CA2177471A1 - Ifn-.gamma.receptor .beta.-chain and derivatives thereof - Google Patents

Abstract

The present invention concerns new receptor subunit polypeptides. More particularly, the invention concerns novel transmembrane proteins which belong to the interferon receptor family and which are species-specific cofactors needed for signal transduction of interferon-.gamma.
(IFN-.gamma.).

Description

~177~71 WO 9S/1~036 PCTIUS9411~277 ~ VK ~CEIAIN ~ND DERlVATrVTS l~REOP
Field o~ the Invention The present invention concerns new receptor subunit polypeptldes.
More particularly, the invention concerns novel transmembrane protelns which belong to the interferon receptor family and which are species-speclEic cofactors needed for signal transduction of interferon-y (IFN-y)-B~ckground o~ th~ Invention Interferons (IFNs) are a diverse group of cytokines exerting a wide variety of biological activities on a wide range of cell types.
There are three known types of interferons, and they are produced by different cel 1 types under different conditions . In response to viral infection, lymphocytes synthesize primarily interferon-o~ (also known as leukocyte interferon), whereas infection of fibroblasts usually induces interferon-l~ ~also known as fibroblast interferon) . Interferons- and -~ are structurally and functionally related proteins, which are collectively referred to as type I interferons. In contrast, IFN-y (immune interferon) is scarcely related to the type I interferons 1n its amino acid sequence and is synthesized by lymphocytes in response to mitogens. ~ence, IFN-y is also referred to as type II interferon.
IFNs-o~ and -~ mediate their biological effects through binding to a presumably common receptor that is expressed ubiquitously [Uze et al., Cell 60, 22~-234 (1990)] . IFN-y binds to a different receptor [Aguet et al., Cel ~ 55, 273-280 (1988) ], but the two signaling pathways involve coromon elements. Receptor binding of IFNs-o and -~ stimulates tyrosine kinase phosphorylation, probably mediated by the non-receptor tyrosine kinase tyk-2 [Velazquez et al., Cell 70, 313-322 (1992) ¦, of at least two cytoplasmic proteins, pll3 and p91, which are then translocated to the nucleus and form a compleY with another latent cytoplasmic protein, p48 [SchIndler et a~., Science 257, 809-813 (1992) ] . This complex binds to a consensus promoter element of IFN-inducible genes (ISRE) and stimulates their transcription [Levy et al., Genes Dev. 3, 1362-1371 (1989) ] . Receptor bLnding of IFN-y also stimulates tyrosine phosphorylation of p91, presumably at the same residues, but mediated through a di~ferent kinase (Schindler et al., supra). In this case, phosphorylated p91 probably complexes with still unidentified proteins [Pearse et al., Proc. Natl. l~cad. Sci. USA 90, 4314-4318 (1993) ], and this complex binds to the GAS sequence found in IFN-y induced genes [~ew et al., Mol. Cell. Biol. 11, 182-191 (1991);
Pell~grini and Schindler, Trends Biochem. $ci (1993) ] . Further WO95/16036 ~1 7 7~ 71 PCT/IJS94/14277 elucidation of how these early slgnaling events are linked to the receptor, however, necessitates the identification of additional constituents of the two receptor systems.
The human IFN-y receptor (huIFN-yR) expressed in mouse cells and 5 vice versa is nonfunctional, even though the binding properties sf the transfected receptor proved indistinguishable from those of the resident functional receptor [Aguet et al., Cell 55, 273-280 (1988)i Gray ~t al., Proc. Natl. Acad. Sci. USA 86, 8497-8501 ~1989); Hemmi et al., Proc Natl. Acad. Sci. USA 86, 9901-9905 (1989) ] . It has been 10 proposed that a so far unidentified species-specific cofactor encoded on human chromosome 21 or mouse chromosome 16 is needed for functionality of the IFN-yR ~Jung et al. Proc. Natl Acad. Sci. USA 84, 4151-4155 (1987), Hibino et al., J. ~Biol. Chem 266, 6948-6951 (1991) ]
It would be desirable to identify the putative species-specific 15 cofactor required for signaling IFN-y biological activity. It would further be desirable to determine the amino acid sequence and the encoding nucleotide sequence of this polypeptide, which would enable its production by recombinant DNA technology or chemical synthesis. It would additionally be desirable to produce functional derivatives, and 20 antagonists of a native-sequence cofactor, which could be used to enhance or block IFN-y biological activity.
8u~ry of the Invention The present invention is based on the cloning and expression of a 25 novel cofactsr required for signal transduction of IPN-y More specifically, a cDNA encoding a novel transmembrane protein was obtained, sequencedi and expressed to produce a polypeptide, which was identified as a species-specific cofactor required for signal transduction of IFN-y, And will hereinafter be designated as IFN-y 30 receptsr ~-chain It is beLieved that this new polypeptide sr a close homologue thereof is also a constituent of other receptors, such as IFN-o~/~ receptor, the erythropoietin (EP0) receptor, the IL-10 and possibly other cytokine receptors.
In one aspect, the present invention concerns an isolated IFN-y 35 receptor ~-chain polypeptide, which is a native IFN-y receptor ~-chain or a functional derivative thereof. In a specific ~nho~ nt, the polypeptide is devoid of a functional transmembrane domain, and optionally of part or whole of the cytoplasmic domain. Certain IFN-y receptor 13-chain polypeptides of the present invention are 40 characterized by comprising the LEVLD sequence motif in their cytoplasmic domains. In a further smbodiment, the IFN-y receptor ~-chain polypeptide is associated with an IFN-y receptor o-chain, with an IFN-o~ or -3 receptor or an EPO receptor and/or is fused to a ~77471 ~0 95/1603G ~ PCTrUS94/14277 heterologous polypeptide. The heterologous polypeptide may comprise an immunoglobulin sequence, which is- preferably fused to a transmembrane domain deleted or inactivated IFN-y receptor ~-chain, to yield a fusisn protein which signals or inhibits IFN-y biological action.
The IFN-y receptor ,~-chain, including functional derivatives, such as fragments thereof (which also may be synthesized by chemical methods) can be fused (by recombinant expression or in vitro covalent methods) to an immunogenic polypeptide and this fusion polypeptide, in turn, used to immunize an animal to raise antibodies against an IFN-y receptor ~3-chain subunit epitope. Anti-IFN-y receptor ¦3-chain antibodies are recovered from the serum of immunized animals.
Alternatively, monoclonal antibodies are prepared from cells of the immunized animal in conventional faShion. Antibodies identified by routine screening will bind to an IFN-y receptor 13-chain but will not substantially cross-react with any other known receptor subunits.
Immobilized anti-IFN-y ~-chain antibodies are useful particularly in the detection (in vitro or in vivo) or purification of IFN-y receptor ~B-chain by passing a mixture contalning a ~-chain over a column to which the antibodies are bound.
Substitutional, deletional, or insertional variants of the IFN-y receptor ~3-chain polypeptides are prepared by in vitro or recombinant methods and screened for immuno-crossreactivity with a native IFN-y receptor ¦3-chain and for IFN-y re~eptor agonist or antagonist activity ~i.e. for the ability to signal or inhibit IFN-y biological activity) .
The IFN-y receptor ¦3-chain polypeptides are also derivatized in vitro to prepare ~ 7(~ 3-chain and labeled ~-chain, particularly for purposes of detection of IFN-y receptor ~-chain or its antibodies, or for affinity purification of anti-IFN-y receptor ~-chain antibodies.
The IFN-y receptor ~-chain polypeptides and the antibodies specifically binding such polypeptldes are formulated into physiologically acceptable vehicles, especially for therapeutic use.
Such vehicles include sustained-release formulations.
In a further aspect, the present invention concerns an isolated nucleic acid molecule encoding an IFN-y receptor ~-chain polypeptide.
Such nucleic acid molecule preferably comprises a nucleotide sequence able to hybridize, under stringent conditions, to the complement of a nucleotide sequence encoding a native IFN-y receptor ~-chain, such as the murine IFN-y receptor ~-chain having the amino acid sequence shown in Figure 2A, or the native human IFN-y receptor ~-chain having the amino acid sequence shown in Figure 5.
In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence greater than about 65~ homologous with the amino acid sequence 2177~7~
WO 9S/16036 PCT/US9.111~1277 shown ln Figure 2A or with the amino acid sequence shown ln Figure 5.
In a further embodlment, the nucleic acid molecule is selected from the group consisting of:
(a) a cDNA clone having a nucleotide sequence derived from the 5 coding region of a native IFN-y receptor ~-chain genei (b) a DNA sequence able to hybridize under strIngent conditions to a clone of (a); and (c) a genetic variant of any of the DNA sequences of (a) and (b) which encodes a protein possessing a biological property of a 10 naturally occurring IFN-y receptor ~-chain molecule.
In a further aspect, the invention concerns an expression vector comprising a nucleic acid molecule encoding an IFN-y receptor ~-chain polypeptide operably linked to control sequences recognized by a host cell transformed with the vector.
In another aspect, the invention concerns a host cell transformed with the vector above.
In a different aspect, the invention concerns a method of uslng a nucleic acid molecule encoding an IFN-y receptor 13-chain comprising expressing it in a cultured host cell transformed with a vector comprising the nucleic acid molecule to be expressed operably linked to control sequences recognized by the host ceLl transformed with the vector, and recovering IFN-y receptor ~-chain from the host cell.
In a further aspect, the invention concerns a method for producing an IFN-y receptor ~-chain comprising inserting into the DNA
of a cell containing nucleic acid encoding the IEN-y receptor ~-chain a transcription modulatory element in sufficient proximity and orientation to the nucleic acid molscule to influence its transcription. The DNA of the cell in which the IFN-y receptor ~-chain is produced may additionally contain DNA encoding an IFN-y receptor o~-chain or other cytokine receptors or their chains/subunits.
The invention further concerns a method of determining the presence of an IFN-y receptor ~-chain, comprlsing hybridizing DNA
encoding the ~-chain to a test sample nucleic acid and determining the presence of IFN-y receptor ~-chain DNA.
In a further aspect, the invention concerns a method for obtaining cells having increased or decreased transcription of a nucleic acid encoding an IFN-y receptor ~-chain, comprising:
(a) providing cells obtaining the IFN-y receptor ¦3-chain:
(b) introducing into the cells a transcription modulating element: and (c) screering the cells for a cell in which the transcription of the ,~-chain nucleic :acid is increased or decreased.

~177~7~
WO 9S/16036 PCTll~S94/1-1277 In a still further aspect, the invention concerns an antagonlst of a native IEN-y receptor 13-chai~- Such antagonists are capable of blocking the biological action of IFN-y and other natlve polypeptldes (such as lnterleukins, EPO, IFNs-~/13), the signal transduction of which involves a native IEN-y receptor ~-chain or a close homologue (functional derivative) thereof In yet another aspect, the invention concerns a pharmaceutical compositlon comprising an IFN-y receptor ¦3-chain polypeptide, or an antagonist of such polypeptide, or an antibody specifically binding an IFN-y receptor rz-chain polypeptide or an antagonist thereof, and a pharmaceutically acceptable carrier Br$e~ Description of the Dr~wing~
Figure l IFN-y-inducible Tac antigen reporter construct and its expression in COSN 31 cells stably expressing the murine IFN-y receptor, in the absence or presence of the novel murine IFN-y receptor 3-chain (A) The reporter construct pUMS (GT~ ,-Tac was designed for tight IFN-y inducible expression of the human Tac antigen An artificial promoter consisting of the hexamer repeat (GAAAGT)~ followed by the TATA box and the Cap site of the rabbit ~-globln promoter (R3G) was placed in front of a cDNA encoding the human Tac antigen. An sv40 enhancer was placed about 100Dbp upstream of the artificial promoter UMS, transcriptional stop site(s) [McGeady et zl, DNA S, 289-298 (1986) ]; pBR, pBR322-derived segment extending from gl72 to 4178 ~Watson, Gene 70, 399-403 (1988) ] . (B) COSN 31 cells stably transfected with the murine IFN-y receptor expression plasmid and the Tac antigen reporter construct were monitored or IFN-y-inducible Tac antigen expression by cytofluorometry. Cells were incubated for 48 hours at 37OC with 200 U/ml of either human (solid bold line) or murine IFN-y (dotted bold line), or left untreated (thin line) Background staining of the FITF-conjugated rabbit-anti mouse IgG F (ab ' ) z antibody was ~ t~r~i ne~l in the absence of anti-Tac antigen antibodies and is represented as a thin dotted line (C) Tac antigen expression in COSN 31 cells transiently reconstituted with a cDNA encoding the murine IFN-y receptor ~ chain COSN 31 cells were transfected with the expression plasmid pAGS-Cl9 encoding the murine IFN-y receptor (muIFN-yR) ~-chain. Transfected cells were incubated for 48 hours at 370C
with 200 U/ml of either human (solid bold line) or rlurine IFN-y (dotted bold line) or left untreated (thin line) Fxpression of Tac antigen and background staining was monitored as above Background staining was consistently slightly increased in transiently transfected cells as compared to untransfected cells (B) .

WO 95/16036 217~ ~ 71 PCTIUS94/14277 Figure 2 ~A) Nucleotidé and inferred amino acid sequences of the muIFN-yR 3-subunit .(SEQ. ID..~Os: l and 2) . ~B) Amino acid sequence al1gnment of the presumed extracellular portion of the muIFN-yR ~-chain ~SEQ. ID. NO: 6) with the duplicated extracellular domains of the type I IFN- receptor ~muIFN--R1/R2, SEQ. ID. NOs: 3 and q), and with the known llgand binding chain of muIFN-yR ~muIFN-yR, SEQ. ID
NO: 5), identified lt as a member of the IFN receptor family [Bazan, Cell 6l, 753-754 ~l990)~
Figure 3. Functionzlity of the muIEN-yT ¦~-chain in HEp-2 cells 0 expressing the muIFN-yR a-chain. ~A,B) Cytofluorometry of IFNy-induced MHC class I ~A) or MHC class II ~B) antigen expresslon in hEp-2 cells permanently transformed with the muIFN-yR -chain alone ~Hep243.7) or together with the muJrll-yR ¦3-chain IHep-2#6) . Cells were incubated for 60 or 84 hours at 370C with 200 U/ml of either human ~solid bold line) 1~ or murine IFN-y ~dotted bold line) or left untreated ~thin line) .
Background staining of the FITC-conjugated rabbit-anti mouse IgG F~ab'), antibody was determined in the absence of anti-Tac antigen antibodies and is represented as a thin doted line. ~C) Northern blot analysis of mRNA from HEp-2 cells expressing the muIFN-yR -chain alone ~HEp-

2#43.7~ or together with the muIFN-yR ~-chain ~HEp-2#6) using IRF-1 cDNA as a hybridization probe. Cells were incubated for 24 hours at 370C in medium in the absence ~a) or presence of 200 U/ml of either human ~b) or murine IFN-y ~c) . Ten ug of total RNA were used per lane and hybridization was carried out according to standard procedures using a random labeled ~PCT-derived IRF-l probe amplified from total C05-7 cell RNA using oIigonucleotide primers specific for the conserved regions between murine and human IRF-1. Hybridization with the rat GAPDH cDNA prsbe ~Fort et al., Nucl. Acids. Res. l3, 1431-1442 (1985)]
revealed no significant difference in the amount of RNA loaded.
(D) Antiviral response of HEp-2 #43.7 (squares) versus hEp-2#6 cells (circles) upon treatment with human (open symbols) or murine (closed symbols) IFN-y. Cells were incubat0d in 96-wells (2 x 10' cells/well) for 24 hours at 370C with 3-fold serial dilutions of L~ ` in.nt human or muIFN-y and subsequently challenged with vesicular stomatitis virus (VsV; Indiana strain) at a multiplicity of infection of lO '. The cytoplasmic effect (CPE) of VSV was quantified after 36 hours at 37C
by staining with crystal violet and determining A,g~. Full protection (lOO~) from the CPE corresponds to the difference between the absorbance of untreated, uninfected cells and untreated, VSV-infected cells. Indicated values are means + SD of trlplicates. (E) Growth inhibitory effect of murine (closed symbols) versus human (open symbols) IFN-y in HEp-2 cells expressing both muIFN-yR subunits. The ~77~7~
t~o 95/16036 PCT/US94/14277 cells/well, cultured for 72 hours at 37OC at varlous concentrations of human (open symbols) or murine (closed symbolsl and counted. Indicated values are means + SD of duplicates.
Figure 4. Nucleotide sequence of human IFN-yR ~-chain (SEQ. ID.
5 NO: 7) Figure 5. Deduced amino acid sequence of human IFN-R -chain (SE:Q. ID. NO: B) .
D--t~il~d Description o~ the Invention A. Definitions _ _ IFN-y receptors have been purified from different human [Aguet, M. ~ Merlin, G., J. Bxp. Med. 165, 988-999 (1987); Noviclc, D. et al., J. Biol. Ch~m. 262, 8483-8487 ~1987~; Calderon, J. et al., Proc. Natl.
Acad. Sci. USA 85, 4837-4841 (1988)] and murine IBasu, M. et al., Proc.
Natl. Acad. SC1: USA 85, 6282-6286 (1988) ~ cell types, and have been characteri:~ed as 90- to 95-kDa single chzin integral membrane glycoproteins that display certain structural heteLogeneity due to cell speci~ic glycosylation. The primary sequence of human IFN-y receptor has been elucidated by Aguet et al, Cell 55, 273-280 (1988), who cloned, expressed and sequenced a 2.1 kb human IFN-y receptor cDNA from a Raju c~ll expression library prepared in Agtll. The cloning and expression of the cDNA for the murine interferon gamma (IFN-y) receptor was reported by Gray, P. W. et al., Proc. Natl. Acad. Sci. USA 86, 8497-8501 (1989), and by Hemmi et al., Proc. Natl. Acad. Sci. USA 86, 9901-9~05 ~1989). For the purpose of the present lnvention, the terms "interferon-y receptor", "IFN-y receptor", "interferon-y receptor ~-chain" and "IFN-yR o-chain" are used interchangeably and refer to a family of polypeptide molecules that comprise the human IFN-y receptor reported by Aguet et al. (1988), supra, the murine IFN-y receptor reported by Gray et al. (1989), supra, or Hemmi et al., supra, their equivalents in any animal species, and the functional derivatives of such native sequence IFN-y receptors.
The terms "IFN-y receptor ¦3-chain", "IFN-yR ,~-chain", "IFN-y receptor ~-chain polypeptide", "IFN-y receptoL ~-subunit", and their grammatical variants derine the native murine IFN-y receptor ~-chain having amino acids 1 thLough 314 as set forth in Figure 2A, the native human IFN-y receptor ~3-chain as shown in Figure 5, their equivalents in any animal species, and functional derivatives of such native sequence polypeptides .
A "functional derivative" of a native polypeptide is a compound having a qualitatlve biological activity in com~on with the native polypeptide. A functional derivative of an IFN-y receptor c~-chain polypeptide is a compound that has a qualitative biological activity in WO 95116036 ~1 ~ 7 ~ 71 PCT/US94/1.1277 common with the native human IFN-y receptor of Aguet et al supra or with the native murine IFN-y receptor of Gray et al. supra, or i~emm~
et al., supra. A functional derivative of an IFN-y receptor ~-chain has a qualitative biological activity in common wlth the native murine 5 IFN-y receptor ¦3-chain of Figure 2A or with the native human IFN-y receptor ~-chain of Figure 5. "Functional derlvatives" include, but are not limited to, fragments of native IFN-y receptor ~-chain polypeptides ~or ~Y-chains) from any animal species (including humans), and derivatives of native (human and non-human) IFN-y receptor ,B-chain 10 polypeptides (or o~-chains) and their fragments, provided that they have a biological activity In com~non wlth a native IFN-y receptor ~-chain (or o:-chain) . "Fragments" comprise regions within the sequence of a mature native IFN-y receptor o~- or ~5-chain. The term "derivative" lS
used to define amiDo acid sequence and glycosylation variants, and 15 covalent modifications of a native IFN-y receptor o~- or ¦3-chain polypeptide, whereas the term "variant" refers to amino acld sequence and glycosylation variants witbin this definltion. Preferably, the functional derivatives are polypeptides which have at least about 65 amino acid sequence identity, more preferably about 75~ amino acid 20 sequence identity, even more preferably at least about 85~ amino acid sequence identity, most preferably at least about 95~ amino acid sequence identity of a native sequence IFN-y receptor - or ~-chain.
Identity or homology with respect to an IFN-y receptor ot- or ~3-chain is defined herein as the percentage of amino acid residues in the 25 candidate sequence that are identical with the residues of a corresponding native IFN-y receptor (x- or ~-chain, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor 30 insertions shall be construed as reducing identity or homology.
"Biological activity" in the context of the definition of "functional derivatives" is de~ ed as either l) immunological cross-reactivity with at least one epitope of a native IEN-y receptor o~- or ~-chain, or 2~ the possession of at least one adhesive, regulatory or 35 effector function qualitatively in common with a native IFN-y receptor o~- or ~-chain.
Immunologically cross-reactive as used herein means that the candidate (poly) peptide is capable of competitively inhibiting the qualitative biological. activity of a native IFN-y o~- or ~-chain having 40 this activity with polyclonal antibodies or antisera raised against the known active molecule. Such antibodies and antisera are prepared in conventional fashion by injecting an animal such as a goat or rabbit, for example, subcutaneously with the known native IFN-y receptor ~- or ô

~ 7 1 WO 95116036 PCTlUSg4/14277 ~-chain in complete Feud's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freud' s .
Included within the scope of the IFN-y receptor ~-subunit herein is the murine IFN-y receptor ~-subunit as set forth ln Figure 2A, with 5 or without the 18 amino 2cids signal sequence, and with or without the initiating methionine, as well as fragments, glycosylation, unglycosylated or completely or partially deglycosylated variants, amino acid sequence variants and covalent derivatives of the native murine receptor ~-chain, provided that they possess a biological 10 activity in common with the native murine IFN-y ~-chain of Figure 2A.
Further included within the scope of the IEN-y receptor ~-subunits herein is the human IFN-y receptor ,~-subunit as set forth in Figure 5, with or without the signal sequence~ and with or without the inltiating methionine, as well as fragments, glycosylation, unglycosylated or 15 completely or partially deglycosylated variants, amino acid sequence variants and covalent derivatives of the native human receptor ~-chain, provided that they possess a biological activity in common with the native human IFN-y ~-chain of Figure 5. While the native IFN-y receptor ~-chains are membrane bound polypeptides, soluble forms, such 20 as those forms lacking a functional transmembrane domain, are also included within this definition. The IFN-y receptor ~-chain fragments within the scope of the present invention preferably have at least lS
and preferably at least ~0 amino acid residues, or have at least about S amino acid residues comprising an immune epitope or other 25 biologically active site of the IFN-y receptor ~-subunit.
The term "amino acid sequence variant" refers to molecules wlth some differences in their amino acid sequences as compared to a native sequence IEN-y receptor ~-chain or a fragment thereof. Ordinarily, the amino acid sequence variants will possess at least about 65 ~, 30 preferably at least about 75~, more preferably at least about 85~, most preferably at least about 95~ homology with the extracellular domain of a native IFN-y receptor ¦3-chain or, alternatively, are encoded by DNA
capable, under stringent conditions, of hybridizing to the complement of the extracellular domain of a native IFN-y receptor ~-chain. A
35 prefelred group of the amino acid sequence variants retains the sequence motif within the native sequence identified as responsible for signaling IFN-y biological action, such as the LEV3:D sequence motif in the cytoplasmic domain of the murine IFN-y receptor ~-chain sequence, or have only conservative amino acid alterations within this region.
40 The amino acid alterations may be substitutions, insertlons, deletions or 2ny desired corbinations of such changes in a native amino 2cid sequence .
_g _ ~77~1 O

Substitutional variants are those that have at least one amino acid residue in a native sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. .
Insertional variants are those with one or more amino acids inserted immediately ad]acent to an amino acid at a particular position in a native amino acid sequence . Im~ediately ad~ acent to an amino acid means connected to either the ~x-carboxy or o~-amino functional group of the amino acid.
Deletional variants are those with one or more amino acids in the native amino acid sequence removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the molecule.
The term "glycosylation variant" lS used to refer to an IFN-y receptor Q-chain molecule having a glycosylation profile different from that of a native IFN-y receptor ¦~-chain. Glycosylation of polypeptides is typicaLly either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side-chain of an ~sparagine residue. The tripeptide sequences, asparagine-X-serine and asparagine-X-threonine, wherein X is any amino acid except proline, are recognition sequences -for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be involYed in O-linked glycosylation.: Any difference in the location and/or nature of the carbohydrate moieties present ln a variant or fragment as compared to its native counterpart is within the scope herein.
The glycosylation pattern of native polypeptides can be ~:~.t~.rmi n~.~ by well known techniques of analytical chemistry, including PPAE chromatography [Hardy, M.R. et al., Anal. i3iochem. 170, 54-62 ~1988 ) ~, methylatlon analysis to determine glycosyl-linkage composition [Lindberg, b., Meth. Enzymol. 28 l78_195 (1972J; Waeghe, T.J. et al., Carbohydr. Res. 123, 231-304 51983~ 3, NMR spectroscopy, mass spectrometry, etc.
"Covalent derivatives" include modifications of a native IFN-y receptor ~-chain or a fragment thereof with an organic proteinaceous or non-proteinaceous derlvatizing agent, and post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reactlng with selected side-chains or terminal _ _ _ _ _ _ . . . .. _ _ _ _ _ _ _ _ . .. _ .

%~ ~747:~
WO 95116036 - PCTIUS94/1~277 residues, or by harnesslng rn~hani~ c of post-translational modifications that function in selected recombinant host cells.
Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide_ Glutaminyl and 5 asparaginyl residues are frequently post-translationally deamidated to the corresponding ylutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues may be present in the IFN-y receptor 13-chain molecules as defined in the present invention. Other post-translational 10 modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the -amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)] .
"Isolated" IFN-y receptor ~-chain nucleic acid or polypeptide is a nucleic acid or polypeptide that i5 identifled and separated from contaminant nucleic acids or polypeptides present in the animal or human source of the IFN-y receptor ,~-chain nucleic acid or polypeptide.
~he nucleic acid or polypeptide may be labeled for diagnostic or probe purposes, using a label as described and defined further below in discussion of diagnostic assays. The isolated IFN-y receptor ~-chain may be associated with any IFN-y receptor -chain, or alternatively, truncated forms of the - and 13-chains may be associated with each other, or with a full length form of the other chain.
IFN-y receptor ~-chain "nucleic acid" is defined as RNA or DNA
containing greater than about lS bases that encodes an IFN-y receptor ~-chain as hereinabove defined, is complementary to a nucleic acid molecule encoding an IFN-y receptor ~-chain, hybridizes to such nucleic acid and remains stably bound under stringent conditions, or encodes a polypeptide sharing at least about 65 qi sequence identity, preferably at least about 75~ sequer~ce identity, more preferably at least about 85~ sequence identity, most preferably at least about 95~ sequence identity with a native IFN-y receptor ~-chain polypeptide, and preferably with the translated amino acid sequence shown in Figure 2A
herein.
"Stringent conditions" are those that (l) employ low ionic strength and high temperature for washing, for example, O. 015 sodium chloride/O . OOlS M sodium citrate/O . l~ sodium dodecyl sulfate at 50oc, or ~2) employ during hybridization a denaturing agent, such as formamide, for example, 50~ (vol/vol) formamide with O.l~ bovine serum albumin/O . lg Ficoll/O . l~ polyvinylpyrrolidone/50 nM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,~ 75 mM sodium citrate at 420C. Another example is use of 50~ formamide, 5 x SSC (0.75 M NaCl, WO 95/16036 ~17 7 ~ 71 PCT/US9~1/1-1277 O.075 M sodium citrate), 50 mM sodium phosphate (pH 6/B), 0.1% sodium pyrophosphate, 5 x Denhardt's so~Lltlon, s~nicated salmon sperm DNA l50 ug/ml), 0.1% SD5, and 10% dextran sulfate at 420C, with washes at 42C
in O 2 x SSC and O.1% SDS.
The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable ~or prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and possibly, other as yet poorly understood sequences .Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancer.
Nucleic ~cid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. Por example, DNA for a presequence or a secretory leader 15 operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide: a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequencei or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, ir the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.
The terms "DNA sequence encoding", "DNA encoding" and "nucleic acid encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic i-cid. The order of these deoxyribonucleotides deter~nines the order of amino acids along the polypeptLde chain. The DNA sequence thus codes for the amino acid sequence .
The terms "replicable expression vector" and "expression vector"
refer to a piece of DNA, usually double-stranded, which may have inserted into it a plece of foreign DNA. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell.
The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate .
independently of the host chromosomal DNA, and several copies of the vector and its inserted (foreign) DNA may be generated. In addition, the vector contains the necessary elements that permit translating the foreign DNA into a polypeptide. Many molecules of the polypeptide encoded by the foreign DNA can thus be rapidly synthesized_ .

~7~4~1 In the context of the present invention the expressions "ceLl", "cell line'-, and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed ~host~ cells" include the primary subject cell and 5 cultures derived therefrom without regard for the number of transfers.
It is also understood that all progeny may not be precisely identlcal in DNA content, due to deliberate or inadvertent mutatlons. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct 10 designations are intended, it will be clear irom the context.
An "exogenous" element is defined herein to mean nucleic acid sequence that is foreign to the cell, or homologous to the cell but in a position within the host cell nucleic acid ln which the element lS
ordinarily not found.
Antibodies ~Abs) and immunoglobulins ~Igs) are glycoproteins having the same structural characteristics. While antibodies exhiblt binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, 20 produced at low levels by the lymph system and at increased levels by myelomas .
Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150, 000 daltons, composed of two identical light ~L) chains and two identical heavy (H) chains.
25 Each light ch~in is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes Each heavy and light ch~in also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain ~V~) followed by a number 30 of constant domains. Each light chain has a variable domain at one and ~VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed 35 to form an interface between the light and heavy chain variable domains [Clothia et al., J. Mol. Biol. 186, 651-663 ~1985); Novotny and Haber, Proc. Natl. Acad. sci. USA 82, 4592-4596 ~1985) ] .
The variability is not evenl~ distributed through the variable regions of antibodies. It is concentrated in three segments called 40 complementarity determining regions ~CDRs) or hypervariable regions both in the light chain and the heavy chain variable regions. The more highly conserved portions of variable domains are called the framework ~FR) . The variable domains of native heavy and light chains each WO 95/16036 2 ~ 7 7 4 7 1 PCTI~S94/1.J277 comprise four FR regions, largely adoptiDg a ~-sheet configuration, connected by three CDRS, which f~rm loops c~nnecting, and in some cases forming part of, the ~-sheet structure The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs 5 from the other chain, contribute to the formation of the antlgen binding site of antibodies [see Kabat, E.A. et al., Sequences of Protelns of Immunological Interest National Institute of Health, bethesda, MD (1987)1. The constant domains are not involved directly 1n binding an antibody to an antigen, but exhibit varlous effector 10 functions, such as participation of the antibody in antibody-dependent cellular toxicity.
Papain digestion of antibodies produces two identlcal antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual "Fc" fragment, whose name reflectr its 15 ability to crystallize readily. Pepsin treatment yields ~n F(ab')~
fragment that has two antigen combining sites and is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete =
antigen recognition and binding site. This region consists of a dimer 20 of one heavy and one llght chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the Vj_VL dimer. Collectively, the six CDRs confer antigen binding specificity to :the antibody. However, even a single variable 25 domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (C~l) of the heavy chain. Fab' 30 fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain C~l domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments 35 originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other, chemical couplings of antibody f ragments are also known .
The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct g0 types, called kappa and lambda (A), based on the amino acid sequences of their constant domains.

W0 9~116036 ~ 4 7 ~ PCTIUS9.1/1~277 Depending on the amino acid sequence of the constant region of their heavy chalns, immunoglobulirs can be assigned to different classes There are five ma30r classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into 5 subclasses ~isotypes), e.g. IgG-l, IgG-2, IgG-3, and IgG-4; IgA-l and IgA-2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called o~, delta, epsilon, y, and u, respectively The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
lO IgA-l and IgA-2 are monomeric subclasses of IgA, which usually is in the form of dimers or larger polymers. Immunocytes in the gut produce mainly polymeric IgA (also referred to poly-IgA including dimers and higher polymers) Such poly-IgA contains a disulfide-linked polypeptide called the "joining" or "J" chain, and can be transported 15 through the glandular epithelium together with the J-chain-containing polymeric IgM (poly-IgM), comprising five subunits.
The term "~tibody" is used in the broadest sense and specifically covers single anti-IFN-y receptor ~3-chain monoclonal antibodies (including agonist and antagonist antibodies) and anti-IFN-y 20 receptor ~-chain antibody compositions with polyepitopic specificity.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that 25 may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a 30 single det~rmin=nt on the antigen. In addition to their specificity, the lrLonoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other ir~lmunoglobulins.
The monoclonal antibodies herein include hybrid and recombinant antibodies produced by splicing a variable . (including hypervariable) 35 domain of an anti-IFN-y receptor ~3-chain antibody with a constant domain (e.g. "humanized" antibodies), only one of which is directed against IFN-y receptor ~i-chain, or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with heterologous proteins, regardless of species of origin or 40 immunoglobulin class Dr subclass designation, as well as antibody fragments (e.g., Fab, ~(ab')2, and Fv), so long as they exhibit the desired biological activity. [See, e.g. Cabilly, et al., U.S. Pat. No.

~177~ ~
WO 95~16036 7 1 PCTIUS94/142~7 4, al6, 567; Mage & I,amoyi, in Monoclonal Antibody Production Techniques and Applicatisn:i, pp.79-97 (Marcel ~Dekker, Inc., New York, 1987~ .3 Thus, the modifier "monoclonal" indicates the character of the antibody as being obtalned from a substantially homogeneous population 5 of antibodies, and is not to be construed as requiring production of the antibody by any p~rticular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler & Milstein, Nature 256:495 (1975), ~or may be made by recorbinant DNA methods [Cabilly, èt al., U.S. Pat. No. 4,816,5673 .
"Humanized" forms of non-human (e.g. murine) antibodies are immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other anti;gen-binding subsequences of antibodies) which contain minimal sequence derived from non-human 15 immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary ~ rrinin7 region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and 20 capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by cQrresponding non-human residues.
Furthermore, humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optlmize 25 ~ntibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The 30 humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin .
B. Isolation of DNA encodlnq IFN-y receptor ~-chain For the purpose of the present invention, DNA encoding an IFN-y 35 receptor ~-chain can be obtained from any cDNA library prepared from tissue believed to possess the IFN-y receptor ~-chain mRNA and to express it at a detectable level. For erample, a cDNA library prepared from mouse Ei-cell leukemia cells, such as that described in the examples, is a good source o~ IFN-y receptor ~3-chain cDNA. The IFN-y 40 receptsr 13-chain gene can also be obtained from a genomic library, such as a human genomic cosmid library.
Identification of IFN-y receptor ¦3-chain DNA is most conveniently accomplished by probing human or other mammalian cDNA or genomic 7 :~
WO 9~/16036 PCTNS9411.1277 libraries by labeled ollgonucleotide sequences seleGted from the 5-chain sequence depicted in Figure_2A in accord with known crlterla, among which is that the sequence should be sufficient in length and sufficiently unambiguous that false positives are minlmized.
Typical~y, a '2P-labeled oligonucleotide having about 30 to 50 bases is sufficient, particularly if the oligonucleotide contains one or more codons for methionine or tryptophan. Isolated nucleic acid will be DNA
that is identified and separated from contaminant nucleic acid encoding other polypeptides from the source of nucleic acid. The nucleic acid may be labeled for diagnostic purposes.
An alternative means to isolate the gene encoding an IFN-y receptor ~-chain DNA is to use polymerase chain reaction (PCF~) methodology as described in U.S. Patent No. 4, 683,195r issued 28 July 1987, in section 14 of Sambrook et al., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press. New York, 1989, or in Chapter 15 of Current Protocols in Molecular Biology, Ausubel et al. eds., Greene Publishing Associates and Wiley-Interscience 1991_ Another alternative is to chemically synthesize the gene encoding an IFN-y receptor ~-chain using one of the methods described in Engels and Uhlmann, Agnew. Chem. Int. Ed. Fngl. 28, 716 ~1989) . These methods include triester, phosphite, phosphoramidite and H-phosphonate methods, PCP~ and other autoprimer methods, and oligonucleotide syntheses on solid supports.
C. Amino Acid Sequence Variants of IFN-y receptor ~-chain Amino acid sequence variants of IFN-y receptor ,~-chain are prepared by methods known in the art by introducing appropriate nucleotide changes into the IFN-y receptor r2-chain DNA, or by in vitro synthesis of the desired polypeptide. There are two principle variables in the construction of amino acid sequence variants: the location of the mutation site and the nature of the mutation. With the exception of naturally occurring alleles, which do not require the manipulation of the DNA sequence encoding the IFN-y receptor r2-chain, the amino acid sequence variants of IFN-y receptor ~-chain are preferably constructed by mutating the DNA, either to arrive at an allele or an amino acid sequence variant that does not occur in nature.
In general, the mutations will be created within the extracellular domain of a native IFN-y receptor ~3-chain. Sites OL regions that appear to be important for the signal transduction of IFN-y or another polypeptide (e . g . cytoklne) the signal transduction of which involves the activation of IFN-y receptor ~-chain, will be selected in in vitro studies of biological activity, such as the antiviral response of IFN-y. Sites at such locations will then be modified in series, e.g. by 21~7~71 ~

(1) substituting first with conservative choices and then wlth more radlcal selectlons depenslng upon- the results achieved, ~2~ deletlng the taryet residue or residues~ or (3) inserting residues of the same or different class adjacent to the located site, or comblnatlons of 5 options 1-3.
one helpful technique is called "alanine scanning" (rl~nnin7h~m and Wells, science 244, 108I-1~085 [1989] ) . Here, a residue or group of target residues is identiiied and substituted by alanlne or polyalanine. Those domains demonstratlng functional sensitivity to the 10 alanine substitutions are then reflned by introducing further or other substituents at or for the sites of alanine substitution.
After identifying the desired mutatlon (s), the gene encoding an IFN-y r~ceptor ¦3-chain variant can be obtained by chemlcal synthesls as hereinabove described~.
More preferably, DNA encoding an IFN-y receptor ~-chain amino acid sequence variant is prepared by site-dlrected mutagenesis of DNA
that encodes an earlier prepared variant or a nonvariant version of IFN-y receptor ~-chain. Site-dirested (site-specific) mutagenesis allows the productlon of IFN-y receptor 13-chain variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of ad]acent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. ~ypically, a primer of about 20 to Z5 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered. In general, the techniques of site-specific mutagenesis are well known in the art, as exemplified by publications such as, Edelman et al., DNA 2, 183 (1983) . As will be appreciated, the site-specific mutagenesis technique typically employs a phage vector that exists in both a single-stranded ~nd double-stranded fsrm. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage, for example, as disclosed by Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam (1981). This and other phage vectors are commercially available and their use is well )cnown to those s3cilled in the art. A versatile and efficient procedure for the construction of oligodeoxyribonucleotide directed site-specific mutations in DNA frayments using M13-derived vectors was published by Zoller, M.J. and Smith, M., Nucleic Acids Res.
10,_6~87_6500 [19823 ) . Also, plasmid vectors that contain a single-stranded phage origin of replication (Veira et al., Meth. En~ymol. 153,

3 ~19873 ) may be employed to obtain single-stranded DNA.
Alternatively, nucleotide substitutions are introduced by synthesixing ~7~71 ~0 95ll6036 PCT/US94/lJ~77 the appropriate DNA fragment in vitro, and ampllfying lt by PCR
procedures known in the art.
In general, site-specific mutagenesis herewith is performed by first obtaining a single-stranded vector that includes withln its sequence a DNA sequence that encodes the relevant protein. An oligonucleotlde primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al., Proc Natl. Acad. Sci. USA 75, 5765 ~1978) . This primer is then annealed with the single-stranded protein sequence-containing vector, and subjected to DNA-polymerizing enzymes such as, E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate host cells such as JPlO1 cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement. Thereafter, the mutated region may be removed and placed in an appropriate expression vector for protein production.
The PCR technique may also be used in creating amino acid sequence variants of the IEN-y receptor ~-chain. When small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template. Eor introduction of a mutation into a plasmid DNA, one of the primers is designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer must be identlcal to a stretch of sequence of the opposite strand of the plasmid, but this sequence can be loc~ted anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer i5 located within 200 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primers can be easily sequenced. PCR amplification using a primer pair like the one ~ust described results in a population of DNA fragments that differ at the position of the mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone.
If the ratio of template to product material is extremely low, the vast majority of product DNA fragments incorporate the desired - 40 mutation(s). This product material is used to replace the corresponding region in the plasmid that served as PCR template using standard DNA technology. Mutations at separate positions can be introduced simultaneously by either using a mutant second primer or _ Lg_ ~177~ 71 WO 95/16036 PCT/US94/1~277 performing a second PCP with different mutant prlmers and llgating the two resulting Pc~ fragments simul~aneously to the vector fragment in a three (or more)-part ligatlon.
In a specific example of PCR mutagenesis, template plasmid DNA (1 5 ug) is lineari~ed by dlgestion with a restrict1on endonuclease that has a unique recognition site in the plasmid DNA outside of the region to be amplified. Of this material, 100 ng is added to a PCR mixture containing PCR buffer, .which contains the four deoxynucleotide tri-phosphates and is included in the GeneAmp~ kits (obtained from Perkin-10 Elmer Cetus, Norwalk, CT and Emeryville, CA), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 ul. The reaction mixture is overlayered with 35 ul mineral oil. The reaction is denatured for 5 minutes at 100C, placed briefly on ice, and then 1 ul Thermus aquaticus (Taq) DNA polymerase (5 units/ 1), purchased from 15 Perkin-Elmer Cetus, Norwalk, CT and Emeryville, CA) is added below the mineral oil layer. The reaction mixture is then inserted into a DNA
Thermal Cycler (purchased from Perkin-Elmer Cetus) programmed as f ollows:
2 min 550C, 20 30 sec. 720C, then 19 cycles of the following:
30 sec 9~oC, 30 sec. 55C, and 30 sec. 720C.
At the end of the program, the reaction vial is removed from the thermal cycler and the aqueous phase transferred to a new vial, extracted with phenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA is recovered by standard procedures. This material is subsequently subjected to appropriate treatments for insertion into a 3 0 vector .
Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al. [Gene 34, 315 (1905) ~ .
The starting material is the plasmid (or vector) comprising the IFN-y receptor 13-chain DNA to be mutated. The codon(s) within the IEN-y receptor ,~-chain to be mutated are identified. There must be a unique restrictLon endonuclease site on each side of the identified mutation site (s) . If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the IFN-y receptor ~-chain DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to lineari~e it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction site but cDntaining the desired mutation(s) is synthesized ~7~71 using standard procedures. The two strands are synthesized separately and then hybridi~ed together using standard tcrhniq~cq This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3 ' and 5 ' ends that are compatible with the ends of the linc~rl7~ plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated I~N-y receptor ~-chain DNA sequence.
Additionally, the so called phagemid display method may be useful in making amino acid sequence variants of IFN-y receptor ~-chains of the present invention. This method involves ~a~ constructing a replicable expression vector comprising a first gene encoding an IFN-y receptor ~-chain to be mutated, a second gene encoding at least a portion of a natural or wild-type phage coat protein wherein the first and second genes are heterologous, and a transcrLption regulatory element operably linked to the first and second genes, thereby forming a gene iusion encoding a fusion protein; ~b) mutating the vector at one or more selected positions within the first gene thereby forming a family of related plasmidsi (c) transforming suitable host cells with the plasmids; (d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein: ~e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles ~nt~inin1 at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle; ~f) contacting the phagemid particles with a suitable antigen so that at least a portion of the phagemid particles bind to the antigen; and ~g) separating the phagemid particles that bind from those that do not.
Steps (d) through (g) can be repeated one or more times Preferably in this method the plasmid is under tight control of the transcription regulatory element, and the culturing conditions are adjusted so that the amount or number of phagemid particles displaying more than one copy of the iusion protein on the surface of the particle is less than about l~. Also, preferably, the amount of phagemid particles displaying more than one copy of the fusion proteln is less than lO~ of the amount of phagemid particles displaying a single copy of the fusion protein. Most preferably, the amount is less than 20~ Typically in this method, the expression vector will further contain a secretory signal sequence fused to the D~A encoding each subunit of the - 40 polypeptide and the transcription regulatory element will be a promoter system Preferred promoter systems are selected from lac Z, ArL, tac, T7 polymerase, tryptophan, and alkaline phosphatase promoters and combinations thereof_ l~lso, normally the method will employ a helper ~1~7471 WO 95/l6036 PCT/US94/1~77 phage selected from M13K07, M13R408, M13-VCS, and Phl X 174. The preferred helper phage is M131i07,_and the pre~erred coat proteln 15 the M13 Phage gene III c~oat protein. The preferred host 15 1;. coli, and protease-deficient strains of IS. coli.
Further details of the foregolng and similar mutagenesis techniques are found in general textbooks, such as, for example, sambrook et al., supra, and Curr~nt ~rotocols ln Molecular Biology, ~usubel et al. eds., supra.
}unino acid substitution variants have at least one amino acid residue in a native IFN-y receptor ¦~-chain molecule removed and a different residue inserted in its place. The sites of great interest for substitutional mutagenesis include sites identified as important for signal transduction and/or ligard binding, such domains within the extracellular domain, or the ~EV~D sequence motif at amino acid positions 280-28q of the murine IFN-y receptor i3-chain and its equivalent in the native receptors from other species, including humans, and sites where the amino acids found in the native IFN-y receptor ~-chains from various species are substantially different in terms of side-chain bulk, charge and/or hydrophobicity.
Other sites of interest are those in which part1cular residues of the native IFN-y receptor ~-chains from various species are identical.
These positions may be important for the biological activity of the IFN-y receptor ¦3-chain. Further important sites for mutagenesls include motifs common in various members of the interferon receptor family, such as the two cysteine pairs and conserved proline, tryptophan and tyrosine residues boxed in Figure 2B.
Naturally occurrfng amiho acids are divided into groups based on common side chain properties:
(l) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophobic: cys, ser, thri ~3) acidic: asp, glui

(4) basic: asn, gln, his, lys" argi

(5) residues that influence chain orientatiOn: gly, proi and

(6) aromati~ trp, tyr, phe.
Conservative substitutions involve ~ rhAnri n~ a member within one group for another member within the same group, whereas non-conservative substitutions will entail r~chAnqi nq a member of one of these classes for another. Non-conservative substitutions within the short cytoplasmic domain of the IFN-y receptor ¦3-chain, and especially within the region responsible for signal transduction, such as the LEVLD sequence motif at amino acid positions 280-284 of the murine IFN-y receptor ~-chain, are expected to result in significant changes in ~O 95/16036 ~ 1 7 7 ~ ~ 1 PC~IU59J/1.1277 the biological properties of the obtalned varlant, and may result ln IFN-y receptor ~-chain variants whlch block the biological activity of IFN-y, i.e. are antagonists of the biological action of the corresponding native IFN-y receptor ~-chain, or the signaling potential of which surpasses that of the corresponding native IFN-y receptor ,~-chain. Similarly, non-conservative substitutions within regions of the IFN-y receptor ~-chain extracellular domain that participate in signal transduction and/or ligand binding are expected to result in significant changes in the biological properties of a native IFN-y receptor 13-chain. Amino acid positions that are conserved among various species and/or various receptors of the IFN receptor family are generally substituted in a relatively conservative manner if the goal is to retain biological activity.
Amino acid sequence deletions generally range from about l to 30 residues, more preferably about l to lO residues, and typically are contiguous. Deletions may be introduced into regions not directly involved in signal transduction and/or ligand binding, to modify the biological activity of the IFN-y receptor ~-chain. Deletions from the regions that are directly involved in signal transduction and/or ligand binding will be more likely to modify the biological activity of the mutated IFN-y receptor ~-chain more significantly, and may potentially yield IFN-y receptor ~-chain antagonists. The number of consecutive deletions will be selected so as to preserve the tertiary structure of the IFN-y receptor ~-chain in the affected domain, e.g. beta-pleated sheet or alpha helix.
Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence lnsertions of single or multiple amino acid residues. Intrasequence insertions (i.e.
insertions within the IFN-y receptor ~-chain amino acid sequence~ may range generally from about l to lO residues, more preferably l to S
residues, more preferably l to 3 residues. Examples of terminal insertions include the IFN-y receptor ~-chain with an N-terminal methionyl residue, an artifact of its direct expression in bacterial ,. inAnt cell culture, and fusion of a heterologous N-terminal signal sequence to the N-terminus of the IFN-y receptor ~-chain molecule to facilitate the secretion of the mature IFN-y receptor ~-chain from recombinant host cells. Such signal sequences will generally be obtained ~rom, and thus homologous to, the intended host - 40 cell specles. suitable sequences include STII or Ipp for E. coli, alpha factor for yeast, and viral signals such as herpes gD for ; An cells .

other insertional variants of the native IFN-y receptor ~-chain molecules include the fusion~, to the N- or C-terminus of the IFN-y receptor ~-chain of immunogenic polypeptides, e g. bacterial polypeptides such as beta-lactamase or an enzyme encoded by the E coli trp locus, or yeast protein, and C-terminal fusions with proteins having a long half-life such as immunoglobulin regions (preferably immunoglobulin constant regions~, albumin, or ferritin, as described in WO 89/02922 published 6 April 1989.~
Since it is often difficult to predict in advance the characteristics of a variant IFN-y receptor ¦3-chain, it will be appreciated that some screening will be needed to select the optimum variant .
D. Insertion of DNA into a,Cloning Vehicle once the nucleic.acid encoding a native or variant IFN-y receptor ~-chain is available, lt is generally ligated into a replicable expression vector for further cloning (amplification of the DNA), or for expression.
Expression and cloning vectors are well known in the art and contain a nucleic acid ~sequence that enables the vector to replicate in one or more selected host cells. The selection of the appropriate vector will depend on 1) whether it is to be used for DNA amplification or for DNA express1on, 2) the size Df the DNA to be inserted into the vector, and 3) the host cell to be transformed with the vector. Each vector contains various components depending on its function ~amplification of DNA of expression of DNA) and the host cell for which it is compatible. The vector components generally include, but ar not limited to, one or more of the following: a signal sequence, an origin of replication, one or:more mar3cer genes, an enhancer element, a promoter, and a transcription termination sequence.
(i) Signal Sequence Component In general, the signal sequence may be a component of the vector, or it may be a part of the IFN-y receptor ~-chain that is inserted into the vector. The native IFN-y receptor ~-chain encodes a signal sequence at the amino terminus (5' end of the DNA) of the polypeptide tha~ is cleaved during~ post-translational processing of the polypeptide to form a mature IFN-y receptor ~-chain. In the murine IFN-y receptor ,~-chain this signal sequence is 18 amino acids long (Figure 2B~ .
Native IFN-y receptor ~-chain is however not secreted from the host cell as it contains a merbrane anchoring domain between the extracellular do=main and the cytoplasmic domain (amino acid residues 225 to 248 of the mature murine IFN-y receptor ~-chain) . Thus, to form a secreted version of an IFN-y receptor ~-chain, the membrane anchoring domain (also referred to as trar~ rane domain) is ordinarily deleted ,, .: .. , :.. :.. .. : .. ...... . .. .......

~1~7~1 WO 95116036 PC'r/lJS94114277 or otherwise lnactivated (for example by point mutation(s) ) Generally, the cytoplasmic domain-is also deleted along with the membrane anchoring domain. In the present case, however, the cytoplasmic domain of the IFN-y receptor ~-chain may play an important 5 role in the signal transduction mediated by this receptor subunit (in addition to the extracellular domains o~ both receptor subunits), therefore it is desirable to retain the cytoplasmic domain if the full biological activity is to be preserved. The truncated (or transmembrane domain-inactivated~ IFN-y receptor 13-chain variants may 10 be secreted from the cell, provided that the DNA encoding the truncated variant retains the amino terminal signal sequence.
Included within the scope of this invention are IFN-y receptor ~-chains with the native signal sequence deleted and replaced with a heterologous signal sequence. The heterologous signal sequence 15 selected should be one that LS recognized and processed (i.e. cleaved by a signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process the native IFN-y receptor ~-chain signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, 20 from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the native IFN-y receptor ~-chain signal sequence may be substituted by the yeast invertase, alpha factor, or acid phosphatase leaders. In mammalian cell expression the native signal sequence is satisfactory, although 25 other mammalian signal sequences may be suitable.
(ii) Oriqin of Replication Component Both expression and cloning vectors contain a nucleic acid sequence that enabled the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that 30 enables the vector to replicate independently of the host chromosomes, and includes origins of replication or autonomously replicating sequences. 5uch sequence are well known ~or a variety of bacteria, yeast and viruses. The origin of replication ~irom the well-known plasmid pBF(322 is suitable or most gram negative bacteria, the 2u 35 plasmid origin for yeast and various viral origins (SV90, polyoma, adenovirus, VSv or BPV) are useful for cloning vectors in mammalian cells. Origins of replication are not needed for ~1iAn expreSSiOn vectors (the SV40 origin may typically be used only because it contains the early promoter) . Most expression vectors are "shuttle" vectors, 40 i.e. they are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned in ~. coli and then the same vector is transfected into yeast or 1 1- An cells for expression even though it ~177~71 0 WO 95/16036 ~ PCTNS94/14277 is not capable of replicating lndependently of the host cell ch r omo s ome .
D~IA is also cloned by insertion into the host genome. This is readily accomplished using Bacillus species as hosts, for example, by 5 including in the vector a DNA sequence that is complementary to a sequence found in Bacillus genomic DNA. Transection of Bacillus with this vector results in homologous recorlbination with the genome and insertion of the DNA encoding the desired heterologous polypeptide.
However, the recovery of genomic DNA l5 more complex than that of an 10 exogenously replicated vector because restriction enzyme digestion is required to excise the encoded polypeptide molecule.
( iii ) Selection Gene Component Expression and cloning vectors should contain a selection gene, also termed a selectable marker. This is a gene that encodes a proteln 15 necessary for the survival or growth of a host cell transformed with the vector. The presence of this gene ensures that any host cell which deletes the vector will not obtain an advantage in growth or reproduction over transformed hosts Typical selection genes encode proteins that ~a) confer resistance to antibiotics or other toxins, 20 e.g. ampicillin, neomycin, methotrexate or tetracycline, (b) complement ~uxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g. the gene encoding D-alanine racemase for bacilli.
one example of a selection scheme utilizes a drug to arrest 25 growth of a host cel 1 . Those cells that are successfully transformed with a heterologous gene express a protein rrnferri nrJ drug resistance and thus survive the selection regimen. ~xamples of such dominant selection use the drugs neomucin lSouthern et al., J. Molec. Appl.
Genet 1, 327 (1982) ], mycophenolic acid [Mulligan et al., Science 209, 1422 (1980) ~, or hygromycin [Sudgen et al., Mol. Cel.. Blol. 5, 410-413 (1985) ] The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G~18 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively .
other examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR) or thymidine kinase. Such markers enable the identification of cells which were competent to take up the desired nucleic acid. The mammalian cell transformants are placed under selection pressure which only the transformants are uniquely adapted to survive by virtue of havlng taken up the marker Selection pressure is imposed by culturing the transformants under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to amplification of both the ~17~
WO 95/16036 PCT/I~S94/1~277 selection gene and the DNA that encDdes the deslred polypeptlde.
Amplification is the process by which genes in greater demand for the production of a protein cr~ tical or growth are reiterated in tandem within the chromosomes of successive generations of recorbinant cells.
5 Increased quantities of the desired polypeptide (either a p75-containing chimeric polypeptide or a segment thereof ~ are synthesized from the amplified DNA.
For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture 10 medium which lacks hypoxanthine, glycine, and thymidine. An appropriate host cell irr this case is the Chinese hamster ovary (CHO) cell line deicient in DHFR activity, prepared and propagated as described by Urlaub and Chasin, PrQc. Nat'l. Acad. Sci. USA 77, 4Z16 (1980~ . A particularly useful DHFR is a mutant DHFR that is highly resistant to MTX (EP 117, 060) . This selection agent can be used with any otherwise suitable host, e.g. ATCC No. CCL61 CHO-K1, notwithstanding the presence of endogenous DHFR. The DNA encoding DH~R
and the desired polypeptide, respectively, then is amplified by exposure to an agent (methotrexate, or MTX) that inactivates the DHFR.
2 0 One ensures that the cell requires more DHFR ( and consequently amplifies all exogenous DNA) by selecting only for cells that can grow in successive rounds of ever-greater MTX concentration. Alternatively, hosts co-transformed with genes encoding the desired polypeptide, wild-type DHFR, and another selectable marker such as the neo gene can be identified ùsing a seleGtion agent for the selectable marker such as G418 and then selected and smplified using methotrexate in a wild-type host that contains endogenous DHFR. (See also U.S. Patent No.
4, 965, 199) .
A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature 282:39; Kingsman et al., 1979, Gene 7:141, ~ or Tschemper et al.., 1980, Gene 10:157). The trpl gene E~rovides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics 85:12) . The presence of the trpl lesion in the yeast host cell genome then provides an effec~ive environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2 deficient yeast strains (ATCC
20, 622 or 38, 626) are complemented by known plasmids bearing the Leu2 gene .
(iv) Promoter Component Expression vectors, unlike cloning vectors, should contain a promoter which is recognized by the host organism and is operably linked to the nucleic acid encoding the desired polypeptide. Promoters . 2~7471 WO 95/16036 PCT/IJS9~ 277 are untranslated sequences located upstream from the start codon of a structural gene (generally within-about 100 to 1000 bp) that control the transcription and translation of nucleic acid under their control.
They typlcally fall into two classes, inducible and constitutive.
Inducible promoters are promoters that initiate increased Levels of transcription from DNA under their control in response to some change in culture conditions, e.g. the presence or absence of a nutrient or a change in temperature. At this time a large number of promoters recognized by a variety of potential host cells are well known. These promoters are operably linked to DNA encoding the desired polypeptide by removing them from their gene of origin by restriction enzyme digestion, followed by insertion 5 to the start codon for the polypeptide to be expressed. This }s not to say that the genomic promoter for IFN-y receptor ~-chain is not usable However, heterologous promoters generally will result in greater transcription and higher yields of expressed IF~-y receptor ~-chain as compared to the native IFN-y receptor i3-chain promoter.
Promoters suitable for use with prokaryotic hosts include the 13-lactamase and lactose promoter systems (Chang t al., Nature 275 615 ~19781; and Goeddel et al, Nature 281:544 ~1979~), alkaline phosphatase, a tryptophan ~trp~ promoter system ~Goeddel, Nuclelc Acids Res. 8:q057 ~1980~ and EPO Appln. Publ. No. 36,776~ and hybrid promoters such as the tac promoter ~H. de Boer et al., Proc. Nat 1.
Acad. Sci. USA 80:21-25 ~1983~ ~ . However, other known bacterial promoters are suitable. Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to DNA encoding NT-4 ~Siebenlist et al. . Cell 20:269 ~1980~ ~ using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno ~S.D.
sequence operably linked to the DNA encoding NT-4.
Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase ~Hitzeman et al. J Biol Chem.
255:2073 ~1980~ ~ or other glycolytlc enzymes ~Hess et al., J. Adv.
Enzyme Reg. 7:149 (1978~; and Holland, Biochemistry 17:4900 ~1978~, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, WO 9~l~6036 217 7 ~ ~ 1 PCTIUS94111277 and enzymes responsible for maltose and galactose utillzation.
Suitable vectors and promoters fo~ use ln yeast expression are furthec described in R . Hitzeman et al., EP 73, 657A. Yeast enhancers also are advantageously used with yeast promoters.
Promoter sequences are known for eukaryotes Virtually all eukaryotic genes have an P.T-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CXCAAT region where X may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3 ' end of the coding sequence All of these sequences are suitably inserted into mammalian expression vectors.
~FN-y receptor ~-chain transcriptlon from vectors in mammalian host cells may be controlled by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2, 211, 504 published 5 July 1989~, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (Sv40), from heterologous mammalian promoters, e_g the actin promoter or an immunoglobulin promoter, from heat shock promoters, and from the promoter normally associated with the IFN-y receptor 13-chain sequence, provided such promoters are compatible with the host cell systems The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication IFiers et al., Nature 273:113 ~1978), Mulligan and Berg, Scienc~ 209, 1422-1427 (1980)i Pavlakis et al., Proc. Natl. Acad. Sci USA 78, 7398-7402 (1981) ] The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restrictLon fragment [Greenaway et al., Gene 18, 355-360 (1982) ] A system for expressing DNP. in mammalian hosts using the bovine papilloma virus as a vector is disclosed in US 4, 419, 446 A
modification of this system is described in US 4, 601, 978 See also, Gray et al., Nature 295, 503-508 (19821 on expressing cDNA encoding human immune interferon in monkey cells; Reyes et dl., Nature 297, 598-601 (1982) on expressing human ~-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus;
Canaani and Berg, Proç. Natl. Acad. Sci. USA 79, 5166-5170 (1982) on expression of the human interferon 131 gene in cultured mouse and rabbit cells; and Gorman et al, Proc Natl. Acad. $ci, USA 79, 6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa WO 95/16036 ~ ~ 7 7 ~ ~ 1 PCT,'US94/14277 cells, and mouse HIN-3T3 cells using the Rous sarcoma virus long terminal repeat as a promoter.
The actual plasmid used ln the course of clonlng the murine IFN-y receptor ~-chain contains the promoter of the murine 3-hydroxy-3-5 methylqlutaryl coenzyme A reductase gene [Gautier et al., Nucleic AcidsRes 17, 8389 ~1989) ], whereas the reporter plasmid [pUMS (GT) ~-Tac]
used during expression cloning contained an artificial multimerized IFN-y-inducible promoter element [McDonald et al., Cell 60, 767-779 ( 1990) ] .
(v) Enhancer Elerlent Component Transcription of a DNA encoding the IFN-y receptor ~-chains of the present invention by higher eukaryotes is often increased by inserting an enhancer sequence into.the vector Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a 15 promoter to increase its transcription Enhancers are relatively orientation and position independent having been found 5 ' [Laimins et al., I?roc Natl Acad. Sci USA 78, 993 (1981)] and 3' [Lusky et al., Mol Cel Biol. 3, 1108 (1983) ] to the transcription unlt, within an intron [Banerji et al., Cell 33, 729 (19831] as well as within the 20 .coding sequence itself [Osborne et al, Mol. CelBiol ~, 1293 (198~) ] . Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, o~-fetoprotein and insulin). Typically, however, one will use an ennancer from a eukaryotic cell virus.
Examples include the SV~0 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origln, and adenovirus enhancers. See also Yaniv, Nature 297, 17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5 ' or 3 ' to the IFN-y receptor :3-chain DNA, but is preferably located at a site 5' from the promoter (vi ) Transcription Termination Component Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain se~uences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3' untranslated regions of eukaryotic or viral DNAs or cDNAs These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the IFN-y receptor ~-chain . The 3 ' untranslated regions also include transcriptlon terminatLon sites.

O 95/16036 ~ 7 1 PCT~lS9~tl4277 Construction of suitable vectors containing one or more of the above listed components, the desifed coding and control sequences, employs standard ligation techniques. Isolated plasmids or DNA
fragments are c~eaved, tailored, and religated in the form desired to generate the plasmids required.
For analysis to confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform E coli K12 strain 294 (ATCC 31, 446~ and successful transformants selected by ampicillin or tetracycline resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion, and/or sequenced by the method of Messing et al., Nucleic Acids ~es. 9, 309 (1981) or by the method of Maxam et al., Methods in Enzymology 65, 499 (1980) .
Particularly useful in the practice of this invention are expression vectors that provide for the transient expression in 1 l ~n cells of DNA encoding an IFN-y receptor ~-chain. In general, transient expression involves the use of an expression vector that lS
able to replicate e~ilciently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector. Transient systems, comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by clones DNAs, as well as for the rapid screening of such polypeptides for desired biological or physiological properties. Thus, transient expression systems are particularly useful in the invention for purposes of identifying analogs and variants of the ~EN-y receptor ~-chain.
Other methods, vectors, and host cells suitable ior adaptation to the synthesis of the IFN-y receptor ,~-chains in L~ i n~nt vertebrate cell culture are described in Getting et al., Nature 293, 620-625 (1981); Mantel et al., Nature 281, 40-46 (1979); Levinson et al.; EP
117, 060 and EP 117, 058. A particulariy usefui plasmid for mammalian cell culture expression of the IFN-y ~-chain is pRK5 (EP 307,247) E. Selection and Transformation of HPst Cells Suitable host cells for cloning or expressing the vectors herein are the prokaryote, yeast or higher eukaryote cells described above.
Suitable prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. A preferred clon1ng host is E. coli 294 (A~CC 31, 446) although other gram negative or gram positive prokaryotes such as E. coli B, E. coli ~1776 lATCC 31,537), E. coli W3110 (ATCC 27, 325), Pseudomonas species, or Serratia Marcesans are suitable WO95116036 ~ 77~ 71 PCT,~IS9~/1.1277 In addition to prokaryotes~ eukaryotic microbes such as filamen~ous fungi or yeast are suLtable hosts for vectors herein.
Saccharomyces cerevisi~e, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of 5 other genera, species and strains are commonly available and useful herein, such as S. pombe [Beach and Nurse, Nature 290, lqO ~1981) ], Kluyveromyces lactis [Louvencourt et al., J. Bacteriol. 737 ~1983) ];
yarrowia ~EP 402,226); Pichia pastoris ~EP 183,070), Trichoderma reesia (EP 244,234), Neurospora crassa [Case ee al., Proc. Natl. Acad. sci.
USA 76, 5259-5263 ~1979) ]; and Aspergillus hosts such as A. nidulans [Ballance et al., 3iochem. Biophys. Res. Commun. lI2, 284-239 ~1983);
Tilburn et al., Gene 26, 2~5-221 ~1983); Yelton et al., Proc. Natl.
Acad. Sci. USA 81, 1470-1474 ~1984) ~ and A. niger [Kelly and Hynes, EM30 J. 4, 475-479 ~19~85) ~ .
Suitable host cells may also derive from multicellular organisms.
Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture, although cells from mammals such as humans are preferred. Examples of invertebrate cells include plants and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda ~caterplllar), Aedes aeqypti ~mosquit~o), Aedes albopictus ~mosqulto), Drosophila melangaster ~frultfly), and Bombyx mori host cells have been identified. See, e.g. Luckow et al., Bio/Technolo~y 6, 47-55 ~1988);
Miller et al., in Genetic F.ngi n~.~rl n~, Setlow, J. K. et al ., eds ., Vol .
8 ~Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature 315, 592-594 ~1985) . A variety of such viral strains are publicly available, e.g. the L-l variant of Autographa californica NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells .
Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts. Typically, plant cells ~re transfected by incubation with certain strains of the bacterium Agrobacterium tumefaciens, which has been previously manipulated to contain the IEN-y receptor ~-chain DNA. During incubation of the plant cell culture with A. tumefaciens, the DNA encoding IFN-y receptor E~-chain is transferred to the plant cell host such that it is transfected, and will, under appropriate conditions, express the IFN-y receptor p-chain DNA. In addition, regulatory and signal sequences compatible with plant cells are available, such as the nopaline ~ynthase promoter and polyadhenylation signal sequences. Depicker et ~77~71 WO 95116036 PCT~US9-1ll 1277 al., ~. Mol Appl. Gen. 1, 561 (198Z) . In addition, DNA segments isolatcd from the upstream region~of the T-DNA 780 gene are capable of activating or increasing transcription levels of plant-expressible genes in 1. i ni3nt DNA-containing plant tissue See EP 321,196 published 21 June 1989.
However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture ~tissue culture) is per se well known See Tissue Culture, Academic Fress, Kruse and Patterson, editors (1973) . Examples of useful 1 i ~n host cell lines are monkey kidney CVl line transformed by SC40 (COS-7, ATCC CRL 1651~:
human embryonic kidney cell line 1293 or 293 cells subcloned for growth in suspension culture, Graham et al., ~. Gen Virol. 36, 59 (1977)1;
baby hamster kidney cell5 9BHK, ATCC CCL 10 ~; Chinese hamster ovary cells/-DHFR ~CH0, Urlaub and Chasin, Proc. Natl. ~cad. Sci. USA 77, q216 ~1980) ]; mouse sertolli cells [TM4, Mather, Biol . Reprod. 23, 243-251 (lsao) ]; monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells ~VER0-76, ATCC CRL-1587); human cervical carcinoma cel 1.~ (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065~; mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells ~Mather et al., Annals N.Y.
Acad. sci. 383, 44068 (1982)1; MRC 5 cells; FS4 celLs; and a human hepatoma celL line (Hep G2) Preferred host cells are human embryonic kidney 293 and Chinese hamster ovary cells.
Particularly preferred host cells for the purpose of the present invention are vertebrate cells producing the IFN-y receptor C~-subunit, chains of the IFN-o~ receptors, and/or other cytokine receptor or EP0 receptor .
Host cells are transfected and preferably transformed with the 3 0 above-described expression or cloning vectors and cultured in conventional nutrient media modified as is appropriate for inducing promoters or selecting transfarmants containing amplified genes.
F. Culturing the Host Cells Prokaryotes cells used to produced the IFN-y :3-chain polypeptides of this invention are cultured in suitable media as describe generally in Sambrook et al., supra.
Mammalian cells can be cultured in a variety of media.
Commercially available media such as Ham' s F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 )Sigma), and Dulbecco's Modified Eagle's Medium ~DMEM, Sigma) are suitable for culturing the host celLs. In addition, any of the media described in Ham and Wallace, Meth. Enzymol. S8, 44 (1979); Barnes and Sato, Anal. Biochem.
02, 255 ~1980), US 4,767,704; 4,657,866; 4,927,762; or 4,560,655; Wo 90/03430; Wo 87~00195 or Us Pat. Re. 30, 985 may be used as culture medla for the host cells. Any of- these media may be supplem~nted as necessary with hormones and/or other growth factors ~such as insulln, transferrin, or epidermal growth factor~, salts ~such as sodium 5 chloride, calcium, magnesium, and phosphate), buffers Isuch as HEPES), nucleosides ~such as adenosine and thymidine~, antibiotics ~such as Gentamycin~M drug) trace elements (defined as inorganic compounds usually present at final concentratlons in the micromolar range), and glucose or an equivalent energy source. Any other necessary lO supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH and the like, suitably are those previously used with the host celI selected fo~ cloning or expression, as the case may be, and will be apparent to the ordinary artisan.
The host cells referred to in this disclosure encompass cells in in vitro cell culture as well as cells that are within a host animal or plant .
It is further envisioned that the IEN-y receptor ~-chain of this invention may be produced by homologous recombination, or with recombinant production methods utilizing control elements introduced into cells already containing DNA encoding the IEN-y receptor ~-chain.
G. Detecting Gene Amplification/Expression Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. .
Acad. Sci. USA 77, 520I-5205 ~l9801 ], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Various labels may be employed, most commonly radioisotopes, particularly ~2p, However, other techniques may also be employed, such as using biotin-modified nucleotides for introduction into a polynucleotide. The biotin then serves as a site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA
hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to the surface, so that upon the formatisn of duplex on the surface, the presence of antibody bound to the duplex can be detected.
Gene expresslon, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. With immunohistochemical staining

7~
WO95/16036 ~ 'l PCTIIJS94/14277 techniques, a cell sample is prepared, typlcally by dehydration and fixation, followed by reaction wi~h labeled eintibodies specific for the gene product coupled, where the labels are usually visually detectable, such as enzymatic labels, fluorescent labels, luminescent labels, and the like. A particularly sensitive staining technique suitable for use in the present invention is described by Hse et al, Am. J. Clin.
Pharm. 75, ?34-738 (1980~
Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodles may be prepared against a native IEN-y ~-chain polypeptide, or against a synthetic peptide based on the DNA sequence provided herein as described further hereinbelow .
H. Purificatlon of the IEN-y G-chain The IFN-y receptor ~-chain preferably is recovered frDm the cell culture medium as a secreted polypeptide, although it also may be recovered from host cell lysates when directly expressed in a form including the membrane anchoring domain, and without a secretory signal .
When tbe IFN-y receptor ~3-subunit is expressed in a L~=~, ' in~nt cell other than one of human origin, the IEN-y receptor ~3-chain is completely free of proteins or polypeptides of human origin. However, it is necessary to purify the ~-chain from recombinant cell proteins or polypeptides to obtain preparations that are substantially homogenous as to the IEN-y receptor ~3-chain. As a first step, the culture medium or lysate is centrifuged to remove particulate cell debris. The membrane and soluble protein fractions are then separated. The IEN-y receptor ~-chain may then be purified from the soluble protein fraction and from the membrane fraction of the culture lysate, depending on whether the IEN-y receptor ~-chain is membrane bound. The following procedures are exemplary of suitable purification procedures:
fractionation on immunoaffinity or ion-exchange columns; ethanol preclpltation; reverse phase HPLC; chromatography on silica or on a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE;
ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-7~; and protein ~ Sepharose columns to remove contaminants such as IgG;
IFN-y receptor ,B-chain functional derivatives in which residues have been deleted, inserted and/or substituted are recovercd in the same fashion as the native receptor chains, taking into account of any substantial changes in properties occasioned by the alteration. Eor example, fusion of the IFN-y receptor ~-chain with another protein or polypeptide, e.g. a bacterial or viral antigen, facilitates WO 95/16036 ~17 7 4 7 ~ PCT/US94/14~77 purification; an immunoaffinity coLumn containing antibody to the antigen can be used to~ absorb the- fusion. Immunoaffinity columns such as a rabbit polyclonal anti-IEN-y receptor i3-chain column can be employed to ~bsorb IFN-y receptor ~3-chain variant by binding to at least one remaining immune epitope. A protease inhibltor, such as phenyl methyl sulfonyl fluoride (PMSF) also may be useful to inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the growth of adventitious contaminants. One skilled in the art will appreciate that purification methods suitable for native IFN-y receptor ;3-chain may require modification to account for changes in the character of the IFN-y receptor p-chain or its variants upon expression in recombinant cell culture.
I. Covalent Modifications of IFN-y receptor ¦3-chain Covalent modifications of IFN-y receptor ~-chain are included within the scope herein. Such modifications are traditionaIly introduced by reacting targeted amino acid residues of the IFN-y receptor ~-chain with an organic de~ivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected Le~ ' in~nt host cells_ The resultant covalent derivatives are useful in programs~ directed at identifying residues important for biological activity, for i ~ ys of the IEN-y receptor 13-chain, or for the preparation of anti-IFN-y receptor ~-chain antibodies for immunoaffinity purification of the recombinant. For example, complets inactivation of the biological activity of the protein after reaction with ninhydrin would suggest that at least one arginyl or lysyl residue is critical for its activity, whereafter the individual residues which were modified under the conditions selected are identified by isolation of a peptide fragment containing the modified amino acid residue. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.
Cysteinyl residues most commQnly are reacted with Q-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, Q-bromo-~- ~5-imidozoyl ) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with diethylpyro-carbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chaln. Para-bL~ , h~.na~yl bromide also is useful;

WO 9S/16036 PCT,'US94/14277 the reaction 15 preferably performed in 0. lM sodium cacodylate at pH
6Ø - _ ~
Lysinyl and amino terminal residues are reacted with succinlc or other carboxyllc acid anhydrides. Derivatization with these agents has the effect of reversing the charge o~ the lysinyl residues. Other suitable reagents for derivatizing ~-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphatei pyridoxal; chloroborohydridei trinitrobenzenesulfonic acid; O-methylisourea; 2, 4-pentanedione; and transaminase-catalyzed reaction with glyoxylate Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cycloheYanedione, and ninhydrin~ Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pl~. of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginlne epsilon-amino group.
The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatlc diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively~ ~yrosyl residues are iodinated using l2~I or ~3~I to prepare labeled proteins for use in radini ~qsAy, the chloramine T method described above being suitable Carboxyl side groups (aspartyl or glutamyl~ are selectively modified by reaction with carbodiimides ~R'-N=C=N-R' ) such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylpentyl) ~:~rhr3~ . Furthermore, aspartyl and 3 0 glutamyl residue5 are converted to asparaginyl and glutaminyl residUes by reaction with ammonium ions.
Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of 3 5 the5e resldues fall5 within the scope of this invention .
other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o~-amino groups of lysine, arginine, and histidine 5ide chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Franclsco, pp. 79-86 [1983] ), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group. The molecules may further be covalently linked to nonprotein-ceous polymers, e. g. p~ly;thylene ~ycol, polypropylene 2~7~ O
WO 95/16036 PCT/'US94/14277 glycol or polyoxyalkylenes, in the manner set forth U.S. patents q, 640, 835; 4, 496, 689; 4, 301,144i 4, 670, 417; 4, 791,192 or 4,179, 337 .
Derivatization with bifunctional agents is useful for preparing intramolecular aggregates of the IEN-y receptor 13-chain with 5 polypeptides as well as for cross-linking the IEN-y receptor ~-chain to a water insoluble support matrlx or surface for use in assays or affinity purification. In addition, a study of interchain cross-links will provide direct illformation on conformational structure. Commonly used cross-linking agents include 1, l-bis (diazoacetyl) -2-phenylethane, 10 glutaraldehyde, N-hydroxysuccinimide esters, homobifunctional imidoesters, and bifunctional maleimides. Derivatizing agents such as methyl-3- [ (p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates which are .capable of forming cross-links in the presence of light. Alternativcly, reactive water insoluble matrices such as 15 cyanogen bromide activated carbohydrates and the systems reactive substrates described in U.S. Patent Nos. 3,959,642; 3,969,287;
3, 691, 016; 4,195,12B; 4, 247, 642; 4, 229, 537; 4, 055, 635; and 4, 330, 440 are employed for protein immobilization and cross-linking.
Certain post-translational modifications are the result of the 20 action of recombinant host cells on the expressed polypeptide.
Glutaminyl and aspariginyl residues are frequently post-translationally deamidated to the cr-rrecrnn~li n~ glutamyl and aspartyl residues.
Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of 25 this inventi~n.
Other post-translatlonal modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o~-amino groups of lysine, ~rginine, and histidine side chains IT~E Creighton, Proteins:
30 Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983) 3 .
Other derivatives comprise the novel peptides of this invention covalently bonded to a nonproteinaceous polymer. ~he nonpro~in~
polymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymer 35 not otherwise found in nature. ~lowever, polymers which exist in nature and are produced by recombinant or in vitro methods are useful, as are polymers which are isolated from nature. ~Iydrophilic polyvinyl polymers fall within the scope of this invention, e . g . polyvinylalcohol and polyvlnylpyrrolidone. Particularly useful are polyvinylalkylene 40 ethers such a polyethylene glycol, polypropylene glycol.
`rhe IEN-y receptor ~-chain may be linked to various nonproteinaceous pol~mers, such as polyethylene glycol, polypropylene -WO 95r16036 ~17 ~ 4 71 PCTlUSg4ll~277 glycol or polyoxyalkylenes, in the manner set forth in U S. Patent Nos 4, 640, 835; 4, 496, 689; 4, 301,144; ~, 670, 417; 4,791,192 or 4,179, 337 .
The IFN-y receptor B-chain may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial 5 polymerization, in colloidal drug delivery systems (e.g. liposomes, albumin miCrospheres, mic~ ns, nano-particles and nanocapsules) or in macroemUlsions . Such techniques are disclosed in Remington ' 5 Pharmaceutical Sciences, 16th Edition, =0501, A., Ed (1980) .
J. IFN-y receptor ~-chain-immunoqlobulin chimeras (i n~1h~cin~) Immunoglobulins ~Ig) and certain varlants thereof are known and many have been prepared in Lf inAnt cell culture. For example, see U.S. Patent 4,745,055; EP 256,654; Eaulkner et al., Nature 298:286 (1982); EP 120,694; EP 125,023; Morrison, J. Immun. 123:793 ~1979);
~ohler et al, Proc. Nat'l. Acad. sci. USA 77:2197 ~1980); Raso et al., Cancer Res. 41:2073 ~1981); ~qorrlson et al., Ann. Rev. Immunol. 2:239 (1984); Morrison, Science 229:1202 ~1985); Morrison et al., Proc.
Nat'l. Acad. Sci. USA 81:6851 (1984); EP 255,694; EP 266,663; and WO
88/03559. ~eassorted immunoglobulin chains also are known. See for example U.S. patent 4,444,878; Wo 88/03565; and EP 68,763 and references cited therein. The immunoglobulin moiety in the chimeras of the present invention may be obtained from IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA, IgE, IgD or IgM, but preferably IgG-1 or IgG-3.
Chimeras constructed from a receptor sequence linked to an appropriate immunoglobulin constant domain sequence (immunoadhe5in51 are known in the art. T --ihf.ei n~ reported in the literature include fusions of the T cell receptor [Gascoigne et al., Proc.
Natl.Acad. Sci. USA 84, 2936-2940 ~1987) ~; CD4 [Capon et al., Nature 337, 525-531 ~1989); ~raunecker et al, Nature 339, 68-70 (1989);
~ettmeissl et al., DNA Cell Biol. USA 9, 347-353 (1990); Byrn et al., Nature 344, 667-670 11990) ]; L-selectin (homing receptor) [Watson et al, J. cell. Biol 110, 2221-~229 (1990); Watson et al., Nature 349, 164-167 (1991)]; CD44 [Aruffo et al., Cell 61, 1303-1313 ~1990)]; CD28 and B7 [Linsley et al., J. Exp. Med. 173, 721-730 (1991) ]; C~L~-4 [Lisley et al., J. Exp. Med. 174, 561-569~(1991~]; CD22 [Stamenkovic et al, Cell 66. 1133-1144 ~1991)]; TNF receptor [Ashkenazi et al., Proc.
Natl. ~cad. Sci. USA 88, 10535-10539 (1991); Lesslauer et al., Eur. J.
Immunol. 27, 2883-2886 ~1991); Peppel et al., J. Exp. Med. 174, 1483-1489 ~1991) ]; NP receptors [Bennett et al., J. Biol. Chem. 266, 23060-23067 ~1991) ]; IgE receptor o-chain [F~idgway and Gorman, J. Cell. Biol.
S, abstr. 1448 ~1991) ]; HGF receptor [Mark, M.~. et al., 1992, J.

WO 95/16036 21 ~ 7 ~ 71 PCT,'llS94114277 BloL Chem. submitted], where the asterisk ( ~ ) indicates that the receptor i5 member of the lmmunoglobulin superfamily.
Ordinarily, when preparing the IFN-y receptor ,~-chaln-immunoglobulin chimeras of the present invention, the nucleic acid 5 encoding the desired IFN-y receptor ,~-chain extracellular domain sequence will be fused C-terminally to nucleic acid encoding the N-terminus of an immunoglobulin constant domain sequence, however N-terminal fusions are also possible.
Typically, in such fusions the encoded chimeric polypeptide will l0 retain at least functionally active hinge, CH2 and CH3 domains of the constant region of an immunoglobulin heavy chain. Fusions are also made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CHl of the heavy chain or the corresponding region of the light chain.
15 The precise site at which the fusion is made is not criticali particular sites are well known and may be selected in order to optimize the biological activity, secretion or binding characteristics of the IFN-y receptor ~-chain-immunoglobulin chimeras.
In some embodiments, the IFN-y receptor ¦3-chain-immunoglobulin 20 chimeras are assembled as monomers, or hetero- or homo-multimers, and particularly as dimers or tetramers, essentially as illustrated in WO
91/08298 .
In a preferred embodiment, an the IFN-y receptor ,~-chain extracellular domain sequence is fused to the N-terminus of the C-25 terrlinal portion of an antibody (in particular the Fc domain),containing the effector functions of an immunoglobulin, e.g.
immunoglobulin G~ tIgG-l) . It is possible to fuse the entire hea~ry chain constant region to the IFN-y receptor ~-chain extracellular domain sequence. However, more preferably, a sequence beginning in the 30 hinge region just upstream of the papain cleavage site (which defines IgG Fc chemically; resldue 216, taking the first residue of heavy chaln constant region to be 114 [~Cobet et al., supral, or analogous sites of other immunoglobulins) is used in the fusion. In a particularly preferred embodiment, the IFN-y receptor 13-chain amino acid sequence is 35 fused to the hinge region and CH2 and CH3 or CHl, hinge, CH2 and CH3 domains of an IgG-l, IgG-2, or IgG-3 heavy chain. The precise site at which the fusion is made is not critical, and the optimal site can be determined by routine experimentation.
In some embodiments, the IFN-y receptor ~-chain-immunoglobulin 40 chimeras are assembled as hetero-multimers, and particularly as hetero-dimers or -tetramers Generally, these assembled immunoglobulins wLll have known unit structures. A basic four chain structural unit is the form in which IgG, IgD, and IgE exist. A four-chain unit is repeated ~7Z~4~

in the higher molecular weight immunoglo~ulinsi IgM generally exists as a pentamer of basic four-chain units held together by disulfide bonds.
IgA globulin, and occasionally IgG globulin, may also exist in multimeric form in serum In the case of multimer, each four-chain 5 unit may be the same or different.
Various exemplary assembled IFN-y receptor ¦~-chain-immunog~obulin chimeras within the scope herein are schematically diagrammed below:
( a ) ACL--ACL;
(b) ACN-- [ACN~ ACL--ACN, ACL VNCN, or VLCL AC5];
0 (c) ACL--ACN-- [ACL--ACN, ACL VNCN, VLCL AC8, or VLCL VNCN] i (d) ACL--VNCN_ ~ACN~ or ACL VNCN, or VLCL ACN];
(e) VLCL--ACN-- [ACL--VNCN~ ~ . rl` L--ACR]; and ( f ) ~A Y ] = [VLCL VNC~]
wherein each A represents identical or different IFN-y receptor ,~-chain amino acid sequences;
VL is an immunoglobulin light chain variable domain;
VN is an immunoglobulin heavy chain variable domain;
CL i5 an immunoglobulin light chain constant domain;
CN is an immunoglobulin heavy chain constant domain;
n is an integer greater than l;
Y designates the residue of a covalent cross-linking agent In the interests of brevity, the foregoing structures only show key features; they do not indicate joining (;r) or other domains of the immunoglobulins, nor are disulfide bonds shown. However, where such domains are required for blnding activity, they shall be constructed as being present in the ordinary locations which they occupy in the immunoglobulin molecules.
Alternatively, the IFN-y receptor ,~-chain extracellular domain sequences can be inserted between immunoglobulin heavy chain and light chain sequences such that an immunoglobulin comprising a chimeric heavy chain is obtained. In this embodiment, the IFN-y receptor ~-chain sequences are fused to the 3 ' end of an immunoglobulin heavy chain in each arm of an immunoglobulin, either between the hinge and the CH2 domain, or between the CH2 and CH3 domains. similar constructs have been reported by Hoogenboom, H. R. et zl., Mol. Immunol. 28, 1027-1037 ( l99I) .
Although the presence of an irmunoglobulin light chain in not ~0 required ln the immunoadhesins of the present invention, an immunoglobulin light chain might be present either covalently associated to an IFN-y receptor ~-chain-immunoglobulin heavy chain fusion polypeptide, or directly fused to the IFN-y receptor ~-chain ~177~17~ 0 extracellular domain. In the former case, DNA encodlng an immunoglobulin light chain l5 typically coexpressed with the DNA
encoding the IFN-y receptor ¦3-chain-immunoglobulin heavy chain fusion protein. Upon secretlon, the hybrLd heavy chain and the lLght chaln 5 will be cova~ently associated to provide an immunoglobulin-like structure comprising two disulfide-linked immunoglobulin heavy chain-light chain pairs Methods suitable for the preparation of such structures are, for example, disclosed in U.S E'atent No 4, 816, 567 issued 28 March 1989.: .
K. Glycosylation variants of the IFN-y receptor ~-chain The native IFN-y receptor ~-chains are glycoproteins. Variants having a glycoslation patte~n which differs from that of any native amino acid sequence which might be ~resent in the molecules of the present invention are within the scope herein. For ease, changes in the glycosylation pattern of a native polypeptide are usually made at the DNA level, essentially using the techniques discussed hereinabove with respect to the amino acid sequence variants.
Chemical or enzymatic coupling of glycosydes to the IEN-y receptor l~-chain of the molecules of the present invention may also be used to modify or increase the number or profile of carbohydrate substituents These procedures are advantageous in that they do not require production of the polypeptide that is capable of O-linked (or N-linked) glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to ~a) arginine and histLdine, (b) free carboxyl groups, (c) free hydroxyl groups such as those of cysteine, (d) free sulfhydryl groups such as those of serine, threonine, or hydroxyproline, ~e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan or (f) the amide group of glutamine. These methods are described in WO 87/05330 (published 11 September 1987), and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306.
Carbohydrate moieties present on a polypeptide may also be removed chemically or enzymatically. Chemical deglycosylation requires exposure to trifluoromethanesulfonic acid or an equivalent compound.
This treatment results in the cleavage of most or all sugars, except the linking sugar, while leaving the polypeptide intact. Chemical deglycosylation is described by I~Aki .~"i n et al., Arch. Biochem.
Biophys. 259, 52 (1987) and by Edge et al., Anal. Biochem. 118, 131 (1981) . Carbohydrate moieties can be re~oved by a variety of endo- and exoglycosidases as described by Thotakura et 41., Meth. E:nzymol. 138, 350 ~1987) . Glycosylation is suppressed by tunicamycin as described by Duskin et al., J. Biol. Chem. 257, 3105 ~1982) . Tunicamycin blocks the formation of protein-N-glycosydase linkages.

~O 95/16036 ~ ~ 7 7 ~ ~ ~ PCT/US94/14277 Glycosylation variants can also be produced by selecting appropriate host cel 1s of recombinant productlon Yeast, for example, introduce glycosylation which varies significantly from that of r- 1 iAn systems. Similarly, mammalian cells having a different ~; species (e.g. hamster, murine, insect, porcine, bovine or ovine) or tissue (e.g lung, liver, lymphoid, mesenchymal or epidermal) origin than the source of the native IFN-y receptor ~-chain, are routinely screened for the ability to introduce variant glycosylation.
L. IFN-y receptor 3-chain antibody preparation (i) Polyclonal antibodies Polyclonal antibodies to the IFN-y receptor ~-chain generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the IFN-y receptor ~-chain and an adjuvant. It may be useful to con]ugate the IFN-y receptor 13-chain or a fragment containing 15 the target amino acid sequence to a protein that is immunogenic in the species to be immuni~ed, e.g. keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxye:~lrrinimi~p 20 (through lysine residues), glytaraldehyde, succinic anhydride, SOCl2, or RIN=C=NR, where R and R' are different alkyl groups.
Animals are immunized against the immunogenic conjugates or derivatives by combining l mg of l ug of conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and 25 injecting the solution intradermally at multiple sites. One month later the animals are boosted with l/5 to l/l0 the original amount of con~ugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. 7 to 14 days later the animals are bled and the serum is assayed for anti-IEN-y receptor r2-chain antibody titer. Animals are 30 boosted until the titer plateaus. Preerably, the animal boosted with the conjugate of the same IFN-y receptor ~-chain, but conjugated to a different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are used to enhance the 35 immune response (ii) Monoclonal antibodies Mnnnrl nnAl antibodies are obtained from a population of substantially homogeneous antibodies, i e., the individual antibodies comprising the population are ide~tical except for possible naturally 40 occurring mutations that may be present in minor amounts. ~hus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

21~7471 ~

For example, the anti-IFN-y receptor ~-chain monoclonal antlbodies of the invention may be made using the hybridoma method first described by Kohler & Milstein, Nature 256:495 (1975), or may be made by recombinant DNA methods ~Cabilly, et al, U s. Pat No.
5 q,816,567]
In the hybridoma method, a mouse or other appropriate host animal, such as hamster is immunized as hereinabove described to elicit lymphocytes that produce or are capable of produclng antlbodies that will speclfically bind to the protein used for immunization Alternatively, lymphocytes may be immunized in vitro I.ymphocytes then are fused with myeloma cells using a suitable fusiny agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, pp 59-103 (Academic Press, 1986) ] .
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-ll mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells avaiIable from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies ~ozbor, J. Immunol. 133:30~1 (1984);
Brodeur,, Monoclonal ~ntibody~ Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)]
Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibordies directed against IFN-y receptor ~-chain Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an _ vitro binding assay, such as radioi ,oa~s~y ~RIA) or enzyme-linked i ~hsr~rhent assay (E~ISA) The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis o~ Munson & Pollard, Anal Biochem 107:220 (1930l. ~

.

~ ~17~
WO 9511C036 PCTll~S94/1~277 After hybridoma cells are identifled that produce antlbodles of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods. Goding, Monoclonal Antibodies: Principles and Practice, pp.59-104 (Academic Press, 1986) . Suitable culture media for this purpose include, for example, Dulbecco ' s Modified l~agle ' s Medium or RPMI-1640 medium. In addition, the hybridoma cells may be grown ln vivo as ascites tumors in an animal.
The monoclonal antibodies secreted by the subclones are sultably separated from the Gulture medium, ascites fluid, or serum by conventional immunogIobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional procedures ~e.g., by using oligonucleotide probes that are capable~ of binding specifically to genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected lnto host cel l s such as simian COS
cells, Chinese hamster ovary tCHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison, et al., Proc. Nat. ~cad. Sci. 8l, 6851 ~1984), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid" antibodLes are 3 0 prepared that have the binding specificity of an anti-selectin ligand monoclonal antibody herein.
Typically such non-immun~oglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining s1te of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an IFN-y receptor 13-chain and another antigen-combining site having specificity f or a di f f erent antigen .
Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. I~or example, immunoto~ins may be constructed - 4 ~

WO 95/16036 ~17 7 ~ 7 ~ PCT/US94/14277 Examples of suitable reagents for this purpose include lminothiolate and methyl-4-mercaptobutyrimidate~
For diagnostic applications, the antibodies of the lnvention typically will be labeled with a detectable molety. The detectable 5 moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal For example, the detectable moiety may be a radioisotope, such as 3H, "C, "P, 'Ss, or '25I, a fluorescent or chemiluminescent compound, such as fluorescein isothlocyanate, rhodamine, or luciferin; radioactive isotopic labels, such as, e.g, 10 IZ~I, "P, '~C, or 'H, or an enzyme, such as alkaline phosphatase, beta-qalactosidase or horseradish peroxidase.
Any method known in the art for separately con3ugating the ~ntibody to the detectable moiety may be employed, including those methods described by Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974); Paln, et al., J. Immunol. Ileth.
40:219 ~1981); and Nygren, ~. Histochem. and Cytochem. 30:407 ~19B2) .
The antibodies of the present inventlon may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 ~CRC Press, Inc., 1987) .
Competitive binding assays rely on the ability of a labeled standard ~which may be an IFN-y receptor ~-chain or an immunologically reactive portion thereof) to compete with the test sample analyte ~I~N-25 y rec~ptor ,~-chain) for binding with a limited amount of antibody. The amount of IFN-y receptor ¦3-chain in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate deterr~iining the amount of standard that becomes bound, the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound. .
Sandwich assays involve the use oi two antibodies, each capable of binding to a different im~unogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex. David & Greene, U.S. Pat No. 4,376,110.
The second antibody may itself be labeled with a detectable moiety ~direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in whlch case the detectable moiety is an enzyme.

.

~177~7~
WO 95/16036 ~ PCTIUS94/14277 ~iii) Humanized antibodies Methods for humanizing non-t~uman antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human These 5 non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable domain.
Humanization can be essentially performed following the method of Ninter and co-workers [Jones et al_, Nature 321, 522-525 (1986);
Riechmann et al., Nature 332, 323-327 ~1988~i Verhoeyen et al., Science 239, 1534-1536 (1988) ], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies ~Cabilly, supra), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human 15 species In practice, humanized antibodies ar~ typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analoyous sites in rodent antibodies_ It is important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological 20 properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences.
~hree dimensional immunoglobulin models are commonly available and are 25 fariliar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate ~mrLunoglobulin sequences. InspeCtion of these displays permits analysis of the likely role of the residues in the functioning o~ the candidate immunoglobulin sequence, i.e. the0 analysis of residues that influence the ability of the candidate nnl nhulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen (s), is achieved. In general, the CDR residues are 35 directly and most substantially involved in influencing antigen binding .
Alternatively, it is now possible to produce transgenic ~nimals (e.g. mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous 40 immunoglobulin production. For e~ample, it has been described that the homozygous deletion of the antibody heavy chain joining region ~J~) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line WO 95116036 21~ 7 ~ 7 ~ PCTIUS94/1.1277 immunoglobulin gene array in such germ-llne mutant mice will result ln the production of human antibodies upon antigen challenge . See, e . g .
Jakobovits et al_, Proc. Natl. Ac~d. Sci. USA 90, 2551-255 ~1993~;
Jakobovits et al., Nature 362, 255=258 ~1993).
(iv~ Bispecific:antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificlties for at least two different antigens. In the present case, one of the binding specificities is for an IFN-y receptor il-chain, the other one is for any other antigen, and preferably for an other receptor or receptor subunits. For example, bispecific antibodies specifically blnding an IFN-y receptor ~-chain and an IFN-y receptor oL-chain, a chain of ~nother cytokine receptor (i.e. a TNF receptor, an Il.-2 receptor), or of an EP0 receptor are within the scope of the present invention.
Methods for making bispecific antibodies are known in the art.
Traditionally, the recor~binant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305, 537-539 (lg83) ) .
Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. : The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in PCT application publication No. W0 93/08829 (published 13 Iqay 1993), and in Traunecker et al., EMB0 10, 3655-3659 (1991) .
According to a different and more preferred approach, antibody variable domains with the desired binding specificitLes (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at~ least part of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CHl) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are cotransfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide. chains in one expression vector when the expression of at least two WO 95116036 ~ 1 7 7 ~ 7 L PCT~US94/14277 polypeptide chains in equal ratios results in high yields or when the ratios are of no particular signlficance. In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair ~providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation.
For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymoloqy l2l, 210 (1986).
~v) Heterocon3ugate antibodies Heterocon~ugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4, 676, 980), and for treatment of HIV lnfection (PCT application publici~tion Nos. WO 9l/00360 and WO 9:2/200373; EP 03089) .
Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in IJ.S. Patent No. 4, 676, 980, along with a number of cross-linking techniques.
M. Pharmaceutical compositions and administration The IFN-y receptor ~-chain polypeptides of the present invention as well as the anti-IFN-y 13-chain antibodles, elther in monospecific or bispecific or heteroconjugate form, are useful in signaling, enhancing or blocking IFN-y biological activity. They may also be useful in signaling, enhancing or blocking the bio1c~i~Al activities of other biologically active polypeptides, such as other cytokines or EP0.
The known biological activities of IFN-y are multifold, and include antimicrobial activity against a variety of viruses, bacteria, parasites and fungii antitumor activity alone or in combination with other agents of similar activity ~especially in the treatment of colon tumor, non-small cell lung carcinoma, small cell lung carcinoma, breast tumor, sarcomas, melanomas); immunoregulatory activities, such as enhancing the host antibody response to specific antigens, which enables the use of IFN-y as vaccine adjuvant. Re~ inAnt human gamma interieron (Actimmune~, Genentech, South San Francisco, California) is - 40 1 rriA11y available as an i , '~-1Atory drug for the treatment of chronic granulomatous diaease ~-hArAI-t~-ri7~ by severe, recurrent infections of the skin, lymph nodes, liver, lungs, and bones due to phagocyte disfunction. IFN-y receptor ~-chains or anti-IFN-y receptor WO 95/16036 ~1 7 7 ~ 71 PCT/'IJS94/11277 antibodies of agonist chara~ter may mimic these are other IFN-y a~tivities .
Other IFN-y receptor ~-chain polypeptides and antagonist anti-IFN-y receptor ¦3-chain antibodies, alone or ln association with an o~-5 chain, may block IFN-y biological activity. This antagonist activity i5 believed to be useful in the treatment of pathological conditions associated with endogenous IFN-y production, such as inflammatory bowel disease (including ulcerative colitis and Crohn's disease) and liver damage, such as fulminant hepatic failure.
Therapeutic formulations of the present invention are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiQlogically acceptable carriers, excipients or stAhil;7~.r~ ~Remington's Pharmacgutical Sciences 16th edition, Osol, A. Ed. ~1980~ ), in the form of lyophilized cake or aqueous solutions.
Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acidsi antioxidants including ascorbic acidi low molecular weight ~less than about 10 residues) polypeptidesi proteins, such as serum albumin, gelatin or immunoglobuLinsi hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysinei monosaccharides, ~ii CAl-~hA~i~l c and other carbohydrates including glucose, mannose, or dextrinsi chelating agents such as E:DTAi sugar alcohols such as mannitol or sorbitoli salt-forming counterions such as sodiumi and/or nonionic surfactants such as Tween, Pluronics or Pl;G.
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microc~psules and poly- (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nAnn~Arclll es) or in macroemulsions . Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.
The formulations to be used for ir VIVO administration must be sterile. This is readily A~ 1 i .h~`i by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
Therapeutic compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic in j ection needle .

.

WO 9S/16036 ~ PCTIUS94/14277 The molecules of the present lnvention optlonally are combined with or administered in concert with other cytokines, such as TNF, lymphotoxin, IL-2, hepatocyte growth factor (hGF), EPO, conventional antitumor agents, such as S-fluorouracil (S-FU) or Etoposide (VP-16), etc The route of administration is in accord with known methods, e.g injection or infusion by intravenous, intraperitoneal, intracerebral, intL .,..1 ar, intraocular, intraarterial or intralesional routes, topical administratlon, or by sustained release systems.
suitable examples of sustained release preparatlons include semipermeable polymer matrices in the form of shaped articles, e.g.
films, or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Patent 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (U. Sidman et al, Biopolymers, 22 (1): 547-556 (1983) ), poly (2-hydroxyethyl-methacrylate) (R Langer, et al., ~. Biomed. Mater. Res. lS: 167-277 (1981) ) and R. Langer, Chem. Tech. 12: 98-lOS (1982) ), ethylene vinyl acetate (~r~. Langer et al, Id. ) or poly-D- (-) -3-hydroxybutyric acid (EP
133, 988A) . Sustained release compositions also include liposomes .
Liposomes containing a molecule within the scope of the present invention are prepared by methods known per se: DE 3,218,121A; Epstein et al, Proc. Natl. Acad. sci. USA, 82: 3688-3692 (1985); ~Iwang et al, Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1930); EP SZ322A; EP 36676A;
EP 88046A; EP 143949A; EP 142641A; Japanese patent application 83-118008; U.S. patents 4, 485, 045 and 4, 544, 545, and EP 102, 324A.
ordinarily the liposomes are of the small (about 200-800 Angstroms) l~nil:~m~ r type in which the lipid content is greater than about 30 mol. g cholesterol, the selected proportion being adjusted for the optimal NT-4 therapy An effective amount of a molecule of the present invention to be employed therapeutically will depend, for example, upon the therapeutic ob]ectives, the route of administration, and the conditlon of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from about 1 ug/kg to up to 100 mg/kg or more, depending on the factors mentioned above. Typically, the clinician will administer a molecule of the present invention until a dosage is reached that provides the required biological effect. The progress of this therapy is easily monitored by conventional assays.
The following examples are offered by ways of illustration and not by way of limitation.

WO 95/16036 21 7 7 ~ 7 ~ PCT/US94/14277 E =LEs The following experlmental procedures were used in Examples 1 and 2 herein below.
5 Pla~mid Constructions The expression vector pHMG-A7 ' containing the ent1re coding region of the murine IFN-yR cDNA was described previously (Hemmi et al., Proc. Natl. Acad. sci. USA 86, 9901-9905 (1989) ) .
The reporter plasmid pUMS (GT) ,-Tac was derived from the plasmid pUMS-UASGH (Sailer et al., Gene Expr. 2, 329-337 (1992) ) which~was cut with BamHI and EcoRI to excise the insert encoding the human growth hormone (GH) and blunted. A HindIII fragment of plasmid pKCR.Tac-2.A
(Nikaido et dl., Nature 311, 631-63S (1984) ) containing the coding region of the human IL 2 receptor -chain (Tac antigen) was blunted and ligated into the above vector to generate pUMS-UAS-Tac. This construct was cleaved with Clal and HindIII: to excise the UAS-sequence and an oligomerized hexamer (GA~AGT) ~, synthesized to carry Clal-dnd HindIII-~
compatible overhangs, was inserted to generate pUMS(GT),-Tac'. An EcoRI
fragment containing the simian virus 40 (SV40) enhancer was excised from the plasmid 61P IKuhl et al., Cell 50, 1057-1069 (1987) ), blunted ~nd lig~ted into the partially Pvul-digested, blunted pUMS(GT),-Tac~ to generate pUMS (GT) ,-Tac (SV40 ), hereafter pUMS (GT) 8-Tac.
The expression plasmid pCDM8-Tac was derived from pCDMa (Seed and Aruffo, Proc. Natl. Acad. Sci. USA 84, 3365-3369 (1987) ) by releasing its BstXl-stuffer and inserting the blunted HindIII fragment of plasmid pKCR . Tac-2 .A.
Generation o~ the cell Line CO3N 31 COSN cells (a subline of COS~ cells, provided by Dr. S. Nagata) were grown in D-MEM (Gibco) supplemented with 10g FCS. Approximately 2 3U x 10' exponentially growing cells were cotransfected by the calcium phosphate precipitation method (Graham and Erb, virology 52, 456-~67 (1973) ) with 10ug Qiagen-purifled pUMS(GT),-Tac, 10ug pHMGA7', and 2ug of pSV2neo DNA (Southern and Berg, J. Mol. Appl. Genet. 1, 327-341 (1982) ) . G418-resistant colonies were pooled and incubated for 48 hr at 370C with 500 units/ml recombinant huIFN-y, which cross-reacts with the simian IFN-yR Cells expressing the Tac 12 antigen were enriched by two consecutive rounds of panning (Seed and Aruffo, Supra), using an anti-Tac Mab ~Becton Dickinson) . In a third round of panning, uninduced cells with constitutive Tac antigen expression were eliminated. In a fourth round, the cells were enriched for the expression of the muIEN-yR using a Mdb against the muIFN-yR (Basu et al., J. Interferon Res. 9, 551-562 (1989) ) . Adherent cells were subse~uently subcloned and individual colonies screened for muIFN-yR

WO 95/16036 X 1 7 ~ ~ ~1 PCT/US94/14277 expression with iodinated muIFN-y ~Aguet and Merlin, J. Exp Med 165, 988-999 (1987~ ) Positive colonies were verlfied by ~ytofluorometry to express the muIFN-y~ and to induce the Tac antigen in response to human but not muIFN-y. The experiments described herein were carried out with one subclone designated COSN 31.
Before using COSN 31 cells for expression cloning, we verified their capaclty to support episomal plasmid replication. Exponentially growing cells were transiently transfected by electroporation, essentially as descrlbed by Gearing ~Gearing et al., EMBO J 8, 3667-3676 ~1389) . Briefly, 2 x 10~ were resuspended in 180ul phosphate-buffered saline, pH 7.2 ~PBS) prior to adding Sug pCDM8-Tac DNA in 20uI
H2O and electroporated at 300 V, 125uFD After culture for 72 hr at 37OC the cells were detached by treatment with 20 mM EDTA in PBS for 20 minutes at 37C, washed once with PBS, pelleted and lysed in 1. 6 ml 0 6~s SDS, 10 mM EDTA for 30 minutes at room temperature. NaC1 was added to a final concentration of lM and the lysate incubated on ice for 24 hr prior to phenol-extraction and ethanol precipitation of extrachromosomal DNA To distinguish between transfected Dpnl-methylated and replicated unmethylated plasmid DNA, the extracted DNA
was Dpnl-digested prior to transformation of MC 1061/p3 E. coli host cells ~Seed and Aruffo, Supra). Colony counts clearly illustrated that COSN 31 cells had retained the ability of episomal replication of pCDM8 -Tac .
8cr~ening of cDNA librAry The pAGS-3 cDNA library, which was derived from oligo ~dT) primed poly ~A~ mRNA from the murine early B-cell line Y16, was kindly provided by Dr. S. Takaki ~Takaki et al., EMBO J. 9, q367-4374 ~1990) ) The library was divided into six pools of approximately 3x105 independent colonies. For the first round of enrichment, 3 x 5ug DNA
from each pool was transfected separately into 3 x 106 subconfluent COSN
31 cells by electroporatlon as described above and seeded into three

8.5 cm-Petri dishes. After 24 hr at 37OC, fresh medium containing 200 U/ml muIFN-y was added and the cells cultured for another 48 hr. The panning procedure was performed according to ~Aruffo and Seed, Proc.
Natl. Acad. Scl USA 84, 8573-3577 (1987) ) . Briefly, cells were detached by incubation in PBS, 20 mM EDTA, incubated for 1 hr on ice in BSS ~140 mM NaCl, 1.0 mM CaC12, 5.4 mM KCI, 0.8 mM MgSO" 0.3 mM Na2HPO,, 0 4 mM KH2PO" pH 7.0), 5~ FCS containing a mouse Mab to the human Tac antigen ~Becton Dlckinson), washed and incubated for 90 minutes at room temperature on bacteriological Petri dishes previously coated with af finity purified rabbit anti-mouse IgG immunoglobulin . The plates were gently washed three times with BSS, 2% FCS. DNA was extracted from adherent cells as described above and amplified in MC1061 E. coli WO 95/16036 2 1 7 ~ ~ 7 1 PCS/US94/14277 host cells ~Seed and Aruffo, Supra) . The subsequent rounds of transfection and enrichment were carried out separately for each of Lhe six original cDNA pools with 10 COSN 31 cells transfected with Sug DNA
Cyto~lu~,~ L~y COSN 31 or HEp-2 cells cultured to subconfluency in 10 cm2 wells under conditions indicated in the figure legends were detached by treatment with PBS, 10 mM EDTA, washed with culture medium and incubated for 90 minutes at 40 with mouse Mabs specific for the human Tac ~ntigen (Becton Dickinson), or common human MHC class I or class II
antigen ~f.r~.rrin~nts (monoclonal antibodies W6/32 and L243, Serotec and Becton Dickinson, respectively). The cells were washed by centrifugation, incubated for 60 minutes at 40 with a FITC-conjugated rabbit anti-mouse IgG second antibody (Serotec), and washed again prior to cytofluorometry (Epics XL, Coulter) . Expression of ~the muIFN-yR (Y-chdin was monitored accordingly, using a rat-anti muIFN-yR Mab (Basu et al., Supra) and FlTC-conjugated rabbit-antl-rat IgG E ~ab' ) 2 antibodies.
Antivir~l ~say Human or murine IFN-y was assayed on human HEp-2 ~ATCC) or murine L929 cells challenged with vesicular stomatitis virus ~VSV) . One unit/ml ~U/ml) of IFN is defined as the concentration that ~esults in 50~ protection from the cytopdthic effect.
Example 1 The cloninq of murine IFN-y receptor ~-chain Expreesion Clonlng ~tr~tegy To identify the putative species-specific accessory component needed for the functionality of the IFN-y receptor ~IFI~I-yR), we designed a complementation approach based on the known cDNA expression cloning strategy in COS cells [Aruffo and Seed, Proc. Natl. Acad Sci.
USA 84, 8573-8577 ~1987) i Seed and~Aruffo, Proc. Natl. Acad. Sci. USA
84, 3365-3369 ~987~ ] . C057 cel1s were stably cotransfected wlth the cDNA expression plasmid pHMG-A7 ' encoding the murine IFN-y receptor (muIFN-yR) (Hemmi et al., Proc. Natl. Acad. Sci. USA 86, 9901-9905 (1989) ) and the reporter plasmid pUMS(GT),-Tac (Figure lA) which consisted of an artificial multimerized IFN-y-inducible promoter element ~MacDonald et al., Cell 60, 767-779 ~1990) ) linked to a cDNA
encoding the human Tac antigen ~IL-2 receptor o~-chain, CD25) ~Nikaido et al., Nature 311: 531-635 rl984~) A COS cell line ~COSN 31) was isolated which stably expressed the muIFN-yR, but responded only to human and not to murine IFN-y ~muIFN-y) by expressing the Tac antigen ~Figure lB) .

WO 95/16036 ~ 17 7 ~ ~ ~ PCT/US94/1~277 Cloning o~ a rDNA Encoding the IVN-yR Accessory Component (IFN-yP~ ~-ch~in ) COSN 31 cells were transiently transformed with pools of the murine eaLly ,B-cell-derived cDNA library pAGS-3 (Takaki et al., EMBO J.

9, 436i-4374 ~1990) ) and cells responsive to muIFN-y in teLms of Tac antigen express1on were enriched by panning. After four rounds of enrichment, one of six pools gave rise to significant muIFN-y-induced adherence of COSN 31 cells to the panning plate. The proportion of cells adhering to the panning plates at this stage was about five times above background, which amounted to about 0 5~ of cells, due to some constitutive expression of the Tac antigen. Two out of 24 cDNA clones picked randomly from the cDNA recovered from these fourth round cells were able to render COSN 31 cells sensitive to muIFN-y, and were identical in teLms of their insert size ~clones pAGS.Cl9 and pAGS.C2) .
A third positive clone ~pAGS.M17~, isolated from the same cDNA pool after a fifth round of panning, contained a smaller insert. Transient expression in COSN 31 cells of all three cDNA clones resulted in muIFN-y-induced adherence of about 20-30~ of the cells to the panning plates, reflecting the transfection efficiency. Figure lC shows a cytofluorometric analysis of murine versus human IFN-y- ~huIFN-y) induced Tac antigen expression in COSN 31 cells transiently expressing pAGS.Cl9 cDNA. About 30~ of the cells showed muIFN-y-induced Tac antigen expression. The level of expression was similar to the one observed with huIFN-y.
rDNA rhAr----t~i ~ r And Sequ~nce All three cDNA clones isolated from the pAGS-3 cDNA library had lost the restriction sites flanking the irsert, probably due to rearrangements known to occur frequently during episomal replication in COS cells (caIos et al., Proc. Natl. Acad. Sci. USA 80, 3015-3019 ~1983) ) . All three clones contalned a seemingly common 1.1 kb HindIII
- Xbal fragment which contained part of the insert. This HindIII -Xbal fragment from the pAGS.CL9 clone was verified to contain part of the insert by sequencing and Northern blot hybridization to mouse spleen RNA, and used as a probe to screen an oligo ~dT) primed murine Agtll cDNA library described previously ~Hemmi et al., Proc. Natl.
Acad. Sci. USA 86, 9901-9905 (1989) ) . A positive clone was isolated ~Al.cl9) which contained a 1283 bp EcoRI insert. The inserts from both the pAGS.Cl9 and the Al.Cl9 clone were sequenced by the chain termination method ~Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463-5467 ~1977) ) using sequence-specific oligonucleotide primers and contained an identical open reading frame of 996 bp. The nucleotide and inferred amino acid sequence of the Al.C19 clone are shown in Figure 2A. The first ATG of the largest open reading frame WO95/16036 21 77~ 71 PCT/US94/14277 ~nucleotides 94-961 is: emoedded in a typlcal consensus sequence for translation initiation (~ozak, Nu~leic ~cids Res. 15, 8125-8148 (1987) ) A translation product starting at this position would consist of 332 amino acids, starting with a presumed signal peptide of 18 amino acids (von Heijne, Nucleic Acids Res. 14, q683-4690 (1986) ) Hydrop~thy analysis revealed the presence of an additional hydrophobic stretch encompassiny amino acid residues 225 to 248 of the mature protein. This putative transmembrane anchoring domain would subdivide the mature protein into an extracellular domain of 224 and a cytoplasmic domain of ~6 amino acids.
We did not find extensive nucleotide or amino acid sequence similarity to any known genes or proteins. However, amino acid sequence alignment of the putative extracellular portion of the muIFN-yR ~-chain with the two duplicated extracellular domains of the type I
IFN receptor, as well as with the known ligand binding chain of muIFN-yR, identifies it as a member of the IFN receptor family (Bazan, Cell 61, 753-754 (1990) ), to which the IL-10 receptor has also been assigned recently (Yue Ho et al., Proc. Natl. Acad. Sci. USA, 90 (23): 11267-11271 (1993) ) . Common motifs include notably two cystein pairs and conserved proline, tryptophan and tyrosine residues (Figure 2B) .
,.r. and chromo om~l Loc~tion o~ the IFN-yR ~-ch~in The insert of the A1.C19 cDNA clone was subcloned into Bluescript and used as a probe for Northern blot hybridization of RNA from different organs. A single transcript of about 2.0 kb was detected in RNA from spleen, liver, kidney, lung and brain. The Al.Cl9 insert which contained one internal Scal and no EcoRV site, hybridized to only two genomic Scal and one EcoRV DNA fragments, suggesting that the transcript was most likely the product of a single gene.
In man, the cofactor for the IFN-yR was proposed to be encoded on chromosome 21 (Jung et al., Proc. Natl. Acad. Sci. USA 84, 4151-4155 (1987) ) . To verify this suggestion, the Al.Cl9 insert was used as a probe to isolate a full length cDNA encoding the huIFN-yR ~3-chain from a human ugtll cDNA library constructed with Namalwa cell mRNA (Sailer et al, Nucleic Acids Res. 20, 2374 (1992a) ) . The insert from this human clone was sequenced and found to encode the human counterpart of the muIFN-yR ~-chain. The nucleotide and deduced amino acid sequences of huIFN-yR ~-chain are depicted in Figures 4 and 5. Hybridization of the labeled insert to Southern blots of genomic DNA from the ~AVRDdAl 9 mouse/human hybrid cell line (Coriell Cell Repositories) containing chromosome 21 as the only human chromosome resulted in a pattern indistinguishable from that observed with genomic DNA from human cells, indicating that the gene encoding the huIFN-y receptor 13-chain cDNA is indeed contained on human chromosome 21.

WO 9~/16036 21 7 ~ ~ 71 PCT/US94/14277 Example 2 The bioloqical function of murine IFN-y receptor ~-chain Responsiven--ss to muI~N-y o~ h~n HEp-2 cells ~ q both muIE'N-yR
ch~-inr To investigate its functionality, expression constructs encoding the novel receptor subunit were stably transfect~d into a previously described human HEp-2 cell line expressing the muIFN-yR -chain (Hemmi et al., supra) . The murine IFN-yR -chain expressed 1n these cells (subline ~Ep-2xmuIFN-yR#43.7) was able to bind muIFN-y with high affinity, but was nonfunctional, since these cells responded only to human, but not murine IFN-y in terms of inducible expression of MHC
class I and class II antigens, antiviral response and growth inhibition (Hemmi et al., ~).
HEp-2xmuIFN-yR~z#43. 7 cells were stably transfected with either the original expression plasmid pAGS.C19, or the expression plasmid pHMG.Cl9 containing the Al clg insert driven by the 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter (Gautier et al., Nucleic Acids Res. l7, 8389 (1989); Hem~i et ~l., supra) . Figure 3 shows the response to murine versus huIFN-y of parental HEp-2xmuIFN-yR#43 . 7 cells, and one subline o~ these cells stably transfected with the pHMG.C19 expression plasmid encoding the muIFN-yR 13-chain (HEp-2xmuIFN-yR/~#6) . Incubation of HEp-2xmuIFN-yR/~#6 cells with either human or muIFN-y resulted in a 4-5-fold increase of MHC class l antigen expression ( Figure 3A) and a de novo expression of MHC class Il antigens (Figure 3E), whereas parental cells expressing only the muIFN-yR -chain were insensitive to muIFN-y.
Analysis of three additional response markers confirmed these results: Figure 3C shows that HEp-2xmuIFN-yR#43.7 cells respond only to huIFN-y, but not muIFN-y in terms of IFN regulatory factor 1 (~RF-1) mRNA induction (Miyamoto et al~., Cell 54, 903-913 ~1988) ), whereas HEp-2xmuIFN-yR/~#6 cells become fully responsive to muIFN-y as well Finally, the results depicted in Figures 3D and 3E illustrate that HEp-2xmulFN-yR~#6 cells expressing both murlne receptor subunits respond equally well to the antiviral and anti-proliferative eifects of muIFN-y and huIFN-y.
These results were confirmed with an independent clone of HEp-2xmuIFN-yR#43.7 cells transfected with the expression plasmid pHMG.C19 (HEp-2xm~1IFN-yRol/3#10), and, with regard to MHC class I and class II
antigen expression, for several clones transfected with the original expression plasmid pAGS.C19.

WO 95116036 2~ 1 7 ~ 4 7 ~ PCTI~JS9411.1277 Clearly, expression of the muIFN-yR accessory or ~-chain in human HEp-2 cells that already express the muIFN-yR ~-chain rendered these cells as sensitive to murine as to huIFN-y with regard to all response markers tested.
The indistinguishable antlviral response to murine as compared to human IFN-y was in contrast to previous reports from our and another laboratory (Hemmi et al., Proc. Natl. Acad. Sci. USA 89: 2737-2741 (1992); Cook et al., Proc Natl. Acad. Sci. USA 89: 11317-11321 (1992) ), according to which mouse/human somatic cell hybrids containing human chromosome 21 and expressing the huIFN-yR -chain were not fully protected from the cytopathic effect of vesicular stomatitis virus (VSV) by huIFN-y, suggesting the requirement of still another species-specific cofactor to mediate the antiviral effect of huIFN-y in mouse cells. This discrepancy remains unexplained and might be due to a different compatibility of the mouse receptor in human cells than vice versa, or to insufficient expression of the possibly rate-limiting ~-chain .
Antibodies raised against the novel receptor subunit should help clarifying this latter point and also, how the 13-chain interacts with the a-chain and whether it is involved in ligand-binding. Experiments with human/mouse hybrid IFN-yR a-chains suggested that species-specific interaction with the putative ~-chain involves the extracellular portions of both subunits ~Gibbs et al, Mol. Cell. Biol. 11, 5860-5866 (1991); Hemmi et al., supra; Hibino et al, ~ Biol. Chem. 267, 37~1-3749 (1992); Kalina et al., J. Virol. 67, 1702-1706 (lg93) ), but it remains unclear whether they become linked together through the dimeric ligand (Ealick et al., Science 252, 698-702 (1991)), or whether they interact directly. Chemical cross-linking experiments suggested that IFN-y-binding can induce dimerization of the a-subunit of the receptor (Greenlund et al., J. Biol. Chem. 268, 18103-18110 (I993) ), but it is not clear whether th~is homodimer represents the functional IFN-yR and there is no biochemical information so far on the involvement of the ¦3-subunit.
Still, the sequence of events described for the growth hormone receptor, where binding of the bivalent ligand to one receptor subunit triggers ligand binding to the second subunit ~Tartaglia and Goeddel, Science 256, 1677-1680 ~1992) ), might serve as a paradigm for the IFN-yR. The absence of detectable IFN-y-binding to cells derived from mice lacking the IFN-yR a-chain (Huang et al., Science 259: 17~2-17~5 (1993) ) suggested that the ~-chain, provided it was still normally expressed in these cells, is unable to bind IFN-y on its own. The relatively short cytoplasmic domain of the 8-chain contains a motif-WO 95/16036 2 ~ 7 7 ~ 7 I PCT/US94/1.1277 LEVL(D) which is reminiscent of the conserved box 2 region of some cytokine receptors, including not~bly the murine IL-2 receptor ~-chaln and the murine erythropoietin receptor (Murakami et al., Proc Natl.
Acad. Sci USA 88, 11349-lI353 (1991) ) . Mutations within this domain 5 suggested that it is crucial in the mitogenic responses mediated through these receptors (Miura et al., Mol. Cell Biol_~ 13, 1788-1795 (1993) ) . Thi5 lS of interest in view of recent findings suggesting that erythropoietin and IFN-y-mediated signaling pathways may share common steps since both involve activation of the JA~2 tyrosine kinase (silvennoinen et al., Science 261~ 1736-1739 (1993) i Wltthuhn et al, Cell 74, 227-236 ~1993) ) and further references therein) . Obviously, the biological responses to these cytokines dlfer, suggestlng that, in addition to the comrLon motif5 and signaling components, more specific signaling elements remain to be identified. Certain cytokine receptors, the IL-3, IL-5 2nd GM-CSF receptors (l~itamura et al, Cell 66, 1165-1174 (1991); Tavernier et al, Cell 66: 1175-1184 (1991) ), and also the IL-6, LIF, oncostatin M and CNTF receptors (Gearing et al., Science 255, 1434-1437 (1992); Taga et al., Proc Natl. Acad. Sci USA 89, 10998-11001 (1992) ) share common subunits. Likewise, the discrepancy between the phenotype of mice lacking IL-2 (Schorle et al, Nature 352, 62162-62164 (1991) ), and the X-linked severe corbined immunodeficiency, which is due to a truncation of the IL-2 receptor ysubunit (Noguchi et al., Cell 73, 147-157 (1993) ), might be e~plained by a presumed role of this subunit in other signaling systems. While it is tempting to speculate that IFN-yR ~-subunit might also be a constituent of other receptors, the o~-chain has at least two, so far unique cytoplasmic domains (a membrane-proximal domain r~n~ , ~ccinq 48 amino acids and C-terminal YNI~ stretch) that are essertial for biologlci~l responsiveness to IFN-y (Farrar et al., ;~ Biol Chem 266, 19626-19635 (1991); Farrar et al., Proc. Natl Acad. Sci. USA 89, 11706-11710 (1992) ) and might be involved ir interaction with more specific signaling elements.
All citations throughout the specification and all references cited thereir are hereby expressly incorporated by reference. ~lthough the foregoing refers to particular preferred embodiments, it will be understood th2t invention is not so limited. It will occur to those ordinarily skilled in the art that various modifications can be made Without diverting from the overall concept of the invention. All such modifications are intended to be withln the scope of the present 4 0 invention .

~1~7 SEQUENCE LISTING
(1) GENERAL lN~'Uhl__IlUN:
(i) APPLICANT: Aguet, Nichel Bohni Ruth He~ni, Silvio (ii) TITLE OF INVENTION: Receptor Subunit Polypeptide~
(iii) Nt1NBER OF SEQUENCES: 8 (iv) ~U~;~UNL~ ; ADDRESS:
A) ~nn~ECCEE: Genentech, Inc.
B) STREET: 460 Point San Bruno Blvd C) CITY: South S~n Francisc~
D) STATE: California E ) COUNTRY: USA
F) ZIP: 94080 (v) COMPDTER READABLE FOR0:
A) NEDIDM TYPE: 5.25 inch, 360 E~b floppy disk B ) COMPUTER : I BN PC, ,~ t ; hl ~
C) OPERATING SYSTEN- PC-DOS/NS-DOS
~D) SOFT~ARE: patin (r.f.n~ntf.rh) (vi) CDRRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/DS94/14277 (B) FILING DATE: 07 -DEC- 1994 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION t~NBER: 08/164596 (B) FILING DATE: 09-DEC-1993 (viii) ATTORNEY/AGENT l~u~
(A) NANE: Love, Richard B.
(B) REGISTRATION N[~3ER: 34,659 (C) REFERENCE/DOC~ET N~MBER- 866PCT
(iX) TET.Err)MMrT~TTr~TION INFORMATION
(A) TELEPHONE: 415/225-5530 (B) TELEFAX: 415/952-9881 (C) TELEX: 910/371-7168 (2) lNr'U~lAIlU~ FOR SEQ ID NO:l:
(i) 'E~2~ENCE rH~rT~T~TIcs:
A LENGTH: 1283 ba~e~
50B TYPE: nucleic ~cid C ST~ ingle D' TOPOLOGY: linear (xi) SEQTJENCE DESCRIPTION: SEQ ID NO:l:
GAATTCGGCA rr~Ar~r~rr~r~n U~ ~l~ GCCATGGCCG ~l~ i 50 60AGTCTGAGCG GCGTCCACCC ~ u ~ ~ 100 CTTTGCCACT ~ 150 .U CAGACTCGTT TTCCCAGCTT ~u~ l. Tr7~rrr2~7~r 200 GCTTCACCTG TACAATGATG AGCPGATTCT AAcI~rGGGAG CCGTCACCTT 250 RECTIFIED SHEET (RULE 91) ISA/EP

1 ~ 7 7 WO 95/16036 2 7 ~ ~ PCT/US94111277 rrDrrDDT~r.D rrr7~Dr7~rr~D~ GTGGTCTACC AGGTGGDATA TAGCTTCATC 300 5 GATGGCTCTT ~GS~ATAGGTT GCTGGAGCCG PDrTrTDrrr ACATCACAGA 350 GACADAC'TGT GACTTAACAG rDc~rrr~r~rrr CTTGAAGCTT TTrrrPrDrC 400 CATTCDCAGT ~LlL ~ L-LJG GTGCGAGCCA AGCGAGGGAA CCTCACTTCC 450 15 AAGTGGGTGG GGCTGGAGCC ATI~rc~DAcAc TATGAGAATG TTACTGTTGG 5 0 0 ACCTCCGAD~A AACATCTCGG 'I`GACCCCAGG ADDAGGTTCC CTCGTCATAC 550 20 ACTTCTCCCC LL_~L_LlLL~A'r GTGTTCCACG GGGCDACTTT TCAGTATCTT 600 25 GTCCACTACT GGGAZAAGTC AGADACCCDA /'Prr'`~rPr.r. TrGADGGCCC 650 ATTGT~TACA AACTGAGGCA CDACTGATTT TGAAAAACDA ADD~AATCCGA 750 CCACATGGGC TCTTGAGC~DA 1~7L~lL LlLil rDrr.7-D7\r~7~ CDGCAPATGc 800 35 CTCCGCC~DGG CTGCAGCAAG TCATCCTGAT LLLLiL~iLiL~C A'l~l ~ LCAl 850 l~i~lLiLL~L~L~ rrTr7rrçrr LiL_Ll~iLl LL~ l ~ L '`'I " ~ I CP-aATACCAA 900 AGCCGAGTGA AGTACT&'GTT TCZ. GGCTCCG CCAAACATCC CGGAPCAAAT 950 rrp~r~pr~TpT rTPDDrr7~rr CAGACCAATT CATCTTAGAG GTCTTGGACA 1000 AGGACGGTTC 7~rrr~`7rr~DÇ GACTCCTGGG ACTCCGTGTC AATTATTTCT 1050 TrTrrprD7~ prr7~rrr7~çA ~ rP~Dr~rrrT r.AZ~rrDr.Grr 1100 AGGGTCTCTG CTTGCCCAGÇ Prr~ÇrPrrrP TCAGTGCACC rr.Dr~r~ÇPT 1150 rrrrDrr.çrr CCAGGACTGG GGAAGATGGT GTAGTTTTGT TcTTTaTGAG 1200 TTTTCTGGAT GrTprrDrTp TTTAAAAGGA TTCCACAGAA A~ACCCTGTC 1250 TCGGADAAAA prD~DD.`Drr TCGTGCCGAA TTC 1283 (2) ll!l~ UICD'L~llUI~ FOR SEQ ID NO:2:
(i) SEQCIE~CE ~r~TDnDrTR~TsTIcs (A) LEr~GTE~: 332 a=Lino arid~

REGllFlEI~ SH~ET ffW~E 9~) ISA/EP

WO 95/16036 217 7 4 7 1 PCTIUS94/1.1277 ( B ) TYPE: amino ac l d ( D ) TOPOLOGY: 1 inear (xi) SEQIJENCE J~8U~l~llU~: SEQ ID NO:2:

Det Arg Pro Leu Pro Leu Trp Leu Pro Ser Leu Leu Leu Cys Gly Leu Gly Ala Ala Ala Ser Ser Pro Asp Ser Phe Ser Gln Leu Ala Ala Pro Leu Asn Pro Arg Leu His Leu Tyr Asn Asp Glu Gln Ile Leu Thr Trp Glu Pro Ser Pro Ser Ser Asn Asp Pro Arg Pro Val Val Tyr Gln Val Glu Tyr Ser Phe Ile A8p Gly Ser Trp His Arg Leu Leu Glu Pro Asn Cys Thr Asp Ile Thr Glu Thr Lys Cys A8p Leu Thr Gly Gly Gly Arg Leu Lys Leu Phe Pro His Pro Phe Thr 95 . 100 105.
Val Phe Leu Arg Val Arg Ala Lys Arg Gly Asn Leu Thr Ser Lys Trp Val Gly Leu Glu Pro Phe Gln His Tyr Glu A8n Val Thr Val 125 . 130 135 Gly Pro Pro Lys Asn Ile Ser Val Thr Pro Gly Lys Gly Ser Leu Val Ile His Phe Ser Pro Pro Phe A8p Val Phe Hi8 Gly Ala Thr Phe Gln Tyr Leu Val His Tyr Trp Glu Lys Ser Glu Thr Gln Gln Glu Gln Val Glu Gly Pro Phe Lys Ser A8n Ser Ile Val Leu Gl 185 . 190 195 Asn Leu Lys Pro Tyr Arg Val Tyr Cys Leu Gln Thr Glu Ala Gln Leu Ile Leu Lys Asn Lys Ly8 Ile Arg Pro His Gly Leu Leu Ser Asn Val Ser Cy8 His Glu Thr Thr Ala AGn Ala Ser Ala Arg Leu Gln Gln Val Ile Leu Ile Pro Leu Gly Ile Phe Ala Leu Leu Leu 245 . 250 255 Gly Leu Thr Gly Ala Cys Phe Thr Leu Phe Leu Lys Tyr Gln Ser 260 ~ 265 270 60 Arg Val Lys Tyr Trp Phe Gln Ala Pro Pro Asn Ile Pro Glu Gln le Glu Glu Tyr Leu Lys Asp Pro Asp Gln Phe Ile Leu Glu Val 65eu Asp Lys A8p Gly Ser Pro Lys Glu Asp Ser Trp Asp Ser Val RECT~FIED SHEET (RULE 91) ISA/EP

2~ 7~
WO 95/16036 ~ PCTIUS94/14277 Ser Ile Ile Ser Ser Pro Glu Lys Glu Arg ASp Asp Val Leu Gln Thr Pro (2~ lN~U~_~llUN FOR SEQ ID NO:3:
(i) SEQUENCE rT~'`~D~T~TSTICS:
(A) LENGTH: 202 amino acids (B) TYPB: anino aci~
(D) TOPOLOGY: linear (xi) SEQUBNCE L~;S~:~lrLl~: SEQ ID NO:3:
Glu Asn Leu Ly~ Pro Pro Glu Asn Ile Asp Val Tyr Ile Ile As Asp Asn Tyr Thr Leu Lys Trp Ser Ser llis Gly Glu Ser Met Gly Ser Val Thr Phe Ser Ala Glu Tyr Arg Thr Lys ASp Glu Ala Lys Trp Leu Lys Val Pro Glu Cys Gln His Thr Thr Thr Thr s Ly Cy Glu Phe Ser Leu Leu Asp Thr Asn Val Tyr Ile Lys Thr Gln Phe Arg Val Arg Ala Glu Glu Gly Asn Ser Thr Ser Ser Trp Asn Glu Val Asp Pro Phe Ile Pro Phe Tyr Thr Ala His Met Ser Pro Pro Glu Val Arg Leu Glu Ala Glu Asp Lys Ala Ile Leu Val E{is Ile Ser Pro Pro Gly Gln Asp Gly Asn Met Trp Ala Leu Glu Lys Pro 45 Ser Phe Ser Tyr Thr Ile Arg Ile Trp Gln Lys Ser Ser Ser ASp ys Lys Thr Ile Asn Ser Thr ffl Tyr Val Glu Lys Ile Pro Glu 50eu Leu Pro Glu Thr Thr Tyr Cys Leu Glu Val Lys Ala Ile E~is ro Ser Leu Lys LyS ~is Ser Asn Tyr Ser Thr Val Gln Cys Ile er Thr Thr Val Ala Asn Lys 60 (2) INFORD5ATION FOR SEQ ID NO:4:
(i) SEQUBNCE ~TDI~D~T~I~T~TICS:
(A) LENGTH: 200 amino acids (B) TYPE a~lno acld 65 (D) TOPOLOGY iinear (xi) SEQUBNCE Llk;~ llUN: SEQ ID NO:4:

RECriFlED SHEi~ (FULE 91) ISA/EP

WO 95/16036 ~ ~ 7 ~ ~ 71 PCTI'US9411~77 Met Pro Val Pro Gly AGn Leu Gln Val AGP Ala Gln Gly Lys Ser Tyr Val Leu LYL Trp Asp Tyr I~e Ala Ser Ala AGp Val Leu Phe Arg Ala Gln Trp Leu Pro Gly Tyr Ser Lys Ser Ser Ser Gly Ser HiG Ser AGp LYG Trp LyG Pro Ile Pro Thr CyG Ala AGn Val Gln 5 Thr Thr His CYG Val Phe Ser Gln AGP Thr Val r Thr Gl Thr 65 70 Ty Y
he Phe Leu HiG Val Gln Ala Ser Glu Gly AGn HiG Thr Ser Phe Trp Ser Glu Glu LYG Phe Ile AGp Ser Gln LYG HiG Ile Leu Pro Pro Pro Pro Val Ile Thr Val Thr Ala Net Ser AGP Thr Leu Leu Val Tyr Val AGn CYG Gln AGp Ser Thr CYG AGP Gly Leu AGn Tyr Glu Ile Ile Phe Trp Glu Asn Thr Ser Asn Thr LyG Ile Ser Net Glu Lys AGp Gly Pro Glu Phe Thr Leu LYG AGn Leu Gln Pro Leu Thr Val Tyr Cys Val Gln Ala Arg Val Leu Phe Arg Ala Leu Leu AGn LyG Thr Ser Asn Phe Ser Glu LYG Leu CYG Glu LyG Thr Ar ro Gly Ser Phe Ser 45 (2) IXFORMATION FOR SEQ ID NO:5:
(i) SEQ~ENCE r~rT~TCTICS:
(A) LENGTH: 228 amino acidG
(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQ~IENCE Jl:;'iCl~l~lllJI~: SEQ ID X0:5:
A1P Leu Thr Ser Thr Glu AGp Pro Glu Pro Pro Ser Val Pro Val l 5 10 15 Pro Thr AGn Val Leu Ile LyG Ser Tyr Asn Leu AGn Pro Val Val 60 CYG Trp Glu Tyr Gln AGn llet Ser Gln Thr Pro Ile Phe Thr Val Gln Val LYG Val Tyr Ser Gly Ser Trp Thr ASp Ser CYG Thr Asn Ile Ser Asp HiG CYG ryG AGn Ile Tyr Glu Gln Ile Net ffl Pro RECTIFIED SHEET (RULE 91) I SAJEP

~7~7:L
WO 95116036 PCI/~JS94/14277 A~p Val Ser Ala Trp Ala Arg Val LYG Ala LYG Val Gly Gln Lys 5 Glu Ser AGp Tyr Ala Arg Ser LYG Glu Phe Lou Met Cys Leu Lys Gly Lys Val Gly Pro Pro Gly Leu Glu Ile Ar Ar 6 fi Glu 110 g g Ly Ly Glu Gln Leu Ser Val Leu Val Phe His Pro Glu Val Val Val Asn Gly Glu Ser Gln Gly Thr De~ Phe Gly Asp Gly Ser Thr Cys Tyr Thr Phe Asp Tyr Thr Val Tyr Val Glu His AGn Arg Ser Gly Glu 20 Ile Leu His Thr LYG HiG Thr Val Glu LYG Glu Glu CYG AGn Glu Thr Leu CYG Glu Leu AGn Ile Ser Val Ser Thr Leu AGP Ser Ar 185 190 = 195 Tyr Cys Ile Ser Val Asp Gly Ile Ser Ser Phe Trp Gln Val Arg Thr Glu Lys Ser Lys Anp Val Cys Ile Pro Pro Phe His AGp AGP

Arg LYG Asp 35 (2) INFOR~ATION FOR SEQ ID NO:6:
(i~ SEQUENCE rT~7~v~ " ( ..j (A) LENGTH: 223 amino acids (B) TYPE: amino acid 4 0 (D) TOPOLOGY: l inear (xi) SEQIJENCE L/Es~ lJr~: SEQ ID NO:6:
Ala Ala Ser Pro Asp Ser Phe Ser Gln Leu Ala Ala Pro Leu AGn Pro Arg Leu HiG Leu Tyr AGn AGP Glu Gln Ile Leu Thr Trp Glu 50 Pro Ser Pro Ser Ser Asn Asp Pro Ar Pro Val Val Gln Val g Tyr Glu Tyr Ser Phe Ile AGP Gly Ser Trp His Arg Leu Leu Glu Pro Asn CYG Thr Asp Ile Thr Glu Tnr LYG Cys AGP Leu Thr Gly Gly Gly Arg Leu LyG Leu Phe Pro HiG Pro Phe Thr Val Phe Leu Arg Val Arg Ala LyG Arg Gly AGn Leu Thr Ser LyG Trp Val Gly Leu 65 Glu Pro Phe Gln HiG Tyr Glu Asn Val Thr Val Gly Pro Pro Ly8 Asn Ile Ser Val Thr Pro Gly LYG Gly Ser Leu Val Ile His Phe RECrl~IED SHEET (RULE 91) ISA/EP

~17~7~ 0 Ser Pro Pro Phe Asp Val Phe Hie Gly Ala Thr Phe Gln ~yr Leu Val Hio Tyr Trp Glu Lye Ser Glu Thr Gln Gln Glu Gln Val Glu 155 = ~ 160 165 Gly Pro Phe Lys Ser Asn Ser Ile Val Leu Gly Asn Leu Lye Pro

10 Tyr Arg Val Tyr Cy~ Leu Gln Thr Glu Ala Gln Leu Ile Leu Lys Asn Lys Lys Ile Arg Pro Hie Gly Leu Leu Ser Asn Val Ser Cy~

His Glu Thr Thr Ala Asn Ala Ser Ala Arg Leu Gln Gln t2) INFOR~ATION FOR SEQ ID NO:7 (i) SEQIJENCE rTl7~D~ lL~
(A LENGTH 1197 basee (3 TYPE: nucleic acid (C STP~ : eingle 25 (D TOPOLOGY: linear (xi) SEQI~ENCE JJk;~iL~tl~llLN SEQ ID NO:7:
30 GAATTCCGGG r.rr~crTr~~ LLL~l~LL-LL GACCCCGAGC ~ ;l [ ~'l lil. 50 CCGCGACCTG Lrrrrrrarr GAGCGCCCGG L~L~ ~i~ CCGACGCTGC 100 ,LLl L~l4LLliLl~ CTCGGAGTCT l ~ ,~LLLLLL ~ 150 CCGCCAGACC ~l~ Ll-~LLI~ L-~l`iLL~i~1 CCTCAGCACC CGAAGATTCG 200 CCTGTACAAC r.rAr.~r~rr TCCTGAGTTG r,r.~r.rr~r.Tr. GCCCTGAGCA 250 45 PTPr.r~rr~~ LLLl~ TACCAAGTGC AGmAAATA rArrr~ r.T 300 AAATGGTTCA rrrrrr.P(~PT L L~ hIA GGGGTGAATT rTprZ~rpr~T 350 r~r~rr~rD GAGTGTGACT TCACTGCCGC CAGTCCCTC~ GCAGGCTTCC 400 CAATGG~TTT CAATGTCACT CTACGCCTTC GAGCTGAGCT GGGAGCACTC 4 5 0 ~ 11~1~7--Ll GGGTGACi~AT LLLll iL-lll rr7~t~PrTPTr GGAATGTGAC 500 5,~11~WL~/ r(~Ar~ r~ TTGAGGTGAC CCCAGGAGAA L`LLl_~,LlCI' 550 TCATCAGGTT LlCLl--~LCL TTTGArATCG CTGATACCTC CI~CGGCCTTT 600 l~L~LL~ CTGGGAAAAA GGAGGAATCC AACAGGTCAA 650 AGGCCCTTTC ArA~r~r~rT CL~ ~11 GGATAACTTA A~ACCCTCCA 700 RECrlFlED SHEET (RULE 91) ISA/EP

~17747~
WO 95/16036 ~ PCII~JS94/1~277 GAGTGTACTG TTTACAAGTC rDr.r.r~r~Dr TGCTTTGGAA CAA. AGTAAC 750 5 ATCTTTAGAG TCGGGCATTT D~lr,r7~7~rATD TCTTGCTACG ATACAATGGC 800 - AGATGCCTCC ACTGAGCTTC AGCAAGTCAT ~l~lUC GTGGGA~CAT 850 lllU~ll~ L~LX~ r GrArr'`rrrT ~~ l~Ll GGTCCTGA~A 900 TATAGAGGCC TGATTA~TA CTGGTTTCAC ACTCCACCAA GCATCCCATT 950 prZ~r.~TDr'`D GAGTATTTAA AAGACCCAAC TCAGCCCATC TTAGAGGCCT 1000 2 0 TGGACAPGGA rArrTrDrr~ A. GGATGAGC TCTGGGACTC TGTGTCCATT 1050 A;~.l.~.l.l~ rr~r7~ r-r~A r.rA~r'`~r~T GTTCTCCA~A CGCTTTGAAC 1100 CA~GCATGG r.rrTArrrrA ~L~ U~l GGAAGAGATC . AGCCATCGG 1150 AGCTGCTAGA Ul 1~l~ J GACTTTCCAG AGACCAGTCC GGAATTC 1197 (2~ 8 lUN FOR SEQ ID NO:8:
(i) SEQUENCE r~ rT~TcTIcs (A) LENGTH: 337 alDino acids (B) TYPE: ~mino acid (D) TOPOLOGY: linear (xi) SEQUENCE D~ llur~: S~Q ID NO:8:
Met Arg Pro Thr Leu Leu Trp Ser Leu Leu Leu Leu Leu Gly Val ~5 Phe Ala Ala Ala Ala Ala Ala Pro Pro Asp Pro Leu Ser Gln Leu Pro Ala Pro Gln His Pro Lys Ile Arg LeU Tyr Asn Ala Glu Gln Val Leu Ser Trp Glu Pro Val Ala Leu Ser Asn Ser Thr Arg Pro Val Val Tyr Gln Val Gln Phe Lys Tyr Thr Asp Ser Lys Trp Phe Thr Ala Asp Ile Met Ser Ile Gly Val Asn Cys Thr Gln Ile Thr 80 a5 90 6 0 Ala Thr Glu Cys Asp Phe Thr Ala Ala Ser Pro Ser Ala Gly Phe Pro Met Asp Phe Asn Val Thr Leu Arg Leu Arg Ala Glu Leu Gly Ala Leu His Ser Ala Trp Val Thr Met Pro Trp Phe Gln His Tyr RECT~FIED SHEET (RULE 91) ISAIEP

o WO 95/16036 21 ~ 7 4 71 PCT/IIS94/14277 Arg Asn Val Thr Val Gly Pro Pro Glu Asn Ile Glu Val Thr Pro 5 Gly Glu Gly Ser Leu Ile Ile Ar~ Phe Ser Ser Pro Phe Asp Ile Ala Asp Thr Ser Thr Ala Phe Phe (:ys Tyr Tyr Val His Tyr Trp Glu Lys Gly Gly Ile Gln Gln Val Lys Gly Pro Phe Arg Ser Asn Ser Ile Ser Leu Asp Asn Leu Lys Pro Ser Arg Val Tyr CYG Leu Gln Val Gln Ala Gln Leu Leu Trp Asn Lys Ser Asn Ile Phe Arg Val Gly His Leu Ser Asn Ile Ser Cys Tyr Asp Thr Net Ala Asp Ala Ser Thr Glu Leu Gln Gln Val Ile Leu Ile Ser Val Gly Thr 245 250 ;155 Phe Ser Leu Leu Ser Val Leu Ala GIy Ala Cys Phe Phe Leu Val Leu Lys Tyr Arg Gly Leu Ile Lys Tyr Trp Phe His Thr Pro Pro Ser Ile Pro Leu Gln Ile Glu Glu Tyr Leu Lys Asp Pro Thr Gln 35 Pro Ile Leu Glu Ala Leu Asp Lys Asp Ser Ser Pro Lys Asp Glu 305 ~ 310 315 Leu Trp Asp Ser Val Ser Ile Ile Ser Phe Pro Glu Lys Glu Gln Glu Asp Val Leu Gln Thr Leu RECriFlED SHEET (RULE 91) ISAIEP

Claims (32)

Claims:

1. An isolated IFN-.gamma. receptor .beta.-chain polypeptide.

2. The polypeptide of claim 1 that is native.

3. The polypeptide of claim 2 comprising amino acids 1-314 of the amino acid sequence shown in Figure 2A.

4. The polypeptide of claim 3 having its transmembrane anchoring domain deleted or inactivated.

5. The polypeptide of claim 4 from which the cytoplasmic domain has been deleted.

6. The polypeptide of claim 2 comprising the human IFN-.gamma.
receptor .beta.-chain amino acid sequence shown in Figure 5.

7. The polypeptide of claim 6 having its transmembrane anchoring domain deleted or inactivated.

8. The polypeptide of claim 7 from which the cytoplasmic domain has been deleted.

9. The polypeptide of claim 1 associated with an IFN-.gamma.
receptor .alpha.-chain.

10. The polypeptide of claim 1 fused to a heterologous polypeptide.

11. The polypeptide of claim 4 fused to a heterologous polypeptide.

12. The polypeptide of claim 7 fused to a heterologous polypeptide.

13. The polypeptide of claim 10 wherein said heterologous polypeptide comprises an immunoglobulin sequence.

14. A composition comprising a polypeptide of claim 2, which composition is substantially free of other proteins of the same animal species in which said polypeptide naturally occurs.

15. An antibody that is capable of specific binding the IFN-.gamma.
receptor .beta.-chain polypeptide of claim 1.

16. A hybridoma cell line producing the antibody of claim 15.

17. An isolated nucleic acid molecule encoding an IFN-.gamma.
receptor .beta.-chain polypeptide.

18. The molecule of claim 17 comprising a nucleotide sequence able to hybridize, under low stringency conditions, to the complement of a nucleotide sequence encoding a native IFN-.gamma. receptor .beta.-chain.

19. The molecule of claim 18 comprising a nucleotide sequence able to hybridize, under stringent conditions, to the complement of a nucleotide sequence encoding a protein having the amino acid sequence shown in Figure 2A or Figure 5.

20. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an IFN-.gamma. receptor .beta.-chain polypeptide having an amino acid sequence greater than about 65% homologous with the IFN-.gamma. receptor .beta.-chain amino acid sequence shown in Figure 2A or Figure 5.

21. An isolated nucleic acid molecule selected from the group consisting of:
(a) a cDNA clone having a nucleotide sequence derived from the coding region of a native IFN-.gamma. receptor .beta.-chain gene;
(b) a DNA sequence able to hybridize under stringent conditions to a clone of (a); and (c) a genetic variant of any of the DNA sequences of (a) and (b) which encodes a polypeptide possessing a biological property of a naturally occurring IFN-.gamma. receptor .beta.-chain molecule.

22. The nuclelc acid molecule of claim 17 which is DNA and comprises a sequence encoding the amino acid sequence LEVLD.

23. The nuclelc acid molecule of claim 17 further comprising a promoter operably linked to the nucleic acid molecule.

24. An expression vector comprising the nucleic acid molecule of claim 17 operably linked to control sequences recognized by a host cell transformed with the vector.

25. A host cell transformed with the vector of claim 24.

26. A method of using a nucleic acid molecule encoding an IFN-.gamma.
receptor .beta.-chain comprising expressing said nucleic acid molecule in a cultured host cell transformed with a vector comprising said nucleic acid molecule operably linked to control sequences recognized by the host cell transformed with the vector, and recovering IFN-.gamma. receptor .beta.-chain from the host cell.

27. A method for producing an IFN-.gamma. receptor .beta.-chain comprising inserting into the DNA of a cell containing nucleic acid encoding the IFN-.gamma. receptor .beta.-chain a transcription modulatory element in sufficient proximity and orientation to the nucleic acid molecule to influence the transcription thereof.

28. The method of claim 27 wherein the DNA of said cell contains nucleic acid encoding an IFN-.gamma. receptor .alpha.-chain.

29. A method of determining the presence of an IFN-.gamma. receptor .beta.-chain, comprising hybridizing DNA encoding said .beta.-chain to a test sample nucleic acid and determining the present of IFN-.gamma. receptor .beta.-chain DNA.

30. A method of amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase reaction with nucleic acid encoding an IFN-.gamma. receptor .beta.-chain.

31. An antagonist of a native IFN-.gamma. receptor .beta.-chain polypeptide.

32. A pharmaceutical composition comprising an IFN-.gamma. receptor .beta.-chain polypeptide, an antagonist of a native IFN-.gamma. .beta.-chain polypeptide, or an antibody specifically binding an IFN-.gamma. receptor .beta.-chain polypeptide or an antagonist of a native IFN-.gamma. receptor .beta.-chainpolypeptide, and a pharmaceutically acceptable carrier.