WO1993011234A1 - Region de domaine cytoplasmique du recepteur a l'interleukine-4 humaine, utilisee comme antagonistes de il-4 - Google Patents

Region de domaine cytoplasmique du recepteur a l'interleukine-4 humaine, utilisee comme antagonistes de il-4 Download PDF

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WO1993011234A1
WO1993011234A1 PCT/US1992/009897 US9209897W WO9311234A1 WO 1993011234 A1 WO1993011234 A1 WO 1993011234A1 US 9209897 W US9209897 W US 9209897W WO 9311234 A1 WO9311234 A1 WO 9311234A1
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human
amino acid
receptor
seq
polypeptide
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PCT/US1992/009897
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English (en)
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Nobuyuki Harada
Kenji Izuhara
Atsushi Miyajima
Maureen C. Howard
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Schering Corporation
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Priority to JP5510142A priority Critical patent/JPH07505047A/ja
Priority to EP92925279A priority patent/EP0615546A1/fr
Publication of WO1993011234A1 publication Critical patent/WO1993011234A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • 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
    • A61P37/08—Antiallergic agents
    • 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 interleukin-4 that are based upon a critical region of the 5 cytoplasmic domain of the human interleukin-4 receptor.
  • Interleukin-4 is a protein which affects a broad spectrum of hematopoietic cells [Strober et al., Pediatr. Res. 24:549 (1988)]. IL-4 enhances a number of activities in 0 human beings, including macrophage function, IgGl and IgE production, and the proliferation of immunoglobulin- stimulated B cells, antigen-stimulated T cells and erythropoietin-stimulated red blood cell progenitors. It also increases the proliferation of IL-3-stimulated mast cells.
  • mast cells play a central role in allergic reactions.
  • Mast cells are granule-containing connective tissue cells which are located proximally to capillaries throughout the body, with especially high concentrations in the lungs, skin and gastrointestinal and 0 genitourinary tracts.
  • mast cells degranulate and release chemical mediators such as histamine, serotonin, heparin, prostaglandins etc. to produce an allergic reaction.
  • Allergic reactions e.g., to dust, pollen or organic 5 detritus
  • More serious reactions e.g., asthma or food or drug allergies, may cause severe discomfort or medical problems.
  • Some very severe reactions such as anaphy lactic shock can be life threatening.
  • 46 million Americans suffered from some allergy 25 million from hayfever, 9 million from asthma and 12 million from other allergies.
  • the cloned IL-4 receptor cDNAs express high affinity binding sites on transfected COS7 cells and encode a binding protein of approximately 130-140 kilodaltons, as measured by ⁇ 25I-IL-4 cross -linking. Despite extensive characterization of the biological properties of IL-4 and its receptor, little is known about the mechanism of signal transduction induced by IL-4.
  • antagonists of IL-4 may be useful for the treatment of allergies. In view of the substantial number of individuals afflicted by allergies, there is a great need for such antagonists.
  • the present invention fills this need by providing
  • IL-4 antagonists compositions and methods for inhibiting the biological activity of human IL-4.
  • this invention provides antagonists of human IL-4 that mimic or comprise an amino acid sequence of a region of the cytoplasmic domain of the human IL-4 receptor, which region has an amino acid sequence defined by the sequence of SEQ ID NO: 1.
  • compositions comprising one or more antagonists of human IL-4 that mimic or comprise an amino acid sequence of a region of the cytoplasmic domain of the human IL-4 receptor, which region has an amino acid sequence defined by the sequence of SEQ ID NO: 1, and a physiologically acceptable carrier.
  • This invention still further provides methods for inhibiting the biological activity of human IL-4 comprising contacting cells bearing receptors for human IL-4 with an antagonist of human IL-4 that mimics or comprises an amino acid sequence of a region of the cytoplasmic domain of the human IL-4 receptor, which region has an amino acid sequence defined by the sequence of SEQ ID NO: 1.
  • the antagonists are polypeptides which contain from about 20 to about 41 amino acid residues and comprise the amino acid sequence defined by SEQ ID NO: 3.
  • Fig. 1 shows a side-by-side comparison of amino acid sequences of regions of the cytoplasmic domains of the mouse and human IL-4 receptors which are critical to the biological activity of IL-4. Also shown schematically are five synthetic polypeptides, the amino acid sequences of which are based upon the human receptor sequence.
  • Fig. 2 is a graphical representation of the effect of varying amounts of the five synthetic polypeptides of Fig. 1 on the proliferation of Ba/F3 cells transfected with human IL-4 receptor cDNA. Percent maximal proliferation rate is shown as a function of polypeptide concentration.
  • Fig. 3 is a graphical representation of the effect of certain synthetic polypeptides on the rate of proliferation of Ba/F3 cells expressing various kinds of receptors. Percent maximal proliferation rate is shown as a function of polypeptide concentration.
  • IL-4 antagonists of this invention can potentially be used to treat any medical condition caused by IL-4, such as allergies. They can also be used to elucidate the mechanism of action of IL-4 and to identify cellular elements involved in the induction of biological activity by IL-4. The understanding of the mechanism and the identification of such elements can provide bases for the rational design of drugs that can augment or inhibit the biological activity of IL-4.
  • the term "antagonist” is defined as a substance that blocks or inhibits one or more of the known biological activities of IL-4.
  • One such biological activity, the stimulation of cell proliferation, is illustrated herein.
  • Fig. 1 The conserved, critical region of the mouse and human IL-4 receptors is shown in Fig. 1 , where the sequences of the two proteins are aligned to show maximum homology. Standard single-letter abbreviations are used, with connecting lines showing homologous amino acid residues. The full amino acid sequences of the critical region in the human and mouse IL-4 receptors are also defined in SEQ ID NOs: 1 and 2, respectively.
  • Fig. 1 Also shown schematically in Fig. 1 are five synthetic polypeptides which have amino acid sequences based on the human sequence, except for additional or substitute amino acid residues that are specifically indicated.
  • the complete amino sequences of polypeptides 1 through 5 in Fig. 1 are defined by the sequences of SEQ ID NOs: 3 through 7, respectively.
  • polypeptides having amino acid sequences corresponding to the sequence of the critical region of the human IL-4 receptor can be taken up by cells and thereby inhibit the proliferative activity of IL-4.
  • the mechanism by which this inhibition occurs is not known, but an understanding of the mechanism of action is not essential to the practice of this invention. It is hypothesized that this region is involved in interactions with intracellular components of a signal transduction pathway.
  • polypeptides As explained in the Example below, two polypeptides have been shown to inhibit the stimulation of cell proliferation by IL-4.
  • One polypeptide has an amino acid sequence corresponding to a critical region of the human IL-4 receptor, as defined in SEQ ID NO: 1.
  • the other inhibitory polypeptide has a sequence corresponding to the 20 amino- terminal residues of the sequence defined by SEQ ID NO: 1.
  • the sequence of this smaller polypeptide is defined by SEQ ID NO: 3.
  • any polypeptide comprising the smaller critical sequence (defined by SEQ ID NO: 3) will inhibit the cell proliferative activity of IL-4.
  • this invention encompasses not only the two exemplary polypeptides, but also others that are intermediate in length (i.e., those which contain in addition to the 20-residue core sequence, one or more of amino acid residues 21-40 of SEQ ID NO: 1) and inhibit a biological activity of IL-4.
  • the 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. 85:2149 (1963); Science 232:341 (1986)] and Atherton et al. (Solid Phase Peptide Synthesis: A Practical Approach, 1989, IRL Press, Oxford).
  • 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.
  • the alpha-amino protecting group is removed using trifluoroacetic acid (TFA) or HC1 in dioxane at a temperature between 0° and 25°C.
  • Dimethylsulfide is added to the TFA after the introduction of methionine (Met) to suppress possible S-alkylation.
  • the remaining protected amino acids are coupled stepwise in the required
  • Various activating agents can be used for the coupling reactions including DCC, N,N'-diisopropyl- carbodiimide, benzotriazol- 1 -yl-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate (BOP) and DCC- hydroxybenzotriazole (HOBt).
  • BOP benzotriazol- 1 -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 Yoo et al. [J. Bio 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 sequence defined by SEQ ID NO: 3.
  • Such other polypeptides are a part of this invention if they (a) have an amino acid sequence that is substantially identical to the sequence defined by SEQ ID NO: 3 and (b) have the ability to inhibit one or more of the biological activities of IL-4.
  • Substantial identity of amino acid sequences means that the sequences are identical or differ by one or more amino acid alterations (deletions, additions, substitutions) that do not substantially impair inhibitory activity.
  • polypeptide antagonists produced in prokaryotic expression systems may also contain an additional N-terminal methionine residue, as is well known in the art. Any polypeptide antagonist meeting the substantial identity requirement is included, whether post-translationally modified, e.g., glycosylated, or not.
  • Polypeptides, polypeptide mimetics or analogs used in this invention should preferably produce at least about 50% inhibition of one of the biological activities of IL-4 in cells bearing IL-4 receptors. More preferably, the degree of inhibition will be at least about 70% and, most preferably, at least about 90%.
  • the IL-4 antagonists of this invention also include antibodies or fragments thereof which may interact with the defined critical region.
  • the use and generation of fragments of antibodies is well known, e.g., Fab fragments [Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)], Fv fragments [Hochman et al., Biochemistry 12 : 1 130 (1973); Sharon et al., Biochemistry 15: 1591 (1976); Ehrlich et al., U.S. Patent No. 4,355,023] and antibody half molecules (Auditore-Hargreaves, U.S. Patent No. 4,470,925).
  • 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 defined 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 humarf, 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 humarf, 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.
  • 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 84:3439 (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 :192 (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 341 :544 (1989)] can also be prepared using the sequence information.
  • the antibody-based IL-4 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 the IL-4 antagonists of the present invention.
  • Such compositions which can be used to treat IL-4-related diseases, 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
  • compositions useful for parenteral administration of such drugs are well known; e.g. Remington's Pharmaceutical Science, 15th Ed. (Mack).
  • 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.
  • the IL-4 antagonists must be taken up by the target cells, it may be desirable to incorporate the antagonists into vehicles that can facilitate such uptake.
  • the antagonists can be incorporated into liposomes.
  • the polypeptide antagonists can also be delivered by standard gene therapy techniques, includir-z, 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. 20:180-199 (1992).
  • Determination of the appropriate dosage of an antagonist of the invention 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.
  • Ba/F3 cells (kindly provided by Dr. Mary Collins, IRC-Chester Beatty Laboratories, London) were maintained in RPMI1640 medium supplemented with 10% fetal calf serum (FCS), 10 mM HEPES (pH 7.4), 50 ⁇ g/ml streptomycin and 50 U/ml penicillin, and silk worm-derived recombinant mouse IL-3 (100 U/ml) [Miyajima et al., Gene 55:273 (1987)].
  • a unit of IL-3 was defined as the amount of protein per milliliter that produced 50% saturating activity in the MC/9 assay [Yokota et al, Proc. Natl. Acad. Sci. USA 52 :1070 (1984)].
  • Stable transfectants of Ba/F3 cells were cultured in the same medium supplemented with 400 ⁇ g/ml G418.
  • n eo -resistant gene was introduced into a mammalian expression vector, pME18S, containing the human IL-4 receptor cDNA [Galizzi et al, Int. Immunol 2:669 (1990)].
  • Vector pME18Sne ⁇ IL-4R-N was constructed by cutting pME18Sne ⁇ IL-4 with Nhel and NotI, and filling in with Klenow fragment followed by ligation.
  • pME18SneoIL-4R-M2 For the construction of pME18SneoIL-4R-M2, a 1.65 kilobase MscI-EcoRI fragment of human IL-4R was prepared by EcoRI digestion and partial MscI cleavage and inserted into the EcoRI and NotI sites of pME18Sne ⁇ vector in which the NotI end was filled in with klenow fragment.
  • pME18SneoIL-4R-Ml For the construction of pME18SneoIL-4R-Ml, a 1.15 kilobase MscI-EcoRI fragment of human IL-4 receptor cDNA was ligated into pME18Sne ⁇ vector as described above.
  • pME18Sne ⁇ IL-4R-P was constructed by inserting a 1.4 kilobase Plel (filled in)-EcoRI fragment of human IL-4 receptor cDNA into the EcoRI and NotI (filled in) sites ofpMEI8Sne ⁇ as described above.
  • ⁇ ME18SIL-4R-S was constructed by inserting a 0.9 kilobase Sau3AI (filled in)-EcoRI fragment of human IL-4 receptor cDNA into EcoRI and NotI (filled in) sites of pME18Sne ⁇ vector as described above.
  • unique EcoRV and Sspl restriction sites were generated using an in vitro mutagenesis kit (Promega). Briefly, the EcoRI-Xbal-digested human IL-4 receptor cDNA was inserted into the pSELECT vector, and single-stranded template was isolated. Annealing of the mutagenic oligonucleotides and second-strand synthesis was performed according to the manual provided. The following oligonucleotides were synthesized on an Applied Biosynthesis 380A DNA synthesizer:
  • pSELECTIL-4R-E contained an EcoRV site
  • pSELECT!L-4R-ES contained an EcoRI site and a Sspl site.
  • pME18S/ze ⁇ IL-4R-lDl was constructed by isolating EcoRI-MscI fragment and the
  • pME18SneoIL- 4R-ID2 was constructed by isolating the EcoRI-EcoRV fragment and the Sspl-Xbal fragment from pSELECTIL-4R-ES and inserting them into the EcoRI-Xbal cleaved pMEl&Sneo vector as described above.
  • the mutant cDNAs were sequenced by the dideoxy sequencing method to confirm the introduced mutation.
  • Plasmid DNAs were transfected into Ba/F3 cells by the electroporation method. Briefly, ten million cells growing exponentially were harvested, washed twice with PBS and resuspended in PBS (1 x 10 ⁇ cells/ml). One hundred micrograms of cDNA linearized with Kpnl and 400 ⁇ g of tRNA were added to 0.8 ml of cell suspension and kept on ice for 15 minutes. Electroporation was carried out at 960 ⁇ F and 400 V using a Gene pulser (Bio-Rad). After an electric pulse was applied, cells were kept on ice for 10 minutes, and cultured with Ba/F3 culture medium as described above. After 2 days culture, transfectants were selected in 1.5 mg/ml G418. Stable transfectants were maintained in 400 ⁇ g/ml G418.
  • Radiolabeling of E. •. '/-derived human IL-4 and binding assays were carried out as previously described [Galizzi et al, Int. Immunol 2:669 (1990)]. Briefly, exponentially growing cells were harvested, washed twice with binding medium (RPMI 1640 containing 2% BSA, 20 mM HEPES, pH 7.4, and 0.5% NaN3), and resuspended in binding medium. Aliquots of cells were incubated with various concentrations of 12 ⁇ I-IL-4 in 200 ⁇ l of binding medium for 3 hr at 4°C. Free and cell-bound 125I-IL-4 were separated by centrifugation through an oil gradient as described previously [Lowenthal et al, J. Immunol. 140:456 (1988)]. Nonspecific binding was measured using a 150-fold molar excess of unlabeled IL-4. Binding data were analyzed with the LIGAND program.
  • Proliferation assays were performed by incubating cells (1 x 10 5 ) in microtiter plates at 37°C in 100 ⁇ l of RPMI 1640 supplemented with 10% FCS with various concentrations of recombinant human 1L-4 or murine IL-4. To test the specificity of these responses, some experiments included the addition of monoclonal antibodies which specifically blocked human IL-4 (provided from Dr. John Abrams in DNAX Research Institute) added at a final concentration of 100 ⁇ g/ml.
  • IL-4 internalization was measured as described previously [Galizzi et al, J. Biol. Chem. 264:6984 (1989)] with slight modifications.
  • Ba/F3 transfectants (1 x 10? cells/ml) were initially incubated for 5 minutes at 37°C in RPMI 1640 medium containing 2% BSA, 20 mM HEPES (pH 7.4), and 100 ⁇ M chloroquine to prevent subsequent degradation of internalized IL-4. Cells were then incubated at 4°C with 150 pM ! 25l-IL-4. After 3 hr incubation, cells were washed twice with ice-cold medium and resuspended at 4 x 10 cells/ml in prewarmed (37°C) medium.
  • the other aliquot was immediately centrifuged through the oil layer, and the radioactivity in the supernatant was measured to determine the level of dissociated IL-4.
  • the level of cell surface-bound IL-4 was calculated by subtracting the level of dissociated IL-4 from the level of cell surface-bound and dissociated IL-4.
  • Ba/F3 cells were transfected by electroporation with an expression plasmid, pME18SneohIL-4R containing the G418 resistance gene, and stable transfectants were subsequently selected with G418.
  • Several clones were examined for responsiveness to human-IL-4. It was found that whereas the original Ba/F3 cells responded only to murine IL-4, several stable transfectants responded in a dose-dependent manner to both human and murine IL-4.
  • Anti-human IL-4 antibody blocked the effect of human IL-4 on Ba/F3 stable transfectants, but had no effect on murine IL-4-induced Ba/F3 growth.
  • human IL-4 receptor cDNAs were constructed which were deleted in various regions of the cytoplasmic domain, and these mutants were introduced into the expression vector, pME18S «e ⁇ . While the full length human IL-4 receptor cDNA has 569 amino acid residues in the cytoplasmic domain, the five deletion mutants, designated
  • N-, M-2-, P-, M-1-, and S-mutants had 374, 266, 176, 99, and 8 amino acid residues in the cytoplasmic domain, respectively. These mutant cDNAs were then transfected into Ba/F3 cells, and several neomycin resistant stable transfectants were characterized.
  • the dissociation constants of human IL-4 receptors expressed by the mutant transfectants were consistent with the values on transfectants expressing the full length human IL-4 receptor cDNA, indicating that the cytoplasmic domain of the IL-4 receptor is not essential for high affinity IL-4 binding.
  • Transfectants expressing N- and M-2-mutants displayed human IL4 receptors exhibiting the predicted molecular sizes (110 and 100 kilodaltons, respectively). However, transfectants from P-, M-1-, and S-mutants expressed shorter human IL-4 receptor than the predicted molecular sizes (73, 65 and 55 kilodaltons, respectively).
  • the size difference (approximately 18 kilodaltons) between the predicted molecular weight and observed molecular weight in these three mutant human IL-4 receptors may be due to glycosylation within the cytoplasmic domain. Indeed, there is one potential N-glycosylation site located between the P- and M-2- restriction sites. Interestingly, two additional cross -linking bands (70 and 80 kilodaltons) were also observed which had been reported previously [Galizzi et al, J. Biol Chem. 265:439 (1990)]. These two bands appeared at constant molecular weight in all the mutant human IL-4 receptors, suggesting that they are more likely to be unaltered proteins which associate with the IL-4 receptor rather than degradation products of the full-length receptor as originally predicted.
  • polypeptides comprising the amino acid sequence of the core of this critical region (defined by SEQ ID NO: 3) are able to enter cells and to inhibit transduction of the proliferation signal of IL-4 bound to cells transfected with wild-type human IL-4 receptor cDNA.
  • Fig. 2 This is shown in Fig. 2, the data of which were produced by carrying out a proliferation assay as described above using Ba/F3 cells transfected with pME18SneohIL-4R.
  • the transfected cells were incubated in the presence of the indicated concentrations of polypeptide 1 (open squares), 2 (filled squares), 3 (open triangles), 4 (filled triangles) and 5 (filled circles), as defined in the legend to Fig. 1 and in SEQ ID NOs: 3 through 7, respectively, of the Sequence Listing.
  • polypeptide No. 1 SEQ ID NO: 3
  • polypeptide No. 3 produced significant inhibition of the stimulation of proliferation by IL-4. Inhibition was complete at the higher concentrations of this polypeptide, which contained the core sequence of the critical receptor region.
  • the other polypeptides showed essentially no inhibitory activity, although the activities were occasionally variable.
  • Ba/F3 transfectants expressing human IL-4 receptors were stimulated as described above with 10 ng/ml human IL-4 in the presence of either the critical region polypeptide (open squares) or the C-terminal polypeptide (filled triangles), at the indicated concentrations.
  • Ba/F3 transfectants expressing chimeric receptors which had the human IL-4 receptor extracellular domain and the human IL-2 receptor ⁇ chain in the cytoplasmic domain and transduced the human IL-2 signal upon IL-4 binding were stimulated with 10 ng/ml human IL-4 in the presence of the critical region polypeptide (filled squares).
  • Ba/F3 transfectants expressing a human IL-2 receptor ⁇ chain were stimulated with 10 ng/ml human IL-2 (open triangles), and parental Ba/F3 cells were stimulated with 10 ng/ml mouse IL-3, both in the presence of varying amounts of the critical region polypeptide (filled circles).
  • the critical region polypeptide inhibited proliferation much more than did the C-terminal polypeptide in cells that expressed the human IL-4 receptor and were stimulated by IL-4.
  • the critical region polypeptide had relatively little effect on any of the other cells.

Abstract

Antagonistes de IL-4 humaine basés sur une région critique du domaine cytoplasmique du récepteur à l'IL-4 humaine. L'invention concerne également des compositions et des procédés d'inhibition de l'activité biologique de l'IL-4 humaine.
PCT/US1992/009897 1991-11-27 1992-11-24 Region de domaine cytoplasmique du recepteur a l'interleukine-4 humaine, utilisee comme antagonistes de il-4 WO1993011234A1 (fr)

Priority Applications (2)

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JP5510142A JPH07505047A (ja) 1991-11-27 1992-11-24 ヒトインターロイキン−４受容体の細胞質ドメインの，ｉｌ−４のアンタゴニストとしての部分
EP92925279A EP0615546A1 (fr) 1991-11-27 1992-11-24 Region de domaine cytoplasmique du recepteur a l'interleukine-4 humaine, utilisee comme antagonistes de il-4

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US80362191A 1991-11-27 1991-11-27
US07/803,621 1991-11-27

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WO1996011213A1 (fr) * 1994-10-07 1996-04-18 Amgen Boulder Inc. Inhibiteurs d'il-4 dimeres
US5599905A (en) * 1988-10-31 1997-02-04 Immunex Corporation Interleukin-4 receptors

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WO1990005183A1 (fr) * 1988-10-31 1990-05-17 Immunex Corporation Recepteurs d'interleukine-4
EP0419091A1 (fr) * 1989-09-07 1991-03-27 Schering Corporation Récepteurs de l'interleukine-4

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WO1990005183A1 (fr) * 1988-10-31 1990-05-17 Immunex Corporation Recepteurs d'interleukine-4
EP0419091A1 (fr) * 1989-09-07 1991-03-27 Schering Corporation Récepteurs de l'interleukine-4

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CELL. vol. 59, 1 December 1989, CAMBRIDGE, NA US pages 837 - 845 Hatakeyama, M. et al.; 'A restricted cytoplasmic region of IL-2 receptor Beta chain is essential for growth signal transduction but not for ligand binding and internalization.' *
JOURNAL OF BIOLOGICAL CHEMISTRY. (MICROFILMS) vol. 267, no. 32, 15 November 1992, BALTIMORE, MD US pages 22752 - 22758 Harada N;Yang G;Miyajima A;Howard M; 'Identification of an essential region for growth signal transduction in the cytoplasmic domain of the human interleukin-4 receptor.' *
THE JOURNAL OF EXPERIMENTAL MEDECINE vol. 171, no. 3, 1 March 1990, THE ROCKEFELLER UNIV. PRESS pages 861 - 873 Idzerda, R.L. et al.; 'Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily.' *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5599905A (en) * 1988-10-31 1997-02-04 Immunex Corporation Interleukin-4 receptors
US5717072A (en) * 1988-10-31 1998-02-10 Immunex Corporation Antibodies that are immunoreactive with interleukin-4 receptors
US5767065A (en) * 1988-10-31 1998-06-16 Immunex Corporation Use of interleukin-4 receptors to inhibit biological responses mediated by interleukin-4
US5856296A (en) * 1988-10-31 1999-01-05 Immunex Corporation DNA encoding interleukin-4 receptors
US6391581B1 (en) 1988-10-31 2002-05-21 Immunex Corporation DNA encoding interleukin-4 receptors
US6548655B1 (en) 1988-10-31 2003-04-15 Immunex Corporation Interleukin-4 receptors
WO1996011213A1 (fr) * 1994-10-07 1996-04-18 Amgen Boulder Inc. Inhibiteurs d'il-4 dimeres

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CA2124338A1 (fr) 1993-06-10
AU3139693A (en) 1993-06-28
JPH07505047A (ja) 1995-06-08
EP0615546A1 (fr) 1994-09-21

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