Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
  • C07K14/7156—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interferons [IFN]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• 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 mterferon- ⁇ (IFN- Y ) •
• Interferons 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 cell types under different conditions. In response to viral infection, lymphocytes synthesize primarily interferon- ⁇ (also known as leukocyte interferon) , whereas infection of fibroblasts usually induces mterferon- ⁇ (also known as fibroblast interferon) . Interferons- ⁇ and - ⁇ are structurally and functionally related proteins, which are collectively referred to as type I interferons.
• IFN- ⁇ immunodeficiency interferon
• IFNs- ⁇ and - ⁇ mediate their biological effects through binding to a presumably common receptor that is expressed ubiquitously [Uze et al . , Cell 60, 225-234 (1990)].
• I N- ⁇ binds to a different receptor
• IFN- lnducible genes This complex binds to a consensus promoter element of IFN- lnducible genes (ISRE) and stimulates their transcription [Levy et al . , Genes Dev. 2_, 1362-1371 (1989)] .
• Receptor binding of IFN- ⁇ also stimulates tyrosine phosphorylation of p91, presumably at the same residues, but mediated through a different kinase (Schindler et al . , supra) .
• phosphorylated p91 probably complexes with still unidentified proteins [Pearse et al . , Proc. Natl. Acad. Sci.
• the human IFN- ⁇ receptor (huIFN- ⁇ R) expressed in mouse cells and vice versa is nonfunctional, even though the binding properties of the transfected receptor proved indistinguishable from those of the resident functional receptor [Aguet et al . , Cell 55, 273-280 (1988); Gray et al . , Proc. Natl. Acad. Sci. USA 86, 8497-8501 (1989); Hemmi et al . , Proc. Natl. Acad. Sci. USA 86, 9901-9905 (1989)] .
• the present invention is based on the cloning and expression of a novel cofactor required for signal transduction of IFN- ⁇ . More specifically, a cDNA encoding a novel transmembrane protein was obtained, sequenced and expressed to produce a polypeptide, which was identified as a species-specific cofactor required for signal transduction of IFN- ⁇ , and will hereinafter be designated as IFN- ⁇ receptor ⁇ -chain. It s believed that this new polypeptide or a close homologue thereof is also a constituent of other receptors, such as IFN- / ⁇ receptor, the erythrop ietm (EPO) receptor, the IL-10 and possibly other cytokine receptors.
• IFN- ⁇ receptor erythrop ietm
• the present invention concerns an isolated IFN- ⁇ receptor ⁇ -cham polypeptide, which is a native IFN- ⁇ receptor ⁇ -cham or a functional derivative thereof.
• the polypeptide is devoid of a functional transmembrane domain, and optionally of part or whole of the cytoplasmic domain.
• Certain IFN- ⁇ receptor ⁇ -chain polypeptides of the present invention are characterized by comprising the LEVLD sequence motif in their cytoplasmic domains.
• the IFN- ⁇ receptor ⁇ - cham polypeptide is associated with an IFN- ⁇ receptor -cham, with an IFN-oc or - ⁇ receptor or an EPO receptor and/or is fused to a heterologous polypeptide.
• the heterologous polypeptide may comprise an lmmunoglobulm sequence, which is-preferably fused to a transmembrane domain deleted or inactivated IFN- ⁇ receptor (.-chain, to yield a fusion protein which signals or inhibits IFN- ⁇ biological action.
• the IFN- ⁇ 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 lmmunogenic polypeptide and this fusion polypeptide, in turn, used to immunize an animal to raise antibodies against an IFN- ⁇ receptor ⁇ -chain subunit epitope.
• Ant ⁇ -IFN- ⁇ receptor ⁇ -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- ⁇ receptor ⁇ -chain but will not substantially cross-react with any other known receptor subunits.
• Immobilized ant ⁇ -IFN- ⁇ ⁇ -cham antibodies are useful particularly in the detection (in vi tro or in vivo) or purification of IFN- ⁇ receptor ⁇ -chain by passing a mixture containing a ⁇ -chain over a column to which the antibodies are bound.
• Substitutional, deletional, or insertional variants of the IFN- ⁇ receptor ⁇ -chain polypeptides are prepared by m vi tro or recombinant methods and screened for lmmuno-crossreactivity w th a native IFN- ⁇ receptor ⁇ -chain and for IFN- ⁇ receptor agonist or antagonist activity (i.e.
• the IFN- ⁇ receptor ⁇ -chain polypeptides are also derivatized in vi tro to prepare immobilized ⁇ -chain and labeled ⁇ -chain, particularly for purposes of detection of IFN- ⁇ receptor ⁇ -chain or its antibodies, or for affinity purification of ant ⁇ -IFN- ⁇ receptor ⁇ -cham antibodies.
• the IFN- ⁇ receptor ⁇ -cham polypeptides and the antibodies specifically binding such polypeptides are formulated into physiologically acceptable vehicles, especially for therapeutic use.
• physiologically acceptable vehicles include sustained-release formulations.
• the present invention concerns an isolated nucleic acid molecule encoding an IFN- ⁇ 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- ⁇ receptor ⁇ -cham, such as the murine IFN- ⁇ receptor ⁇ -chain having the amino acid sequence shown in Figure 2A, or the native human IFN- ⁇ receptor ⁇ -chain having the amino acid sequence shown in Figure 5.
• 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 shown in Figure 2A or with the amino ac d sequence shown in Figure 5.
• the nucleic acid molecule is selected from the group consisting of:
• the invention concerns an expression vector comprising a nucleic acid molecule encoding an IFN- ⁇ receptor ⁇ -chain polypeptide operably linked to control sequences recognized by a host cell transformed with the vector.
• the invention concerns a host cell transformed with the vector above.
• the invention concerns a method of using a nucleic acid molecule encoding an IFN- ⁇ receptor ⁇ -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- ⁇ receptor ⁇ -chain from the host cell.
• the invention concerns a method for producing an IFN- ⁇ receptor ⁇ -chain comprising inserting into the DNA of a cell containing nucleic acid encoding the IFN- ⁇ receptor ⁇ -cham a transcription modulatory element in sufficient proximity and orientation to the nucleic acid molecule to influence its transcription.
• the DNA of the cell in which the IFN- ⁇ receptor ⁇ -cham is produced may additionally contain DNA encoding an IFN- ⁇ receptor - chain or other cytokine receptors or their chains/subunits.
• the invention further concerns a method of determining the presence of an IFN- ⁇ receptor ⁇ -chain, comprising hybridizing DNA encoding the ⁇ -cham to a test sample nucleic acid and determining the presence of IFN- ⁇ receptor ⁇ -chain DNA.
• the invention concerns a method for obtaining cells having increased or decreased transcription of a nucleic acid encoding an IFN- ⁇ receptor ⁇ -cham, comprising:
• the invention concerns an antagonist of a native IFN- ⁇ receptor ⁇ -chaian.
• Such antagonists are capable of blocking the biological action of IFN- ⁇ and other native polypeptides (such as interleukins, EPO, IFNs- ⁇ / ⁇ ), the signal transduction of which involves a native IFN- ⁇ receptor ⁇ -chain or a close homologue (functional derivative) thereof.
• the invention concerns a pharmaceutical composition
• a pharmaceutical composition comprising an IFN- ⁇ receptor ⁇ -cham polypeptide, or an antagonist of such polypeptide, or an antibody specifically binding an IFN- ⁇ receptor ⁇ -chain polypeptide or an antagonist thereof, and a pharmaceutically acceptable carrier.
• FIG. 1 IFN- ⁇ - ⁇ nduc ⁇ ble Tac antigen reporter construct and its expression in COSN 31 cells stably expressing the murine IFN- ⁇ receptor, in the absence or presence of the novel murine IFN- ⁇ receptor ⁇ -chain.
• the reporter construct pUMS (GT) 8 -Tac was designed for tight IFN- ⁇ inducible expression of the human Tac antigen.
• An artificial promoter consisting of the hexamer repeat (GAAAGT) B followed by the TATA box and the Cap site of the rabbit ⁇ -globm promoter (R ⁇ G) was placed in front of a cDNA encoding the human Tac antigen.
• An SV40 enhancer was placed about lOOObp upstream of the artificial promoter.
• COSN 31 cells were transfected with the expression plasmid pAGS-C19 encoding the murine IFN- ⁇ receptor (muIFN- YR) (.-chain. Transfected cells were incubated for 48 hours at 37°c with 200 U/ml of either human (solid bold line) or murine IFN- ⁇ (dotted bold line) or left untreated (thin line) . Expression 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) .
• Figure 2. Nucleotide and inferred ammo acid sequences of the muIFN- ⁇ R ⁇ -subunit (SEQ. ID.
• FIG. 3 Functionality of the muIFN- ⁇ T ⁇ -chain in HEp-2 cells expressing the muIFN- ⁇ R ⁇ -chain.
• A,B Cytofluorometry of IFN ⁇ -mduced MHC class I (A) or MHC class II (B) antigen expression in HEp-2 cells permanently transformed with the muIFN- ⁇ R ⁇ -chain alone (Hep243.7) or together with the muI "** N- ⁇ R ⁇ -chain Hep-2#6) .
• Cells were incubated for 60 or 84 hours at 37°C with 200 U/ml of either human (solid bold line) or murine IFN- ⁇ (dotted bold line) or left untreated (thin line) .
• Figure 5 Deduced amino acid sequence of human IFN-R -chain (SEQ. ID. NO: 8) .
• IFN- ⁇ receptors have been purified from different human [Aguet, M. & Merlin, G. , J. Exp. Med. 165, 988-999 (1987); Novick, D. et al . , J. Biol. Chem. 262, 8483-8487 (19871; Calderon, J. et al . , Proc. Natl. Acad. Sci. USA 85, 4837-4841 (1988)] and murine [Basu, M. et al . , Proc. Natl. Acad. Sci.
• ⁇ nterferon- ⁇ receptor ⁇ nterferon- ⁇ receptor
• IFN- ⁇ receptor ⁇ nterferon- ⁇ receptor ⁇ - chain
• IFN- ⁇ R ⁇ -chain a family of polypeptide molecules that comprise the human IFN- ⁇ receptor reported by Aguet et al . (1988), supra, the murine IFN- ⁇ 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- ⁇ receptors.
• IFN- ⁇ receptor ⁇ -cham The terms "IFN- ⁇ receptor ⁇ -cham", “IFN- ⁇ R ⁇ -chain”, “IFN- ⁇ receptor ⁇ -chain polypeptide”, “IFN- ⁇ receptor ⁇ -subunit”, and their grammatical variants define the native murine IFN- ⁇ receptor ⁇ -chain having amino acids 1 through 314 as set forth in Figure 2A, the native human IFN- ⁇ receptor ⁇ -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 qualitative biological activity in common with the native polypeptide.
• a functional derivative of an IFN- ⁇ receptor ⁇ -chain polypeptide is a compound that has a qualitative biological activity in common with the native human IFN- ⁇ receptor of Aguet et al . , supra or with the native murine IFN- ⁇ receptor of Gray et al . , supra, or Hemmi et al . , supra.
• a functional derivative of an IFN- ⁇ receptor ⁇ -chain has a qualitative biological activity in common with the native murine IFN- ⁇ receptor ⁇ -cham of Figure 2A or with the native human IFN- ⁇ receptor ⁇ -cham of Figure 5.
• “Functional derivatives” include, but are not limited to, fragments of native IFN- ⁇ receptor ⁇ -cham polypeptides (or ⁇ -chains) from any animal species (including humans) , and derivatives of native (human and non-human) IFN- ⁇ receptor ⁇ -cham polypeptides (or ⁇ -chains) and their fragments, provided that they have a biological activity m common with a native IFN- ⁇ receptor ⁇ -chain (or ⁇ -chain) .
• “Fragments” comprise regions within the sequence of a mature native IFN- ⁇ receptor ⁇ - or ⁇ -chain.
• the term “derivative” is used to define amino acid sequence and glycosylation variants, and covalent modifications of a native IFN- ⁇ receptor ⁇ - or ⁇ -chain polypeptide, whereas the term “variant” refers to ammo acid sequence and glycosylation variants within this definition.
• the functional derivatives are polypeptides which have at least about 65% amino acid sequence identity, more preferably about 75% ammo acid sequence identity, even more preferably at least about 85% ammo acid sequence identity, most preferably at least about 95% amino acid sequence identity of a native sequence IFN- ⁇ receptor ⁇ - or ⁇ -chain.
• Identity or homology with respect to an IFN- ⁇ receptor ⁇ - or ⁇ - chain is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native IFN- ⁇ receptor ⁇ _.- 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 insertions shall be construed as reducing identity or homology.
• “functional derivatives” is defined as either 1) lmmunological cross- reactivity with at least one epitope of a native IFN- ⁇ receptor ⁇ - or ⁇ -cham, or 2) the possession of at least one adhesive, regulatory or effector function qualitatively m common with a native IFN- ⁇ receptor ⁇ - or (.-chain.
• Immunologically cross-reactive means that the candidate (poly)pept de is capable of competitively inhibiting the qualitative biological activity of a native IFN- ⁇ ⁇ - or (.-chain having 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- ⁇ receptor ⁇ - or ⁇ -cham in complete Feud's ad uvant, followed by booster mtraperitoneal or subcutaneous injection incomplete Freud's.
• the murine IFN- ⁇ receptor ⁇ -subunit as set forth n Figure 2A, with or without the 18 ammo acids 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 ⁇ -cham, provided that they possess a biological activity in common with the native murine IFN- ⁇ ⁇ -chain of Figure 2A.
• the human IFN- ⁇ receptor ⁇ -subunit as set forth in Figure 5, with or without the 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 human receptor ⁇ -chain, provided that they possess a biological activity in common with the native human IFN- ⁇ ⁇ -chain of Figure 5. While the native IFN- ⁇ receptor ⁇ -chams are membrane bound polypeptides, soluble forms, such as those forms lacking a functional transmembrane domain, are also included within this definition.
• the IFN- ⁇ receptor ⁇ -chain fragments within the scope of the present invention preferably have at least 15 and preferably at least 30 amino acid residues, or have at least about 5 amino acid residues comprising an immune epitope or other biologically active site of the IFN- ⁇ receptor ⁇ -subunit.
• amino acid sequence variant refers to molecules with some differences in their am o acid sequences as compared to a native sequence IFN- ⁇ receptor ⁇ -cham or a fragment thereof.
• amino acid sequence variants will possess at least about 65 %, 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- ⁇ receptor ⁇ -chain or, alternatively, are encoded by DNA capable, under stringent conditions, of hybridizing to the complement of the extracellular domain of a native IFN- ⁇ receptor ⁇ -chain.
• a preferred group of the ammo acid sequence variants retains the sequence motif within the native sequence identified as responsible for signaling IFN- ⁇ biological action, such as the LEVLD sequence motif in the cytoplasmic domain of the murine IFN- ⁇ receptor ⁇ -cham sequence, or have only conservative amino acid alterations within this region.
• the ammo acid alterations may be substitutions, insertions, deletions or any desired combinations of such changes in a native amino acid sequence.
• Substitutional variants are those that have at least one amino acid residue in a native sequence- removed and a different ammo 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 ammo acids have been substituted in the same molecule.
• Insertional variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native amino acid sequence. Immediately adjacent to an amino acid means connected to either the ⁇ -carboxy or ⁇ -amino functional group of the amino acid.
• Deletional variants are those with one or more ammo acids in the native ammo acid sequence removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the molecule.
• glycosylation variant is used to refer to an IFN- ⁇ receptor ⁇ -chain molecule having a glycosylation profile different from that of a native IFN- ⁇ receptor ⁇ -chain.
• Glycosylation of polypeptides is typically either N-linked or O-linked.
• N-l ked refers to the attachment of the carbohydrate moiety to the side-chain of an asparagine residue.
• the tripeptide sequences, asparagme-X-serme and asparagine-X-threonme, wherein X is any amino acid except prol e, 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 se ⁇ ne or threomne, although 5-hydroxyprol ⁇ ne or 5-hydroxylys ⁇ ne may also be involved in 0- lmked glycosylation. Any difference in the location and/or nature of the carbohydrate moieties present in a variant or fragment as compared to its native counterpart is within the scope herein.
• glycosylation pattern of native polypeptides can be determined by well known techniques of analytical chemistry, including HPAE chromatography [Hardy, M.R. et al . , Anal. Biochem. 170, 54-62 (1988)], methylation analysis to determine glycosyl-linkage composition [Lindberg, B., Meth. Enzymol. 28. 178-195 (1972); Waeghe, T.J. et al . , Carbohydr. Res. 123, 281-304 (1983)], NMR spectroscopy, mass spectrometry, etc.
• Covalent derivatives include modifications of a native IFN- ⁇ receptor ⁇ -cham or a fragment thereof with an organic protemaceous or non-protemaceous derivatiz g agent, and post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted am o acid residues with an organic derivatizmg 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 recombinant host cells. Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues.
• these residues are deamidated under mildly acidic conditions. Either form of these residues may be present m the IFN- ⁇ receptor ⁇ -chain molecules as defined in the present invention.
• Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the ⁇ -ammo groups of lysine, arginine, and histidme side chains [T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)] .
• IFN- ⁇ receptor ⁇ -chain nucleic acid or polypeptide is a nucleic acid or polypeptide that is identified and separated from contaminant nucleic acids or polypeptides present in the animal or human source of the IFN- ⁇ receptor ⁇ -cham nucleic acid or polypeptide.
• the 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- ⁇ receptor ⁇ -chain may be associated with any IFN- ⁇ receptor ⁇ -chain, or alternatively, truncated forms of the ⁇ - and ⁇ -chains may be associated with each other, or with a full length form of the other chain.
• nucleic acid is defined as RNA or DNA containing greater than about 15 bases that encodes an IFN- ⁇ receptor ⁇ -chain as hereinabove defined, is complementary to a nucleic acid molecule encoding an IFN- ⁇ receptor ⁇ -chain, hybridizes to such nucleic acid and remains stably bound under stringent conditions, or encodes a polypeptide sharing at least about 65 % sequence identity, preferably at least about 75% sequence identity, more preferably at least about 85% sequence identity, most preferably at least about 95% sequence identity with a native IFN- ⁇ receptor ⁇ -chain polypeptide, and preferably with the translated ammo acid sequence shown in Figure 2A herein.
• “Stringent conditions” are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 sodium chlor ⁇ de/0.0015 M sodium c ⁇ trate/0.1% sodium dodecyl sulfate at 50°C, or (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovme serum albumin/0.1% F ⁇ coll/0.1% polyv ⁇ nylpyrrol ⁇ done/50 nM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C.
• a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovme serum albumin/0.1% F ⁇ coll/0.1% polyv ⁇ nylpyrrol ⁇ done/50 nM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C.
• control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence m a particular host organism.
• control sequences that are suitable for 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 acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
• DNA for a presequence or a secretory leader is 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 sequence; or
• a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
• "operably linked” means that the DNA sequences being linked are contiguous and, in 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.
• DNA sequence encoding refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide chain. The DNA sequence thus codes for the amino acid sequence.
• replicable expression vector and "expression vector” refer to a piece of DNA, usually double-stranded, which may have inserted into it a piece 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 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.
• 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.
• the expressions "cell”, “cell line”, and “cell culture” are used interchangeably, and all such designations include progeny.
• the words “transformants” and “transformed (host) cells” include the primary ⁇ subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are mcluded. Where distinct designations are intended, it will be clear from 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 in which the element is ordinarily not found.
• Antibodies (Abs) and immunoglobulins (Igs) are glycoprote s having the same structural characteristics. While antibodies exhibit 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, produced at low levels by the lymph system and at increased levels by myelomas.
• Native antibodies and immunoglobulins are usually heterotetrameric glycoprotems of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different lmmunoglobulm isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
• V H variable domain
• Each light chain has a variable domain at one and (V 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 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)] .
• variable domains of antibodies each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which foxm loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
• the CDRs in each chain are held together m close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies [see Kabat, E.A. et al .
• Papain digestion of antibodies produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an 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 of one heavy and one light 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 V H -V. dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable 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 Threadl) of the heavy chain.
• Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain C H 1 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 originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other, chemical couplings of antibody fragments are also known.
• immunoglobulins The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda ( ⁇ ) , based on the ammo acid sequences of their constant domains. Depending on the ammo acid sequence of the constant region of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2.
• immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG-1, Ig
• the heavy chain constant regions that correspond to the different classes of immunoglobulins are called ⁇ , delta, epsilon, y, and ⁇ , respectively.
• the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
• IgA-1 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) .
• poly-IgA contains a disulfide-linked polypeptide called the "joining" or “J” chain, and can be transported through the glandular epithelium together with the J-chain-containmg polymeric IgM (poly-IgM) , comprising five subumts.
• J disulfide-linked polypeptide
• antibody is used in the broadest sense and specifically covers single ant ⁇ -IFN- ⁇ receptor ⁇ -chain monoclonal antibodies (including agonist and antagonist antibodies) and ant ⁇ -IFN- ⁇ receptor ⁇ -chain antibody compositions with polyepitopic specificity.
• monoclonal antibody 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 may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
• each monoclonal antibody is directed against a single determinant on the antigen.
• the monoclonal antibodies are advantageous in that they are synthesized by the hyb ⁇ doma culture, uncontammated by other immunoglobulins.
• the monoclonal antibodies herein include hybrid and recombinant antibodies produced by splicing a variable (including hypervariable) domain of an ant ⁇ -IFN- ⁇ receptor (.-chain antibody with a constant domain (e.g. "humanized” antibodies), only one of which is directed against IFN- ⁇ receptor ⁇ -cham, 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 lmmunoglobulm class or subclass designation, as well as antibody fragments (e.g., Fab, F(ab') 2 , and Fv) , so long as they exhibit the desired biological activity. [See, e.g.
• the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the present invention may be made by the hyb ⁇ doma method first described by Kohler & Milstem, Nature 256:495 (1975), or may be made by recombinant DNA methods [Cabilly, et al . , U.S. Pat. No. 4,816,567] .
• Humanized forms of non-human (e.g. murine) antibodies are immunoglobulins, lmmunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human lmmunoglobulin.
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining 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 capacity.
• CDR complementary determining region
• 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 optimize antibody performance.
• 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 lmmunoglobulin and all or substantially all of the FR regions are those of a human lmmunoglobulin consensus sequence.
• the humanized antibody optimally also will comprise at least a portion of an lmmunoglobulin constant region (Fc), typically that of a human lmmunoglobulin.
• Fc lmmunoglobulin constant region
• DNA encoding an IFN- ⁇ receptor ⁇ -chain can be obtained from any cDNA library prepared from tissue believed to possess the IFN- ⁇ receptor ⁇ -cham mRNA and to express it at a detectable level.
• a cDNA library prepared from mouse B-cell leukemia cells such as that described in the examples, is a good source of IFN- ⁇ receptor ⁇ -chain cDNA.
• the IFN- ⁇ receptor ⁇ -chain gene can also be obtained from a genomic library, such as a human genomic cosmid library.
• IFN- ⁇ receptor ⁇ -chain DNA is most conveniently accomplished by probing human or other mammalian cDNA or genomic libraries by labeled oligonucleotide sequences selected from the ⁇ - chain sequence depicted in F ⁇ gure-2A in accord with known criteria, among which is that the sequence should be sufficient in length and sufficiently unambiguous that false positives are minimized.
• oligonucleotide sequences selected from the ⁇ - chain sequence depicted in F ⁇ gure-2A in accord with known criteria, among which is that the sequence should be sufficient in length and sufficiently unambiguous that false positives are minimized.
• a 32 P-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 nucle
• Another alternative is to chemically synthesize the gene encoding an IFN- ⁇ receptor ⁇ -cham using one of the methods described in Engels and Uhlmann, Agnew. Chem. Int. Ed. Engl. 28, 716 (1989) . These methods include t ⁇ ester, phosphite, phosphoramidite and H-phosphonate methods, PCR and other autop ⁇ mer methods, and oligonucleotide syntheses on solid supports.
• Amino Acid Sequence Variants of IFN- ⁇ receptor (--chain Amino acid sequence variants of IFN- ⁇ receptor ⁇ -chain are prepared by methods known in the art by introducing appropriate nucleotide changes into the IFN- ⁇ receptor ⁇ -cham DNA, or by in vi tro 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- ⁇ receptor ⁇ -cham, the ammo acid sequence variants of IFN- ⁇ 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.
• the mutations will be created within the extracellular domain of a native IFN- ⁇ receptor ⁇ -chain.
• Sites or regions that appear to be important for the signal transduction of IFN- ⁇ or another polypeptide (e.g. cytokme) the signal transduction of which involves the activation of IFN- ⁇ receptor ⁇ -cham will be selected in m vitro studies of biological activity, such as the antiviral response of IFN- ⁇ .
• Sites at such locations w ll then be modified in series, e.g. by (1) substituting first with conservative choices and then with more radical selections depending upon- the results achieved, (2) deleting the target residue or residues, or (3) inserting residues of the same or different class adjacent to the located site, or combinations of options 1-3.
• alanine scanning (Cunningham and Wells, Science 244, 1081-1085 [1989]) .
• a residue or group of target residues is identified and substituted by alanine or polyalanme.
• Those domains demonstrating functional sensitivity to the alanine substitutions are then refined by introducing further or other substituents at or for the sites of alanine substitution.
• the gene encoding an IFN- ⁇ receptor ⁇ -chain variant can be obtained by chemical synthesis as hereinabove described. More preferably, DNA encoding an IFN- ⁇ receptor ⁇ -cham amino acid sequence variant is prepared by site-directed mutagenesis of DNA that encodes an earlier prepared variant or a nonva ⁇ ant version of IFN- ⁇ receptor ⁇ -cham.
• Site-directed (site-specific) mutagenesis allows the production of IFN- ⁇ receptor ⁇ -cham variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent 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.
• a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
• 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) .
• the site-specific mutagenesis technique typically employs a phage vector that exists in both a single-stranded and double-stranded form.
• 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 known to those skilled in the art.
• site-specific mutagenesis herewith is performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant protein.
• An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al . , Proc. Natl. Acad. Sc . USA 75, 5765 (1978) .
• This primer is then annealed with the single-stranded protein sequence-containing vector, and subjected to DNA-polymenzing enzymes such as, E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand.
• 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 JPlOl 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 IFN- ⁇ receptor ⁇ -cham.
• 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.
• 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 identical to a stretch of sequence of the opposite strand of the plasmid, but this sequence can be located anywhere along the plasmid DNA.
• sequence of the second primer is located within 200 nucleotides from that of the first, such that 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 just 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.
• the ratio of template to product material is extremely low, the vast majority of product DNA fragments incorporate the desired 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 performing a second PCR with different mutant primers and ligating the two resulting PCR fragments simultaneously to the vector fragment in a three (or more) -part ligation.
• template plasmid DNA (1 ⁇ g) is linearized by digestion with a restriction endonuclease that has a unique recognition site in the plasmid DNA outside of the region to be amplified.
• 100 ng is added to a PCR mixture containing PCR buffer, which contains the four deoxynucleotide tri- phosphates and is included in the GeneAmp R kits (obtained from Perkm- Elmer Cetus, Norwalk, CT and Emeryville, CA) , and 25 pmole of each oligonucleotide primer, to a final volume of 50 ⁇ l.
• the reaction mixture is overlayered with 35 ⁇ l mineral oil.
• Thermus aquaticus (Tag) DNA polymerase (5 units/ 1), purchased from 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 follows:
• 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 vector.
• the starting material is the plasmid (or vector) comprising the IFN- ⁇ receptor ⁇ -chain DNA to be mutated.
• the codon(s) withm the IFN- ⁇ receptor ⁇ -cham to be mutated are identified.
• the plasmid is cut at these sites to linearize it.
• a double- stranded oligonucleotide encoding the sequence of the DNA between the restriction site but containing the desired mutation (s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques.
• 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 linearized plasmid, such that it can be directly ligated to the plasmid.
• This plasmid now contains the mutated IFN- ⁇ receptor ⁇ -cham DNA sequence.
• the so called phagemid display method may be useful in making amino acid sequence variants of IFN- ⁇ receptor ⁇ -cha s of the present invention.
• This method involves (a) constructing a replicable expression vector comprising a first gene encoding an IFN- ⁇ 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 transcription regulatory element operably linked to the first and second genes, thereby forming a gene fusion encoding a fusion protein; (b) mutating the vector at one or more selected positions within the first gene thereby forming a family of related plasmids; (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
• Steps (d) through (g) can be repeated one or more times.
• 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 fusion protein on the surface of the particle is less than about 1%.
• the amount of phagemid particles displaying more than one copy of the fusion protein is less than 10% of the amount of phagemid particles displaying a single copy of the fusion protein. Most preferably, the amount is less than 20%.
• the expression vector will further contain a secretory signal sequence fused to the DNA encoding each subunit of the polypeptide and the transcription regulatory element will be a promoter system.
• Preferred promoter systems are selected from lac Z, ⁇ PL , tac, T7 polymerase, tryptophan, and alkaline phosphatase promoters and combinations thereof.
• the method will employ a helper phage selected from M13K07, M13R408, M13-VCS, and Phi X 174.
• the preferred helper phage is M13K07,.and the preferred coat protein is the M13 Phage gene III coat protein.
• the preferred host is E. coli , and protease-deficient strains of E. coll . Further details of the foregoing and similar mutagenesis techniques are found in general textbooks, such as, for example, Sambrook et al . , supra, and Current Protocols in Molecular Biology, Ausubel et al . eds. , supra.
• Amino acid substitution variants have at least one amino acid residue in a native IFN- ⁇ 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 ligand binding, such domains within the extracellular domain, or the LEVLD sequence motif at amino acid positions 280-284 of the murine IFN- ⁇ receptor ⁇ -chain and its equivalent in the native receptors from other species, including humans, and sites where the amino acids found in the native IFN- ⁇ receptor ⁇ -chams from various species are substantially different in terms of side-chain bulk, charge and/or hydrophobicity.
• sites of interest are those in which particular residues of the native IFN- ⁇ receptor ⁇ -chains from various species are identical. These positions may be important for the biological activity of the IFN- ⁇ receptor ⁇ -cham. Further important sites for mutagenesis include motifs common in various members of the interferon receptor family, such as the two cysteine pairs and conserved prolme, tryptophan and tyrosme residues boxed in Figure 2B.
• Naturally occurring ammo acids are divided into groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, lie; (2) neutral hydrophobic: cys, ser, thr;
• Non-conservative substitutions withm the short cytoplasmic domain of the IFN- ⁇ receptor (.-chain, and especially within the region responsible for signal transduction, such as the LEVLD sequence motif at am o acid positions 280-284 of the murine IFN- Y receptor ⁇ -chain, are expected to result in significant changes in the biological properties of the obtained variant, and may result in IFN- ⁇ receptor ⁇ -cham variants which block the biological activity of IFN- ⁇ , i.e.
• Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably about 1 to 10 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- ⁇ receptor ⁇ -cham. 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- ⁇ receptor ⁇ -cham more significantly, and may potentially yield IFN- ⁇ receptor ⁇ -chain antagonists. The number of consecutive deletions will be selected so as to preserve the tertiary structure of the IFN- ⁇ receptor ⁇ -cham in the affected domain, e.g. beta-pleated sheet or alpha helix.
• Am o acid insertions include ammo- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as mtrasequence insertions of single or multiple amino acid residues.
• Intrasequence insertions i.e. insertions within the IFN- ⁇ receptor ⁇ -chain amino acid sequence
• terminal insertions include the IFN- ⁇ receptor ⁇ -cham with an N-terminal methionyl residue, an artifact of its direct expression in bacterial recombinant cell culture, and fusion of a heterologous N-terminal signal sequence to the N-termmus of the IFN- ⁇ receptor ⁇ -chain molecule to facilitate the secretion of the mature IFN- ⁇ receptor ⁇ - cham from recombinant host cells.
• signal sequences will generally be obtained from, and thus homologous to, the intended host cell species. Suitable sequences include ⁇ TII or Ipp for E. col , alpha factor for yeast, and viral signals such as herpes gD for mammalian cells.
• IFN- ⁇ receptor ⁇ -cham molecules include the fusion to the N- or C-termmus of the IFN- ⁇ receptor ⁇ -cham of lmmunogenic 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 lmmunoglobulin regions (preferably lmmunoglobulin constant regions) , albumin, or ferritin, as described in WO 89/02922 published 6 April 1989.
• lmmunogenic polypeptides e.g. bacterial polypeptides such as beta-lactamase or an enzyme encoded by the E. coli trp locus, or yeast protein
• C-terminal fusions with proteins having a long half-life such as lmmunoglobulin regions (preferably lmmunoglobulin constant regions) , albumin, or ferrit
• nucleic acid encoding a native or variant IFN- ⁇ receptor ⁇ -cham is available, it 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 expression, 2) the size of 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 marker genes, an enhancer element, a promoter, and a transcription termination sequence.
• Signal Sequence Component generally include, but ar not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
• the signal sequence may be a component of the vector, or it may be a part of the IFN- ⁇ receptor ⁇ -chain that is inserted into the vector.
• the native IFN- ⁇ receptor ⁇ -chain encodes a signal sequence at the ammo terminus (5' end of the DNA) of the polypeptide that is cleaved during post-translational processing of the polypeptide to form a mature IFN- ⁇ receptor ⁇ -chain.
• this signal sequence is 18 amino acids long ( Figure 2B) .
• Native IFN- ⁇ receptor ⁇ -chain is however not secreted from the host cell as it contains a membrane anchoring domain between the extracellular domain and the cytoplasmic domain (am o acid residues
• the membrane anchoring domain (also referred to as transmembrane domain) is ordinarily deleted or otherwise inactivated (for example by point mutation (s)) .
• the cytoplasmic doma - is also deleted along with the membrane anchoring domain.
• the cytoplasmic domain of the IFN- ⁇ receptor ⁇ -chain may play an important role in the signal transduction mediated by this receptor subunit (in addition to the extracellular domains of 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- ⁇ receptor ⁇ -chain variants may be secreted from the cell, provided that the DNA encoding the truncated variant retains the ammo terminal signal sequence.
• IFN- ⁇ receptor ⁇ - chains with the native signal sequence deleted and replaced with a heterologous signal sequence.
• the heterologous signal sequence selected should be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell.
• the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillmase, lpp, or heat-stable enterotox II leaders.
• a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillmase, lpp, or heat-stable enterotox II leaders.
• yeast secretion the native IFN- ⁇ receptor ⁇ -cham signal sequence may be substituted by the yeast vertase, alpha factor, or acid phosphatase leaders.
• the native signal sequence is satisfactory, although other mammalian signal sequences may be suitable, (li) Origin 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.
• this sequence is one that enables the vector to replicate independently of the host chromosomes, and includes origins of replication or autonomously replicating sequences.
• origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast and viruses.
• the origin of replication from the well-known plasmid pBR322 is suitable for most gram negative bacteria, the 2 ⁇ plasmid origin for yeast and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
• Origins of replication are not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter) .
• Most expression vectors are "shuttle" vectors, 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 E. coli and then the same vector is transfected into yeast or mammalian cells for expression even though it is not capable of replicating independently of the host cell chromosome.
• DNA is also cloned by insertion into the host genome. This is readily accomplished using Bacillus species as hosts, for example, by including in the vector a DNA sequence that is complementary to a sequence found in Bacillus genomic DNA. Transfection of Bacillus with this vector results in homologous recombination with the genome and insertion of the DNA encoding the desired heterologous polypeptide. However, the recovery of genomic DNA is more complex than that of an exogenously replicated vector because restriction enzyme digestion is required to excise the encoded polypeptide molecule, (m) Selection Gene Component
• Selection genes also termed a selectable marker. This is a gene that encodes a protein 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, e.g. ampicillm, neomycin, methotrexate or tetracycline, (b) complement auxotrophic 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 growth of a host cell. Those cells that are successfully transformed with a heterologous gene express a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomucin [Southern 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.. Biol. 5, 410-413 (1985)].
• bacterial genes under eukaryotic control employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin) , xgpt (mycophenolic acid) , or hygromycin, respectively.
• 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.
• DHFR dihydrofolate reductase
• 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 having 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 selection gene and the DNA that encodes the desired polypeptide.
• Amplification is the process by which genes in greater demand for the production of a protein critical for growth are reiterated m tandem within the chromosomes of successive generations of recombinant cells. Increased quantities of the desired polypeptide (either a p75- contaming chimeric polypeptide or a segment thereof) are synthesized from the amplified DNA.
• cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium which lacks hypoxanthine, glycme, and thymidine.
• An appropriate host cell in this case is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub and Chasin, Proc. Nat'l. Acad. Sci. USA 77, 4216 (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.
• an agent metalhotrexate, or MTX
• MTX metalhotrexate
• hosts co-transformed with genes encoding the desired polypeptide, wild- type DHFR, and another selectable marker such as the neo gene can be identified using a selection agent for the selectable marker such as
• G418 and then selected and amplified 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 (St chcomb et al . , 1979, Nature
• the trpl gene provides 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 effective environment for detecting transformation by growth in the absence of tryptophan.
• Leu2 deficient yeast strains ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.
• 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 are untranslated sequences located upstream from the start codon of a structural gene (generally with -about 100 to 1000 bp) that control the transcription and translation of nucleic acid under their control. They typically fall into two classes, mducible 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.
• 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 is not to say that the genomic promoter for IFN- ⁇ receptor ⁇ -chain is not usable. However, heterologous promoters generally will result in greater transcription and higher yields of expressed IFN- ⁇ receptor ⁇ -chain as compared to the native IFN- ⁇ receptor ⁇ -cham promoter.
• Promoters suitable for use with prokaryotic hosts include the ⁇ - lactamase and lactose promoter systems (Chang et al . , Nature 275: 615 (1978); and Goeddel et al . , Nature 281:544 (1979)), alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res. 8 ⁇ :4057 (1980) and EPO Appln. Publ . No. 36,776) and hybrid promoters such as the tac promoter (H. de Boer et al . , Proc. Nat'l. Acad. Sci. USA 0:21-25 (1983)) .
• 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 Sh e-Dalgarno (S.D.) sequence operably linked to the DNA encoding NT-4.
• Sh e-Dalgarno S.D.
• 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 glycolytic enzymes (Hess et al . , J. Adv. Enzyme Reg.
• 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, and enzymes responsible for maltose and galactose utilization.
• Suitable vectors and promoters for use m yeast expression are further 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 AT-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.
• IFN- ⁇ receptor ⁇ -chain transcription 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), bovme 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 lmmunoglobulin promoter, from heat shock promoters, and from the promoter normally associated with the IFN- ⁇ receptor ⁇ -chain sequence, provided such promoters are compatible with the host cell systems.
• viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovme
• 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 [Fiers et al . , Nature 273: 113 (1978), Mulligan and Berg, Science 209, 1422-1427 (1980); 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 Hindlll E restriction fragment [Greenaway et al . , Gene 18, 355-360 (1982)].
• the actual plasmid used in the course of cloning the murine IFN- ⁇ receptor ⁇ -cham contains the promoter of the murine 3-hydroxy-3- methylglutaryl coenzyme A reductase gene [Gautier et al . , Nucleic Acids Res. 17, 8389 (1989)], whereas the reporter plasmid [pUMS (GT) 8 -Tac] used during expression cloning contained an artificial multimerized IFN- ⁇ - ⁇ nduc ⁇ ble promoter element [McDonald et al . , Cell 60, 767-779 (1990) ] . (v) Enhancer Element Component
• Enhancers are cis- acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent having been found 5' [Laimins et al . , Proc. Natl. Acad. Sci. USA 78, 993 (1981)] and 3' [Lusky et al . , Mol Cel.. B ol . 3 , 1108 (1983)] to the transcription unit, within an intron [Banerji et al . , Cell 33, 729 (1983)] as well as within the coding sequence itself [Osborne et al . , Mol. Cel.. Biol. 4_, 1293
• enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotem and insulin) .
• an enhancer from a eukaryotic cell virus. Examples include the SV40 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 origin, 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- ⁇ receptor ⁇ -chain DNA, but is preferably located at a site 5' from the promoter.
• Expression vectors used in eukaryotic host cells will also contain sequences 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- ⁇ receptor ⁇ -chain. The 3' untranslated regions also include transcription termination sites. Construction of suitable vectors containing one or more of the above listed components, the desired coding and control sequences, employs standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required.
• the ligation mixtures are used to transform E. coli K12 strain 294 (ATCC 31,446) and successful transformants selected by ampicill 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 Res. $_, 309 (1981) or by the method of Maxam et al . , Methods n Enzymology 65, 499 (1980) .
• transient expression involves the use of an expression vector that is able to replicate efficiently 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.
• transient expression systems are particularly useful in the invention for purposes of identifying analogs and variants of the IFN- ⁇ receptor ⁇ -chain.
• plasmid for mammalian cell culture expression of the IFN- ⁇ ⁇ -cham is pRK5 (EP 307,247) .
• E. Selection and Transformation of Host 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 cloning host is E.
• E. coli 294 ATCC 31,446 although other gram negative or gram positive prokaryotes such as E. coli B, E. coli X1776 (ATCC 31,537), E. coli W3110 (ATCC 27,325), Pseudomonas species, or Serratia Marcesans are suitable.
• eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for vectors herein. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species and strains are commonly available and useful herein, such as S.
• pombe [Beach and Nurse, Nature 290, 140 (1981)], Kluyveromyces lactis [Louvencourt et al . , J . Bacteriol . 737 (1983)]; yarrowia (EP 402,226); Pichia pastor s (EP 183,070), Trichoderma reesia (EP 244,234), Neurospora crassa [Case et al . , Proc. Natl. Acad. Sci. USA 76, 5259-5263 (1979)]; and Asperqillus hosts such as A. nidulans [Ballance et al . , Biochem. Biophys. Res. Commun.
• 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.
• viruses 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.
• plant cells are transfected by incubation with certain strains of the bacterium Agrobacterium tumefaciens, which has been previously manipulated to contain the IFN- ⁇ receptor ⁇ -chain DNA.
• Agrobacterium tumefaciens the DNA encoding IFN- ⁇ receptor ⁇ - chain is transferred to the plant cell host such that it is transfected, and will, under appropriate conditions, express the IFN- ⁇ receptor ⁇ -chain DNA.
• regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadhenylation signal sequences.
• DNA segments isolated from the upstream region"of the T-DNA 780 gene are capable of activating or increasing transcription levels of plant-expressible genes in recombinant DNA-containing plant tissue. See EP 321,196 published 21 June 1989.
• mice sertolli cells [TM4, Mather, Biol. Reprod. 23, 243- 251 (1980)]; monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (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.
• 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- ⁇ receptor ⁇ -subunit, chains of the IFN- ⁇ /- ⁇ receptors, and/or other cytokine receptor or EPO receptor.
• Host cells are transfected and preferably transformed with the above-described expression or cloning vectors and cultured in conventional nutrient media modified as is appropriate for inducing promoters or selecting transf ⁇ rmants containing amplified genes.
• F. Culturing the Host Cells Prokaryotes cells used to produced the IFN- ⁇ ⁇ -cham 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.
• any of the media described in Ham and Wallace, Meth. Enzymol. 58, 44 (1979); Barnes and Sato, Anal ⁇ Biochem. 102, 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 media for the host cells.
• Any of-these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transfemn, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine) , antibiotics (such as GentamycinTM drug) trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
• growth factors such as insulin, transfemn, or epidermal growth factor
• salts such as sodium chloride, calcium, magnesium, and phosphate
• buffers such as HEPES
• nucleosides such as adenosine and thymidine
• antibiotics such as GentamycinTM drug
• trace elements defined as inorganic compounds usually present at final concentrations in the microm
• the culture conditions such as temperature, pH and the like, suitably are those previously used with the host cell selected for 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.
• the IFN- ⁇ 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 IFN- ⁇ 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, 5201-5205 (1980)], 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 32 P.
• other techniques may also be employed, such as using biotin-modifled 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.
• 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 formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
• Gene expression may be measured by immunological methods, such as lmmunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.
• immunological methods such as lmmunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.
• lmmunohistochemical staining techniques a cell sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies 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, 734-738 (1980) .
• Antibodies useful for lmmunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native IFN- ⁇ ⁇ -chain polypeptide, or against a synthetic peptide based on the DNA sequence provided herein as described further hereinbelow.
• the IFN- ⁇ receptor ⁇ -cham preferably is recovered from 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.
• the IFN- ⁇ receptor ⁇ -subunit When the IFN- ⁇ receptor ⁇ -subunit is expressed in a recombinant cell other than one of human origin, the IFN- ⁇ receptor ⁇ -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 IFN- ⁇ receptor ⁇ -chain.
• the culture medium or lysate is centrifuged to remove particulate cell debris.
• the membrane and soluble protein fractions are then separated.
• the IFN- ⁇ receptor ⁇ -cham may then be purified from the soluble protein fraction and from the membrane fraction of the culture lysate, depending on whether the IFN- ⁇ receptor ⁇ -chain is membrane bound.
• the following procedures are exemplary of suitable purification procedures: fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation exchange resin such as DEAE; chromatofocusmg; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example,
• IFN- ⁇ receptor ⁇ -chain functional derivatives in which residues have been deleted, inserted and/or substituted are recovered in the same fashion as the native receptor chains, taking into account of any substantial changes in properties occasioned by the alteration.
• fusion of the IFN- ⁇ receptor ⁇ -cham with another protein or polypeptide e.g. a bacterial or viral antigen, facilitates purification; an immunoaffmity column containing antibody to the antigen can be used to absorb the" fusion.
• Immunoaffinity columns such as a rabbit polyclonal ant ⁇ -IFN- ⁇ receptor ⁇ -chain column can be employed to absorb IFN- ⁇ receptor ⁇ -chain variant by binding to at least one remaining immune epitope.
• a protease inhibitor 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.
• PMSF phenyl methyl sulfonyl fluoride
• purification methods suitable for native IFN- ⁇ receptor ⁇ -cham may require modification to account for changes in the character of the IFN- ⁇ receptor ⁇ -cham or its variants upon expression in recombinant cell culture.
• I Covalent Modifications of IFN-y receptor ⁇ -cham
• Covalent modifications of IFN- ⁇ receptor ⁇ -chain are included within the scope herein. Such modifications are traditionally introduced by reacting targeted amino acid residues of the IFN- ⁇ receptor ⁇ -cham with an organic derivatizmg 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 recombinant host cells.
• the resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays of the IFN- ⁇ receptor ⁇ -chain, or for the preparation of ant ⁇ -IFN- ⁇ receptor ⁇ -chain antibodies for immunoaff ity purification of the recombinant.
• Cystemyl residues most commonly are reacted with ⁇ -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, ⁇ -bromo- ⁇ - (5- ⁇ m ⁇ dozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-n ⁇ tro-2-pyr ⁇ dyl disulfide, methyl 2-pyr ⁇ dyl disulfide, p-chloromercunbenzoate, 2-chloromercur ⁇ -4- mtrophenol, or chloro-7-n ⁇ trobenzo-2-oxa-l, 3-d ⁇ azole.
• 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 chain.
• Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0.
• Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
• Other suitable reagents for derivatiz g ⁇ -ammo-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O- methylisourea; 2, 4-pentaned ⁇ one; and transaminase-catalyzed reaction with glyoxylate.
• Arg yl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2, 3-butaned ⁇ one, 1,2- cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
• tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
• aromatic diazonium compounds or tetranitromethane Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-n ⁇ tro derivatives, respectively.
• Tyrosyl residues are lodmated using l2 I or I to prepare labeled proteins for use in radioimmunoassay, the chloramine T method described above being suitable.
• 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 these residues falls within the scope of this invention.
• 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 histidme side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 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 nonproteinaceous polymers, e.g. polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth U.S. patents 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
• Derivatization with bifunctional agents is useful for preparing intramolecular aggregates of the IFN- ⁇ receptor ⁇ -cham with polypeptides as well as for cross-linking the IFN- ⁇ receptor ⁇ -cham to a water insoluble support matrix or surface for use in assays or affinity purification.
• Commonly used cross-linking agents include 1, 1-b s (d azoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, homobifunctional lmidoesters, and bifunctional maleimides.
• Derivatizmg agents such as methyl-3-[ (p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates which are capable of forming cross-links in the presence of light.
• reactive water insoluble matrices such as cyanogen bromide activated carbohydrates and the systems reactive substrates described in U.S. Patent Nos. 3,959,642; 3,969,287; 3,691,016; 4,195,128; 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 action of recombinant host cells on the expressed polypeptide.
• Glutaminyl and asparigmyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
• nonproteinaceous polymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymer not otherwise found in nature.
• hydrophilic polyvinyl polymers fall within the scope of this invention, e.g. polyv ylalcohol and polyvmylpyrrolidone.
• Particularly useful are polyvinylalkylene ethers such a polyethylene glycol, polypropylene glycol.
• the IFN- ⁇ receptor ⁇ -cham may be linked to various nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4",670,417; 4,791,192 or 4,179,337.
• the IFN- ⁇ receptor ⁇ -chain may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) , or in macroemulsions.
• Immunoglobulins and certain variants thereof are known and many have been prepared in recombinant cell culture. For example, see U.S. Patent 4,745,055; EP 256,654; F.aulkner et al . , Nature 298:286 (1982); EP 120,694; EP 125,023; Morrison, J. Im un. 123:793 (1979); Kohler et al . , Proc. Nat'l. Acad. Sci. USA 77:2197 (1980); Raso et al . , Cancer Res. 4.-2073 (1981); Morrison et al . , Ann. Rev. Immunol.
• the lmmunoglobulin 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.
• Immunoadhesins Chimeras constructed from a receptor sequence linked to an appropriate lmmunoglobulin constant domain sequence (immunoadhesins) are known in the art. Immunoadhesins 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); Traunecker et al . , Nature 339, 68-70 (1989); Zettmeissl et al . , DNA Cell Biol. USA 9, 347-353 (1990); Byrn et al .
• TNF receptor [Ashkenazi et al . , Proc. Natl. Acad. 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 ⁇ -cham * [Ridgway and Gorman, J. Cell. Biol. 115, abstr.
• HGF receptor Mark, M.R. et al . , 1992, J ⁇ Biol. Chem. submitted], where the asterisk (*) indicates that the receptor is member of the lmmunoglobulin superfamily.
• the nucleic acid encoding the desired IFN- ⁇ receptor ⁇ -chain extracellular domain sequence will be fused C-termmally to nucleic acid encoding the N- terminus of an lmmunoglobulin constant domain sequence, however N- terminal fusions are also possible.
• the encoded chimeric polypeptide will retain at least functionally active hinge, CH2 and CH3 domains of the constant region of an lmmunoglobulin heavy chain. Fusions are also made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CHI of the heavy chain or the corresponding region of the light chain.
• the precise site at which the fusion is made is not critical; particular sites are well known and may be selected m order to optimize the biological activity, secretion or binding characteristics of the IFN- ⁇ receptor ⁇ -chain-immunoglobulm chimeras.
• the IFN- ⁇ receptor ⁇ -chain-immunoglobulm chimeras are assembled as monomers, or hetero- or homo-multimers, and particularly as dimers or tetramers, essentially as illustrated in WO 91/08298.
• an the IFN- ⁇ receptor ⁇ -cham extracellular domain sequence is fused to the N-terminus of the C- terminal portion of an antibody (in particular the Fc domain) , containing the effector functions of an lmmunoglobulin, e.g. lmmunoglobulin G, (IgG-1) . It is possible to fuse the entire heavy chain constant region to the IFN- ⁇ receptor ⁇ -chain extracellular domain sequence. However, more preferably, a sequence beginning in the hinge region just upstream of the papam cleavage site (which defines
• IgG Fc chemically; residue 216, taking the first residue of heavy chain constant region to be 114 [Kobet et al . , supra] , or analogous sites of other immunoglobulins) is used in the fusion.
• the IFN- ⁇ receptor ⁇ -cham amino acid sequence is fused to the hinge region and CH2 and CH3 or CHI, hinge, CH2 and CH3 domains of an IgG-1, 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.
• the IFN- ⁇ receptor ⁇ -cham-immunoglobulin chimeras are assembled as hetero-multimers, and particularly as hetero- dimers or -tetramers.
• these assembled immunoglobulins will 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 in the higher molecular weight immunoglobulins; 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 unit may be the same or different.
• IFN- ⁇ receptor ⁇ -chain-immunoglobulm chimeras within the scope herein are schematically diagrammed below:
• V L is an lmmunoglobulin light chain variable domain
• Vache is an lmmunoglobulin heavy chain variable domain
• C L an lmmunoglobulin light chain constant domain
• C H an lmmunoglobulin heavy chain constant domain
• n is an integer greater than 1;
• Y designates the residue of a covalent cross-linking agent.
• the foregoing structures only show key features; they do not indicate joining (J) or other domains of the immunoglobulins, nor are disulfide bonds shown. However, where such domains are required for binding activity, they shall be constructed as being present in the ordinary locations which they occupy in the lmmunoglobulin molecules.
• the IFN- ⁇ receptor ⁇ -chain extracellular domain sequences can be inserted between lmmunoglobulin heavy chain and light chain sequences such that an lmmunoglobulin comprising a chimeric heavy chain is obtained.
• the IFN- ⁇ receptor ⁇ -chain sequences are fused to the 3' end of an lmmunoglobulin heavy chain in each arm of an lmmunoglobulin, 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 al . , Mol. Immunol. 28, 1027-1037 (1991) .
• an lmmunoglobulin light chain might be present either covalently associated to an IFN- ⁇ receptor ⁇ -chain-immunoglobulin heavy chain fusion polypeptide, or directly fused to the IFN- ⁇ receptor ⁇ -cham extracellular ⁇ omain.
• DNA encoding an lmmunoglobulin light chain is typically coexpressed with the DNA encoding the IFN- ⁇ receptor ⁇ -chain-immunoglobulm heavy chain fusion protein.
• the hybrid heavy chain and the light chain Upon secretion, the hybrid heavy chain and the light chain will be covalently associated to provide an lmmunoglobulin-like structure comprising two disulfide-linked lmmunoglobulin heavy chain- light chain pairs.
• Methods suitable for the preparation of such structures are, for example, disclosed in U.S. Patent No. 4,816,567 issued 28 March 1989.
• the native IFN- ⁇ receptor ⁇ -chams are glycoprotems.
• Variants having a glycoslation pattern which differs from that of any native amino acid sequence which might be present in the molecules of the present invention are within the scope herein.
• 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 ammo acid sequence variants.
• Chemical or enzymatic coupling of glycosydes to the IFN-y receptor ⁇ -cham 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-lmked (or N-linked) glycosylation.
• the sugar (s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free hydroxyl groups such as those of cysteine, (d) free sulfhydryl groups such as those of serine, threonine, or hydroxyprol e, (e) aromatic residues such as those of phenylalanme, tyrosme, or tryptophan or (f) the amide group of glutam e.
• arginine and histidine free carboxyl groups
• free hydroxyl groups such as those of cysteine
• free sulfhydryl groups such as those of serine, threonine, or hydroxyprol e
• aromatic residues such as those of phenylalanme, tyrosme, or tryptophan
• f the amide group of glutam e.
• 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 the cleavage of most or all sugars, except the linking sugar, while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al . , Arch. Biochem. Biophys. 259, 52 (1987) and by Edge et al . , Anal. Biochem. 118, 131 (1981) . Carbohydrate moieties can be removed by a variety of endo- and exoglycosidases as described by Thotakura et al . , Meth.
• Glycosylation variants can also be produced by selecting appropriate host cells of recombinant production. Yeast, for example, introduce glycosylation which varies significantly from that of mammalian systems. Similarly, mammalian cells having a different species (e.g. hamster, murine, insect, porcine, bovine or ovine) or tissue (e.g.
• Polyclonal antibodies to the IFN- ⁇ receptor ⁇ -chain generally are raised in animals by multiple subcutaneous (sc) or mtrape ⁇ toneal dp) injections of the IFN- ⁇ receptor ⁇ -chain and an adjuvant. It may be useful to conjugate the IFN- ⁇ receptor ⁇ -chain or a fragment containing the target amino acid sequence to a protein that is immunogenic in the species to be immunized, e.g.
• Animals are immunized against the immunogenic conjugates or derivatives by combining 1 mg of 1 ⁇ g of conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
• 1 mg of 1 ⁇ g of conjugate for rabbits or mice, respectively
• 3 volumes of Freund's complete adjuvant injecting the solution intradermally at multiple sites.
• the animals are boosted with 1/5 to 1/10 the original amount of conjugate 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 ant ⁇ -IFN- ⁇ receptor ⁇ -chain antibody titer. Animals are boosted until the titer plateaus.
• the animal boosted with the conjugate of the same IFN- ⁇ receptor ⁇ -cham, 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 immune response.
• Monoclonal antibodies are 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 may be present in minor amounts.
• the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.
• the ant ⁇ -IFN- ⁇ receptor ⁇ -cham monoclonal antibodies of the invention may be made using the hybridoma method first described by Kohler & Milste , Nature 256: 495 (1975), or may be made by recombinant DNA methods [Cabilly, et al . , U.S. Pat. No. 4, 816,567] .
• lymphocytes 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 producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Godmg, Monoclonal Antibodies: Principles and Practice,, pp.59-103 (Academic Press, 1986)].
• a suitable fusing agent such as polyethylene glycol
• 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.
• a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
• the culture medium for the hybridomas typically will include hypoxanthine, aminopterm, and thymidine (HAT medium) , which substances prevent the growth of HGPRT-deflcient 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.
• preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells available 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 [Kozbor, J. Immunol. 133:3001 (1984); Brodeur, , Monoclonal Antibody 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 antibodies directed against IFN- ⁇ receptor ⁇ -cham.
• the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by lmmunoprecipitation or by an iri vitro binding assay, such as radioimmunoassay (RIA) or enzyme- linked lmmunoabsorbent assay (ELISA) .
• RIA radioimmunoassay
• ELISA enzyme- linked lmmunoabsorbent assay
• the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson & Pollard, Anal. Biochem. 107:220 (1980) .
• 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 Eagle's Medium or RPMI-1640 medium.
• the hybridoma cells may be grown m vivo as ascites tumors in an animal.
• the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional lmmunoglobulin 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.
• the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce lmmunoglobulin 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. Acad. Sci. 81, 6851 (1984), or by covalently joining to the lmmunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulm polypeptide.
• “chimeric” or “hybrid” antibodies are prepared that have the binding specificity of an anti-selectin ligand monoclonal antibody herein.
• non-immunpglobulin 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 site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an IFN- ⁇ receptor ⁇ -chain and another antigen-combining site having specificity for a different antigen.
• Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
• lmmunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond.
• suitable reagents for this purpose include lmmothiolate and methyl-4-mercaptobutyr ⁇ m ⁇ dateT
• the antibodies of the invention typically will be labeled with a detectable moiety.
• the detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal.
• the detectable moiety may be a radioisotope, such as 3 H, C, 32 P, 35, S, or 12 I, a fluorescent or chemilummescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; radioactive isotopic labels, such as, e.g., 125 I, 32 P, 14 C, or 3 H, or an enzyme, such as alkaline phosphatase, beta- galactosidase or horseradish peroxidase.
• any method known in the art for separately conjugating the antibody 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); Pain, et al . , J . Immunol. Meth.
• the antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and lmmunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987) .
• ком ⁇ онентs rely on the ability of a labeled standard (which may be an IFN- ⁇ receptor ⁇ -chain or an lmmunologically reactive portion thereof) to compete with the test sample analyte (IFN- Y receptor ⁇ -chain) for binding with a limited amount of antibody.
• the amount of IFN- ⁇ receptor ⁇ -chain in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies.
• 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 of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected.
• 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.
• the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti- lmmunoglobulm antibody that is labeled with a detectable moiety (indirect sandwich assay) .
• sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. (111) Humanized antibodies
• a humanized antibody has one or more am o acid residues introduced into it from a source which is non-human. These 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 Winter and co-workers [Jones et al . , Nature 321, 522-525 (1986); Riechmann et al . , Nature 332, 323-327 (1988); Verhoeyen et al . , Science 239, 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
• 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 species.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• 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.
• Three dimensional lmmunoglobulin models are commonly available and are familiar to those skilled in the art.
• Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate lmmunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate lmmunoglobulin sequence, i.e. the analysis of residues that influence the ability of the candidate lmmunoglobulin to bind its antigen.
• 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 ant ⁇ gen(s), is achieved.
• the CDR residues are directly and most substantially involved in influencing antigen binding.
• transgenic animals e.g. mice
• transgenic animals e.g. mice
• J H antibody heavy chain joining region
• Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
• one of the binding specificities is for an IFN- ⁇ receptor ⁇ -chain
• the other one is for any other antigen, and preferably for an other receptor or receptor subunits.
• bispecific antibodies specifically binding an IFN- ⁇ receptor ⁇ -chain and an IFN- ⁇ receptor ⁇ -cham, a chain of another cytokme receptor (i.e. a TNF receptor, an IL-2 receptor), or of an EPO receptor are within the scope of the present invention.
• Methods for making bispecific antibodies are known in the art.
• bispecific antibodies are based on the coexpression of two lmmunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305, 537-539 (1983)) . Because of the random assortment of lmmunoglobulin 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. WO 93/08829 (published 13 May 1993), and in Traunecker et al . , EMBO 10, 3655-3659 (1991) .
• antibody variable domains with the desired binding specificities are fused to lmmunoglobulin constant domain sequences.
• the fusion preferably is with an lmmunoglobulin 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 (CHI) containing the site necessary for light chain binding, present at least one of the fusions.
• CHI first heavy chain constant region
• the bispecific antibodies are composed of a hybrid lmmunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid lmmunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm.
• Heteroconjugate 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 infection (PCT application publication Nos. WO 91/00360 and WO 92/200373; EP 03089) .
• Heteroconjugate antibodies may be made using any convenient cross- lmking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent No. 4,676,980, along with a number of cross-linking techniques.
• IFN- ⁇ receptor ⁇ -chain polypeptides of the present invention as well as the ant ⁇ -IFN- ⁇ ⁇ -chain antibodies, either in monospecific or bispecific or heteroconjugate form, are useful in signaling, enhancing or blocking IFN- ⁇ biological activity. They may also be useful in signaling, enhancing or blocking the biological activities of other biologically active polypeptides, such as other cytokines or EPO.
• IFN- ⁇ immunoregulatory activities, such as enhancing the host antibody response to specific antigens, which enables the use of IFN- ⁇ as vaccine adjuvant.
• Recombinant human gamma interferon (Act ⁇ mmune R , Genentech, South San Francisco, California) is commercially available as an immunomodulatory drug for the treatment of chronic granulomatous disease characterized by severe, recurrent infections of the skin, lymph nodes, liver, lungs, and bones due to phagocyte disfunction.
• IFN- ⁇ receptor ⁇ -chains or ant ⁇ -IFN- ⁇ receptor antibodies of agonist character may mimic these are other IFN- ⁇ activities.
• IFN- ⁇ receptor ⁇ -cham polypeptides and antagonist anti- IFN- ⁇ receptor ⁇ -cham antibodies may block IFN- ⁇ biological activity.
• This antagonist activity is believed to be useful in the treatment of pathological conditions associated with endogenous IFN- ⁇ 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 physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical 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 acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccha ⁇ des and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or PEG.
• buffers such as phosphate, citrate and other organic acids
• antioxidants including ascorbic acid
• the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by mterfacial polymerization, for example, hydroxymethylcellulose or gelat - microcapsules and poly- (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
• colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
• macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
• the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
• 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 injection needle.
• the molecules of the present invention optionally are combined with or administered n concert with other cytokmes, such as TNF, lymphotoxin, IL-2, hepatocyte growth factor (HGF) , EPO, conventional antitumor agents, such as 5-fluorouracil (5-FU) or Etoposide (VP-16) , etc.
• the route of administration is in accord with known methods, e.g. injection or infusion by intravenous, mtraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or mtralesional routes, topical administration, or by sustained release systems.
• sustained release preparations 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 .
• 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); Hwang et al . , Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980); EP 52322A; 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.
• the liposomes are of the small (about 200-800 Angstroms) unilamelar type in which the lipid content is greater than about 30 mol. % 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 objectives, the route of administration, and the condition 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 ⁇ g/kg to up to 100 mg/kg or more, depending on the factors mentioned above.
• 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 expression vector pHMG-A7 ' containing the entire coding region of the murine IFN- ⁇ R cDNA was described previously (Hemmi et al . , Proc. Natl. Acad. Sci. USA 86, 9901-9905 (1989)) .
• the reporter plasmid pUMS (GT) 8 -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.
• GH human growth hormone
• the expression plasmid pCDM8-Tac was derived from pCDM8 (Seed and Aruffo, Proc. Natl. Acad. Sci. USA 84, 3365-3369 (1987)) by releasing its BstXl-stuffer and inserting the blunted Hindlll fragment of plasmid pKCR.Tac-2.A. Generation of the Cell Line COSN 31
• COSN cells (a sublme of COS7 cells, provided by Dr. S. Nagata) were grown in D-MEM (Gibco) supplemented with 10% FCS. Approximately 2 x 10 6 exponentially growing cells were cotransfected by the calcium phosphate precipitation method (Graham and Erb, Virology 52, 456-467 (1973)) with lO ⁇ g Qiagen-purified pUMS (GT) ⁇ -Tac, lO ⁇ g pHMGA7' , and 2 ⁇ g of pSV2neo DNA (Southern and Berg, J. Mol. Appl. Genet. 1 , 327-341 (1982)) .
• G418-res ⁇ stant colonies were pooled and incubated for 48 hr at 37oc with 500 units/ml recombinant huIFN- ⁇ , which cross-reacts with the simian IFN- ⁇ R.
• 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, unmduced cells with constitutive Tac antigen expression were eliminated. In a fourth round, the cells were enriched for the expression of the muIFN- ⁇ R using a Mab against the muIFN- ⁇ R (Basu et al . , J.
• COSN 31 cells had retained the ability of episomal replication of pCDM8-Tac.
• 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, 4367-4374 (1990)) .
• the library was divided into six pools of approximately 3xl0 5 independent colonies. For the first round of enrichment, 3 x 5 ⁇ g DNA from each pool was transfected separately into 3 x 10 6 subconfluent COSN 31 cells by electroporation as described above and seeded into three 8.5 cm-Pet ⁇ dishes.
• DNA was extracted from adherent cells as described above and amplified in MC1061 E. coli host cells (Seed and Aruffo, Supra) . The subsequent rounds of transfection and enrichment were carried out separately for each of the six original cDNA pools with 10 6 COSN 31 cells transfected with 5 ⁇ g DNA.
• 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- ⁇ R ⁇ - chain was monitored accordingly, using a rat-anti muIFN- ⁇ R Mab (Basu et al . , Supra) and FITC-conjugated rabbit-anti-rat IgG F(ab')2 antibodies.
• Human or murine IFN- ⁇ was assayed on human HEp-2 (ATCC) or murine L929 cells challenged with vesicular stomatitis virus (VSV) .
• VSV vesicular stomatitis virus
• U/ml concentration that results in 50% protection from the cytopathic effect.
• COS7 cells were stably cotransfected with the cDNA expression plasmid pHMG-A7 ' encoding the murine IFN- ⁇ receptor (muIFN- ⁇ R) (Hemmi et al . , Proc. Natl. Acad.
• COS cell line (COSN 31) was isolated which stably expressed the muIFN- ⁇ R, but responded only to human and not to murine IFN- ⁇ (muIFN- ⁇ ) by expressing the Tac antigen ( Figure IB) .
• COSN 31 cells were transiently transformed with pools of the murine early ⁇ -cell-derived cDNA library pAGS-3 (Takaki et al . , EMBO J 9_, 4367-4374 (1990)) and cells responsive to muIFN- ⁇ in terms of Tac antigen expression were enriched by panning. After four rounds of enrichment, one of six pools gave rise to significant muIFN- ⁇ - ⁇ nduced 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.
• Figure IC shows a cytofluorometric analysis of murine versus human IFN- ⁇ - (huIFN- ⁇ ) induced Tac antigen expression in COSN 31 cells transiently expressing pAGS.C19 cDNA. About 30% of the cells showed muIFN- ⁇ - ⁇ nduced Tac antigen expression. The level of expression was similar to the one observed with huIFN- ⁇ .
• This HindiII - Xbal fragment from the pAGS.C19 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 ⁇ gtll cDNA library described previously (Hemmi et al . , Proc. Natl.
• the first ATG of the largest open reading frame (nucleotides 94-96) is embedded in a typical consensus sequence for translation initiation (Kozak, Nucleic Acids 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, 4683-4690 (1986)) .
• Hydropathy analysis revealed the presence of an additional hydrophobic stretch encompassing am o 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 66 ammo acids.
• the insert of the ⁇ l.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 ⁇ l.C19 insert which contained one internal Seal and no EcoRV site, hybridized to only two genomic Seal and one EcoRV DNA fragments, suggesting that the transcript was most likely the product of a single gene.
• the cofactor for the IFN- ⁇ R was proposed to be encoded on chromosome 21 (Jung et al . , Proc. Natl. Acad. Sci. USA 84, 4151-4155 (1987)) .
• the ⁇ l.C19 insert was used as a probe to isolate a full length cDNA encoding the huIFN- ⁇ R ⁇ -chain from a human ⁇ gtll 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- ⁇ R ⁇ -cham.
• HEp-2xmuIFN- ⁇ R ⁇ #43.7 cells were stably transfected with either the original expression plasmid pAGS.C19, or the expression plasmid pHMG.C19 containing the ⁇ l.C19 insert driven by the 3-hydroxy-3- methylglutaryl coenzyme A reductase promoter (Gautier et al . , Nucleic Acids Res. 17, 8389 (1989); Hemmi et al . , supra) .
• Figure 3 shows the response to murine versus huIFN- ⁇ of parental HEp-2xmuIFN- ⁇ R ⁇ #43.7 cells, and one subline of these cells stably transfected with the pHMG.C19 expression plasmid encoding the muIFN- ⁇ R ⁇ -chain (HEp-2xmuIFN- ⁇ R ⁇ / ⁇ #6) .
• Figure 3C shows that HEp-2xmuIFN- ⁇ R ⁇ #43.7 cells respond only to huIFN- ⁇ , but not muIFN- ⁇ in terms of IFN regulatory factor 1 (IRF-1) mRNA induction (Miyamoto et al: , Cell 54, 903-913 (1988)), whereas HEp- 2xmuIFN- ⁇ R ⁇ / ⁇ #6 cells become fully responsive to muIFN- ⁇ as well.
• IRF-1 IFN regulatory factor 1
• muIFN- ⁇ R accessory or ⁇ -chain rendered these cells as sensitive to murine as to huIFN- ⁇ with regard to all response markers tested.
• Antibodies raised against the novel receptor subunit should help clarifying this latter point and also, how the ⁇ -chain interacts with the ⁇ -chain and whether it is involved in ligand-binding.
• Experiments with human/mouse hybrid IFN- ⁇ R ⁇ -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 . , J. Biol. Chem. 267, 3741-3749 (1992); Kalma et al . , J. Virol.
• the relatively short cytoplasmic domain of the 8-cham contains a motif- LEVL(D) which is reminiscent of the conserved box 2 region of some cytokme receptors, including notably the murine IL-2 receptor ⁇ -cham and the murine erythropoietin receptor (Murakami et al . , Proc. Natl. Acad. Sci. USA 88, 11349-11353 (1991)) . Mutations within this domain suggested that it is crucial in the mitogenic responses mediated through these receptors (Miura et al . , Mol. Cell Biol. 13, 1788-1795 (1993)).
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• Leu lie Leu Lys Asn Lys Lys lie Arg Pro His Gly Leu Leu Ser 215 220 225
• RECT1FIED SHEET (RULE 91) Ser lie lie Ser Ser Pro Glu Lys Glu Arg Asp Asp Val Leu Gin 320 325 330
• Lys Lys Thr lie Asn Ser Thr Tyr Tyr Val Glu Lys lie Pro Glu 155 160 165
• Tyr Val Leu Lys Trp Asp Tyr lie Ala Ser Ala Asp Val Leu Phe 20 25 30
• Pro Pro Pro Pro Val lie Thr Val Thr Ala Met Ser Asp Thr Leu Leu 110 115 120
• CAATGGATTT CAATGTCACT CTACGCCTTC GAGCTGAGCT GGGAGCACTC 450

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