP2284192A3 (fr) * 2002-11-08 2011-07-20 Ablynx N.V. Anticorps de Camelidae pour administration sublinguale
US9243065B2 (en) 2002-11-08 2016-01-26 Ablynx N.V. Polypeptide constructs including VHH directed against EGFR for intracellular delivery
US9320792B2 (en) 2002-11-08 2016-04-26 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
US9371381B2 (en) 2002-11-08 2016-06-21 Ablynx, N.V. Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor
US9725522B2 (en) 2002-11-08 2017-08-08 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
WO2006099701A1 (fr) * 2005-03-21 2006-09-28 Ivan Ivanov Inhibiteur de l’interferon-gamma humain endogene
US7973133B2 (en) 2005-03-21 2011-07-05 Ivan Ivanov Inhibitor of endogenous human interferon-gamma

Also Published As

Publication number Publication date
EP0668873A1 (fr) 1995-08-30
EE9400365A (et) 1996-04-15
AU5669694A (en) 1994-06-22
CA2149785A1 (fr) 1994-06-09
JPH07508763A (ja) 1995-09-28

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