CA2124338A1 - Region of cytoplasmic domain of the human interleukin-4 receptor, as antagonists of il-4 - Google Patents

Abstract

Antagonists of human IL-4 are provided which are based upon a critical region of the cytoplasmic domain of the human IL-4 receptor. Also provided are compositions and methods for inhibiting the biological activity of human IL-4.

Description

2 1 2 ~ ~ 3 ~ PCI/US9t/0989/

REGION ~F CYTOPLASMIC DOMAIN OF THE HUMAN INTERLEUKIN-4 RECEPTOR, AS

This invention rela~es to antagonists of human interleukin-4 that are based upon a critical region of the 5 cytoplasmic domain of the human interleukin-4 receptor.
BAC~CGRQUND OF THE INVENTIQN
Interleukin-4 (IL-4) is a protein which affects a broad spectrum of hematopoietic cells [SIrober et al., Pediatr.
Res. 24:549 (1988)]. IL-4 enhances a number of activities in 10 humar; beings~ including macrophage function, IgG 1 and IgE
production, and the proliferation of immunoglobulin-stimulated B cells, antigen-stimulated T cells and erythropoietin-stimula~ed red blood cell progenitors. I~ also -increases the proliferation of IL-3-stimulated mast cells.
1~ Together with IgE, 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 especiahy high concentrations in the lungs, skin and gastrointestinal and 2 0 genitourinary tracts. Following exposure to an an~igenic substance, 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 2 5 detritus, may cause minor discomfort through allergic rhinitis, sneezing or tearing, or more serious problems. More serious reactions, e.g., asthma or food or drug allergies, may cause severe discomfort or mcdical problenls. Some very severe reactions such as anaphylactic shock can be life threatening.

wo 93/11234 ~ PCr/US92~0g897 z ~ 3 3 8 2 In 1989, 46 million Americans suffered from some allergy: 25 million from hayfever, 9 million from asthma and 12 million from other allergies.
IL-4 exerts biological effects through cell surface-specific receptors on target cells. Binding analyses have demonstrated that relatively small numbers (up to about 5,000 receptors/cell) of a single class of high affinity IL-4 receptor (Kd = 20-100 pM) are expressed on many types of ~nurine and human cells of hemopoietic and nonhemopoietic origin. See, e.g., papers by Ohara et al. [Nature 325:537 (1987)], Nakajima et al. ~J. Imm~nol. 139:774 (1987)], Park et ul. lProc. Natl. Acad. Sci. USA 84:1669 (1987)], Park let al.
[J. Exp. Med. 166:476 (1~87)], Cabrillat e~ al. ~Biochem. Biophys.
Res. Commun. 149:995 (1987)~ and Lowenthal et al.
[J. Immunol. 140:456 (1988)].
Cross-linking studies have led to the cha~acterization of a family of IL-4 binding proteins having molecular weights of 140, 80 and 70 kilodaltons lPark et al., J. Exp. Med. 166:476 (1987~; Park et al., ~. Cell; Bioch~m. Suppl.
12A:111 (1988)]. The relationship between these various binding proteins, however, is unclear.
Recently, cDNAs encoding mouse and human IL-4 receptor components have been isolated [Mosley et al., Cell 59:335 (1989); Harada et al., Proc. Natl. Acad. Sci. USA 87:857 (1990); Idzerda et al., J. Exp. Med. 171:861 (1990~; G~lizzi et al., Intl. Immu~eol. 2:669 (1990)], and it has been discovered tl~at these recep~ors belong to the recently described eytokine receptor family [Bazan, Biochem. Biophys. Res. Commun.
164:788 (1989)1. The cloned IL-4 receptor cDNAs express high 3 0 a~finity binding sites on transfected COS7 cells and encode a binding protein of approxin~ately 130-140 kilodaltons, as measured by 1251-IL-4 cross-linking.

wo 93/11234 2 1 2 ~ 3 3 ~3 Pcr/US92/09897 Despite extensive characterization of the biological properties of IL-4 and itS receptor, little is known about ~he mechanism of signal transduction induced by IL-4.
Because of the induction by IL-4 of IgE production, 5 mast cell proliferation and other biological effects, antagonists of IL-4 may be useful for the treatment of allergies. In view of the substan~ial number of individuals afflicted by allergies, there is a great need for such antagonists.
SU~IMARY OF l`HE INVENTION
The present invention fills this need by providing IL-4 antagonists, compositions and methods for inhib;ting the , biological activity of human IL-4.
More particularly, this invention provides antagonists of human IL-4 that mimic or comprise an amino 15 acid sequence of a region of the cytoplasmic domain of the human IL-4 receptor, which region has an amino ~cid sequence defined by the sequence of SEQ ID NO: l.
This invention further provides pharmaceutical compositions comprising one or more antagonists of human 20 IL-4 that mimie or comprise an amino acid sequence of a region of the cytoplasmic domain of ~he human IL-4 recep~or, which region has an amino acid sequence defined by the sequence of.SEQ ID NO: l, and a physiologically acceptable carrier.
2 5 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 4 PCMlS92/09897 3 ~3 4 human IL-4 receptor, which region has an amino acid sequence defined by the sequence of SEQ ID NO: 1.
In one embodimen~ of this invention, the antagonists are polypeptides which contain from about 20 to 5 about 41 amino acid residues and comprise the amino acid sequence deffned by SEQ I~ NO: 3.
BREF DESCRIPTI()N O~ THE FIGURES
This invention can be more readily understood by reference to the accompanying Figures, in which:
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 --15 based upon the human receptor sequence.
~ ig. 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 Iransfected with human IL-4 receptor cDNA. Percent maximal proliferation rate is shown as 2 0 a function of polypeptide concentration.
Fig. 3 is a graphical representation of ~he effect of certain synthetie polypeptides on the rate of proliferation of Ba/~3 cells expressing various kinds of receptors. Percent maximal proliferation rate is shown as a function of 2 5 polypeptide concentration.
, .
DESCRIPTION OF T~JVENl`ION
All references cited herein are hereby incorporated in their entirety by reference.

wos3/11234 2 1 2 ~ 3 3 ~ Pcr/uss2/09897 The IL-4 antagonists of this invention can potentially be used to treat any medical condition caused by IL-4, sueh as allergies. They can also be used to elucidate the mechanism of action of IL-4 and to identify cellular elements 5 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.
As used herein, the term "antagonist" is defined as 10 a substance that blocks or inhibits one or more of the Icnown biological activities of IL-4. One such biological activity, the stirnulation of cell proliferation, is illustrated herein.
- Through DNA deletion studies described below, it has surpnsingly been found that there is a critical region in the cytoplasmic domain of the h~man IL-4 receptor, interaction with which by as yet uncharacterized intracellular material(s) appears to be essential for the induction of cellular proliferation by IL-4. This critical region is highly conserved in mouse and human IL-4 receptors but lack,s homology with 2 0 other cytokine receptors.
The conserved, critical region of the mouse an~ ' human IL-4 receptors is shown in Fig. 1, where the sequences of the two proteins are aligned to show maximum homology.
Standard single-le~ter abbreviations are used, with connecting lines showing homologous amino acid residues. The full amino acid sequences of tbe critical region in the human and mouse ~L-4 receptors are also defined in SEQ ID NOs: I and 2, respectively.
Also shown sGhematically in ~ig. l are five 3 0 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 wo ~3/11234 Pcr/US92/09897 O ~ 3 ~

Fig. 1 are defined by the sequences of SEQ ID NOs: 3 through 7, respectively .
Surprisingly, it has been found that polypeptides having amino acid sequences corresponding to the sequence of the critical region of the human IL-4 receptor c~n 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 intrace~llular components of a signal transduction pathway.
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 inhibitor~
polypeptide has a sequence colTesponding to the 20 amino-terminal residlles of the sequence defined by SEQ ID NO: 1.
The sequence of this smaller polypeptide is defined by SEQ ID
NO:3.
From the foregoing~ it should be clear that any ' polypeptide comprising the smaller critical sequence (defined by SEQ ID NO: 3) will inhibit the cell proliferative activity of IL-4. Thus this invention encompasses no~ only the two 2 5 exemplary polypeptides, bu~ also others that are intermediate in length (i.e., those which contain in addition to th~
20-residue eore 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 3 0 be syntbesized by a suitable method such as by exclusive solid phase syntbesis, partial solid phase methods, fragment condensation or classical solution synthesis~ The polypeptides WO 93/11234 ~ i 2 ~ ~ 3 ~ PCT/US92/09X97 are preferably prepared by solid phase peptide synthesis as described, e.g., by Merrifield ~J. Am. Chem. Soc.85:2149 (1963); Science ~32:341 (19g6)] and Atherton et al. (~Solid Phase Peptide Synthesis: A Practical Approach, 1989, IRL
5 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 10 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 proteeting groups are ~hose 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), substitùted benzyloxycarbonyl and 9-fluorenylmethyloxycarbonyl (Fmoc)~, aliphatic urethane protecting groups~(e.g., ~
t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, ~ `cyclohexyloxycarbonyl) and alkyl type protecting groups (etg., benzyl, triphenylmethyl). T'~ preferred protecting group is Boc. The side-chain protec~ng groups for Tyr include 2 5 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 3 0 group for Asp is cyclohexyl. The side-chain protecting groups for Thr and Ser include acetyl, benzoyl, trityl, ~etrahydropyranyl, 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, TQS, Cbz, WO 93/1 1234 . PCl /US9~/09897 ~12~8 adamantyloxycarbonyl and Boc. The preferred protecting group for Arg is Tos. The side-chain amino group of Lys may be protected with Cbz, 2-CI-Cb7, 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 calTied 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 resulling polypeptide will have a free carboxyl group at the C-terminus.
Alternatively, when a benzhydrylamine or p^methylbenz-hydrylamine resin is used, an amide bond is f~rmed and the resulting polypeptide will have a carboxamide group at the 2 0 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., lg84.
The C-terminal amino acid, protected at the side-chain if necessary and at the alpha-amino group, is coupled to the benzhydrylamine resin using various activating agents including dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide and carbonyldiimidazole. Following the attachment to the resin support, the alpha-amino 3 0 protecting group is removed using tri~luoroacetic acid (TFA) or HCl in dioxane at a temperature between 0 and 25C.
Dimethylsulfide is added to the TFA after the intr~duction of WO93/11234 Pcr/l~S9~/09897 2 ~ t ~- ~
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- 1 -yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) and DCC-hydroxybenzotriazole (HOBt). Each protected amino acid is used in excess (>2.0 equivalents~, and the couplings are usually carried out in N-methylpyrrolidone (NMP) or in DMF, CH2Cl~ ~
or mi~tures thereof. The extent of completion of the coupling -reaction is monitored at each stage, e.g., by the ninhydrin reaction as described by Kaiser et al.,Anal. Biochem.,34:S9S ~:
(1970). In cases where incomplete coupling is found, the coupling reaction is repeated. The coupling reactions can be performed automatically with commercially available ~ ~instruments. - --After the entire assembly of the desired 2 0 polypeptide, the polypeptide-resin is cleaved with a reagent such as liquid HF for 1-2 hours at 0C, 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 HP to prevent cations forrned during the 5 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.
Side-chain to side-chain cyclization on the solid support typically requires the use of an orthogonal protection 3 0 scheme which enables selective cleavage of the side-chain functions of acidic amino acids (e.g., Asp) and the basic amino acids (e.g~, Lys). The 9-fluorenylmethyl (Fm) protecting group -~

wo ~3/l 1234 PCr/USg2/09897 212 ~ 8 10 for the side-chain of Asp and the 9-fluorenylmethyloxy-carbonyl (Fmoc) protecting group for the side-chain of L~ ~ can ~`be used for this purpose. In these cases, the side-chain protecting groups of the Boc-protected polypeptide-resin are se.ectively removed with piperidine in DMF. Cyclization is achieved on the solid support using various activating agents including DCC, DCC/HOBt or BOP. The HF reaction is carried out on the cyclized polypeptide-resin as described above.
Recombinant DNA methodology can also be used to prepare polypeptide antagonists. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Man~al, 1989, Cold Spring Harbo: Press, Cold Spring Harbor, New York. The known genetic code, ~ailored 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.
lJ. Am. Chem. Soc. 103:3185 (1981)~, the method of Yoo et al.
[J. Biol. Chem. 764:17078 (1989)], or other well known methods can be ~sed for such synthesis. The resulting 2 0 oligonucleotides can be inserted into an app~opriate vector and expressed in a compatible host organism. Alternatively, standard molecular biology techniques can be used to pern~it engineering of an appropriate gene for efficient expression, including tandemly repeated segments having convenient 2 5 protease sites for later cleavage and processing.
The polypeptides can be purified using HPLC, gel filtration, ion exchange and partition chromatography, countercurrent distribution or other known methods.
The present invention also encompasses 3 0 polypepti~de analogs and mimetics, as well as other polypeptides comp~ising amino acid sequences which differ slightly from the sequence defined by S~Q ID NO: 3. Such wo 93/l 1234 Pcr/uss2/os8s7 2 1 2 .~

other polypep~ides 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. For example, 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 be2ring IL-4 receptors. More preferably, the degree of inhibition will be at least about 70% and, most preferably, at least about 90%.
The I~-4 antagonists of this invention also inclltde 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, Pracfice and Theory of Enzy~me Immunoassays (Elsevier, Amsterdam, 198~)], Fv fragments [Hochman et al., Biochemistry 12:1130 ~1973); Sharon et al., Biochemistry 15:1591 (1976); Ehrlich et al., ~J.S Patent No. 4,355,023~ and antibody half molecules (Auditore-Hargreaves, U.S. Patent No. 4,470,925,~.
3 ~ 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 WO93/11234 ~1~4~3~ 12 Pcr/uss2/os8s7 of the defined polypeptide ant~gonists as the antigen.
Preferably, 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 5 host animal.
A wide variety of suitable adjuvants is well known in the art. 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 10 molecule such as keyhole limpet hemocyanin or a mammalian serum protein such as human or bovine gammaglobulin, or humart, bovine or rabbit serum albumin. Preferably, but not necessarily, the protein carrier will be foreign to the host animal in which antibodies against the polypeptides are to be 1 5 elicited.
Once a hybridoma producing the desired monoclonal antibody is obtained, the above~mentioned antibody fragments can be made.
Alternatively, DNA encoding the antibody can be 2 0 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 anothcr species lLiu et al., Proc. Natl. Acad. Sci.
USA 84:3439 (1987)]. For example, the CDRs &om a rodent 2 5 monoclonal antibody can be grafted onto a human antibody, thereby "humanizing" the rodent antibody [Riechmann ~ al., Nature332:323 ~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, 3 0 e.g., to humanize a mouse monoclonal antibody against the p55 (Tac) subunit of the human interleukin-2 receptor lQueen WO 93/1~234 . PCl/US92/09897 ? '1~;? 13 ' ' ' '' et al., Proc. Natl. Acad. Sci. USA 86:10029 (1989)]. Fragments of such humanized antibodies can also be made.
Once the CDRs of the heavy and light chains of the ;
monoclonal antibody have been identified, such 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 85:5879 ( 1 988) l.
Single-domain antibodies comprising isolated heavy-chain variable domains [Ward et al., Nature 341:544 (19g9)~ can also be prepared using the sequence information.
Because of their smaller size and more ready uptake by cells, the antibody-based IL-4 antagonists used in 2 0 this invention are preferably antibody fragments, BABS, mimetic compounds or single-domain antibodies. The use ,of humanized antibody sequences is also prefer~ed.
Pharmaceutical compositions can be prepared using the IL-4 antagonists of the present invention. Such 2 5 compositions, which can be used to treat IL-4-related diseases, can be prepared by admixing an effective amount of one or mor of the antagonists and a physiologically acceptable carrier.
Useful pharmaceutical carriers can be any 3 0 compatible, non-toxic substance suit;lble for delivering the compositions of the invention to a patient. Sterile water, alcohol, fats, waxes, and inert solids may be included in a ,:

WO 93/11234 PCI/US92tO9897 21~

carrier. Pharmaceutically acceptable adjuvants (buffering agents, dispersing agents) may also be incorporated into the pharmaceutical composition. Generally, compositions useful for parenteral administration of such drugs are well known; e.g.
Remington's Pharmaceutical Science, l~th Ed. (Mack Publishing Company, Easton, PA, 1980). Single-dose packaging will often be preferred, e.g., in sterile form.
Alternatively, compositions of the invention may be introduced into a patient's body by implantable drug delivery systems lUrquhart e~ al., Ann. Rev. Pharmacol.
Toxicol. 24:199 (1984)]. Such carriers are well known to those skilled in the art.
- Because 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. For example, the antagonists can be incorporated into liposomes.
The polypeptide antagonists can also be delivered by standard gene therapy techniques, includi~, e.g., direct DNA injection into tissues, the use of recombinant viral vectors and implanta~ion of transfected cells. See, e.g., Rosenberg, J. Clin.
Oncol. 10:180-199 (1992).
~etermination of the appropriate dosage of an antagonist of the invention for a particular situation is within the skill of the ar~. Generally, treatment is initiated with 2 5 smaller dosages that are less than optimum. Thereafter, the dosage is increased by small insrements 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.
3 0 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 `

WO 93/11234 PCltllS92/09897 21 2 ~ f~ 3 ~} 1 5 clinician, taking into account such factors as age, condition and size of the patient and severity of the symptom(s) being treated .
EXAMPLE
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.
Gçneral Approaçh DNA mutation analyses were used to identify the critical signal transduction region of the cytoplasmic domain of the human lL-4 recep20r. Consistent with previous reports using murine CTLL cells [Idzerda et al., J. Æxp. Med. 171:861 ~`
( 1 990)] , the full-length human ~L^4 receptor was shown to be capable of growth signal transduction when stably transfected into the murine pro-B cell line, Ba/F3. By systematically deleting discrete regions of the in~racellular domain of the IL-4 receptor, a shor~ segment encoding 41 amino acid 2 0 residues was identified which is critical for signal transduc~ion in this system. These results also demonstrate that high affinity binding to IL-4 can still be obserJed on stable transfectan~s expressing a mutant IL-4 receptor cDNA which is not capable of growth signal transduction, and that such cells 2 5 are capable of internalizing IL-4.
Ma~erials and Metho~s Cell Cult!lre Ba/F3 cells (kindly provided by Dr. Mary Collins, IRC-Chester Beatty Laboratories, London) were maintained in RPMIl640 medium supplemented with 10% fetal calf serum ' ~.

WO 93/11~34 PCr/US92/09897 21 ~33~

(FCS), 10 mM HEPES (pH 7.4), 50 llg/ml streptomycin and 50 U/ml penicillin, and silk worm-derived recombinant mouse IL-3 (100 U/ml) [Miyajima et al., Gene 58:273 (1987)]. A unit of IL-3 was defined as the amount of protein per milliliter that 5 produced 50% saturating activity in the ~Ct9 assay [Yokota et al., Proc. Natl. Acad. Sci. USA 81:1070 (1984)~. Stable transfectants of Ba/F3 cells were cultured in the same medium supplemented with 400 11 g/ml G4 18 .
Plasmid CQn~truction The neo-resistant gene was introduced into a mammalian expression vector, pME 1 8S, containing the human IL-4 receptor cDNA ~Galizzi et al., Int. Immunol. 2:669 (1990)].
Vector pME1 8SneoIL-4R-N was constructed by cutting pME18SneoIL-4 with NheI and NotI, and filling }n with Klenow fragment followed by ligation. For the constructi~n of pMEl 8SneoIL-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 pME18Sneo vector in which the Notl end was filled in with 2 0 klenow fragment.
For the construction of pME18SneoIL-4R-Ml, a r.15 kilobase MscI-EcoRI ~ragment of human IL-4 receptor cDNA
was ligated into pME18Sneo vector as described above.
pMEl 8SneoIL-4R-P was constructed by inserting a 1.4 2 5 kilobase PleI (filled in)-EcoRI fragment of human IL-4 receptor cDNA into tbe EcoRI and Notl (filled in) sites ofpME18Sneo as described above. pME18SIL-4R-S was constructed by inserting a 0.9 kilobase Sau3AI (filled in)-EcoRI
fragment of human IL-4 receptor cDNA inlo EcoRI and Notl ~filled in) sites of pMElBSneo vector as described above.

wo 93/1l234 Pcr/uss2/o9897 212 ~ 3~38 To construct internal deletion mutants of human L-R, 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 5 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:
1 0 EcoRV, AGAGCAGCAGGGATATCI~CCAGGAGGGAA~
SspI~ CATGGGGGAGTCAAATAl-rCl~CCACCl'I`C, and used to construct two mutant cDNAs; pSELECTIL-4R-E
contained an EcoRV site and pSELECTIL-4R-ES contained an EcoRI site and a SspI site. pME18SneoIL-4R-ID1 was 1~ constructed by isolating EcoRI-MscI fragment and the EcoRV-XbaI fragment from pSELECTIL-4R-E and inserting into the the EcoRI-XbaI-cleaved pME18Sneo vector. pME18SneoIL-4R-ID2 was constructed by isolating the EcoRI-EcoRV fragment and the SspI-XbaI fragment from pSELECTIL-~R^ES and 20 inser~ing them into the EcoRI-XbaI cleaved pME18Sneo vector as described above. The mutant cDNAs were sequenced b~ ,the dideoxy sequencing method to confirm the in~roduced mutation.
Transfection 2 5 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 107 cells/ml). One hundred micrograms of cDNA linearized with KpnI and 400 llg of tRNA
were added to 0.8 ml of cell suspension and kept on ice for 15 minutes. Elect~oporation was carried out at 960 ,uF and 400 V
using a Gene pulser (Bio-Rad). After an electric pulse was wo 93/11234 Pcr/US92/09897 ~12433~ 1 8 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 G4 18. Stable transfectants were maintained in 400 '~1 g/ml G4 18 .
Bindin~ Assays Radiolabeling of E. . 'i-derived human IL-4 and binding assays were carried out as previously described ~Galizzi ef al., Inr. 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 bindling medium. Aliquots of cells were incubated with various - concentrations of l25I-IL-4 in 200 ~l of binding medium for 3hr at 4C. Free and cell-bound 125I-IL-4 were separated by centrifugation :hrough an oil gradient as described previously [Lowenthal et al., J. Immunol. 140:456 (1988)1. Nonspeci~lc :~
binding was measured using a 1 50-fold molar excess of unlabeled IL-4. Binding data were analyzed with the LIGAND
program. :
2o 125I IL-4 closs-linkin&

~ells (1 x 107~ were incubated with 1~0 pM of -~
125I-IL-4 in binding medium for 2 hr at 4C. Cells were then ' washed twicç with RPMI 1640 containing 50 mM HEPES
(pH 7.4), resuspended with 1 ml of PBS containing 50 mM
2 5 HEPES (pH 8.0), and bis-(sulfosuccinimidyl) suberate (BS3, Pierce) to a final concentration of 0.2 mM. After 3() minutes at 4C, the reaction was stopped by the addition of 50 ~11 of l M
TrisHCl (pH 8.0), and lysed with 100 ~l of 1% Triton X-100 containing 140 mM NaCI, 50 mM HEPES (pH 7.4), 2 mM
3 0 phenylmethyl-sulfonyl fluoride, 1 ~lM Pepstatin A, 1 mM
iodoacetamide, 1 mM l,10-o-phenanthroline, and 5 mM EDTA
for 30 minutes at 4C. Cell lysates were then centrifuged at WO 93/1 1234 ~, 1 2 ~ 3 ~ ~ PCr/U!~i92/09897 l 2,0û0 x g for l 5 minutes, and the supenatants were analyzed by SDS-PAGE as previously described [Galizzi et al., Int.
Immunol. 2:669 (1~90)].

Proliferation _Assav$
Proliferation assays were performed by incubating cells (1 x 105) in microtiter plates at 37C in lO0 ~Ll of RPMI
1640 supplemented with l 0% FCS with various concentrations of recombinant human IL-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 concentra~ion of l 00 `
llg/ml. After 42 hr incubation, lO 1ll of a 5 mglml solution of 3- ~ 4,5 -dimethythiazol-2-yl 1 -2,5 -diphenyltetra701ium -~
bromide-tetrazolium (MTT) were added to each well followed by a further incubation at 37C for 6 hr. The optical density was measured as previously described [Lowenthal et al., `~
J. Immunol. 140:456 ( l 988)1-IL-4 Internalizati~n 2 0 IL-4 internali~ation was measured as described -previously [Galizzi et al., J. Biol. Cherrl. 264:6984 (1989)] with slight modifications. Ba/F3 transfectants (l x 107 cellslml) were initially incubated for S minutes at 37C in RPMI 1640 medium containing 2% BSA, 20 mM HEPES (pH 7.4), and 100 2 5 IlM chloroquine to prevent subsequent degradation of internalized IL-4. Cells were then incubated at 4C with 150 pM 125I-IL-4. After 3 hr incubation, cells were washed twiee with ice-cold medium and resuspended at 4 x 107 cells/ml in prewarmed (37C) medium.

PCr/US92J09897 WO 93/11234 2 1 2 L~i 3 3 ~ 2 0 At various times, two aliquots (50 ~Ll) of the cell suspension were removed. One aliquot was adjusted to pH 3.2 for 10 minutes by the addition of 150 1ll of 0.1 M glycine-HCl, 0.15 M NaCI buffer (pH 2.7). The cells were then centrifuged 5 through an oil layer. The radioactivity levels in both the cell pellet and in the supernatant above the oil layer were measured to determine the amount of acid-resistant (i.e., -internalized) IL-4 and the amount of acid-sensitive (i.e., cell surface-bound and dissociated) IL-4, respectively. The other 10 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 1~ dissociated IL-4.
Re~Llts Ex~ession o F~nction~l Human IL-4 Receptor~ on a Murin~
Pro-B C~ll Line.
~ .
Idzerda et al. [J. Exp. Med. 171:861 (1990)] have 2 0 shown that expression of the cloned human IL-4 receptor cDNA in the murine T cell line CTLL conferred responsiven'ess to human IL-4. An IL-3 dependent murine proB cell line, Ba/F3, was sel~ted as a recipient cell for human IL-4 receptor trans~ections, since Ba/F3 cells prolifera~e in response to 2 5 murine IL-4 as measured by the Ml-r method, grow very rapidly, and incorporate exogenous DNA efficiently.
Ba/F3 cells were transfected by electroporation with an expression plasmid, pME I 8SneohIL-4R containing the G418 resistance gene, and stable transfectants were 30 subsequently selected with G418. Several clones were examined for responsiYeness to human-IL-4. It was found th?t whereas the original Ba/F3 cells responded only to wo 93/11234 ~12 ~ 3 ~ 8 Pcr/uss2/098s7 murine IL-4, several stable transfectants responded in a dose-dependent manner to both human and murine IL-4.
Anti-human IL^4 anti~ody blocked the effect of human IL-4 on Ba/F3 stable transfectants, but had no effect on murine 5 IL-4-induced Ba/F3 growth.
Binding Of 1 25I labeled human lL-4 (1 25I-hIL-4) to the human IL-4 receptor expressed on stable transfectants was examined by Scatchard analysis. On the parental cells, no ,;
significant binding Of 125 I-hIL-4 was observed. However, the ~stable transfectant, P-2, expressed 1 80 human IL-4 ~:receptors/cell with a dissociation constant for human IL-4 of 23 p~I, a binding affinity which was consistent with values -previously reported for native human IL-4 receptors lGalizzi et al., J. Biol. Chem. 264:6984 (1989)].
A second clone, F-~, expressed 1 40 human IL4 receptors/cell with a dissociation constant of 25 pM.
Collectively, these results demonstrated that the human IL-4 receptor could be expressed on the cell surface of murine Ba/F3 cells, and that this receptor was functional in terms of 20 its ability to transduce a biological response to human IL-4.
E~ ~ of Hum~n IL-4 ~çcep~or Terminal Deletion Mutants - To dissect the region of the cytoplasmic domain responsible for transducing the above human IL-4-mediated 25 biological response, a series of human IL-4 receptor cDNAs was constructed which were deleted in various regions of the cyt~plasmic domainj and these mutants were introduced into the expression vector, pME18Sneo. While the full length human IL-4 receptor cDNA has 569 amino acid residues in the 3 0 cytoplasmic domain, the five deletion mutants, designated N-, M-2-, P^, M-l-, and S-mu~ants, had 374, 266, 176, 99, and 8 amino acid residues in the cytoplasmic domain, respectively.

wo 93/1 1234 Pcr/uss2/09897 ~ 1 5~ X 2 2 These mutant cDNAs were then transfected into Ba/F3 cells, and several neomycin resistant stable transfectants were characterized.
It was found that the Ba/F3 transfectants which :~
expressed the N-mutant and M-2-mutant cDNAs proliferated -in response to human IL-4, whereas transfectants with the :
shorter M- l -mutant and S-mutant cDNAs failed to respond tO
human IL-4. The response of Ba/F3 transfectants expressing the P-mutant cDNA to human IL-4 was lower than that of the ~-~
N- or M-2-mutants, although still significantly above background. These results suggested that the critical region of the c~toplasmic domain~ of the human IL-4 receptor for growth signal transduction in pro B cells was loeated between the Sau3A l site (S-) and the PleI site (P-), and probably close to the PleI site.
This conclusion was dependent on all five deletion mutants expressing significant numbers of high affinity binding sites for human IL-4. To test this, human IL-4 receptor expression level was analyzed on each of the deletion 2 0 mutant s~able transfec~ants by both binding assay and affinity cross-linking using l 25 I-hIL-4. Equilibrium binding studies showed that. all transfectants expressed high affinity binding sites for human IL-4, as can be seen in Table l.

.. :

WO 93/11234 ~ 1 2 4 3 3 ~ PCI/US92/09897 23 ::

Tsble 1 Expression of Human IL-4 Receptors on Ba/F3 Cells Transfecte~
with Tn~ncated Human IL-4R Plasmids Cells Receptor Numbers Kd(pM) (si~s/cell) Wild-type c!one 2 180 23 N-mutant clone 5 2,468 1 1 3 M-2-mutant clone 4 542 21 P-mutant clone 3 136 39 4 814 ` 41 M-1-mutant clone 4 599 34 S-mutant clone 5 1,500 22 11 2,720 ,6 2 0 ~ - .
The dissociation constants of human IL-4 receptors expressed by the mutant transfectants were eonsistent with the values on transfectants expressing the full length human IL-4 receptor cDNA, indicating tha~ the cytoplasmic domain of 2 5 the lL-4 receptor is not essential for high affinity Il,-4 binding.
In chemical cross-linking experiments, an appropriate complex of truncated human IL-4 receptor and ~-1 25I-hIL-4 was detectable on cach of the transfectants.
3 0 Cross-linlcing of 1 25 I-hIL-4 to the intact human IL-4 reeeptor WO 93/11234 . PCr/US92~09897 21C1 43~8 24 ':

showed a l 30 kilodalton band which could not be ~?
distinguished from the cross-linking band expressed on a human hemopoietic cell line, TF1 cells. The five mutant cDNAs, N-, M-2-, P-, M- l - and S-, were calculated to encode proteins which were approximately 20, 30, 40, 47 and 57 kilodaltons shorter, respectively, than the intact human IL-4 receptor.
Transfectants expressing N- and M-2-mutan~s displayed human IL4 receptors exhibiting the predicted molecular sizes - ~`(1 lO and lO0 kilodaltons, respectively). However, transfectants from P-, M-l-, and S-mutants expressed shorter human ~L-4 receptor than the predicted molecular sizes (73 65 and SS kilodaltons, respectively).
The size difference (approximately l 8 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 2 0 also observed which had been reported previously [Galizzi et al., J. Biol. Chem. 265:439 (l990)~. 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 2 5 rather than degradation products of the full-length receptor as -`
originally predicted.
~r~i~ ~nd~unction of Human IL-4 Rççe~tor Internal De!etion Mutants The above results suggested that the critical signal 3 0 transducing region of the IL-4 receptor for IL-4-induced growth in pro B cells was located between the P- and M-l-sites of the cytoplasmic domain of this receptor.

-wo 93/l 1234 Pcr/us92/o9897 2~4-'~`8 To further define this region, two internal deletion mutant cDNAs were constructed. Mutant ID- 1 lacked all residues between arnino acids 433 and 473, and lD-2 lacked all residues between amino acids 394 ~nd 432 from the carboxyl terminus. Several stable Ba/F3 transfectants expressing ID-l and ID-2 mutant human IL-4 receptor cDNAs were obtained and examined for human IL-4 induced proliferation, 125 I-hIL-4 binding and cross-linking. Further detailed analysis in this manner will allow more precise definition of the boundaries of the critical segments.
A number of transfectants expressing ID-2 c:DNA
were ~able to respond to hIL-4, while~ transfectants expressing ID-l cDNA failed to respond to hum~n IL-4. Scatchard analysis revealed that transfectants expressing ID- 1 and ID-2 mutant cDNAs exhibited comparable numbers of human IL-4 binding sites per cell. The results of this analysis are shown in -Table 2. ~
, .
Table 2 Expression of Human IL-4 Receptors on Ba/F3 Cells Trans~ected with _ Intemally Deleted Human IL-4R P!asmids ~ _ Cells ReceptorNumbcrs Kd(pM) (sites/cell) ID-1 -mutant ~0 clone 2 172 66 ID-2-mutant clone 4 689 27 .... . . . . _ _ _ wo 93/11234 Pcr/us92/os8s7 212~33~ 26 ~

The results of chemical cross-linking studies showed that transfectants with ID- l and ID-2 mutant human IL-4 receptor cDNAs both expressed truncated human IL-4 receptors. This indicated that the critical region of the human 5 IL-4 receptor for growth signal transduction in pro B cells is located between amino acids 433 and 473 from the carboxyl terminus. This region is moderately conserved between murine and human IL-4 receptors. -Li~and Internalization by Mutant Human IL-4 Receptors Previous studies showed that human IL-4 is -internalized after binding to the human IL-4 receptor [Galizzi et al., J. Biol. Chem. 264:6984 (1989)~. To evaluate the ~ -relationship between ligand internalization and growth signal transduction, ligand internalization was analyzed in each of the ~ -mutant Ba/F3 transfectanls described above. As a result of --these analyses, it was found that each of the above transfectants expressing mutant human IL-4 receptor cDNAs, ~`
including those incapable of growth signal transduction, was `
able to internalize l25I-hIL-4, reaching plateau internalization ; `
2 0 at approximately 10 minutes. These results indicated that ligand internalization does not correlate with growth signal transduction and, indeed, that the cytoplasmic domain of the human IL-4 receptor is not required for ligand internalization.
Poly~epti~ Antagonis~of IL-4 As has been explained above, there is a critical `~
region of the human IL-4 receptor cytoplasmic domain which `~
is essential for growth signal transduction following the ` .`
binding of IL-4 to the high affinily receptor on the surface of the cell. Surprisingly, it has been found that polypeptides 30 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 --'', `` ' ~

wo 93/1 1234 2 1 2 4 3 i'' ~ Pcr/usg2/0989?

inhibit transduction of the proliferation signal of IL-4 bound to cells transfected with wild-type human IL-4 receptor cDNA.
This is shown in Fig. 2, the data of which were produced by carrying out a proliferation assay as described 5 above using Ba/F3 cells transfected with pME l 8SneohIL-4R.
The transfected cells were incubated in the presence of the indicated concentrations of polypeptide l (open squares), 2 (filied squares), 3 (open triangles), 4 (filled triangles) and 5 (filled circles), as defined in the legend to Fig. 1 and in SEQ ID
10 NOs: 3 through 7, respectively, of the Sequence Listing.
As can be seen in Fig. 2, polypeptide No. 1 (SEQ ID NO: 3~ produced significant inhibition of the stimulation of proliferation by IL-4. Inhibition was complete ~;at the higher concentrations of this polypeptide, which 15 contained the core sequence of the critical receptor region. In the present experiment, the other polypeptides showed essentially no inhibitory activity, although the activities were occasionally variable.
To further demonstrate inhibitory~ activity by an 2 0 antagonist of the invention, the effect of varying levels of a synthetic polypeptide having an amino acid sequence defi~ed by SEQ ID NO: l (critical region polypeptide) was investigated.
This polypeptide contained the core sequence (SEQ ID NO: 3) plus additional C-terminal aniino acid residues. The effect of 2 5 another synthetic polypeptide having an amino acid sequence corresponding to that of the 30 C-terminal residues of the human IL-4 receptor ~C-terminal polypeptide) was also investigated. The results are shown in Fig. 3.
Ba/F3 transfectants expressing human IL-4 3 0 receptors were stimulated as described above with lO ng/ml human IL-4 in the presence of either the critical region polypeptide (open squares) or the C-terminal polypeptide 212~3~'~ 28 (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 l O ng/ml human IL-4 in the presence of the critical region polypeptide (filled squares). Ba/F3 transfectants expressing a human IL-2 receptor ,B chain were -stimulated with 10 ng/ml human IL-2 (open triangles), and parental Ba/F3 cells were stimulated with 1 0 ng/ml mouse IL-3, both in the presence of varying amounts of the critical region polypeptide (filled circles). ~;-. ~
All of the cells were incubated for 24 hours. .~TT
was added during the last 4 hours of the incubation, after ;
which the optical densities (O.D.) were measured. Proliferation rate (%) = (a-b)/(c-b) x lO0, where ~ ~:
a = O.D. produced by a factor in the presence of a polypeptide, - -`
b = O.D. in the presence of medium alone, and c = O.D. produced by a factor in the absence of any polypeptide.
As can be seen in Fig. 3, 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 cr~tical region polypeptide had relatively little effect on any of the other 2 5 cells.
Many modifications and variations of this invention can be made without departing from its spirit and scope, as will become apparent to those skilled in the art. The specific embodiments described herein are offered by way of 3 0 example only, and the invention is to be limited only by the terms of the appended claims.

. ~

WO93/11234 2 1 2 4 3 3 ~ PCI/~JS92/09897 `.

SEQUENCE LISTING

(1 ) GENERAL INFORMATION: .
(i) APPLICANT: Harada, Nobuyuki Izuhara, Kenji :
Miyajima, Atsushi Howard, Maureen 10 (ii) TITLE OF INVENTION: Antagonists of Human Interleukin-4 (iii) NUMBER OF SEQUENCES: 7 1 5 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: DNAX Research Institute (B) STREET: 901 California Avenue (C) CITY: Palo Alto .
(D) STATE: California 2 5 (E) CC)UNTRY: USA
.

(F) 71P: 94304 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Apple Macintosh ,~

WO g3/11234 Pcr/us92/og897 ~3 ~ s (C) OPERATING SYSTEM: Macintosh 6Ø5 (D~ SOFTWARE: Microsoft Word 4.00B :~

5 (vi) CURRENTAPPLICATION DATA:
.
(A) APPLICATION NUMBER~

(8) FILING DATE: -:

(~) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/803,621 (B) FILING DATE: 27-NOV-91 (viii) ATTORNEY/AGENT INFORMATION- ;
(A) NAME: Ching, Edwin P.

(B) REGISTRATION NUMBER: 34,090 2 ~ (C) REFERENt: EIDOCKET NUMBER: DX0245K

~ix) TELECOMMUNICATION INFORMATION:

(A~ TELEPHONE: 415-496 1204 (B) TELEFAX: 415-496-1200 (C) TELEX:

WO 93/11234 ~ 1 2 ~ 3 3 3 PCr/US92/09897 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids (B~ TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Pro Glu Ser Ile Ser Val Val Arg Cys Val Glu Leu Phe Glu Ala Pro 1 s l0 15 Val Glu Cys Glu Glu Glu Glu Glu Val Glu Glu Glu Lys Gly Ser Phe Cys Ala Ser Pro Glu Ser Ser Arg Asp (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE GHAP~ACTERISTICS:
(A) LENGTH: 44 amino acids (B) TYPE: amino acid 3 0 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
;~ ~
: ` ~

WO93/11234 . PCI/~S92/09897 : ~
~ t ' ~ 3 2 Pro Glu Asn Val Ser Val Ser Val Val Arg Cys Met Giu Leu ?he Glu 1 5 10 '5 Ala Pro Val Gln Asn Val Glu Glu Glu Glu Asp Glu Iie Vai 'ys Glu 5 Asp Leu Ser Met Ser Pro Glu Asn Ser Gly Gly Cys (2) INFORMATION FOR SEQ ID NO: 3:

10(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 2 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ' -Pro Glu Ser Ile Ser Val Val Arg Cys Val Glu Leu Phe Glu Ala Pro 1 5 10 1;
Val Glu Cys Glu ..
(2) INFORMATION FOR SEQ ID NO: 4:

(i) S~QUENGE CHARACTERISTICS:

(A) LENGTH 20 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear :

WO93/11234 ~ 3 3~ PCl/US92~09897 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
5 Val Glu Leu Phe ~_~ Ala Pro Val Glu Cys Glu Glu Glu G'u Glu Val Glu Glu Glu Lys 10 (2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) 1 YPE: amino acid (D) TOPOLOGY: linear 20 (ii) MOLECULE TYPE: peptide ~ .
(xi) SEQUENCE DESCP~IPTION: SEQ ID NO: 5:
Val Glu Leu Phe Glu Ala Pro Val Glu Cys Glu Glu Glu Glu Glu ~al 2 5 Glu Glu Glu Glu (2) INFORMATION FOR SEQ ID NO: 6:

3 0 (i) SEQUENCE C:HARACTERISTICS:

(A) LENGTH: 20 amino acids ~B) TYPE: amino acid WO 93/11234 . PCI/US92~09897 21 24~ 34 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Glu Glu Glu Val Glu Glu Glu Lys Gly Ser Phe Cys Ala Ser Pro Glu Ser Ser Asp Arg .

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS: -(A) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 7:
2 5 Glu Glu Glu Val t;lu Glu Glu Glu Gly Ser Phe Cys Ala Ser Pro Glu Ser Ser Asp Arg ,

Claims (9)

WHAT IS CLAIMED IS:

1. 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.

2. The antagonist of claim 1 which is a polypeptide that contains from about 20 to about 41 amino acid residues and comprises the amino acid sequence defined by SEQ ID
NO: 3.

3. The polypeptide of claim 2 which has an amino acid sequence defined by SEQ ID NO: 1 or SEQ ID NO: 3.

4. A pharmaceutical composition 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 pharmaceutically acceptable carrier.

5. The pharmaceutical composition of claim 4 in which the antagonist is a polypeptide that contains from about 20 to about 41 amino acid residues and comprises the amino acid sequence defined by SEQ ID NO: 3.

6. The pharmaceutical composition of claim 5 in which the polypeptide has an amino acid sequence defined by SEQ ID NO: 1 or SEQ ID NO: 3.

7. A method 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.

8. The method of claim 7 in which the antagonist is a polypeptide that contains from about 20 to about 41 amino acid residues and comprises the amino acid sequence defined by SEQ ID NO: 3.

9. The method of claim 8 in which the polypeptide has an amino acid sequence defined by SEQ ID NO: 1 or SEQ ID
NO: 3.