MXPA99000688A - Muleines de interleuquina-4 de gran afini - Google Patents

Abstract

The present invention relates to a recombinant human IL-4 mutein, numbered according to wild-type IL-4, in which the mutein comprises at least one amino acid substitution at the conjugation surface of alpha helices A or C of the Wild IL-4, by which the mutein is conjugated with, the IL-4Ralpha receptor with at least greater affinity than the native IL-4. The substitution is preferably selected from the group of positions comprising, in helix A, positions 13 and 16 and in helix C, positions 81 and 89. A more preferred embodiment in the mutein of recombinant human IL-4 in that the substitution at position 13 is Thr to Asp. Also described are pharmaceutical compositions, amino acid and polynucleotide sequences that encode muteins, transformed host cells, antibodies to muteins and therapeutic methods.

Description

INTERLEUQUINE MUTEINS-4 OF GREAT AFINITY Background 1 Field of the invention The invention relates generally to the fields of pharmacology and immunology. More specifically, the invention deals with novel vanants of the cytokine family and, in particular, with the human nterleuquine 4 (IL-4) 2 Description of the Related Technology Interleukin 4 is a 15 kDa glycoprotein secreted by activated T cells (How ard v colab, J Exp Med 155 914 (1982)), mast cells (Btown \ colab, CeU 50 S09 1 1937)) and baseline (Scdet \ colab Proc Nati Acad Sci USA 88 2835 (1991)), which regulates a broad spectrum of cellular functions in hematopoietic and non-hematopoic cells > The sequence of IL-4 is divided in US Patent No. 5 017 691 Interleukin 4 L-41 is a pleiotropic cytokine activity on the cells of the immune system, those of the endothelium and those of fibroblastic nature The in? ttro effects reported with the administration of IL-4 include proliferation of B and T cells, transfer of the class of immunoglobulin in B cells, stimulation of the production of adhesion molecules to the cell surface in cells endotehales and stimulation of the release of 1L 6 In T cells, IL-4 stimulates the proliferation of T cells after mitogenes and regulates the production of IFN and reduces it. In monocytes, IL-4 induces an expression of n of MHC class II molecules releases tP? induced by lipopolysaccharides and the expression of CD23 In endotehal cells (CE), IL 4 induces the expression of VC AM- l and the release of IL-6 and reduces the expression of IC AM 1 Maher DW \ colab Human? nterleuk? n -4 An immunomodulatoi w ith potential thei apeutic applications [Immune modulator with potential therapeutic applications], Progressin Growth Factor Research, 3 43-56 (1991) Treatment with IL-4 was investigated because of its ability to stimulate the proliferation of activated T cells by exposure to IL 2 For example, IL-4 demonstrated antineoplastic activity in animal models of renal carcinomas and induced tumor regression in mice (Basco, M v colab., Lovv doses of IL-4 injected peplymphaticallv m tumor-bearing mice mhibit the grovvth of poorly and apparently nonnmunogenic tumors and induces tumor specific im une memory [Low doses of IL 4, peplinfatically injected to tumor bearing mice, inhi The growth of tumors that are not immunogenic and apparently non-immunogenic and induce a specific immune memory for the tumor], J Immunol, 145: H6-43 (1990)) However, its toxicity limits the posology in humans in renal cancer and advanced malignant melanoma], J. Immunotherapy, 15: 147-153 (1994)). In general, the general structure and function of BL-4 and the 5 related monomer ligands containing 4 antiparallel helical-domains (A-D) are currently known. The three-dimensional structure of IL-4 has been resolved (Powers v. Co., Science 256: 1673 (1992)). The protein contains 4 helices-left and two b-sheets. The IL-4 receptor consists of at least two chains. The first IL-4R chain, EL-4Ra, shares significant homology with the b chain of IL-2R and other members of the superfamily of growth factor receptors (Ldzerda et al., J. E.xp. Med. 171: 861 (1990)). A second DL-4R chain has been identified, that of IL-2R, also known as the 'common chain', gc (Russell et al., Science 262: 1877 (1993)). The two conjugation sites are probably involved in a sequential double conjugation event, which results in a 1: 1: 1 ternary complex. It is believed that the region of IL-4 likely responsible for conjugation with BL-4Ra would be located in one or both helices A and C, while it is believed that the interactive region with gc would be located in helix D. The current theory holds that the first conjugating event involves the ligand in contact with DL-4Ra, the primary conjugating component. No cell signaling activity is associated with this event. The second conjugating event occurs when the IL-4 / IL-4Ra complex obtains a second string,? C. After this second conjugating event, signaling occurs and cellular activity is established. Antagonism of type IL-4 is found _ wild when conjugating interactions mediated by the second region (necessary for cellular activity) decrease or eliminate, while retaining the conjugation to EL-4Ra. Agonism occurs when a candidate ligand interacts constructively, through the first and second regions, with both receptor components. IL-4 antagonists have been published. IL-4 mutants that function as antagonists include the mutein IL-4 antagonist, IL-4 Y124D (Kruse, N .; Tony, HP; Sebald, W., Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement [Conversion of human interleukin-4 to a high affinity antagonist by an individual amino acid replacement], Embo J., 11: 3237-44 (1992)) and a double IL-4 mutein [R121D / Y124D] (Tony, H., and colab., Design of human interleukin-4 antagonists in inhibiting interleukin-4-dependent and interleukin-13-dependent responses in T-cells and B-cells with efficiency], Eur. J. Biochem. 225: 659-664 (1994)). The individual mutein is a substitution of tyrosine for aspartic acid at position 124 of helix D. The double mutein is a substitution of arginine for aspartic acid at position 121 and for tyrosine for aspartic acid in the position 124 of the helix D. The variations in this section of the helix D are positively related to changes in the interactions in the second conjugating region. Mutant variants of EL-4 that demonstrate agonism or antagonism of wild-type BL-4 may be useful for the treatment of conditions associated with one of the effects pleiotropics of IL-4. For example, IL-4 antagonists would be useful in the treatment of conditions exacerbated by BL-4 production, including asthma, allergy or other conditions related to the inflammatory response. IL-4 agonists may be useful for the treatment of conditions where the presence of BL-4 is associated with the alleviation or attenuation of a disease, for example, an autoimmune disease such as rheumatoid arthritis, multiple sclerosis, diabetes mellitus dependent on insulin, etc. These • Autoimmune diseases are characterized by a polarization in the production of collaborating T cell populations, types 1 and 2 (Tcl, Tc2). Innocent CD4 + T cells differentiate into subgames Tcl or Tc2, depending on the cytokine that is present during the stimulus. An IL-4 agonist would ideally shift production to the T cell type collaborator that would be desired, for example, towards Tc2, thus achieving a therapeutic effect. PCT / US93 / 03613 discloses a variant of IL-4 with a sequence Phe-Leu or Tyr-Leu in an alpha helical domain and a negatively charged amino acid within two amino acids innierely upstream or downstream of the Phe-Leu sequence or Tyr-Leu, the variant B exhibiting increasing affinity for the IL-4 receptor by virtue of a neutral amino acid replaced by the negatively charged amino acid. It also discloses that the specific substitution of Trp-Leu or Phe-Leu within an a-helix of EL-4, within 2 residues of a negatively charged residue, results in a better affinity. The variant is a fusion protein of EL-4 (with diphtheria toxin). , with the wild-type EL-4, in which the mutein comprises at least one amino acid substitution on the conjugation surface of the helices A or C alpha of the wild-type IL-4, and in which the mutein is conjugated with the IL-4Ra receptor with an affinity at least greater than the native EL-4. The substitution is preferably selected from the group of positions comprising, in helix A, positions 13 and 16 and in helix C, positions 81 and 89. A more preferred embodiment is the recombinant human IL-4 mutein in that the substitution at position 13 is Thr to Asp. The pharmaceutical compositions are also described, amino acid and polynucleotide sequences encoding the muteins, transformed host cells, antibodies to the muteins and methods of treatment. The invention also concerns a test for determining the ability of a mutein to be conjugated to a receptor, comprising the steps of: introducing the conjugating portion of a receptor chain, first, into a streptavidin-coated FlashPlate®; the conjugating portion of a receptor chain having a peptide ear capable of being conjugated by streptavidin; second, introduction into the FlashPlate of a radiolabelled native ligand that has affinity for the conjugating portion of a receptor chain; introduction in the FlashPlate, third, of a mutein ligand that has an affinity for the conjugating portion of a receptor chain; the measurement of the amount of signal emitted by the FlashPlate after allowing it to equilibrate and, finally, the calculation of the relative affinity of the mutein ligand against the native ligand. In a preferred mode, the method uses the IL-4Ra receptor chain. The invention also concerns a recombinant human EL-4 antagonist mutein, numbered according to wild-type DL-4, in which the mutein comprises: (a) substitutions R121D and Y124D in helix D of wild-type DL-4, and (b) at least one amino acid substitution at the conjugation surface of the helices A or C alpha of wild-type IL-4, in which the mutein is conjugated to the IL-4Ra receptor at least with greater affinity than ELL-4. native The substitution is preferably selected from the group of positions comprising, in helix A, Thr to Asp. Also disclosed are pharmaceutical compositions, amino acid sequences and polynucleotides that encode muteins, transformed host cells, antibodies to muteins and methods of treatment.

Brief description of the figures Figure 1 is a schematic view of the ligand / receptor architecture for IL-4. EL-4 is a four-helix bundle protein presented here in an 'extreme' view of the four helices (A, B, C and D from the N and C ends, respectively). The primary conjugating component of the DL-4 receptor is IL-4Ra, which interacts with the ligand of LfL-4 through helices A and C of IL-4. The formation of the IL-4 / IL-4Ra / LL-4R complex? induces signaling in the target cell. Figure 2 is an abscissa and ordinate graph of the competitive conjugation of T13D-IL-4 [R12ID / Y 124D]. We present the ability of T13D-DL-4 [R121D / Y124D], •, to compete with I125-IL-4 in a solid-phase conjugation test, relative to that of IL-4 [R 121D / Y124D], or. The value of KL < ? determined using this assay for T13D-IL-4 [R 121D / Y124D] was 0.28 nM and for LL-4 [R121D / Y124D] 5.0 nM. Figure 3 is a similar abscissa and ordinate graph, showing the antagonism of E -4 by T13D-rL-4 [R121D Y124D], or. The ability of T13D-EL-4 [R12 lD / Y124D] to compete with the proliferation of PHA blasts induced by IL-4 is presented in relation to D -4 [R121D Y124D], •. The IC50 determined from this test for T13D-LL-4 [R121D Y124D] was 2 nM and for IL-4 [R121D / Y124D], 13 nM. Figure 4 is the list of the amino acid sequence of EL-4Ra-STX. Figure 5 is a complete sequence listing of the mutein T13D-IL-4. Figure 6 is a complete sequence listing of the mutein T13D-IL-4 [R 121D / Y124D]. novel EL-4 muteins that present higher affinities for wild EL-4 receptors.

As used herein, "wild-type IL-4" means human interleukin-4, whether native or recombinant, with amino acid sequence 129 of normal occurrence in native human IL-4, as disclosed in the patent. from the USA No. 5,017,691, incorporated herein by reference. As used herein, "IL-4 mutein" means a polypeptide in which specific substitutions have been made in the mature human interleukin-4 protein, specifically the helices A or C and more preferably at the amino acids that comprise their conjugating surfaces. The conjugating surface of helix A has generally been shown to be from about the amino acid position 5 to about 16, and the helix C from the approximate position 77 to the approximate position 89. These changes to either of the helices increase the affinity of the mutein resulting by IL-4Ra, whose mutein can be an agonist or an antagonist of wild-type IL-4, depending on the final nature of the additional substitutions to the molecule. As used herein, "IL-4 antagonist mutein" means a polypeptide in which substitutions of specific amino acids have been made in the mature human interleukin-4 protein. Specifically, the antagonist muteins presented in this document contain at least three different substitutions. The pair of substitutions "IL-4 [R121D Y124D]" occurs in all the antagonistic muteins contained herein and refers to a dorsal pair of substitutions in helix D, R121D (Arg to Asp) and Y124D (Tyr to Asp). In addition, a third substitution has been introduced into the conjugation surfaces of helix A or C in a position that increases the conjugating affinity of the mutein to the alpha receptor chain. Outside of these changes, the most preferred IL-4 antagonist muteins have an amino acid sequence identical to that of wild-type IL-4 in the other unsubstituted residues. The IL-4 muteins of this invention can also be characterized by insertions, deletions, substitutions and amino acid modifications at one or more sites in or to the other residues of the native IL-4 polypeptide chain. In accordance with this invention, any of We prefer to make conservative modifications and substitutions at other B -4 positions (ie, those that exert a minimal effect on the secondary or tertiary structure of the mutein). Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978) and by Argos in Embo J., 8: 779-785 (1989). For example, amino acids that belong to one of the following groups represent conservative changes: - wing, pro, gly, gln, asn, ser, thr; 10 - cys, ser, tyr, thr; - val, ile, leu, met, ala, phe; - lys, arg, his; - phe, tyr, trp, his; and - asp, glu, tyr. 15 We also prefer modifications or substitutions that eliminate crossed intermolecular linkage sites or incorrect formation of disulfide bonds. For example, it is known that IL-4 has six cys residues, in wild positions 3, 24, 46, 65, 99 and 127, one or more of which may be involved in cross-linking interactions. Substitutions should be selected in a manner that preserves the tertiary structure of the wild protein as much as possible. ^^ By declaring "numbered according to the wild IL-4" we mean identifying a ^^ amino acid chosen with reference to the position in which such an amino acid normally occurs in wild-type IL-4. In cases where insertions or cancellations are made to the IL-4 antagonist mutein, an individual skilled in the art will appreciate that, for example. Be (S) that normally occurs at position 125 when numbered according to wild-type IL-4, can shift in position in the mutein. However, the location of the displaced Ser (S) can be easily determined by inspection and correlation of the surrounding amino acids with those surrounding Ser in wild-type IL-4. The EL-4 muteins of the present invention can be produced by any method adequate known in technology. Such methods include constructing a DNA sequence which codes for the IL-4 muteins of this invention and which expresses such sequences in a but less preferably, by chemical synthesis or a combination of chemical synthesis and recombinant DNA technology. In one embodiment of a recombinant method for producing a mutein of this invention, a DNA sequence is constructed by isolating or synthesizing an ALDN sequence encoding wild-type IL-4 and then changing the codon for threonine 13 to a codon for aspartatic acid by site-specific mutagenesis. This technique is well known. See, for example, Mark et al., Site-specific mutagenesis of the human fibroblast 10 interferon gene [Mutagenesis of the human, site-specific fibroblast interferon gene], Proc. Nati Acad. Sci. USA 81: 5662-66 (1984); U.S. Patent No. 4,588,585, incorporated herein by reference. Another method to construct a DNA sequence encoding the IL-4 muteins of ^ fc this invention, it would be by chemical synthesis. For example, a gene encoding the desired IL-4 mutein 15 can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides have been designed based on the amino acid sequence of the desired IL-4 mutein and preferably by selecting the codons favored in the host cell in which the recombinant mutein will be produced. Regarding this, it is well recognized that the genetic code is degenerate: that an amino acid can be encoded by more than one codon. For example, Phe 20 (F) is encoded by two codons, TTC or TTT, Tyr (Y) is encoded by TAC or TAT and HIS (H) is encoded by CAC or CAT. Tf () is encoded by a single codon, TGG. Agree ^^ With this, it will be understood that, for a given DNA sequence encoding a particular EL-4 mutein, there will be many degenerate DNA sequences that will encode for that EL-4 mutein. For example, it will be appreciated that, in addition to the preferred DNA sequence for the mutein T13D-IL-4 [R 121D Y124D] presented in SEQ ID NO: 9, there will be many degenerate DNA sequences encoding the presented LL-4 mutein. These degenerate DNA sequences are considered to be within the scope of this invention. Accordingly, the "corresponding degenerate variants" in the context of this invention, mean all DNA sequences that code for a particular mutein. also DNA sequences that encode a signal sequence. Such a signal sequence, if present, should be one recognized by the cell chosen for the expression of the mutein of EL-4. It can be prokaryotic, eukaryotic or a combination of both. It may also be the signal sequence of native IL-4. The inclusion of a signal sequence depends on whether it is desired to secrete the IL-4 mutein from the recombinant cells in which it is produced. If the chosen cells are prokaryotic, it is generally preferred that the DNA sequence does not encode a signal sequence but includes an N-terminal methionine to direct expression. If the chosen cells are eukaryotic, it is generally preferred that a signal sequence be encoded and more preferably, that the signal sequence of wild-type IL-4 be used.

Normal methods can be applied to synthesize a gene encoding an IL-4 mutein according to this invention. For example, the complete amino acid sequence can be used to construct a back-translated gene. A DNA oligomer containing a nucleotide sequence encoding DL-4 mutein can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5 'or 3' overhangs for complementary assembly. Once armed (by synthesis, site-directed mutagenesis or other methods), the DNA sequences encoding an LCL-4 mutein of this invention will be inserted into an expression vector and will be operably linked to a control sequence. of the appropriate expression for the expression of the DL-4 mutein in the desired transformed host. The appropriate assembly can be confirmed by determination of nucleotide sequences, restriction mapping and expression of a biologically active polypeptide in the appropriate host. As is well known in the art, to obtain high levels of expression of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences, which are functional in the chosen expression host. host / expression vector. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising SV40 expression control sequences, bovine papillomavirus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as those from £ L »Coli, including col El, pCRl, pER32z, pMB9 and its derivatives, plasmids from larger amplitude hosts, such as RP4, phage DNA, such as the numerous lambda phage derivatives, eg, NM989, and other DNA phages, such as M 13 and filamentous DNA phages from a single 10 strand. Useful expression vectors for yeast cells include plasmid 2m and its derivatives. Useful vectors for insect cells include pVL 941. We prefer pFastBac 1 (GibcoBRL, Gaithersburg, MD). Cate and colab., Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells [Isolation of the bovine and human genes for Müller inhibitory substance and B expression of the human gene in animal cells], Cell, 45: 685-98 (1986). In addition, any of a variety of expression control sequences can be used in these vectors. Such useful expression control sequences include those associated with structural genes for the aforementioned expression vectors. Examples of useful expression control sequences include, for example, early and late triggers of SV40 or adenoviruses, the lac system, the tf system, the TAC or TRC system, the main activating and activating regions of lambda phage, for example PL, the control regions of the fd coat protein, the activating enzyme for 3-phosphoglycerate kinase or other enzymes B glycolytics, activators of acid phosphatase, for example, PhoA, those of the yeast-binding system, the polyhedron activator of Baculovirus and other sequences that control the Expression of genes from prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof. Any suitable host can be used to produce the EL-4 muteins of this invention, including bacteria, fungi (including yeasts), plant, insect, mammalian or other animal cells or appropriate cell lines, as well as animals or plants. transgenic. More particularly, these hosts can include well-known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces,, African green, such as COS 1, COS 7, BSC 1, BSC 40 and BNT 10, and human cells, as well as plant cells in tissue culture. For the expression of animal cells, we prefer those of OHC and COS 7 in cultures and, particularly, the cell line of OHC, CHC (DHFR-). Of course, it should be understood that not all vectors and "expression control sequences" will work equally well to express the DNA sequences described herein, In the same way, not all hosts function equally well with the same expression system. An individual with experience in the technology can make a selection between these vectors, expression control sequences and hosts without much experimentation.For example, when selecting a vector, the host must be considered because the vector must replicate within it. consider the copy number of the vector, the ability to control such a copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, For example, preferred vectors for use in this invention include those that allow The DNA encoding the EL-4 muteins is amplified in copy number. Expandable res are well known in the art and include, for example, vectors capable of being amplified by DHFR amplification (see, for example, Kaufman, U.S. Patent 4,470,461, Kaufman and Sharp, Construction of a modular dihydrafolate reducíase cDNA gene : Analysis of meanings used for efficient expression [Construction of a modular dihydrofolate reductase cDNA gene: Analysis of signals used for efficient expression], Mol. Cell. Biol., 2: 1304-19 (1982)) or glutamine synthetase ("GS") amplification (see, for example, U.S. Patent 5,122,464 and published European application EP0338841). When selecting an expression control sequence, a variety of factors should be considered, including, for example, the relative potency of the sequence, the possibility of controlling it, and its compatibility with the actual DNA sequence encoding the IL- mutein. 4 of this invention, especially with respect to potential secondary structures. Hosts should be selected based on their compatibility with the chosen vector, the toxicity of the product for which the DNA sequences of this invention encode, their encoded by the DNA sequences. Within these parameters, an individual skilled in the art can select various combinations of vector / expression control sequence / host, which express the desired DNA sequences on fermentation or in large-scale animal culture using, for example, using OHC cells or COS 7 cells. The IL-4 muteins obtained according to the present invention can be glycosylated or non-glycosylated, depending on the host used to produce the mutein. If bacteria are chosen as hosts, then the produced EL-4 mutein will not be glycosylated. On the other hand, eukaryotic cells will glycosylate IL-4 muteins although perhaps not in the same way that native IL-4 is glycosylated. The IL-4 mutein produced by the transformed host can be purified according to any suitable method. Several methods for purifying IL-4 are known. See, for example, United States patents 5,013,824; 5,017,691 and WO9604306-A2. We prefer purification by immunoaffinity. See, for example, Okamura et al., Human f? Broblastoid interferon: Immunosorbent column chromatography and N-terminal amino acid sequence [Human fibroblast interferon: Nano-absorbent column chromatography and N-terminal amino acid sequence], Bjochem., 19: 3831 -35 (1980). The biological activity of the DL-4 muteins of this invention can be tested by any suitable method known in the art. Such tests include neutralization of antiviral activity by anti-convolutions, induction of protein kinase, activities of oligoadenylate 2,5-A synthetase or phosphodiesterase, as described in EP-B 1 -41313. Such tests also include immunomodulatory assays (see, for example, U.S. Patent 4,753,795), growth inhibition tests, T cell proliferation, induction of IL-6 and induction of MCP-1 in endothelial cells and measurement of the conjugation to cells expressing interleukin-4 receptors. See also Spits, H .; Yssel, H .; Takebe, Y., et al. Recombinant intérleukin-4 promotes the growth of human T cells [Recombinant interleukin-4 promotes the growth of human T cells], J. Immunol. 139: 1 142-47 (1987). The IL-4 mutein of this invention will be administered at a dose approximately parallel or greater than that employed in the treatment with recombinant IL-4 or wild-type native. indication that is being treated. It will be appa to those skilled in the art that the effective amount of IL-4 mutein will depend, among other factors, on the disease, the dose, the IL-4 mutein administration schedule, whether the mutein of LL-4 is administered alone or together with other therapeutic agents, the serum half-life of the composition and the general health of the patient. Preferably, the IL-4 mutein is administered as a composition that includes a pharmaceutically acceptable carrier agent. A "pharmaceutically acceptable carrier" means a carrier agent that does not cause adverse effects in the patients to whom it is administered. Such pharmaceutically acceptable carriers are well known in the art.

We prefer 2% ASH / SSTF at a pH of 7.0. The IL-4 muteins of the present invention can be formulated into compositions Pharmaceuticals using well-known methods. See, for example, Remington's 15 Pharmaceutical Science, by E.W. Martin, incoforado here by refee, that describes suitable formulas. The pharmaceutical composition of the EL-4 mutein can be formulated in a variety of forms, including liquid, gel, lyophilized or any other suitable form. The preferred form will depend on the particular indication that is being addressed and will be evident to persons with experience in the technology. The pharmaceutical composition of the EL-4 mutein can be administered orally, by aerosol, intravenously, intramuscularly, intraperitoneally, intradermally or subcutaneously, or in ^ any other acceptable form. The preferred mode of administration will depend on the particular indication in question and will be evident to persons with experience in the technology. The pharmaceutical composition of the IL-4 mutein can be administered together with other therapeutic agents. These agents can be incubated as part of the same pharmaceutical composition or can be administered separately from the D -4 mutein, either concurly or in accordance with any other acceptable therapeutic program. In addition, the pharmaceutical composition of the DL-4 mutein can be used as an adjuvant to other treatments. ,, immunomodulation in any suitable animal, preferably a mammal and more preferably the human. As previously noted in the Background section, IL-4 exerts many effects, some of which include stimulation of T cell proliferation, diffeiation of helper T cells, induction of activation and proliferation of human B cells and class transfer of immunoglobulins directed by lymphokine. Effects on the lymphoid system include increasing the expression of the MHC class II antigen (tyoelle, R., et al., Increased expression of antigens on resting B cells: A new role for B cell growth factor B cells at rest: a new role for B cell growth factor], PNAS USA 81: 6149-53 (1984)) and CD 23 on B cells (Kikutani, H., and colab., Molecular structure of human lymphocyte receptor for immunoglobulin [Molecular structure of human lymphocyte receptors for immunoglobulin], Cell 47: 657-61 (1986)). Therefore the biology of B -4 suggests that there may be a significant role in the development of allergy and allergic inflammatory diseases, including asthma. Collaborating T cells of type 1 (Tc l) and type 2 (Tc2) participate in the immune response. The stimulated Tc2 cells secrete DL-4 and block the progression of Tcl. Accordingly, any disease in which Tc2 is involved is susceptible to treatment with a BL-4 antagonist and, likewise, any disease in which Tc 1 is involved is susceptible to treatment with an IL-4 agonist. The use of the DNA sequences encoding IL-4 muteins of this invention in therapeutic applications with genes is also contemplated. Therapeutic applications with genes contemplated for BL-4 antagonists include the treatment of diseases in which IL-4 is expected to cause or exacerbate an existing clinical condition, for example a condition associated with inflammation (asthma) or allergies. Agonist indications would include autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, and insulin-dependent diabetes mellitus. These autoimmune diseases are characterized by a polarization in the production of collaborating T cell populations towards type 1 collaborative T (Tcl). Local delivery of IL-4 muteins, both agonists and antagonists, using gene therapy, can provide the therapeutic agent to the target area while avoiding the potential toxicity problems associated with the non-specific administration of agonists. Treatment methodologies with genes are contemplated both in vitro and in vivo.

Basic of Gene Therapy], Science, 260: 926-31 (1993). These methods include: 1) Direct transfer of the gene. See, for example, Wolff and c? It.o '. Direct gene transfer? 5 mouse muscle in vivo [Direct gene transfer to mouse muscle in vivo], Scieno ?, 247: 1405-68 (1990); ^ 2) Transfer of DNA mediated by liposomes. See, for example, Caplen and colab., Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis [Transfer of CFTR gene mediated by liposomes, to the nasal epithelium of patients with 10 mucoviscidosis], Nature Med., 3: 39-46 (1995); Crystal, The gene as a drug, Nature Med., 1: 15-17 (1995); Gao and Huang, A novel cationic liposome reagent for ef ?? cient transfection of mammalian cells [A novel cationic liposome reagent for the efficient transfection of mammalian cells], Biochem. Biophvs. Res. Comm., 179: 280-85 (1991). 15 3) Transfer of DNA mediated by retroviruses. See, for example, Kay et al., In vivo gene therapy of hemophilia B: Sustained partial correction in factor-deficient dogs [Gene therapy of hemophilia B, in vivo: Durable partial correction in dogs with factor IX deficiency], Science, 262: 1 17-19 (1993); Anderson, Human gene therapy [Gene Therapy in the Human Being !, Science, 256: 808-13 (1992). 20 4) DNA transfer mediated by DNA virus. Such DNA viruses include adenoviruses (preferably vectors based on Ad-2 or Ad-5), herpetic viruses (preferably vectors based on single-hepe virus) and parvoviruses (preferably "defective" or non-self-contained parvovirus-based vectors, more preferably, vectors based on adeno-associated viruses, and more preferably, AAV-2-based vectors). See, for example, Ali and • colab., The use of DNA viruses as vectors for gene therapy, Gene Therapy, 1: 367-84 (1994); U.S. Patent 4,797,368, hereby incorporated by reference, and U.S. Patent 5,139,941, hereby incorporated by reference. The choice of a particular vector system to transfer the gene of interest will depend from a variety of factors. One important is the nature of the cellular target recipient.

Although retroviral vectors have been extensively studied and have been used in numerous applications of gene therapy, they are generally not suitable for infecting non-dividing cells. In addition, retroviruses exhibit an oncogenic potential. Adenoviruses have the advantage of presenting a broad range of hosts, they can infect resting or terminally differentiated cells, such as laurons or hepatocytes, and enovus does not appear to be found in the genome. In the case of chromosomes, the risk of insertion mutagenesis is greatly reduced. Ali and colab., Before, p. 373, 5 Adeno-associated viruses exhibit advantages similar to those of vectors based on ^ p adenovirus. However, they exhibit site-specific integration in human chromosome 19. Ali and colab., Before, p. 377. According to this embodiment, the genetic therapy with DNA encoding IL-4 muteins of this invention is administered to a patient in need thereof concurrently with the patient. diagnosis or immediately after it. The skilled technologist will appreciate that any suitable gene therapy vector, containing tnutein DNA of IL-4, can be used in accordance with this embodiment. The techniques for constructing such a vector are well known. See, for example, Ohno et al., Before, p. 784; flB Chang et al., before, p. 522. The introduction of the vector containing mutein DNA from IL-F to the recipient site can be achieved using known techniques, for example, those described in Ohno et al., Before, p. 784. For this invention to be better understood, the following examples are presented, for purposes of illustration only, which should not be construed as in any way limiting the scope of the invention. 20 Examples In general. The alanine substitutions were introduced into the wild-type DL-4 sequence by site-directed mutagenesis at the positions corresponding to the predicted residues, on the surface of helices A and C of IL-4 (Smith Lf; Redßeld C; Boyd J; Lawrence GM; Edwards RG; Smith RA and Dobson CM, Human interleukin 4. The solution structure of a four- 25 helix bundle protein [Interleukin 4 human. The solution of the structure of a four-helix bundle protein], J. Mol. Biol., 224 (4): 899-904 (1992)). These are the most likely residues to facilitate the interaction of IL-4 with IL-4Ra (Figure 1); the effects on the conjugating affinity of the LL-4 muteins with alanine substitution for LL-4Rcx, indicate that the substituted residue might be involved in the conjugating interaction. The residues that, upon being substituted with alanine, give intermediate effects on the affinity, were extensively substituted to identify the changes that improved the affinity. The mutations that must be related to a greater power. The muteins were generated by site-directed mutagenesis, expressed in the baculovirus system, purified to homogeneity, determined quantitatively by amino acid analysis and evaluated in receptor conjugation tests. The amino acid sequence of mature human IL-4 (SEQ ID NO: 1) used in this study is presented below. His at position 1 represents the N-terminus of the mature polypeptide. The helices A, C and D are indicated above their respective initial points and underlined. The amino acids in which the appropriate substitutions gave higher affinity variants are indicated in bold: A:? His Lys Cys Asp lie Thr Leu Gln Glu lie lie Lvs Thr Leu Asn 1 5 10 15 Be Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr 20 25 30 Asp lie Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe 35 40 45 Cys Arg Ala Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu 50 55 60 C :? Lys Asp Thr Arg Cys Leu Gly Wing Thr Wing Gln Gln Phe His Ara 65 70 75 His Lvs Gln Leu lie Ara Phe Leu Lvs Ara Leu ASD Ara Asn Leu 80 85 90 Trp Gly Leu Wing Gly Leu Asn Ser Cys Pro Val Lys Glu Wing Asn 95 100 105 D :? Gln Ser Thr Leu Glu Asn Phe Leu Glu Ara Leu Lys Thr lie Met Ara G u Lvs Tyr Ser Lvs Cys Ser Ser (SEQ ID NO: l) 125 The mutations examined in this study were introduced into a known antagonist variant of human IL-4, which contained two substitutions in helix D, R121D and Y124D (Tony HP et al., Design of human interleukin-4 antagonists inhibiting interleukin-4). - dependent and interleukin-13-dependent responses in T-cells and B-cells with high efficiency 10 [Design of human interleukin 4 antagonists that inhibit interleukin-4 dependent and interleukin-13 dependent responses in T and B cells, with great efficiency], Eur. I Biochem. 225 (2): 659-65 (1994), this mutein is designated "IL-4 [R121D / Y124D]"). The muteins were expressed in a baculovirus system, purified to homogeneity and evaluated in a solid-phase DL-4R receptor conjugation test. The biological significance of the better affinity for IL-4R was evaluated in T cell proliferation tests.

^^ As the mutein IL-4IR121D / Y124D] is an antagonist of EL-4, the best affinity for EL- ^^ 4Ra should result in a reduction of the IC50 for the higher affinity mutein antagonist (Cl50 is defined as the antagonist concentration necessary to inhibit a defined agonist response of 50%). Example 1. Production of muteins. The muteins were generated by site-directed mutagenesis, using primers containing codons corresponding to the desired mutation, essentially as described by Kunkel TA; Roberts fD and Zakour RA, Rapid and efficient site- specific mutagenesis without phenotypic selection [Fast and efficient site-specific mutagenesis, without phenotypic selection], Methods Enzymol 154: 367-382 (1987). In few In the words, the human IL-4 cDNA, which contains the restriction enzyme sites Bam Hl and Xba I, was subcloned into the phage vector M 13 mpl9 (New England Biolabs, Beverly, MA) using the same sites. The wild-type IL-4 cDNA was obtained using the polymerase chain reaction (PCR) of a cDNA library generated from mRNA isolated from pheral human blood lymphocytes, induced for 24 hours with phorbol 12-myristate 13-acetate (10 ng / ml). The used PCR primaries were, for the 5 'end of the IL-4 open reading frame, '-CGC GGA TCC ATG GGT CTC ACC TCC-3 '(SEQ ID NO: 2); '-CGC TCT AGA CTA GCT CGA ACÁ CTT TGA AT-3 * (SEQ ID NOS).

Restriction enzyme sites BamHl (5 'end) and Xbal (3' end) were incubated in each oligonucleotide, which are indicated in italics. The CPR conditions used were one minute at 94 ° C, one minute at 58.7 ° C and one minute at 72 ° C for 25 cycles. The correct IL-4 cDNA sequence obtained in this way was confirmed by determining the sequences with the use of the Sequenase sequence kit (Amersham Life Sciences, Arlington Heights, Ill.) As described by the manufacturer. The single-strand DNA, which contains uracil (U-DNA) was obtained by transforming the E. coli strain CJ236 (Bio-Rad Laboratories, Hercules, CA) with M 13 mpl9 containing IL-4 cDNA. Site-directed mutagenesis used general primaries containing 15 nucleotide homologs to the 5 'U-DNA template at or to the codons intended for mutagenesis, the nucleotides that caused the desired change and also 10 5 nucleotides homologous to the template U- 3 'DNA of the ultimately altered nucleotide. The Arg-121 mutations were introduced to Asp and Tyr-124 to Asp in helix D to the wild-type IL-4 sequence. The uracil DNA for this variant, designated IL-4 [R 121 D / Y 124D] was generated as described above. All mutations generated in these studies were generated using the template IL-4 [R121D Y124D]. 0 The primaries were phosphorylated using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) using the manufacturer's protocol. Then the phosphorylated primary was quenched to the U-DNA template followed by extension with T7 DNA polymerase (Bio-Rad Laboratories, Hercules, CA), as described by the manufacturer. Cells of strain E. coli DH5a '(GibcoBRL, Gaithersburg, MD) were transformed with 5 ml of reaction mixture and plated on "LB medium" with 0.7% agar. After incubation at 37 ° C, the plates were expanded by selecting three individual plates from each mutagenesis reaction and transferring to 2 ml of "LB media" and allowing to proliferate overnight at 37 ° C. Single-stranded DNA was isolated using an M13 purification kit (Qiagen, Inc., Chats vorth, CA) according to the manufacturer's protocol, and clones containing the desired mutein IL-4 mutation of the DNA were identified. replicative formula (double-stranded form of phage M 13), corresponding to plates containing the correct mutated sequence of IL-4, was isolated using the Miniprep plasmid kit from Qiagen (Qiagen, Inc., Chatsworth, CA). The LL-4 mutein DNA was isolated using Bam Hl and Xba I of the replicative purified form of DNA and subcloning to the pFastBac ™ l plasmid vector (GibcoBRL, Gaithersburg, MD). After subcloning, the recombinant baculovirus DNA (hereinafter referred to as Bacmid) was generated by transforming pFastBac ™ l containing the mutein cDNA into the DH lOBac ™ strain of E. coli (GibcoBRL, Gaithersburg, MD) as described by the manufacturer. Muteins were expressed in Spodoptera frugiperda (Sf) 9 cells using Bac-to-Bac% baculovirus expression system (GibcoBRL, Gaithersburg, MD). All incubations in insect cells occurred at 28 ° C. Briefly, cultures of 2 ml of Sf 9 cells were transfected with 5 ml of recombinant Bacmid using CellFECTILN ™ (GibcoBRL, Gaithersburg, MD). The supernatant was harvested 60 hours after transfection and used to infect a 100 to 200 ml culture of 1 x 10 6 Sf 9lm cells in Grace's medium (GibcoBRL, Gaithersburg, MD). Following the manufacturer's protocol, the supernatants were harvested at 48 to 60 hours after infection by centrifugation at 5000 fm for 10 minutes in a Sorvall® RC-5B centrifuge, using a GSA rotor (Dupont Instrument Co., Wilmington, DE) and were subjected to viral titer test (typically> 1 x 108 plaque forming units / ml were obtained). For protein production, 2 to 3 x 106 cells 5/9 / ml were infected in 500 ml of SF900 II medium (GibcoBRL, Gaithersburg, MD), at a multiplicity of infection between 4 and 10 and the supernatant was harvested at 60 to 72 hours after infection by centrifugation at 5000 fm for 10 minutes, in a Sorvall® RC-5B centrifuge, using a GSA rotor (Dupont Instrument Co., Wilmington, DE) and filtered through a control unit. 0.2 mM sterile filter. Example 2. Purification of muteins. Anti-human IL-4 monoclonal anti-viruses 0400.1 and C400.17 were generated using normal protocols generated from mice, using recombinant human IL-4 (Genzyme Diagnostics, Cambridge, MA) as supernatants of Sf 9 cells generated from cell infection. Sf 9 by recombinant baculovirus, containing the respective DL-4 mutein, were loaded onto a 1 ml 5 Sepharose column associated with anti-IL-4 MAb, washed with 100 mM NaHCO 3, 500 mM NaCl, pH 8, 3, washed with water to extract the salt and eluted with eight column volumes of »100 mM glycine, pH 3.0. The fractions were collected in siliconized vials containing 0.1 volume of Tris 1, pH 8.0. The mutein protein was further purified by reverse phase chromatography using a Dynamax®-300? Column. C8 (Rainin Instrument Co., Woburn, 10 MA) with a gradient of 0 to 100% buffer A to B (buffer A, water, buffer B, acetonitrile, 0.1% trifluoroacetic acid). The fractions were evaluated by SDS-PAGE and those containing mutein were lyophilized for storage and resuspended in sterile saline with phosphate buffer for the assays (STF: 10 mM NaP04, 137 mM NaCl, pH 7.6). The mutein ^ purified in this manner was typically a single band as observed by SDS-PAGE 15 (silver stain) and was determined quantitatively by amino acid analysis (with a typical accuracy of> 90%). Example 3. Receptor conjugation tests. In order to determine the effects of substitution on the ability of IL-4 muteins to conjugate with IL-4Ra (Ldzerda, R.L. and colab., Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily [The human interleukin 4 receptor confers capacity of _ biological response and defines a novel receptor superfamily], J. Exp. Med. 171: 861-883 (1990)), a conjugation test of solid phase receptors was developed. Briefly, the affinity of the muteins was measured based on their ability to displace radiolabeled IL-4 from conjugated IL-4Ra with a solid surface. Figure 2 shows the ability of T13D-EL-4 [R 121D Y124D] to compete with I125-IL-4 in a solid-phase conjugation test compared to that of EL-4 [[R121D / Y124D]. The test was formatted using 96-well FlashPlates® plates (DuPont NEN®, Boston, MA) coated with streptavidin; the extracellular domain of EL-4Ra was conjugated with these plaques by virtue of a peptide ear obtained in the extracellular domain of IL-4Ra that is conjugated with streptavidin.

FlashPlates plates contain a scintillation agent impregnated in their plastic material,. samples containing radiolabelled EL-4 and antagonist mutein at E -4 at each hole and incubated Jiasta obtain equilibrium. As the unconjugated radiolabelled compound does not induce a signal! of scintillation, no washing step was necessary before evaluating the amount of conjugate radioactivity in each hole. Accordingly, the measured radioactivity represents the amount of radiolabelled IL-4 conjugated to E -Ra and, when considering the amount of non-radiolabeled aggregated IL-4 antagonist mutein, allows the affinity of the antagonist mutein to non-radiolabelled IL-4 to be calculated by BL-4Ra. An internal standard was generated in each test by measuring the affinity of IL-4 [R121D / Y 124D] in parallel with each mutein. Therefore, specific relative measures of such affinity could be obtained, which allowed the evaluation of the effect of individual substitutions on affinity for IL-4R. The extracellular domain of IL-4Ra was recovered using PCR from an Igtl 1 Jurkat cell collection (Stratagene Cloning Systems, La Jolla, CA). The PCR primaries used to isolate the extracellular domain of LL-4Ra were, for the 5 'end of the open reading frame of IL-4Ra, 'GGC ATG GAT CCA TGG GGT GGC TTT GCT CTG G 3' (SEQ ID NO: 4); and for the 3 'end of the extracellular domain of EL-4Ra, 'AAG CCG CTA GCG CTG TGC TGC TCG AAG GGC 3' (SEQ ID NOS).

The CPR conditions used were one minute at 94 ° C, one minute at 65.8 ° C and one minute at 72 ° C for 30 cycles. The resulting PCR product was digested with the restriction enzymes BamHl and £ co47III and subcloned into a pBluescript® vector (Stratagene Cloning Systems, La Jolla, CA) containing DNA with codons corresponding to the Ser-Ala-Tf sequence. -Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 6) digested with the same enzymes. It has been reported that this sequence is conjugated with streptavidin (Schmidt TG and Skerra, A., The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the a functional FIV fragment Ig), Protein Eng, 6 ( 1): 109-122 (1992)). The extracellular domain of IL-4R generated in this way, encoded the peptide sequence Ser-Ala-Tf-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 6) at the C-terminus of the extracellular domain and it was designated sDL-4Ra-STX (Met in position 1 is the N-terminus of mature EL-4Ra) and is SEQ ID NO: 7.

The sIL-4Ra-STX protein was produced using the baculovirus system in a manner identical to that used for the IL-4 antagonist muteins, purified by affinity chromatography with a matrix associated with IL-4 as described (Kruse N et al. .Two distinct functional sites of human interleukin 4 are identified by variants impaired in either receptor binding or receptor activation [Two distinctive functional sites of human interleukin 4 are identified by impaired variants in conjugation with receptors or in receptor activation], EMBO J., 12 (13): 5121-9 (1993)), and stored in STF. The EL-4 matrix was generated by associating the IL-4 produced in E. coli with Sepharose 4B CNBr, as described by the manufacturer (Pharmacia, Uppsala, Sweden). Streptavidin-coated FlashPlates® plates (DuPont NEN®, Boston, MA) were coated with 100 ml of 2 mg / ml sDL-4Ra-STX, in 100 mM Tris, 0.1% ASB, pH 7.0 per 2 hours at 20 ° C, washed with STF, 0.1% ASB, pH 7.6, and incubated with 200 pM I 125-IL-4 (DuPont NEN®, Boston, MA) and varying concentrations of antagonist mutein EL-4 in 100 ml of STF, 0.1% ASB, pH 7.6, for 1.5 hours at 20 ° C, in quadruplicate. The tests were repeated at least twice. As an internal reference, BL-4 [R121D and 124D] was titrated in parallel with each mutein in the same FlashPlate® plate. Conjugated radioactivity was measured in a TopCount® scintillation counter (Packard Instrt Co., Meriden, CT) and the values of j were calculated using the ligand program (Munson, PJ and Rodbard, D., Computerized analysis of ligand binding data [Computer analysis of ligand conjugation data], Meth.Enzymol., 92: 543-576 (1983)); the error in the values of Kj. expressed as% CV, fell between 2 and 20%. The results were expressed as the ratio of Kj of mutein / Ka of IL-4 [R121D / Y124D], using data acquired from the interior of each test plate. The differences between the measured ICj values reflect a relative increase in the affinity of the respective mutein for EL-4Ra (ie, I j for mutein / Kj for IL-4 [R121D Y124D] < 1) or a relative decrease in the affinity of the respective mutein for lL-4Ra (ie, mutein Kj / KLj for LL-4 [R121 D / Y124D] > 1). using Ficoll-Paque Plus (Pharmacia, Uppsala, Sweden) essentially as described by Kruse, N .; Tony, H.P. and Sebald, W. Conversion of human interleukin-4 into a high affinity antigonist by a single amino acid replacement [Conversion of human interleukin-4 to a high affinity antagonist, by a single amino acid substitution], Embo J. 11: 3237 -44 (1992). Peripheral blood mononuclear cells, purified, were incubated for 7 days with 10 mg / ml of phytohaemagglutinin (Sigma Chemical Co., St. Louis, MO), harvested by centrifugation and washed in RPMI 1640 medium (GibcoBRL, Gaithersburg, MD). 5 x 104 pit activated T cells (PHA blasts) were incubated with varying amounts of EL-4 or mutein in RPMI 1640 medium containing 10% fetal bovine serum, 10 mM HEPES, pH 7.5, L-glutamine 2 mM, 100 units of penicillin G / ml and 100 mg of streptomycin sulfate ml in 96-well plates for 48 hours at 37 ° C; were pulsed with 1 m3 H3-thymidine (DuPont NEN®, Boston, MAVhoyo for 6 hours, harvested and radioactivity was measured in a TopCount® scintillation counter (Packard Instrument Co., Meriden, CT) Example 5. Effect of alanine substitution on the affinity of IL-4 for IL-4Ra The effects of alanine substitution of residues exposed to the surface of helices A and C of DL-4, on the affinity of IL-4 by D -4Ra, are presented in Table I.

Table I. Results of Alanine Screening of Residues Exposed on the Surface of Helices A and C of EL-4 * * All mutations are superimposed on the spine of IL-4 [R 121 D / Y 124D] 4 ^ ^ Since alanine substitution does not generally cause structural changes in the fold of the protein, the effects on activity, in this case affinity, can be attributed to the loss of the substituted side chain (Cunningham, BC, Wells, JA, High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis [Mapping of high-resolution epitopes, of interactions with hGH receptors, by alanine scanning mutagenesis], Science 244 (4908): 1081-5 (1989 )). The nominal changes in affinity (for example, approximately double) that are obtained as a consequence of the substitution of alanine, suggest that the residue substituted position are not involved in the interaction; the extensive effects (for example, more than 100 times) suggest that the residue / position replaced ^^ are critical for interaction (Lowman HB et al., Selecting high-affinity binding proteins by monovalent phage display [Selection of high-affinity conjugating proteins by monovalent phage display], Biochemistry 30 (45): 10832-8 (1991 )). Lowman and colab., (ibid) found that residues exhibiting intermediate effects in conjugation, as a consequence of alanine substitution were involved in the interaction studied, in addition to the most sensitive residues. By applying these findings to the results of this alanine scan to propellers A and C of EL-4, the most critical residues for the interaction of IL-4 with IL-4Ra are Glu-9 and Arg-88; The intermediate residues would include De-5 > Asn 89 > Lys-84 - Arg-81 > Thr-13-GIn-78-Arg-85-Lys-77; residues probably not involved in the interaction include Gln-8, Ile-1 1, BL-4 and LTL-4Ra. These results define a probable conjugation surface of IL-4 for D -4Ro, which cμand? it is considered in the context of the structure of EL-4 (Smith LJ et al., Human interleukin 4. The solution structure of a four-helix bundle protein [Solution of the structure of a four-bundle protein] propellers], J. Mol. Biol., 224 (4): 899-904 (1992)) comprises adjacent portions of the helices A and C of IL-4 * and extends for about three helical turns in each helix, i.e. , positions 5 to 16 in helix A and 77 to 89 in C. The residues most likely involved in contact with the alpha receptor of IL-4 are, in helix A: De-5, Glu-9, Thr - 13 and Ser- 16; in the C helix: Lys-77, Gln-78, Arg-81, Lys-84, Arg-85, Arg-88 and Asn-89. Structurally, the critical center of interaction seems to be composed of residues Glu-9, Arg-88 and Asn-89 and the observed alanin effect generally decreases as one moves away from this portion of the molecule. Accordingly, this analysis describes a conjugation surface of LL-4 for IL-4Ra. Example 6. Replacement muteins in selected positions. In a previous analysis of growth hormone, substitutions were found for residues that, when substituted with alanine, have an intermediate effect on affinity and that were shown to improve the affinity of growth hormone for its receptor (Lowman HB et al., Selecting high-affinity binding proteins by monovalent phage display [Selection of high affinity conjugating proteins by monovalent phage display], Biochemistry 30 (45): 10832-8 (1991)). It was shown that the nature of the substitution of a given residue could not be foreseen, resulting in a better affinity. Therefore, in the analysis presented here, all the substitutions that did not have inherent structural effects (for example, exclusive of Cys, Gly, Pro) or that passed through easy oxidation reactions (for example, exclusive of Met), were introduced in the Target positions: Table II. Waste addressees and their substitutions * . Therefore, cysteine, glycine, methionine and proline were excluded from this substitution analysis. Residues were chosen to continue the analysis if, when substituted with alanine, they showed a reduction of 5 to 80 times in the affinity or any increase in it; _ ^ these limits were chosen based on the results obtained by Lowman and colab. (ibid.) In addition Ser-16 was selected to continue the analysis due to the observed increase in affinity that resulted from alanine substitution, which suggested that other substitutions in this position could also yield improvements in such affinity. Example 7. Substitutions that gave improvement in affinity for IL-4Rcx. Ten muteins were generated containing substitutions at individual positions of helices A and C in combination with spine 1L-41R121D / Y124D]; Competitive conjugation tests were performed such that each mutein was compared in parallel with EL-4 [R121D / Y124D], which allowed direct comparison to be made and, consequently, to reach conclusions about the effect of each substitution on affinity of the specific mutein by EL-4Ra. 15 All substitutions that gave a better affinity are presented in Table IE. The ratio "Kd of IL-4 [R121D / Y124D] KLd of mutein" indicates the relative increase in affinity observed as a consequence of each substitution.

Table III The majority of the substitutions were deleterious or did not exert an effect on affinity for DL-4Ra (the data are not presented). However, several substitutions improved the affinity, the most notable being that of Thr-13 to Asp, which led to a surprising 18-fold increase in affinity for IL-4Ra (Figure 2). The amino acid Ser-16 was unique in this analysis since most of the substitutions gave rise to modest increases in affinity. ^ B similar competitive conjugation frames were obtained for the other muteins in relation to their relative affinity (no data presented). The improvements in affinity are relative to the mother protein IL-4 [R121D / Y124D]. It is expected that the combination of these substitutions in a protein could cause combined increases in affinity; for example, [T13D N89I] -IL-4 [R 121 D Y124D] could produce a mutein with an affinity 36 times greater than IL-4 [R 121 D / Y 124D]. The residues Thr-13 and Ser-16 gave the greatest improvements in affinity when it was identified ^^ the appropriate substitution, suggesting that other residues that, when substituted with alanine, give similar effects, such as mutein TBA or S 16A (decrease of 6.4 times and increase of 2.5 times, respectively) would also probably yield IL-4 variants with higher affinity when substituted appropriately. For the current series of alanine-substituted residues, this would include: Ile-1 1, Lys-77, Gln-78, Lys-84 and Arg-85. In the case of growth hormone, which has been studied extensively, the 0 individual substitutions that would give increases in affinity were of the order of 1, 5 to 5 times (Lowman HB; Wells JA, Affinity maturation of human growth hormone by monovalent phage display [Affinity maturation of human growth hormone by monovalent phage display], J. Mol. Biol 234 (3): 564-78 (1993) ). However, substitutions that have -, J.Biol.Chem. 270 (40): 23754-60 (1995)) or affinity (human ciliary neurotrophic factor [FNTC] (Saggio I et al., CNTF variants with increased biological potency and receptor selectivity 5 defines a functional site of receptor interaction [The variants FNTC with biological potency and ^^ selectivity of larger receptors, define a functional site of interaction with receptors], EMBO J. 14 (13): 3045-54 (1995)) have recently been identified. For IL-3, a mutation increased biological activity in vitro approximately 26-fold; for FNTC, a single substitution increased the affinity approximately 32 fold. For other cytokines in the In the literature, most substitutions generally resulted in absence of affinity / activity loss or activity. Therefore, the absolute effect of any given substitution on affinity and / or activity is unpredictable. Example 8. Effect of T13D substitution on biological activity. The IL-4 antagonist mutein, IL-4 [R121D / Y124D] is an antagonist of EL-4 (Tony HP, Shen BJ, Reusch P and Sebald W, Design of human interleukin-4 antagonists inhibiting interIeukin-4-dependent and interleukin-13-dependent responses in T-cells and B-cells with high efficiency [Design of human interleukin 4 antagonists that inhibit the interleukin-4 dependent and interleukin-13 dependent responses in T and B cells, with great efficiency] , Eur. J. Biochem 225 (2): 659-65 (1994)), which was used as the 'basal' peptide in this study due to its inability to stimulate EL-4 activities. It is believed that this mutein is antagonistic by virtue of its ability to conjugate LDL-4Ra but not implicate gc in a competent manner of signaling, thus blocking EL-4 to conjugate with its complex. ^ like receptors. Accordingly, the biological effects of IL-4 [R 121 D and 124D] (antagonism to IL-4) are isolated to their interactions, measured by affinity, with D-4Ra. In order to demonstrate that affinity for receptors is related to biological potency, the mutein T13D-EL-4 [R121D / Y124D] was evaluated for its ability to inhibit the proliferation of PHA blasts induced by IL-4 (Figure 3) . The observed IC50 (concentration at which 50% inhibition is observed) in relation to IL-4 [R121D Y124D], proved to be approximately proportional to the relative change observed in receptor affinity. In conclusion, although it is conjugated with higher affinity to IL-4Ra, T13D-IL-4 [R121D and 124D] remains an IL-4 antagonist. The IC50 for T13D-1L-4 [R121D / Y124D] versus IL-4 [R121D and 124D] is approximately proportional to the values of the ICj of EL-4 [R121D / Y124D] (0.28 nM versus 5 , 0 nM, respectively); the IC50 of T13D-IL-4 [R12ID Y124D] is approximately 5 to 10 times smaller than the IC50 of IL-5 4 [R121D.124D] (2 nM versus 13 nM, respectively). The numerical differences ^^ Specific in the relative effect may be a consequence of the particular conditions of each test: 1, 5 hours of incubation for the solid phase conjugation test at 20 ° C versus 48 hours of incubation at 37 ° C for the test of proliferation. The ability of other muteins evaluated in this study to compete with EL-4 in biological tests was also proportional to its K relative to 1L-4 [R121D? '124D] (no data presented). These results indicate that conjugation to EL-4Ra is an event that can be separated from activation of the EL-4 receptor; this activation requires the heterodimerization of EL-4Ra and of at least one other subunit (for example gc). Consequently, the modification to IL-4 in the helices ^ A and C modulate the affinity of EL-4 for IL-4R and does so proportionally to the ability of said mutein to antagonize IL-4 in a biological context. This affinity effect must also be translated, by virtue of the mechanism of interaction of EL-4 with its receptor, in an increase in the potency of the agonist peptides derived from D -4. The theories of this invention can also be adapted to other cytokines. The most obvious goal is EL-13 since the IL-13 receptor complex also uses IL-4Ra (Zurawski S.M. colab., The primary binding subunit of the human interleukin-4 receptor is also a component of the interleukin-13 receptor [The primary conjugating subunit of the human inlterleuquina-4 receptor is also a component of the interleukin-13 receptor], J Biol. Chem. 270: 13869-78 (1995)). Therefore, by mutating the helices A and C of BL-13 to more closely resemble those of EL-4 an increase in the conjugating affinity should be obtained by EL-25 4Ra. An alignment of the two interleukins allows the identification of positions that would be analogous to a target mutation site, for example, Thr 13 in EL-4. The conjugating surfaces of the two interleukins are compared in the EV Table below.

Waste positions Sequence Helix A: hIL-4 5-17 ... ITLQEIIKTLN2L ... hIL-13 4-16 ... TALRELIEELVNI ...

Propeller C: hIL-4 74-91 ... HKQLIRFLKRLDRNLW ... hIL-13 59-74 ... TQRML2GFCPHKV2AG ... * Alignment taken from: Bamborough, P., Duncan, D. and Richards, WG, Predictive Modeling of the 3-D Structure of Interleukin-13 [Predictive modeling of the 3-dimensional structure of interleukin-13], Protein Engineering 7: 1007-82 (1994).

The most critical residues of EL-4, mediators of the interactions with EL-4Ra, ^^ identified by the alanine exploration, Glu-9 and Arg-88, are presented in bold letters, as well as the corresponding residues in DL-13 based on the alignment of this sequence. How I know mentioned previously, IL-13 uses the IL-4Ra chain in its receptor complex (Zurawski S.M. et al., Before). Accordingly, the change of helices A and C of IL-13 to more closely resemble those of EL-4 should cause an increase in the conjugating affinity for IL-4Ra.

In addition, the substitution of positionally equivalent EL-13 residues, with residues that have been shown to increase the affinity of IL-4 for IL-4R (the positions are indicated with double underlining for EL-4 and EL-13), should also result in a greater affinity of EL- 13 for ^^ its complex receiver and, therefore, to a better power. Sequences The following biological sequences are contained herein: SEQ ID NO: 1: amino acid sequence, mature human IL-4; SEQ ED NO: 2: nucleotide sequence, primary PCR; 20 SEQ ED NO: 3: nucleotide sequence, primary PCR; SEQ ED NO: 4: nucleotide sequence, primary PCR; SEQ ED NO: 5: nucleotide sequence, primary PCR; SEQ ED NO: 6: amino acid sequence of the peptide ear for streptavidin: SEQ ID NO: 7: amino acid sequence of sIL-4Ra-STX; :: secuenca e amno c os, nuce t os e - - 1 1. Other embodiments of the invention will be apparent to individuals with experience in the technology. The concept and experimental approach described here should be applicable to other cytokines that use heterologous multimer receptor systems, in particular EL-2 and related cytokines (eg, EL-7, EL-9, IL-10, IL-13 and IL). -15), interferon alfa and interferon gamma. (1. GENERAL INFORMATION: (i) APPLICANTS: Shanafelt, Armen; Greve, Jeffrey; Roczniak, Steven (ü) 'TITLE OF THE INVENTION: High affinity IL-4 muteins (iii) SEQUENCE NUMBER: 9 (iv) ADDRESS TO SEND CORRESPONDENCE: (A) RECIPIENT: Bayer Coforation, Pharmaceutical Division (B) STREET : 400 Morgan Lane (C) CITY: West Haven (D) STATE: Connecticut (E) COUNTRY: United States of America (F) POSTAL CODE: 06 16-4175 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Diskette, 3.5 inches, 1, 44 Mb storage (B) COMPUTER: Compatible with EBM CP (C) OPERATING SYSTEM: PC-DOS v.6.30 (D) PROGRAM: Word for Windows 6.0 (vi) CURRENT REQUEST DATA: (A) REQUEST NUMBER: 08 / 687.803 ( B) SUBMISSION DATE: JULY 19, 1996 (vii) ATTORNEY / AGENT INFORMATION: (A) NAME: Huw R. Jones (B) REGISTRATION NUMBER: 33,916 (C) REFERENCE NUMBER / REWARD: WH5020 (viii) INFORMATION ABOUT TELECOMMUNICATIONS: (A) PHONE NUMBER: (203) 812-23 17 (B) TELEFAX: (203) 812-5492 (2) INFORMATION FOR NO. SEQUENCE IDENTIFICATION: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 129 (B) TYPE: amino acid (C) FTLAMENT: a cord (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (A) ) DESCRIPTION: human interleukin-4 protein (iii) HYPOTHETICAL: no (iv) ANTISENTED: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1: His Lys Cys Asp lie Thr Leu Gln Glu lie lie Lys Thr Leu Asn 1 5 10 15 Ser Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr 20 25 t 30 Asp l ie Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe 35 40 45 Cys Arg Wing Wing Thr Val Leu Arg Gln Phe Tyr Ser His His Glu 50 55 60 Lys Asp Thr Arg Cys Leu Gly Wing Thr Wing Gln Gln Phe His Arg 65 70 75 Trp Gly Leu Wing Gly Leu Asn Ser Cys Pro Val Lys Glu Ala Asn 95 100 105 Gln Ser Thr Leu Glu Asn Phe Leu Glu Arg Leu Lys Thr lie Met 110 115 120 Arg Gla Lys Tyr Ser Lys Cys Ser Ser 125 (2) INFORMATION FOR NO. SEQUENCE IDENTIFICATION: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 (B) TYPE: nucleic acid (C) FILAMENT: a bead (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA ( A) DESCRIPTION: primary CPR 5 ', IL-4 (iii) HYPOTHETICAL: no (iv) ALNOSTENTTDO: no (xi) DESCRIPTION OF THE SEQUENCE: SEQ ED NO. 2: CGCGGATCCA TGGGTCTCAC CTCC 24 (2) INFORMATION FOR NO. SEQUENCE IDENTIFICATION: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 (B) TYPE: nucleic acid (C) FILAMENT: a cord (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA ( A) DESCRIPTION: CPR primary 3 ', LTL-4 (iii) HYPOTHETICAL: no (iv) ANTI-STIMULUS: no (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO. 3: CGCTCTAGAC TAGCTCGAAC ACTTTGAAT 29 (2) INFORMATION FOR NO. SEQUENCE IDENTIFICATION: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 (B) TYPE: nucleic acid (C) FILAMENT: a cord (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA ( A) DESCRIPTION: primary CPR 5 ', EL-4Ra (ED) (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO. 4: . GGCATGGATC CATGGGGTGG CTTTGCTCTG G 31 (2) INFORMATION FOR NO. SEQUENCE IDENTIFICATION: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 (B) TYPE: nucleic acid (C) FILAMENT: a string (iii) HYPOTHETICAL: no (iv) ANTI-SUIT: no (xi) DESCRIPTION OF THE SEQUENCE: SEQ ED NO. 5: AAGCCGCT AG CGCTGTGCTG CTCGAAGGGC 30 (2) INFORMATION FOR NO. SEQUENCE IDENTIFICATION: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 (B) TYPE: amino acid (C) FTLAMENTOSITY: a cord (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (A) ) DESCRIPTION: ear for streptavidin (iii) HYPOTHETICAL: no (iv) ANTISENTT: no (xi) SEQUENCE DESCREPTION: SEQ ID NO. 6: Ser Ala Tf Arg His Pro Gln Phe Gly Gly 10 1 5 10 (2) INFORMATION FOR NO. SEQUENCE IDENTIFICATION: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 197 (B) TYPE: amino acid (C) F LAMENT: a cord (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ( A) DESCRIPTION: sIL-4Ra-STX (iii) HYPOTHETICAL: no (iv) ANTISENTED: no (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 7: Met Lys Val Leu Gln Glu Pro Thr Cys Val Ser Asp Tyr Met Ser 1 5 10 15 lie Ser Thr Cys Glu Trp Lys Met Asn Gly Pro Thr Asn Cys Ser 20 25 30 Thr Glu Leu Arg Leu Gly Wing Gly Cys Val Cys His Leu Leu Met 35 40 45 Asp Asp Val Val Ser Wing Asp Asn Tyr. hr Leu Asp Leu Trp Wing 50 55 60 Gly Gln Gln Leu Leu Trp Lys Gly Ser Phe Lys Pro Ser Glu His 65 70 75 Val Lys Pro Arg Wing Pro Gly Asn Leu Thr Val His Thr Asn Val 80 85 90 Be Asp Thr Leu Leu Leu Thr Trp Ser Asn Pro Tyr Pro Pro Asp 95 100 105 Asn Tyr Leu Tyr Asn His Leu Thr Tyr Wing Val Asn l ie Trp Ser 110 115 120 or 140 145 150 Ser Tyr Arg Wing Arg Val Arg Wing Trp Wing Gln Cys Tyr Asn Thr 155 160 165 Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser Tyr 170 175 180 Arg Glu Pro Phe Glu Gln His Ser Wing Trp Arg His Pro Gln Phe 185 190 - 195 Gly Gly 197 (2) INFORMATION FOR NO. SEQUENCE IDENTIFICATION: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 462 (B) TYPE: nucleic acid (C) FELAMENTOSITY: a cord (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA ( A) DESCRIPTION: IL-4 / T13D (iü) 1 HIPO 'THETICS: no (iv) ANTISENTED: no (xi) DESCRIPTION OF SEC: uE ATG GGT CTC ACC CC CAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGT GCC GGC AAC TT GTC CAC GGA CAC AAG TGC GAT ATC ACC 90 Ala Cys Ala Gly Asn Phe Val His Gly His Lys Cys Asp He Thr 20 25 30 TTA CAG GAG ATC ATC AAA GAT TTG AAC AGC CTC ACÁ GAG CAG AAG 135 Leu Gln Glu He He Lys Asp Leu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTA ACÁ GAC ATC TTT GCT GCC TCC 180 Thr Glu Leu Thr Val Thr Asp He Phe Wing Wing Ser 50 55 60 AAG AAC ACA ACT GAG AAG GAA ACC TC TGC AGG GCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala Wing Thr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGC CTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 80 85 90 GGT GCG ACT GCA CAG CAG TC CAC AGG CAC AAG CAG CTG, ATC CGA 315 Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu lie Arg 95 100 105 AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 405 Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGG CTA AAG ACG ATC ATG AGA GAG AAA TAT TCA AAG 450 Phe Leu Glu Arg Leu Lys Thr He Met Arg Glu Lys Tyr Ser Lys 140 145 150 TGT TCG AGC TAG_462_Cys Ser Ser End (2) INFORMATION FOR NO. SEQUENCE IDENTIFICATION: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 462 (B) TYPE: nucleic acid (C) FILAMENT: a string (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA ( A) DESCRIPTION: IL-4 / T13D [R121D / Y124D] (iii ') i HYPOTHETICAL: no (iv) > ANTI-SENSE: no (xi) i DESCRIPTION OF THE SEC IUE ATG GGT CTC ACC TCC CAA CTG CTT CCC CCT CTG TTC TTC CTG CTA 45 Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 GCA TGT GCC GGC AAC TT GTC CAC GGA CAC AAG TGC GAT ATC ACC '90 Ala Cys Ala Gly Asn Phe Val His Gly His Lys Cys Asp He Thr 20 25 30 TTA CAG GAG ATC ATC AAA GAT TTG AAC AGC CTC ACÁ GAG CAG AAG 135 Leu Gln Glu He He Lys Asp Leu Asn Ser Leu Thr Glu Gln Lys 35 40 45 ACT CTG TGC ACC GAG TTG ACC GTA ACÁ GAC ATC TTT GCT GCC TCC 180 Thr Leu Cys Thr Glu Leu Thr Val Thr Asp He Phe Wing Wing Ser 50 55 60 AAG AAC AC ACT GAG AAG GAA ACC TTC TGC AGG GCT GCG ACT GTG 225 Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala Wing Thr Val 65 70 75 CTC CGG CAG TTC TAC AGC CAC CAT GAG AAG GAC ACT CGC TGC CTG 270 Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu 80 85 90 GGT GCG ACT GCA CAG CAG TTC CAC AGG CAC AAG CAG CTG ATC CGA 315 Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu He Arg 95 100 '105 AAT TCC TGT CCT GTG AAG GAA GCC AAC CAG AGT ACG TTG GAA AAC 4OS Asn Ser Cys Pro Val Lys Glu Wing Asn Gln Ser Thr Leu Glu Asn 125 130 135 TTC TTG GAA AGG CTA AAG ACG ATC ATG GAC GAG AAA GAC TCA AAG 459 Phe Leu. Glu Arg Leu Lys Thr He Met Asp Glu Lys Asp Ser Lys 140 145 150 TGT TCG AGC TAG_462_Cys Be Ser End

Claims (8)

1. A recombinant human IL-4 mutein, numbered according to wild-type IL-4, said mutein comprises at least one amino acid substitution at the conjugation surface of alpha A or C helices of said wild-type EL-4, by which said mutein is conjugated with the IL-4Ra receptor with affinity at least greater than the native EL-4.

2. The recombinant human EL-4 mutein of claim 1, wherein said substitution is selected from the group of positions comprising, in helix A, positions 13 and 16 and in helix C, positions 81 and 89.

3. The recombinant human EL-4 mutein of claim 2, wherein said substitution at position 13 is Thr to Asp.

4. A pharmaceutical composition comprising the recombinant human DL-4 mutein of claim 1 in combination with a pharmaceutically acceptable carrier.

5. An amino acid sequence encoding the recombinant human IL-4 mutein of claim 1.

6. The recombinant human IL-4 mutein of claim 1 encoded by the amino acid sequence of SEQ ED NO: 8.

7, A purified and isolated polynucleotide sequence encoding the recombinant human EL-4 mutein of claim 1.

8. A host cell transformed with the purified and isolated polynucleotide sequence of claim 7. . 10. A transformed host cell capable of expressing the recombinant human JL-4 mutein of claim 9. 1. A method of treating a human in need thereof, comprising administering a pharmaceutically effective amount of the composition of claim 4. 12. A test for determining the ability of a mutein to be conjugated with a receptor, comprising the steps of: (a) introducing into a streptavidin-coated FlashPlate® plate, the conjugating portion of a receptor chain; said conjugating portion of a receptor chain having a kappa peptide capable of being conjugated by streptavidin; (b) introducing into said FlashPlate plate a radiolabelled native ligand with affinity for said conjugating portion of a receptor chain; (C) introducing into said FlashPlate plate a muteinic ligand with affinity for said conjugating portion of a receptor chain; (d) measuring the amount of signal emitted by the FlashPlate plate after allowing it to equilibrate, and ^^ (e) calculating the relative affinity of the muteinic ligand against the native ligand. 13. The method of claim 12 wherein said receptor chain is DL-4Ra. 14. The method of claim 12 wherein said peptide ear comprises SEQ ID NO: 6 or a degenerate variant thereof. 15. The method of claim 12 wherein said peptide ear has been extracted from the receptor chain and the receptor chain has been biotinylated. 17. A recombinant human IL-4 antagonist mutein, numbered according to wild type IL-4, wherein said mutein comprises: (a) substitutions R121D and Y 124D in helix D of said wild-type IL-4, and (b) al minus an amino acid substitution at the conjugation surface of the alpha A or C helices of said wild-type IL-4, wherein said mutein is conjugated to the EL-4Ra receptor with at least one greater affinity than the native EL-4. 18. The recombinant human IL-4 antagonist mutein of claim 17, wherein said substitution is selected from the group of positions comprising, in helix A, positions 13 and 16 and in helix C, positions 81 and 89 . 19. The recombinant human IL-4 antagonist mutein of claim 18 wherein said substitution at position 13 is Thr to Asp. 20. A pharmaceutical composition comprising the recombinant human EL-4 antagonist mutein of claim 17, in combination with a pharmaceutically acceptable carrier. 21. An amino acid sequence encoding the recombinant human DL-4 antagonist mutein of claim 17. 22. The recombinant human EL-4 antagonist mutein of claim 17 encoded by the amino acid sequence of SEQ ID NO: 9. 23. A purified and isolated polynucleotide sequence encoding the recombinant human EL-4 antagonist mutein of claim 17. . 25. L-a recombinant human IL-4 mutein of claim 19, encoded by the DNA sequence of SEQ ED NO: 9 or a stably hybridizable variant thereof. 26. A transformed host cell capable of expressing the recombinant human IL-4 mutein of claim 26. 27. A method for treating a human in need thereof, comprising administering a pharmaceutically effective amount of the composition of claim 20. A recombinant human IL-4 mutein, numbered according to wild-type IL-4, in which the mutein comprises at least one amino acid substitution at the conjugation surface of alpha-helices A or C of wild-type EL-4, by which mutein is conjugated to the IL-4Ra receptor with at least greater affinity than the native IL-4. The substitution is preferably selected from the group of positions comprising, in helix A, positions 13 and 16 and in the helix C, positions 81 and 89. A more preferred embodiment is the recombinant human EL-4 mutein in which the substitution at position 13 is Thr to Asp. Also described are pharmaceutical compositions, amino acid and polynucleotide sequences that encode muteins, transformed host cells, anti-muteins and therapeutic methods.