EP1185641A1 - Recepteur de cytokine zcytor10 de souris - Google Patents

Recepteur de cytokine zcytor10 de souris

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Publication number
EP1185641A1
EP1185641A1 EP00928962A EP00928962A EP1185641A1 EP 1185641 A1 EP1185641 A1 EP 1185641A1 EP 00928962 A EP00928962 A EP 00928962A EP 00928962 A EP00928962 A EP 00928962A EP 1185641 A1 EP1185641 A1 EP 1185641A1
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EP
European Patent Office
Prior art keywords
leu
ala
pro
amino acid
ser
Prior art date
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EP00928962A
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German (de)
English (en)
Inventor
Scott R. Presnell
Donald C. Foster
Angela K. Hammond
Si Lok
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Zymogenetics Inc
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Zymogenetics Inc
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Publication of EP1185641A1 publication Critical patent/EP1185641A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Hormones and polypeptide growth factors control proliferation and differentiation of cells of multicellular organisms. These diffusable molecules allow cells to communicate with each other and act in concert to form cells and organs, and to repair damaged tissue.
  • hormones and growth factors include the steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.
  • CSF CSF
  • neutrophils which stimulates development of neutrophils.
  • cytokines are useful in restoring normal blood cell levels in patients suffering from anemia, thrombocytopenia, and neutropenia or receiving chemotherapy for cancer.
  • the demonstrated in vivo activities of these cytokines illustrate the enormous clinical potential of, and need for, other cytokines, cytokine agonists, and cytokine antagonists.
  • the present invention addresses these needs by providing new a hematopoietic cytokine receptor, as well as related compositions and methods.
  • the present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
  • affinity tag is used herein to denote a poiypeptide segment that can be attached to a second poiypeptide to provide for purification or detection of the second poiypeptide or provide sites for attachment of the second poiypeptide to a substrate.
  • any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
  • Affinity tags include a poly- histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzvmol.
  • an "isolated" poiypeptide or protein is a poiypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated poiypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • the term “isolated” does not exclude the presence of the same poiypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • ortholog denotes a poiypeptide or protein obtained from one species that is the functional counterpart of a poiypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • bp base pairs
  • nt nucleotides
  • kb kilobases
  • a “secretory signal sequence” is a DNA sequence that encodes a poiypeptide (a "secretory peptide") that, as a component of a larger poiypeptide, directs the larger poiypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger peptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • the sequence of the mouse zcytorlO poiypeptide was deduced from a single clone that contained its corresponding polynucleotide sequence.
  • the clone was obtained from murine embryo and placenta libraries.
  • Other libraries that might also be searched for such sequences include PBL, thymus, spleen, lymph node, human erythroleukemia cell lines (e.g., TF-1), Raji cells, acute monocytic leukemia cell lines, other lymphoid and hematopoietic cell lines, and the like.
  • nucleotide sequence of a representative mouse zcytorlO-encoding DNA is described in SEQ ID NO: l (from nucleotide 215 to 1285), and its deduced 357 amino acid sequence is described in SEQ ID NO:2.
  • SEQ ID NO:34 from nucleotide 74 to 1151
  • SEQ ID NO:35 The nucleotide sequence of a representative mouse zcytorlO-encoding DNA is described in SEQ ID NO:34 (from nucleotide 74 to 1151), and its deduced 359 amino acid sequence is described in SEQ ID NO:35.
  • SEQ ID NO: 2 Analysis of the mouse zcytorlO poiypeptide encoded by the DNA sequence of SEQ ID NO: 1 revealed an open reading frame encoding 357 amino acids (SEQ ID NO:2) comprising a predicted secretory signal peptide of 14 amino acid residues (residue 1 (Met) to residue 14 (Gly) of SEQ ID NO:2), and a mature poiypeptide of 343 amino acids (residue 15 (Cys) to residue 357 (Leu) of SEQ ID NO:2).
  • the mouse zcytorlO receptor further comprises a cytokine-binding domain of approximately 200 amino acid residues (residues 15 (Cys) to 230 (Pro) of SEQ ID NO:2); a domain linker (residues 114 (Lys) to 121 (Nal) of SEQ ID ⁇ O:2); a penultimate strand region (residues 177 (Ala) to 185 (Arg) of SEQ ID NO:2); a transmembrane domain (residues 231 (Leu) to 251 (Leu) of SEQ ID NO:2); complete intracellular signaling domain (residues 252 (Arg) to 357 (Leu) of SEQ ID NO:2) which contains a "Box I" signaling site (residues 260 (Leu) to 267 (Pro) of SEQ ID NO:2), and a "Box H” signaling site (residues 298 (Thr
  • the mouse zcytorlO receptor further comprises a cytokine-binding domain of approximately 200 amino acid residues (residues 17 (Ala) to 232 (Pro) of SEQ ID NO:35); a domain linker (residues 116 (Lys) to 123 (Val) of SEQ ID NO:35); a penultimate strand region (residues 179 (Ala) to 187 (Arg) of SEQ ID NO:35); a transmembrane domain (residues 233 (Leu) to 253 (Leu) of SEQ ID NO:35); complete intracellular signaling domain (residues 254 (Arg) to 359 (Leu) of SEQ ID NO:35) which contains a "Box I" signaling site (residues 262 (Leu) to 269 (Pro) of SEQ ID NO:35), and a "Box IT signaling site (residues 300 (Thr) to 304 (
  • conserved receptor features in the encoded receptor include (as shown in SEQ ED NO: 35) a conserved Tip residue at positions 137 and 161, and a conserved Arg residue at position 187.
  • the corresponding polynucleotides encoding the mouse zcytorlO poiypeptide regions, domains, motifs, residues and sequences described above are as shown in SEQ ID NO: 34.
  • Block 1 corresponds to amino acid residues 25 (Gly) to amino acid residue 230 (Pro) of SEQ ED NO:2.
  • Block 1 defines a common extracellular cytokine binding domain between the variant forms of zcytorlO (SEQ ID NO:2 and SEQ ID NO:35).
  • Motif 1 The first motif, referred to hereinafter as "Motif 1,” is described in SEQ ID NO:43, and corresponds to amino acid residues 34 (Leu) to amino acid residue 41 (Tip) of SEQ ED NO:2.
  • the fourth motif is Nal-Thr-Nal (NTN), and corresponds to amino acid residues 131 (Nal) to amino acid residue 133 (Nal) of SEQ ID ⁇ O:2.
  • the fifth motif referred to hereinafter as “Motif 5,” is described in SEQ ID ⁇ O:2.
  • SEQ ID NO: 4 and SEQ ID NO: 39 also provide all RNA sequences encoding SEQ ID NO : 2 and SEQ ID NO: 35 respectively by substituting U for T.
  • mouse zcytorlO polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 1071 of SEQ ID NO : 4 and nucleotide 1 to nucleotide 1077 of SEQ ID NO: 39 and their RNA equivalents are contemplated by the present invention.
  • Table 1 sets forth the one-letter codes used within SEQ ID NO: 4 and SEQ ID NO: 39 to denote degenerate nucleotide positions.
  • any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY).
  • WSN can, in some circumstances, encode arginine
  • MGN degenerate codon for arginine
  • AGY serine
  • polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:35. Variant sequences can be readily tested for functionality as described herein.
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2).
  • the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequences disclosed in SEQ ID NO:4 and SEQ ID NO: 39 serve as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein. Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ED NO: l, SEQ ED
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, CA), as well as sites on the Internet, are available tools for analyzing a given sequence and calculating T m based on user defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences, >50 base pairs, is performed at temperatures of about 20-25°C below the calculated T m . For smaller probes, ⁇ 50 base pairs, hybridization is typically carried out at the T m or 5-10°C below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.
  • Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42°C in a solution comprising: about 40-50% formamide, up to about 6X SSC, about 5X Denhardt's solution, zero up to about 10% dextran sulfate, and about 10-20 ⁇ g/ml denatured commercially- available carrier DNA.
  • stringent conditions include temperatures of 20-70°C and a hybridization buffer containing up to 6x SSC and 0-50% formamide; hybridization is then followed by washing filters in up to about 2X SSC.
  • a suitable wash stringency is equivalent to 0.1X SSC to 2X SSC, 0.1% SDS, at 55°C to 65°C.
  • Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence.
  • the washes following hybridization are performed at increasing degrees of stringency to remove non-hybridized polynucleotide probes from hybridized complexes.
  • Stringent hybridization and wash conditions depend on the length of the probe, reflected in the Tm, hybridization and wash solutions used, and are routinely determined empirically by one of skill in the art.
  • RNA is isolated from a tissue or cell that produces large amounts of mouse zcytorlO RNA.
  • tissue and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include PBLs, spleen, thymus, and lymph tissues, Raji cells, human erythroleukemia cell lines (e.g., TF-1), acute monocytic leukemia cell lines, other lymphoid and hematopoietic cell lines, and the like.
  • Total RNA can be prepared using guanidinium isothiocyanate extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al.,
  • Poly (A)+ RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69: 1408-12, 1972).
  • cDNA Complementary DNA
  • genomic DNA can be isolated.
  • Polynucleotides encoding mouse zcytorlO polypeptides are then identified and isolated by, for example, hybridization or polymerase chain reaction (PCR) (Mullis, U.S. Patent No. 4,683,202).
  • a full-length clone encoding mouse zcytorlO can be obtained by conventional cloning procedures.
  • Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron.
  • Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library.
  • Expression libraries can be probed with antibodies to mouse zcytorlO, receptor fragments, or other specific binding partners.
  • the polynucleotides of the present invention can also be synthesized using DNA synthesis machines. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a DNA or a DNA fragment, then each complementary strand is made separately, for example via the phosphoramidite method known in the art.
  • the production of short polynucleotides 60 to 80 bp is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them.
  • special strategies are usually employed for producing longer polynucleotides (longer than about 300 bp). For example, synthetic DNAs (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length.
  • One method for building a synthetic DNA involves producing a set of overlapping, complementary oligonucleotides. Each internal section of the DNA has complementary 3' and 5' terminal extensions designed to base pair precisely with an adjacent section. After the DNA is assembled, the process is completed by ligating the nicks along the backbones of the two strands.
  • synthetic DNAs can be designed with terminal sequences that facilitate insertion into a restriction endonuclease site of a cloning vector.
  • Alternative ways to prepare a full-length DNA are also known in the art. See Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al., Proc. Natl. Acad. Sci.
  • a cDNA can also be cloned using PCR (Mullis, supra.), using primers designed from the representative mouse zcytorlO sequence disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to mouse zcytorlO poiypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • a polynucleotide sequence for the rat ortholog of mouse zcytorlO receptor has been identified and is shown in SEQ ED NO: 15 and the corresponding amino acid sequence shown in SEQ ID NO: 16.
  • Analysis of the rat zcytorlO poiypeptide encoded by the DNA sequence of SEQ ED NO: 15 revealed a partial sequence encoding 110 amino acids (SEQ ID NO: 16) comprising the rat intracellular cytokine signaling domain including part of the transmembrane domain transmembrane domain (residues 1 (Ala) to 12 (I__eu) of SEQ ID NO: 16); a functional intracellular signaling domain (residues 13 (Arg) to 113 (Leu) of SEQ ID NO: 16) which contains a "Box r signaling site (residues 21 21 (Leu) to 28 (Pro) of SEQ ID NO: 16), and a "Box II" signaling site (residues 59 (
  • rat and mouse amino acid sequences contain corresponding structural features described above.
  • the complete rat sequence can be obtained by performing routine 5' RACE using primers within SEQ ID NO: 15.
  • the corresponding polynucleotides encoding the rat zcytorlO poiypeptide regions, domains, motifs, residues and sequences described above are as shown in SEQ ID NO:15.
  • Cytokine receptor subunits are characterized by a multi-domain structure comprising an extracellular domain, a transmembrane domain that anchors the poiypeptide in the cell membrane, and an intracellular domain.
  • the extracellular domain may be a ligand-binding domain
  • the intracellular domain may be an effector domain involved in signal transduction, although ligand-binding and effector functions may reside on separate subunits of a multimeric receptor.
  • the ligand-binding domain may itself be a multi-domain structure.
  • Multimeric receptors include homodimers (e.g., PDGF receptor ⁇ and ⁇ isoforms, erythropoietin receptor, MPL, and G-CSF receptor), heterodimers whose subunits each have ligand-binding and effector domains (e.g., PDGF receptor ⁇ isoform), and multimers having component subunits with disparate functions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSF receptors). Some receptor subunits are common to a plurality of receptors.
  • the AIC2B subunit which cannot bind ligand on its own but includes an intracellular signal transduction domain, is a component of IL-3, GM-CSF, and IL-5 receptors.
  • Many cytokine receptors can be placed into one of four related families on the basis of the structure and function.
  • Hematopoietic receptors for example, are characterized by the presence of a domain containing conserved cysteine residues and the WSXWS motif (SEQ ID NO: 3). Cytokine receptor structure has been reviewed by Urdal, Ann. Reports Med. Chem. 26:221-228, 1991 and Cosman, Cytokine 5:95-106, 1993.
  • the cytokine receptor superfamily is subdivided into several families, for example, the immunoglobulin family (including CSF-1, MGF, EL-1, and PDGF receptors); the hematopoietin family (including IL-2 receptor ⁇ -subunit, GM-CSF receptor ⁇ -subunit, GM-CSF receptor ⁇ - subunit; and G-CSF, EPO, IL-3, IL-4, IL-5, IL-6, IL-7, and EL-9 receptors); TNF receptor family (including TNF (p80) TNF (p60) receptors, CD27, CD30, CD40, Fas, and NGF receptor) .
  • the immunoglobulin family including CSF-1, MGF, EL-1, and PDGF receptors
  • the hematopoietin family including IL-2 receptor ⁇ -subunit, GM-CSF receptor ⁇ -subunit, GM-CSF receptor ⁇ - subunit; and G-CSF, EPO, IL
  • mice zcytorlO sequence suggests that it is a member of the same receptor subfamily as the EPO and growth hormone receptors.
  • Certain receptors in this subfamily e.g., G-CSF
  • Other members of the subfamily e.g., EL-6, EL-11, and LIF receptors
  • combine with a second subunit e.g., a ⁇ -subunit to bind ligand and transduce a signal.
  • Specific ⁇ -subunits associate with a plurality of specific cytokine receptor subunits.
  • the ⁇ -subunit gpl30 (Hibi et al., Cell 63:1149-1157, 1990) associates with receptor subunits specific for EL-6, EL-11, and LEF (Gearing et al., EMBO J. 10:2839-2848, 1991; Gearing et al., U.S. Patent No. 5,284,755).
  • Oncostatin M binds to a heterodimer of LEF receptor and gpl30.
  • CNTF binds to trimeric receptors comprising CNTF receptor, LEF receptor, and gpl30 subunits.
  • ZcytorlO shows sequence and structural homology to EL-2R ⁇ (gamma common receptor; ⁇ c), EL-3R discussed above, and IL-7R that are known to form heterodimeric or multimeric complexes with other cytokine receptor subunits.
  • IL-7R ⁇ heterodimerizes with gamma common to form the receptor for the EL- 7 ligand.
  • TSLP-R another heterodimeric receptor
  • TSLP-R has also been shown to heterodimerize with IL-7R ⁇ to form the receptor for a novel ligand, TSLP (Levine, SD et al., J. Immunol. 162:677-683, 1999; Isaksen, DE et al., J.
  • zcytorlO forms heterodimerizes or forms multimers with other receptor subunits in the gamma common receptor family, creating receptors for other novel cytokines.
  • These cytokines may have functions that overlap those of the gamma common-interacting cytokine, as is the case with EL7 and TSLP.
  • the effects may be quite divergent, or present at different times or under different conditions. Therefore it is important to identify cytokines that interact with zcytorlO in combination with other cytokine receptor subunits.
  • An assay cell line can be created by transfection of zcytorlO and an additional cytokine receptor subunit into a cell line such as BaF3, described herein.
  • a cell line such as BaF3, described herein.
  • Known cytokines and a collection of conditioned media from at least 100 cell lines, as well as tissue preparations, and purified cytokine preparations can be rapidly tested for the ability to support proliferation of this co-transfected cell line.
  • a sample that contains such an activity is further evaluated in the presence of neutralizing antibodies against gamma common receptor (e.g., anti-IL-2 receptor monoclonal antibodies from PharMingen International, San Diego, CA) to confirm that the endogenous gamma common in the BaF3 cells is not participating in the receptor complex.
  • neutralizing antibodies against gamma common receptor e.g., anti-IL-2 receptor monoclonal antibodies from PharMingen International, San Diego, CA
  • a cell line which produces an activity that supports non-gamma common-mediated proliferation, can then be used to produce a cDNA library for ligand cloning.
  • Such Baf3 assay cell lines can be created with zcytorlO co-expressed with other receptor complexes including but not limited to zcytorlO receptor in combination with an cytokine receptor fusion comprising one or more of the IL-2 receptor components (EL- 2R ⁇ , EL-2R ⁇ , EL-2R ⁇ ), zcytorlO receptor with one or more of the EL-4/IL-13 receptor family receptor components (EL-4R ⁇ , IL-13R , EL-13R ⁇ '), as well as other Interleukin receptors (e.g., EL-15 R ⁇ , IL-7R ⁇ , IL-9R , IL-21R (Zalphal l receptor; commonly owned US Pat. Application No. 09/404,641).
  • SEQ ED NO:l represents a single allele of mouse zcytorlO and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO:l, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ED NO:2.
  • cDNAs generated from alternatively spliced mRNAs, such as SEQ ID NO:34, which retain the properties of the mouse zcytorlO poiypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
  • the present invention also provides isolated mouse zcytorlO polypeptides that are substantially similar to the polypeptides of SEQ ID NO:2, or SEQ ED NO:35, and their orthologs.
  • the term "substantially similar” is used herein to denote polypeptides having at least 70%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO:2, or SEQ ED NO:35 or their orthologs.
  • Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:2 or SEQ ID NO:35 or its orthologs.) Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio.
  • Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.
  • the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zpepl4.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat' I Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
  • the ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:181 (1974)), which allows for amino acid insertions and deletions.
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other FASTA program parameters set as default.
  • the BLOSUM62 table (Table 3) is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat' I Acad. Sci. USA 59:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed below), the language "conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • Variant or substantially homologous mouse zcytorlO polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the poiypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • the present invention thus includes polypeptides of from about 489 to about 568 amino acid residues that comprise a sequence that is at least 80%, preferably at least 90%, and more preferably 95% or more identical to the corresponding region of SEQ ID NO:2 or SEQ ID NO:35.
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the mouse zcytorlO poiypeptide and the affinity tag. Suitable sites include thrombin cleavage sites and factor Xa cleavage sites. Table 4
  • Auxiliary domains can be fused to mouse zcytorlO polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen).
  • a mouse zcytorlO poiypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain.
  • Poiypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34: 1-9, 1996.
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, tr ⁇ n.s-3-methylproline, 2,4-methanoproline, cw-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, ⁇ //o-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
  • the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395- 403, 1993).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for mouse zcytorlO amino acid residues.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991).
  • site-directed mutagenesis or alanine-scanning mutagenesis Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991.
  • single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g. ligand binding and signal transduction) as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699-4708, 1996.
  • Sites of ligand-receptor, protein-protein or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899- 904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.
  • the identities of essential amino acids can also be inferred from analysis of homologies with related receptors.
  • Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule.
  • Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity and computer analysis using available software (e.g., the Insight ⁇ ® viewer and homology modeling tools; MSI, San Diego, CA), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., Current Opin. Struct. Biol. 6:3-10, 1996).
  • modifications to molecules or identifying specific fragments determination of structure will be accompanied by evaluating activity of modified molecules.
  • Amino acid sequence changes are made in zcytorlO polypeptides, including zcytorlO soluble receptors and heterodimeric receptor polypeptides, so as to minimize disruption of higher order structure essential to biological activity.
  • zcytorlO polypeptides, zcytorlO soluble receptors and heterodimeric receptor polypeptides comprise one or more helices
  • changes in amino acid residues will be made so as not to disrupt the helix geometry and other components of the molecule where changes in conformation abate some critical function, for example, binding of the molecule to its binding partners.
  • amino acid sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995).
  • Other techniques that are well known in the art compare folding of a variant protein to a standard molecule (e.g., the native protein). For example, comparison of the cysteine pattern in a variant and standard molecules can be made.
  • Mass spectrometry and chemical modification using reduction and alkylation provide methods for determining cysteine residues which are associated with disulfide bonds or are free of such associations (Bean et al., Anal. Biochem. 201:216-226, 1992; Gray, Protein Sci.
  • SEQ ED NO:2 and SEQ ID NO:35 can be generated (Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88: 1-18, 1986 and Triquier et al., Protein Engineering JX: 153-169, 1998).
  • the profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored.
  • zcytorlO poiypeptide hydrophilic regions include (1) amino acid number 150 (Arg) to amino acid number 155 (Asp) of SEQ ID NO:2; (2) amino acid number 254 (Arg) to amino acid number 259 (Ala) of SEQ ID NO:2; (3) amino acid number 296 (Ala) to amino acid number 301 (Glu) of SEQ ID NO:2; (4) amino acid number 297 (Arg) to amino acid number 302 (Asp) of SEQ ID NO:2; and (5) amino acid number 310 (Lys) to amino acid number 315 (Glu) of SEQ ID NO:2.
  • the corresponding zcytorlO hydrophilic peptides of SEQ ED NO:35 are also included with comparison of the above hydrophilic peptides SEQ ED NO:2 in reference to SEQ ID NO:35.
  • hydrophilicity or hydrophobicity will be taken into account when designing modifications in the amino acid sequence of a zcytorlO polypeptides, zcytorlO soluble receptors and heterodimeric receptor polypeptides, so as not to disrupt the overall structural and biological profile.
  • hydrophobic residues selected from the group consisting of Val, E_eu and He or the group consisting of Met, Gly, Ser, Ala, Tyr and Tip.
  • residues tolerant of substitution could include such as shown in SEQ ID NO: 2 and SEQ ED NO:35. Cysteine residues will be relatively intolerant of substitution.
  • the identities of essential amino acids can also be inferred from analysis of sequence similarity between class I cytokine receptor family members with zcytorlO polypeptides, including zcytorlO soluble receptors and heterodimeric receptors. Using methods such as "FASTA" analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions.
  • An alternative approach to identifying a variant zcytorlO, zcytorlO soluble receptors and heterodimeric receptor polynucleotides on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ED NO: 1, or SEQ ED NO:34 as discussed above.
  • Other methods of identifying essential amino acids in the polypeptides of the present invention are procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et al., Proc. Natl Acad. Sci.
  • the present invention also includes functional fragments of zcytorlO polypeptides, zcytorlO soluble receptors and heterodimeric receptor polypeptides and nucleic acid molecules encoding such functional fragments.
  • a "functional" zcytorlO poiypeptide includes zcytorlO soluble receptors and heterodimeric receptors or fragment thereof defined herein is characterized by its proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to a either soluble or immobilized anti- zcytorlO antibody, a zcytorlO ligand or cytokine receptor subunit.
  • the zcytorlO receptor is characterized by a class I cytokine receptor structure.
  • the present invention further provides fusion proteins encompassing: (a) homodimeric or multimeric poiypeptide molecules comprising an extracellular or intracellular domain described herein; and (b) functional fragments comprising one or more of these domains.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encode zcytorlO polypeptides, zcytorlO soluble receptors and heterodimeric receptor polypeptides.
  • DNA molecules having the nucleotide sequence of SEQ ID NO:l or fragments thereof can be digested with Bal3l nuclease to obtain a series of nested deletions.
  • DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for zcytorlO polypeptides, including zcytorlO soluble receptors and heterodimeric receptor activity, or for the ability to bind anti-zcytorlO antibodies or zcytorlO receptor.
  • zcytorlO polypeptides including zcytorlO soluble receptors and heterodimeric receptor activity
  • oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired zcytorlO polypeptides, including zcytorlO soluble receptors and heterodimeric receptor fragment.
  • particular fragments of zcytorlO polypeptides, including zcytorlO soluble receptors and heterodimeric receptor polynucleotides can be synthesized using the polymerase chain reaction.
  • Variants of the disclosed mouse zcytorlO DNA and poiypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-91, 1994, Stemmer. Proc. Natl. Acad. Sci. USA 91:10747-51. 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process.
  • Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized mouse zcytorlO receptor polypeptides in host cells.
  • Preferred assays in this regard include cell proliferation assays and biosensor-based ligand-binding assays, which are described below.
  • Mutagenized DNA molecules that encode active receptors or portions thereof e.g., ligand-binding fragments, signaling domains, and the like
  • polypeptides may include additional amino acids from, for example, part or all of the transmembrane and intracellular domains.
  • polypeptides may also include additional poiypeptide segments as generally disclosed herein such as labels, affinity tags, and the like.
  • any mouse zcytorlO poiypeptide, including variants, soluble receptors, and fusion polypeptides or proteins one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.
  • the mouse zcytorlO polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred.
  • a DNA sequence encoding a mouse zcytorlO poiypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • a secretory signal sequence also known as a leader sequence, prepro sequence or pre sequence
  • a secretory signal sequence also known as a leader sequence, prepro sequence or pre sequence
  • the secretory signal sequence may be that of mouse zcytorlO, or may be derived from another secreted protein (e.g., t- PA) or synthesized de novo.
  • the secretory signal sequence is operably linked to the mouse zcytorlO DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized poiypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the poiypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No.
  • a signal fusion poiypeptide can be made wherein a secretory signal sequence derived from amino acid 1 (Met) to amino acid 19 (Gly) of SEQ ID NO:2, or amino acid 1 (Met) to amino acid 16 (Ala) of SEQ ID NO:35, is operably linked to another poiypeptide using methods known in the art and disclosed herein.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • Such constructs have numerous applications known in the art.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein.
  • Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.
  • Cultured mammalian cells are suitable hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981 : Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No.
  • CRL 1573 Graham et al., J. Gen. Virol. 36:59-72, 1977
  • Chinese hamster ovary e.g. CHO-K1; ATCC No. CCL 61
  • Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland.
  • strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288.
  • Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
  • a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
  • Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • hygromycin resistance multi-drug resistance
  • puromycin acetyltransferase can also be used.
  • Alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa calif ornica nuclear polyhedrosis virus (AcNPV).
  • AcNPV Autographa calif ornica nuclear polyhedrosis virus
  • a second method of making recombinant mouse zcytorlO baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al., J Virol 67:4566-79, 1993).
  • This system which utilizes transfer vectors, is sold in the Bac-to-BacTM kit (Life Technologies, Rockville, MD).
  • This system utilizes a transfer vector, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the mouse zcytorlO poiypeptide into a baculovirus genome maintained in R, coli as a large plasmid called a "bacmid.” See, Hill-Perkins, M.S.
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed mouse zcytorlO poiypeptide, for example, a Glu- Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985).
  • a transfer vector containing mouse zcytorlO is transformed into E. Coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.
  • Recombinant virus that expresses mouse zcytorlO is subsequently produced.
  • Recombinant viral stocks are made by methods commonly used in the art.
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda.
  • Another suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435).
  • Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 ⁇ TM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells. Procedures used are generally described in available laboratory manuals (King, L.
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
  • Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • a preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311 ; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No.
  • Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.
  • Pichia methanolica as host for the production of recombinant proteins is disclosed in WEPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
  • DNA molecules for use in transforming R. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a R. methanolica alcohol utilization gene (AUG1 or A UG2).
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • a preferred selectable marker for use in Pichia methanolica is a R. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine.
  • host cells For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a poiypeptide of interest into P. methanolica cells. It is preferred to transform P.
  • methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.). When expressing a mouse zcytorlO poiypeptide in bacteria such as E.
  • the poiypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence.
  • the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured poiypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the poiypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell.
  • R. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C.
  • Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
  • a preferred culture medium for R. methanolica is Y ⁇ PD (2% D-glucose, 2% BactoTM Peptone (Difco Laboratories, Detroit, MI), 1% BactoTM yeast extract (Difco Laboratories), 0.004% adenine and
  • a mouse zcytorlO cytokine receptor (including transmembrane and intracellular domains) is produced by a cultured cell, and the cell is used to screen for ligands for the receptor, including the natural ligand, as well as agonists and antagonists of the natural ligand.
  • a cDNA or gene encoding the receptor is combined with other genetic elements required for its expression (e.g., a transcription promoter), and the resulting expression vector is inserted into a host cell.
  • Cells that express the DNA and produce functional receptor are selected and used within a variety of screening systems.
  • Mammalian cells suitable for use in expressing the novel receptors of the present invention and transducing a receptor-mediated signal include cells that express a ⁇ -subunit, such as gpl30, and cells that co-express gpl30 and LIF receptor (Gearing et al., EMBO J. 10:2839-2848, 1991; Gearing et al., U.S. Patent No. 5,284,755).
  • Preferred cells of this type include the human TF-1 cell line (ATCC number CRL-2003) and the DA-1 cell line (Branch et al., Blood 69: 1782, 1987; Broudy et al., Blood 75:1622-1626, 1990).
  • suitable host cells can be engineered to produce a ⁇ -subunit or other cellular component needed for the desired cellular response.
  • the murine cell line BaF3 (Palacios and Steinmetz, Cell 4 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol.
  • a baby hamster kidney (BHK) cell line, or the CTLL-2 cell line can be transfected to express the mouse gpl30 subunit, or mouse gpl30 and LIF receptor, in addition to mouse zcytorlO. It is generally preferred to use a host cell and receptor(s) from the same species, however this approach allows cell lines to be engineered to express multiple receptor subunits from any species, thereby overcoming potential limitations arising from species specificity. In the alternative, species homologs of the mouse receptor cDNA can be cloned and used within cell lines from the same species, such as a mouse cDNA in the BaF3 cell line.
  • Cells expressing functional mouse zcytorlO are used within screening assays.
  • a variety of suitable routine assays are high throughput and well known in the art. These assays are based on the detection of a biological response in the target cell.
  • One such assay is a cell proliferation assay.
  • Cells are cultured in the presence or absence of a test compound, and cell proliferation is detected by, for example, measuring incorporation of tritiated thymidine or by colorimetric assay based on the metabolic breakdown of Alymar BlueTM (AccuMed, Chicago, EL) or 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Mosman, J. Immunol. Meth. 65: 55-63, 1983).
  • An alternative assay format uses cells that are further engineered to express a reporter gene. The reporter gene is linked to a promoter element that is responsive to the receptor-linked pathway, and the assay detects activation of transcription of the reporter gene.
  • a preferred promoter element in this regard is a serum response element, or SRE (see, for example, Shaw et al., Cell 56:563- 572, 1989).
  • a preferred such reporter gene is a luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987). Expression of the luciferase gene is detected by luminescence using methods known in the art (e.g., Baumgartner et al., J. Biol. Chem. 269:19094- 29101, 1994; Schenborn and Goiffin, Promega Notes 41:11, 1993). Luciferase assay kits are commercially available from, for example, Promega Corp., Madison, WI.
  • Target cell lines of this type can be used to screen libraries of chemicals, cell- conditioned culture media, fungal broths, soil samples, water samples, and the like.
  • a bank of cell- or tissue-conditioned media samples can be assayed on a target cell to identify cells that produce ligand. Positive cells are then used to produce a cDNA library in a mammalian cell expression vector, which is divided into pools, transfected into host cells, and expressed. Media samples from the transfected cells are then assayed, with subsequent division of pools, retransfection, subculturing, and re- assay of positive cells to isolate a clonal cell line expressing the ligand. Media samples conditioned by kidney, liver, spleen, thymus, other lymphoid tissues, or T-cells are preferred sources of ligand for use in screening procedures.
  • a natural ligand for mouse zcytorlO can also be identified by mutagenizing a cytokine-dependent cell line expressing mouse zcytorlO and culturing it under conditions that select for autocrine growth. See WIPO publication WO 95/21930. Within a typical procedure, cells expressing mouse zcytorlO are mutagenized, such as with EMS. The cells are then allowed to recover in the presence of the required cytokine, then transferred to a culture medium lacking the cytokine.
  • Surviving cells are screened for the production of a ligand for mouse zcytorlO, such as by adding soluble (ligand-binding) receptor poiypeptide to the culture medium or by assaying conditioned media on wild-type cells and transfected cells expressing the mouse zcytorlO.
  • Preferred cell lines for use within this method include cells that are transfected to express gpl30 or gpl30 in combination with LEF receptor.
  • Preferred such host cell lines include transfected CTLL-2 cells (Gillis and Smith, Nature 268:154-156, 1977) and transfected BaF3 cells.
  • a secretion trap method employing mouse zcytorlO soluble receptor poiypeptide can be used to isolate a mouse zcytorlO ligand (Aldrich, et al, Cell 87: 1161-1169, 1996).
  • a cDNA expression library prepared from a known or suspected ligand source is transfected into COS-7 cells.
  • the cDNA library vector generally has an SV40 origin for amplification in COS-7 cells, and a CMV promoter for high expression.
  • the transfected COS-7 cells are grown in a monolayer and then fixed and permeabilized.
  • Tagged or biotin-labeled mouse zcytorlO soluble receptor is then placed in contact with the cell layer and allowed to bind cells in the monolayer that express an anti-complementary molecule, i.e., a mouse zcytorlO ligand.
  • a cell expressing a ligand will thus be bound with receptor molecules.
  • An anti-tag antibody (anti-Ig for Ig fusions, M2 or anti-FLAG for FLAG-tagged fusions, streptavidin, and the like) which is conjugated with horseradish peroxidase (HRP) is used to visualize these cells to which the tagged or biotin-labeled mouse zcytorlO soluble receptor has bound.
  • the HRP catalyzes deposition of a tyramide reagent, for example, tyramide-FITC.
  • a tyramide reagent for example, tyramide-FITC.
  • a commercially-available kit can be used for this detection (for example, Renaissance TSA-DirectTM Kit; NEN Life Science Products, Boston, MA).
  • Cells which express mouse zcytorlO receptor ligand will be identified under fluorescence microscopy as green cells and picked for subsequent cloning of the ligand using procedures for plasmid rescue as outlined in Aldrich, et al, supra., followed by subsequent rounds of secretion trap assay until single clones are identified.
  • the activity of mouse zcytorlO poiypeptide can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses.
  • An exemplary device is the CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell, H.M. et al., Science 257: 1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228:84-108, 1997; Arimilli, S.
  • the microphysiometer can be used for assaying eukaryotic, prokaryotic, adherent or non-adherent cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including agonists, ligands, or antagonists of the mouse zcytorlO poiypeptide.
  • the microphysiometer is used to measure responses of a mouse zcytorlO-expressing eukaryotic cell, compared to a control eukaryotic cell that does not express mouse zcytorlO poiypeptide.
  • Mouse zcytorlO-expressing eukaryotic cells comprise cells into which mouse zcytorlO has been transfected, as described herein, creating a cell that is responsive to mouse zcytorlO-rnodulating stimuli, or are cells naturally expressing mouse zcytorlO, such as mouse zcytorlO- expressing cells derived from lymphoid, spleen, thymus tissue, PBLs, lung, liver, heart or testis.
  • mice zcytorlO-modulated responses Differences, measured by an increase or decrease in extracellular acidification, in the response of cells expressing mouse zcytorlO, relative to a control, are a direct measurement of mouse zcytorlO-modulated cellular responses. Moreover, such mouse zcytorlO-modulated responses can be assayed under a variety of stimuli.
  • a method of identifying agonists and antagonists of mouse zcytorlO poiypeptide comprising providing cells expressing a mouse zcytorlO poiypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting an increase or a decrease in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • Antagonists and agonists, including the natural ligand for zcytorlO poiypeptide can be rapidly identified using this method.
  • hybrid receptor polypeptides include the use of hybrid receptor polypeptides. These hybrid polypeptides fall into two general classes. Within the first class, the intracellular domain of mouse zcytorlO, comprising approximately residues 252 (Arg) to 357 (I_eu) of SEQ ID NO:2, or residues 254 (Arg) to 359 (Leu) of SEQ ID NO:35, is joined to the ligand-binding domain of a second receptor. It is preferred that the second receptor be a hematopoietic cytokine receptor, such as mpl receptor (Souyri et al., Cell 63: 1137-1147, 1990). The hybrid receptor will further comprise a transmembrane domain, which may be derived from either receptor.
  • the hybrid receptor will further comprise a transmembrane domain, which may be derived from either receptor.
  • a DNA construct encoding the hybrid receptor is then inserted into a host cell.
  • Cells expressing the hybrid receptor are cultured in the presence of a ligand for the binding domain and assayed for a response.
  • This system provides a means for analyzing signal transduction mediated by mouse zcytorlO while using readily available ligands. This system can also be used to determine if particular cell lines are capable of responding to signals transduced by mouse zcytorlO.
  • a second class of hybrid receptor polypeptides comprise the extracellular (ligand-binding) domain of mouse zcytorlO (approximately residues 15 (Cys) to 230 (Pro) of SEQ ID NO:2, or 17 (Ala) to 232 (Pro) of SEQ ED NO:35) with a cytoplasmic domain of a second receptor, preferably a cytokine receptor, and a transmembrane domain.
  • the transmembrane domain may be derived from either the mouse zcytorlO receptor or second receptor.
  • Hybrid receptors of this second class are expressed in cells known to be capable of responding to signals transduced by the second receptor.
  • the mouse zcytorlO may play a role in early thymocyte development and/or immune response regulation. These processes involve stimulation of cell proliferation and differentiation in response to the binding of one or more cytokines to their cognate receptors.
  • agonists including the natural ligand
  • antagonists have enormous potential in both in vitro and in vivo applications.
  • Compounds identified as receptor agonists are useful for stimulating proliferation and development of target cells in vitro and in vivo.
  • agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture.
  • Agonists are thus useful in specifically promoting the growth and/or development of T-cells, B-cells, and other cells of the lymphoid and myeloid lineages, and hematopoietic cells in culture. Assays for determining growth and development of these cell lineages are well known in the art.
  • Agonist ligands for mouse zcytorlO may be useful in stimulating cell- mediated immunity and for stimulating lymphocyte proliferation, such as in a mouse model for use in studying the treatment of infections involving immunosuppression, including certain viral infections. Additional uses include use in a mouse model for studying tumor suppression, where malignant transformation results in tumor cells that are antigenic. Agonist ligands could be used to induce cytotoxicity, which may be mediated through activation of effector cells such as T-cells, NK (natural killer) cells, or LAK (lymphoid activated killer) cells, or induced directly through apoptotic pathways, ans as such applied in mouse models for human disease. Agonist ligands may also be useful in a mouse model for studying potential treatments for leukopenias by increasing the levels of the affected cell type, and for studies involving enhancing the regeneration of the T-cell repertoire after bone marrow transplantation.
  • Antagonist or agonist ligands or compounds may find utility in the suppression of the immune system, and provide a useful mouse model for studying the treatment of autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, diabetes mellitis, inflammatory bowel disease, Crohn's disease, etc. Immune suppression can also be used to reduce rejection of tissue or organ transplants and grafts and to treat T-cell specific leukemias or lymphomas by inhibiting proliferation of the affected cell type.
  • Mouse zcytorlO may also be used within diagnostic systems for the detection of circulating levels of both human and mouse ligand.
  • antibodies or other agents that bind to mouse zcytorlO can be used to detect circulating receptor polypeptides.
  • Elevated or depressed levels of ligand or receptor polypeptides may be indicative of pathological conditions, including cancer.
  • Soluble receptor polypeptides may contribute to pathologic processes and can be an indirect marker of an underlying disease; as such, a mouse model expressing mouse zcytorlO soluble receptors can be utilized as a model to study a human pathologic process.
  • soluble EL-2 receptor in human serum have been associated with a wide variety of inflammatory and neoplastic conditions, such as myocardial infarction, asthma, myasthenia gravis, rheumatoid arthritis, acute T-cell leukemia, B-cell lymphomas, chronic lymphocytic leukemia, colon cancer, breast cancer, and ovarian cancer (Heaney et al., Blood 87:847-857, 1996).
  • a ligand-binding poiypeptide of a mouse zcytorlO receptor can be prepared by expressing a truncated DNA encoding the mouse zcytorlO cytokine binding domain (approximately residue 15 (Cys) to 230 (Pro) of the murine receptor (SEQ ID NO:2); or approximately 17 (Ala) to 232 (Pro) of SEQ ED NO:35) or the corresponding region of a mouse paralog or non-mouse receptor. It is preferred that the extracellular domain be prepared in a form substantially free of transmembrane and intracellular poiypeptide segments.
  • ligand-binding poiypeptide fragments within the mouse zcytorlO cytokine binding domain can also serve as mouse zcytorlO soluble receptors for uses described herein.
  • the receptor DNA is linked to a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide or a mouse zcytorlO secretory peptide.
  • a C-terminal extension such as a poly-histidine tag, substance P, FlagTM peptide (Hopp et al., Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co., New Haven, CT) or another poiypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the receptor poiypeptide.
  • a receptor extracellular domain can be expressed as a fusion with immunoglobulin heavy chain constant regions, typically an
  • F c fragment which contains two constant region domains and lacks the variable region.
  • Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two receptor polypeptides are arrayed in close proximity to each other. Fusions of this type can be used to affinity purify the cognate ligand from solution, as an in vitro assay tool, to block signals in vitro by specifically titrating out ligand, and as antagonists in vivo by administering them parenterally to bind circulating ligand and clear it from the circulation.
  • a mouse zcytorlO-Ig chimera is added to a sample containing the ligand (e.g., cell-conditioned culture media or tissue extracts) under conditions that facilitate receptor-ligand binding (typically near-physiological temperature, pH, and ionic strength).
  • the chimera-ligand complex is then separated by the mixture using protein A, which is immobilized on a solid support (e.g., insoluble resin beads).
  • the ligand is then eluted using conventional chemical techniques, such as with a salt or pH gradient.
  • the chimera itself can be bound to a solid support, with binding and elution carried out as above. Collected fractions can be re-fractionated until the desired level of purity is reached.
  • mouse zcytorlO soluble receptors can be used as a "ligand sink," i.e., antagonist, to bind ligand in vivo or in vitro in a murine model for therapeutic or other applications where the presence of the ligand is not desired.
  • the mouse zcytorlO soluble receptors can be used as an antagonist to bind human ligand in vitro or in vivo for therapeutic or other applications.
  • mouse zcytorlO soluble receptors can be used as a direct antagonist of the ligand in vivo, and may aid in reducing progression and symptoms associated with the disease.
  • mouse zcytorlO soluble receptor can be used to slow the progression of cancers that over- express zcytorlO receptors, by binding ligand in vivo that would otherwise enhance proliferation of those cancers. Similar in vitro applications for a mouse zcytorlO soluble receptor can be used, for instance, as a negative selection to select cell lines that grow in the absence of mouse zcytorlO ligand.
  • mouse zcytorlO soluble receptor can be used in vivo or in diagnostic applications to detect zcytorlO ligand-expressing cancers in vivo or in tissue samples, including human cancers and tissues that express a human orthologous ligand.
  • the mouse zcytorlO soluble receptor can be conjugated to a radio-label or fluorescent label as described herein, and used to detect the presence of the human or mouse ligand in a tissue sample using an in vitro ligand-receptor type binding assay, or fluorescent imaging assay.
  • a radio-labeled mouse zcytorlO soluble receptor could be administered in vivo to detect ligand-expressing solid tumors through a radio- imaging method known in the art.
  • mouse zcytorlO receptor As a cytokine receptor, a role for the mouse zcytorlO receptor in proliferation, differentiation, and/or activation of immune cells, and in development and regulation of immune responses is suggested.
  • the interaction of mouse zcytorlO with its ligand may stimulate proliferation and development of myeloid cells and may, like EL-2, EL-6, LIF, IL-11 and OSM (Baumann et al., J. Biol. Chem. 268:8414-8417, 1993), induce acute-phase protein synthesis in hepatocytes.
  • polypeptides of the present invention it is preferred to purify the polypeptides of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • a purified poiypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Expressed recombinant mouse zcytorlO polypeptides can be purified using fractionation and/or conventional purification methods and media.
  • Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
  • Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl- Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica- based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used.
  • These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
  • These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.
  • the polypeptides of the present invention can be isolated by exploitation of their biochemical, structural, and biological properties.
  • immobilized metal ion adsorption (EVIAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • a fusion of the poiypeptide of interest and an affinity tag may be constructed to facilitate purification.
  • an affinity tag e.g., maltose-binding protein, an immunoglobulin domain
  • poiypeptide fusions or hybrid mouse zcytorlO proteins
  • regions or domains of the inventive mouse zcytorlO in combination with those of other mouse or human cytokine receptor family proteins, or heterologous proteins (Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511-5, 1994, and references therein).
  • These methods allow the determination of the biological importance of larger domains or regions in a poiypeptide of interest.
  • Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a poiypeptide, and can be applied to polypeptides of unknown structure.
  • Fusion polypeptides or proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them.
  • a polynucleotide encoding one or more components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain(s) conferring a biological function may be swapped between mouse zcytorlO of the present invention with the functionally equivalent domain(s) from another cytokine family member.
  • Such domains include, but are not limited to, the secretory signal sequence, extracellular cytokine binding domain, transmembrane domain, and intracellular signaling domain, Box I and Box II sites, block 1, mammalian signaling motif, and motifs 1-6, as disclosed herein.
  • Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known family proteins, depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.
  • Standard molecular biological and cloning techniques can be used to swap the equivalent domains between the mouse zcytorlO poiypeptide and those polypeptides to which they are fused (e.g., human zcytorlO or other cytokine receptors).
  • a DNA segment that encodes a domain of interest e.g., a mouse zcytorlO domain described herein, is operably linked in frame to at least one other DNA segment encoding an additional poiypeptide (for instance a domain or region from another cytokine receptor, such as IL-7R, IL-3R, IL2R, EPO receptor, or the like), and inserted into an appropriate expression vector, as described herein.
  • DNA constructs are made such that the several DNA segments that encode the corresponding regions of a poiypeptide are operably linked in frame to make a single construct that encodes the entire fusion protein, or a functional portion thereof.
  • a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal poiypeptide followed by a cytokine binding domain, followed by a transmembrane domain, followed by an intracellular signaling domain.
  • fusion proteins can be expressed, isolated, and assayed for activity as described herein.
  • Mouse zcytorlO polypeptides or fragments thereof may also be prepared through chemical synthesis, mouse zcytorlO polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
  • Polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Methods for synthesizing polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595, 1970. After the entire synthesis of the desired peptide on a solid support, the peptide-resin is with a reagent which cleaves the poiypeptide from the resin and removes most of the side-chain protecting groups. Such methods are well established in the art. The activity of molecules of the present invention can be measured using a variety of assays that measure cell differentiation and proliferation. Such assays are well known in the art.
  • Proteins of the present invention are useful for example, in treating lymphoid, immune, inflammatory, spleenic, blood or bone disorders, and can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model.
  • host cells expressing a zcytorlO soluble receptor poiypeptide can be embedded in an alginate environment and injected (implanted) into recipient animals.
  • Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation and diffusion chambers are a means to entrap transfected mammalian cells or primary mammalian cells.
  • non-immunogenic "encapsulations” permit the diffusion of proteins and other macromolecules secreted or released by the captured cells to the recipient animal. Most importantly, the capsules mask and shield the foreign, embedded cells from the recipient animal's immune response. Such encapsulations can extend the life of the injected cells from a few hours or days (naked cells) to several weeks (embedded cells).
  • Alginate threads provide a simple and quick means for generating embedded cells.
  • the materials needed to generate the alginate threads are known in the art.
  • 3% alginate is prepared in sterile H2O, and sterile filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered. An approximately 50% cell suspension (containing about 5 x 10 ⁇ to about 5 x 10 ⁇ cells/ml) is mixed with the 3% alginate solution.
  • One ml of the alginate/cell suspension is extruded into a 100 mM sterile filtered CaCl2 solution over a time period of -15 min, forming a "thread".
  • the extruded thread is then transferred into a solution of 50 mM CaCl2, and then into a solution of 25 mM CaCl2.
  • the thread is then rinsed with deionized water before coating the thread by incubating in a 0.01% solution of poly-L-lysine.
  • the thread is rinsed with Lactated Ringer's Solution and drawn from solution into a syringe barrel (without needle). A large bore needle is then attached to the syringe, and the thread is intraperitoneally injected into a recipient in a minimal volume of the Lactated Ringer's Solution.
  • viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for review, see T.C. Becker et al., Meth. Cell Biol. 43: 161-89, 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44-53, 1997).
  • adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to high- titer; (iii) infect a broad range of mammalian cell types; and (iv) can be used with a large number of different promoters including ubiquitous, tissue specific, and regulatable promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
  • adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary).
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
  • tissue e.g., liver
  • adenoviral vectors containing various deletions of viral genes can be used in an attempt to reduce or eliminate immune responses to the vector.
  • Such adenoviruses are El deleted, and in addition contain deletions of E2A or E4 (Lusky, M. et al., J. Virol. 72:2022-2032, 1998; Raper, S.E. et al., Human Gene Therapy 9:671- 679, 1998).
  • deletion of E2b is reported to reduce immune responses (Amalfitano, A. et al., J. Virol. 72:926-933, 1998).
  • by deleting the entire adenovirus genome very large inserts of heterologous DNA can be accommodated.
  • the adenovirus system can also be used for protein production in vitro.
  • the cells By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
  • adenovirus vector infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Gamier et al., Cytotechnol. 15:145-55, 1994).
  • an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins may also be effectively obtained.
  • mouse zcytorlO agonists including the natural ligand substrate/ cofactor/ etc.
  • antagonists have enormous potential in both in vitro and in vivo applications.
  • Compounds identified as mouse zcytorlO agonists are useful for stimulating growth of immune and hematopoietic cells in vitro and in vivo.
  • mouse zcytorlO and agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture.
  • Agonists are thus useful in specifically promoting the growth and/or development of T-cells, B-cells, and other cells of the lymphoid and myeloid lineages in culture.
  • mouse zcytorlO soluble receptor, agonist, or antagonist may be used in vitro in an assay to measure stimulation of colony formation from isolated primary bone marrow cultures. Such assays are well known in the art.
  • Antagonists are also useful as research reagents for characterizing sites of ligand-receptor interaction.
  • mouse zcytorlO antagonists include anti-mouse zcytorlO antibodies and soluble mouse zcytorlO receptors, as well as other peptidic and non-peptidic agents (including ribozymes).
  • Mouse zcytorlO can also be used to identify modulators (e.g, antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of mouse zcytorlO. In addition to those assays disclosed herein, samples can be tested for inhibition of mouse zcytorlO activity within a variety of assays designed to measure mouse zcytorlO binding, oligomerization, or the stimulation inhibition of mouse zcytorlO-dependent cellular responses. For example, mouse zcytorlO-expressing cell lines can be transfected with a reporter gene construct that is responsive to a mouse zcytorlO-stimulated cellular pathway.
  • modulators e.g, antagonists
  • Reporter gene constructs of this type are known in the art, and will generally comprise a mouse zcytorlO-DNA response element operably linked to a gene encoding an assay detectable protein, such as luciferase.
  • DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563- 72, 1989). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem.
  • a method of identifying agonists of mouse zcytorlO poiypeptide comprising providing cells responsive to a mouse zcytorlO poiypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a increase in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • third cell containing the reporter gene construct described above, but not expressing zaplphal 1 receptor, can be used as a control cell to assess non-specific, or non-mouse zcytorlO-mediated, stimulation of the reporter.
  • Agonists including the natural ligand, are therefore useful to stimulate or increase mouse zcytorlO poiypeptide function.
  • a mouse zcytorlO ligand-binding poiypeptide such as the cytokine binding domain disclosed herein, can also be used for purification of ligand.
  • the poiypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting medium will generally be configured in the form of a column, and fluids containing ligand are passed through the column one or more times to allow ligand to bind to the receptor poiypeptide.
  • the ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HC1), or pH to disrupt ligand-receptor binding.
  • an assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument may be advantageously employed (e.g., BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ; or SELDF M technology, Ciphergen,
  • receptor, antibody, member of a complement/anti- complement pair or fragment is immobilized onto the surface of a receptor chip.
  • Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-240, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993.
  • a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell.
  • a test sample is passed through the cell.
  • a ligand, epitope, or opposite member of the complement/anti- complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
  • Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949) and calorimetric assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et al conflict Science 245:821-25, 1991).
  • Mouse zcytorlO polypeptides can also be used to prepare antibodies that bind to mouse zcytorlO epitopes, peptides or polypeptides.
  • the mouse zcytorlO poiypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
  • antigenic, epitope-bearing polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a zcytorlO poiypeptide (e.g., SEQ ID NO:2).
  • Polypeptides comprising a larger portion of a zcytorlO poiypeptide, i.e., from 10 to 30 residues up to the entire length of the amino acid sequence are included.
  • Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein. Suitable antigens include the mouse zcytorlO poiypeptide encoded by SEQ ID NO:2 from amino acid number 15 (Cys) to amino acid number 357 (I u), or a contiguous 9 to 343 amino acid fragment thereof.
  • suitable antigens include the mouse zcytorlO poiypeptide encoded by SEQ ID NO:2 from amino acid number 17 (Ala) to amino acid number 359 (I-eu), or a contiguous 9 to 343 amino acid fragment thereof.
  • Preferred peptides to use as antigens are the cytokine binding domain, intracellular signaling domain, Box I and Box II sites, block 1, mammalian signaling motif, and motifs 1-6, disclosed herein, and mouse zcytorlO hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot, determined for example, from a Hopp/Woods hydrophilicity profile based on a sliding six-residue window, with buried G, S, and T residues and exposed H, Y, and W residues ignored.
  • Mouse zcytorlO hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: (1) amino acid number 150 (Arg) to amino acid number 155 (Asp) of SEQ ID NO:2; (2) amino acid number 254 (Arg) to amino acid number 259 (Ala) of SEQ ID NO:2; (3) amino acid number 296 (Ala) to amino acid number 301 (Glu) of SEQ ID NO:2; (4) amino acid number 297 (Arg) to amino acid number 302 (Asp) of SEQ ID NO:2; and (5) amino acid number 310 (Lys) to amino acid number 315 (Glu) of SEQ ID NO:2.
  • the corresponding zcytorlO hydrophilic peptides of SEQ ID NO:35 are also included with comparison of the above hydrophilic peptides SEQ ID NO:2 in reference to SEQ ID NO:35.
  • conserved motifs, and variable regions between conserved motifs of mouse zcytorlO are suitable antigens.
  • Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example. Current Protocols in Immunology, Cooligan, et al.
  • polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a mouse zcytorlO poiypeptide or a fragment thereof.
  • the immunogenicity of a mouse zcytorlO poiypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of mouse zcytorlO or a portion thereof with an immunoglobulin poiypeptide or with maltose binding protein.
  • the poiypeptide immunogen may be a full-length molecule or a portion thereof.
  • poiypeptide portion is "hapten-like"
  • such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included.
  • Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non- human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody).
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. Moreover, human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
  • Antibodies are considered to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with related poiypeptide molecules.
  • a threshold level of binding is determined if anti-mouse zcytorlO antibodies herein bind to a mouse zcytorlO poiypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-mouse zcytorlO) poiypeptide. It is preferred that the antibodies exhibit a binding affinity (K a ) f. ⁇ 7 _ 1 & _ 1 of 10 M or greater, preferably 10 M or greater, more preferably 10 M or greater, and most preferably 10 9 M -1 or greater.
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).
  • anti-mouse zcytorlO antibodies do not significantly cross-react with related poiypeptide molecules is shown, for example, by the antibody detecting mouse zcytorlO poiypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.).
  • known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family, Screening can also be done using non-mouse mouse zcytorlO, and mouse zcytorlO mutant polypeptides.
  • antibodies can be "screened against" known related polypeptides, to isolate a population that specifically binds to the mouse zcytorlO polypeptides.
  • antibodies raised to mouse zcytorlO are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to mouse zcytorlO will flow through the matrix under the proper buffer conditions. Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to known closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995).
  • assays known to those skilled in the art can be utilized to detect antibodies which bind to mouse zcytorlO proteins or polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant mouse zcytorlO protein or poiypeptide.
  • Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to mouse zcytorlO protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled mouse zcytorlO protein or peptide).
  • Genes encoding polypeptides having potential mouse zcytorlO poiypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli.
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • random peptide display libraries can be used to screen for peptides that interact with a known target that can be a protein or poiypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • a known target can be a protein or poiypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Patent NO. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and Ladner et al., US Patent NO.
  • Random peptide display libraries can be screened using the mouse zcytorlO sequences disclosed herein to identify proteins which bind to mouse zcytorlO.
  • binding polypeptides which interact with mouse zcytorlO polypeptides can be used for tagging cells; for isolating paralog and ortholog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity, e.g., for blocking interaction between ligand and receptor, or viral binding to a receptor. The binding polypeptides can also be used for diagnostic assays for determining circulating levels of mouse zcytorlO polypeptides or zcytorlO orthologs, e.g.
  • binding polypeptides can also act as zcytorlO "antagonists" to block zcytorlO binding and signal transduction in vitro and in vivo.
  • anti-mouse zcytorlO-binding polypeptides would be useful for inhibiting zcytorlO activity or protein binding.
  • Antibodies to mouse zcytorlO may be used for tagging cells that express mouse zcytorlO; for isolating mouse zcytorlO by affinity purification; for diagnostic assays for determining circulating levels of mouse zcytorlO polypeptides or zcytorlO orthologs, e.g. in human samples; for detecting or quantitating soluble zcytorlO polypeptides as marker of underlying pathology or disease in a mouse model, or in human samples expressing zcytorlO orthologs; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zcytorlO activity in vitro and in vivo.
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cof actors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • antibodies to mouse zcytorlO or fragments thereof may be used in vitro to detect denatured mouse zcytorlO or fragments thereof in assays, for example, Western Blots or other assays known in the art.
  • Antibodies to mouse zcytorlO are useful for tagging cells that express the receptor and assaying mouse zcytorlO expression levels, for affinity purification, within diagnostic assays for determining circulating levels of soluble receptor polypeptides, analytical methods employing fluorescence-activated cell sorting. Divalent antibodies may be used as agonists to mimic the effect of the mouse zcytorlO ligand.
  • Antibodies herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic, in murine models to study therapeutic applications, or in therapeutic applications.
  • antibodies or binding polypeptides which recognize mouse zcytorlO of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (i.e., a mouse zcytorlO receptor). More specifically, anti-mouse zcytorlO antibodies, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the zcytorlO molecule.
  • Suitable detectable molecules may be directly or indirectly attached to polypeptides that bind mouse zcytorlO ("binding polypeptides," including binding peptides disclosed above), antibodies, or bioactive fragments or portions thereof.
  • Suitable detectable molecules include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
  • Suitable cytotoxic molecules may be directly or indirectly attached to the poiypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium- 188 or yttrium-90 (either directly attached to the poiypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance). Binding polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin.
  • the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the binding poiypeptide or antibody portion.
  • biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
  • binding polypeptide-toxin fusion proteins or antibody-toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues).
  • a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest.
  • the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule.
  • domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue-specific delivery of generic anti-complementary- detectable/ cytotoxic molecule conjugates.
  • mouse zcytorlO binding polypeptide-cytokine or antibody-cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, blood, lymphoid, colon, and bone marrow cancers), if the binding polypeptide-cytokine or anti-mouse zcytorlO antibody targets the hyperproliferative cell (See, generally, Hornick et al., Blood 89:4437-47, 1997). They described fusion proteins enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine.
  • target tissues for example, blood, lymphoid, colon, and bone marrow cancers
  • Suitable anti-mouse zcytorlO antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediates improved target cell lysis by effector cells.
  • Suitable cytokines for this purpose include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM- CSF), for instance.
  • mouse zcytorlO binding poiypeptide or antibody fusion proteins described herein can be used for enhancing in vivo killing of target tissues by directly stimulating a zcytorlO-modulated apoptotic pathway, resulting in cell death of hyperproliferative cells expressing zcytorlO or orthologous sequences that cross-react with the antibody or binding poiypeptide, such as human zcytorlO.
  • bioactive binding poiypeptide or antibody conjugates described herein can be delivered orally, intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action.
  • Polynucleotides encoding mouse zcytorlO polypeptides are useful within gene therapy applications where it is desired to increase or inhibit mouse zcytorlO activity. If a mammal has a mutated or absent zcytorlO gene, the zcytorlO gene can be introduced into the cells of the mammal.
  • a gene encoding a mouse zcytorlO poiypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell.
  • defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30, 1992; and a defective adeno-associated virus vector (Samulski et al., X Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).
  • HSV1 herpes simplex virus 1
  • a mouse zcytorlO gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 62:1120, 1988; Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No.
  • the vector can be introduced by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988).
  • the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • liposomes Molecular targeting of liposomes to specific cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters
  • proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.
  • DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.
  • Mouse models employing mzctyorlO can be used to study the application, safety, efficacy, and perfect such gene therapy techniques and applications discussed in the paragraphs above.
  • Antisense methodology can be used to inhibit mouse zcytorlO gene transcription, such as to inhibit cell proliferation in vivo.
  • Polynucleotides that are complementary to a segment of a mouse zcytorlO-encoding polynucleotide e.g., a polynucleotide as set froth in SEQ ED NO:l
  • Such antisense polynucleotides are used to inhibit expression of mouse zcytorlO polypeptide-encoding genes in cell culture or in a mouse model for use in studying human disease, and studying the application, safety, efficacy, and perfection of antisense therapy methods.
  • mouse zcytorlO poiypeptide can be used as a target to introduce gene therapy into a cell.
  • This application would be particularly appropriate for introducing therapeutic genes into cells in which mouse zcytorlO is normally expressed, for example, lymphoid tissue and PBLs, or cancer cells which may express mouse zcytorlO poiypeptide.
  • viral gene therapy such as described above, can be targeted to specific cell types in which express a cellular receptor, such as mouse zcytorlO poiypeptide, rather than the viral receptor.
  • Antibodies, or other molecules that recognize mouse zcytorlO molecules on the target cell's surface can be used to direct the virus to infect and administer gene therapeutic material to that target cell. See, Woo, S.L.C, Nature Biotech. 14: 1538, 1996; Wickham,
  • a bispecific antibody containing a virus-neutralizing Fab fragment coupled to a mouse zcytorlO-specific antibody can be used to direct the virus to cells expressing the mouse zcytorlO receptor and allow efficient entry of the virus containing a genetic element into the cells. See, for example, Wickham, T.J., et al., J.
  • the mouse zcytorlO gene a probe comprising mouse zcytorlO DNA or RNA or a subsequence thereof can be used to determine the location of the murine zcytorlO gene on a mouse chromosome, of if a mouse zcytorlO ortholog gene is present on a human chromosome, or if a mutation has occurred.
  • Detectable chromosomal aberrations at the mouse zcytorlO gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, fluorescence in situ hybridization methods, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid. ; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
  • molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis, fluorescence in situ hybridization methods, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid. ; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
  • Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250:245-50, 1990). Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., HuntsviUe, AL). These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence-tagged sites (STSs), and other nonpolymorphic and polymorphic markers within a region of interest.
  • STSs sequence-tagged sites
  • the precise knowledge of a gene's position can be useful for a number of purposes, including: 1 ) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular human zcytorlO ortholog gene might have. Sequence tagged sites (STSs) can also be used independently for chromosomal localization.
  • STSs are based solely on DNA sequence they can be completely described within an electronic database, for example, Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences.
  • dbSTS Database of Sequence Tagged Sites
  • GenBank National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov
  • mouse zcytorlO mutant and transgenic mice can be used as a mouse model for human genetic diseases.
  • Over-expression or under-expression of the native mzyctorlO locus may result in a murine phenotype that corresponds to a human heritable disease state.
  • defects or mutations in the mouse zcytorlO locus itself may result in a murine phenotype that corresponds to a human heritable disease state.
  • Molecules of the present invention such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a mouse zcytorlO genetic defect, corresponding to a defect in the human ortholog, and aid in the understanding of human disease.
  • mice engineered to express the mouse zcytorlO gene referred to as "transgenic mice,” and mice that exhibit a complete absence of mouse zcytorlO gene function, referred to as “knockout mice,” can also be generated (Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature 366:740-42, 1993; Capecchi, M.R., Science 244: 1288-1292, 1989; Palmiter, R.D. et al. Annu Rev Genet. 20: 465-499, 1986).
  • transgenic mice that over-express mouse zcytorlO, either ubiquitously or under a tissue-specific or tissue-restricted promoter can be used to ask whether over-expression causes a phenotype.
  • over-expression of a wild- type mouse zcytorlO poiypeptide, poiypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which mouse zcytorlO expression is functionally relevant and may indicate a therapeutic target for the mouse zcytorlO, its agonists or antagonists.
  • a preferred transgenic mouse to engineer is one that expresses a "dominant-negative" phenotype, such as one that over-expresses the mouse zcytorlO extracellular cytokine binding domain (approximately amino acids 15 (Cys) to 230 (Pro) of SEQ ID NO:2, or amino acids 17 (Ala) to 232 (Pro) of SEQ ID NO:35) with a transmembrane domain attached (for example, approximately amino acids 15 (Cys) to 251 (Leu) of SEQ ED NO:2, or amino acids 17 (Ala) to 254 (Leu) of SEQ ED NO:35) if the mouse zcytorlO transmembrane were used).
  • a "dominant-negative" phenotype such as one that over-expresses the mouse zcytorlO extracellular cytokine binding domain (approximately amino acids 15 (Cys) to 230 (Pro) of SEQ ID NO:2, or amino acids
  • knockout mouse zcytorlO mice can be used to determine where mouse zcytorlO is absolutely required in vivo.
  • the phenotype of knockout mice is predictive of the in vivo effects of that a mouse zcytorlO antagonist, such as those described herein, may have.
  • These mice may be employed to study the mouse zcytorlO gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human diseases.
  • transgenic mice expression of mouse zcytorlO antisense polynucleotides or ribozymes directed against mouse zcytorlO, described herein can be used analogously to transgenic mice described above.
  • Cytokine Receptor was designated mouse zcytorlO.
  • a 433 base pair probe was generated using primers ZC 14603 (SEQ ED NO:5), and ZC14606 (SEQ ID NO:6), 0.5 ng cDNA encoding the original EST (EST631772) as a template in a PCR reaction.
  • KlenTaqTM polymerase (Clontech) and buffer were used.
  • the PCR reaction conditions were as follows: followed by 30 cycles of 95°C for 30 sec, 60° C for 15 sec, 72°C for 1 min.; followed by 72°C for 10 min.; followed by a 10°C soak.
  • a sample of the PCR reaction product was run on a 1.5% agarose gel. A band of the expected size of approximately 400 bp was seen.
  • the PCR product was purified using a chromaspin 400 column (Clontech).
  • the purified product was radioactively labeled with 32 P-dCTP using Rediprime HTM (Amersham), a random prime labeling system, according to the manufacturer's specifications.
  • the probe was then purified using a Nuc-TrapTM column (Stratagene, La Jolla, CA) according to the manufacturer's instructions.
  • mice MTN and Mouse Embryo MTN (Clontech).
  • the full length mouse probe was generated by PCR from the mouse cDNA plasmid as template and oligos ZC 17,213 (SEQ ID NO: 11) and ZC 17,314 (SEQ ID NO: 12) as primers.
  • PCR conditions were as follows: 35 cycles at 95°C for 1 minute, 55 for lminute, 72 for 2 minutes; 72°C for 10 minutes; 4°C overnight
  • a sample of the PCR reaction product was run on 1% low melting point agarose gel (Boehringer Mannheim, Indianapolis, IN). A band of the expected size of 1.9 kb was seen.
  • the 1.9 kb PCR fragment was gel purified using a commercially available kit (Qiaquick Gel Extraction KitTM; Qiagen,
  • the blots were then washed 4 times for 15 minutes in 2X SSC/1% SDS at 25°C, followed by three30 minutes washes in 0.1X SSC/0.1% SDS at 50°C.
  • a transcript of approximately 1.35 kb was detected in heart, spleen, lung, liver and testis.
  • a weak band was observed in mouse seven-day embryo.
  • Rat ZcytorlO Sequence A rat EST was identified as an ortholog of the murine zcytorlO receptor.
  • the EST contained the zcytorlO transmembrane domain as well as the entire intracellular region through the stop codon. Oligos were designed, ZC 24,055 (SEQ ID NO:13) and ZC 23,711 (SEQ ID NO:14). A rat kidney cDNA library (Clontech) was screened using the above oligos in a PCR reaction with the following conditions: 35 cycles at 95degrees for 1 minute, 55 degrees for 1 minute, and 72 degrees for 1 minute;
  • the PCR reaction product was run on 1% low melting point agarose gel (Boehringer Mannheim). A band of the expected size of approximately 300 bp was seen.
  • the 300 bp PCR fragment was gel purified using a commercially available kit (Qiaquick Gel Extraction KitTM; Qiagen). Sequencing confirmed the PCR fragment to be the intracellular and transmembrane portion of the rat zcytorlO.
  • the rat cDNA sequence is shown in SEQ ID NO: 15 and corresponding amino acid sequence shown in SEQ ID NO: 16.
  • the transmembrane, intracellular domain, class I cytokine motifs and the like correlate with those shown as shown in the mouse zcytorlO sequence (SEQ ID NO:2).
  • Rat ZcytorlO Tissue Distribution Rat Multiple Tissue Northern Blots were probed to determine the tissue distribution of rat zcytorlO expression.
  • An approximately 250 bp PCR derived probe was amplified using rat cDNA (Example 3) as a template and oligonucleotide ZC23711 (SEQ ID NO:14) and ZC23712 (SEQ ID NO: 17) as primers.
  • PCR reaction conditions were as follows: 30 cycles of 90°C for 1 min., 55°C for 1 min., 72°C for 1.5 min.; 72°C for 10 min.; 4°C overnight.
  • a sample of the PCR reaction product was run on 1% low melting point agarose gel (Boehringer Mannheim). A band of the expected size of 250 bp was seen.
  • the 250 bp PCR fragment was gel purified using a commercially available kit (Qiaquick Gel Extraction KitTM; Qiagen).
  • the probe was radioactively labeled using the MULTIPRIMETM labeling kit (Amersham) according to the manufacturer's instructions.
  • the probe was purified using a NUCTRAPTM push column (Stratagene). EXPRESSHYBTM (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots.
  • Hybridization took place overnight at 60°C using about 10 6 cpm/ml of labeled probe. The blots were then washed one time in 6X SSC and 0.1% SDS at room temperature, followed by 5 washes in 6X SSC and 0.1% SDS at 60°C.
  • a transcript of approximately 1.3 kb was seen in stomach, small intestine, lung, testis, skin, brain, kidney, spleen, thymus, and liver.
  • a larger transcript of about 3.0 kb was seen in skeletal muscle and all the above tissues with the exception of spleen. For testis there was an additional transcript at 1.0 kb and there was also a larger 6.0 kb transcript seen for thymus and testis.
  • the extracellular and transmembrane domains of the MPL receptor were isolated from a plasmid containing the MPL receptor (Souyri et al., Cell 63:1137-1147, 1990) (designated PHZ1/MPL plasmid) using PCR with primers ZC17,212 (SEQ ID NO:18) and ZC17,313 (SEQ ID NO:19).
  • the PCR reaction conditions were as follows: 95°C for 1 min.; 35 cycles at 95°C for 1 min., 45°C for 1 min., 72°C for 2 min.; followed by 72°C at 10 min.; then a 10°C soak.
  • the PCR product was run on a 1% low melting point agarose (Boerhinger Mannheim, Indianapolis, IN) and the approximately 1.5 kb MPL receptor fragment isolated using QiaquickTM gel extraction kit (Qiagen) as per manufacturer' s instructions.
  • the intracellular domain of zcytorlO was isolated from a plasmid containing zcytorlO receptor cDNA using PCR with primers ZC17,315 (SEQ ID NO:20) and ZC17,314 (SEQ ED NO:21).
  • the polynucleotide sequence corresponding to the zcytorlO receptor intracellular domain coding sequence is shown in SEQ ID NO:l.
  • the reaction conditions were as per above.
  • the PCR product was run on a 1% low melting point agarose (Boerhinger Mannheim) and the approximately 350 bp zcytorlO fragment isolated using Qiaquick gel extraction kit (Qiagen) as per manufacturer's instructions.
  • Each of the isolated fragments described above were mixed at a 1: 1 volumetric ratio and used in a PCR reaction using ZC17,212 (SEQ ID NO: 18) and ZC17,314 (SEQ ED NO:21) to create the MPL-zcytorlO chimera.
  • the reaction conditions were as follows: 95°C for 1 min.; 35 cycles at 95°C for 1 min., 55°C for 1 min., 72°C for 2 min.; followed by 72°C at 10 min.; then a 10°C soak.
  • the entire PCR product was run on a 1 % low melting point agarose (Boehringer Mannheim) and the approximately 1.9 kb MPL-zcytorlO chimera fragment isolated using Qiaquick gel extraction kit (Qiagen) as per manufacturer's instructions.
  • the MPL-zcytorlO chimera fragment was digested with EcoRI (BRL) and Xbal (Boerhinger Mannheim) as per manufacturer's instructions.
  • the entire digest was run on a 1% low melting point agarose (Boehringer Mannheim) and the cleaved MPL-zcytorlO chimera isolated using QiaquickTM gel extraction kit (Qiagen) as per manufacturer's instructions.
  • the resultant cleaved MPL-zcytorlO chimera was inserted into an expression vector as described below.
  • Recipient expression vector pZP-5Z was digested with EcoRI (BRL) and Hindm (BRL) as per manufacturer's instructions, and gel purified as described above. This vector fragment was combined with the EcoRI and Xbal cleaved MPL-zcytorlO chimera isolated above and a Xbal/HindEQ linker fragment in a ligation reaction. The ligation was run using T4 Ligase (BRL), at 15°C overnight. A sample of the ligation was electroporated in to DH10B ElectroMAXTM electrocompetent E. coli cells (25 ⁇ F, 200 ⁇ , 2.3V).
  • Transformants were plated on LB+Ampicillin plates and single colonies screened by PCR to check for the MPL-zcytorlO chimera using ZC 17,212 (SEQ ID NO: 18) and ZC 17,314 (SEQ ID NO:21) using the PCR conditions as described above. Confirmation of the MPL-zcytorlO chimera sequence was made by sequence analyses. The insert was approximately 1.9 kb, and was full-length. The plasmid DNA was designated pZP-5Z/MPL-zcytorl0.
  • MPL-zcytorlO Chimera Based Proliferation in BAF3 Assay Using Alamar Blue A. Construction of BaF3 Cells Expressing MPL-zcytorlO Chimera
  • BaF3 an interleukin-3 (IL-3) dependent pre-lymphoid cell line derived from murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985; Mathey- Prevot et al., Mol. Cell. Biol.
  • IL-3 interleukin-3
  • pZP-5Z/MPL-zcytorlO DNA was prepared and purified using a Qiagen Maxi Prep kit (Qiagen) as per manufacturer's instructions.
  • BaF3 cells for electroporation were washed once in RPMI media and then resuspended in RPMI media at a cell density of 10 ⁇ cells/ml.
  • One ml of resuspended BaF3 cells was mixed with 30 ⁇ g of pZP-5Z/MPL-zcytorl0 plasmid DNA (Example 5) and transferred to separate disposable electroporation chambers (GIBCO BRL). Following a 15 minute incubation at room temperature the cells were given two serial shocks (800 lFad/300 V.; 1180 lFad/300 V.) delivered by an electroporation apparatus (CELL-PORATORTM; GIBCO BRL).
  • the electroporated cells were transferred to 50 ml of complete media and placed in an incubator for 15-24 hours (37°C, 5% CO2). The cells were then spun down and resuspended in 50 ml of complete media containing GeneticinTM (Gibco) selection (500 ⁇ g/ml G418) in a T-162 flask to isolate the G418-resistant pool. Pools of the transfected BaF3 cells, hereinafter called BaF3/MPL-zcytorl0 cells, were assayed for signaling capability as described below.
  • BaF3/MPL-zcytorl0 cells (Example 6A) were spun down and washed in the complete media, described above, but without mIL-3 (hereinafter referred to as "mIL-3 free media"). The cells were spun and washed 3 times to ensure the removal of the mIL-3. Cells were then counted in a hemacytometer. Cells were plated in a 96-well format at 5000 cells per well in a volume of 100 ⁇ l per well using the mEL-3 free media.
  • TPO thrombopoietin
  • mIL-3 free media 1000 ng/ml, 500 ng/ml, 250 ng/ml, 125 ng/ml, 62 ng/ml, 30 ng/ml, 15 ng/ml, 7.5 ng/ml concentrations.
  • 100 ⁇ l of the diluted TPO was added to the BaF3/MPL-zcytorl0 cells.
  • the total assay volume is 200 ⁇ l.
  • Negative controls were run in parallel using mIL-3 free media only, without the addition of TPO.
  • Alamar Blue (Accumed, Chicago, EL) was added at 20 ⁇ l/well. Alamar Blue gives a fluourometric readout based on number of live cells, and is thus a direct measurement of cell proliferation in comparison to a negative control. Plates were again incubated at 37°C, 5% CO for 24 hours. Plates were read on the FmaxTM plate reader (Molecular Devices Sunnyvale, CA) using the SoftMaxTM Pro program, at wavelengths 544 (Excitation) and 590 (Emmission).
  • Results showed no proliferation of the Baf3/Mpl-zcytorl0 chimera cell line in response to TPO suggesting that the intracellular portion of the zcytorlO molecule is incapable of signaling as a homodimer.
  • Example 7 Construction of Mouse ZcytorlO-mpl Poiypeptide Chimera: ZcytorlO Extracellular Domain Fused to the Mpl Intracellular Signaling Domain and TM Domain
  • the extracellular domains of the zcytorlO receptor were isolated from a plasmid containing the zcytorlO receptor using PCR with primers ZC17,213 (SEQ ED NO: 11) and ZC17.204 (SEQ ID NO:22).
  • the reaction conditions were as follows: 95°C for 1 min.; 35 cycles at 95°C for 1 min., 45°C for 1 min., 72°C for 2 min.; followed by 72°C at 10 min.; then a 10°C soak.
  • the PCR product was run on a 1% low melting point agarose (Boerhinger Mannheim, Indianapolis, IN) and the approximately 800 bp zcytorlO receptor fragment, isolated using QiaquickTM gel extraction kit (Qiagen) as per manufacturer's instructions.
  • MPL The intracellular and transmembrane domains of MPL were isolated from a plasmid containing MPL receptor cDNA (PHZ1/MPL plasmid) (Example 5) using PCR with primers ZC17,205 (SEQ ID NO:23) and ZC17,206 (SEQ ID NO:24).
  • the reaction conditions were run as per above.
  • the PCR product was run on a 1% low melting point agarose (Boerhinger Mannheim) and the approximately 450 bp MPL fragment isolated using Qiaquick gel extraction kit (Qiagen) as per manufacturer's instructions.
  • Qiaquick gel extraction kit Qiagen
  • Each of the isolated fragments described above were mixed at a 1: 1 volumetric ratio and used in a PCR reaction using ZC 17,213 (SEQ ID NO: 11) and ZC17,206 (SEQ ED NO:24) to create a ZcytorlO-mpl chimera.
  • the reaction conditions were as follows: 95°C for 1 min.; 35 cycles at 95°C for 1 min., 55°C for 1 min., 72°C for 2 min.; followed by 72°C at 10 min.; then a 10°C soak.
  • the entire PCR product was run on a 1 % low melting point agarose (Boehringer Mannheim) and an approximately
  • ZcytorlO-mpl chimera fragment isolated using Qiaquick gel extraction kit (Qiagen) as per manufacturer's instructions.
  • the ZcytorlO-mpl chimera fragment was digested with EcoRI (BRL) and Xbal (Boerhinger Mannheim) as per manufacturer's instructions. The entire digest was run on a 1% low melting point agarose (Boehringer Mannheim) and the cleaved ZcytorlO-mpl chimera isolated using QiaquickTM gel extraction kit (Qiagen) as per manufacturer's instructions.
  • QiaquickTM gel extraction kit QiaquickTM gel extraction kit
  • Recipient expression vector pZP-5Z was digested with EcoRI (BRL) and HindHI (BRL) as per manufacturer's instructions, and gel purified as described above. This vector fragment was combined with the EcoRI and Xbal cleaved ZcytorlO-mpl chimera isolated above and a Xbal HindEH linker fragment in a ligation reaction. The ligation was run using T4 Ligase (BRL), at 15°C overnight. A sample of the ligation was electroporated in to DH10B ElectroMAXTM electrocompetent E. coli cells (25 ⁇ F, 200 ⁇ , 2.3V).
  • the entire zcytorlO receptor was isolated from a plasmid containing zcytorlO receptor cDNA using PCR with primers ZC17,213 (SEQ ID NO: 11) and ZC17.314 (SEQ ID NO:21).
  • the reaction conditions were as follows: 95°C for 1 min; 35 cycles at 95°C for 1 min, 55°C for 1 min, 72°C for 2 min; followed by 72°C at 10 min; then a 10°C soak.
  • the PCR product was run on a 1% low melting point agarose
  • the purified zcytorlO cDNA was digested with EcoRI (BRL) and Xbal (Boehringer Mannheim) as per manufacturer's instructions. The entire digest was run on a 1 % low melting point agarose (Boerhinger Mannheim) and purified the cleaved zcytorlO fragment using Qiaquick gel extraction kit (Qiagen) as per manufacturer's instructions. The resultant cleaved zcytorlO was inserted into an expression vector as described below.
  • Recipient expression vector pZP-5N was digested with EcoRI (BRL) and Xbal (Boerhinger Mannheim) as per manufacturer's instructions, and gel purified as described above. This vector fragment was combined with the EcoRI and Xbal cleaved zcytorlO fragment isolated above in a ligation reaction. The ligation was run using T4 Ligase (BRL), at 15°C overnight. A sample of the ligation was electroporated in to DH10B electroMAXTM electrocompetent E. coli cells (25 ⁇ F, 200 ⁇ , 2.3V).
  • Transformants were plated on LB+Ampicillin plates and single colonies screened by PCR to check for the zcytorlO sequence using ZC17.213 (SEQ ID NO: 11) and ZC17,314 (SEQ ID NO:21) using the PCR conditions as described above. Confirmation of the full-length zcytorlO sequence was made by sequence analyses. The insert was approximately l.lkb, and was full-length.
  • BaF3 cells expressing the ZcytorlO-MPL receptor were constructed as per Example 6 A, using 30 ⁇ g of the zcytorlO expression vector, described in Example 7.
  • the BaF3 cells expressing the pZP-5Z/zcytorl0 receptor plasmid were designated as BaF3/Zcytorl0-mpl. These cells were used to screen for a zcytorlO activity as described below in Examples 10 and 18.
  • BaF3 cells expressing the full-length zcytorlO receptor were constructed as per Example 6A, using 30 ⁇ g of the zcytorlO expression vector, described in Example 8.
  • the BaF3 cells expressing the pZP-5Z/zcytorl0 receptor plasmid were designated as BaF3/zcytorl0. These cells were used to screen for a zcytorlO activity as described below in Examples 10 and 18.
  • Example 10 Screening for zcytorlO activity using BaF3/zcytorlQ-MPL cells and Baf3/zcytorl0 cells using an Alamar Blue Proliferation Assay
  • Baf3/zcytorl0-mpl chimera cells and Baf3/zcytorl0 cells were spun down and washed independently mIL-3 free media (Example 6). The cells were spun and washed 3 times to ensure the removal of the mEL-3. Cells were then counted in a hemacytometer. Cells were plated in a 96-well format at 5000 cells per well in a volume of 100 ⁇ l per well using the mIL-3 free media.
  • conditioned media samples from a variety of cell lines were screened. 100 ⁇ l of each conditioned media sample was added to the BaF3/MPL-zcytorl0 chimera cells as well as the Baf3/zcytorl0 cells. The total assay volume was 200 ⁇ l. All known cytokines were also screened at a concentration of 250 ng/ml on both cell lines. Negative controls were run in parallel using mIL-3 free media only. Mouse IL-3 at a concentration of 250 pg/ml was used as a positive control. The assay plates were incubated at 37°C, 5%
  • Alamar Blue gives a fluourometric readout based on number of live cells, and is thus a direct measurement of cell proliferation in comparison to a negative control. Plates were again incubated at 37°C, 5% CO 2 for 24 hours. Plates were read on the FmaxTM plate reader (Molecular Devices Sunnyvale, CA) using the SoftMaxTM
  • Results showed no proliferation of on either the Baf3/zcytorl0-mpl chimera cell line or the Baf3/zcytorl0 cell line in response to conditioned media samples or the known ligands. This result suggested that the zcytorlO receptor may not signal as a homodimer. The actual receptor-signaling complex may require another receptor subunit not present in BaF3 cells. See example 18 and Example 19 below.
  • Example 11
  • Receptors zcvtorlOCEE, zcytorlOCFLG, zcvtorlOCHIS and zcvtorlO-Fc4
  • An expression vector is prepared for the expression of the soluble, extracellular domain of the zcytorlO poiypeptide, pC4zcytorlOCEE, wherein the construct is designed to express a zcytorlO poiypeptide comprised of the predicted initiating methionine and truncated adjacent to the predicted transmembrane domain, and with a C-terminal Glu-Glu tag (SEQ ID NO:25).
  • a zcytorlO DNA fragment comprising the zcytorlO extracellular cytokine binding domain (amino acid 15 (Cys) to 230 (Pro) of SEQ ED NO:2) is created using PCR, and purified for example, as described in Example 7.
  • the excised DNA is subcloned into a plasmid expression vector that has a signal peptide, e.g., the native zcytorlO signal peptide, and attaches a Glu-Glu tag (SEQ ID NO:25) to the C-terminus of the zcytorlO polypeptide-encoding polynucleotide sequence.
  • Such an expression vector mammalian expression vector contains an expression cassette having a mammalian promoter, multiple restriction sites for insertion of coding sequences, a stop codon and a mammalian terminator.
  • the plasmid can also have an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene and the SV40 terminator.
  • Restriction digested zcytorlO insert and previously digested vector are ligated using standard molecular biological techniques, and electroporated into competent cells such as DH10B competent cells (GIBCO BRL, Gaithersburg, MD) according to manufacturer's direction and plated onto LB plates containing 50 mg/ml ampicillin, and incubated overnight. Colonies are screened by restriction analysis of DNA prepared from individual colonies. The insert sequence of positive clones is verified by sequence analysis. A large scale plasmid preparation is done using a QIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.
  • the same process is used to prepare the zcytorlO soluble receptors with a C-terminal his tag, composed of 6 His residues in a row; and a C-terminal flag (SEQ ED NO:26) tag, zcytorlOCFLAG.
  • the aforementioned vector has either the HIS or the FLAG® tag in place of the glu-glu tag (SEQ ED NO:25).
  • An expression plasmid containing all or part of a polynucleotide encoding zcytorlO is constructed via homologous recombination.
  • a fragment of zcytorlO cDNA was isolated using PCR that includes the polynucleotide sequence from extracellular domain of the zcytorlO receptor.
  • Primers used in PCR for the production of the zcytorlO fragment are from 5' to 3' end: (1) about 40 bp of the vector flanking sequence (5' of the insert) and about 17 bp corresponding to the 5' end of the zcytorlO extracellular domain; and (2) about 40 bp of the 5' end of the Fc4 polynucleotide sequence (SEQ ED NO:27) and about 17 bp corresponding to the 3' end of the zcytorlO extracellular domain.
  • the fragment of Fc-4 for fusion with the zcytorlO is generated by PCR in a similar fashion.
  • the two primers used in the production of the Fc4 fragment include: (1) a 5' primer consisting of about 40 bp of sequence from the 3' end of zcytorlO extracellular domain and about 17 bp of the 5' end of Fc4 (SEQ ED NO:27); and (2) a 3' primer consisting of about 40 bp of vector sequence (3' of the insert) and about 17 bp of the 3' end of Fc4 (SEQ ID NO:27).
  • PCR amplification of the each of the reactions described above is then performed using conditions standard in the art.
  • An exemplary expression vector is derived from the plasmid pCZR199 (deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, designated No. 98668), that is cut with Smal (BRL).
  • the expression vector was derived from the plasmid pCZR199, and is a mammalian expression vector containing an expression cassette having the CMV immediate early promoter, a consensus intron from the variable region of mouse immunoglobulin heavy chain locus, multiple restriction sites for insertion of coding sequences, a stop codon and a human growth hormone terminator.
  • the expression vector also has an E.
  • coli origin of replication a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene and the SV40 terminator.
  • the expression vector used was constructed from pCZR199 by the replacement of the metallothionein promoter with the CMV immediate early promoter.
  • Competent yeast cells (S. cerevisiae) are combined with approximately 1 ⁇ g each of the zcytorlO and Fc4 inserts, and 100 ng of Smal (BRL) digested expression vector and electroporated.
  • the yeast/DNA mixtures are electropulsed at, for example, 0.75 kV (5 kV/cm), "infinite" ohms, 25 ⁇ F.
  • To each cuvette is added 600 ⁇ l of 1.2 M sorbitol and the yeast was plated in aliquots onto URA-D plates and incubated at 30°C. After about 48 hours, the Ura+ yeast transformants from a single plate are picked, DNA isolated, and transformed into electrocompetent E.
  • coli cells e.g., DH10B, GibcoBRL
  • plasmid DNA is isolated using the Qiagen Maxi kit (Qiagen) according to manufacturer's instructions.
  • BHK 570 cells (ATCC No. CRL- 10314), DG-44 CHO, or other mammalian cells are plated at about 1.2X10 6 cells/well (6- well plate) in 800 ⁇ l of appropriate serum free (SF) media (e.g., DMEM, Gibco/BRL High Glucose) (Gibco BRL, Gaithersburg, MD).
  • SF serum free
  • the cells are transfected with expression plasmids containing zcytorlOCEE, zcytorlOCFLG, zcytorlOCHIS or zcytorlO-Fc4 (Example 11), using LipofectinTM (Gibco BRL), in serum free (SF) media according to manufacturer's instruction.
  • Single clones expressing the soluble receptors are isolated, screened and grown up in cell culture media, and purified using standard techniques.
  • An expression plasmid containing a polynucleotide encoding a zcytorlO soluble receptor fused C-terminally to maltose binding protein (MBP) is constructed via homologous recombination.
  • the fusion poiypeptide contains an N-terminal approximately 388 amino acid MBP portion fused to the zcytorlO soluble receptor (amino acid 15 (Cys) to amino acid 230 (Pro) of SEQ ID NO:2).
  • a fragment of zcytorlO cDNA (SEQ ID NO:l) is isolated using PCR as described herein.
  • Two primers are used in the production of the zcytorlO fragment in a standard PCR reaction: (1) one containing about 40 bp of the vector flanking sequence and about 25 bp corresponding to the amino terminus of the zcytorlO, and (2) another containing about 40 bp of the 3' end corresponding to the flanking vector sequence and about 25 bp corresponding to the carboxyl terminus of the zcytorlO.
  • Two ⁇ l of the 100 ⁇ l PCR reaction is run on a 1.0% agarose gel with 1 x TBE buffer for analysis, and the expected approximately fragment is seen.
  • the remaining PCR reaction is combined with the second PCR tube and precipitated with 400 ⁇ l of absolute ethanol.
  • the precipitated DNA used for recombining into the Smal cut recipient vector pTAP98 to produce the construct encoding the MBP-zcytorlO fusion, as described below.
  • Plasmid pTAP98 is derived from the plasmids pRS316 and pMAL-c2.
  • the plasmid pRS316 is a Saccharomyces cerevisiae shuttle vector (Hieter P. and Sikorski, R., Genetics 122:19-27, 1989).
  • pMAL-C2 (NEB) is an E. coli expression plasmid. It carries the tac promoter driving MalE (gene encoding MBP) followed by a His tag, a thrombin cleavage site, a cloning site, and the rrnB terminator.
  • the vector pTAP98 is constructed using yeast homologous recombination.
  • lOOng of EcoRI cut pMAL-c2 is recombined with l ⁇ g Pvul cut pRS316, l ⁇ g linker, and l ⁇ g Seal /EcoRI cut pRS316 are combined in a PCR reaction.
  • PCR products are concentrated via 100% ethanol precipitation.
  • Competent yeast cells S. cerevisiae
  • Competent yeast cells are combined with about 10 ⁇ l of a mixture containing approximately 1 ⁇ g of the zcytorlO receptor PCR product above, and 100 ng of Smal digested pTAP98 vector, and electroporated using standard methods and plated onto URA-D plates and incubated at 30°C.
  • the Ura+ yeast transformants from a single plate are picked, DNA isolated, and transformed into electrocompetent E. coli cells (e.g., MCI 061, Casadaban et. al. J. Mol. Biol. 138, 179-207), and plated on MM/CA +AMP 100 mg/L plates (Pryor and Leiting, Protein Expression and Pruification 10:309-319, 1997).using standard procedures. Cells are grown in MM/CA with 100 ⁇ g/ml Ampicillin for two hours, shaking, at 37°C. 1ml of the culture is induced with ImM IPTG.
  • each culture is mixed with 250 ⁇ l acid washed glass beads and 250 ⁇ l Thorner buffer with 5% ⁇ ME and dye (8M urea, 100 mM Tris pH7.0, 10% glycerol, 2mM EDTA, 5% SDS). Samples are vortexed for one minute and heated to 65°C for 10 minutes. 20 ⁇ l are loaded per lane on a 4%-12% PAGE gel (NO VEX). Gels are run in IXMES buffer. The positive clones are designated pCZR225 and subjected to sequence analysis.
  • One microliter of sequencing DNA is used to transform strain BL21.
  • the cells are electropulsed at 2.0 kV, 25 ⁇ F and 400 ohms. Following electroporation, 0.6 ml MM/CA with 100 mg/L Ampicillin. Cells are grown in MM CA and induced with ITPG as described above., The positive clones are used to grow up for protein purification of the huzcytorl0/MBP-6H fusion protein using standard techniques.
  • Example 14 ZcytorlO Soluble Receptor Polyclonal Antibodies
  • Polyclonal antibodies are prepared by immunizing female New Zealand white rabbits with the purified huzcytorl0/MBP-6H poiypeptide (Example 13), or the purified recombinant zcytorlOCEE soluble receptor (Example 11).
  • the rabbits are each given an initial intraperitoneal (IP) injection of 200 mg of purified protein in Complete Freund's Adjuvant (Pierce, Rockford, IL) followed by booster IP injections of 100 mg purified protein in Incomplete Freund's Adjuvant every three weeks. Seven to ten days after the administration of the third booster injection, the animals are bled and the serum is collected. The rabbits are then boosted and bled every three weeks.
  • IP intraperitoneal
  • the zcytorlO-specific polyclonal antibodies are affinity purified from the rabbit serum using an CNBr-SEPHAROSE 4B protein column (Pharmacia LKB) that is prepared using about 10 mg of the purified huzcytorlO/MBP-6H poiypeptide per gram CNBr-SEPHAROSE, followed by 20X dialysis in PBS overnight.
  • ZcytorlO-specific antibodies are characterized by an ELISA titer check using 1 mg/ml of the appropriate protein antigen as an antibody target.
  • the lower limit of detection (LLD) of the rabbit anti-zcytorlO affinity purified antibodies is determined using standard methods.
  • ZcytorlO soluble receptor Monoclonal antibodies are prepared by immunizing male BalbC mice (Harlan Sprague Dawley, Indianapolis, IN) with the purified recombinant soluble zcytorlO proteins described herein. The mice are each given an initial intraperitoneal (IP) injection of 20 mg of purified protein in Complete Freund's Adjuvant (Pierce, Rockford, EL) followed by booster EP injections of 10 mg purified protein in Incomplete Freund's Adjuvant every two weeks. Seven to ten days after the administration of the third booster injection, the animals are bled and the serum is collected, and antibody titer assessed.
  • IP intraperitoneal
  • Splenocytes are harvested from high-titer mice and fused to murine SP2/0 myeloma cells using PEG 1500 (Boerhinger Mannheim, UK) in two separate fusion procedures using a 4: 1 fusion ratio of splenocytes to myeloma cells (Antibodies: A Laboratory Manual, E. Harlow and D. Lane, Cold Spring Harbor Press).
  • specific antibody-producing hybridomas are identified by ELISA using purified recombinant zcytorlO soluble receptor protein (Example 6C) as an antibody target and by FACS using Baf3 cells expressing the zcytorlO sequence (Example 8) as an antibody target.
  • the resulting hybridomas positive by both methods are cloned three times by limiting dilution.
  • Example 16 Assessing ZcytorlO Receptor Heterodimerization using ORIGEN assay
  • Soluble zcytorlO receptor zcytorlOCFLAG (Example 11), or g ⁇ l30 (Hibi, M. et al., Cell 63:1149-1157, 1990) are biotinylated by reaction with a five-fold molar excess of sulfo-NHS-LC-Biotin (Pierce, Inc., Rockford, IL) according to the manufacturer's protocol.
  • Soluble zcytorlO receptor and another soluble receptor subunit for example, soluble EL-7R ⁇ (sIL-7R ⁇ ) or IL-2 receptor- ⁇ (sIL-2R ⁇ ) (R&D Systems, Minneapolis, MN), or soluble zalphal l receptor (IL-21R; commonly owned US Pat. Application No.
  • biotinylated and Ru-BPY-NHS-labeled forms of the soluble zcytorlO receptor can be respectively designated Bio-zcytorlO receptor and Ru-zcytorlO; the biotinylated and Ru-BPY-NHS-labeled forms of the other soluble receptor subunit can be similarly designated.
  • Assays can be carried out using conditioned media from cells expressing a ligand that binds zcytorlO heterodimeric receptors, or using purified ligands.
  • Preferred ligands are those that can bind class 1 heterodimeric cytokine receptors such as, IL-2, IL-4, IL-7, IL-9, IL-15, zalphal l Ligand (IL-21) (commonly owned US Pat. Application No. 09/522,217), TSLP (Levine, SD et al., ibid.; Isaksen, DE et al., ibid.; Ray, RJ et al., ibid.; Friend, SL et al., ibid.).
  • class 1 heterodimeric cytokine receptors such as, IL-2, IL-4, IL-7, IL-9, IL-15, zalphal l Ligand (IL-21) (commonly owned US Pat. Application No. 09/522,217), TSLP (Levine, SD et al., ibid.; Isaksen, DE et al., ibid.; Ray, RJ
  • cytokines or conditioned medium are tested to determine whether they can mediate homodimerization of zcytorlO receptor and if they can mediate the heterodimerization of zcytorlO receptor with the soluble receptor subunits described above.
  • TBS-B 50 ⁇ l of conditioned media or TBS-B containing purified cytokine
  • TBS-B 20 mM Tris, 150 mM NaCl, 1 mg/ml BSA, pH 7.2
  • e.g., 400 ng/ml of Ru-zcytorlO receptor and Bio-zcytorlO 400 ng/ml of Ru-zcytorlO receptor and e.g., Bio-gpl30, or 400 ng/ml of e.g., Ru-EL2R ⁇ and Bio-zcytorlO.
  • Example 17 Construct for generating a zcytorlO receptor Heterodimer
  • a vector expressing a secreted human zcytorlO heterodimer is constructed.
  • the extracellular cytokine-binding domain of zcytorlO is fused to the heavy chain of IgG gamma 1 (IgG ⁇ l) (SEQ ID NO:28 and SEQ ID NO:29), while the extracellular portion of the heteromeric cytokine receptor subunit (E.g., an EL- 2 receptor component (IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ ), an EL-4/EL-13 receptor family receptor components (EL-4R ⁇ , IL-13R ⁇ , IL-13R ⁇ '), interleukin receptor subunits (e.g., IL-15 R ⁇ , EL-7R ⁇ , IL-9R ⁇ ); or zalphal l receptor (IL-21R)) is fused to a human kappa light chain (human K light chain) (SEQ ID NO:30 and SEQ ID NO:31).
  • IgG gamma 1 and human K light chain fusion vectors The heavy chain of IgG ⁇ l is cloned into the Zem229R mammalian expression vector (ATCC deposit No. 69447) such that any desired cytokine receptor extracellular domain having a 5' EcoRI and 3' Nhel site can be cloned in resulting in an N-terminal extracellular domain-C-terminal IgG ⁇ l fusion.
  • the IgG ⁇ l fragment used in this construct is made by using PCR to isolate the IgG ⁇ l sequence from a Clontech hFetal Liver cDNA library as a template.
  • PCR products are purified using methods described herein and digested with Mlul and EcoRI (Boerhinger-Mannheim), ethanol precipitated and ligated with oligos ZC11,440 (SEQ ID NO: 32) and ZC11,441 (SEQ ID NO:33), which comprise an MluL ⁇ coRI linker, into Zem229R previously digested with and EcoRI using standard molecular biology techniques disclosed herein.
  • the human K light chain (SEQ ID NO:30 and SEQ ID NO:31) is cloned in the Zem228R mammalian expression vector (ATCC deposit No.
  • any desired cytokine receptor extracellular domain having a 5' EcoRI site and a 3' Kpnl site can be cloned in resulting in a N-terminal cytokine extracellular domain-C-terminal human K light chain fusion.
  • a special primer is designed to clone the 3' end of the desired extracellular domain of a cytokine receptor into this Kpnl site:
  • the primer is designed so that the resulting PCR product contains the desired cytokine receptor extracellular domain with a segment of the human K light chain up to the Kpnl site (SEQ ID NO:36).
  • This primer preferably comprises a portion of at least 10 nucleotides of the 3' end of the desired cytokine receptor extracellular domain fused in frame 5' to SEQ ID NO:36.
  • the human K light chain fragment used in this construct is made by using PCR to isolate the human K light chain sequence from the same Clontech human Fetal Liver cDNA library used above. PCR products are purified using methods described herein and digested with Mlul and EcoRI (Boerhinger-Mannheim), ethanol precipitated and ligated with the MluI/EcoRI linker described above, into Zem228R previously digested with and EcoRI using standard molecular biology techniques disclosed herein.
  • the resulting PCR product is digested with EcoRI and Nhel, gel purified, as described herein, and ligated into a previously EcoRI and Nhel digested and band-purified Zem229R/IgG ⁇ l described above.
  • the resulting vector is sequenced to confirm that the zcytorlO/IgG gamma 1 fusion (zcytorlO/Chl IgG) is correct.
  • a separate construct having a heterodimeric cytokine receptor subunit extracellular domain fused to K light is also constructed as above.
  • the EL-2R ⁇ /human K light chain construction is performed as above by PCRing from, e.g., a lymphocyte cDNA library (Clontech) using standard methods, and oligos that provide EcoRI and Kpnl restriction sites.
  • the resulting PCR product is digested with EcoRI and Kpnl and then ligating this product into a previously EcoRI and Kpnl digested and band-purified Zem228R/human K light chain vector described above.
  • the resulting vector is sequenced to confirm that the cytokine receptor subunit/human K light chain fusion is correct.
  • Approximately 15 ⁇ g of each of vectors above, are co-transfected into mammalian cells, e.g., BHK-570 cells (ATCC No. CRL-10314) using LipofectaminePlusTM reagent (Gibco/BRL), as per manufacturer's instructions.
  • the transfected cells are selected for 10 days in DMEM + 5%FBS (Gibco/BRL) containing 1 ⁇ M of methotrexate (MTX) (Sigma, St. Louis, MO) and 0.5 mg/ml G418 (Gibco/BRL) for 10 days.
  • the resulting pool of transfectants is selected again in 10 ⁇ m of MTX and 0.5 mg/ml G418 for 10 days.
  • the resulting pool of doubly selected cells is used to generate protein.
  • Three Factories (Nunc, Denmark) of this pool are used to generate 10 L of serum free conditioned medium. This conditioned media is passed over a 1 ml protein-A column and eluted in about 10, 750 microliter fractions. The fractions having the highest protein concentration are pooled and dialyzed (10 kD MW cutoff) against PBS. Finally the dialyzed material is submitted for amino acid analysis (AAA) using routine methods.
  • AAA amino acid analysis
  • Example 18 Determination of receptor subunits that heterodimerize or multimerize with zcytorlO receptor.
  • the BaF3/MPL-zcytorl0 chimera cells (Example 6) are transfected with an additional heterodimeric cytokine receptor subunit serve as a bioassay cell line to measure signal transduction response of heterodimeric zcytorlO receptor complexes to the luciferase reporter in the presence of
  • TPO TPO (Example 6).
  • the BaF3/MPL-zcytorl0 cells do not signal, suggesting that zcytorlO receptor must heterodimerize to signal.
  • Transfection of the BaF3/MPL-zcytorl0 cell line with and additional MPL-class I cytokine receptor fusion that signals in the presence of the TPO ligand determines which heterodimeric cytokine receptor subunits are required for zcytorlO receptor signaling.
  • Use of MPL-receptor fusions for this purpose alleviates the requirement for the presence of a natural ligand for the zcytorlO receptor.
  • MPL-class I cytokine receptor fusions are made as per Example 5 using the extracellular domain and transmembrane domains of the MPL receptor and the intracellular signaling domain of the desired class I cytokine receptor.
  • the BaF3/MPL- zcytorlO bioassay cell line co-transfected with an individual MPL-class I cytokine receptor fusions as per Example 6 to form a BaF3/MPL-zcytorlO/MPL-class I cytokine receptor cell line.
  • Receptor complexes include but are not limited to zcytorlO receptor in combination with an MPL-cytokine receptor fusion comprising one or more of the IL-2 receptor components (IL-2R ⁇ , EL-2R ⁇ , IL-2R ⁇ ), zcytorlO receptor with one or more of the EL-4/IL-13 receptor family receptor components (EL-4R ⁇ , EL-13R ⁇ , IL- 13R ⁇ '), as well as other Interleukin receptors (e.g., IL-15 R ⁇ , IL-7R ⁇ , IL-9R ⁇ , IL-21R (Zalphal 1 receptor)).
  • Each independent receptor complex cell line is then assayed in the presence of TPO (example 6) and proliferation measured using routine methods (e.g., Alamar Blue assay as described in Example 6).
  • the BaF3/MPL-zcytorl0 bioassay cell line serves as a control for the background luciferase activity, and is thus used as a baseline to compare signaling by the various receptor complex combinations.
  • a BaF3/MPL-class I cytokine receptor cell line can be constructed to control for MPL-class I cytokine receptor homodimerization effects for those class I cytokine receptors known to signal upon homodimerization.
  • the TPO in the presence of the correct receptor complex is expected to increase proliferation of the BaF3/MPL- zcytorlO/MPL-class I cytokine receptor cell line approximately 5 fold over background or greater in the presence of TPO.
  • Example 19 Reconstitution of zcytorlO receptor in vitro
  • BHK 570 cells ATCC No. CRL-10314 transfected, using standard methods described herein, with a luciferase reporter mammalian expression vector plasmid serve as a bioassay cell line to measure signal transduction response from a transfected zcytorlO receptor complex to the luciferase reporter in the presence of zcytorlO Ligand.
  • BHK cells do not endogenously express the zcytorlO receptor.
  • An exemplary luciferase reporter mammalian expression vector is the KZ134 plasmid which was constructed with complementary oligonucleotides ZC12,749 (SEQ ID NO:37) and ZC12,748 (SEQ ID NO:38) that contain STAT transcription factor binding elements from 4 genes.
  • a modified c-fos Sis inducible element m67SIE, or hSIE
  • the p21 SIE1 from the p21 WAF1 gene (Chin, Y.
  • oligonucleotides contain Asp718-XhoI compatible ends and were ligated, using standard methods, into a recipient firefly luciferase reporter vector with a c-fos promoter (Poulsen, L.K. et al., J.
  • the KZ134 plasmid is used to stably transfect BHK, or BaF3 cells, using standard transfection and selection methods, to make a BHK/KZ134 or BaF3/KZ134 cell line respectively.
  • the bioassay cell line is transfected with zcytorlO receptor alone, or co- transfected with zcytorlO receptor along with one of a variety of other known receptor subunits.
  • Receptor complexes include but are not limited to zcytorlO receptor only, various combinations of zcytorlO receptor with one or more of the EL-2 receptor components (EL-2R , IL-2R ⁇ , IL-2R ⁇ ), zcytorlO receptor with one or more of the EL- 4/IL-13 receptor family receptor components (EL-4R ⁇ , IL-13R ⁇ , IL-13R ⁇ '), as well as other Interleukin receptors (e.g., IL-15 R ⁇ , IL-7R ⁇ , IL-9R ⁇ , IL-21R (zalphal l)).
  • Each independent receptor complex cell line is then assayed in the presence of cytokine- conditioned media or purified cytokines and luciferase activity measured using routine methods.
  • the untransfected bioassay cell line serves as a control for the background luciferase activity, and is thus used as a baseline to compare signaling by the various receptor complex combinations.
  • the conditioned medium or cytokine that binds the zyctorlO receptor in the presence of the correct receptor complex is expected to give a luciferase readout of approximately 5 fold over background or greater.
  • a similar assay can be performed wherein the Baf3/zcytorl0-mpl and Baf3/zcytorl0 (Example 10) cell lines are co-transfected as described above and proliferation measured.

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Abstract

L'invention concerne de nouveaux polypeptides, polynucléotides codant les polypeptides, ainsi que des compositions et procédés connexes destinés à un nouveau récepteur de cytokine de souris, zcytor10 de souris. On peut utiliser les polypeptides dans le cadre des procédés destinés à détecter des ligands qui stimulent la prolifération et/ou le développement de cellules hématopoïétiques, lymphoïdes et myéloïdes. Les polypeptides du récepteur de liaison des ligands peuvent également être utilisés pour bloquer l'activité du ligand. Les polynucléotides codant le récepteur zcytor10 de souris peuvent être utilisés pour identifier un orthologue humain. L'invention concerne également des procédés de production de la protéine, les utilisations associées et les anticorps correspondants.
EP00928962A 1999-05-11 2000-05-11 Recepteur de cytokine zcytor10 de souris Withdrawn EP1185641A1 (fr)

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ES2377188T3 (es) * 2002-01-18 2012-03-23 Zymogenetics, Inc. Nuevo ligando de citocina ZCYTOR17
US7217797B2 (en) 2002-10-15 2007-05-15 Pdl Biopharma, Inc. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
US7217798B2 (en) 2003-10-15 2007-05-15 Pdl Biopharma, Inc. Alteration of Fc-fusion protein serum half-lives by mutagenesis
US7365168B2 (en) 2002-10-15 2008-04-29 Pdl Biopharma, Inc. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis

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