Human interferon-epsilon: a type I interferon

The invention concerns a human interferon-.epsilon., originally designated PRO655, and its variants and derivatives. The interferon is related to but distinct from members of the IFN-.alpha. family and from IFNs-.beta. and -.gamma.. Nucleic acid encoding the polpypeptide, and methods and means for their recombinant production are also included.

FIELD OF THE INVENTION
 The present invention relates generally to the identification of a novel
 member of the type I interferon family. More specifically, the present
 invention concerns the isolation of a novel nucleic acid encoding a new
 and distinct type I interferon, termed interferon-epsilon (IFN-.epsilon.).
 BACKGROUND OF THE INVENTION
 Interferons are relatively small, single-chain glycoproteins released by
 cells invaded by viruses or certain other substances. Interferons are
 presently grouped into three major classes, designated leukocyte
 interferon (interferon-alpha, .alpha.-interferon, IFN-.alpha.), fibroblast
 interferon (interferon-beta, .beta.-interferon, IFN-.beta.), and immune
 interferon (interferon-gamma, .gamma.-interferon, IFN-.gamma.). In
 response to viral infection, lymphocytes synthesize primarily
 .alpha.-interferon (along with a lesser amount of a distinct interferon
 species, commonly referred to as omega interferon, IFN-.omega.), while
 infection of fibroblasts usually induces .beta.-interferon. .alpha.- and
 .beta.-interferons share about 20-30 percent amino acid sequence homology.
 Thus, the gene for human IFN-.beta. lacks introns, and encodes a protein
 possessine 29% amino acid sequence identity with human IFN-.alpha.I,
 suggesting that IFN-.alpha. and IFN-.beta. genes have evolved from a
 common ancestor (Taniguchi et al., Nature 285, 547-549 (1980)). By
 contrast, IFN-.gamma. is not induced by viral infection, rather, is
 synthesized by lymphocytes in response to mitogens, and is scarcely
 related to the other two types of interferons in amino acid sequence.
 Interferons-.alpha., .beta. and .omega. are known to induce MHC Class I
 antigens, and are referred to as type I interferons, while IFN-.gamma.
 induces MHC Class II antigen expression, and is also referred to as type
 II interferon.
 A large number of distinct genes encoding different species of IFNs-.alpha.
 have been identified. Alpha interferon species identified previously fall
 into two major classes, I and II, each containing a plurality of discrete
 proteins (Baron et al., Critical Reviews in Biotechnology 10, 1790190
 (1990); Nagata et al., Nature 287, 401-408 (1980); Nagata et al., Nature
 284, 316-320 (1980); Streuli et al., Science 209, 1343-1347 (1980);
 Goeddel et al., Nature 290, 20-26 (1981); Lawn et al., Science 212,
 1159-1162 (1981); Ullrich et al., J. Mol. Biol. 156, 467-486 (1982);
 Weissmann et al., Phil. Trans. R. Soc. Lond. B299, 7-28 (1982); Lund et
 al., Proc. Natl. Acad. Sci. 81, 2435-2439 (1984); Capon et al., Mol. Cell.
 Biol. 5, 768 (1985)). The various IFN-.alpha. species include IFN-.alpha.A
 (IFN-.alpha.2), IFN-.alpha.B, IFN-.alpha.C, IFN-.alpha.C1, IFN-.alpha.D
 (IFN-.alpha.1), IFN-.alpha.E, IFN-.alpha.F, IFN-.alpha.G, IFN-.alpha.H,
 IFN-.alpha.I, IFN-.alpha.J1, IFN-.alpha.J2, IFN-.alpha.K, IFN-.alpha.L,
 IFN-.alpha.4B, IFN-.alpha.5, IFN-.alpha.6, IFN-.alpha.74, IFN-.alpha.76
 IFN-.alpha.4a), IFN-.alpha.88, and alleles of these species. According to
 our current knowledge, the IFN-.alpha. family consists of 13 expressed
 alleles producing 12 different proteins that exhibit remarkably different
 biological activity profiles. Pestka, S., Semin. Oncol. 24(suppl. 9),
 S9-4-S9-17 (1997).
 Interestingly. while only a single human IFN-.beta. gene has been
 unequivocally identified, bovine IFN-.beta. is encoded by a family of five
 or more homologous, yet distinct genes.
 Interferons were originally produced from natural sources, such as buffy
 coat leukocytes and fibroblast cells, optionally using known inducing
 agents to increase interferon production. Interferons have also been
 produced by recombinant DNA technology.
 The cloning and expression of recombinant IFN-.alpha.A (rIFN-.alpha.A, also
 known as IFN-.alpha.2) was described by Goeddel et al., Nature 287, 411
 (1980). The amino acid sequences of rIFNs-.alpha.A, B, C, D, F, G, H, K
 and L, along with the encoding nucleotide sequences, are described by
 Pestka in Archiv. Biochem. Biophys. 221, 1 (1983). The amino acid
 sequences and the underlying nucleotide sequences of rIFNs-.alpha.E, I and
 J are described in British Patent Specification No. 2,079,291, published
 Jan. 20, 1982. Hybrids of various IFNs-.alpha. are also known, and are
 disclosed, e.g. by Pestka et al., supra. Nagcata et al., Nature 284, 316
 (1980), described the expression of an IFN-.alpha. gene, which encoded a
 polypeptide (in non-mature form) that differs from rIFN-.alpha.D by a
 single amino acid at position 114. Similarly, the cloning and expression
 of an IFN-.alpha. gene (designated as rIFN-.alpha.2) yielding a
 polypeptide differing from rIFN-.alpha.A by a single amino acid at
 position 23, was described in European Patent Application No. 32 134,
 published Jul. 15, 1981.
 The cloning and expression of mature rIFN-.beta. is described by Goeddel et
 al., Nucleic Acids Res. 8, 4057 (1980).
 The cloning and expression of mature rIFN-.gamma. are described by Gray et
 al., Nature 295, 503 (1982).
 IFN-.omega. has been described by Capon et al., Mol. Cell. Biol. 5, 768
 (1985).
 IFN-.tau. has been identified and disclosed by Whaley et al., J. Biol.
 Chem. 269, 10864-8 (1994).
 All of the known IFNs-.alpha., -.beta., and -.gamma. contain multiple
 cysteine residues. These residues contain sulfhydryl side-chains which are
 capable of forming intermolecular disulfide bonds. For example, the amino
 acid sequence of mature recombinant rIFN-.alpha.A contains cysteine
 residues at positions 1, 29, 98 and 138. Wetzel et al., Nature 289, 606
 (1981), assigned intramolecular disulfide bonds between the cysteine
 residues at positions 1 and 98, and between the cysteine residues at
 positions 29 and 138.
 Antibodies specifically binding various interferons are also well known in
 the art. For example, anti-.alpha.-interferon agonist antibodies have been
 reported by Tsukui et al., Microbiol. Immunol. 30, 112901139 (1986);
 Duarte et al., Interferon-Biotechnol. 4, 221-232 (1987); Barasoaian et
 al., J. Immunol. 143, 507-512 (1989); Exley et al., J. Gen. Virol. 65,
 2277-2280 (1984); Shearer et al., J. Immunol. 133, 3096-3101 (1984); Alkan
 et al., Ciba Geigy Foundation Symposium 119, 264-278 (1986); Noll et al.,
 Biomed. Biochim. Acta 48, 165-176 (1989); Hertzog et al., J. Interferon
 Res. 10 (Suppl. 1) (1990); Kontsek et al., J. Interferon Res. (special
 issue) 73-82 (1991), and U.S. Pat. No. 4,423,147 issued Dec. 27, 1983.
 The actions of type I interferons appear to be mediated by binding to the
 IFN-.alpha. a receptor complex on the cell surface. This receptor is
 composed of at least two distinct subunits identified as IFN-.alpha.R1
 (Uze et al., Cell 60, 225-234 [1990]) and IFN-.alpha.R2 (Novick et al.
 Cell 77, 391-400 [1994]), each having 2 and 3 spliced variants,
 respectively. IFN-.alpha.R2 is the binding subunit of the known type
 interferons, whereas IFN-.alpha.R1 contributes to higher affinity binding
 and signaling. The engagement of receptors by ligand binding activates
 Janus family kinases (JAK) and protoplasmic latent signal transducers and
 activators of transcription (STAT) proteins by tyrosine phosphorylation.
 Activated STATs translocate to the nucleus in forms of complexes and
 interact with their cognitive enhancer elements of IFN-stimulated genes
 (ISGs). leading to a corresponding transcription activation and biological
 responses. Darnell et al., Science 264, 1415-21 (1994). However, despite
 similarities in their binding properties, the biological responses
 stimulated by type I interferons are significantly different.
 Interferons have a variety of biological activities, including antiviral,
 immunoregulatory and antiproliferative properties, and are, therefore, of
 great interest as therapeutic agents in the control of cancer, and various
 viral diseases. Interferons have been implicated in the pathogenesis of
 various autoimmune diseases, such as systemic lupus erythematoses,
 Behcet's disease, insulin-dependent diabetes mellitus (IDDM, also referred
 to as type I diabetes). It has been demonstrated in a transgenic mouse
 model that .beta. cell expression of IFN-.alpha. can cause insulitis and
 IDDM, and IFN-.alpha. antagonists (including antibodies) have been
 proposed for the treatment of IDDM (WO 93/04699, published Mar. 18, 1993).
 Impaired IFN-.gamma. and IFN-.alpha. production has been observed in
 multiple sclerosis (MP) patients. An acid-labile IFN-.alpha. has been
 detected in the serum of many AIDS patients, and it has been reported that
 the production of IFN-.gamma. is greatly suppressed in suspensions of
 mitogen-stimulated mononuclear cells derived from AIDS patients. For a
 review see, for example, Chapter 16, "The Presence and Possible Pathogenic
 Role of Interferons in Disease", In: Interferons and other Regulatory
 Cytokines, Edward de Maeyer (1988, John Wilet and Sons publishers). Alpha
 and beta interferons have been used in the treatment of the acute viral
 disease herpes zoster (T. C. Merigan et al., N. Engl. J. Med. 298, 981-987
 (1978); E. Heidemann et al., Onkologie 7, 210-212 (1984)), chronic viral
 infections, e.g. hepatitis B infections (R. L. Knobler el al., Neurology
 34, 1273078 (1984); M. A. Faerkkilae et al., Act. Neurol. Sci. 69, 184-185
 (1985)). rIFN-.alpha.-2a (Roferon.RTM., Roche) is an injection formulation
 indicated in use for the treatment of hairy cell leukemia and AIDS-related
 Kaposi's sarcoma. Recombinant IFN-.alpha.-2b (Intron.RTM. A. Schering) has
 been approved for the treatment of hairy cell leukemia, selected cases of
 condylomata acuminata, AIDS-related Kaposi's sarcoma, chronic hepatitis
 Non-A, Non-B/C, and chronic helatitis B infections is certain patients.
 IFN-.gamma.-1b (Actimmune.RTM., Genentech. Inc.) is commercially available
 for the treatment of chronic granulomatous disease.
 For further information about the biologic activities of type I IFNs see,
 for example, Pfeffer, Semin. Oncol. 24(suppl 9), S9-63-S9-69 (1997).
 SUMMARY OF THE INVENTION
 Applicants have identified a cDNA clone (designated in the present
 application as "DNA50960") that encodes a novel human interferon
 polypeptide, which is now designated as human IFN-.epsilon..
 In one embodiment, the invention provides an isolated nucleic acid molecule
 comprising DNA having at least a 95% sequence identity to (a) a DNA
 molecule encoding a novel human interferon polypeptide originally
 designated PRO655, and hereinafter also referred to as IFN-.epsilon.,
 comprising the sequence of amino acids from about 22 to 189 of FIG. 1 (SEQ
 ID NO:1), or (b) the complement of the DNA molecule of (a). In one aspect,
 the isolated nucleic acid comprises DNA encoding a new interferon
 polypeptide having at least amino acid residues 22 to 189 of FIG. 1 (SEQ
 ID NO:1), or is complementary to such encoding nucleic acid sequence, and
 remains stably bound to it under at least moderate, and optionally, under
 high stringency conditions. In another embodiment, the isolated nucleic
 acid molecule encodes the full-length polypeptide represented in FIG. 1
 (SEQ. ID. NO:1), with or without the putative signal peptide at amino
 acids 1-21, and with or without the initiating methionine, or is the
 complement of such DNA molecule. In a further embodiment, the isolated
 nucleic acid molecule comprises DNA having at least a 95% sequence
 identity to (a) DNA molecule encoding the same mature polypeptide encoded
 by the human interferon protein cDNA in ATCC Deposit No.209509
 (DNA50960-1224), deposited on Dec. 3, 1997.
 In another embodiment, the invention provides a vector comprising DNA (as
 hereinabove defined) encoding a novel interferon-.epsilon. polypeptide. A
 host cell comprising such a vector is also provided. By way of example,
 the host cells may be CHO cells, E. coli, or yeast (including
 Saccharomyces cerevisiae and other yeast strains). A process for producing
 the new interferon polypeptides of the present invention is further
 provided and comprises culturing host cells under conditions suitable for
 expression of the desired interferon polypeptide, and recovering the
 interferon from the cell culture.
 In another embodiment, the invention provides novel, isolated
 interferon-.epsilon. polypeptides. In particular, the invention provides
 isolated a native interferon-.epsilon. polypeptide, which in one
 embodiment, includes an amino acid sequence comprising residues 22 to 189
 of FIG. 1 (SEQ ID NO:1). In another embodiment, the IFN-.epsilon.
 polypeptide has at least about 95% sequence identity with the native human
 IFN-.epsilon. polypeptide specifically disclosed in the present
 application. and preferably retains the pair of cysteine residues at amino
 acid positions 32 and 142. Both glycosylated and unglycosylated forms of
 the IFN-.epsilon. polypeptides are included.
 In another embodiment, the invention provides chimeric molecules
 comprising, a novel interferon-.epsilon. polypeptide herein fused to a
 heterologous polypeptide or amino acid sequence. An example of such a
 chimeric molecule comprises an interferon-.epsilon. polypeptide fused to
 an epitope tag sequence or an immunoglobulin heavy or light chain constant
 region sequence, e.g. the Fc region of an immunoglobulin.
 In another embodiment, the invention provides an antibody which
 specifically binds to a novel interferon-.epsilon. polypeptide disclosed
 herein. Optionally, the antibody is a monoclonal antibody.
 In a further aspect, the present invention concerns compositions comprising
 an effective amount of an IFN-.epsilon. polypeptide, or an agonist
 thereof, in admixture with a pharmaceutically acceptable carrier. The
 composition may, for example, be used for the inhibition of neoplastic
 cell growth, e.g. for the treatment of various tumors, including cancers,
 such as leukemias, AIDS-related Kaposi's sarcoma, etc. In a particular
 embodiment, the composition comprises a cytostatic amount of an
 IFN-.epsilon. polypeptide, or an agonist thereof. In a preferred
 embodiment, the composition comprises a growth inhibitory amount of an
 IFN-.epsilon. polypeptide, or an agonist thereof. In another preferred
 embodiment, the composition comprises a cytotoxic amount of an
 IFN-.epsilon. polypeptide, or an agonist thereof. In yet another preferred
 embodiment, the composition comprises IFN-.epsilon. in an amount capable
 of evoking apoptosis of a target cell. Optionally, the compositions may
 contain one or more additional growth inhibitory and/or cytotoxic and/or
 other chemotherapeutic agents. In a further embodiment, the compositions
 may be used to treat viral infections, such as, the acute viral disease
 zoster, chronic viral infections, e.g. chronic hepatitis non-A, non-B and
 chronic hepatitis B infections, etc. In a still further embodiment, the
 compositions are used to upregulate the immune system.
 In another aspect, the invention concerns a method for inhibiting the
 growth of a tumor cell comprising exposing the cell to an effective amount
 of an IFN-.epsilon. polypeptide, or an agonist thereof. In a particular
 embodiment, the agonist is an anti-IFN-.epsilon. agonist antibody. In
 another embodiment, the agonist is a small molecule that mimics the
 biological activity of a native IFN-.epsilon. polypeptide. The treatment
 may be performed in vitro or in vivo.
 In yet another aspect, the invention concerns a method for treating a viral
 infection comprising administering a therapeutically effective amount of
 an IFN-.epsilon. polypeptide, or an agonist thereof.
 In a further aspect, the invention concerns a method for upregulation of
 the immune system comprising administering a therapeutically effective
 amount of an IFN-.epsilon. polypeptide, or an agonist thereof.
 In a still further embodiment, the invention concerns an article of
 manufacture, comprising:
 a container; and
 a composition comprising an active agent contained within the container;
 wherein the composition is effective for inhibiting neoplastic cell
 growth, e.g. growth of tumor cells, and/or to cause apoptosis of such
 cells, and the active agent in the composition is an IFN-.epsilon.
 polypeptide, or an agonist thereof. In a particular embodiment, the
 aoonist is an anti-IFN-.epsilon. agonist antibody. In another embodiment,
 the agonist is a small molecule that mimics the biological activity of a
 native IFN-.epsilon. polypeptide.
 Similarly, articles of manufacture comprising IFN-.epsilon. in an amount
 effective to treat viral infections and/or to upregulate the immune system
 are within the scope of the invention.
 In a further embodiment, the invention concerns a method for screening
 compounds for anti-tumor activity. In one aspect, the screening assay is
 designed to identify agonists of a native IFN-.epsilon. polypeptide by
 testing the ability of a candidate compound to inhibit the growth of a
 tumor cell the growth of which has been inhibited by a native
 IFN-.epsilon. polypeptide, or a fragment thereof. In another embodiment,
 the screening assay is designed to identify compounds that are capable of
 enhancing the expression level of a native IFN-.epsilon. polypeptide in a
 biological cell sample in which the expression of level of the native
 protein has been determined to be subnormal.
 In yet another embodiment, the invention concerns a method for the
 prognosis or diagnosis of tumor in a mammal, comprising determining in a
 test sample taken from the mammal, the expression level of an
 IFN-.epsilon. polypeptide, and comparing the result with the expression
 level of the same polypeptide in a test sample taken from a healthy mammal
 of the same species. under identical conditions. Subnormal expression of
 any of the IFN-.epsilon. gene may be indicative that the mammal tested has
 a tendency to develop a tumor, or has already developed tumor.
 The invention firther concerns compositions comprising an effective amount
 of an IFN-.epsilon. antagonist, e.g. an antagonist anti-IFN-.epsilon.
 antibody or a small molecule antagonist. Such compositions may be used for
 the treatment of conditions associated with the overexpression of
 IFN-.epsilon.. Without limitation, such conditions include autoimmune
 diseases, such as systemic lupus erythematoses, Behcet's disease, and
 insulin-dependent diabetes mellitus (IDDM, also referred to as type I
 diabetes). Methods for treating such conditions are also within the scope
 of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 1. Definitions
 The terms "interferon-.epsilon. (IFN-.epsilon.)", "IFN-.epsilon.
 polypeptide", "PRO655 polypeptide" and "PRO655" when used herein encompass
 native sequence IFN-.epsilon. and IFN-.epsilon. variants (which are
 further defined herein). The novel IFN-.epsilon. polypeptide, originally
 designated PRO655, may be isolated from a variety of sources, such as from
 human tissue types or from another source, or prepared by recombinant or
 synthetic methiods, or by any combination of these or similar techniques.
 A "native sequence interferon-.epsilon. (IFN-.epsilon.)",or "native
 sequence IFN-.epsilon. polypeptide " or "native sequence PRO655
 polypeptide" or "native sequence PRO655", which terms are used
 interchangeably, comprises a polypeptide having the same amino acid
 sequence as an IFN-.epsilon. polypeptide derived from nature. Such native
 sequence IFN-.epsilon. can be isolated from nature or can be produced by
 recombinant or synthetic means. The term "native sequence IFN-.epsilon."
 specifically encompasses naturally-occurring truncated forms of the
 IFN-.epsilon. polypeptide, naturally-occurring variant forms (e.g.
 alternatively spliced forms) and naturally-occurring allelic variants of
 the native sequence interferon polypeptide herein. In one embodiment of
 the invention, the native sequence IFN-.epsilon. is a mature or
 full-length native sequence IFN-.epsilon. comprising amino acids 22 to 208
 of FIG. 1 (SEQ ID NO:1).
 "IFN-.epsilon. variant" means an active IFN-.epsilon. as defined below
 encoded by a nucleic acid comprising DNA having at least about 80% nucleic
 acid sequence identity to (a) a DNA molecule encoding an IFN-.epsilon.
 polypeptide, with or without its signal sequence, or (b) the complement of
 the DNA molecule of (a). In a particular embodiment, the "IFN-.epsilon.
 variant" has at least about 80% amino acid sequence identity with the
 IFN-.epsilon. having the deduced amino acid sequence shown in FIG. 1 (SEQ
 ID NO:1) for a full-length native sequence IFN-.epsilon.. Such
 IFN-.epsilon. variants include, for instance, IFN-.epsilon. polypeptides
 wherein one or more amino acid residues are added, or deleted at the N- or
 C-terminus of the sequence of FIG. 1 (SEQ ID NO:1). Preferably, the
 nucleic acid or amino acid sequence identity is at least about 85%, more
 preferably at least about 90%, and even more preferably at least about
 95%.
 "Percent (%) amino acid sequence identity" with respect to the
 IFN-.epsilon. sequences identified herein is defined as the percentage of
 amino acid residues in a candidate sequence that are identical with the
 amino acid residues in the IFN-.epsilon. sequence, after aligning the
 sequences and introducing gaps, if necessary, to achieve the maximum
 percent sequence identity, and not considering any conservative
 substitutions as part of the sequence identity. Alignment for purposes of
 determining percent amino acid sequence identity can be achieved in
 various ways that are within the skill in the art, for instance, using
 publicly available computer software such as BLAST, ALIGN or Megalign
 (DNASTAR) software. Those skilled in the art can determine appropriate
 parameters for measuring alignment, including any algorithms needed to
 achieve maximal alignment over the full length of the sequences being
 compared. In a preferred embodiment, alignment is done using the ALIGN
 software.
 "Percent (%) nucleic acid sequence identity" with respect to the
 IFN-.epsilon. coding sequences identified herein is defined as the
 percentage of nucleotides in candidate sequence that are identical with
 the nucleotides in the IFN-.epsilon. coding sequence, after aligning the
 sequences and introducing gaps, if necessary, to achieve the maximum
 percent sequence identity. Alignment for purposes of determining percent
 nucleic acid sequence identity can be achieved in various ways that are
 within the skill in the art, for instance, using publicly available
 computer software such as BLAST, ALIGN or Megalign (DNASTAR) software.
 Those skilled in the art can determine appropriate parameters for
 measuring alignment, including any algorithms needed to achieve maximal
 alignment over the full length of the sequences being compared.
 Preferably, the ALIGN software is used to determine nucleic acid sequence
 identity.
 In a particularly preferred embodiment, percent (%) amino acid sequence
 identity" with respect to the IFN-.epsilon. polypeptides identified herein
 is defined as the percentage of amino acid residues in a candidate
 sequence that are identical with the amino acid residues in the
 IFN-.epsilon. sequence, after aligning the sequences and introducing gaps,
 if necessary, to achieve the maximum percent sequence identity, and not
 considering any conservative substitutions as part of the sequence
 identity. The % identity values used herein are generated by WU-BLAST-2
 which was obtained from [Altschul et al., Methods in Enzymology, 266:
 460-480 (1996); http://blast.wustl/edu/blast/README.html]. WU-BLAST-2 uses
 several search parameters, most of which are set to the default values.
 The adjustable parameters are set with the following values: overlap
 span=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP
 S2 parameters are dynamic values and are established by the program itself
 depending upon the composition of the particular sequence and composition
 of the particular database against which the sequence of interest is being
 searched; however, the values may be adjusted to increase sensitivity. A %
 amino acid sequence identity value is determined by the number of matching
 identical residues divided by the total number of residues of the "longer"
 sequence in the aligned region. The "longer" sequence is the one having
 the most actual residues in the aligned region (gaps introduced by
 WU-Blast-2 to maximize the alignment score are ignored).
 The term "positives", in the context of sequence comparison performed as
 described above. includes residues in the sequences compared that are not
 identical but have similar properties (e.g. as a result of conservative
 substitutions). The % value of positives is determined by the fraction of
 residues scoring a positive value in the BLOSUM 62 matrix divided by the
 total number of residues in the longer sequence, as defined above.
 In a similar manner, in a particularly preferred embodiment, "percent (%)
 nucleic acid sequence identity" with respect to the coding sequence of
 IFN-.epsilon. is defined herein as the percentage of nucleotide residues
 in a candidate sequence that are identical with the nucleotide residues in
 the IFN-.epsilon. coding sequence. The identity values used herein were
 generated by the BLASTN module of WU-BLAST-2 set to the default
 parameters, with overlap span and overlap fraction set to 1 and 0.125,
 respectively.
 "Isolated," when used to describe the various polypeptides disclosed
 herein, means polypeptide that has been identified and separated and/or
 recovered from a component of its natural environment. Contaminant
 components of its natural environment are materials that would typically
 interfere with diagnostic or therapeutic uses for the polypeptide, and may
 include enzymes, hormones, and other proteinaceous or non-proteinaceous
 solutes. In preferred embodiments, the polypeptide will be purified (1) to
 a degree sufficient to obtain at least 15 residues of N-terminal or
 internal amino acid sequence by use of a spinning cup sequenator, or (2)
 to homogeneity by SDS-PAGE under non-reducing or reducing conditions using
 Coomassie blue or, preferably, silver stain. Isolated polypeptide includes
 polypeptide in situ within recombinant cells, since at least one component
 of the IFN-.epsilon. natural environment will not be present. Ordinarily,
 however, isolated polypeptide will be prepared by at least one
 purification step.
 An "isolated" nucleic acid molecule encoding IFN-.epsilon. is a nucleic
 acid molecule that is identified and separated from at least one
 contaminant nucleic acid molecule with which it is ordinarily associated
 in the natural source of the IFN-.epsilon.-encoding nucleic acid. An
 isolated nucleic acid molecule is other than in the form or setting in
 which it is found in nature. Isolated nucleic acid molecules encoding
 IFN-.epsilon. therefore are distinguished from the IFN-.epsilon.-encoding
 nucleic acid molecule as it exists in natural cells. However, an isolated
 nucleic acid molecule encoding IFN-.epsilon. includes nucleic acid
 molecules contained in cells that ordinarily express IFN-.epsilon. where,
 for example, the nucleic acid molecule is in a chromosomal location
 different from that of natural cells.
 The term "control sequences" refers to DNA sequences necessary for the
 expression of an operably linked coding sequence in a particular host
 organism. The control sequences that are suitable for prokaryotes, for
 example, include a promoter, optionally an operator sequence, and a
 ribosome binding site. Eukaryotic cells are known to utilize promoters,
 polyadenylation signals, and enhancers.
 Nucleic acid is "operably linked" when it is placed into a functional
 relationship with another nucleic acid sequence. For example, DNA for a
 presequence or 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. Generally, "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, the synthetic
 oligonucleotide adaptors or linkers are used in accordance with
 conventional practice.
 "Stringency" of hybridization reactions is readily determinable by one of
 ordinary skill in the art, and generally is an empirical calculation
 dependent upon probe length, washing temperature, and salt concentration.
 In general, longer probes require higher temperatures for proper
 annealing, while shorter probes need lower temperatures. Hybridization
 generally depends on the ability of denatured DNA to reanneal when
 complementary strands are present in an environment below their melting
 temperature. The higher the degree of desired homology between the probe
 and hybridizable sequence, the higher the relative temperature which can
 be used. As a result, it follows that higher relative temperatures would
 tend to make the reaction conditions more stringent, while lower
 temperatures less so. For additional details and explanation of stringency
 of hybridization reactions, see Ausubel et al., Current Protocols in
 Molecular Biology, Wiley Interscience Publishers, (1995).
 "Stringent conditions" or "high stringency conditions", as defined herein,
 may be identified by those that: (1) employ low ionic strength and high
 temperature for washing, for example 0.015 M sodium chloride/0.0015 M
 sodium citrate/0.1% sodium dodecyl sulfate at 50.degree. C.; (2) employ
 during hybridization a denaturing agent, such as formamide, for example,
 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
 polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM
 sodium chloride, 75 mM sodium citrate at 42.degree. C.; or (3) employ 50%
 formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate). 50 mM sodium
 phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's
 solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10%
 dextran sulfate at 42.degree. C., with washes at 42.degree. C. in
 0.2.times.SSC (sodium chloride/sodium citrate) and 50% formamide at
 55.degree. C., followed by a high-stringency wash consisting of
 0.1.times.SSC containing EDTA at 55.degree. C.
 "Moderately stringent conditions" may be identified as described by
 Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold
 Spring Harbor Press, 1989, and include the use of washing solution and
 hybridization conditions (e.g., temperature, ionic strength and %SDS) less
 stringent that those described above. An example of moderately stringent
 conditions is overnight incubation at 37.degree. C. in a solution
 comprising: 20% formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium
 citrate), 50 mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution,
 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,
 followed by washing the filters in 1.times.SSC at about 37-50.degree. C.
 The skilled artisan will recognize how to adjust the temperature, ionic
 strength, etc. as necessary to accommodate factors such as probe length
 and the like.
 The term "epitope tagged" when used herein refers to a chimeric polypeptide
 comprising an IFN-.epsilon. polypeptide fused to a "tag polypeptide". The
 tag polypeptide has enough residues to provide an epitope against which an
 antibody can be made, yet is short enough such that it does not interfere
 with activity of the polypeptide to which it is fused. The tag polypeptide
 preferably also is fairly unique so that the antibody does not
 substantially cross-react with other epitopes. Suitable tag polypeptides
 generally have at least six amino acid residues and usually between about
 8 and 50 amino acid residues (preferably, between about 10 and 20 amino
 acid residues).
 "Tumor", as used herein, refers to all neoplastic cell growth and
 proliferation, whether malignant or benign, and all pre-cancerous and
 cancerous cells and tissues.
 The terms "cancer" and "cancerous" refer to or describe the physiological
 condition in mammals that is typically characterized by unregulated cell
 growth. Examples of cancer include but are not limited to, carcinoma,
 lymphoma, blastoma, sarcoma, and leukemia. More particular examples of
 such cancers include breast cancer, prostate cancer, colon cancer,
 squamous cell cancer, small-cell lung cancer, non-small cell lung cancer,
 ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic
 cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal
 cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer,
 vulval cancer, thyroid cancer, hepatic carcinoma and various types of head
 and neck cancer.
 "Treatment" is an intervention performed with the intention of preventing
 the development or altering the pathology of a disorder. Accordingly,
 "treatment" refers to both therapeutic treatment and prophylactic or
 preventative measures. Those in need of treatment include those already
 with the disorder as well as those in which the disorder is to be
 prevented. In tumor (e.g. cancer) treatment, a therapeutic agent may
 directly decrease the pathology of tumor cells, or render the tumor cells
 more susceptible to treatment by other therapeutic agents, e.g. radiation
 and/or chemotherapy. Similarly, in the treatment of virus infections, the
 therapeutic agent may treat the infection directly, or increase the
 efficacy of other antiviral treatments, e.g. by upregulating the immune
 system of the patient.
 "Chronic" administration refers to administration of the agent(s) in a
 continuous mode as opposed to an acute mode, so as to maintain the initial
 biological effect for an extended period of time.
 The "pathology" of cancer includes all phenomena that compromise the
 well-being of the patient. This includes, without limitation, abnormal or
 uncontrollable cell growth, metastasis, interference with the normal
 functioning of neighboring cells, release of cytokines or other secretory
 products at abnormal levels, suppression or aggravation of inflammatory or
 immunological response, etc.
 "Mammal" for purposes of treatment refers to any animal classified as a
 mammal, including humans, domestic and farm animals, and zoo, sports, or
 pet animals, such as horses, sheep, cows, pigs, dogs, cats, etc.
 Preferably, the mammal is human.
 An "effective amount" of an IFN-.epsilon. polypeptide disclosed herein or
 an agonist thereof, in reference to inhibition of neoplastic cell growth,
 is an amount capable of inhibiting, to some extent, the growth of target
 cells. The term includes an amount capable of invoking a growth
 inhibitory, cytostatic and/or cytotoxic effect and/or apoptosis of the
 target cells.
 A "therapeutically effective amount", in reference to the treatment of
 tumor, refers to an amount capable of invoking one or more of the
 following effects: (1) inhibition, to some extent, of tumor growth,
 including, slowing down and complete growth arrest; (2) reduction in the
 number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e.,
 reduction, slowing down or complete stopping) of tumor cell infiltration
 into peripheral organs; (5) inhibition (i.e., reduction, slowing down or
 complete stopping) of metastasis; (6) enhancement of anti-tumor immune
 response, which may, but does not have to, result in the regression or
 rejection of the tumor; and/or (7) relief, to some extent, of one or more
 symptoms associated with the disorder.
 In "effective amount" in the context of antiviral treatment is an amount
 capable of at least partial killing of the target virus population.
 A "therapeutically effective amount" in the context of antiviral activity
 is an amount capable of invoking one or more of the following effects: (1)
 at least partial killing of the virus causing the infection; (2)
 enhancement of anti-viral immune response; (3) relief, to some extent, of
 one or more symptoms associated with the disorder.
 "Carriers" as used herein include pharmaceutically acceptable carriers,
 excipients, or stabilizers which are nontoxic to the cell or mammal being
 exposed thereto at the dosages and concentrations employed. Often the
 physiologically acceptable carrier is an aqueous pH buffered solution.
 Examples of physiologically acceptable carriers include buffers such as
 phosphate, citrate, and other organic acids; antioxidants including
 ascorbic acid; low molecular weight (less than about 10 residues)
 polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins;
 hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as
 glycine, glutamine, asparagine, arginine or lysine; monosaccharides,
 disaccharides, 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.TM., polyethylene glycol (PEG), and
 PLURONICS.TM..
 Administration "in combination with" one or more further therapeutic agents
 includes simultaneous (concurrent) and consecutive administration in any
 order.
 The term "cytotoxic agent" as used herein refers to a substance that
 inhibits or prevents the function of cells and/or causes destruction of
 cells. The term is intended to include radioactive isotopes (e.g.
 I.sup.131, I.sup.125, Y.sup.90 and Re.sup.186), chemotherapeutic agents,
 and toxins such as enzymatically active toxins of bacterial, fungal, plant
 or animal origin, or fragments thereof.
 A "chemotherapeutic agent" is a chemical compound useful in the treatment
 of tumor, e.g. cancer. Examples of chemotherapeutic agents include
 adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside
 ("Ara-C"), cyclophosphamide thiotepa, busulfan, cytoxin, taxoids, e.g.
 paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, N.J.), and
 doxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, Rnace), toxotere,
 methotrexate, cisplatin melphalan, vinblastinc, bleomycin, etoposide,
 ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine,
 carboplatin, teniposide, daunomycin, carminomycin, aminopterin,
 dactinomycin, mitomycins, esperamicins (see U.S. Pat. No. 4,675,187),
 melphalan and other related nitrogen mustards. Also included in this
 definition are hormonal agents that act to regulate or inhibit hormone
 action on tumors such as tamoxifen and onapristone.
 A "growth inhibitory agent" when used herein refers to a compound or
 composition which inhibits growth of a cell, especially tumor, e.g. cancer
 cell, either in vitro or in vivo. Thus, the growth inhibitory agent is one
 which significantly reduces the percentage of the target cells in S phase.
 Examples of growth inhibitory agents include agents that block cell cycle
 progression (at a place other than S phase), such as agents that induce G1
 arrest and M-phase arrest. Classical M-phase blockers include the vincas
 (vincristine and vinblastine), taxol, and topo II inhibitors such as
 doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those
 agents that arrest G1 also spill over into S-phase arrest, for example,
 DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,
 mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
 Further information can be found in The Molecular Basis of Cancer,
 Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation,
 oncogens, and antineoplastic drugs" by Murakami et al. (WB Saunders:
 Philadelphia, 1995), especially p. 13.
 The term "cytokine" is a generic term for proteins released by one cell
 population which act on another cell as intercellular mediators. Examples
 of such cytokines are lymphokines, monokines, and traditional polypeptide
 hormones. Included among the cytokines are growth hormone such as human
 growth hormone, N-methionyl human growth hormone, and bovine growth
 hormnone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;
 prorelaxin; glycoprotein hormones such as follicle stimulating hormone
 (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);
 hepatic growth factor; fibroblast growth factor; prolactin; placental
 lactooen; tumor necrosis factor-.alpha. and -.beta.; mullerian-inhibiting
 substance; mouse gonadotropin-associated peptide; inhibin; activin;
 vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve
 growth factors such as NGF-.beta.; platelet-growth factor; transforming
 growth factors (TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like
 growth factor-I and -II; erythropoietin (EPO); osteoinductive factors;
 interferons such as interferon-.alpha., -.beta., and -.gamma.; colony
 stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
 granulocyte-macrophage-CSF (GM-CSF); and granulocyte-(G-CSF); interleukins
 (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
 IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-.alpha. or
 TNF-.beta.; and other polypeptide factors including LIF and kit ligand
 (KL). As used herein, the term cytokine includes proteins from natural
 sources or from recombinant cell culture and biologically active
 equivalents of the native sequence cytokines.
 The term "prodrug" as used in this application refers to a precursor or
 derivative form of a pharmaceutically active substance that is less
 cytotoxic to tumor cells compared to the parent drug and is capable of
 being enzymatically activated or converted into the more active parent
 form. See, e.g. Wilman, "Prodrugs in Cancer Chemotherapy", Biocheniical
 Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and
 Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery,"
 Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
 (1985). The prodrugs of this invention include, but are not limited to,
 phosphate-containing prodrugs, thiophosphate-containing prodrugs,
 glycosylated prodrugs or optionally substituted phenylacetamide-containing
 prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be
 derivatized into a prodrug form for use in this invention include, but are
 not limited to, those chemotherapeutic agents described above.
 "Active" or "activity" for the purposes herein refers to form(s) of
 IFN-.epsilon. or to other polypeptides (e.g. antibodies) or organic or
 inorganic small molecules, peptides, etc. which retain the biological
 and/or immunological activities/properties of a native or
 naturally-occurring IFN-.epsilon. which retain the biologic and/or
 immunologic activities of native or naturally-occurring IFN-.epsilon.. A
 preferred biological activity is the ability to activate components of the
 JAC-STAT signaling pathway, and typical activities include, but are not
 limited to, antiviral, immunoregulatory or antiproliferative properties.
 "Biological activity" in the context of an antibody or another molecule
 that can be identified by the screening assays disclosed herein (e.g. an
 organic or inorganic small molecule, peptide, etc.) is used to refer to
 the ability of such molecules to invoke one or more of the effects listed
 hereinabove in connection with the definition of a "therapeutically
 effective amount." In a specific embodiment, "biological activity" is the
 ability to inhibit neoplastic cell growth or proliferation. A preferred
 biological activity is inhibition, including slowing or complete stopping,
 of the growth of a target tumor (e.g. cancer) cell. Another preferred
 biological activity is cytotoxic activity resulting in the death of the
 target tumor (e.g. cancer) cell. Yet another preferred biological activity
 is the induction of apoptosis of a target tumor (e.g. cancer) cell. In a
 still further embodiment "biological activity" is an antiviral or
 immunoregulatory activity.
 The phrase "immunological property" means immunological cross-reactivity
 with at least one epitope of an IFN-.epsilon. polypeptide.
 "Immunological cross-reactivity" as used herein means that the candidate
 polypeptide is capable of competitively inhibiting the qualitative
 biological activity of an IFN-.epsilon. polypeptide having this activity
 with polyclonal antisera raised against the known active IFN-.epsilon.
 polypeptide. Such antisera are prepared in conventional fashion by
 injecting goats or rabbits, for example, subcutaneously with the known
 active analogue in complete Freund's adjuvant, followed by booster
 intraperitoneal or subcutaneous injection in incomplete Freunds. The
 immunological cross-reactivity preferably is "specific", which means that
 the binding affinity of the immunologically cross-reactive molecule (e.g.
 antibody) identified, to the IFN-.epsilon. polypeptide is significantly
 higher (preferably at least about 2-times, more preferably at least about
 4-times, even more preferably at least about 6-times, most preferably at
 least about 8-times higher) than the binding affinity of that molecule to
 any other known native polypeptide.
 The term "antagonist" is used in the broadest sense, and includes any
 molecule that partially or fully blocks, inhibits, or neutralizes a
 biological activity of a native IFN-.epsilon. polypeptide disclosed
 herein. In a similar manner, the term "agonist" is used in the broadest
 sense and includes any molecule that mimics a biological activity of a
 native IFN-.epsilon. polypeptide disclosed herein.
 A "small molecule" is defined herein to have a molecular weight below about
 500 daltons.
 The term "antibody" is used in the broadest sense and specifically covers
 single anti-IFN-.epsilon. monoclonal antibodies (including agonist,
 antagonist, and neutralizing antibodies) and anti-IFN-.epsilon. antibody
 compositions with polyepitopic specificity. The term "monoclonal antibody"
 as used herein refers to an antibody obtained from a population of
 substantially homogeneous antibodies, i.e., the individual antibodies
 comprising the population are identical except for possible
 naturally-occurring mutations that may be present in minor amounts.
 "Native antibodies" and "native immunoglobulins" are usually
 heterotetrameric glycoproteins 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 among the heavy chains of different
 immunoglobulin isotypes. Each heavy and light chain also has regularly
 spaced intrachain disulfide bridges. Each heavy chain has at one end a
 variable domain (V.sub.H) followed by a number of constant domains. Each
 light chain has a variable domain at one end (V.sub.L) 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.
 The term "variable" refers to the fact that certain portions of the
 variable domains differ extensively in sequence among antibodies and are
 used in the binding and specificity of each particular antibody for its
 particular antigen. However, the variability is not evenly distributed
 throughout the variable domains of antibodies. It is concentrated in three
 segments called complementarity-determining regions (CDRs) or
 hypervariable regions both in the light-chain and the heavy-chain variable
 domains. The more highly conserved portions of variable domains are called
 the framework (FR). The variable domains of native heavy and light chains
 each comprise four FR regions, largely adopting a .beta.-sheet
 configuration, connected by three CDRs, which form loops connecting, and
 in some cases forming part of, the .beta.-sheet structure. The CDRs in
 each chain are held together in 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 et al., NIH Publ. No.
 91-3242, Vol. I, pages 647-669 (1991)). The constant domains are not
 involved directly in binding an antibody to an antigen, but exhibit
 various effector functions, such as participation of the antibody in
 antibody-dependent cellular toxicity.
 The term "hypervariable region" when used herein refers to the amino acid
 residues of an antibody which are responsible for antigen-binding. The
 hypervariable region comprises amino acid residues from a "complementarity
 determining region" to "CDR" (i.e. residues 24-34 (L1), 50-56 (L2) and
 89-97 (L3) in the light chain variable domain and 31-15 (H1), 50-65 (H2)
 and 95-102 (H3) in the heavy chain variable domain; Kabat et al.,
 Sequences of Proteins of Immunological Interest, 5th Ed. Public Health
 Service, National Institute of Health, Bethesda, Md. [1991]) and/or those
 residues from a "hypervariable loop" (i.e. residues 26-32 (L1), 50-52 (L2)
 and 91-96 (L3) in the light chain variable domain and 26-32 (H1) 53-55
 (H2) and 96-101 (H3) in the heavy chain variable domain; Clothia and Lesk,
 J. Mol. Biol. 196:901-917 [1987]). "Framework" or "FR" residues are those
 variable domain residues other than the hypervariable region residues as
 herein defined.
 "Antibody fragments" comprise a portion of an intact antibody, preferably
 the antigen binding or variable region of the intact antibody. Examples of
 antibody fragments include Fab, Fab', F(ab').sub.2, and Fv fragments;
 diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062
 [1995]); single-chain antibody molecules; and multispecific antibodies
 formed from antibody fragments.
 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').sub.2 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.sub.H -V.sub.L 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 (CH1) 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 CH1 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').sub.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.
 The "light chains" of antibodies (immunoglobulins) from any vertebrate
 species can be assigned to one of two clearly distinct types, called kappa
 (.eta.) and lambda (.lambda.), based on the amino acid sequences of their
 constant domains.
 Depending on the amino acid sequence of the constant domain of their heavy
 chains, inimunoglobulins 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.,
 IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains
 that correspond to the different classes of immunoglobulins are called
 .alpha., .delta., .epsilon., .gamma., and .mu., respectively. The subunit
 structures and three-dimensional configurations of different classes of
 immunoglobulins are well known.
 The term "monoclonal antibody" as used herein refers to an antibody
 obtained from a population of substantially homogeneous antibodies, i.e.,
 the individual antibodies comprising the population are identical except
 for possible naturally occurring mutations that may be present in minor
 amounts. Monoclonal antibodies are highly specific, being directed against
 a single antigenic site. Furthermore, in contrast to conventional
 (polyclonal) antibody preparations which typically include different
 antibodies directed against different determinants (epitopes), each
 monoclonal antibody is directed against a single determinant on the
 antigen. In addition to their specificity, the monoclonal antibodies are
 advantageous in that they are synthesized by the hybridoma culture,
 uncontaminated by other immunoglobulins. 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. For example, the monoclonal antibodies to be used in accordance
 with the present invention may be made by the hybridoma method first
 described by Kohler et al., Nature, 256:495 [1975], or may be made by
 recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The
 "monoclonal antibodies" may also be isolated from phage antibody libraries
 using the techniques described in Clackson et al., Nature, 352:624-628
 [1991] and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
 The monoclonal antibodies herein specifically include "chimeric" antibodies
 (immunoglobulins) in which a portion of the heavy and/or light chain is
 identical with or homologous to corresponding sequences in antibodies
 derived from a particular species or belonging to a particular antibody
 class or subclass, while the remainder of the chain(s) is identical with
 or homologous to corresponding sequences in antibodies derived from
 another species or belonging to another antibody class or subclass, as
 well as fragments of such antibodies, so long as they exhibit the desired
 biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl.
 Acad. Sci. USA, 81:6851-6855 [1984]).
 "Humanized" forms of non-human (e.g., murine) antibodies are chimeric
 immunoglobulins immunoglobulin chains or fragments thereof (such as Fv,
 Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of
 antibodies) which contain minimal sequence derived from non-human
 immunoglobulin. For the most part, humanized antibodies are human
 immunoglobulins (recipient antibody) in which residues from a 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. In some instances, Fv FR residues of
 the human immunoglobulin are replaced by corresponding non-human residues.
 Furthermore, humanized antibodies 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 maximize
 antibody performance. In general the humanized antibody will comprise
 substantially all of at least one, and typically two, variable domains, in
 which all or substantially all of the CDR regions correspond to those of a
 non-human immunoglobulin and all or substantially all of the FR regions
 are those of a human immunoglobulin sequence. The humanized antibody
 optimally also will comprise at least a portion of an immunoglobulin
 constant region (Fc), typically that of a human immunoglobulin. For
 further details, see Jones et al., Nature, 321:522-525 (1986); Reichmann
 et al., Nature, 332:323-329 [1988]; and Presta, Curr. Op. Struct. Biol.,
 2:593-596 (1992). The humanized antibody includes a PRIMATIZED.TM.
 antibody wherein the antigen-binding region of the antibody is derived
 from an antibody produced by immunizing macaque monkeys with the antigen
 of interest.
 "Single-chain Fv" or "sFv" antibody fragments comprise the V.sub.H and
 V.sub.L domains of antibody, wherein these domains are present in a single
 polypeptide chain. Preferably, the Fv polypeptide further comprises a
 polypeptide linker between the V.sub.H and V.sub.L domains which enables
 the sFv to form the desired structure for antigen binding. For a review of
 sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
 Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
 The term "diabodies" refers to small antibody fragments with two
 antigen-binding sites, which fragments comprise a heavy-chain variable
 domain (V.sub.H) connected to a light-chain variable domain (V.sub.L) in
 the same polypeptide chain (V.sub.H -V.sub.L). By using a linker that is
 too short to allow pairing between the two domains on the same chain, the
 domains are forced to pair with the complementary domains of another chain
 and create two antigen-binding sites. Diabodies are described more fully
 in, for example, EP 404,097; WO 93/11161: and Hollinger et al., Proc.
 Natl. Acad. Sci. USA, 90:6444-6448 (1993).
 An "isolated" antibody is one which has been identified and separated
 and/or recovered from a component of its natural environment. Contaminant
 components of its natural environment are materials which would interfere
 with diagnostic or therapeutic uses for the antibody, and may include
 enzymes, hormones, and other nonproteinaceous solutes. In preferred
 embodiments, the antibody will be purified (1) to greater than 95% by
 weight of antibody as determined by the Lowry method, and most preferably
 more than 99% by weight, (2) to a degree sufficient to obtain at least 15
 residues of N-terminal or internal amino acid sequence by use of a
 spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing
 or nonreducing conditions using Coomassie blue or, preferably, silver
 stain. Isolated antibody includes the antibody in situ within recombinant
 cells since at least one component of the antibody's natural environment
 will not be present. Ordinarily, however, isolated antibody will be
 prepared by at least one purification step.
 The word "label" when used herein refers to a detectable compound or
 composition which is conjugated directly or indirectly to the antibody so
 as to generate a "labeled" antibody. The label may be detectable by itself
 (e.g. radioisotope labels or fluorescent labels) or, in the case of an
 enzymatic label, may catalyze chemical alteration of a substrate compound
 or composition which is detectable.
 By "solid phase" is meant a non-aqueous matrix to which the antibody of the
 present invention can adhere. Examples of solid phases encompassed herein
 include those formed partially or entirely of glass (e.g., controlled pore
 glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene,
 polyvinyl alcohol and silicones. In certain embodiments, depending on the
 context, the solid phase can comprise the well of an assay plate; in
 others it is a purification column (e.g., an affinity chromatography
 column). This term also includes a discontinuous solid phase of discrete
 particles, such as those described in U.S. Pat. No. 4,275,149.
 A "liposome" is a small vesicle composed of various types of lipids,
 phospholipids and/or surfactant which is useful for delivery of a drug
 (such as a PRO211, PRO228, PRO538, PRO172, or PRO182 polypeptide or an
 antibody thereto and, optionally, a chemotherapeutic agent) to a mammal.
 The components of the liposome are commonly is arranged in a bilayer
 formation, similar to the lipid arrangement of biological membranes.
 As used herein, the term "immunoadhesin" designates antibody-like molecules
 which combine the binding specificity of a heterologous protein (an
 "adhesin") with the effector functions of immunoglobulin constant domains.
 Structurally, the immunoadhesins comprise a fusion of an amino acid
 sequence with the desired binding specificity which is other than the
 antigen recognition and binding site of an antibody (i.e., is
 "heterologous"), and an immunoglobulin constant domain sequence. The
 adhesin part of an immunoadhesin molecule typically is a contiguous amino
 acid sequence comprising at least the binding site of a receptor or a
 ligand. The immunoglobulin constant domain sequence in the immunoadhesin
 may be obtained from any imnimunoglobulin, such as IgG-1, IgG-2, IgG-3, or
 IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
 2. Compositions and Methods of the Invention
 a. Full-Length human IFN-.epsilon. polypeptide
 The present invention provides newly identified and isolated nucleotide
 sequences encoding novel human interferon polypeptides originally referred
 to as PRO655, and now renamed as "IFN-.epsilon.". In particular,
 Applicants have identified and isolated cDNA encoding a novel polypeptide,
 as disclosed in further detail in the Examples below. Using BLAST and
 FastA sequence alignment computer programs, Applicants found that a
 full-length native sequence PRO655 polypeptide (shown in FIG. 1 and SEQ ID
 NO:1) has about 35-40% amino acid sequence identity with the sequence of
 various human IFN-.alpha. species. Specifically, the sequence identity is
 about 33% and 37% to IFN-.alpha.2 and IFN-.beta., respectively. The
 sequence identity with IFN-.alpha.14 is 38%. The homology is highest
 within the 22-189 amino acid region of the sequence of FIG. 1 (SEQ ID
 NO:1). At the nucleotide level, the sequence identity with the coding
 sequence of IFN-.alpha. is about 60%. Accordingly, we have concluded that
 PRO655 is a newly identified, novel member of the human interferon family
 which may possess antiviral, immunoregulatory and/or antiproliferative
 activities typical of the human interferon family. The relationship of
 this distinct, novel human interferon to some known IFN-.alpha. species
 and IFN-.beta. is illustrated in FIGS. 5 and 7.
 b. IFN-.epsilon. Variants
 In addition to the full-length native sequence IFN-.epsilon. described
 herein, it is contemplated that IFN-.epsilon. variants can be prepared.
 IFN-.epsilon. variants can be prepared by introducing appropriate
 nucleotide changes into the DNA encoding IFN-.epsilon., or by synthesis of
 the desired polypeptide. Those skilled in the art will appreciate that
 amino acid changes may alter post-translational processes of the
 IFN-.epsilon., such as changing the number or position of glycosylation
 sites or altering the membrane anchoring characteristics.
 It is well known that interferons tend to oligomerize. Although the
 etiology of these oligomers is not entire understood, it is believed, that
 certain oligomeric forms result from two or more interferon molecules
 becoming irreversibly associated with one another through intermolecular
 covalent bonding, such as by disulfide linkages. This problems has been
 observed particularly with respect to leukocyte and fibroblast
 interferons. (See, e.g. U.S. Pat. No. 4,816,566) Accordingly, it may be
 desirable to prepare amino acid variants of the native IFN-.epsilon.
 polypeptides of the present invention in which one or more cysteine
 residues are deleted or substituted by residues of other amino acids which
 are incapable of disulfide bond formation. Preferred variants
 substantially retain, mimic or antagonize the biological activity of the
 IFN-.epsilon. from which they are derived. As noted before, the native
 IFN-.epsilon. sequence includes cysteine residues at positions 53, 163 and
 175 in the sequence of FIG. 1 (SEQ ID NO:1). In a preferred embodiment, at
 least one of the cysteine residues at positions 53, 163, and 175 is
 replaced by amino acid residues that are incapable of forming
 intermolecular disulfide bonds.
 Variations in the native full-length sequence IFN-.epsilon. or in various
 domains of the IFN-.epsilon. described herein, can be made, for example,
 using any of the techniques and guidelines for conservative and
 non-conservative mutations set forth, for instance, in U.S. Pat. No.
 5,364,934. Variations may be a substitution, deletion or insertion of one
 or more codons encoding IFN-.epsilon. that results in a change in the
 amino acid sequence of IFN-.epsilon. as compared with the native sequence
 IFN-.epsilon.. Optionally the variation is by substitution of at least one
 amino acid with any other amino acid in one or more of the domains of
 IFN-.epsilon.. Guidance in determining which amino acid residue may be
 inserted, substituted or deleted without adversely affecting the desired
 activity may be found by comparing the sequence of IFN-.epsilon. with that
 of homologous known protein molecules and minimizing the number of amino
 acid sequence changes made in regions of high homology. Amino acid
 substitutions can be the result of replacing one amino acid with another
 amino acid having similar structural and/or chemical properties such as
 the replacement of a leucine with a serine, i.e., conservative amino acid
 replacements. Insertions or deletions may optionally be in the range of 1
 to 5 amino acids. The variation allowed may be determined by
 systematically making insertions, deletions or substitutions of amino
 acids in the sequence and testing the resulting variants for activity in
 the in vitro assay described in the Examples below.
 The variations can be made using methods known in the art such as
 oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,
 and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids
 Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)],
 cassette mutagenesis [Wells et al., Giene, 34:315 (1985)], restriction
 selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA,
 317:415 (1986)] or other known techniques can be performed on the cloned
 DNA to produce the IFN-.epsilon. variant DNA.
 Scanning amino acid analysis can also be employed to identify one or more
 amino acids along a contiguous sequence. Among the preferred scanning
 amino acids are relatively small, neutral amino acids. Such amino acids
 include alanine, glycine, serine, and cysteine. Alanine is typically a
 preferred scanning amino acid among this group because it eliminates the
 side-chain beyond the beta-carbon and is less likely to alter the
 main-chain conformation of the variant. Alanine is also typically
 preferred because it is the most common amino acid. Further, it is
 frequently found in both buried and exposed positions [Creighton, The
 Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1
 (1976)]. If alanine substitution does not yield adequate amounts of
 variant, an isomeric amino acid can be used.
 c. Modifications of IFN-.epsilon.
 Covalent modifications of IFN-.epsilon. are included within the scope of
 this invention. One type of covalent modification includes reacting
 targeted amino acid residues of the IFN-.epsilon. polypeptide with an
 organic derivatizing agent that is capable of reacting with selected side
 chains or the N- or C-terminal residues of IFN-.epsilon.. Derivatization
 with bifunctional agents is useful, for instance, for crosslinking
 IFN-.epsilon. to a water-insoluble support matrix or surface for use in
 the method for purifying anti-IFN-.epsilon. antibodies, and vice-versa.
 Commonly used crosslinking agents include e.g.,
 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide
 esters, for example, esters with 4-azidosalicylic acid, homobifunctional
 imidoesters, including disuccinimidyl esters such as
 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as
 bis-N-maleimido-1,8-octane and agents such as
 methyl-3-[(p-azidophenyl)dithio]propioimidate.
 Other modifications include deamidation of glutaminyl and asparaginyl
 residues to the corresponding glutamyl and aspartyl residues,
 respectively, hydroxylation of proline and lysine, phosphorylation of
 hydroxyl groups of seryl or threonyl residues, methylation of the
 .alpha.-amino groups of lysine, arginine, and histidine side chains [T. E.
 Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &
 Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal
 amine, and amidation of any C-terminal carboxyl group.
 Another type of covalent modification of the IFN-.epsilon. polypeptide
 included within the scope of this invention comprises altering the native
 glycosylation pattern of the polypeptide. "Altering the native
 glycosylation pattern" is intended for purposes herein to mean deleting
 one or more carbohydrate moieties found in native sequence IFN-.epsilon.,
 and/or adding one or more glycosylation sites that are not present in the
 native sequence IFN-.epsilon., and/or altering the nature (profile) of the
 sugar moieties attached to the polypeptide at various glycosylation sites.
 Addition of glycosylation sites to the IFN-.epsilon. polypeptide may be
 accomplished by altering the amino acid sequence. The alteration may be
 made, for example, by the addition of, or substitution by, one or more
 serine or threonine residues to the native sequence IFN-.epsilon. (for
 O-linked glycosylation sites). The IFN-.epsilon. amino acid sequence may
 optionally be altered through changes at the DNA level, particularly by
 mutating the DNA encoding the IFN-.epsilon. polypeptide at preselected
 bases such that codons are generated that will translate into the desired
 amino acids.
 Another means of increasing the number of carbohydrate moieties on the
 IFN-.epsilon. polypeptide is by chemical or enzymatic coupling of
 glycosides to the polypeptide. Such methods are described in the art,
 e.g., in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston,
 CRC Crit. Rev. Biochem., pp. 259-306 (1981).
 Removal of carbohydrate moieties present on the IFN-.epsilon. polypeptide
 may be accomplished chemically or enzymatically or by mutational
 substitution of codons encoding for amino acid residues that serve as
 targets for glycosylation. Chemical deglycosylation techniques are known
 in the art and described, for instance, by Hakimuddin, et al., Arch.
 Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,
 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on
 polypeptides can be achieved by the use of a variety of endo- and
 exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350
 (1987).
 Another type of covalent modification of IFN-.epsilon. comprises linking
 the IFN-.epsilon. polypeptide to one of a variety of nonproteinaceous
 polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or
 polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;
 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. For example,
 PEGylated variants are expected to have a longer half-life and/or shorter
 clearance than the corresponding, non-PEGylated IFN-.epsilon. polypeptide.
 The IFN-.epsilon. molecules of the present invention may also be modified
 in a way to form a chimeric molecule comprising IFN-.epsilon. fused to
 another, heterologous polypeptide or amino acid sequence. In one
 embodiment, such a chimeric molecule comprises a fusion of the
 IFN-.epsilon. with a tag polypeptide which provides an epitope to which an
 anti-tag antibody can selectively bind. The epitope tag is generally
 placed at the amino- or carboxyl-terminus of the IFN-.epsilon.. The
 presence of such epitope-tagged forms of the IFN-.epsilon. can be detected
 using an antibody against the tag polypeptide. Also, provision of the
 epitope tag enables the IFN-.epsilon. to be readily purified by affinity
 purification using an anti-tag antibody or another type of affinity matrix
 that binds to the epitope tag.
 Various tag polypeptides and their respective antibodies are well known in
 the art. Examples include poly-histidine (poly-his) or
 poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and
 its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)];
 the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto
 [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the
 Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et
 al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides
 include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)];
 the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an
 .alpha.-tubulin epitope peptide [Skinner et al., J. Biol. Chem.,
 266:15163-15166 (1991)]; and the T7 gene 10 protein pcptide tag
 [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
 In another embodiment, the chimeric molecule may comprise a fusion of the
 IFN-.epsilon. with an immunoglobulin or a particular region of an
 immunoglobulin. For a bivalent form of the chimeric molecule, such a
 fusion could be to the Fc region of an IgG molecule, to form an
 "immnunoadhesin" as hereinbefore defined. The fusion is preferably to a
 heavy chain constant region sequence, e.g., a hinge, CH2 and CH3 regions,
 or the CH1, hinge, CH2 and CH3 regions of an IgG immunoglobulin.
 Immunoadhesins are expected to have a longer half-life and/or slower
 clearance than the corresponding IFN-.epsilon. polypeptide.
 d. Preparation of IFN-.epsilon.
 The description below relates primarily to production of IFN-.epsilon. by
 culturing cells transformed or transfected with a vector containing
 nucleic acid encoding IFN-.epsilon.. It is, of course, contemplated that
 alternative methods, which are well known in the art, may be employed to
 prepare IFN-.epsilon.. For instance, the IFN-.epsilon. sequence, or
 portions thereof, may be produced by direct peptide synthesis using
 solid-phase techniques [see, e.g., Stewart et al., Solid-Phase Peptide
 Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J.
 Am. Chem. Soc., 85:2149-2154 (1963)]. In vitrio protein synthesis may be
 performed using manual techniques or by automation. Automated synthesis
 may be accomplished, for instance, using an Applied Biosystems Peptide
 Synthesizer (Foster City, Calif.) using manufacturer's instructions.
 Various portions of IFN-.epsilon. may be chemically synthesized separately
 and combined using chemical or enzymatic methods to produce the
 full-length IFN-.epsilon..
 i. Isolation of DNA Encoding IFN-.epsilon.
 DNA encoding IFN-.epsilon. may be obtained from a cDNA library prepared
 from tissue believed to possess the IFN-.epsilon. mRNA and to express it
 at a detectable level. Accordingly, human IFN-.epsilon. DNA can be
 conveniently obtained from a cDNA library prepared from human tissue, such
 as described in the Examples. The IFN-.epsilon.-encoding gene may also be
 obtained from a genomic library or by oligonucleotide synthesis.
 Libraries can be screened with probes (such as antibodies to IFN-.epsilon.
 or oligonucleotides of at least about 20-80 bases) designed to identify
 the gene of interest or the protein encoded by it. Screening the cDNA or
 genomic library with the selected probe may be conducted using standard
 procedures, such as described in Sambrook et al., Molecular Cloning: A
 Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).
 An alternative means to isolate the gene encoding IFN-.epsilon. is to use
 PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A
 Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
 The Examples below describe techniques for screening a cDNA library. The
 oligonucleotide sequences selected as probes should be of sufficient
 length and sufficiently unambiguous that false positives are minimized.
 The oligonucleotide is preferably labeled such that it can be detected
 upon hybridization to DNA in the library being screened. Methods of
 labeling are well known in the art, and include the use of radiolabels
 like .sup.32 P-labeled ATP, biotinylation or enzyme labeling.
 Hybridization conditions, including moderate stringency and high
 stringency, are provided in Sambrook et al., supra.
 Sequences identified in such library screening methods can be compared and
 aligned to other known sequences deposited and available in public
 databases such as GenBank or other private sequence databases. Sequence
 identity (at either the amino acid or nucleotide level) within defined
 regions of the molecule or across the full-length sequence can be
 determined through sequence alignment using computer software programs
 such as ALIGN, DNAstar, and INHERIT which employ various algorithms to
 measure homology.
 Nucleic acid having protein coding sequence may be obtained by screening
 selected cDNA or genomic libraries using the deduced amino acid sequence
 disclosed herein for the first time, and, if necessary, usingy
 conventional primer extension procedures as described in Sambrook et al.,
 supra, to detect precursors and processing intermediates of mRNA that may
 not have been reverse-transcribed into cDNA.
 ii. Selection and Transformation of Host Cells
 Host cells are transfected or transformed with expression or cloning
 vectors described herein for IFN-.epsilon. production and cultured in
 conventional nutrient media modified as appropriate for inducing
 promoters, selecting transformants, or amplifying the genes encoding the
 desired sequences. The culture conditions, such as media, temperature, pH
 and the like, can be selected by the skilled artisan without undue
 experimentation. In general, principles, protocols, and practical
 techniques for maximizing the productivity of cell cultures can be found
 in Mammalian Cell Biotechnology: a Practical Approach, M. Butler. ed. (IRL
 Press, 1991) and Sambrook et al., supra.
 Methods of transfection are known to the ordinarily skilled artisan, for
 example, CaPO.sub.4 and electroporation. Depending on the host cell used,
 transformation is performed using standard techniques appropriate to such
 cells. The calcium treatment employing calcium chloride, as described in
 Sambrook et al., supra, or electroporation is generally used for
 prokaryotes or other cells that contain substantial cell-wall barriers.
 Infection with Agrobacterium tumefaciens is used for transformation of
 certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and
 WO 89/05859 published Jun. 29, 1989. For mammalian cells without such cell
 walls, the calcium phosphate precipitation method of Graham and van der
 Eb, Virology, 52:456-457 (1978) can be employed. General aspects of
 mammalian cell host system transformations have been described in U.S.
 Pat. No. 4,399,216. Transformations into yeast are typically carried out
 according to the method of Van Solingen et al., J. Bact., 130:946 (1977)
 and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However,
 other methods for introducing DNA into cells, such as by nuclear
 microinjection, electroporation, bacterial protoplast fusion with intact
 cells, or polycations, e.g., polybrene, polyornithine, may also be used.
 For various techniques for transforming mammalian cells, see Keown et al.,
 Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature,
 336:348-352 (1988).
 Suitable host cells for cloning or expressing the DNA in the vectors herein
 include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes
 include but are not limited to Eubacteria, such as Gram-negative or
 Gram-positive organisms, for example, Enterobacteriaceae such as E. coli.
 Various E. coli strains are publicly available, such as E. coli K12 strain
 MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
 (ATCC 27,325) and K5 772 (ATCC 53,635).
 In addition to prokaryotes, eukaryotic microbes such as filamentous fungi
 or yeast are suitable cloning or expression hosts for
 IFN-.epsilon.-encoding vectors. Saccharomyces cerevisiae is a commonly
 used lower eukaryotic host microorganism.
 Suitable host cells for the expression of glycosylated IFN-.epsilon. are
 derived from multicellular organisms. Examples of invertebrate cells
 include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as
 plant cells. Examples of useful mammalian host cell lines include Chinese
 hamster ovary (CHO) and COS cells. More specific examples include monkey
 kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651): human
 embryonic kidney line (293 or 293 cells subcloned for growth in suspension
 culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster
 ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,
 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23
 243-251 (1980)); human lung cells (W138, ATCC CCL, 75); human liver cells
 (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). The
 selection of the appropriate host cell is deemed to be within the skill in
 the art.
 iii. Selection and Use of a Replicable Vector
 The nucleic acid (e.g., cDNA or genomic DNA) encoding IFN-.epsilon. may be
 inserted into a replicable vector for cloning (amplification of the DNA)
 or for expression. Various vectors are publicly available. The vector may,
 for example, be in the form of a plasmid, cosmid, viral particle, or
 phage. The appropriate nucleic acid sequence may be inserted into the
 vector by a variety of procedures. In general, DNA is inserted into an
 appropriate restriction endonuclease site(s) using techniques known in the
 art. Vector components generally include, but are not limited to, one or
 more of a signal sequence, an origin of replication, one or more marker
 genes, an enhancer element, a promoter, and a transcription termination
 sequence. Construction of suitable vectors containing one or more of these
 components employs standard ligation techniques which are known to the
 skilled artisan.
 IFN-.epsilon. may be produced recombinantly not only directly, but also as
 a fusion polypeptide with a heterologous polypeptide, which may be a
 signal sequence or other polypeptide having a specific cleavage site at
 the N-terminus of the mature protein or polypeptide. In general, the
 signal sequence may be a component of the vector, or it may be a part of
 the IFN-.epsilon.-encoding DNA that is inserted into the vector. The
 signal sequence may be a prokaryotic signal sequence selected, for
 example, from the group of the alkaline phosphatase, penicillinase, lpp,
 or heat-stable enterotoxin II leaders. For yeast secretion the signal
 sequence may be, e.g., the yeast invertase leader, alpha factor leader
 (including Saccharoimyces and Kluyveroimyces .alpha.-factor leaders, the
 latter described in U.S. Pat. No. 5,010,182), or acid phosphatase leader,
 the C. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990),
 or the signal described in WO 90/13646 published Nov. 15, 1990. In
 mammalian cell expression, mammalian signal sequences may be used to
 direct secretion of the protein, such as signal sequences from secreted
 polypeptides of the same or related species, as well as viral secretory
 leaders.
 Both expression and cloning vectors contain a nucleic acid sequence that
 enables the vector to replicate in one or more selected host cells. Such
 sequences are well known for a variety of bacteria, yeast, and viruses.
 The origin of replication from the plasmid pBR322 is suitable for most
 Gram-negative bacteria, the 2.mu. plasmid origin is suitable for yeast,
 and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are
 useful for cloning vectors in mammalian cells.
 Expression and cloning vectors will typically contain a selection gene,
 also termed a selectable marker. Typical selection genes encode proteins
 that (a) confer resistance to antibiotics or other toxins e.g.,
 ampicillin, neomycin, methotrexate, or tetracycline, (b) complement
 auxotrophic deficiencies, or &ccirc ; supply critical nutrients not
 available from complex media, e.g., the gene encoding D-alanine racemase
 for Bacilli.
 An example of suitable selectable markers for mammalian cells are those
 that enable the identification of cells competent to take up the PRO655
 nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell
 when wild-type DHFR is employed is the CHO cell line deficient in DHFR
 activity, prepared and propagated as described by Urlaub et al., Proc.
 Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in
 yeast is the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et
 al., Nature, 282:39 (1979), Kingsman et al., Gene, 7:141 (1979); Tschemper
 et al., Gene, 10:157 (1980)]. The trp 1 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, Genetics, 85:12 (1977)].
 Expression and cloning vectors usually contain a promoter operably linked
 to the nucleic acid sequence encoding IFN-.epsilon. to direct mRNA
 synthesis. Promoters recognized by a variety of potential host cells are
 well known. Promoters suitable for use with prokaryotic hosts include the
 .beta.-lactamase and lactose promoter systems [Chang et al., Nature,
 275:615 (1978): Goeddel et al., Nature, 281:544 (1979)], alkaline
 phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids
 Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac
 promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].
 Promoters for use in bacterial systems also will contain a Shine-Dalgarno
 (S.D.) sequence operably linked to the DNA encoding IFN-.epsilon..
 Examples of 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., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as
 enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
 decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,
 phosphoglucose isomcrase, and glucokinase.
 Other yeast promoters, which are inducible promoters having the additional
 advantage of transcription controlled by growth conditions, are the
 promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid
 phosphatase, degradative enzymes associated with nitrogen metabolism,
 metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes
 responsible for maltose and galactose utilization. Suitable vectors and
 promoters for use in yeast expression are further described in EP 73,657.
 IFN-.epsilon. transcription from vectors in mammalian host cells is
 controlled, for example, by promoters obtained from the genomes of viruses
 such as polyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5,
 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
 sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian
 Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin
 promoter or an immunoglobulin promoter, and from heat-shock promoters,
 provided such promoters are compatible with the host cell systems.
 Transcription of a DNA encoding the IFN-.epsilon. by higher eukaryotes may
 be increased by inserting an enhancer sequence into the vector. Enhancers
 are cis-acting elements of DNA, usually about from 10 to 300 bp, that act
 on a promoter to increase its transcription. Many enhancer sequences are
 now known from mammalian genes (globin, elastase, albumin,
 .alpha.-fetoprotein, and insulin). Typically, however, one will use 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. The enhancer may
 be spliced into the vector at a position 5' or 3' to the IFN-.epsilon.
 coding sequence, but is preferably located at a site 5' from the promoter.
 Expression vectors used in eukaryotic host cells (yeast, fungi, insect,
 plant, animal, human, or nucleated cells from other multicellular
 organisms) 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 IFN-.epsilon..
 Still other methods, vectors, and host cells suitable for adaptation to the
 synthesis of IFN-.epsilon. in recombinant vertebrate cell culture are
 described in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,
 Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.
 iv. 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. Alternatively, antibodies may be employed that
 can recognize specific duplexes, including DNA duplexes. RNA duplexes, and
 DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn
 may be labeled and the assay may be carried out where the duplex is bound
 to a surface, so that upon the formation of duplex on the surface, the
 presence of antibody bound to the duplex can be detected.
 Gene expression, alternatively, may be measured by immunological methods,
 such as immunohistochemical staining of cells or tissue sections and assay
 of cell culture or body fluids, to quantitate directly the expression of
 gene product. Antibodies useful for immunohistochemical staining and/or
 assay of sample fluids may be either monoclonal or polyclonal, and may be
 prepared in any mammal. Conveniently, the antibodies may be prepared
 against a native sequence IFN-.epsilon. polypeptide or against a synthetic
 peptide based on the DNA sequences provided herein or against exogenous
 sequence fused to IFN-.epsilon. DNA and encoding a specific antibody
 epitope.
 v. Purification of IFN-.epsilon. Polypeptide
 Forms of IFN-.epsilon. may be recovered from culture medium or from host
 cell lysates. If membrane-bound, it can be released from the membrane
 using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic
 cleavage. Cells employed in expression of IFN-.epsilon. can be disrupted
 by various physical or chemical means, such as freeze-thaw cycling,
 sonication, mechanical disruption, or cell lysing agents.
 It may be desired to purify IFN-.epsilon. from recombinant cell proteins or
 polypeptides. The following procedures are exemplary of suitable
 purification procedures: by fractionation on an ion-exchange column;
 ethanol precipitation, reverse phase HPLC; chromatography on silica or on
 a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium
 sulfate precipitation; gel filtration using, for example, Sephadex G-75;
 protein A Sepharose columns to remove contaminants such as IgG; and metal
 chelating columns to bind epitope-tagged forms of the IFN-.epsilon..
 Various methods of protein purification may be employed and such methods
 are known in the art and described for example in Deutscher, Methods in
 Enzymology, 182 (1990); Scopes, Protein Purification: Principles and
 Practice, Springer-Verlag, New York (1982). The purification step(s)
 selected will depend, for example, on the nature of the production process
 used and the particular IFN-.epsilon. produced.
 e. Uses for IFN-.epsilon.
 Nucleotide sequences (or their complement) encoding IFN-.epsilon. have
 various applications in the art of molecular biology, including uses as
 hybridization probes, in chromosome and gene mapping and in the generation
 of anti-sense RNA and DNA. IFN-.epsilon. encoding nucleic acid will also
 be useful for the preparation of IFN-.epsilon. polypeptides by the
 recombinant techniques described herein.
 The full-length native sequence gene encoding IFN-.epsilon. (DNA50960, FIG.
 2, SEQ ID NO:2), or portions thereof, may be used as hybridization probes
 for a cDNA library to isolate the full-length gene or to isolate still
 other genes (for instance, those encoding naturally-occurring variants of
 IFN-.epsilon. or IFN-.epsilon. from other species) which have a desired
 sequence identity to the IFN-.epsilon. sequence disclosed in FIG. 2 (SEQ
 ID NO:2). Optionally, the length of the probes will be about 20 to about
 50 bases. The hybridization probes may be derived from the nucleotide
 sequence of SEQ ID NO:2 or from genomic sequences including promoters.
 enhancer elements and introns of native sequence IFN-.epsilon.. By way of
 example, a screening method will comprise isolating the coding region of
 the IFN-.epsilon. gene using the known DNA sequence to synthesize a
 selected probe of about 40 bases. Hybridization probes may be labeled by a
 variety of labels, including radionucleotides such as .sup.32 P or .sup.35
 S, or enzymatic labels such as alkaline phosphatase coupled to the probe
 via avidin/biotin coupling systems. Labeled probes having a sequence
 complementary to that of the IFN-.epsilon. gene of the present invention
 can be used to screen libraries of human cDNA, genomic DNA or mRNA to
 determine which members of such libraries the probe hybridizes to.
 Hybridization techniques are described in further detail in the Examples
 below.
 The probes may also be employed in PCR techniques to generate a pool of
 sequences for identification of closely related IFN-.epsilon. sequences.
 Nucleotide sequences encoding an IFN-.epsilon. polypeptide can also be used
 to construct hybridization probes for mapping the gene which encodes that
 IFN-.epsilon. and for the genetic analysis of individuals with genetic
 disorders. The nucleotide sequences provided herein may be mapped to a
 chromosome and specific regions of a chromosome using known techniques,
 such as in situ hybridization, linkage analysis against known chromosomal
 markers, and hybridization screening with libraries. Other interferons,
 e.g. IFNs-.alpha.1, .alpha.8, .alpha.10, .alpha.14, .alpha.16, .alpha.21,
 .beta.1, and omega1 have been mapped to Chromosome 9.
 The novel human interferon-.epsilon. (PRO655) can also be used in assays to
 identify and purify its receptor, and to identify other proteins or
 molecules involved in the ligand/receptor binding interaction. By such
 methods, inhibitors of the receptor/ligand binding interaction can be
 identified. Proteins involved in such binding interactions can also be
 used to screen for peptide or small molecule inhibitors or agonists of the
 binding interaction. Screening assays can be designed to find lead
 compounds that mimic the biological activity of a native PRO655 interferon
 or a receptor for PRO655. Such screening assays will include assays
 amenable to high-throughput screening of chemical libraries, making them
 particularly suitable for identifying small molecule drug candidates.
 Small molecules contemplated include synthetic organic or inorganic
 compounds. The assays can be performed in a variety of formats, including
 protein-protein binding assays, biochemical screening assays, immunoassays
 and cell based assays, which are well characterized in the art.
 Nucleic acids which encode IFN-.epsilon. (PRO655) or its modified forms can
 also be used to generate either transgenic animals or "knock out" animals
 which, in turn, are useful in the development and screening of
 therapeutically useful reagents. A transgenic animal (e.g., a mouse or
 rat) is an animal having cells that contain a transgene, which transgene
 was introduced into the animal or an ancestor of the animal at a prenatal,
 e.g., an embryonic stage. A transgene is a DNA which is integrated into
 the genome of a cell from which a transgenic animal develops. In one
 embodiment, cDNA encoding IFN-.epsilon. (PRO655) can be used to clone
 genomic DNA encoding PRO655 in accordance with established techniques and
 the genomic sequences used to generate transgenic animals that contain
 cells which express DNA encoding PRO655. Methods for generating transgenic
 animals, particularly animals such as mice or rats, have become
 conventional in the art and are described, for example, in U.S. Pat. Nos.
 4,736,866 and 4,870,009. Typically, particular cells would be targeted for
 IFN-.epsilon. transgene incorporation with tissue-specific enhancers.
 Transgenic animals that include a copy of a transgene encoding
 IFN-.epsilon. introduced into the germ line of the animal at an embryonic
 stage can be used to examine the effect of increased expression of DNA
 encoding IFN-.epsilon.. Such animals can be used as tester animals for
 reagents thought to confer protection from, for example, pathological
 conditions associated with its overexpression. In accordance with this
 facet of the invention, an animal is treated with the reagent and a
 reduced incidence of the pathological condition, compared to untreated
 animals bearing the transgene, would indicate a potential therapeutic
 intervention for the pathological condition.
 Alternatively, non-human homologues of IFN-.epsilon. (PRO655) can be used
 to construct a IFN-.epsilon. "knock out" animal which has a defective or
 altered gene encoding IFN-.epsilon. as a result of homologous
 recombination between the endogenous gene encoding IFN-.epsilon. and
 altered genomic DNA encoding IFN-.epsilon. introduced into an embryonic
 cell of the animal. For example, cDNA encoding IFN-.epsilon. can be used
 to clone genomic DNA encoding IFN-.epsilon. in accordance with established
 techniques. A portion of the genomic DNA encoding IFN-.epsilon. can be
 deleted or replaced with another gene. such as a gene encoding a
 selectable marker which can be used to monitor integration. Typically,
 several kilobases of unaltered flanking DNA (both at the 5' and 3' ends)
 are included in the vector [see e.g., Thomas and Capecchi. Cell, 51:503
 (1987) for a description of homologous recombination vectors]. The vector
 is introduced into an embryonic stem cell line (e.g., by electroporation)
 and cells in which the introduced DNA has homologously recombined with the
 endogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)].
 The selected cells are then injected into a blastocyst of an animal (e.g.,
 a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in
 Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.
 Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can
 then be implanted into a suitable pseudopregnant female foster animal and
 the embryo brought to term to create a "knock out" animal. Progeny
 harboring the homologously recombined DNA in their germ cells can be
 identified by standard techniques and used to breed animals in which all
 cells of the animal contain the homologously recombined DNA. Knockout
 animals can be characterized for instance, for their ability to defend
 against certain pathological conditions and for their development of
 pathological conditions due to absence of the IFN-.epsilon. (PRO655)
 polypeptide.
 The novel IFN-.epsilon. (PRO655) human interferon polypeptides of the
 present invention have antiviral, antiproliferative and/or
 immunoregulatory activities. Thus, IFN-.epsilon., including variants and
 derivatives of the native protein, may be used for the treatment of
 malignant or non-malignant conditions associated with unwanted cell
 proliferation, or viral diseases. More particularly, IFN-.epsilon. may be
 useful for the treatment of diseases characterized by tumorigenic or
 neoplastic cell growth, malignant hematological systemic diseases, viral
 disease, asthma, carcinomas, sarcomas, myelomas, melanomas, lymphomas,
 papillomas, degenerative diseases, allergic diseases psoriasis and pain.
 Dosages can be calculated based upon the specific activity of
 IFN-.epsilon. as compared to the specific activities of other, known
 interferons, which have been used to treat similar conditions.
 The IFN-.epsilon. polypeptides and their agonists may also be used as
 adjuncts to chemotherapy. It is well understood that chemotherapeutic
 treatment results in suppression of the immune system. Often, although
 successful in destroying the tumor cells against which they are directed,
 chemotherapeutic treatments result in the death of the subject due to such
 side effects of the chemotherapeutic agents. Administration of the
 IFN-.epsilon. polypeptides or their agonists may prevent this side effect
 as a result of their ability to upregulate the subject's immune system. In
 general, patients suffering from immunesuppression due to any underlying
 cause, including HIV infection (or AIDS), may benefit from treatment with
 the IFN-.epsilon. polypeptides or agonist thereof.
 f. Anti-IFN-.epsilon. Antibodies
 The present invention further provides anti-IFN-.epsilon. antibodies.
 Exemplary antibodies include polyclonal, monoclonal, humanized,
 bispecific, and heteroconjugate antibodies.
 i. Polyclonal Antibodies
 The anti-IFN-.epsilon. antibodies may comprise polyclonal antibodies.
 Methods of preparing polyclonal antibodies are known to the skilled
 artisan. Polyclonal antibodies can be raised in a mammal, for example, by
 one or more injections of an immunizing agent and, if desired, an
 adjuvant. Typically, the immunizing agent and/or adjuvant will be injected
 in the mammal by multiple subcutaneous or intraperitoneal injections. The
 immunizing agent may include the IFN-.epsilon. polypeptide or a fusion
 protein thereof. It may be useful to conjugate the immunizing agent to a
 protein known to be immunogenic in the mammal being inmmunized. Examples
 of such imrnunogenic proteins include but are not limited to keyhole
 limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean
 trypsin inhibitor. Examples of adjuvants which may be employed include
 Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,
 synthetic trehalose dicorynomycolate). The immunization protocol may be
 selected by one skilled in the art without undue experimentation.
 ii. Monoclonal Antibodies
 The anti-IFN-.epsilon. antibodies may, alternatively, be monoclonal
 antibodies. Monoclonal antibodies may be prepared using hybridoma methods,
 such as those described by Kohier and Milstein, Nature, 256:495 (1975). In
 a hybridoma method, a mouse, hamster, or other appropriate host animal, is
 typically immunized with an immunizing agent to elicit lymphocytes that
 produce or are capable of producing antibodies that will specifically bind
 to the immunizing agent. Alternatively, the lymphocytes may be immunized
 in vitro.
 The immunizing agent will typically include the anti-IFN-.epsilon.
 polypeptide or a fusion protein thereof. Generally, either peripheral
 blood lymphocytes ("PBLs") are used if cells of human origin are desired,
 or spleen cells or lymph node cells are used if non-human mammalian
 sources are desired. The lymphocytes are then fused with an immortalized
 cell line using a suitable fusing agent, such as polyethylene glycol, to
 form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and
 Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are
 usually transformed mammalian cells, particularly myeloma cells of rodent,
 bovine and human origin. Usually, rat or mouse myeloma cell lines are
 employed. The hybridoma cells may be cultured in a suitable culture medium
 that preferably contains one or more substances that inhibit the growth or
 survival of the unfused, immortalized cells. For example, if the parental
 cells lack the enzyme hypoxanthine guanine phosphoribosyl transterase
 (HGPRT or HPRT), the culture medium for the hybridomas typically will
 include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which
 substances prevent the growth of HGPRT-deficient cells.
 Preferred immortalized cell lines 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. More preferred immortalized cell lines are murine myeloma lines,
 which can be obtained, for instance, from the Salk Institute Cell
 Distribution Center, San Diego, Calif., and the American Type Culture
 Collection, Rockville, Md. 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 et al.,
 Monoclonal Antibody Production Techniques and Applications, Marcel Dekker,
 Inc., New York, (1987) pp. 51-63].
 The culture medium in which the hybridoma cells are cultured can then be
 assayed for the presence of monoclonal antibodies directed against
 anti-IFN-.epsilon.. Preferably, the binding specificity of monoclonal
 antibodies produced by the hybridoma cells is determined by
 immunoprecipitation or by an in vitro binding assay, such as
 radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
 Such techniques and assays are known in the art. The binding affinity of
 the monoclonal antibody can, for example, be determined by the Scatchard
 analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
 After the desired hybridoma cells are identified, the clones may be
 subcloned by limiting dilution procedures and grown by standard methods
 [Goding, supra]. Suitable culture media for this purpose include, for
 example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
 Alternatively, the hybridoma cells may be grown in vivo as ascites in a
 mammal.
 The monoclonal antibodies secreted by the subclones may be isolated or
 purified from the culture medium or ascites fluid by conventional
 immunoglobulin purification procedures such as, for example, protein
 A-Sepharose, hydroxylapatite chromatography, gel electrophoresis,
 dialysis, or affinity chromatography.
 The monoclonal antibodies may also be made by recombinant DNA methods, such
 as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal
 antibodies of the invention can be readily isolated and sequenced using
 conventional procedures (e.g., by using oligonucleotide probes that are
 capable of binding specifically to genes encoding the heavy and light
 chains of murine antibodies). The hybridoma cells of the invention serve
 as a preferred source of such DNA. Once isolated, the DNA may be placed
 into expression vectors, which are then transfected into host cells such
 as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells
 that do not otherwise produce immunoglobulin protein, to obtain the
 synthesis of monoclonal antibodies in the recombinant host cells. The DNA
 also may be modified, for example, by substituting the coding sequence for
 human heavy and light chain constant domains in place of the homologous
 murine sequences [U.S. Pat. No. 4,816,567; Morrison et al., supra] or by
 covalently oining to the immunoglobulin coding sequence all or part of the
 coding sequence for a non-immunoglobulin polypeptide. Such a
 non-immunoglobulin polypeptide can be substituted for the constant domains
 of an antibody of the invention, or can be substituted for the variable
 domains of one antigen-combining site of an antibody of the invention to
 create a chimeric bivalent antibody.
 The antibodies may be monovalent antibodies. Methods for preparing
 monovalent antibodies are well known in the art. For example, one method
 involves recombinant expression of immunoglobulin light chain and modified
 heavy chain. The heavy chain is truncated generally at any point in the Fc
 region so as to prevent heavy chain crosslinking. Alternatively, the
 relevant cysteine residues are substituted with another amino acid residue
 or are deleted so as to prevent crosslinking.
 In vitro methods are also suitable for preparing monovalent antibodies.
 Digestion of antibodies to produce fragments thereof, particularly, Fab
 fragments, can be accomplished using routine techniques known in the art.
 iii. Humanized and Human Antibodies
 The anti-IFN-.epsilon. antibodies of the invention may further comprise
 humanized antibodies or human antibodies. Humanized forms of non-human
 (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin
 chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
 antigen-binding subsequences of antibodies) which contain minimal sequence
 derived from non-human immunoglobulin. Humanized antibodies include 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. In
 some instances, Fv framework residues of the human immunoglobulin are
 replaced by corresponding non-human residues. Humanized antibodies may
 also comprise residues which are found neither in the recipient antibody
 nor in the imported CDR or framework sequences. In general, the humanized
 antibody will comprise substantially all of at least one, and typically
 two, variable domains, in which all or substantially all of the CDR
 regions correspond to those of a non-human immunoglobulin and all or
 substantially all of the FR regions are those of a human immunoglobulin
 consensus sequence. The humanized antibody optimally also will comprise at
 least a portion of an immunoglobulin constant region (Fc), typically that
 of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986);
 Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op.
 Struct. Biol., 2:593-596 (1992)].
 Methods for humanizing non-human antibodies are well known in the art.
 Generally, a humanized antibody has one or more amino acid residues
 introduced into it from a source which is non-human. These 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. Accordingly, such "humanized" antibodies are chimeric antibodies
 (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human
 variable domain has been substituted by the corresponding sequence from a
 non-human species. In practice, 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.
 Human antibodies can also be produced using various techniques known in the
 art, including phage display libraries [Hoogenboom and Winter, J. Mol.
 Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The
 techniques of Cole et al. and Boerner et al. are also available for the
 preparation of human monoclonal antibodies (Cole et al., Monoclonal
 Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et
 al., J. Immunol., 147(1):86-95 (1991)].
 iv. Bispecific Antibodies
 Bispecific antibodies are monoclonal, preferably human or humanized,
 antibodies that have binding specificities for at least two different
 antigens. In the present case, one of the binding specificities is for the
 IFN-.epsilon., the other one is for any other antigen, and preferably for
 a cell-surface protein or receptor or receptor subunit. In a further
 embodiment, one specificity is for IFN-.epsilon., while the other
 specificity is for type I interferon, preferably IFN-.alpha..
 Methods for making bispecific antibodies are known in the art.
 Traditionally, the recombinant production of bispecific antibodies is
 based on the co-expression of two immnunoglobulin heavy-chain/light-chain
 pairs, where the two heavy chains have different specificities [Milstein
 and Cuello, Nature, 305:537-539 (1983)]. Because of the random assortment
 of immunoglobulin heavy and light chains, these hybridomas (quadromas)
 produce a potential mixture of ten different antibody molecules, of which
 only one has the correct bispecific structure. The purification of the
 correct molecule is usually accomplished by affinity chromatography steps.
 Similar procedures are disclosed in WO 93/08829, published May 13, 1993,
 and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
 Antibody variable domains with the desired binding specificities
 (antibody-antigen combining sites) can be fused to immunoglobulin constant
 domain sequences. The fusion preferably is with an immunoglobulin
 heavy-chain constant domain, comprising at least part of the hinge, CH2,
 and CH3 regions. It is preferred to have the first heavy-chain constant
 region (CH1) containing the site necessary for light-chain binding present
 in at least one of the fusions. DNAs encoding the immunoglobulin
 heavy-chain fusions and, if desired, the immunoglobulin light chain, are
 inserted into separate expression vectors, and are co-transfected into a
 suitable host organism. For further details of generating bispecific
 antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210
 (1986).
 v. Heteroconjugate Antibodies
 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. Pat. No. 4,676,980],
 and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089].
 It is contemplated that the antibodies may be prepared in vitro using
 known methods in synthetic protein chemistry, including those involving
 crosslinking agents. For example, imnmunotoxins may be constructed using a
 disulfide exchange reaction or by forming a thioether bond. Examples of
 suitable reagents for this purpose include iminothiolate and
 methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
 Pat. No. 4,676,980.
 6. Effector Function Engineering
 It may be desirable to modify the antibody of the invention with respect to
 effector function, so as to enhance the effectiveness of the antibody in
 treating cancer, for example. For example cysteine residue(s) may be
 introduced in the Fc region, thereby allowing interchain disulfide bond
 formation in this region. The homodimeric antibody thus generated may have
 improved internalization capability and/or increased complement-mediated
 cell killing and antibody-dependent cellular cytotoxicity (ADCC). See
 Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.
 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor
 activity may also be prepared using heterobifunctional cross-linkers as
 described in Wolff et al. Cancer Research 53 :2560-2565 (1993).
 Alternatively, an antibody can be engineered which has dual Fc regions and
 may thereby have enhanced complement lysis and ADCC capabilities. See
 Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).
 7. Immunoconjugates
 The invention also pertains to immunoconjugates comprising an antibody
 conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin
 (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal
 origin, or fragments thereof), or a radioactive isotope (i.e., a
 radioconjugate).
 Chemotherapeutic agents useful in the generation of such immunoconjugates
 have been described above. Enzymatically active toxins and fragments
 thereof which can be used include diphtheria A chain, nonbinding active
 fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas
 aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
 Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins
 (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,
 sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,
 phenomycin, enomycin and the tricothecenes. A variety of radionuclides are
 available for the production of radioconjugated antibodies. Examples
 include .sup.212 Bi, .sup.131 I, .sup.131 In, .sup.90 Y, and .sup.186 Re.
 Conjugates of the antibody and cytotoxic agent are made using a variety of
 bifunctional protein coupling agents such as
 N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT),
 bifunctional derivatives of imidoesters (such as dimethyl adipimidate
 HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as
 glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)
 hexanediamine), bis-diazonium derivatives (such as
 bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene
 2,6-diisocyanate), and bis-active fluorine compounds (such as
 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be
 prepared as described in Vitetta et al., Science 238: 1098 (1987).
 Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
 triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for
 conjugation of radionucleotide to the antibody. See WO 94/11026.
 In another embodiment, the antibody may be conjugated to a "receptor" (such
 streptavidin) for utilization in tumor pretargeting wherein the
 antibody-receptor conjugate is administered to the patient, followed by
 removal of unbound conjugate from the circulation using a clearing agent
 and then administration of a "ligand" (e.g. avidin) which is conjugated to
 a cytotoxic agent (e.g. a radionucleotide).
 8. Immunoliposomes
 The antibodies disclosed herein may also be forrnulated as immunoliposomes.
 Liposomes containing the antibody are prepared by methods known in the
 art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA,
 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980);
 and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced
 circulation time are disclosed in U.S. Pat. No. 5,013,556.
 Particularly useful liposomes can be generated by the reverse phase
 evaporation method with a lipid composition comprising
 phosphatidylcholine, cholesterol and PEG-derivatized
 phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters
 of defined pore size to yield liposomes with the desired diameter. Fab'
 fragments of the antibody of the present invention can be conjugated to
 the liposomes as described in Martin et al., J. Biol. Chem. 257: 286-288
 (1982) via a disulfide interchange reaction. A chemotherapeutic agent
 (such as Doxorubicin) is optionally contained within the liposome. See
 Gabizon et al., J. National Cancer Inst. 81(19)1484 (1989).
 g. Uses for anti-IFN-.epsilon. Antibodies
 The anti-IFN-.epsilon. antibodies of the invention have various utilities.
 For example, anti-IFN-.epsilon. antibodies may be used in diagnostic
 assays for IFN-.epsilon., e.g., detecting its expression in specific
 cells, tissues, or serum. Various diagnostic assay techniques known in the
 art may be used, such as competitive binding assays, direct or indirect
 sandwich assays and immunoprecipitation assays conducted in either
 heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual
 of Techniques, CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in
 the diagnostic assays can be labeled with a detectable moiety. The
 detectable moiety should be capable of producing, either directly or
 indirectly, a detectable signal. For examnple, the detectable moiety may
 be a radioisotope, such as .sup.3 H, .sup.14 C, .sup.32 P, .sup.35 S, or
 .sup.125 I, a fluorescent or chemiluminescent compound, such as
 fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as
 alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any
 method known in the art for 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., 40:219 (1981); and Nygren, J. Histochem.
 and Cytochem., 30:407 (1982).
 Anti-IFN-.epsilon. antibodies also are useful for the affinity purification
 of IFN-.epsilon. from recombinant cell culture or natural sources. In this
 process, the antibodies against IFN-.epsilon. are immobilized on a
 suitable support, such a Sephadex resin or filter paper, using methods
 well known in the art. The immobilized antibody then is contacted with a
 sample containing the IFN-.epsilon. to be purified, and thereafter the
 support is washed with a suitable solvent that will remove substantially
 all the material in the sample except the IFN-.epsilon., which is bound to
 the immobilized antibody. Finally, the support is washed with another
 suitable solvent that will release the IFN-.epsilon. from the antibody.
 Agonist antibodies specifically binding IFN-.epsilon. mimic its biological
 activities and thus are potentially useful for the treatment of the same
 or similar conditions as those targeted by IFN-.epsilon. itself. Such
 conditions include, for example, neoplastic cell growth, e.g. tumor
 (including cancer), viral infections, autoimmune diseases and, in general,
 conditions where the upregulation of the immune system is desirable.
 Antagonist antibodies specifically binding IFN-.epsilon. may be used to
 treat conditions associated with the overexpression of IFN-.epsilon.. Such
 conditions might include various autoimmune diseases, such as systemic
 lupus erythematoses, Behcet's disease, and insulin-dependent diabetes
 mellitus (IDDM, also referred to as type I diabetes).
 H. Animal Models for Testing Anti-Tumor Activity
 A variety of well known animal models can be used to further understand the
 role of IFN-.epsilon. in the development and pathogenesis of tumors, and
 to test the efficacy of candidate therapeutic agents, including
 antibodies, and other agonists of the native polypeptide, including small
 molecule agonists. The in vivo nature of such models makes them
 particularly predictive of responses in human patients. Animal models of
 tumors and cancers (e.g. breast cancer, colon cancer, prostate cancer,
 lung cancer, etc.) include both non-recombinant and recombinant
 (transgenic) animals. Non-recombinant animal models include, for example,
 rodent, e.g., murine models. Such models can be generated by introducing
 tumor cells into syngeneic mice using standard techniques, e.g.
 subcutaneous injection, tail vein injection, spleen implantation,
 intraperitoneal implantation, implantation under the renal capsule, or
 orthopin implantation, e.g. colon cancer cells implanted in colonic
 tissue. (See, e.g. PCT publication No. WO 97/33551, published Sep. 18,
 1997).
 Probably the most often used animal species in oncological studies are
 immunodeficient mice and, in particular, nude mice. The observation that
 the nude mouse with hypo/aplasia could successfully act as a host for
 human tumor xenografts has lead to its widespread use for this purpose.
 The autosomal recessive nu gene has been introduced into a very large
 number of distinct congenic strains of nude mouse, including, for example,
 ASW, A/He, AKR, BALB/c, B10,LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st,
 NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL. In addition, a wide
 variety of other animals with inherited immunological defects other than
 the nude mouse have been bred and used as recipients of tumor xenografts.
 For further details see, e.g. The Nude Mouse in Oncology Research, E.
 Boven and B. Winograd, eds., CRC Press, Inc., 1991.
 The cells introduced into such animals can be derived from known
 tumor/cancer cell lines, such as, any of the above-listed tumor cell
 lines, and, for example, the B104-1-1 cell line (stable NIH-3T3 cell line
 transfected with the neu protooncogene); ras-transfected NIH-3T3 cells;
 Caco-2 (ATCC HTB-37); a moderately well-differentiated grade II human
 colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or from tumors and
 cancers. Samples of tumor or cancer cells can be obtained from patients
 undergoing surgery, using standard conditions, involving freezing and
 storing in liquid nitrogen (Karmali et al., Br. J. Cancer 48, 689-696
 [1983]).
 Tumor cells can be introduced into animals, such as nude mice, by a variety
 of procedures. The subcutaneous (s.c.) space in mice is very suitable for
 tumor implantation. Tumors can be transplanted s.c. as solid blocks, as
 needle biopsies by use of a trochar, or as cell suspensions. For solid
 block or trochar implantation, tumor tissue fragments of suitable size are
 introduced into the s.c. space. Cell suspensions are freshly prepared from
 primary tumors or stable tumor cell lines, and injected subcutaneously.
 Tumor cells can also be injected as subdermal implants. In this location,
 the inoculum is deposited between the lower part of the dermal connective
 tissue and the s.c. tissue. Boven and Winograd (1991), supra.
 Animal models of breast cancer can be generated, for example, by implanting
 rat neuroblastoma cells (from which the neu oncogen was initially
 isolated), or neu-transformed NIH-3T3 cells into nude mice, essentially as
 described by Drebin et al. PNAS USA 83, 9129-9133 (1986).
 Similarly, animal models of colon cancer can be generated by passaging
 colon cancer cells in animals, e.g. nude mice, leading to the appearance
 of tumors in these animals. An orthotopic transplant model of human colon
 cancer in nude mice has been described, for example, by Wang et al.,
 Cancer Research 54, 4726-4728 (1994) and Too et al., Cancer Research 55,
 681-684 (1995). This model is based on the so-called "METAMOUSE" sold by
 AntiCancer, Inc. (San Diego. Calif.).
 Tumors that arise in animals can be removed and cultured in vitro. Cells
 from the in vitro cultures can then be passaged to animals. Such tumors
 can serve as targets for further testing or drug screening. Alternatively,
 the tumors resulting from the passage can be isolated and RNA from
 pre-passage cells and cells isolated after one or more rounds of passage
 analyzed for differential expression of genes of interest. Such passaging
 techniques can be performed with any known tumor or cancer cell lines.
 For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 are chemically induced
 fibrosarcomas of BALB/c female mice (DeLeo et al., J. Exp. Med. 146, 720
 [1977]), which provide a highly controllable model system for studying the
 anti-tumor activities of various agents (Palladino et al., J. Immunol.
 138, 4023-4032 [1987]). Briefly, tumor cells are propagated in vitro in
 cell culture. Prior to injection into the animals, the cell lines are
 washed and suspended in buffer, at a cell density of about
 10.times.10.sup.6 to 10.times.10.sup.7 cells/ml. The animals are then
 infected subcutaneously with 10 to 100 .mu.l of the cell suspension
 allowing one to three weeks for a tumor to appear.
 In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the
 most thoroughly studied experimental tumors, can be used as an
 investigational tumor model. Efficacy in this tumor model has been
 correlated with beneficial effects in the treatment of human patients
 diagnosed with small cell carcinoma of the lung (SCCL). This tumor can be
 introduced in normal mice upon injection of tumor fragments from an
 affected mouse or of cells maintained in culture (Zupi et al., Br. J.
 Cancer 41, suppl. 4, 309 [1980]), and evidence indicates that tumors can
 be started from injection of even a single cell and that a very high
 proportion of infected tumor cells survive. For further information about
 this tumor model see Zacharski, Haemostasis 16, 300-320 [1986]).
 One way of evaluating the efficacy of a test compound in an animal model is
 implanted tumor is to measure the size of the tumor before and after
 treatment. Traditionally, the size of implanted tumors has been measured
 with a slide caliper in two or three dimensions. The measure limited to
 two dimensions does not accurately reflect the size of the tumor,
 therefore, it is usually converted into the corresponding volume by using
 a mathematical formula. However, the measurement of tumor size is very
 inaccurate. The therapeutic effects of a drug candidate can be better
 described as treatment-induced growth delay and specific growth delay.
 Another important variable in the description of tumor growth is the tumor
 volume doubling time. Computer programs for the calculation and
 description of tumor growth are also available, such as the program
 reported by Rygaard and Spang-Thomsen, Proc. 6th Int. Workshop on
 Immune-Deficieni Animals, Wu and Sheng eds., Basel, 1989, 301. It is
 noted, however, that necrosis and inflammatory responses following
 treatment may actually result in an increase in tumor size, at least
 initially. Therefore, these changes need to be carefully monitored, by a
 combination of a morphometric method and flow cytometric analysis.
 Recombinant (transgenic) animal models can be engineered by introducing the
 coding portion of the genes identified herein into the genome of animals
 of interest, using standard techniques for producing transgenic animals.
 Animals that can serve as a target for transgenic manipulation include,
 without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs,
 and non-human primates, e.g. baboons, chimpanzees and monkeys. Techniques
 known in the art to introduce a transgene into such animals include
 pronucleic microinjection (Hoppe and Wanger, U.S. Pat. No. 4,873,191);
 retrovirus-mediated gene transfer into germ lines (e.g., Van der Putten et
 al., Proc. Natl. Acad. Sci. USA 82, 6148-615 [1985]); gene targeting in
 embryonic stem cells (Thompson et al., Cell 56, 313-321 [1989]);
 electroporation of embryos (Lo, Mol. Cel. Biol. 3, 1803-1814 [1983]);
 sperm-mediated gene transfer (Lavitrano et al., Cell 57, 717-73 [1989]).
 For review, see, for example, U.S. Pat. No. 4,736,866.
 For the purpose of the present invention, transgenic animals include those
 that carry the transgene only in part of their cells ("mosaic animals").
 The transgene can be integrated either as a single transgene, or in
 concatamers, e.g., head-to-head or head-to-tail tandems. Selective
 introduction of a transgene into a particular cell type is also possible
 by following, for example, the technique of Lasko et al., Proc. Natl.
 Acad. Sci. USA 89, 6232-636 (1992).
 The expression of the transgene in transgenic animals can be monitored by
 standard techniques. For example, Southern blot analysis or PCR
 amplification can be used to verify the integration of the transgene. The
 level of mRNA expression can then be analyzed using techniques such as in
 situ hybridization, Northern blot analysis, PCR, or immunocytochemistry.
 The animals are further examined for signs of tumor or cancer development.
 The efficacy of IFN-.epsilon., antibodies specifically binding
 IFN-.epsilon. and other drug candidates, can be tested also in the
 treatment of spontaneous animal tumors. A suitable target for such studies
 is the feline oral squamous cell carcinoma (SCC). Feline oral SCC is a
 highly invasive, malignant tumor that is the most common oral malignancy
 of cats, accounting for over 60% of the oral tumors reported in this
 species. It rarely metastasizes to distant sites, although this low
 incidence of metastasis may merely be a reflection of the short survival
 times for cats with this tumor. These tumors are usually not amenable to
 surgery, primarily because of the anatomy of the feline oral cavity. At
 present, there is no effective treatment for this tumor. Prior to entry
 into the study, each cat undergoes complete clinical examination, biopsy,
 and is scanned by computed tomography (CT). Cats diagnosed with sublingual
 oral squamous cell tumors are excluded from the study. The tongue can
 become paralyzed as a result of such tumor, and even if the treatment
 kills the tumor, the animals may not be able to feed themselves. Each cat
 is treated repeatedly, over a longer period of time. Photographs of the
 tumors will be taken daily during the treatment period, and at each
 subsequent recheck. After treatment, each cat undergoes another CT scan.
 CT scans and thoracic radiograms are evaluated every 8 weeks thereafter.
 The data are evaluated for differences in survival, response and toxicity
 as compared to control groups. Positive response may require evidence of
 tumor regression, preferably with improvement of quality of life and/or
 increased life span.
 In addition, other spontaneous animal tumors, such as fibrosarcoma,
 adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma of dogs, cats, and
 baboons can also be tested. Of these mammary adenocarcinoma in dogs and
 cats is a preferred model as its appearance and behavior are very similar
 to those in humans. However, the use of this model is limited by the rare
 occurrence of this type of tumor in animals.
 I. Screening Assays for Drug Candidates
 Screening assays for drug candidates are designed to identify compounds
 that competitively bind or complex with the receptor(s) of IFN-.epsilon.,
 and signal through such receptor(s) (e.g. IFN-.alpha.R, including both
 subunits, and any other receptor that might be identified hereinafter as
 being involved in IFN-.epsilon. signal transduction). Such screening
 assays will include assays amenable to high-throughput screening of
 chemical libraries, making them particularly suitable for identifying
 small molecule drug candidates. Small molecules contemplated include
 synthetic organic or inorganic compounds, including peptides, preferably
 soluble peptides, (poly)peptide-immunoglobulin fusions, and, in
 particular, antibodies including, without limitation, poly- and monoclonal
 antibodies and antibody fragments, single-chain antibodies, anti-idiotypic
 antibodies, and chimeric or humanized versions of such antibodies or
 fragments, as well as human antibodies and antibody fragments. The assays
 can be performed in a variety of formats, including protein-protein
 binding assays, biochemical screening assays, immunoassays and cell based
 assays, which are well characterized in the art.
 In binding assays, the interaction is binding and the complex formed can be
 isolated or detected in the reaction mixture. In a particular embodiment,
 a receptor of a polypeptide encoded by the gene identified herein or the
 drug candidate is immobilized on a solid phase, e.g. on a microtiter
 plate, by covalent or non-covalent attachments. Non-covalent attachment
 generally is accomplished by coating the solid surface with a solution of
 the polypeptide and drying. Alternatively, an immobilized antibody, e.g. a
 monoclonal antibody, specific for the polypeptide to be immobilized can be
 used to anchor it to a solid surface. The assay is performed by adding the
 non-immobilized component, which may be labeled by a detectable label, to
 the immobilized component, e.g. the coated surface containing the anchored
 component. When the reaction is complete, the non-reacted components are
 removed e.g. by washing, and complexes anchored on the solid surface are
 detected. When the originally non-immobilized component carries a
 detectable label, the detection of label immobilized on the surface
 indicates that complexing occurred. Where the originally non-immobilized
 component does not carry a label, complexing can be detected, for example,
 by using a labeled antibody specifically binding the immobilized complex.
 If the candidate compound interacts with but does not bind to the
 IFN-.epsilon. receptor, its interaction with the receptor can be assayed
 by methods well known for detecting protein-protein interactions. Such
 assays include traditional approaches, such as, cross-linking,
 co-immunoprecipitation, and co-purification through gradients or
 chromatographic columns. In addition, protein-protein interactions can be
 monitored by using a yeast-based genetic system described by Fields and
 co-workers [Fields and Song, Nature (London) 340, 245-246 (1989); Chien et
 al., Proc. Natl. Acad. Sci. USA 88, 9578-9582 (1991)] as disclosed by
 Chevray and Nathans [Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1991)].
 Many transcriptional activators, such as yeast GAL4, consist of two
 physically discrete modular domains, one acting as the DNA-binding domain,
 while the other one functioning as the transcription activation domain.
 The yeast expression system described in the foregoing publications
 (generally referred to as the "two-hybrid system") takes advantage of this
 property, and employs two hybrid proteins, one in which the target protein
 is fused to the DNA-binding domain of GAL4, and another, in which
 candidate activating proteins are fused to the activation domain. The
 expression of a GAL1-lacZ reporter gene under control of a GAL4-activated
 promoter depends on reconstitution of GAL4 activity via protein-protein
 interaction. Colonies containing interacting polypeptides are detected
 with a chromogenic substrate for .beta.-galactosidase. A complete kit
 (MATCHMAKER.TM.) for identifying protein-protein interactions between two
 specific proteins using the two-hybrid technique is commercially available
 from Clontech. This system can also be extended to map protein domains
 involved in specific protein interactions as well as to pinpoint amino
 acid residues that are crucial for these interactions.
 Methods to screen potential agents for their ability to inhibit neoplastic
 cell growth can be designed without detailed knowledge of the precise
 mechanism, although the knowledge of such mechanism may certainly be
 helpful. For example, after it has been determined that neoplastic cell
 growth (e.g. tumor growth) is correlated wit subnormal expression (or
 activity) of a gene identified herein, agents can be screened for their
 ability to increase such gene expression and/or restore normal activity.
 J. Pharmaceutical Compositions
 The IFN-.epsilon. polypeptides of the present invention, agonist antibodies
 specifically binding such polypeptides, as well as other molecules
 identified by the screening assays disclosed hereinbefore, can be
 administered for the treatment of various pathologic conditions discussed
 hereinabove, such as. tumors, including cancers, viral diseases, and as
 immunomodulatory agents, in the form of pharmaceutical compositions.
 Therapeutic formulations of the IFN-.epsilon. polypeptides identified
 herein, or agonists thereof are prepared for storage by mixing the active
 ingredient having the desired degree of purity with optional
 pharmaceutically acceptable carriers, excipients or stabilizers
 (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),
 in the form of lyophilized formulations 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 and methionine; preservatives (such as
 octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
 benzalkoniwm chloride, benzethonium chloride; phenol, butyl or benzyl
 alcohol, alkyl parabens such as methyl or propyl paraben; catechol;
 resorcinol, cyclohexanol; 3-pentanol; and m-cresol); 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,
 histidine, arginine, or lysine; monosaccharides, disaccharides, and other
 carbohydrates including glucose, mannose, or dextrins; chelating agents
 such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol;
 salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein
 complexes); and/or non-ionic surfactants such as TWEEN.TM., PLURONICS.TM.
 or polyethylene glycol (PEG).
 The formulation herein may also contain more than one active compound as
 necessary for the particular indication being treated, preferably those
 with complementary activities that do not adversely affect each other.
 Alternatively, or in addition, the composition may comprise a cytotoxic
 agent, cytokine or growth inhibitory agent. Such molecules are suitably
 present in combination in amounts that are effective for the purpose
 intended.
 The active ingredients may also be entrapped in microcapsules prepared, for
 example, by coacervation techniques or by interfacial polymerization, for
 example, hydroxymethylcellulose or gelatin-microcapsules and
 poly-(methylmethacylate) microcapsules, respectively, in colloidal drug
 delivery systems (for example, liposomes, albumin microspheres,
 microemulsions, nano-particles and nanocapsules) or in macroemulsions.
 Such techniques are disclosed in Remington's Pharmnaceutical Sciences 16th
 edition, Osol, A. Ed. (1980).
 The formulations to be used for in vivo administration must be sterile.
 This is readily accomplished by filtration through sterile filtration
 membranes.
 Sustained-release preparations may be prepared. Suitable examples of
 sustained-release preparations include semipermeable matrices of solid
 hydrophobic polymers containing the antibody, which matrices are in the
 form of shaped articles, e.g. films, or microcapsules. Examples of
 sustained-release matrices include polyesters, hydrogels (for example,
 poly(2-hydroxyethyl-metliacrylate), or poly(vinylalcohol)), polylactides
 (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.
 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable
 lactic acid-glycolic acid copolymers such as the LUPRON DEPOT .TM.
 (injectable microspheres composed of lactic acid-glycolic acid copolymer
 and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While
 polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid
 enable release of molecules for over 100 days, certain hydrogels release
 proteins for shorter time periods. When encapsulated antibodies remain in
 the body for a long time, they may denature or aggregate as a result of
 exposure to moisture at 37.degree. C., resulting in a loss of biological
 activity and possible changes in immunogenicity. Rational strategies can
 be devised for stabilization depending on the mechanism involved. For
 example, if the aggregation mechanism is discovered to be intermolecular
 S--S bond formation through thio-disulfide interchange, stabilization may
 be achieved by modifying sulfhydryl residues, lyophilizing from acidic
 solutions, controlling moisture content, using appropriate additives, and
 developing specific polymer matrix compositions.
 Where antibody fragments are used, the smallest inhibitory fragment which
 specifically binds to the binding domain of the target protein is usually
 preferred. For example, based upon the variable region sequences of an
 antibody, peptide molecules can be designed which retain the ability to
 bind the target protein sequence. Such peptides can be synthesized
 chemically and/or produced by recombinant DNA technology (see. e.g.
 Marasco et al., Proc. Natl. Acad. Sci. USA 90, 7889-7893 [1993]). It is
 noted, however, that for some purposes, such as, to determine the pK
 value, a larger fragment, having a longer circulatory half-life, may be
 preferred.
 H. Methods of Treatment
 It is contemplated that the IFN-.epsilon. polypeptides of the present
 invention and their agonists including antibodies, peptides, and small
 molecule agonists, may be used to treat various tumors, e.g. cancers,
 viral infections, and generally conditions where immunomodulation, e.g.
 upregulation of the immune system, is desirable.
 Exemplary conditions or disorders to be treated include benign or malignant
 tumors (e.g. renal, liver, kidney, bladder, breast, gastric, ovarian,
 colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic
 carcinomas; sarcomas; glioblastomas; and various head and neck tumors);
 leukemias and lymphoid malignancies; other disorders such as neuronal,
 glial, astrocytal, hypothalamic and other glandular, macrophagal,
 epithelial, stromal and blastocoelic disorders; and inflammatory,
 angiogenic and immunologic disorders.
 The anti-tumor agents of the present invention (including the IFN-.epsilon.
 polypeptides disclosed herein and agonists which mimic their activity,
 e.g. antibodies, peptides and small organic molecules), are administered
 to a mammal, preferably a human, in accord with known methods, such as
 intravenous administration as a bolus or by continuous infusion over a
 period of time, by intramuscular, intraperitoneal, intracerobrospinal,
 subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical,
 or inhalation routes. Intravenous administration is preferred.
 Other therapeutic regimens may be combined with the administration of the
 anti-cancer agents of the instant invention. For example, the patient to
 be treated with such anti-cancer agents may also receive radiation
 therapy. Alternatively, or in addition, a chemotherapeutic agent may be
 administered to the patient. Preparation and dosing schedules for such
 chemotherapeutic agents may be used according to manufacturers'
 instructions or as determined empirically by the skilled practitioner.
 Preparation and dosing schedules for such chemotherapy are also described
 in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore,
 Md. (1992). The chemotherapeutic agent may precede, or follow
 administration of the anti-tumor agent of the present invention, or may be
 given simultaneously therewith. The anti-cancer agents of the present
 invention may be combined with an anti-oestrogen compound such as
 tamoxifen or an anti-progesterone such as onapristone (see, EP 616812) in
 dosages known for such molecules.
 It may be desirable to also administer antibodies against tumor associated
 antigens, such as antibodies which bind to the ErbB2, EGFR, ErbB3, ErbB4,
 or vascular endothelial factor (VEGF). Sometimes, it may be beneficial to
 also administer one or more cytokines to the patient. In a preferred
 embodiment, the anti-cancer agents herein are co-administered with a
 growth inhibitory agent. For example, the growth inhibitory agent may be
 administered first, followed by the administration of an anti-cancer agent
 of the present invention. However, simultaneous administration or
 administration of the anti-cancer agent of the present invention first is
 also contemplated. Suitable dosages for the growth inhibitory agent are
 those presently used and may be lowered due to the combined action
 (synergy) of the growth inhibitory agent and the antibody herein.
 For the prevention or treatment of disease, the appropriate dosage of an
 anti-tumor agent herein will depend on the type of disease to be treated,
 as defined above, the severity and course of the disease. whether the
 agent is administered for preventive or therapeutic purposes, previous
 therapy, the patient's clinical history and response to the agent, and the
 discretion of the attending physician. The agent is suitably administered
 to the patient at one time or over a series of treatments.
 For example, depending on the type and severity of the disease, about 1
 .mu.g/kg to 15 mg/kg (e.g. 0.1-20mg/kg) of an antitumor agent is an
 initial candidate dosage for administration to the patient, whether, for
 example, by one or more separate administrations, or by continuous
 infusion. A typical daily dosage might range from about 1 .mu.g/kg to 100
 mg/kg or more, depending on the factors mentioned above. For repeated
 administrations over several days or longer, depending on the condition,
 the treatment is sustained until a desired suppression of disease symptoms
 occurs. However, other dosage regimens may be useful. The progress of this
 therapy is easily monitored by conventional techniques and assays.
 Nucleic acid encoding an IFN-.epsilon. polypeptide may also be used in gene
 therapy. In gene therapy applications, genes are introduced into cells in
 order to achieve in vivo synthesis of a therapeutically effective genetic
 product, for example for replacement of a defective gene. "Gene therapy"
 includes both conventional gene therapy where a lasting effect is achieved
 by a single treatment, and the administration of a therapeutically
 effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic
 agents for blocking the expression of certain genes in vivo. It has
 already been shown that short antisense oligonucleotides can be imported
 into cells where they act as inhibitors, despite their low intracellular
 concentrations caused by their restricted uptake by the cell membrane.
 (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83, 4143-4146 [1986]). The
 oligonucleotides can be modified to enhance the uptake, e.g., by
 substituting their negatively charged phosphodiester groups by uncharged
 groups.
 There are a variety of techniques available for introducing nucleic acids
 into viable cells. The techniques vary depending upon whether the nucleic
 acid is transferred into cultured cells in vitro, or in vivo in the cells
 of the intended host. Techniques suitable for the transfer of nucleic acid
 into mammalian cells in vitro include the use of liposomes,
 electroporation, microinjection, cell fusion, DEAE-dextran, the calcium
 phosphate precipitation method, etc. The currently preferred in vivo gene
 transfer techniques include transfection with viral (typically retroviral)
 vectors and viral coat protein-liposome mediated transfection (Dzau et
 al., Trends in Biotechnology 11, 205-210 [1993]). In some situations it is
 desirable to provide the nucleic acid source with an agent that targets
 the target cells, such as an antibody specific for a cell surface membrane
 protein or the target cell, a ligand for a receptor on the target cell,
 etc. Where liposomes are employed, proteins which bind to a cell surface
 membrane protein associated with endocytosis may be used for targeting
 and/or to facilitate uptake, e.g. capsid proteins or fragments thereof
 tropic for a particular cell type, antibodies for proteins which undergo
 internalization in cycling, proteins that target intracellular
 localization and enhance intracellular half-life. The technique of
 receptor-mediated endocytosis is described, for example, by Wu et al., J.
 Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad.
 Sci. USA 87, 3410-3414 (1990).
 For review of gene marking and gene therapy protocols see Anderson et al.,
 Science 256, 808-813 (1992).
 Techniques for reintroducing cells into a patient after engineering with a
 polynucleotide (RNA or DNA) encoding a polypeptide herein ex vivo (cell
 therapy) are well known in the art.
 10. Articles of Manufacture
 In another embodiment of the invention, an article of manufacture
 containing materials useful for the diagnosis or treatment of the
 disorders described above is provided.
 The article of manufacture comprises a container and a label. Suitable
 containers include, for example, bottles, vials, syringes, and test tubes.
 The containers may be formed from a variety of materials such as glass or
 plastic. The container holds a composition which is effective for
 diagnosing or treating the condition and may have a sterile access port
 (for example the container may be an intravenous solution bag or a vial
 having a stopper pierceable by a hypodermic injection needle). The active
 agent in the composition is an IFN-.epsilon. of the present invention, or
 an agonist or antagonist thereof. The label on, or associated with, the
 container indicates that the composition is used for diagnosing or
 treating the condition of choice. The article of manufacture may further
 comprise a second container comprising a pharmaceutically-acceptable
 buffer, such as phosphate-buffered saline, Ringer's solution and dextrose
 solution. It may further include other materials desirable from a
 commercial and user standpoint, including other buffers, diluents,
 filters, needles, syringes, and package inserts with instructions for use.
 11. Diagnosis and Prognosis of Tumors
 As the IFN-.epsilon. proteins disclosed herein has been found to be
 effective in inhibiting tumor cell proliferation or growth and/or in
 killing tumor cells, their reduced level of expression may be indicative
 of the predisposition of a patient to develop tumor, and/or of the
 development or progression of tumor. Accordingly antibodies directed
 against the proteins disclosed herein may be used as tumor diagnostics or
 prognostics.
 For example, antibodies, including antibody fragments, can be used to
 qualitatively or quantitatively detect the expression of the IFN-.epsilon.
 protein ("marker gene product"). The antibody preferably is equipped with
 a detectable, e.g. fluorescent label, and binding can be monitored by
 light microscopy, flow cytometry, fluorimetry, or other techniques known
 in the art.
 In situ detection of antibody binding to the marker gene product can be
 performed, for example, by immunofluorescence or immunoelectron
 microscopy. For this purpose, a histological specimen is removed from the
 patient, and a labeled antibody is applied to it, preferably by overlaying
 the antibody on a biological sample. This procedure also allows for
 determining the distribution of the marker gene product in the tissue
 examined. It will be apparent for those skilled in the art that a wide
 variety of histological methods are readily available for in situ
 detection.
 In the following examples, IFN-.epsilon. is shown to be widely expressed in
 multiple human tissues, and to activate multiple signaling components in
 the JAK-STAT pathway in a IFNAR-dependent manner. The disclosed results
 also demonstrate that IFN-.epsilon. exhibits anti-growth and
 immunomodulating effects on cells. In addition, as noted before,
 interferons have been implicated in the pathogenesis of various autoimmune
 diseases, such as systemic lupus erythematoses, Behcet's disease,
 insulin-dependent diabetes mellitus (IDDM, also referred to as type I
 diabetes), and antibodies to various interferons the overexpression of
 which has been associated with the development and pathogenesis of such
 diseases have been proposed as potential therapeutics. For example, it has
 been demonstrated in a transgenic mouse model that .beta. cell expression
 of IFN-.alpha. can cause insulitis and IDDM, and IFN-.alpha. antagonists
 (including antibodies) have been proposed for the treatment of IDDM (WO
 93/04699, published Mar. 18, 1993). Accordingly, anti-IFN-.epsilon.
 antibodies might be useful in the treatment of diseases associated with
 the overexpression of IFN-.epsilon..
 The following examples are offered for illustrative purposes only, and are
 not intended to limit the scope of the present invention in any way.
 All patent and literature references cited in the present specification are
 hereby incorporated by reference in their entirety.
 EXAMPLES
 Commercially available reagents referred to in the examples were used
 according to manufacturer's instructions unless otherwise indicated. The
 source of those cells identified in the following examples, and throughout
 the specification, by ATCC accession numbers is the American Type Culture
 Collection, Rockville, Md.
 Example 1
 Isolation of cDNA clones Encoding Human IFN-.epsilon.
 An expressed sequence tag (EST) DNA database (LIFESEQ.TM., Incyte
 Pharmaceuticals, Palo Alto, Calif.) was searched and an EST was identified
 which showed homology to interferon-.alpha.. Possible homology was noted
 between Incyte EST 3728969 (subsequently renamed as DNA49668) and
 mammalian alpha interferons, in particular IFN-.alpha.14. The homology was
 confirmed by inspection.
 The following PCR primers and oligonucleotide probe were synthesized:
 49668.r1:
 TCTCTGCTTCCAGTCCCATGAGTGC (SEQ ID NO:4) 49668.r2:
 GCTTCCAGTCCCATGAGTGCTTCTAGG (SEQ ID NO:5) 49668.p1:
 GGCCATTCTCCATGAGATGCTTCAGCAGATCTTCAGCCTCTTCAGGGCAA (SEQ ID NO:6)
 In order to screen several libraries for a source of a full-length clone,
 DNA from the libraries was screened using the r1 and r2 probes identified
 above. A positive library was then used to isolate clones encoding the
 IFN-.epsilon.-encoding gene using the probe oligonucleotide.
 Three million clones from a size selected (500-4000 bp) oligo dT primed
 cDNA library from human small intestine (LIB 99) constructed in a
 pRK5-based vector screened by hybridization. The cDNA libraries used to
 isolate the cDNA clones were constructed by standard methods using
 commercially available reagents such as those from Invitrogen, San Diego,
 Calif. The cDNA was primed with oligo dT containing a NotI site, linked
 with blunt to SalI hemikinased adaptors, cleaved with NotI, sized
 appropriately by gel electrophoresis, and cloned in a defined orientation
 into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor
 of pRK5D that does not contain the SfiI site; see, Holmes et al., Science,
 253:1278-1280 (1991)) in the unique Xhol and NotI sites. Only one positive
 clone was found out of 3.6.times.10.sup.6 cfu. The clone was sequenced in
 both directions and was found to cover the entire reading frame (ORF). A
 BAC clone (F480) was identified by screening a BAC array panel (Research
 Genetics) with PCR primers generated from the sequence of IFN-.epsilon..
 DNA sequencing of the clone isolated as described above gave the
 full-length DNA sequence for DNA50960 and the derived protein sequence for
 IFN-.epsilon. (PRO655).
 The entire nucleotide sequence of DNA50960 is shown in FIG. 2 (SEQ ID
 NO:2). Clone DNA50960 contains a single open reading frame with an
 apparent translational initiation site at nucleotide positions 621-623
 (FIG. 2). The predicted polypeptide precursor is 208 amino acids long, of
 which 21 N-terminal amino acid residues represent a putative signal
 sequence. Clone DNA50960-1224 (clone F480) has been deposited with ATCC
 and is assigned ATCC deposit no. 209509, deposited on Dec. 3, 1997.
 Using BLAST and FastA sequence alignment computer programs, it was found
 that PRO655 (shown in FIG. 1 and SEQ ID NO:1) has about 35-40% amino acid
 sequence identity with the sequence of various human IFN-.alpha. species.
 The homology is highest within the 22-189 amino acid region of the
 sequence of FIG. 1 (SEQ ID NO:1). At the nucleotide level, the homology
 with the coding sequence of IFN-.alpha. is about 60%. Based upon these
 data as well as the presence of a characteristic sequence beginning at
 amino acid 147 that is typical of type I interferons
 ([FYH][FY].[GNRC][LIVM].{1}[FY]L.{7}[CY]AW), this molecule was identified
 as a member of the type I IFN family (FIG. 7). The sequence of
 IFN-.epsilon. is nearly as divergent from IFN-.alpha. as it is from
 IFN-.beta. family members (33% and 37% sequence identity to
 IFN-.alpha..sub.2a and IFN-.beta., respectively) and thus defines a new
 branch on the type 1 interferon family tree. Molecular modeling suggests
 that IFN-.epsilon. displays similar tertiary structure compared to
 IFN-.alpha. (L. Presta, data not shown). A diagrammatic comparison of
 IFN-.epsilon. with other IFNs is shown in FIG. 7(A).
 Example 2 Use of the Novel Human Interferon Encoding DNA as a Hybridization
 Probe
 The following method describes use of a nucleotide sequence encoding
 IFN-.epsilon. as a hybridization probe.
 DNA comprising the coding sequence of IFN-.epsilon. (as shown in FIG. 2,
 SEQ ID NO:2) is employed as a probe to screen for homologous DNAs (such as
 those encoding naturally-occurring variants of IFN-.epsilon.) in human
 tissue cDNA libraries or human tissue genomic libraries.
 Hybridization and washing of filters containing either library DNAs is
 performed under the following high stringency conditions. Hybridization of
 radiolabeled probe derived from the PRO655-encoding DNA, to the filters is
 performed in a solution of 50% formamide, 5.times.SSC, 0.1% SDS, 0.1%
 sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2.times.Denhardt's
 solution, and 10% dextran sulfate at 42.degree. C. for 20 hours. Washing
 of the filters is performed in an aqueous solution of 0.1.times.SSC and
 0.1% SDS at 42.degree. C.
 DNAs having a desired sequence identity with the DNA encoding full-length
 native sequence IFN-.epsilon. can then be identified using standard
 techniques known in the art.
 Example 3
 Expression of IFN-.epsilon. in E. coli
 This example illustrates preparation of an unglycosylated form of
 IFN-.epsilon. by recombinant expression in E. coli.
 The DNA sequence encoding IFN-.epsilon. (SEQ ID NO:2) is initially
 amplified using selected PCR primers. The primers should contain
 restriction enzyme sites which correspond to the restriction enzyme sites
 on the selected expression vector. A variety of expression vectors may be
 employed. An example of a suitable vector is pBR322 (derived from E. coli,
 see Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin
 and tetracycline resistance. The vector is digested with restriction
 enzyme and dephosphorylated. The PCR amplified sequences are then ligated
 into the vector. The vector will preferably include sequences which encode
 for an antibiotic resistance gene, a trp promoter, a polyhis leader
 (including the first six STII codons, polyhis sequence, and enterokinase
 cleavage site), the IFN-.epsilon. coding region, lambda transcriptional
 terminator, and an argU gene.
 The ligation mixture is then used to transform a selected E. coli strain
 using the methods described in Sambrook et al., supra. Transformants are
 identified by their ability to grow on LB plates and antibiotic resistant
 colonies are then selected. Plasmid DNA can be isolated and confirmed by
 restriction analysis and DNA sequencing.
 Selected clones can be grown overnight in liquid culture medium such as LB
 broth supplemented with antibiotics. The overnight culture may
 subsequently be used to inoculate a larger scale culture. The cells are
 then grown to a desired optical density during which the expression
 promoter is turned on.
 After culturing the cells for several more hours, the cells can be
 harvested by centrifugation. The cell pellet obtained by the
 centrifugation can be solubilized using various agents known in the art,
 and the solubilized IFN-.epsilon. protein can then be purified using a
 metal chelating column under conditions that allow tight binding of the
 protein.
 A specific example of the expression and purification of recombinant
 IFN-.epsilon. in E coil is provided in Example 10 below.
 Example 4
 Expression of IFN-.epsilon. in Mammalian Cells
 This example illustrates preparation of a glycosylated form of
 IFN-.epsilon. (PRO655) by recombinant expression in mammalian cells.
 The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employed as
 the expression vector. Optionally, the IFN-.epsilon.-encoding DNA is
 ligated into pRK5 with selected restriction enzymes to allow insertion of
 the IFN-.epsilon.-encoding DNA using ligation methods such as described in
 Sambrook et al., supra. The resulting vector is called pRK5-IFN-.epsilon.
 (PRO655).
 In one embodiment. the selected host cells may be 293 cells. Human 293
 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in
 medium such as DMEM supplemented with fetal calf serum and optionally,
 nutrient components and/or antibiotics. About 10 .mu.g pRK5-IFN-.epsilon.
 (PRO655) DNA is mixed with about 1 .mu.g DNA encoding the VA RNA gene
 [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 .mu.l of 1
 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl.sub.2. To this mixture is added,
 dropwise, 500 .mu.l of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM
 NaPO.sub.4, and a precipitate is allowed to form for 10 minutes at
 25.degree. C. The precipitate is suspended and added to the 293 cells and
 allowed to settle for about four hours at 37.degree. C. The culture medium
 is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds.
 The 293 cells are then washed with serum free medium, fresh medium is
 added and the cells are incubated for about 5 days.
 Approximately 24 hours after the transfections, the culture medium is
 removed and replaced with culture medium (alone) or culture medium
 containing 200 .mu.Ci/ml .sup.35 S-cysteine and 200 .mu.Ci/ml .sup.35
 S-methionine. After a 12 hour incubation, the conditioned medium is
 collected, concentrated on a spin filter, and loaded onto a 15% SDS gel.
 The processed gel may be dried and exposed to film for a selected period
 of time to reveal the presence of IFN-.epsilon. polypeptide. The cultures
 containing transfected cells may undergo further incubation (in serum free
 medium) and the medium is tested in selected bioassays.
 In an alternative technique, DNA encoding IFN-.epsilon. may be introduced
 into 293 cells transiently using the dextran sulfate method described by
 Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are
 grown to maximal density in a spinner flask and 700 .mu.g
 pRK5-IFN-.epsilon. DNA is added. The cells are first concentrated from the
 spinner flask by centrifugation and washed with PBS. The DNA-dextran
 precipitate is incubated on the cell pellet for four hours. The cells are
 treated with 20% glycerol for 90 seconds, washed with tissue culture
 medium, and re-introduced into the spinner flask containing tissue culture
 medium, 5 .mu.g/ml bovine insulin and 0.1 .mu.g/ml bovine transferrin.
 After about four days, the conditioned media is centrifuged and filtered
 to remove cells and debris. The sample containing expressed IFN-.epsilon.
 can then be concentrated and purified by any selected method, such as
 dialysis and/or column chromatography.
 In another embodiment, the novel interferon polypeptide (IFN-.epsilon.,
 PRO655) was transiently transfected into COS7 cells. 20 .mu.g of a plasmid
 encoding IFN-.epsilon. under control of the CMV IE promoter, was mixed
 with 2 .mu.g of a Green Fluorescent Protein (GFP) expressing plasmid. The
 DNA was introduced into the cells with a commercially available
 transfection reagents following manufacturer's instructions. One day
 post-transfection, the cells were visualized at 425 nM, using a
 fluorescent microscope to ensure a transfection efficiency &gt;25% (25%
 GFP positive). The medium was then removed and the plates were fed 25 ml
 of collection media and incubated at 32.degree. C. for 5 days. Collection
 media: enriched serum-free medium containing 100 ng/ml insulin. Media:
 high-glucose DMEM (Gibco-BRL) with 0.5% fetal bovine serum. Media were
 collected, cells and debris removed by centrifugation and filtration
 through a 0.2 .mu.M sterile filter.
 Epitope-tagged IFN-.epsilon. DNA may also be expressed in host CHO cells.
 The IFN-.epsilon. DNA may be subcloned out of the pRK5 vector. The
 subclone insert can undergo PCR to fuse in frame with a selected epitope
 tag such as a poly-his tag. The poly-his tagged insert can then be
 subcloned into a SV40 driven vector containing a selection marker such as
 DHFR for selection of stable clones. Finally, the CHO cells can be
 transfected (as described above) with the SV40 driven vector. Labeling may
 be performed, as described above, to verify expression. The culture medium
 containing the expressed poly-His tagged IFN-.epsilon. can then be
 concentrated and purified by any selected method, such as by Ni.sup.2+
 -chelate affinity chromatography. Following essentially the protocol
 described, a poly-his tagged human IFN-.epsilon. polypeptide (PRO713) was
 prepared and purified. The different PRO number merely indicates that the
 protein was obtained in a different expression experiment. PRO713 has the
 same amino acid sequence as PRO655, i.e. is encoded by DNA50960.
 Example 5
 Expression of IFN-.epsilon. in Yeast
 The following method describes recombinant expression of IFN-.epsilon. in
 yeast.
 First, yeast expression vectors are constructed for intracellular
 production or secretion of IFN-.epsilon. from the ADH2/GAPDH promoter. DNA
 encoding IFN-.epsilon., a selected signal peptide and the promoter is
 inserted into suitable restriction enzyme sites in the selected plasmid to
 direct intracellular expression of IFN-.epsilon.. For secretion, DNA
 encoding IFN-.epsilon. can be cloned into the selected plasmid, together
 with DNA encoding the ADH2/GAPDH promoter, the yeast alpha-factor
 secretory signal/leader sequence, and linker sequences (if needed) for
 expression of IFN-.epsilon..
 Yeast cells, such as yeast strain AB110, can then be transformed with the
 expression plasmids described above and cultured in selected fermentation
 media. The transformed yeast supernatants can be analyzed by precipitation
 with 10% trichloroacetic acid and separation by SDS-PAGE, followed by
 staining of the gels with Coomassie Blue stain.
 Recombinant IFN-.epsilon. can subsequently be isolated and purified by
 removing the yeast cells from the fermentation medium by centrifugation
 and then concentrating the medium using selected cartridge filters. The
 concentrate containing IFN-.epsilon. may further be purified using
 selected column chromatography resins.
 Example 6
 Expression of IFN-.epsilon. in Baculovirus-infected Insect Cells
 The following method describes recombinant expression of IFN-.epsilon. in
 Baculovirus expression system.
 The IFN-.epsilon.-encoding DNA is fused upstream of an epitope tag
 contained with a baculovirus expression vector. Such epitope tags include
 poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety
 of plasmids may be employed, including plasmids derived from commercially
 available plasmids such as pVL1393 (Novagen). Briefly, the coding sequence
 of IFN-.epsilon. or the desired portion of the coding sequence is
 amplified by PCR with primers complementary to the 5' and 3' regions. The
 5' primer may incorporate flanking (selected) restriction enzyme sites.
 The product is then digested with those selected restriction enzymes and
 subcloned into the expression vector.
 Recombinant baculovirus is generated by co-transfecting the above plasmid
 and BaculoGold.TM. virus DNA (Pharmingen) into Spodoptera frugiperda
 ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available
 from GIBCO-BRL). After 4-5 days of incubation at 28.degree. C., the
 released viruses are harvested and used for further amplifications. Viral
 infection and protein expression is performed as described by O'Reilley et
 al., Baculovirus expression vectors: A laboratory Manual, Oxford: Oxford
 University Press (1994).
 Expressed poly-his tagged IFN-.epsilon. can then be purified, for example,
 by Ni.sup.2+ -chelate affinity chromatography as follows. Extracts are
 prepared from recombinant virus-infected Sf9 cells as described by Rupert
 et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed,
 resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl.sub.2
 ; 0.1 mM EDTA; 10% Glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice
 for 20 seconds on ice. The sonicates are cleared by centrifugation and the
 supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM
 NaCl, 10% Glycerol, pH 7.8) and filtered through a 0.45 .mu.m filter. A
 Ni.sup.2+ -NTA agarose column (commercially available from Qiagen) is
 prepared with a bed volume of 5 mL, washed with 25 mL of water and
 equilibrated with 25 mL of loading buffer. The filtered cell extract is
 loaded onto the columnn at 0.5 mL per minute. The column is washed to
 baseline A.sub.280 with loading buffer, at which point fraction collection
 is started. Next, the column is washed with a secondary wash buffer (50 mM
 phosphate; 300 mM NaCl, 10% Glycerol, pH 6.0), which elutes
 nonspecifically bound protein. After reaching A.sub.280 baseline again,
 the column is developed with a 0 to 500 mM Imidazole gradient in the
 secondary wash buffer. One mL fractions are collected and analyzed by
 SDS-PAGE and silver staining or western blot with Ni.sup.2+
 -NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the
 eluted His.sub.10 -tagged IFN-.epsilon. are pooled and dialyzed against
 loading buffer.
 Alternatively, purification of the IgG tagged (or Fc tagged) IFN-.epsilon.
 can be perforrned using known chromatography techniques, including for
 instance, Protein A or protein G column chromatography.
 A specific protocol for purification of IgG-tagged proteins is as follows:
 The conditioned medium is filtered through a 0.45 micron filter, and
 loaded onto a Sepharose A column (Pharmacia). The column is washed with
 5-6 CV 20 mM NaH.sub.2 PO.sub.4, pH 6.8, and eluted with 3 CV 100 mM
 citric acid pH 3.4. After neutralization with 1 M Tris (pH 9.)) in
 fraction tubes (275 microliters per 1 ml fraction), the IFN-.epsilon.
 protein is desalted on PD-10 column.
 Example 7
 Preparation of Antibodies that Bind IFN-.epsilon.
 This example illustrates preparation of monoclonal antibodies which can
 specifically bind IFN-.epsilon..
 Techniques for producing the monoclonal antibodies are known in the art and
 are described, for instance, in Goding, supra. Immunogens that may be
 employed include purified IFN-.epsilon., fusion proteins containing
 IFN-.epsilon., and cells expressing recombinant IFN-.epsilon. on the cell
 surface. Selection of the immunoyen can be made by the skilled artisan
 without undue experimentation.
 Mice, such as Balb/c, are immunized with the IFN-.epsilon. immunogen
 emulsified in complete Freund's adjuvant and injected subcutaneously or
 intraperitoneally in an amount from 1-100 micrograms. Alternatively. the
 immunogeni is emulsified in MPL-TDM adjuvant (Ribi Immunochemical
 Research, Hamilton, MT) and injected into the animal's hind foot pads. The
 immunized mice are then boosted 10 to 12 days later with additional
 immunogen emulsified in the selected adjuvant. Thereafter, for several
 weeks, the mice may also be boosted with additional immunization
 injections. Serum samples may be periodically obtained from the mice by
 retro-orbital bleeding for testing in ELISA assays to detect
 anti-IFN-.epsilon. antibodies.
 After a suitable antibody titer has been detected, the animals "positive"
 for antibodies can be injected with a final intravenous injection of
 IFN-.epsilon.. Three to four days later, the mice are sacrificed and the
 spleen cells are harvested. The spleen cells are then fused (using 35%
 polyethylene glycol) to a selected murine myeloma cell line such as
 P3X63AgU. 1, available from ATCC, No. CRL 1597. The fusions generate
 hybridoma cells which can then be plated in 96 well tissue culture plates
 containing HAT (hypoxanthine, aminopterin, and thymidine) medium to
 inhibit proliferation of non-fused cells, myeloma hybrids. and spleen cell
 hybrids.
 The hybridoma cells will be screened in an ELISA for reactivity against
 IFN-.epsilon.. Determination of "positive" hybridoma cells secreting the
 desired monoclonal antibodies against IFN-.epsilon. is within the skill in
 the art.
 The positive hybridoma cells can be injected intraperitoneally into
 syngeneic Balb/c mice to produce ascites containing the anti-IFN-.epsilon.
 monoclonal antibodies. Alternatively, the hybridoma cells can be grown in
 tissue culture flasks or roller bottles. Purification of the monoclonal
 antibodies produced in the ascites can be accomplished using ammonium
 sulfate precipitation, followed by gel exclusion chromatography.
 Alternatively, affinity chromatography based upon binding of antibody to
 protein A or protein G can be employed.
 Example 8
 Chromosomal Localization of IFN-.epsilon.
 DNA from BAC clone F480 containing the IFN-.epsilon. gene was labeled with
 digoxigenin dUTP followed by standard fluorescent in situ (FISH)
 hybridization procedure. (Knoll and Lichter, Current Protocols in Human
 Genetics, Dracopoli et al., eds., John Wiley & Sons, New York, 1995. Units
 4.3.1-4.3.29, Current Protocols in Molecular Biology, Ausubel et al.,
 eds., John Wiley & Sons, New York 1997. Units 3.18; 14.7.1-14.7.14.) The
 initial experiment resulted in specific labeling of the short arm of a
 group C chromosome which was believed to be chromosome 9, based on size,
 morphology, and banding pattern. A second experiment was conducted in
 which a biotin-labeled probe which is specific for the heterochromatic
 region of chromosome 9 was co-hybridized with clone F480.
 Measurements of 10 specifically labeled chromosomes 9 demonstrated that
 F480 is located at a position which is 51% of the distance from the
 centromere to the telomere of the 9p, an area which corresponds to
 chromosome 9p21.2-21.3 (FIG. 7(B)). A total of 80 metaphase cells were
 analyzed with 72 exhibiting specific labeling. The identified location is
 near other type I interferons (DeMaeyer, E. and De. Maeyer-Guignard, J.,
 Interferons. The Cytokine Handbook, 2nd ed., 265-288 [1994]). Sequencing
 of the F480 BAC clone indicates that like other type I interferons, the
 IFN-.epsilon. gene has no intervening sequences in its coding region.
 Example 9
 Northern Blot Analysis
 The expression of IFN-.epsilon. in multiple tissues was examined by
 quantitative RT-PCR (TaqMan.RTM. Technology).
 A multi-tissue RNA blot containing 2 .mu.g each of poly(A)+RNA from human
 tissues was purchased from Clontech. An overlapping oligo corresponding to
 codons for amino acid 2-31 in the IFN-.epsilon. precursor was generated.
 The DNA probes were labeled with .alpha.-.sup.32 P=dCTP by random priming
 (Promega). The RNA blot was hybridized with 50% formamide, 5.times.SS, 50
 mM potassium phosphate (pH 7.0), 5.times.Denhardt's solution, 10% dextran
 sulfate at 42.degree. C. for 20 hours. The blot was washed with
 0.1.times.SSC, 0.1% SDS at 50.degree. C. for 30 minutes and exposed in
 Phospholmager.
 The following tissues were exarnined: adult 1) heart, 2) brain, 3)
 placenta, 4) lung, 5) liver, 6) skeletal muscle, 7) kidney, 8) pancreas,
 9) spleen, 10) thymus, 11) prostate, 12) testis, 13) ovary, 14) small
 intestine, 15) colon (mucosal lining), and 16) peripheral blood
 leukocytes, and human fetal tissues: 17) brain, 18) lung, 19) liver, and
 20) kidney. Low levels of constitutive expression were detected in tissues
 of brain, lung, kidney, and small intestine (data not shown).
 Example 10
 Characterization and Biological Activities of IFN-.epsilon.
 Protocols
 Expression and Purification of Recombinant IFN-.epsilon. in E. coli
 IFN-.epsilon. was expressed in the E. coli cytoplasm, using a derivative of
 the tryptophan (trp) promoter vector pHGH207-1 (DeBoer et al., Promoter
 Structure and Function, Rodriguez et al., eds., p. 462, Praeger, New York,
 1982.) A 210 amino acid leader sequence was fused to the amino termini of
 the mature interferon to ensure efficient translation initiation and to
 facilitate purification. This leader encodes the first 6 amino acids of
 the STII signal sequence (Picken et al., H. Infect. Immun. 42, 269-275
 [1983]), followed by 8 histidines, and finally the amino acid sequence
 ASDDDDK for potential cleavage by the protease enterokinase. Downstream of
 the leader and mature IFN-.epsilon. coding sequences was placed the
 .lambda. to transcriptional terminator (Scholtissek and Grosse, Nucl.
 Acids Res. 15, 3185 [1987]).
 The expression plasmid was transformed into the E. coli host 52A7 (W3110
 fhuA(tonA) lon galE rpoHts(htpRts) clpP lacIq) prior to the induction of
 the trp promoter. Cells were first grown in LB containing ampicillin at
 30.degree. C. until a cell density of 2-4 (A.sub.600) was reached. The LB
 culture was then diluted 20 fold into a high cell density tryptophan
 limiting media (per liter: 1.86 g Na.sub.2 HPO.sub.4, 0.93 g NaH.sub.2
 PO.sub.4 H.sub.2 O, 3.57 g (NH.sub.4).sub.2 SO.sub.4, 0.71 g Na.sub.2
 Citrate(H.sub.2 O).sub.2, 1.07 g KCl, 5.36 g yeast extract, 5.36 g
 casamino acids, autoclave, then add MOPS pH7.3 to 110 mM, NgSO.sub.4 to 7
 mM, and glucose to 0.55% w/v). After 5 hours, trans-3-indoleacrylic acid
 was added to 50 .mu.g/mL and then growth was continued for another 16
 hours at 30.degree. C. with shaking.
 E. coli paste from 0.5 liter fermentations was resuspended in 10 volumes
 (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and
 sodium tetrathionate were added to final concentrations of 0.1 M and 0.02
 M, and stirred overnight at 40.degree. C. After centrifugation, the
 supernatant was diluted in metal chelate column buffer (6 M guanidine, 20
 mM Tris, pH 7.4) and subjected to Ni-NTA metal chelate column (Quiagen).
 Eluted IFN-.epsilon. was refolded by diluting the metal chelate purified
 protein slowly into freshly prepared refolding buffer consisting of: 20 mM
 Tris, pH 8.6, 0.3 M NaCl, 5 mM cysteine, 20 mM glycine, 40 .mu.g/m;
 polyethylene glycol (3350 MW) and 1 mM EDTA. The refolded protein was
 chromatographed on a Poros R1/H reversed phase column (PerSeptive).
 Quantitative amino acid analysis was used to determine protein
 concentrations.
 Expression and Purification of IFN-.alpha. receptor (IFNAR) Immunoadhesins
 Mammalian expression vectors encoding IFN-.alpha.R1-IgG1 and
 IFN-.alpha.R2-IgG1 (pRKIFN-.alpha./.beta.-IgG and
 pRKIFN-.alpha./.beta.-IgG) were constructed from plasmids encoding the
 human type 1 interferon receptors (pRKIFN-.alpha./.beta.R1 and
 pRKIFN-.alpha./.beta.-R2) and CD4-IgG1 (pRKCD4.sub.2 Fc.sub.1 - Capon et
 al., Nature 337:525-531 [1989]). The mature IFN-.alpha./.beta.R1-IgG and
 IFN-.alpha./.beta.R2-IgG polypeptide encoded by
 pRKIFN-.alpha./.beta.R1-IgG and pRKIFN-.alpha./.beta.R2-IgG thus contain
 633 and 443 amino acids, respectively. The IFN-.alpha./.beta.R-IgGs were
 expressed in human embryonic kidney 293 cells by transient transfection
 with the respective plasmids, using the calcium phosphate precipitation
 method. The receptor-IgG immunoadhesins were purified to greater than 95%
 homogeneity from serum-free cell supernatants by affinity chromatography
 on Staphylococcus aureus Protein A. The immunoadhesins were eluted with 50
 mM sodium citrate pH 3 / 20% (w/v) glycerol, and the pH was neutralized
 with 0.05 volumes of 3M TRIS HCl (pH 8-9).
 Tyrosine Phosphorylation Assay
 Cells were serum-starved for 6 hours and subjected to treatment of
 cytokines for the indicated period of time, using the indicated
 concentrations. The lysis of cells, immunoprecipitation, Western blot and
 ECL detection were performed as previously described by Zhang et al.,
 Proc. Natl. Acad. Sci. USA 94, 9562-7 (1997). The following antibodies
 were used: JAK1 (Q-19), JAK2 (HR758) Tyk2 (C-20), Stat1 (C-111), Stat2
 (C-20) and Stat3 (C-20) purchased from Santa Cruz Biotechnology (CA).
 Antibody 4G10 was purchased from Upstate Biotechnology. anti-IFN-.alpha.R1
 antibody 2E1.5.2 and anti-IFN-.alpha.R2 antibody 3B7.22.7 were prepared as
 described in Lu J. et al., J. Immunol. 160: 1782-1788 (1998).
 Electrophoretic Mobility Shift Assay (EMSA)
 HelaS3 (ATCCCCL2.2) cells were pretreated with IFN-.gamma. (100 U/ml)
 overnight to increase the expression of p48 (Levy et al., Genes Dev. 3,
 1362-71 [1989]). Cells were treated with IFN-.epsilon..sup.His or
 IFN-.alpha. for 45 minutes and nuclear extract was prepared. The
 preparation of nuclear extract and EMSA followed the protocol described by
 Levy, supra, with modifications (Zhang et al., J. Biol. Chem. 271, 95-3-9
 [1996]). The probe for ESRE (ISG-15) and SIE is based on Darnell et al.,
 Science 264, 1415-21 (1994).
 Cell Culture, FACS analysis and Antiproliferation and Antiviral Assays
 A549 cells (ATCC CCL-185.1, human lung carcinoma) and human 293 cell lines
 (ATCC 45504, kidney epithelial) were growth in "50:50" medium (HAM's F12:
 Dulbecco's Modified Eagle medium), with 10% FBS. Daudi cells. MELT-4 and
 U266 were growth in RPMI1640, supplemented with 10% FBS. Daudi cells (ATCC
 CCL-213, B lymphoblast), MLT-4 (ATCC CRL-1582, T lymphoblast) and U266
 (ATCC TIB-196, lymphoblast) cells were grown in RPMI 1640 with 10% FBS.
 FACS analysis was performed as previously described (Zhang, 1997, supra).
 The anti-MHC I antibody (HLA-A, B, C) was purchased from Pharmacia.
 The antiproliferation assay was performed as described by Evinger and
 Pestka, Methods Enzamol. 79, 362-8 (1981) with the following
 modifications. Daudi cells were treated with different doses of IFNs in
 the presence or absence of antagonistic antibodies in 96-well culture
 plates at 5.times.10.sup.5 cells/ml, and incubated at 37.degree. C. for 72
 hours. One tenth volume of AlamarBlue reagent was added to the culture and
 the cells were incubated for 4 hours before measuring the fluorescent
 intensity as a indicator of cell proliferation (Alamar Biotechnology,
 Sacramento, Calif.).
 Antiviral analysis was performed as described Rubinstein et al., J. Virol.
 37, 755-758 (1981). Briefly, cells were seeded into 96-well culture plates
 and allowed to grow for 24 hours before IFN-.epsilon. treatment. EMCV
 challenge at 1 multiplicity of infection unit/well was performed 24 hours
 after the IFN-.epsilon. treatment and the cells were allowed to be
 infected for another 24 hours. Cell survival was quantified by crystal
 violet dye exclusion.
 Results
 A search of an expressed sequence tag (EST) database for sequences related
 to Type I IFN family members revealed an EST that was predicted to encode
 a polypeptide bearing about 38% amino acid sequence identity to amino
 acids 58-148 of IFN-.alpha.14. Using probes based on the EST, a cDNA was
 cloned and found to encode an ORF of 208 amino acids (FIG. 1) with a
 potential signal sequence of 21 amino acids and a calculated molecular
 weight of 21.9 kD. Analysis of the amino acid sequence revealed that it
 contained homology to Type I IFN family members (e.g. about 33% and 37%
 sequence identity to IFN-.alpha.2 and IFN-.beta., respectively).
 Progressive alignment analysis (Feng, D. F. and Doolittle, R. F., Meth.
 Enzymol. 183, 375-387 [1990]) of the encoded protein and other Type I IFNs
 indicates that the protein sequence defines a new branch of the Type I IFN
 family (FIG. 5). We therefore named this gene product IFN-.epsilon..
 lFN-.epsilon. contains two potential sites for N-linked glycosylation at
 positions 74 and 83 (predicted mature protein, thereafter). A pair of Cys
 residues (Cys32 and Cys 142) that are conserved in all human type I IFNs
 and are known to form a disulfide linkage crucial for activity (Morehead
 et al., Biochemistry 23, 2500-2507 [1984]) are also conserved in
 IFN-.epsilon.. A second pair of cysteines that are present in IFN-.alpha.
 and -.omega. (e.g. Cys1 and Cys98 g in IFN-.alpha.) and form a disulfide
 bridge are not conserved in IFN-.epsilon.. Instead, IFN-.epsilon. has a
 cysteine at position 154 that is not conserved in other Type I IFNs.
 Despite the limited sequence identity to other IFNs, molecular modeling
 suggests that IFN-.epsilon. displays similar tertiary structure compared
 to IFN-.alpha. (L. Presta, data not shown).
 As described earlier. a BAC clone (F480) encodino the IFN-.epsilon. gene
 was isolated. This clone was used to map the chromosomal location by
 fluorescent in situ hybridization (FISH). These studies localized the
 IFN-.epsilon. gene to chromosome 9p21.2-21.3 (FIG. 7A), placing it near
 the human IFN-.omega., IFN-.beta. and a cluster of IFN-.alpha. genes that
 have been mapped to 9p22-p13 (De Maeyer, E. and DeMaeyer-Guignard, J.,
 supra).
 The expression of IFN-.epsilon. in different human tissues was examined by
 real time quantitative RT-PCR (TaqMan.RTM. technology) using
 tissue-specific polyA+RNA from adult humans as templates (FIG. 7B). The
 highest expression of IFN-.epsilon. mRNA was observed in the brain,
 kidney, and small intestine. Several other tissues have lower levels of
 expression, including the lung, liver, spleen, thymus and lymph node,
 whereas heart and bone marrow shoed low levels of mRNA expression. Thus,
 IFN-.epsilon. mRNA is expressed constitutively in various adult tissues.
 We have not detected significant poly(I).poly.COPYRGT. induction of
 IFN-.epsilon. mRNA in human fibroblasts or in Daudi cells. In contrast,
 human Mx gene expression was greatly induced in these cells and IFN-.beta.
 transcription was upregulated in fibroblasts (data not shown).
 IFN-.epsilon. protein was expressed in E. coli with an amino terminal His
 targ and purified by Ni-NTA affinity chromatography. To determine whether
 IFN-.epsilon. can activate known Type I IFN receptors, we treated
 lymphoblast U266 cells with the recombinant IFN-.epsilon. (designated
 IFN-.epsilon..sup.His) of various concentrations and observed a dose
 dependent increase in tyrosine phosphorylation of both receptor subunits.
 IFN-.alpha.R1 and IFN-.alpha.R2 (the long form, or IFN-.alpha.R2c) (FIGS.
 8A-B). This induction of receptor tyrosine phosphorylation was a rapid
 response, starting at less than 1 minute after treatment, peaking at 15
 minutes and decreasing to undetectable levels by one hour (data not
 shown). Similar results were obtained with other cell lines such as the T
 lymphoblast cell line MOLT-4 and the B lymphoblast cell line Daudi (not
 shown).
 IFNs and other cytokines have been shown to activate JAK-STAT signaling
 components after interacting with their receptors. To determine if
 IFN-.epsilon. activates the JAK-STAT pathway, we assayed tyrosine
 phosphorylation of the key components in the IFN signal pathway. As shown
 in FIGS. 8C-D, IFN-.epsilon. stimulated tyrosine phosphorylation of Janus
 kinase members JAK1 and Tyk2, but not JAK2 (not shown). It also induced
 tyrosine phosphorylation of Stat1, Stat2 and Stat3. In addition, we
 examined the formation of transcription factor complexes
 interferon-stimulated gene factor 3 (ISGF3) and serum-induced factor (SIF)
 (Darnell et al., Science 964, 1415-1421 [1994]) upon treatment of HeLa
 cells with IFN-.epsilon. (FIG. 8E). Like IFN-.alpha., IFN-.epsilon.
 stimulated the formation of ISGF3 and SIF. These complexes can be
 specifically competed by excess amounts of cold oligonucleotides and can
 be abolished or supershifted by anti-Stat antibodies. Therefore, like
 other type I IFNs, IFN-.epsilon. activates STAT-1, -2, and -3 and leads to
 the formation of transcription complexes ISGF3 and SIF.
 We used antagonistic antibodies directed against IFN-.alpha.R1 and
 IFN-.alpha.R2 to determine if the known IFN-.alpha. receptor subunits are
 required for IFN-.epsilon. induced activation of JAK-STAT signaling. These
 monoclonal antibodies have previously been shown to inhibit the antiviral
 response of IFN-.alpha. (Lu et al., (1998) supra). These antibodies
 recognize the corresponding receptor specifically and inhibit
 IFN-.alpha.2a-induced State2 tyrosine phosphorylation in MOLT-4 cells
 (FIG. 8F). These same antibodies also inhibited IFN-.epsilon. -induced
 tyrosine phosphorylation of Stat2. In fact, the anti-IFN-.alpha.-R1
 antibody was a more potent inhibitor of IFN-.epsilon. activity than of
 IFN-.alpha.2a activity (FIG. 8F). We conclude from these experiments that
 IFN-.alpha.R1 and IFN-.alpha.R2 are necessary for IFN-.epsilon. to
 stimulate the JAK-STAT pathway. However, we have not ruled out the
 possibility that other receptor component(s) are involved in the
 IFN-.epsilon. -receptor interaction.
 The subtype of human IFN to which murine cells respond is dependent upon
 whether the IFN is species specific and whether the cells express human
 IFN-.alpha.R2 or IFN-.alpha.R2. Expression of human IFN-.alpha.R1 in L929
 cells renders them responsive to human IFN-.alpha.3, but not human
 IFN-.alpha.2 (Gibbs et al., J. Biol. Chem. 271, 28710-28716 [1996]).
 Conversely L929 cells that express human IFN-.alpha.R2 respond to human
 IFN-.alpha.2 but not human IFN-.alpha.3. We took advantage of the species
 specificity of the IFN receptor by analyzing human IFN-.alpha. and
 IFN-.epsilon. induced ISGF3 complex formation in mouse L929 cells stably
 transfected with human IFN-.alpha.R1 or IFN-.alpha.R2 or both. Expression
 of either IFN-.alpha.R alone was not sufficient to confer sensitivity to
 IFN-.epsilon. (FIG. 10). Strikingly, IFN-.epsilon. induced dramatic
 elevation in ISGF3 formation in L929 cells expressing both IFN-.alpha.R1
 and IFN-.alpha.R2. This result indicates that IFN-.epsilon. is a human
 species-specific interferon, it requires both human IFN-.alpha.R subunits
 to signal in murine cells. It also suggests that potential differences
 exist between the interaction of the IFN-.alpha.R with IFN-.epsilon. and
 the interaction of the IFN-.alpha.R with other type I IFNs.
 To explore the biological activities of IFN-.epsilon., we first evaluated
 its antiproliferative effect. Daudi cells were treated with increasing
 concentrations of IFN-.epsilon..sup.His, and cell proliferation was
 measured by Alamar-Blue assay (FIG. 9A) and confirmed by cell counting
 (data not shown). Antibodies that block the functions of IFN-.alpha.R1
 (2E1) and IFN-.alpha.R2 (3B7) were included in the assay. IFN-.epsilon.
 inhibited proliferation of Daudi cells in a dose dependent fashion. The
 antibodies completely (for 3B7) or partially (for 2E1) blocked the growth
 inhibition effect of IFN-.epsilon. at a concentration of 0.5 .mu.g/ml. The
 blocking effect of 2E1 was 100% at concentrations higher than 5 .mu.g/ml
 (data not shown). In contrast, a control antibody against the receptor
 tyrosine kinase HER2 did not block IFN-.epsilon. activity. Second, we used
 several cell lines to evaluate MHCI expression as an indication of immune
 modulation. IFN-.epsilon. increased the expression of MHC I in MOLT-4
 (FIG. 9B), U266 and Daudi cells (data not shown) in dose dependent manner.
 MHC II expression was not induced by IFN-.epsilon. in these cells (data
 not shown). Finally, to test if IFN-.epsilon. exhibits antiviral activity,
 human amniotic WISH cells were challenged with encephalomyocarditis virus
 (EMCV) and the protective effect of IFN-.epsilon. was examined in a
 cytopathic assay. Pretreatment of these cells by IFN-.epsilon. protected
 WISH cells from an EMCV-induced cytopathic effect (FIG. 9C). A similar
 result was obtained using A549 cells challenged with EMCV (data not
 shown).
 Discussion
 In summary, from analysis of sequence homology, chromosomal localization,
 receptor interaction, downstream signaling and biological effects, it can
 be concluded that IFN-.epsilon. belongs to a novel family of Type I IFNs.
 Phylogenetic analysis of the human IFNs indicates that IFN-.epsilon. is
 nearly equally divergent from the IFN-.alpha. cluster, IFN-.omega., and
 IFN-.beta. (FIG. 5). Therefore, IFN-.epsilon. defines a novel type I IFN,
 and is likely to have evolved from a common ancestor by successive gene
 duplication. Like other type I IFNs, the IFN-.epsilon. is intronless (data
 not shown).
 Although IFN-.epsilon. shares common features with other type I IFNs, it is
 unique in several aspects.
 First, IFN-.epsilon. is constitutively expressed in multiple adult tissues
 with a pattern that differs from that of IFN-.alpha. or IFN-.beta. (FIG.
 7A-B). The fact that IFN-.epsilon. transcription was detected in the brain
 suggests that it may play a role in neuronal modulation. Neuronal activity
 and growth were found to be modulated by other type I interferons (Dafny
 et al., Brain Res. 734, 269-274 [1996]; Pliopsys and Massimini,
 Neuroimmunomodulation 2, 31-5 [1995]). In contrast to the IFN-.beta. gene,
 the IFN-.epsilon. gene appears only marginally inducible by
 poly(I).poly.COPYRGT. in human fibroblasts which suggests it may be
 regulated by different inducers.
 Second, the low degree of sequence homology between other type I interferon
 proteins and IFN-.epsilon. has indicated that the receptor interaction
 between IFN-.epsilon. and IFN-.alpha.R might differ from the interaction
 of the same receptor with other type I interferons. Indeed, we have
 experimentally found differences in the interaction between the
 IFN-.alpha.R and IFN-.epsilon. and for other type I interferons.
 IFN-.alpha.R2 has been shown to bind type I interferons in vitro. While we
 observed binding of other type I IFNs to IFN-.alpha.R2 in vitro, we did
 not detect significant binding of IFN-.epsilon. to either IFN-.alpha.R1 or
 IFN-.alpha.R2 alone. In contrast to results with human IFN-.alpha.2 and
 IFN-.alpha.3, we reconstituted IFN-.epsilon. signaling in L929 cells only
 when human IFN-.alpha.1 and human IFN-.alpha.2 were coexpressed (FIG. 10).
 In contrast, expression of either IFN-.alpha.R1 or IFN-.alpha.R2 alone was
 not sufficient to confer sensitivity to IFN-.epsilon.. This result
 indicates that IFN-.epsilon. requires both human IFN-.alpha.R subunits to
 signal in murine cells and suggests potential differences in the
 interactions of the IFN-.alpha.R with IFN-.epsilon. and other type I IFNs.
 To explore the biological activities of IFN-.epsilon., we first evaluated
 the growth inhibitory effect of this interferon. Daudi cells were treated
 with increasing concentrations of IFN-.epsilon..sup.His and cell
 proliferation was measured by an Alamar Blue assay (FIG. 4A), and
 confirmed by cell counting (data not shown). Antibodies that block the
 functions of IFN-.alpha.R1 (2E1) and IFN-.alpha.R2 (3B7) were included in
 the assay. IFN-.epsilon. inhibited proliferation of Daudi cells in a dose
 dependent fashion. Consistent with the result from Stat2 activation (FIG.
 3F), the antibodies completely (for 3B7) or partially (for 2E1) blocked
 the growth inhibition effect of IFN-.epsilon. at a concentration of 0.5
 .mu.g/ml. The blocking effect of 2E1 was 100% at concentrations higher
 than 5 .mu.g/ml (data not shown). In contrast, a control antibody against
 the receptor tyrosine kinase HER2 did not block IFN-.epsilon. activity.
 Second, we used several cell lines the evaluate MHC I expression as a
 indication of immune modulation. IFN.epsilon. induced the expression of
 MHC I in MOLT-4 (FIG. 9B), U266 and Daudi cells (data not shown) in a dose
 dependent manner. MHC II expression was not induced by IFN-.epsilon. in
 these cells (data not shown). Finally, to test if IFN-.epsilon. exhibits
 antiviral activity, human amniotic WISH cells were challenged with
 encephalomycarditis virus (EMCV) and the protective effect of
 IFN-.epsilon. was examined in a cytopathic assay. Pretreatment of these
 cells by IFN-.epsilon. protected WISH cells from EMCV induced cytopathic
 effect (FIG. 9C). A similar result was obtained using 549 cells challenged
 with EMCV (data not shown).
 The specific activities of IFN-.epsilon..sup.His to stimulate these
 biological results are lower than purified IFN-.alpha.R1a (1-2 logs lower
 in JAK-STAT signaling and 2-3 logs lower in biological assays). This could
 indicate either a physiologically relevant and inherently lower specific
 activity of IFN-.epsilon. in these particular assays, or may reflect the
 manner in which the epitope-tagged recombinant protein was prepared. It is
 possible that in vivo, IFN-.epsilon. is expressed at higher levels in some
 tissues and thus the lower potency observed in vitro reflects the
 physiological situation. Alternatively, the nature of ligand-receptor
 interaction is different between IFN-.epsilon. and other Type I IFNs. In
 addition, IFN-.epsilon..sup.His is a His-tagged recombinant protein,
 factors such as protein folding may affect specific activity. Further
 experiments are needed to elucidate the unique character of IFN-.epsilon.,
 which include detailed ligand-receptor interact ion studies and comparison
 of various activities between members of the Type I interferon family.
 Deposit of Material
 The following material has been deposited with the American Type Culture
 Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):