CA1340761C - Interleukin-4-receptors - Google Patents

Abstract

Mammalian Interleukin-4 receptor proteins, DNAs and expression vectors encoding mammalian IL-4 receptors, and processes for producing mammalian IL-4 receptors as products of cell culture, are disclosed.

Description

BACKGROUND OF THE INVENTION
The present invention relates generally to cytokine receptors and, more specifically, to Interleukin-4 receptors.
Interleulcin-4 (IL-4, also known as B cell stimulating factor, or BSF-1) Haas originally characterized by its ability to stimulate the prol:Lferation of B cells in response to low concentrations of <~ntibo~dies directed to surface immunoglobulin.
More recently, IL-~E has lbeen shown to possess a far broader spectrum of biological a~~tivities, including growth co-stimulation of T cells, mast cells, granulocytes, megakaryocytes, and erythrocytes. In addition, IL-4 stimulates the proliferation of several IL-2 and Ih-3 dependent cell lines, induces the expression of class II major histor.ompatibility complex molecules on resting B cells, and enhances the secretion of IgE and IgGi isotypes by stimulated B cells, Both murine and human IL-4 have been definitively characterized by recombinant DNA technology and by purification to honnogene:ity of the natural murine protein (Yokota, et al., Proc. Natl.Acad.Sci. USA 83:5894, 1986; Noma, et al., Nature 319:640, 19E36; and Grabstein, et al., J. Exp.Med. 163:1405, 1986).
The biological activities of IL-4 are mediated by specific cell surface receptors for IL-4 which are expressed on primary cells and in vitro cell lines of mammalian origin. IL-4 binds to the receptor, which then transducer a biological signal to various immune e;ffector cells. Purified IL-4 receptor (IL-4R) compositions will therefore be useful in diagnostic assays for ..
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13~O~1~i IL-4 or IL-4 receptor, and in raising antibodies to IL-4 receptor for use in diagnosis or therapy. In addition, purified IL-4 receptor compositions may be used directly in therapy to bind or scavenge IL-4, providing a means for regulating the biological activities of the cytokine.
Although IL-4 has been extensively characterized, little progress has been made in characterizing its receptor. Numerous studies documentin~~ the existence of an IL-4 receptor on a wide range of cell types have been published; however, structural characterization his been limited to estimates of the molecular weight of the protE~in as determined by SDS-PAGE analysis of covalent complexes formed by chemical cross-linking between the receptor and radio:labele~d IL-4 molecules. Ohara, et al., (Nature 325:537, 1987) and Park, et al. (Proc.Natl.Acad.Sci. USA 84:1669, 1987) first establ,Lshed 'the presence of an IL-4 receptor using radioiodinated recombinant murine IL-4 to bind a high affinity receptor expressed in l.ow numbers on B and T lymphocytes and a wide range of cell:> of tlhe haematopoietic lineage. By affinity cross-linking 125I__IL-4 to IL-4R, Ohara, et al. and Park, et al.
identified receptor proteins having apparent molecular weights of 60,000 and 75,000 c~alton~s, respectively. It is possible that the small receptor sizes observed on the murine cells represents a proteolytically cleaved :Fragment of the native receptor.
Subseguent experiments by Park, et al. (J.
- 1a -t ~t6~~r~~~
Exp. Med. 166:476, 1987) using yeast-derived recombinant human IL-4 radiolabeled with 1251 showed that human IL-4 receptor is present not only on ~ce~ll lines of B, T, and hematopoietic cell lineages, but is also found on human fibroblasts and cells of epithelial and endothelial origin. IL-4 receptors have since been shown to be present on other cell lines, including CBA/N
splenic H cells (Nakajima et al., J. Immunol. 139:774, 1987), Burkitt lymphoma Jijoye cells (Cabrillat et al., Bjochem. &
Biophys. Res. Commun. 149:995, 1987) a wide variety of hemopoietic and nonhe~riopoietic cells (Lowenthal et al., J. Immunol. 140:456, 1988) and murine Lyt-2 /L3T4 thymocytes.
More recently, Park et al. (UCLA Symposia, J. Cell B.tol., Suppl. 12A, 1988) reported that, in the presence of sufficient protease inhibitors, 1251-IL-4-binding plasma membrane receptors of 138-145 k.Da could be identified on several murine cell lines. Considerable controversy thus remains regarding the actual size and structure of IL-4 receptors.
Further study of the structure and biological characteristics of IL-4 receptors and the role played by IL-4 receptors in the responses of various cell populations to IL-4 or other cytokine stimulation, or of the methods of using IL-4 receptors effectively in therapy, diagnosis, or assay, has not been possible because of the difficulty in obtaining sufficient quantities of purified .IL-4 receptor. No cell lines have previously been known to express high levels of IL-4 receptors constitutively and continuously, and in cell lines known to express detectable levels of IL-4 receptor, the _.

~~~~"~~ 1 level of expression i:~ generally limited to less than about 2000 receptors per cell. Thus, efforts to purify the IL-4 receptor molecule for use in biochemical analysis or to clone and express mammalian genes encoding IL-4 receptor have been impeded by lack of purified receptor and a suitable source of receptor mRNA.
SUMMARY OF THE INVENTION
The present invention provides DNA sequences encoding mammalian Interleukin-4 receptors (IL-4R) or subunits thereof. Preferably, such DNA sequences are selected from the group consisting of: (a) cDNA clones having a nucleotide sequence derived from the coding region of a native IL-4R
gene; (b) DNA sequences capable of hybridization to the cDNA
clones of (a) under moderately stringent conditions and which encode biologically active IL-4R molecules; and (c) DNA
sequences which are degenerate, as a result of the genetic code, to the DNA sequences defined in (a) and (b) and which encode biologically ,active IL-4R molecules. The present invention also provides recombinant expression vectors comprising the DNA sequences defined above, recombinant IL-4R
molecules produced using the recombinant expression vectors, and processes for producing the recombinant IL-4R molecules using the expression vectors. Such an expression vector may comprise (1) such a synthetic or cDNA sequence, operably linked to (2) a transcriptional and t ranslational regulatory element derived from a mammalian, microbial, viral or insect gene, said regulatory element including (a) a transcriptional 2a ~~t~~ ~~1 promoter, (b) a sequence encoding suitable mRNA ribosomal binding site andL ( c ) :sequences which cont rol the terminat ion of t ransc ript ion. and t; rans lat ion .
The present invention also provides substantially homogeneous protein compositions comprising mammalian IL-4R.
The full length murinE~ molecule is a glycoprotein having a molecular weight of about 130,000 to about 140,000 Mr by SDS-PAGE. The apparent binding affinity (Ka) for COS
cells transfected with murine IL-4 receptor clones 16 and 18 from the CTLL 19.4 library is 1 to 8 x 109 M 1. The Ka for COS cells transfected with murine IL-4 receptor clones 7B9-2 and 7B9-4 from the murine 7B9 library is 2 x 109 to 1 x lOlOM 1. The mature murine IL-4 receptor molecule has an N-terminal amino acid sequence as follows:
I K V L G E P T C F S D Y I R T S T C E W.
2b ~3~~~~b~.
The human IL-4R mol~scule is believed to have a molecular weight of between about 110,000 and 150,000 Mr and has an N-terminal amino acid sequence, predicted from the cDNA sequence and by analogy to the biochemically deternnined N-terminal sequence of the mature murine protein, as follows: MKVLQEPTCVSDYMSISTCEW.
The present invention also provides compositions for use in therapy, diagnosis, assay of IL-4 receptor, ar in raising ar~bodi~as to IL-4 receptors, comprising effective quantities of soluble receptor proteins prepared according to the foregoing processes. Such soluble recombinant receptor molecules include truncated proteins wherein regions of the receptor molecule not required for IL-4 Minding have been deleted. These anc! other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings.
RI :F DESCRIPTION OF THE DRAWINGS
Figure 1 shows restricl;ion maps. of cDNA clones containing the coding regions (denoted by a bar) of the rnurine and human iL-4R cDNAs. The restriction sites EcoRl, Pvull, Hinc II and Sst I are represented by the letters R, P, H and S, respectively.
Figures 2A-C depict the cDNA sequence and the derived amino acid sequence of the coding region of a murine IL-4 receptor, as dE;rived from clone 7B9-2 of the 7B9 library. The N-terminal isoleucine of the mature protein is designated amino acid number 1. The coding region of the full-length membrane-bound protelin from cNone 7B9-2 is defined by amino acids 1-785. The ATC codon specifying the isoleuane residue constituting the mature N-terminus is underlined at position 1 of the protein sequence; the putative transmembrane region at amino acids 209-232 is also underlined. The sequences of the coding regions of clones 7B9-4 and clones CTLL-18 and CTLL-16 of the CTLL
19.4 library are identical to that of 7B9-2 except as follows. The coding region of CTLL-16 encodes a membrane-bound IL-4-bindinl~ receptor defined by amino acids -25 through 233 (including the putative 25 amino acid signal peptide aequence), but is followed by a TAG
terminator codon (not shown) which ends the open reading frame. The nucleic acid sequence indicates the presence of a splice donor site at this position (indicates! by an arrow in Figure t ) and a splice acceptor site near the 3' end (indicated by a second arrow), suggesting that CTLL-16 was derived from an unspliced mRNA
intermediate. Clones 7B9-4 and CTLL-18 encode amino acids 23 through 199 and -25 through 199, respectively. After amino acid 199, a 1 X14-base pair insert (identical in both clones and shown by an open box in Figure a) introducE~s six nevv amino acids, followed by a termination codon. This form of the receptor is so~uWe.
Figure 3 is a schematic illustration of the mammalian high expression plasmid pCAV/NOT, which is described in greater detail in Example 8.
Figures 4A-C depict the coding sequence of a human IL-4 receptor cDNA from clone T22-8, which was obtained from a cDNA librar)r derived from the T cell line T22. The predicted N-terminal methionine of the mature protein and the transmembrane region are underlined.
Figures 5A-B are a comparison c~f the predicted amino acid sequences of human (top line) and murine (bottom line) IL-4 receptor cDNA clones.

1340~1~i DETAIL DESCRIPTION OF THE INVENTION
As used herein, the terms "IL-~4 receptor" and "IL-4R" refer to proteins having amino acid sequences which are substarrtia.i~yr slm'ctar to the native mammalian Interleukin-4 receptor amino acid sequences disclosed in Figurers 2 and 4, and which are biologically active as defined below, in that they are capable of binding Intc;rleukin-4~ (1L-4) molecules or transduang a biological signal initiated by an IL-4 molecule binding to a all, or cross-reacting with anti-IL-4R
antibodies raised against IL-4R from natural (i.e., nonrecombinant) sources. The native murine IL-4 receptor molecule is thought to have an apparent molecular weight by SDS-PAGE of about 140 kilodaltons (kDa). The terms "IL-4 receptor"
or "IL-4R" include, but are not; limited to, analogs or subunits of native proteins having at least 20 amino acids and which exhibit at least some biological activity in common with IL-4R. As used throughout the specification, the term "mature" means a protein expressed in a form lacking a leader sequence as may be present in full-length transcripts of a native gene.
Various bioequivalent protein and amino acid analogs are de;~cribed in detail below.
The term "substantially similar," when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is to retain biological activity of the IL-4R protein. Alternatively, nucleic acid subunits and analogs are "substantially similar" to the s~yecitic DMA sequences disclosed herein if:
(a) the DNA sequence is derived from the coding region ~of a native: mammalian IL-4R gene; (b) the DNA
sequence is capable of hybridization to DNA sequences of (a) under moderately stringent conditions and which encode biologically active IL-4R molecules; or DIVA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (a) or (b) and which encode biologically active IL-4R
molecules. Substantially similar analog proteins will be greater than about 30 percent similar to the corresponding sequence of the native IL-4R. Set~ences having lesser degrees of similarity but comparable tHOlogical activity 2~re considlered to be equivalents. More preferably, the analog proteins will be greater than about 80 percent siimilar to the corresponding sequence of the native IL-4R, in which case they are defined as; being "substantially identical." In defining nucleic acid sequences, all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered substantially simil~~r to a reference nucleic acid sequence.
Percent similarity may be determined, for example, by c;omparine,~ sequence information using the GAP
computer program, version 6.0, available from the ljryversity of Wisconsin Genetics Computer Group (UWGCG). The GAP
program uti~zes the alignment method of hleedlernan and Wunsch (J. Mol. Biol.
48:443, 1970), as revised by Smith and Waterman (Adv. ,Appl. Math. 2:482, 1981). Briefly, the GAP program defines similarity as the number of aligned symW Is (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program inGude: (1 ) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, 13~~~1~~.
Nucl. Acids Res. 14:6745, 1986 as described by Shwartz and Dayhoff, ed., Atl3s o:f Protein Seduence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979;
(2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
"Recombinant," as used herein, means that a protein is derived from recombinant (e. g., microbial or mammalian) expression systems. "Microbial" refers to recombinant proteins made in bacterial or fungal (e. g., yeast) expression systems. As a pr~~duct_, "recombinant microbial" defines a protein produced :in a microbial expression system which is essentially free of native endogenous substances. Protein expressed in most bacterial cultures, e.g., E. coli, will be free of glycan. ~rotein expressed in yeast may have a glycosylation pat-~ern different from that expressed in mammalian cells .
"Biologically active," as used throughout the specification as a characteristic of IL-4 receptors, means that a particular molecule shares sufficient amino acid sequence similari-~y with the embodiments of the present invention discloss=d herein to be capable of binding detectable quanti-~ies of IL-4, transmitting an IL-4 stimulus to a cell, for example, as a component of a hybrid receptor construct, or cross-reacting with anti-IL-4R antibodies raised against IL-4R from natural (i.e., nonrecombinant) sources. Preferably, biologically active IL-4 receptors within the scope c~f the present invention are capable of binding greater than 0.1 nmoles IL-4 per nmole receptor, and most preferably, greater than 0.5 nmole IL-4 per nmole receptor in standard binding assays (see below).
"DNA sequencE~" refers to a DNA molecule, in the form of a separate fragment or as a component of a larger DNA
construct, which lzas been derived from DNA isolated at least t once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical metho<~s, for example, using a cloning vector.
Such sequences are preferably provided in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA containing the relevant sequences could a=Lso be used. Sequences of non-translated DNA may be present= 5' or 3' from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.
"Nucleotide sequence" refers to a heteropolymer of deoxyribonucleoti<~es. DNA sequences encoding the proteins provided by this invention can be assembled from cDNA
fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being Expressed in recombinant transcriptional unit.
"Recombinant expression vector" refers to a replicable DNA construct used either to amplify or to express DNA which encodes IL-4R and which includes a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriai=a transcription and translation initiation and termination sEquences. Structural elements intended for use in yeast expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, x 5a where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
"Recombinant microbial expression system" means a substantially hom~~geneous monoculture of suitable host microorganisms, for example, bacteria such as E, coli or yeast such as S, cerevisiae, which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit as a component of a resident plasmid.
Generally, cells ~:onstituting the system are the progeny of a single ancestral i~ransformant. Recombinant expression systems as defined herein w ill express heterologous protein upon induction of the regulatory elE:ments linked to the DNA sequence or synthetic gene to be expres:~ed.
The pre:~ent invention therefore provides an isolated DNA
sequence encoding a mammalian IL-4 receptor (IL-4R) capable of binding IL-4, wherein said DNA sequence is selected from the group consisting of:
(a) cDNA clones comprising a nucleotide sequence selected from the sequence presented as nucleotides -75 to 2355 of Figures 2A-2C, nucleotide;> 1 to 2355 of Figures 2A-2C,nucleotides -75 to 2400 of Figure~~ 4A-4C, and nucleotides 1 to 2400 of Figures 4A-4C;
(b) DNA sequences capable of hybridization to a cDNA of (a) under moderately :stringent conditions, and which encode an IL-4R
polypeptide capab7.e of binding IL-4; and .

~.3~076:~
(c) DNA sequences that are degenerate as a result of the genetic code to a DNA defined in (a) or (b), and which encode an IL-4R polypeptide capable of binding IL-4.
In prefE~rred .embodiments:
(a) said DN~~ sequ:snce comprises a nucleotide sequence selected from the group consisting of nucleotides -75 to 2355 of Figures 2A-2C, nucleotides 1 to 2355 of Figures 2A-2C, nucleotides -75 to 2400 of Figures 4A-4C, and nucleotides 1 to 2400 of Figures 4A-4C;
(b) said DNFv sequence comprises a nucleotide sequence selected from the group consisting of nucleotides -75 to 621 of Figure 4A, and nuc:leoti<ies 1 to 621 of Figure 4A;
(c) the DNA sequence encodes an amino acid sequence that is greater than 80% ~;imilar to an amino acid sequence selected from residues -25 to 800, 1 t:o 800, -25 to 207, or 1 to 207 depicted in Figures 4A-4C;
(d) the DNA sequence encodes a soluble human IL-4 receptor, wherein said DNA encoder an amino acid sequence consisting essentially of amino acid residues -25 to 207 depicted in Figures 4A or 1-207 depicted in Figure 4A.
The invention also provides a substantially homogenous biologically active recombinant human interleukin-4 receptor protein substantially free of contaminating endogenous materials and without associated native-pattern glycosylation, which protein has an N-terminal amino acid sequence Met-Lys-Val-Leu-Gln-Glu-Pro-Thr-Cys-Val-Ser-Asp-Tyr-Met-Ser-Ile-Ser-Thr-Cys-Glu-Trp and is 6a ~,3~~'lG
capable of bindin~~ greater than 0.1 nmole interleukin-4 per nmole of the receptor.
Preferably such a protein is in the form of a glycoprotein. In particular embodiments (a) the protein comprises an amino acid sequence that is greater than 80% similar to the sequence of amino acid residues 1-800 depicted in Figures 4A, 4B
and 4C, or especi<~lly (.b) comprises an amino acid sequence consisting essent_Lally of residues 1-800 depicted in Figures 4A, 4B and 4C.
Proteins and Anal<3qs The present invention provides substantially homogeneous recombinant mamma7.ian I1L-4R polypeptides substantially free of contaminating endogenous materials and, optionally, without associated native--patte:rn glycosylation. The native murine and human IL-4 receptor molecules are recovered from cell lysates as glycoproteins having an apparent molecular weight by SDS-PAGE of about 130-145 kilodaltons (kDa). Mammalian IL-4R of the present invention include:c, by way of example, primate, human, murine, canine, feline, bovine, ovine, equine and porcine IL-4R.
Derivatives of IL-~4R wii~hin the scope of the invention also include various st;ructun~al forms of the primary protein which retain biological activity. Due to the presence of ionizable amino and carboxyl. groups, for example, an IL-4R protein may be in the form of acidic: or basic salts, or in neutral form. Individual amino acid residues may also be modified by oxidation or reduction.
6b .._ The primary amino acid structure may be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating amino acid sequence mutants.
Covalent derivatives are prepared by linking particular functional groups to IL-4R amino acid side chains or at the N- or C-termini.
Other derivatives of IL-4R within the scope of this invention include covalent or aggregative conjugates of IL-4R or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugated peptide may be a signal (or leader) polypepti<ie sequence at the N-terminal region of the protein which co-i:ranslationally or post-translationally directs transfer of the p~°otein from its site of synthesis to its site of function inside or outside of the cell membrane or wall (e.g., the yeast a-factor leader). IL-4R protein fusions can comprise peptides added to facilitate purification or identification of IL-4R (e. g., poly-His). The amino acid sequence of IL-4 receptor can also be linked to the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (Hope et al.,, BiolTechnology 6:1204, 1988). The latter sequence is highly antigenic and provides an epitope reversibly bound by a specifj.c monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. This sequence is also ~;pecif~lcally cleaved by bovine mucosal enterokinase at th.e res~~due immediately following the Asp-Lys pairing. Fusion ~~roteins capped with this peptide may also be resistant to intra.cellul.ar degradation in E. coli.
~C
k ._.. 13~~~161 IL-4R derivatives may also be used as immunogens, reagents in receptor-based immunoassays, or as Minding agents for affinity purification procedures of IL-4 or other binding ligands.
IL-4R derivatives may also he obtained by cross-linking agents, such as M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, at cysteine and lysine residues.
IL-4R proteins may also be covalently bound through reactive side groups to various insoluble substrates, such as cyanogen bromide-activated, bisoxirane-activateei, carbonyldiimidazole-activated or tosyl-activated agarose structures, or by adsorbing to f~otyolefin surfaces (with or without glutaraldehyde cross-linking). Once bound to a substrate, IL-4R many be used to selectively bird (far purposes of assay or purification) anti-IL-4R antibodies or IL-4.
The present invention also includes IL-4R with or without associated native-pattern glycosylation. IL-4R expressed in yeast or mammalian expression systems, e.g., COS-7 cells, may be similar or significantly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system. Expression of IL-4R DNAs in bacteria such as E. toll provides non-glycosylated molecules. Functional mutant analogs of mammalian IL-4R having inactivated N-glycosylatior sites can be produced by oligorucleotide synthesis and ligation or by site-specific mutagenesis techniques. These analog proteins can be produced in a homogeneous, reduced-carbohydrate form in good yield using yeast expression systems. N-glycosylation sites in eukaryotic proteins are charac~lerized by the amino acid triplet Asn-At-Z, where A1 is any amino acid except Pro, and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for covalent attachment of carbohydrate. ;such a site can be eliminated by substituting another amino acid for Asn or for residue Z, deleting Asn or Z, or inserting a non-Z amino acid between A~ and Z, or an amino acid other than Asn between A,sn and A~ .
IL-4R derivatives may ;also be obtained by mutations of IL-4R or its subunits.
An IL-4R mutant, as referred to herein, is a polypeptide homologous to IL-4R but which has an amino acid sequence different from native !L-4R because of a deletion, insertion or substitution.
Like most mammalian genes, mammalian IL-4 receptors are presumably encoded by multi-exon genes.
Alternative mRNA
constructs which car be attributed to different mRNA splicing events following transcription, and which share large regions of identity or :;in~ilarity with the cDNAs claimed herein, are considered to be within the scope of the present invention.
Bioequivalent analogs of IL-4R proteins may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or iMemal residues or sequences not needed for biological activity. For example, cysteine residues can be deleted or replaced with other amino acids to prevent formation of incorrect intramolecular disulfide bridges upon renaturation.
Other approaches to rrnrtagenesis inva~lve modification of adjacent dibasic amino acid residues to enhance expression in yeast; systems in which KEX2 protease activity is present. Generally, substitutions should be made conservatively; i.e., the most preferred substitute amino acids are those having physicochemical characteristics resembling those of the residue to be replaced. Similarly, when a deletion or insertion strategy is adopted, the potential effect of the deletion or insertion on biological activity should be coinsiderad.

1340r~~~
Subunits of IL-4R may be conslivcled by deleting terminal or internal residues or sequences.
Particularly preferred subunits include those in which the Iransmembrane region and intracellular domain of IL-4R are deleted or subshluled with hydrophilic residues to facilitate secretion of the rucontor Into tlro cull culluro modiurn. 'fho rosulllng protoin Is a soluble IL-4R molocule which may retain its ability to bind IL-4. Particular examples of soluble IL-4R include polypeptides having substantial IdenUty to the sequence of amino acid residues 1-208 fn Figure 2A, and residues t-207 in Figure 4A.
Mutations in nucleotide sequences constructed for expression of analog IL-4Rs must, of course, preserve the reading Irame phase of the coding sequences and prelerably will not create complementary regions that could hybridlize to produce secondary mRNA
structures, such as loops or halrp(ns, which would adversely allect Iranslation of the receptor mRNA.
Although a mutation site may be predetermined, il is not necessary Ihal the nature of the mulalton per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mulagenesis may be conducted at the target colon and the expressed IL-4R mutants screened for the desired activity.
Not all mutations in Ihn nucleotide sequence which encodes IL-4R will be expressed in the Iinal product, for example, nucleotide su~~bslitutions may be made to enhance expression, primarily to avoid secondary structure loops in the transcribed mRNA (see EPA 75,444A~
or to provide colons that are. more readily translated by the selected host, e.g., the well-known E. coti preference colons for E. coli expression.
Mutations can be introduced at particular loci by synthesizing oligonucleolides containing a mutant sequence, Ilanked by restriction sites enabling ligalion to fragments of the native sequence.
Following Ilgation, the resulting recons;lrucled sequence encodes an analog having the desired amino acid insertion, substitution, or delErlion.
Alternatively, oligonucleotide-~direcled site-specilic mutagenesis procedures can be empbyed to provide an atterecf gene having particular colons altered according to the substitution, deletion, or Insertion required. Exemplary methods of making the alterations set lorth above are disclosed by Walder et al. I;Gene 4.2:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985~, 12-19); Smiih et ai. (Genetic Engineering:
Principles and Methods, Plenum Press, 1981); and U.;i. Patent Nos. 4,518,584 and 4,737,462, EXRtBS. l11r11:_48 The present Invention provides recombinant expression vectors which include synthetic or cDNA-derived ONA Iragmenls trncoding mammalian IL-4R or bioequivalenl analogs operably linked to suitable Iranscripltonal or transl,ational regulatory elements derived from mammalian, microbial, viral or Insect genes. Such regulatory elements Include a Iranscriptional promoter, an optional operator sequence Io control iranscripticrn, a sequence encoding suitable mRNA
ribosomal binding sites, and sequences which control the termination of Iranscription and Iranslallon, as described in detail below.
_. g _ 1340r161 The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. DNA regions are operably linked when they are functionally related tc each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA
for a polypeptide if .it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of secretory leaders, contiguous and in reading frame.
DNA sequences encoding mammalian IL-4 receptors which are to be expressed in a microorganism will preferably contain no introns that could prematurely terminate transcription of DNA into mRNA; however, premature termination of transcription may be desirable, for example, where it would result in mutants having advantageous C-terminal truncations, for example, deletion of a transmembrane region to yield a soluble receptor not bound to the cell membrane. Due to code degeneracy, there can be considerable variation in nucleotide sequences encoding the same amino acid sequence; exemplary DNA embodiments are those corresponding to the nucleotide sequences shown in the Figures. Other embodiments include sequences capable of hybridizing to the sequences of the Figures under moderately stringent conditions (50°C, 2 X SSC) and other sequences hybridizing or degenerate to those described above, which encode biologically active IL-4 receptor polypeptides.
Transformed host cells are cells which have been transformed or transfected with IL-4R vectors constructed using recombinant DNA techniques. Transformed host cells ordinarily express IL-9R, but host cells transformed for purposes of cloning or amplifying IL-4R DNA do not need to express IL-4R. Expres~;ed IL-4R will be deposited in the cell membrane or secreted into the culture supernatant, depending on the IL-4R DNA selected. Suitable host cells for expression o:f mammalian. IL-4R include prokaryotes, yeast or higher eukryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems could also be employed to produce mammalian IL-4R using RNAs derived from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985), the relevant disclosure of which is hereby i:zcorporated by reference.
Prokaryotic expression hosts may be used for expression of IL-4Rs that do not require extensive proteolytic and disulfide processing. Prokaryotic expression vectors generally comprise one or more phenotypic selectable markers, for example a gene encoding proteins conferring antibiotic resistance or sups?lying an autotrophic requirement, and an origin of replica-ion recognized by the host to ensure amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhim~:~rium, and various species within the genera Pseudomonas, Stre~~tomyces, and Staphylococcus, although others may also be employed as a matter of choice.
Useful expre:~sion vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commc=rcially available plasmids comprising genetic elements of the well known cloning vector pBR322 . rt (ATCC 37017). Such corr,mercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed. E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene 2:95, 1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells.
Promoters co~iunonly used in recombinant microbial expression vectors include the (3-lactamase (penicillinase) and lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), the tryptophan (trp) ~~romoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EPA 36,776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p 412, 1982). A particularly useful bacterial c=_xpression system employs the phage 7~ PL
promoter and c1857ts thermolabile repressor. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the 7~ PL promoter include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RRl (ATCC 53082).
Recombinant IL-4R proteins may also be expressed in yeast hosts, prefc=rably from the Saccharomyces genus, such as S. cerevisiae. YE~ast of other genera, such as Pichia or Kluyveromyces may be also employed. Yeast vectors will generally contain an origin of replication from the 2~, yeast plasmid or an autonomously replicating sequence (ARS), promoter, DNA encoding IL-4R, sequences for polyadenylation and transcription termination and a selection gene.
._ Preferably, yeast vectors will include an origin of ,c replication and s~=_lectable marker permitting transformation 13~O~lGs of both yeast and E. coli, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae trpl gene, which provides a selection marker for a :mutant strain of yeast lacking the ability to grow in trytophan, and a promoter derived from a highly expressed ~~east gene to induce transcription of a structural sequence downstream. The presence of the trpl lesion in the yea:~t host cell genome then provides an effective environment f~~r detecting transformation by growth in the absence of trypt~~phan.
Suitable pronoter sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Bi~~l. Chem. 255:2073, 1980) or other glycolytic enzyme: (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceralc~ehyde-3-phosphate dehydrogenase, hexokinase, pyruvate de~~arboxylase, phosphofructokinase, glucose-6-phosphat:e isomerase, 3-phosphoglycerate mutase, pyruvate kinase, t:riosephosphate isomerase, phosphoglucose isomerase, and glucokin,~se. Suitable vectors and promoters for use in yeast Expression are further described in Hitzeman, EPA 73,Ei57.
Preferred yeast ve~~tors can be assembled using DNA
sequences from pBR322 f~~r selection and replication in E.
coli (Ampr gene and origin of replication) and yeast DNA
sequences including a glusose-repressible ADH2 promoter and a,-factor secretion leader. The ADH2 promoter has been described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982). The yeast a-factor leader, which directs secretion of heterologous proteins, can be inserted between the promoter and the structural gene to be expressed. SeE~, e.g., Kurjan et al., Cell 30:933, 1982;
and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984.
The leader sequen<:e may be modified to contain, near its 3' end, one or more useful restriction sites to facilitate fusion of the leader sequence to foreign genes.
Suitable yeast transformation protocols are known to those of skill in the art; an exemplary technique is described by Hinnen et al., Proc. Natl. Acad. Sci. USA
75:1929, 1978, selecting for Trp+ transformants in a selective medium consisting of 0.670 yeast nitrogen base, 0.5o casamino acids, 2o glucose, 10 ~g/ml adenine and 20 ~g/ml uracil.
Host strains transformed by vectors comprising the ADH2 promoter may be grown for expression in a rich medium consisting of to yeast extract, 2o peptone, and to glucose supplemented with 80 ~g/ml adenine and 80 ~g/ml adenine and 80 ~.g/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants are harvested by filtration and held at 4°C prior to further purification.
Various mamm<~lian or insect cell culture systems can be employed to express recombinant protein. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).
Examples of suitaJ~le mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 198L), and other cell lines capable of expressing an appropriate vector including, for example, L
cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK
cell lines. Mammalian expression vectors may comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
.., 13 134~1~~~i.
The transcriptional and translational control sequences in expression vectors t:o be used in transforming vertebrate cells may be provided by viral sources. For example, commonly used promoter~~ and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA ~,equences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DN.A sequence. The early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Hers et al., Nature 273:113, 1978) .
Smaller or larger SV40 fragments may also be used, provided the approximately 250 by sequence extending from the Hind III
site toward the B~~1 I site located in the viral origin of replication is in~~luded. Further, mammalian genomic IL-4R
promoter, control and/or signal sequences may be utilized, provided such con-~rol sequences are compatible with the host cell chosen. Additional details regarding the use of mammalian high ex~~ression vectors to produce a recombinant mammalian IL-4 receptor are provided in Example 8 below.
Exemplary vectors can be constructed as disclosed by Okayama and Berg (Mol. Ce_L1. Biol. 3:280, 1983).
A useful sysi=em for stable high level expression of mammalian receptor cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986).
A particular=_y preferred eukaryotic vector for expression of IL-~1R DNA is disclosed below in Example 2.
This vector, referred t~~ as pCAV/NOT, was derived from the mammalian high expressi~~n vector pDC201 and contains regulatory sequences fr~~m SV40, adenovirus-2, and human cytomegalovirus. pCAV/1VOT containing a human IL-7 receptor ~.340~~~i insert has been c'.eposit;ed with the American Type Culture Collection (ATCC) under deposit accession number 68014.
Purified marrsnalian IL-4 receptors or analogs are prepared by culturing suitable host/vector systems to express the recombinant translation products of the DNAs of the present invention, which are then purified from culture media or cell extracts.
For example, supernatants from systems which secrete recombinant protein, into culture media can be first concentrated using a commerically available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. for example, a suitable affinity matrix can comprise an IL-4 or lectin or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a can on exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising of sulfopropyl or ca:rboxymethyl groups. Sulfopropyl groups are preferred.
Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.c~., silica gel having pendant methyl or other aliphatic g..oups, can be employed to further purify an IL-4R composition. Some or all of the forgoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.
Recombinant protein produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous X
14a ~J~~ ~~~.L
ion exchange or size e~:clusion chromatography steps.
Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of recombinant mammalian IL-4R can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
Fermentation of yeast which express mammalian IL-4R as a secreted protein greatly simplifies purification. Secreted recombinant protein resulting from a large-scale fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296:171, 1984). This reference describes two sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC
column.
Human IL-4R synthesized in recombinant culture is characterized by the presence of non-human cell components, including proteins, in amounts and of character which depend upon the purification steps taken to recover human IL-4R from the culture. These components ordinarily will be of yeast, prokaryotic or non-human higher eukaryotic origin and preferably are prE=_sent in innocuous contaminant quantities, on the order of lE~ss than about 1 percent by weight.
Further, recombinant cell culture enables the production of IL-4R free of proi=eins which may be normally associated with IL-4R as it is found in nature in its species of origin, e.g.
in cells, cell exudates or body fluids.
IL-4R compos_~tions are prepared for administration by mixing IL-4R having the desired degree of purity with physiologically accepta:ole carriers. Such carriers will be nontoxic to recip__ents ,~t the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the I:G-4R with buffers, antioxidants such ' as ascorbic acid, low molecular weight (less than about 10 F~' 14b l3~UrG1 residues) polypept~ides, proteins, amino acids, carbohydrates including glucose,. sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients.
IL-4R compos:_tions may be used to regulate the function of B cells. For exampl~s, soluble IL-4R (sIL-4R) inhibits the proliferation of B cell cultures induced by IL-4 in the presence of anti-..g. sIL-4R also inhibits IL-4 induced IgGl secretion by LPS-activated B cells as determined by isotype specific ELISA anc~ inhibits IL-4 induced Ia expression on murine B cells as determined by EPICS analysis. sIL-4R also inhibits IL-4 induced IgE synthesis and may accordingly be used to treat IgE--induced immediate hypersensitivity reactions, such a:~ alle:rgic rhinitis (common hay fever), bronchial asthma, atopic dermatitis and gastrointestinal food allergy.
IL-4R compositions may also be used to regulate the function of T cel~_s. F«r example, IL-4R inhibits IL-4 induced proliferation o:E T cell lines, such as the CTLL T
cell line. sIL-4R also inhibits functional activity mediated by endogenously pz:oducec~ IL-4. For example, sIL-4R inhibits the generation of allorc~active cytolytic T lymphocytes (CTL) in secondary mixed leukocyte culture when present in culture concomitantly with a monoclonal antibody against IL-2, such as S4B6. Neutralizing agents for both IL-2 and IL-4 are used to inhibit endogenous I:~-2 and IL-4, both of which regulate CTL generation anct are produced in such cultures.
In therapeutic app:Lications, a therapeutically effective quantity of an IL--4 receptor composition is administered to a mammal, preferably a human, in association with a pharmaceutical carrier car diluent.
The following examples are offered by way of illustration, and not by way of limitation.
14c L'VTTA1~T L'C
Example 1 Binding assays for IL-4 receptor A. Radiolab~sling of IL-4. Recombinant murine and human IL-4 were expressf~d in yeast and purified to homogeneity as described by Park,, et al., Proc. Natl. Acad. Sci. USA 84:5267 ( 1987 ) and Park ei. al . , J. Exp. Med. 166: 476 ( 1987 ) respectively. The purified protein was radiolabeled using a commercially avai:Lable enzymobead radioiodination reagent (BioRad). In thi:~ procedure 2.5 ~g rIL-4 in 50 ~.g 0.2 M
sodium phosphate, pH 7.2 are combined with 50 ~1 enzymobead reagent, 2 MCi of sodiu:~ iodide in 20 ~1 of 0.05 M sodium phosphate pH 7.0 <~nd 10 ~l of 2.5o b-D-glucose. After 10 min. at 25°C, sodium az=~de (101 of 50 mM) and sodium metabisulfite (10 ~1 of 5 mg/ml) were added and incubation continued for 5 m:in. at 25°C. The reaction mixture was fractioned by gel filtration on a 2 ml bed volume of Sephadex~ G-25 (S:igma) equilibrated in Roswell Park Memorial Institute (RPMI) 1640 medium containing 2.5% (w/v) bovine serum albumin (BS~~), 0.20 (w/v) sodium azide and 20 mM Hepes pH 7.4 (binding me=dium). The final pool of 1251-IL-4 was diluted to a working stock solution of 2 x 10-8 M in binding medium and stored for up to one month at 4°C without detectable loss o:E receptor binding activity. The specific activity is routinely in the range of 1-2 x 1016 cpm/mmole IL-4.
B. Binding to Adherent Cells. Binding assays done with cells grown in suspension culture (i.e., CTLL and CTLL-19.4) were performed by a phthalate oil separation method (Dower et al., J. Immunol. 132:751, 1984) essentially as described by Park et al., J. Biol. Chem. 261:4177, 1986 and Park et al., supra. Binding assays were also done on COS cells 14d b transfected with ~~ mammalian expression vector containing cDNA encoding an IL-4 receptor molecule. For Scatchard analysis of bindi:zg to adherent cells, COS cells were transfected with ~~lasmid DNA by the method of Luthman et al., Nucl. Acids. Res. 11:1295, 1983, and McCutchan et al.,~J.
Natl. Cancer Inst. 41:351, 1968. Eight hours following transfection, cells were trypsinized, and reseeded in six well plates (Cost;~r, Cambridge, MA) at a density of 1 x 104 COS-IL-4 receptor transfectants/well mixed with 5 x 105 COS
control transfectf=_d cells as carriers. Two days later monolayers were a:~sayed for 1251-IL-4 binding at 4°C
essentially by thE= method described by Park et al., J. Exp.
Med. 166:476, 198'7. Nonspecific binding of 1251-IL-4 was measured in the presence of a 200-fold or greater molar excess of unlabelf=_d IL-4. Sodium azide (0.20) was included in all binding assays to inhibit internalization of 1251-IL-4 by cells at 37°C.
For analysis of inhibition of binding by soluble IL-4R, supernatants from COS cells transfected with recombinant IL-4R constructs werE~ harvested three days after transfection.
Serial two-fold d:ilusions of conditioned media were pre-incubated with 3 :~ 10-1OM125I-IL-4 (having a specific activity of about 1 x 1016 cpm/mmol) for one hour at 37°C
prior to the addi~=ion of 2 x 106 CTLL cells. Incubation was continued for 30 minutes at 37°C prior to separation of free and cell-bound mu:rine 1251-IL-4.
C. Solid Ph~~se Binding Assays. The ability of IL-4 receptor to he stably adsorbed to nitrocellulose from detergent extract; of CTLL 19.4 cells yet retain IL-4 binding activity provided a means of monitoring purification. One ml aliquots of cell extracts (see Example 3), IL-4 affinity column fractions (see Example 4) or other samples are placed on dry BA85/21 nitrocellulose membranes (Schleicher and Schuell, Keene, Nl-~) and allow to dry. The membranes are 14e ~.3~~1~~~1 incubated in tissue culture dishes for 30 minutes in Tris (0.05 M) buffered saline (0.15 M) pH 7.5 containing 3o w/v BSA to block nonspecific binding sites. The membrane is then covered with 4 x 10-11 M125I-IL-4 in PBS + 3o BSA with or without a 200-fold molar excess of unlabeled IL-4 and incubated for 2 hr at 4°C with shaking. At the end of this time, the membranes are washed 3 times in PBS, dried and placed on Kodak X-OmatT°~ AR film for 18 hr at -70°C.
14f ~34o~rm Example 2 ~~1 of CTLL cells with high IL-4 recg~ tour .~ r - ion ~,y fluorescemce activated cell sorting (FACS) The preferred cell lime for obtaining high IL-4 receptor selection is CTLL, a murine IL-2 dependent cytotoxic T cell line (ATCC 1'IB 214). To obtain higher levels of IL-4 receptor expression, CTLL cells (parent cells) were sorted using fluorescence-activated cell sorting and fluorescein-conjugated recombinant murirxe IL-4 (m~IL-d) in which the extensive carbohydrate attached to rmlL-4 by the yeast host is used to a~d~vanEage 15y coupling fluorescein hydrazide to periodate oxidized sugar moieties. The fluorescein-conjugated IIL-4 was prepared by combining aliquots of hyperglycosylated rmlL-4 (300 N.g in 300 pl of 0.1 M citrate-phosphate buffer, pH 5.5) with 30 p.l of 10 mM sodium m-periodiate (Sigma), freshly prepared in I).1 M citrate-phosphate, pH 5.5 and the mixture incubated at 4°C for 30 minutes in the dark. The reaction was quenched with 30 l,~l of 0.1 M glycerol and dialyzed for 18 hours at 4°C against 0.1 M citrate-phosphate pH 5.5. Following dialysis, a 1/10 volume of 100 mM 5-(((2-(carbohydrazino)methyl)thio)acetyl)-aminofluorescein (Molecular Probes, Eugene OR) dissolved in DMSO was addE:d to the sample and incubated at 25°C for 30 minutes. The IL-4-fluorescein was then exhaustively dialyzed at 4°C against PBS, pH 7.4 and protein concentration determined by amino acid analysis. The final product was stored at 4°C
following the addition of 1%
(w/v) BSA and sterile filtration.
In order to sort, CTLL cells (5 x 106) were incubated for 30 min at 37°C in 150 ~I PBS + 1 BSA containing 1 x 10-g M IL-~4-fluoresc:ein urxier sterile conditions. The mixture was then chilled to 4°C, washed once in a large volume of FIBS + 1% BSA and sorted using an EPICS~ C flow cytometer (Coulter Instruments). The cells providing the highest level fluorescence signal (top 1.0%) were collected in bulk and the population expanded in liquid cell culture.
Alternatively, for single cell cloning, cells exhibiting a fluorescence ;signal in the top 1.0% were sorted into 96 well tissue culture microtiter plates at 1 cell per well.
Progress was monitored by doing binding assays with 1251-IL-4 following each round of FACS
selection. Unsorted CTLL cells (CTLL parent) Iyplcally extriblted 1000-2000 IL-4 receptors per cell.
CTLL cells were subjected to 19 rounds of FRCS selection. The final CTLL cells selected (CTLL-19) exhibited 5 x 105 to 1 x 106 IL-4 receptors per cell. At this point the CTLL-19 population was subjected to EPICS~ C-assist~:d single cell cloning and individual clonal populations were expanded and tested for 1251-IL-4 binding. A single clone, designated CTLL-19.4, exhibited 1 x 106 IL-4 receptors per cell and was selected for purification and cloning studies.
While the ca~ulated apparent Ka values are similar for the two lines, CTLL-19.4 expresses approximately 400-fold more rec~:ptors on its surface than does the CTLL parent.
Example 3 eteraent extraction of CTLL cells CTLL 19.4 cells were maintained in RPMI 1640 containing 10% fetal bovine serum:, 50 U/ml penicillin, 50 lrglml streptomycin and 10~ ng/ml of recomt~inant human IL-2.
Cells were grc,wvn to 5 x 105 cells/ml in roller bottles, harvested by centrifugation, washed twice in serum free DMEM and sedimented at 2000 x g for 10 minutes to form a packed pellet (about 2 x 108 cells/ml). To the pellet was added an equal volume of~PBS
containing 1'o Triton0 X-100 and a cocktail of protease inhibitors (2 mM phenylmethysulfonylfluoride, 10 ~M
pepstatin, 10~M, leupeptin, 2 mM o-phenanthroline and 2 mM
EGTA). The cells were mixed with the extraction buffer by vigorous vortexing and the mixture incubated on ice for 20 minutes after which the mixture was centrifuged at 12,000 x g for 20 minutes at 8°C t'~ remove nuclei and other debris. The supernatant was either used immediately or stored at -70°C
until use.
Example 4 IL-4 receptor purification by IL-4 affinity chromatography In order to obtain sufficient quantities of murine IL-4R
to determine its N-terminal sequence or to further characterize human IL-4R, protein obtained from the detergent extraction of cells was further purified by affinity chromatography. Recombinant murine or human IL-4 was coupled to Affigel~-10.(BioRad) according to the manufacturer's suggestions. For example, to a solution of IL-4(3.4 mg/ml in 0.4 ml of 0.1 M H?pes pH 7.4) was added 1.0 ml of washed Affigel~-10. The solution was rocked overnight at 4°C and an aliquot of the supernatant tested for protein by a BioRad protein assay per the manufacturer's instructions using BSA
as a standard. Greater than 950 of protein had coupled to the gel, suggesting that the column had a final load of 1.3 mg IL-4 per ml gel. Glycine ethyl ester was added to a final concentration of 0.05 M to block any unreacted sites on the gel. The gel was washed extensively with PBS-to Triton~
followed by 0.1 Glycine-HCl, pH 3.0 A 0.8 x 4.0 cm column was ul prepared with IL-4-coupled Affigel~ prepared as described (4.0 ml bed volum~=) and washed with PBS containing to Triton~
X-100 for purific,~tion of murine IL-4R. Alternatively, 50 ~1 aliquots of 20o sv.zspension of IL-4 coupled Affigel~ were incubated with 35;~-cysteine/methionine-labeled cell extracts for small-scale affinity purifications and gel electrophoresis.
Aliquots (25 ml) of detergent extracted IL-4 receptor bearing CTLL 19.4 cells were slowly applied to the murine IL-4 affinity column at 4°C (flow rate of 3.0 ml/hr). The column was then washed sequentially with PBS containing to Triton~
X-100, RIPA buffe:= (0.05 M Tris, 0.15 M NaCl, to NP-40, to deoxycholate and e).1% SDS), PBS containing O.lo Triton~ X-100 and lOmM ATP, and PBS with to Triton~ X-100 to remove all contaminating mate rial except the mIL-4R. The column was then eluted with pH 3.0 glycine HCl buffer containing O.lo Tritons X-100 to remove the IL-4R and washed subsequently with PBS containing O.lo Triton~ X-100. One ml fractions were collected for the elution and 2 ml fractions collected during the wash. Immediately following elution, samples were neutralized with f30 ~,1. of 1 M Hepes, pH 7.4. The presence of receptor in the fractions was detected by the solid phase binding assay as descri:oed above, using 1251-labeled IL-4.
Aliquots were removed from each fraction for analysis by SDS-PAGE and the rema=_nder frozen at -70°C until use. For SDS-PAGE, 40 ~1 of each column fraction was added to 40 ~.1 of 2 X
SDS sample buffer (0.125 M Tris HC1 pH 6.8,40 SDS, 200 glycerol, 100 2-mercaptoethanol). The samples were placed in a boiling water bath fo.r 3 minutes and 80 ~1 aliquots applied to sample wells oj_ a 10o polyacrylamide gel which was set up and run according to the method of Laemmli (Nature 227:680, 1970). Following electrophoresis, gels were silver stained .. .., 17 1~40r1~i as previously described by Urdal et al. (Proc. Natl. Acad.
Sci. USA 81:6481, 1984).
Purification by the foregoing process permitted identification by silver staining of polyacrylamide gels of two mIL-4R protein bands averaging 45-55 kDa and 30-40~kDa that were present in fractions exhibiting IL-4 binding activity. Experi::nents in which the cell surface proteins of CTLL-19.4 cells wire radiolabeled and 1251-labeled receptor was purified by affinity chromatography suggested that these two proteins were expressed on the cell surface. The ratio of the lower to higher molecular weight bands increased upon storage of fractions at 4°C, suggesting a precursor product relationship, possibly due to slow proteolytic degradation.
The mIL-4 receptor protein purified by the foregoing process remains capable of binding IL-4, both in solution and when adsorbed to nitrocellulose.
Example 5 Sequencing of IL-4 receptor protein CTLL 19.4 mIL-4 receptor containing fractions from the mIL-4 affinity column purification were prepared for amino terminal protein sequence analysis by fractioning on an SDS-PAGE gel and then transferred to a PVDF membrane. Prior to running the protein fractions on polyacrylamide gels, it was first necessary to remove residual detergent from the affinity purification process. Fractions containing proteins bound to the mIL-4 affinity column from three preparations were thawed and concentrated individually in a speed vac under vacuum to a final volume of 1 ml. The concentrated fractions were then adjusted to pH 2 by the addition of 500 (v/v) TFA and injected onto a Brownlees RP-300 reversed-phase HPLC column (2.1 x 30 mm) equilibrated with 0.1% (v/v) TFA in H20 at a flow rate of 200 E~1/min running on a Hewlett Packard Model 1090M HPLC. The column was washed with O.lo TFA in H20 r5 ~) _L
for 20 minutes post injection. The HPLC column containing the bound protein was then developed with a gradient as follows:
Time % Acetonitrile in O.lo TFA

1 ml fractions were collected every five minutes and analyzed for the presence of protein by SDS-PAGE followed by silver 15 staining.
Each fraction from the HPLC run was evaporated to dryness in a speed vac and then resuspended in Laemmli reducing sample buffer, prepared as described by Laemmli, U. K. Nature 227:680, 1970. Samples were applied to a 5-200 20 gradient Laemmli SDS gel and run at 45mA until the dye front reached the botto:~ of the gel. The gel was then transferred to PVDF paper and stained as described by Matsudaira, J.
Biol. Chem. 262:10035, 1987. Staining bands were clearly identified in fractions from each of the three preparations 25 at approximately 30,000 to 40,000 Mr, The bands fr~~m the previous PVDF blotting were excised and subjected to ~~utomated Edman degradation on an applied Biosystems Model 477A Protein Sequencer essentially as described by March et al. (Nature 315:641, 1985), except 30 that PTH amino acids were automatically injected and analyzed on line with an A~~plied Biosystems Model 120A HPLC using a gradient and detecJtion system supplied by the manufacturer.
The following amino terminal sequence was determined from the results of sequencing: NH2-Ile-Lys-Val-Leu-Gly-Glu-Pro-Thr-18a Cys/Asn-Phe-Ser-A:~p-Tyr-Ile. The bands from the second preparation used :Eor amino terminal sequencing were treated with CNBr using the in situ technique described by March et al. (Nature 315:641, 1985) to cleave the protein after internal methionine residues. Sequencing of the resulting cleavage products yielded the following data, indicating that the CNBr cleaved 1_he protein after two internal methionine residues Cycle _Re:~idues Observed 1 0 1 Va:L, Ser 2 Gly, Leu 3 Ile, Val 4 Ty_r, Ser 5 Arch, Tyr 6 Glu, Thr 7 Ash, A1a 8 Asn, Leu 9 Pr~~, Val 10 Ala 11 Glv.z, Val 12 Ph~=, Gly 13 I1=, Asn 14 Va.l, G1n 15 Ty.r, Ile 16 Ly,s, Asn 17 Val, Thr 18 Thr, Gly When compared with the protein sequences derived from clones 16 and 18 (see Figure 2), the sequences matched as follows:

Sequence 1: (Met)-Val-Asn-Ile-Ser-Arg-Glu-Asp-Asn-Pro-Ala-G lu-1?he-Ile-Va1-Tyr-Asn-Val-Thr Sequence 2: (Met)-Ser-Gly-Val-Tyr-Tyr-Thr-Ala-Arg-Val-Arg-V al-~~rg-Ser-Gln-Ile-Leu-Thr-Gly Identical matc hes were found for all positions of sequence except Asn(2) and sequence 2, except Arg at positions 8, 10, and 12, Ser at poaition 13, and Leu at position 16. The 18b ~34~~t~Gi above sequences c~~rrespond to amino acid residues 137-154 and 169-187 of Figure 2.
In addition, the amino terminal sequence matched a sequence derived from the clone with position 9 being defined as a Cys.
The above data support the conclusion that clones 16 and 18 are derived fr~~m the message for the IL-4 receptor.
x '' 18c Example 6 ~vnthesis of hybrid-subtracted cDNA probe In order to screen a lik>rary for clones encoding a murine IL-4 receptor, a highly enriched IL-4 receptor cDNA probe was olbtained using a subtractive hybridization strategy.
Polyadenylated (polyA+) mRNA was isolated prom two similar cell lines, the parent cell line CTLL (which expresses approximately 2,000 receptors per ceN) and the sorted cell line CTLL 19.4 (which expresses 1 x 106 receptors per cell). The mRNA content of these two cell lines is expected to be identical except for the relative level of IL-4 receptor mRNA,. A radiolabeted single-stranded cDNA
preparation was then made from the mRNA of the sorted cell line CTLL 19.4 by reverse transcription of polyadenylated mRNA from CTLL 19.4 cells by a procedure similar to that described by Maniatis et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory, New York, 1982).
Briefly, polyA+
mRNA was purified as describi,d by March et al. (Nature 315:641-647, 1985) and copied into cDNA by reverse transcriptase using align dT as a primer. To obtain a high level of 32P-labeling of the cDNA,100 ~.Ci of 32P-dCTP (~s.a.=300(t Ci/mmol) was used in a 50 ~.I reaction with non-radioactive dCTP at 10 p.M. After reverse transcription at 42°C for 2 hours, EDTA
was added to 20 mM and the RNA was hydrolyzed by adding NaOHI to 0.2 M and incubating the cDNA mixture at 68°C for 20 minutes. The single-strandE;d cDNA was extracted with a phenol/chloroform (50/50) mixture previously equilibrated with 10 mM Tris-CI, 1 mM EDTA. The aqueous phase was removed to a clean tube and made alkaline again by the addition of NaOH to 0.5 M. The cDNA was then size-fractionated by chromatography on a 6 ml Sephadex~ G50 column in 30mM NaOH and 1 mM EDTA to remove small molewlar weight contanu pants.
The resulting size-fractionated cDNA generated from the sorted CTLL 19.4 cells was then hybridized with an excess of mRNA from the unsorted parental CTLL cells by ethanol-precipitating the cDNA from CTLL 19.4 cells with 30 p.g of polyA+ mRNA isolated from unsorted CTLL cells, resuspending in 16 pl of 0.25 frl NaP04, pH 6.8, 0.2% SDS, 2 mM EDTA and incubating for 20 hours at 68°C. The cDNAs from the; sorted CTLL 19.4 cells that are complementary to mRNAs from the unsorted CTLL cells torm doulble stranded cDNA/mRNA hybrids, which can then be separated from the single stranded cDNA based on their different binding affinities on hydroxyapatite. The mixture was diluted with 30 volumes of 0.02 M NaP04, pH 6.8, bound to hydroxyapatite at room temperature, and single-stranded cDNA was then ehuted from the resin with 0.12 M NaP04, pH
6.8, at 60°C, as described by Sims et al., Nature 3n2:541, 1984. Phosphate buffer was then removed by centrifugation over 2 ml Seph~~dex~ G!i0 spin columns in water. This hybrid subtraction procedure removes a majority of common sequenGSS between CTLL 19.4 and unsorted CTLL
cells, and leaves a single-stranded cDNA pool enriched for radiolabeled IL-4 receptor cDNA which can be used to probe a cDNA ibrary (as described be~bw).

Example 7 ~ypthesis of cDNA library and n, la~ue screening A cDNA library was amstruded from polyadenylated mRNA isolated from CTLL 19.4 cells using standard techniques (Gubler, et all., Gene 25:263, 1983; Ausubel et al., eds., Current Protocols in Molecular Biology, Vol. 1, 1987). Ail.er reverse transcription using oligo dT as primer, the single-stranded cDNA was rendered double-stranded with DNA polymerase I, blunt-ended with T4 DNA
polymerase, methylated with EcoR I methylase to protect EcbR t cleavage sites within the cDNA, and ligated to EcoR 1 tinkers. The resulting constructs were digested w'tth EcoR I
to remove all but one copy of the linkers at each end of the cDNA, and ligated to an equimolar concentration of EcoR I cut and dephosphorylated 7vZAP~ arms ands the resulting ligation mix was packaged in vitro (Gigapadc~) according to the manufadurei"s instructions. Other suitable methods and reagents for generating cDNA libraries in ~. phage vectors are described by Huynh et al., DNA Cloning Techniques: A Practical Approach , IRL Press, Oxford (1984); Meissner et al., Proc. NatL Acad. Sci.
USA 84:4171 (1987), and Ausubel et al., supra. 71ZAP~ is a phage ~, cloning vector similar to ~.gtl1 (U.S. Patent 4,788,135) containing plasmid sequences from pUCl9 (Norrander et al., Gene 26101, 1987), a polylinker site located in a tact gene fragment, and an f1 phage origin of replication permitting recovery of ssDNA
when host bacteria are superinieded wiith f1 helper phage. DNA is excised in the form of a plasmid comprising the foregoing elemc;nts, desi!~nated Bluescript~. Gigapack~ is a sonicated E. colt extract used to package ~, phage DN<~. ~ZAP~~, Bluescript~, and Gigapack~ are registered trademarks of Stratagene, San Diego, CA, USA.
The radiolabeled hybrid-subtracted cDNA from Example 6 was then used as a probe to screen the cDNA library. The amplified library was plated on BB4 cells at a density of 25,000 plaques on each of 20 150 mm plates and incubated overnight at 37°C. All manipulations of 7~ZAP~ and exasion of the Bluescript~ plasmid were as described by Short et al., (Nucl. Acids Res.
16:7583, 1988) and Stratagene product literature. (Duplicate plaque lift filters were incubated with hybrid-subtracted cDNA
probes from Example 6 in hybridization buffer containing 50% formamide, 5 X
SSC, 5 X Denhardt's reagent and 10% dextran sulfate at 42°C for 48 hours as described by Wahl et al., Proc. Natl. Acad.
Sci. USA7E.3683, 1979. Filters wrere then washed at 68°C in 0.2 X SSC.
Sixteen positive plaques were purified for further analysis.
Bluescript~ plasmids c~ntainingi the cDNA inserts were excised from the phage as described by the manufacturer and. trau~.sfurmed into E coG. Plasmid DNA was isolated from individual colonies, digested with EcoR I to releasE; the cDNA inserts and eledrophoresed on standard 1% agarose gels.
Four duplicate gels were blotted onto nylon filters to produce identical Southern blots for analysis with various probes which were (1) radiolaf.~:led cDNA from unsorted CTLL cells, (2) radiolabeled cDNA
from CTLL 19.4 sorted cells, (,'3) hybrid ;subtracted cDNA from CTLL 19.4 sorted cells, and (4) hybrid subtracted cDNA from CTLL 1!x.4 sorted cells after a second round of hybridization to poly A+ mRNA
from an IL-4 receptor negative mouse cell line (LBRM 33 1A5B6). These probes were increasingly enriched for cDNA copies of rnRNA specific for the sorted cell line CTLL 19.4.
Of the 16 positive 13~0't~1 plagues isolated from t:he library, four clones (11A, 14, 16 and 18) showed a paral7_el increase in signal strength with enrichment of the probe.
Restriction mapping (shown in Figure 1) and DNA
sequencing of the isolated CTLL clones indicated the existence of at least t:wo distinct mRNA populations. Both mRNA types have homo7_oqous open reading frames over most of the coding region yet diverge at the 3' end, thus encoding homologous proteins with different COOH-terminal sequences.
DNA sequence from inside the open reading frames of both clones code for protein sequence that is identical to protein sequence derived from ~:equencing of the purified IL-4 receptor described in r~tore detail in Example 5. Clone 16 and clone 18 were used as the prototypes for these two distinct message types. Clone 1.6 contains an open reading frame that encodes a 258-amino acid polypeptide which includes amino acids-25 to 233 of Figure 2A. Clone 18 encodes a 230-amino acid polypeptide, the N-terminal 224 amino acids of which are identical to the N-terminus of clone 16 but diverge at the 3' end with nucleotides CfAAGTAATGAAAATCTG which encode the C-terminal 6 amino acids, Pro-Ser-Asn-G7_u-Asn-Leu, followed by a termination codon TGP.. Both clones were expressed in a mammalian expression system, as described in Example 8.
Example 8 Expression of IL-4R in mammalian cells A. Expression in C.'OS-7 Cells. A eukaryotic expression vector pCAV/NOT, shown in Figure 3, was derived from the mammalian high expression vector pDC201, described by Sims et al., Science 241:585, 1988). pDC201 is a derivative of pMLSV, previously described by Cosman et al., Nature 312:768, 1984. PCAV/NOT is designed to express cDNA sequences inserted at its multiple cloning site (MCS) when transfected into mamma7_ian cells and includes the following components:

SV40 (hatched box:) contains SV40 sequences from coordinates 5171-270 including the origin of replication, enhancer sequences and early and late promoters. The fragment is oriented so that the direction of transcription from the early promoter is as shown by the arrow. CMV contains~the promoter and enhancer regions from human cytomegalovirus (nucleotides-671 to +7 from the sequence published by Boshart et al., Ce11:41:521-530, 1985). The tripartite leader (stippled box) contain; the first exon and part of the intron between the first and ~;econd exons of the adenovirus-2 tripartite leader, the second exon and part of the third exon of the tripartite leader and a multiple cloning site (MCS) containing sites for Xl:~o I, Kpn I, Sma I, Not I, and Bg1 II.
pA (hatched box) contains SV40 sequences from 4127-4100 and 2770-2533 that include the polyadenylation and termination signals for early transcription. Clockwise from pA are adenovirus-2 sequences 10532-11156 containing the VAI and VAII genes (designated by a black bar) followed by pBR322 sequences (solid line) from 4363-2486 and 1094-375 containing the ampicillin resistance gene and origin of replication.
The resulting expression vector was designated pCAV/NOT.
Inserts in clone 16 and clone 18 were both released from Bluescript~ plasmid by digestion with Asp 718 and Not 1. The 3.5 kb insert from clone 16 was then ligated directly into the expression ve~~tor pCAV/NOT also cut at the Asp 718 and Not I sites in thc= polylinker region. The insert from clone 18 was blunt-ended with T4 polymerase followed by ligation into the vector pCAV/NOT cut with Sma I and dephosphorylated.
Plasmid DNA :From both IL-4 receptor expression plasmids were used to tranafect a subconfluent layer of monkey COS-7 cells using DEAF-c~extran followed by chloroquine treatment, as described by Luthman et al. (Nucl. Acids Res. 11:1295, 1983) and McCutch<~n et al. (J. Natl. Cancer Inst. 41:351, y ~f~
1968). The cells were then grown in culture for three days to permit transient expression of the inserted sequences.
After three days, cell culture supernatants and the cell monolayers were assayer. (as described in Example 1) and IL-4 binding was confirmed.
B. Expression in CHO Cells. IL-4R was also expressed in the mammalian CHO cell line by first ligating an Asp718/Notl restriction fragment of clone 18 into the pCAV/NOT vector as described in Example 8. The pCAV/NOT
vector containing the insert from clone 18 was then co-transfected using a standard calcium phosphate method into CHO cells with the dihydrofolate reductase (DHFR) cDNA
selectable marker under the control of the SV40 early promoter. The DHFR sequence enables methotrexate selection for mammalian cells harboring the plasmid. DHFR sequence amplification events in such cells were selected using elevated methotrexate concentrations. In this way, the contiguous DNA sequences are also amplified and thus enhanced expression is achieved. Mass cell cultures of the transfectants secreted active soluble IL-4R at approximately 100 ng/ml.
C. Expression in HeZa Cells. IL-4R was expressed in the human HeLa-EB~VA cell line 653-6, which constitutively expresses EBV nuclear antigen-1 driven from the CMV
immediate-early enhancer/promoter. The expression vector used was pHAV-EO-VEO (described by Dower et al., J. Immunol.
142:4314, 1989), a derivative of pDC201, which contains EBV
origin of replication and allows high level expression in the 653-6 cell line. pHAV-EO-NEO is derived from pDC201 by replacing the ade:novirus major late promoter with synthetic sequences from HIV-1 extending from -148 to +78 relative to the cap site of the viral mRNA, and including the HIV-1 tat gene under the control of the SV-40 early promoter. It also contains a Bgl II-Sma I fragment containing the neomycin resistance gene of pSV2NE0 (Southern & Berg, J. Mol. Appl.
X

9~~0'~~l Genet. 1:332, 1982) inserted into the Bgl II and Hpa I sites and subcloning downstream of the Sa1 I cloning site. The resulting vector ~~ermits selection of transfected cells for neomycin resistan~Je.
A 750 by IL-~~R fragment was released from the Bluescript~ plasmid by digesting with EcoN I and Sst I
restriction enzymes. This fragment of clone 18 corresponds to nucleotides 1-672 of Figure 2A, with the addition of a 5' terminal nucleoti<~e sequence of GTGCAGGCACCTTTTGTGTCCCCA, a TGA stop codon wh:LCh follows nucleotide 672 of Figure 2A, and a 3' terminal nuc:Leotide sequence of CTGAGTGACCTTGGGGGc;TGCGGTGGTGAGGAGAGCT. This fragment was then blunt-ended using T4 polymerase and subcloned into the Sa1 I site of pHA~I-EO-NEO. The resulting plasmid was then transfected into 1=he 653-6 cell line by modified polybrene transfection method as described by Dower et al. (J. Immunol.
142:4314, 1989) with the exception that the cells were trypsinized at 2 days post-transfection and split at a ratio of 1:8 into media containing 6418 (Gibco Co.) at a concentration of :L mg/ml. Culture media were changed twice weekly until neom~~cin-resistant colonies were established.
Colonies were then either picked individually using cloning rings, or pooled i~ogether, to generate several different cell lines. These cel=L lines were maintained under drug selection at a 6418 concent:=ation of 250 ~g/ml. When the cells reached confluency supernatants were taken and tested in the inhibition assay of Example 1B. Cell lines produced from 100 ng/ml to 600 ng/m=L of soluble IL-4R protein.
Example 9 Expression of IL-4R in yeast cells For expression of mIL-4R, a yeast expression vector derived from pIXY:L20 was constructed as follows. pIXY120 is identical to pYaHuGM (ATCC 53157), except that it contains no ~~ ~~~~ ~1 cDNA insert and includes a polylinker/multiple cloning site with an Nco I site. This vector includes DNA sequences from the following sources: (1) a large Sph I (nucleotide 562) to EcoR I (nucleotide 4361) fragment excised from plasmid pBR322 (ATCC 37017), inc7_uding the origin of replication and the ampicillin resist~~nce marker for selection in E. coli; (2) S.
cerevisiae DNA in<:ludin~~ the TRP-1 marker, 2~, origin of replication, ADH2 promoter; and (3) DNA encoding an 85 amino acid signal peptide de rived from the gene encoding the secreted peptide cc-factor (See Kurjan et al., U.S. Patent 4,546,082). An A:~p 718 restriction site was introduced at position 237 in tree a-f,~ctor signal peptide to facilitate fusion to heterologous genes. This was achieved by changing the thymidine residue at nucleotide 241 to a cytosine residue by oligonucleotide-dire~~ted in vitro mutagenesis as described by Craik, BioTechniques, January 1985, pp. 12-19. A
synthetic oligonuc:leotide containing multiple cloning sites and having the fo7_lowing sequence was inserted from the Asp718 site at amino acid 79 near the 3' end of the a-factor signal peptide to a Spe I site in the 2~ sequence:
Asp718 Stu I Nco I
GTACCTTTGGATAAAAG~1GACTACAAGGACGACGATGACAAGAGGCCTCCP.TGGATC
BamHI Sma I Spe I
CCCCGGGACA
GAAACCTATTTTCTCTG~1TGTTCCTGCTGCTACTGTTCTCCGGAGGTACCTAGGGGGCCCT
GTGATC
~Polylinker~~
pBC120 also varies from pYa,HuGM by the presence of a 514 by DNA fragment derived from the single-stranded phage fl containing the origin o:f replication and intergenic region, which has been in:~erted at the Nru I site in the pBR322 sequence. The presence of an fl origin of replication x ~.3~~~~~i permits generation of single-stranded DNA copies of the vector when transformed into appropriate strains of E. coli and superinfected with bacteriophage f1, which facilitates DNA sequencing of the vector and provides a basis for in vitro mutagenesis. To insert a cDNA, pIXY120 is digested with Asp 718 which cleaves near the 3' end of the a-factor leader peptide (nucleotide 237) and, for example, BamH I
which cleaves in the polylinker. The large vector fragment is then purified and ligated to a DNA fragment encoding the protein to be expressed.
To create a secretion vector for expressing mIL-4R, a cDNA fragment encoding mIL-4R was excised from the Bluescript~ plasrr.id of Example 8 by digestion with Ppum I and Bg1 II to release an 831 by fragment from the Ppum I site (see FIGURE) to a:n Bg1 II site located 3' to the open reading frame containing the mIL-4R sequence minus the first two 5' codons encoding I1e and Lys. pIXY120 was digested with Asp 718 near the 0 en~~ of the a-factor leader and BamHI. The vector fragment was ligated to the Ppum I/Bg1 II hIL-4R cDNA
fragment and the following fragment created by annealing a pair of synthetic oligonucleotides to recreate the last 6 amino acids of th~~ a-factor leader and the first two amino acids of mature mIL-4R.
a-f: actor processing-~~
GTA CCT CTA GAT AAA AGA ATC AAG
G:~ GAT CTA TTT TCT TAG TTC CAG
Va.l Pro Leu Asp Lys Arg Ile Lys f-mIL-4R
The oligonucleoti~~e also included a change from the nucleotide sequen~~e TGG ATA to CTA GAT which introduces a Xba x ~J~~r~~l I restriction site, without altering the encoded amino acid sequence.
The foregoing expression vector was then purified and employed to transform a diploid yeast strain of S.
cerevisiae(XV2181) by ~>tandard techniques, such as those disclosed in EPA 165,654, selecting for tryptophan prototrophs. The resulting transformants were cultured for expression of a secreted mIL-4R protein. Cultures to be assayed for biological activity were grown in 20-50 ml of YPD
medium (1% yeast extract, 2o peptone, to glucose) at 37°C to a cell density of 1-5 x 1.08 cells/ml. To separate cells from medium, cells were removed by centrifugation and the medium filtered through a 0.45 ~ cellulose acetate filter prior to assay. Supernatants produced by the transformed yeast strain, or crude extracas prepared from disrupted yeast cells transformed the plasmid, were assayed to verify expression of a biologically active protein.
Example 10 Isolation of fu~_1-length and truncated forms of murine IL-4 receptor cDNAs from unsorted 7B9 cells Polyadenylated RNP, was isolated from 7B9 cells, an antigen-dependent helper T cell clone derived from C57BL/6 mice, and used to construct a cDNA library in a,ZAP
(Stratagene, San Diego), as described in Example 7. The a,ZAP
library was amplified once and a total of 300,000 plagues were screened as described in Example 7, with the exception that the probe was a randomly primed 32P-labeled 700 by EcoR
I fragment isolated from CTLL 19.4 clone 16. Thirteen clones were isolated and characterized by restriction analysis.
Nucleic acid sequence analysis of clone 7B9-2 revealed that it contains a polyadenylated tail, a putative t polyadenylation signal, and an open reading frame of 810 amino acids (shown in fig 2), the first 258 of which are identical to those encoded by CTLL 19.4 clone 16, including the 25 amino acid putative signal peptide sequence. The 7B9-2 cDNA was subcloned into the eukaryotic expression vector, pCAV/NOT, and the resulting plasmid was transfected into COS-7 cells as described in Example 8. COS-7 transfectants were analyzed as set forth in Example 12.
A second cDN.A form., similar to clone 18 in the CTLL 19.4 library, was isolated from the 7B9 library and subjected to sequence analysis. This cDNA, clone 7B9-4, is 376 by shorter than clone 7B9-2 at the 5' end, and lacks the first 47 amino acids encoded by 7B9-2, but encodes the remaining N-terminal amino acids 23-199 (in Fig. 2). At position 200, clone 7B9-4 (like clone 18 from CTLL 19.4) has a 114 by insert which changes the amino acid sequence to Pro Ser Asn Glu Asn Leu followed by a termination codon. The 114 by inserts, found in both clone 7B9-4 and CTLL 19.4 clone 18 are identical in nucleic acid sequ~ance. The fact that this cDNA form, which produces a secret~Jd form of the IL-4 receptor when expressed in COS-7 cells, w,as isolated from these two different cell lines indicates that it is neither a cloning artifact nor a mutant form pecu liar to the sorted CTLL cells.
Example 11 Isolation of human IL-4 receptor cDNAs from PBL and T22 libraries by cross-species hybridization Polyadenylatf=d RNA was isolated from pooled human peripheral blood :Lymphocytes (PBL) that were obtained by standard Ficoll p»rification and were cultured in IL-2 for six days followed by stimulation with PMA and Con-A for eight hours. An oligo c~T primed cDNA library was constructed in ~,gtl0 using techn=~ques described in Example 7. A probe was produced by synthesizing an unlabeled RNA transcript of the 27a 1~~U'~~
7B9-4 cDNA insert using T7 RNA polymerase, followed by 32p_ labeled cDNA synthesis with reverse transcriptase using random primers (Boehringer-Mannheim). This murine single-stranded cDNA probe was used to screen 50,000 plagues from the human cDNA library in 50o formamide/0.4 M NaCl at 42°C, followed by washing in 2 X SSC at 55°C. Three positive plagues were puri:=ied, and the EcoR I inserts subcloned into the Bluescript~ plasmid vector. Nucleic acid sequencing of a portion of clone I?BL-1, a 3.4 kb cDNA, indicated the clone was approximately 67o homologous to the corresponding sequence of the murine IL-4 receptor. However, an insert of 68 bp, containing a termination codon and bearing no homology to the mouse IL-4 receptor clones, was found 45 amino acids downstream of the predicted N-terminus of the mature protein, suggesting that c=_one PBL-1 encodes a non-functional truncated form of the receptor. Nine additional human PBL
clones were obtained by screening the same library (under stringent conditions) with a 32P-labeled random-primed probe made from the clone PBL-1 (the 3.4 kb EcoR I cDNA insert).
Two of these clonE:s, PB:L-11 and PBL-5, span the 5' region that contains the 68 by insert in PBL-1, but lack the 68 by insert and do not extend fully 3' as evidenced by their size, thus precluding functio:zal analysis by mammalian expression.
In order to obtain a co::zstruct expressible in COS-7 cells, the 5' Not I-Hinc II fr~~gment of clones PBL-11 and PBL-5 were separately ligatec~ to the 3' Hinc II-BamH I end of clone PBL-1, and subcloned into the pCAV/NOT expression vector cut with Not I and Bgl II described in Example 8. These chimeric human IL-4R cDNAs containing PBL-11/PBL-1 and PBL-5/PBL1 DNA
sequences have been termed clones A5 and B4, respectively, as further described in Example 12. These constructs were transfected into C:OS-7 cells, and assayed for IL-4 binding in a plate binding a=say substantially as described in Sims et al. (Science 241:_'85, 1'x88). Both composite constructs 27b 13~~~~i encoded protein which exhibit IL-4 binding activity. The nucleotide sequencJe and predicted amino acid sequence of the composite A5 construct correspond to the sequence information set forth in Figures 4A-4C, with the exception that a GTC
codon encodes the amino acid Val at position 50, instead of Ile. No other clones that were sequenced contained this change. The consensus codon from clones PBL-1, PBL-5 and T22-8, however, i:~ ATC and encodes I1e50, as set forth in Figure 4A. The nucleotide and predicted amino acid sequence of the composite B4 construct also shows that the 25 amino acid leader sequence of PBL-11 is replaced with the sequence Met-Gln-Lys-Asp-A_La-Arg-Arg-Glu-Gly-Asn.
Constructs e:~pressing a soluble form of the human IL-4 receptor were made by excising a 5'-terminal 0.8 kb Sma I-Dra III fragment from PBL-5 and the corresponding 0.8 kb Asp718 Dra III fragment From PBL-11, of which the Dra III overhangs were blunt-ended with T4 polymerase. The PBL-5 and PBL-11 fragments were separately subcloned into CAV/NOT cut with Sma I or Asp 718 plus Sma I, respectively; these are called soluble hIL-4R-5 and soluble hIL-4R-11, respectively. In both constructs the final IL-4 receptor amino acid Thr194 codon is followed by the vector-encoded amino acids GlyGlnArgProLeuGlnIleTyrAlaIle before terminating.
A second library made from a CD4+/CD8-human T cell clone, T22 (Acres et al., J Immunol. 138:2132, 1987) was screened (using duplicate filters) with two different probes synthesized as described above. The first probe was obtained from a 220 by Pvu II fragment from the 5' end of clone PBL-1 and the second probe was obtained from a 300 by Pvu II-EcoR I
fragment from the 3' end of clone PBL-1. Five additional cDNA clones were :identified using these two probes. Two of these clones span the 5' region containing the 68 by insert, but neither contain the insert. The third of these clones, T22-8, was approximately 3.6 kb in size and contained an open 27c 1340'~~G!
reading frame of 825 amino acids, including a 25 amino acid leader sequence, ~~ 207 amino acid mature external domain, a 24 amino acid transmemb:rane region and a 569 amino acid cytoplasmic domain. The sequence of clone T22-8 is set forth in Figures 4A-4C. Figures 5A-5B compare the predicted~human IL-4R amino acid sequence with the predicted murine IL-4R
sequence and show approximately 53o sequence identity between the two proteins.
A third soluble human IL-4 receptor construct was made as follows. cDNA clone T22-8 was cleaved at the DraIII site in the Thr194 codon, and repaired with synthetic oligonucleotides t;o regenerate the Thr194 and Lys195 codons, followed by a termination codon, and a NotI restriction site.
A 0.68 kb StyI-Not:I restriction fragment of this clone was then blunt-ended at the StyI site and subcloned into a SmaI-NotI digested pCAV/NOT vector. This cDNA expression vector was designed hIL-~3R-8.
Example 12 Analysis and purification of IL-4 receptor in COS
transfectants Equilibrium bindin~~ studies were conducted for COS cells transfected with rnurine IL-4 receptor clones 16 and 18 from the CTLL 19.4 library. In all cases analysis of the data in the Scatchard coordinate system (Scatchard, Ann. N.Y. Acad.
Sci. 51:660-672, (1949) yielded a straight line, indicating a single class of h=ugh-affinity receptors for murine IL-4. For COS pCAV-16 cells the c,~lculated apparent Ka was 3.6 x 109 M-1 with 5.9 x 105 specific binding sites per cell. A similar apparent Ka was c:~lculated for COS pCAV-18 cells at 1.5 x 109 M-1 but receptor number expressed at the cell surface was 4.2 x 104. Equilibrium binding studies performed on COS cells transfected with =CL-4R DNA clones isolated from the 7B9 cell ;.
27d ~3~~~t~si library also showed high affinity binding of the receptor to IL-4. Specifical:Ly, studies using COS cells transfected with pCAV-7B9-2 demonsvrated that the full length murine IL-4 receptor bound 12'I-IL-4 with an apparent Ka of about 1.4 x 1010 M-1 with 4.5 x 104 specific binding sites per cell. The apparent Ka of CA'J-7B9-4 IL-4R was calculated to be about final assay volumf= of 150 ~l gives approximately 500 inhibition of 1251-IL-4 binding to the IL-4 receptor on CTLL
cells. 1251-IL-4 receptor competing activity is not detected in control pCAV t:ransfected COS supernatants. From quantitative anal:~sis of the dilution of pCAV-18 supernatant required to inhibit 1251-IL-4 binding by 500, it is estimated that approximately 60-100 ng/ml of soluble IL-4 receptor has been secreted by COS cells when harvested three days after transfection. Similar results were obtained utilizing supernatants from COS cells transfected with pCAV-7B9-4.
Conditioned medium from COS cells transfected with pCAV-18 or pCAV-7B9-4 (see Example 8) and grown in DMEM containing 3o FBS was harvested three days after transfection.
Supernatants were centrifuged at 3,000 cpm for 10 minutes, and frozen until :needed. Two hundred ml of conditioned media was loaded onto a column containing 4 ml of muIL-4 Affigel prepared as described above. The column was washed extensively with PBS and IL-4 receptor eluted with 0.1 M
glycine, 0.15 M N~~Cl pH 3Ø Immediately following elution, samples were neutralized with 80 ~1 of 1 M Hepes pH 7.4.
Samples were tested for their ability to inhibit binding of 1251-muIL-4 to CT:LL cells as set forth in example 1B.
Additionally samples were tested for purity by analysis on SDS-PAGE and silver staining as previously described.
Alternative metho~~s for testing functional soluble receptor activity or :LL-4 :oinding inhibition include solid-phase binding assays, as described in Example 1C, or other similar cell free assays which may utilize either radio iodinated or 27e 1340~1~i colorimetrically developed IL-4 binding, such as RIA or ELISA. The protein analyzed by SDS-PAGE under reducing conditions has a rnolecular weight of approximately 37,500 and appears approximately 90o pure by silver stain analysis of gels.
Purified recombinant soluble murine IL-4 receptor protein may also be tested for its ability to inhibit IL-4 induced 3H-thymid=Lne incorporation in CTLL cells. Pursuant to such methods, soluble IL-4 receptor has been found to block IL-4 stimulated proliferation, but does not affect IL-2 driven mitogenic response.
Molecular we__ght estimates were performed on mIL-4 receptor clones transfected into COS cells. Utilizing M2 monoclonal antibody pre.oared against murine CTLL 19.4 cells (see example 13), IL-4 receptor is immunoprecipitated from COS cells transfec:ted with CAV-16, CAV7B9-2 and CAV-7B9-4 and labeled with 35S-cystei:ne and 35S-methionine. Cell associated receptor from CAV-7B9-4 shows molecular weight heterogeneity ranging from 32-39 kDa. Secreted CAV-7B9-4 receptor has molecular weight between 36 and 41 kDa. Cell associated receptor from CAV-16 transfected COS cells is about 40-41 kDa. This is significantly smaller than molecular weight E'stima'tions from crosslinking studies described by Park et al., J Exp. Med. 166:476, 1987; J. Cell.
Biol. Suppl. 12A, 1988. Immunoprecipitation of COS CAV-7B9-2 cell-associated receptor showed a molecular weight of 130-140 kDa, similar to the estimates of Park et al., J. Cell. Biol., suppl. 12A, 1988, estimated to be the full length IL-4 receptor. Similar= mole~~ular weight estimates of cell-associated CAV-16 and C;~1V-7B9-2 IL-4 receptor have also been made based upon cross-linking 125IL-4 to COS cells transfected with these ~~DNAs. Heterogeneity of molecular weight of the ind~_vidual clones can be partially attributed to glycosylation. This data, together with DNA sequence 27f analysis, suggests that: the 7B9-2 cDNA encodes the full length cell-surface IL--4 receptor, whereas both 7B9-4 and clone 18 represent soluble forms of murine IL-4 receptor.
.
27g 1340't~i Receptor characterization studies were also done on COS cells transfected with hIL-4R
containing expression plasmids. The two chimeric human IL-4R molecules A5 and B4 (defined in Example 11) were transfected into COS cells and equilibrium binding studies undertaken. The COS
monkey cell itself has receptors capabl~a of binding hIL-4; therefore the binding ca~ulations performed on COS cells transtected with and overexpressing hIL-4R cDNAs represent background binding from endogenous monkey IL-4R molecules subtracted tram the total binding. COS cells transfected with hIL-4R A5 had 5.3 x 104 hIL-4. birxiing ;;ices with a calculated Ka of 3.48 x 109 M-1. Similarly, the hIL-4R B4 expressed in COS cell;; bound 1251-hIL-4 with an affinity of 3.94 x 109 M-1 exhibiting 3.2 x 104 receptors per cell.
Molecular weight estimates of human IL-4R expressed in COS cells were also performed.
COS cells transtected with clones A5 or B4 in pCAV/NOT were labeled with 35S-cysteine/ methionine and lysed. Human IL-4R was affinity purified from the resulting lysates with hIL-4-coupled Affiget~ (as descrtbed in Example 4). The hIL-4R A5 and B4 eluted from this affinity support migrated at about 140,000 daltons on SDS-PAGiE, agreeing well with previous estimates of hIL-4R
molecular weight by cross-linking (Park et al., J. E,rp. Med 166476, 1987), as well as with estimates of full-length mIL-4R
presented here.
Because no soluble human IL-4R cDNA has thus tar been found occurring naturally, as was the case for the murine receptor (clones 18 and 7B9-4), a truncated form was constructed as described in Example 1t. Following expression in COS cells, supernatants were harvested three days after transtection with soluble hIL-4R-11 and soluble hIL-4R-5 and tested for inhibition of t 2~1-hIL-4 binding to the human B cell Gne Rah. Supernatants from two of the soluble hIL-4R-11 and one of the soluble hIL-4R-5 transfected plates contained 29-149 ng/ml of IL-4R
competing activity into the medium. In addition, the truncated protein could be detected in 35S-methionine/cysteine-labeled COS cell transfectants by affinity purification on hIL-4-coupled Affiget~ as approximately a 44 kDa protein by SDS-PAGE. Supernatants from C4S cells transfected with h1L-4R-8 (encoding soluble truncated lL-4R) when concentrated 25-fold, inhibited human IL-4 binding to Raji cells, and contained approximately 16 nglml of competing activity.
Example 13 $~naration of monoclonal antif.~~jes to IL-4R
Preparations of purified recombinant IL-4 receptor, for example, human or murine IL-4 receptor, transfected COS cells expressing high levels of IL-4 receptor or CTLL 19.4 cells are employed to generate monoclonal antiibodies against IL-4 receptor using conventional techniques, such as those disclosed in IU. S. Patent 4,411,993. Such antibodies are likely to be useful in interfering with 1L-4 binding 1o IL-4 receptors, for example, in ameliorating toxic or other undesired effects of iL-4.
To immunize rats, IL-~s receptor bearing CTLL 19.4 cells were used as immunogen emulsified in complete Freund's adjuvan~t and injected in amounts ranging from 10-100 p!
subcutaneously into Lewis rats. Three weeks later, the irnmunized animals were boosted with additiorra! immunogen emulsified in incomplete Frf~und's acljuvant and boosted every three weeks thereaffer. Serum samples are periodically taken by retro~~ort~ital bleeding or tail-tip excision for testing by dot-blot assay, ~. . - 2 8 -134~Y161 ELISA (enzyme-linked inununosorbent assay), or inhibition of binding of 1251-I7~-4 to extracts of CTLL cells (as described in Example 1). 01=her assay procedures are also suitable.
Following detection of an appropriate antibody titer, positive animals were given a final intravenous injection of antigen in saline. Three to four days later, the animals were sacrificed, :~plenocytes harvested, and fused to the murine myeloma ce_L1 line AG8653. Hybridoma cell lines generated by this procedure were plated in multiple microtiter plates in a HAT selective medium (hypoxanthine, aminopterin, and vhymidine) to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
Hybridoma clones thus generated were screened for reactivity with I:~~-4 receptor. Initial screening of hybridoma supernatants utilized an antibody capture and binding of partia.Lly purified 1251-mIL-4 receptor. Two of over 400 hybridomas screened were positive by this method.
These two monoclonal antibodies, M1 and M2, were tested by a modified antibody capture to detect blocking antibody. Only Ml was able to inhibit 1251-rmIL-4 binding to intact CTLL
cells. Both antibodies are capable of immunoprecipitating native mIL-4R protein from CTLL cells or COS-7 cells transfected with IL-4R clones labeled with 35S-cysteine/methioni:ze. Ml and M2 were then injected into the peritoneal cavities of nude mice to produce ascites containing high c~~ncentrations (>1 mg/ml) of anti-IL-4R
monoclonal antibo~~y. The resulting monoclonal antibody was purified by ammonium sulfate precipitation followed by gel exclusion chromatography, and/or affinity chromatography based on binding sf antibody to Protein G.
~x Example 14 1340'~~1 Use of soluble IL-4R to suppress immune response in vivo Experiments were conducted to determine the effect of soluble IL-4R on allogenic host versus graft (HVG) response in vivo using a p~~pliteal lymph node assay. In this model mice are injected in the footpad with irradiated, allogeneic spleen cells. Irradiated, syngeneic cells are then injected into the contralaveral pad. An alloreactive response occurs in the pad receiving the allogeneic cells, the extent of which can be measured by the relative increase in size and weight of the pop:Liteal lymph node draining the site of antigen deposition.
On day 0 three BALB/C mice were injected in the footpad with irradiated, <~llogeneic spleen cells from c57BL/6 mice and in the contra:Lateral footpad with irradiated, syngeneic spleen cells. On days -1,0 and +1 three mice were injected (intravenously on days -1 and 0, and subcutaneously on day +1) with 100ng of purified soluble IL-4R(sIL-4R) in phosphate buffered saline, i~hree :mice were injected intravenously with 1 ~g of sIL-4R, three mice were injected with 2~g of sIL-4R
and three mice we..a injected with MSA (control). The mean difference in weight of the lymph nodes from the sites of allogeneic and syngeneic spleen cells was approximately 2.5 mg for the mice t..eated with MSA, 1 mg for the mice treated with 100 ng of sIh-4R, and 0.5 mg for mice treated with leg sIL-4R. No detect=able difference in weight of lymph nodes was ascertainable for the mice treated with 2~.g sIL-4R.
Thus, IL-4R signi=icantly (p < 0.5 in all groups, using a two-tailed T test; suppressed the in vivo lymphoproliferative response in a dose dependent fashion relative to control mice.
r ... 29a

Claims (42)

1. An isolated DNA sequence encoding a mammalian IL-4 receptor (IL-4R) capable of binding IL-4, or analog of said IL-4 receptor wherein said DNA sequence is selected from the group consisting of:
(a) cDNA clones comprising a nucleotide sequence selected from the sequence presented as nucleotides -75 to 2355 of Figures 2A-2C, nucleotides 1 to 2355 of Figures 2A-2C,nucleotides -75 to 2400 of Figures 4A-4C, and nucleotides 1 to 2400 of Figures 4A-4C;
(b) DNA sequences capable of hybridization to a cDNA of (a) under moderately stringent conditions, and which encode an IL-4R polypeptide capable of binding IL-4; and (c) DNA sequences that are degenerate as a result of the genetic code to a DNA defined in (a) or (b), and which encode an IL-4R polypeptide capable of binding IL-4.

2. A DNA sequence according to claim 1, wherein said DNA sequence comprises a nucleotide sequence selected from the group consisting of nucleotides -75 to 2355 of Figures 2A-2C, nucleotides 1 to 2355 of Figures 2A-2C, nucleotides -75 to 2400 of Figures 4A-4C, and nucleotides 1 to 2400 of Figures 4A-4C.

3. A DNA sequence according to claim 1, wherein said DNA sequence comprises a nucleotide sequence selected from the group consisting of nucleotides -75 to 621 of Figure 4A, and nucleotides 1 to 621 of Figure 4A.

4. A DNA sequence according to claim 1, which encodes an amino acid sequence that is greater than 80 % similar to an amino acid sequence selected from residues -25 to 800, 1 to 800, -25 to 207, or 1 to 207 depicted in Figures 4A-4C.

5. An isolated DNA sequence encoding a soluble human IL-4 receptor, wherein said DNA encodes an amino acid sequence consisting essentially of amino acid residues -25 to 207 depicted in Figures 4A or 1-207 depicted in Figure 4A.

6. A recombinant expression vector comprising a DNA
sequence according to any one of claims 1 to 5.

7. A process for preparing a mammalian IL-4 receptor or an analog thereof, comprising culturing a host cell comprising a vector according to claim 6 under conditions promoting expression of the receptor or analog thereof.

8. A process according to claim 7, wherein the IL-4 receptor is a human IL,-4 receptor or an analog thereof.

9. A purified mammalian IL-4 receptor protein capable of binding IL-4, wherein said protein is encoded by a DNA
according to claim 1.

10. An IL-4 receptor protein according to claim 9, consisting essentially of murine IL-4 receptor.

11. An IL-4 receptor protein according to claim 9, consisting essentially of human IL-4 receptor.

12. A human IL-4 receptor protein according to claim 11, wherein the IL-4 receptor is in the form of a glycoprotein having a molecular weight of between about 110,000 and 150,000 Mr by SDS-PAGE and a binding affinity (Ka) for human IL-4 of from about 1-8x109M-1.

13. A human IL-4 receptor protein according to claim 12, wherein the IL-4 receptor has an N-terminal amino acid sequence Met-Lys-Val-Leu-Gln-Glu-Pro-Thr-Cys-Val-Ser-Asp-Tyr-Met-Ser-Ile--Ser-Thr-Cys-Glu-Trp.

14. A human IL-4 receptor protein according to claim 11, wherein the transmembrane region and cytoplasmic domain of the native receptor have been deleted.

15. A human IL-4 receptor protein according to claim 11, wherein said protein comprises an amino acid sequence that is greater than 80 % similar to a sequence selected from residues 1-800 depicted in Figures 4A to 4C and residues 1-207 depicted in Figure 4A.

16. A human IL-4 receptor protein according to claim 15, wherein said protein comprises an amino acid sequence consisting essentially of residues 1-207 depicted in Figure 4A.

17. A composition for regulating immune responses in a mammal, comprising an effective amount of a protein according to claim 9, and a pharmaceutically acceptable diluent or carrier.

18. A composition according to claim 17, wherein the protein has a specific binding activity of at least about 0.01 nanomole IL-4/nanomole IL-4 receptor.

19. A composition according to claim 17, wherein the protein consists essentially of a substantially homogeneous human IL-4 receptor in the form of a glycoprotein having a binding affinity (Ka) for human IL-4 of about 1-8x109M 1, and an N-terminal amino acid sequence Met-Lys-Val-Leu-Gln-Glu-Pro-Thr-Cys-Val-Ser-Asp-Tyr-Met-Ser-Ile-Ser-Thr-Cys-Glu-Trp.

20. A composition according to claim 17, wherein said IL-4 receptor protein comprises an amino acid sequence consisting essentially of amino acids 1 to 207 depicted in Figure 4A.

21. Use of a purified IL-4 receptor protein according to claim 9 in a binding assay for detecting IL-4 or IL-4 receptor molecules or interaction thereof.

22 . An antibody that is immunoreactive with a mammalian IL-4 receptor protein according to claim 9.

23. A purified mammalian IL-4 receptor protein according to claim 9, for use in inhibiting an IL-4 induced biological response.

24. The use of IL-4 receptor protein according to claim 9 in preparing a medicament for regulating an immune response in a mammal.

25. The use of claim 24, wherein the IL-4 receptor is a soluble human IL-4 receptor and the mammal to be treated is a human.

26. A substantially homogenous biologically active recombinant human interleukin-4 receptor protein substantially free of contaminating endogenous materials and without associated native-pattern glycosylation, which protein has an N-terminal amino acid sequence Met-Lys-Val-Leu-Gln-Glu-Pro-Thr-Cys-Val-Ser-Asp-Tyr-Met-Ser-Ile-Ser-Thr-Cys-Glu-Trp and is capable of binding greater than 0.1 nmole interleukin-4 per nmole of the receptor.

27. A human IL-4 receptor protein according to claim 26, which comprises an amino acid sequence that is greater than 80 % similar to the sequence of amino acid residues 1-800 depicted in Figures 4A, 4B and 4C.

28. A human IL-4 receptor protein according to claim 27, in the form of a glycoprotein.

29. A human IL-4 receptor protein according to claim 27, wherein said protein comprises an amino acid sequence consisting essentially of residues 1-800 depicted in Figures 4A, 4B and 4C.

30. A recombinant expression vector comprising (1) a synthetic or cDNA-derived DNA sequence defined in any one of claims 1 to 5, operably linked to (2) a transcriptional and translational regulatory element derived from a mammalian, microbial, viral or insect gene, said regulatory element including (a) a transcriptional promoter, (b) a sequence encoding suitable mRNA ribosomal binding site and (c) sequences which control the termination of transcription and translation.

31. An isolated DNA sequence encoding a human interleukin-4 receptor protein wherein said protein comprises an amino acid sequence consisting essentially of amino acid residues -25 to 800 or 1 to 800 depicted in Figures 4A, 4B
and 4C.

32. A pharmaceutical composition for suppressing immune response in a human, which comprises an effective amount of the interleukin-4 receptor protein as defined in any one of claims 26, 27, 28 and 29 together with a pharmaceutically acceptable carrier or diluent.

33. An antibody according to claim 22, wherein said antibody is a monoclonal antibody.

34. An antibody according to claim 33, wherein said monoclonal antibody is immunoreactive with a human IL-4 receptor protein having an amino acid sequence selected from residues 1 to 800 depicted in Figures 4A to 4C and residues 1 to 207 depicted in Figure 4A.

35. An antibody according to claim 33, wherein said monoclonal antibody is immunoreactive with a murine IL-4 receptor protein having the amino acid sequence of residues 1 to 785 depicted in Figures 2A to 2C.

36. The use of a purified IL-4 receptor protein according to claim 9 for binding IL-4.

37. An isolated DNA sequence selected from the group consisting of:
a) a DNA sequence comprising at least 60 consecutive nucleotides of the nucleotide sequence presented in Figures 2A-2C; and b) a DNA sequence comprising at least 60 consecutive nucleotides of the nucleotide sequence presented in Figures 4A-4C.

38. An isolated fragment of an IL-4 receptor protein, selected from the group consisting of:
a) a fragment of the murine IL-4 receptor protein of Figures 2A-2C; and b) a fragment of the human IL-4 receptor protein of Figures 4A-4C;
wherein said fragment is capable of binding IL-4.

39. An isolated polypeptide selected from the group consisting of:
a) an immunogenic polypeptide comprising at least 20 consecutive amino acid residues of the sequence presented as amino acids 1 to 785 of Figures 2A-2C; and b) an immunogenic polypeptide comprising at least 20 consecutive amino acid residues of the sequence presented as amino acids 1 to 800 of Figures 4A-4C.

40. A use according to claim 25, wherein said immune response is an asthmatic response.

41. A use according to claim 25, wherein said immune response is an allergic response.

42. A human IL-4 receptor protein according to claim 14 or 16, for use in treating asthma.