EP1192254A1 - Polypeptide spirale zalpha29 - Google Patents

Polypeptide spirale zalpha29

Info

Publication number
EP1192254A1
EP1192254A1 EP00941509A EP00941509A EP1192254A1 EP 1192254 A1 EP1192254 A1 EP 1192254A1 EP 00941509 A EP00941509 A EP 00941509A EP 00941509 A EP00941509 A EP 00941509A EP 1192254 A1 EP1192254 A1 EP 1192254A1
Authority
EP
European Patent Office
Prior art keywords
seq
residues
zalpha29
polypeptide
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00941509A
Other languages
German (de)
English (en)
Inventor
Darrell C. Conklin
Zeren Gao
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zymogenetics Inc
Original Assignee
Zymogenetics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zymogenetics Inc filed Critical Zymogenetics Inc
Publication of EP1192254A1 publication Critical patent/EP1192254A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons

Definitions

  • Cytokines are polypeptide hormones that are produced by a cell and affect the growth or metabolism of that cell or another cell. In multicellular animals, cytokines control cell growth, migration, differentiation, and maturation. Cytokines play a role in both normal development and pathogenesis, including the development of solid tumors.
  • Cytokines are physicochemically diverse, ranging in size from 5 kDa (TGF- ⁇ ) to 140 kDa (Mullerian-inhibiting substance). They include single polypeptide chains, as well as disulfide-linked homodimers and heterodimers.
  • Cytokines influence cellular events by binding to cell-surface receptors. Binding initiates a chain of signalling events within the cell, which ultimately results in phenotypic changes such as cell division, protease production, cell migration, expression of cell surface proteins, and production of additional growth factors. Cell differentiation and maturation are also under control of cytokines.
  • the hematopoietic factors erythropoietin, thrombopoietin, and G-CSF stimulate the production of erythrocytes, platelets, and neutrophils, respectively, from precursor cells in the bone marrow.
  • Development of mature cells from pluripotent progenitors may require the presence of a plurality of factors.
  • the role of cytokines in controlling cellular processes makes them likely candidates and targets for therapeutic intervention; indeed, a number of cytokines have been approved for clinical use.
  • Interferon-alpha for example, is used in the treatment of hairy cell leukemia, chronic myeloid leukemia, Kaposi's sarcoma, condylomata acuminata, chronic hepatitis C, and chronic hepatitis B (Aggarwal and Puri, "Common and Uncommon Features of Cytokines and Cytokine Receptors: An
  • cytokine erythropoietin has been approved in the United States and other countries for the treatment of dermal ulcers in diabetic patients.
  • the hematopoietic cytokine erythropoietin has been developed for the treatment of anemias (e.g., EP 613,683).
  • G- CSF, GM-CSF, IFN- ⁇ , IFN- ⁇ , and IL-2 have also been approved for use in humans (Aggarwal and Puri, ibid.).
  • Experimental evidence supports additional therapeutic uses of cytokines and their inhibitors.
  • VEGFs Vascular endothelial growth factors
  • WO 95/24473 U.S. Patent No. 5,219,739
  • a soluble VEGF receptor (soluble flt-1) has been found to block binding of VEGF to cell-surface receptors and to inhibit the growth of vascular tissue in vitro (Biotechnology News 16(17):5-6, 1996). Experimental evidence suggests that inhibition of angiogenesis may be used to block tumor development (Biotechnology News, Nov. 13, 1997) and that angiogenesis is an early indicator of cervical cancer (Br. I. Cancer 76:1410-1415, 1997).
  • thrombopoietin has been shown to stimulate the production of platelets in vivo (Kaushansky et al., Nature 369:568-571, 1994) and has been the subject of several clinical trials (reviewed by von dem Borne et al., Bailliere's Clin. Haematol. JJ . :427-445, 1998).
  • Cytokines are used in the laboratory to study developmental processes, and in laboratory and industry settings as components of cell culture media.
  • an isolated polypeptide comprising a sequence of amino acid residues selected from the group consisting of residues 48-62 of SEQ ID NO:2, residues 47-61 of SEQ ID NO:4, residues 63-104 of SEQ ID NO:2, residues 62-103 of SEQ ID NO:4, residues 105-119 of SEQ ID NO:2, residues 104-118 of SEQ ID NO:4, residues 120-137 of SEQ ID NO:2, residues 119-136 of SEQ ID NO:4, residues 138-152 of SEQ ID NO:2, residues 137-151 of SEQ ID NO:4, residues 153-170 of SEQ ID NO:2, residues 152-169 of SEQ ID NO:4, residues 171-185 of SEQ ID NO:2, and residues 170-184 of SEQ ID NO:4.
  • the isolated polypeptide has from 15 to 1500 amino acid residues.
  • the sequence of amino acid residues is operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, a polyhistidine tag, and a peptide as shown in SEQ ID NO:5.
  • the isolated polypeptide comprises at least 30 contiguous residues of SEQ ID NO:2 or SEQ ID NO:4.
  • the isolated polypeptide comprises residues 48-185 or residues 27-190 of SEQ ID NO:6.
  • the isolated polypeptide comprises residues 48-185 of SEQ ID NO:2, residues 47-184 of SEQ ID NO:4, residues 27-190 of SEQ ID NO:2, or residues 26-188 of SEQ ID NO:4.
  • an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as disclosed above; and a transcription terminator.
  • the DNA segment comprises nucleotides 79 to 570 of SEQ ID NO:7.
  • the expression vector further comprises a secretory signal sequence operably linked to the DNA segment.
  • the invention provides a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses the DNA segment.
  • the expression vector further comprises a secretory signal sequence operably linked to the DNA segment, and the polypeptide is secreted by the cell.
  • the invention provides a method of making a protein comprising culturing a cell into which has been introduced an expression vector as disclosed above under conditions whereby the DNA segment is expressed and the polypeptide is produced, and recovering the protein.
  • the expression vector further comprises a secretory signal sequence operably linked to the DNA segment, the polypeptide is secreted by the cell and recovered from a medium in which the cell is cultured.
  • the invention provides a protein produced by the method disclosed above.
  • an antibody that specifically binds to the protein disclosed above.
  • a seventh aspect of the invention there is provided method of detecting, in a test sample, the presence of an antagonist of zalpha29 activity.
  • the method comprises the steps of (a) culturing a cell that is responsive to zal ⁇ ha29; (b) exposing the cell to a zalpha29 polypeptide in the presence and absence of a test sample; (c) comparing levels of response to the zalpha29 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and (d) determining from the comparison the presence of an antagonist of zalpha29 activity in the test sample.
  • Figs. 1A-1D are a Hopp/Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO:2. 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. These residues are indicated in the figure by lower case letters.
  • Fig. 2 is an alignment of representative human (SEQ ID NO:2) and mouse (SEQ ID NO:4) zalpha29 amino acid sequences.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • affinity tag 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.
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • amino-terminal and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • Angiogenic denotes the ability of a compound to stimulate the formation of new blood vessels from existing vessels, acting alone or in concert with one or more additional compounds. Angiogenic activity is measurable as endothelial cell activation, stimulation of protease secretion by endothelial cells, endothelial cell migration, capillary sprout formation, and endothelial cell proliferation. Angiogenesis can also be measured using any of several in vivo assays as disclosed herein.
  • a "complement" of a polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence.
  • the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • corresponding to when applied to positions of amino acid residues in sequences, means corresponding positions in a plurality of sequences when the sequences are optimally aligned.
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
  • expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
  • additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example,
  • An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated polypeptide may be substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • the polypeptides may be prepared in a highly purified form, i.e. greater than 95% pure or greater than 99% pure.
  • the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • “Operably linked” means that two or more entities are joined together such that they function in concert for their intended purposes.
  • DNA segments the phrase indicates, for example, that coding sequences are joined in the correct reading frame, and transcription initiates in the promoter and proceeds through the coding segment(s) to the terminator.
  • “operably linked” includes both covalently (e.g., by disulfide bonding) and non-covalently (e.g., by hydrogen bonding, hydrophobic interactions, or salt-bridge interactions) linked sequences, wherein the desired function(s) of the sequences are retained.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • a "polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.
  • bp base pairs
  • nt nucleotides
  • kb kilobases
  • the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
  • polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides”.
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a "protein” is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • a “secretory signal sequence” is a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • a “segment” is a portion of a larger molecule (e.g., polynucleotide or polypeptide) having specified attributes.
  • a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from the 5' to the 3' direction, encodes the sequence of amino acids of the specified polypeptide.
  • zalpha29 This novel cytokine, termed "zalpha29", was identified by the presence of polypeptide and polynucleotide features characteristic of four-helix-bundle cytokines (e.g., erythropoeitin, thrombopoietin, G-CSF, IL-2, IL-4, leptin, and growth hormone). Analysis of the human zalpha29 amino acid sequence shown in SEQ ID NO:2 indicates the presence of four amphipathic, alpha-helical regions.
  • These regions include at least amino acid residues 48 through 62 (helix A), 105 through 119 (helix B), 138 through 152 (helix C), and 171 through 185 (helix D).
  • residues that are expected to lie within the core of the four-helix bundle occur at positions 48, 51, 52, 55, 58, 59, 62, 105, 108, 109, 112, 115, 116, 119, 138, 141, 142, 145, 148, 149, 152, 171, 174, 175, 178, 181, 182, and 185 of SEQ ID NO:2.
  • Residues 49, 50, 53, 54, 56, 57, 60, 61, 106, 107, 110, 111, 113, 114, 117, 118, 139, 140, 143, 144, 146, 147, 150, 151, 172, 173, 176, 177, 179, 180, 183, and 184 are expected to lie on the exposed surface of the bundle.
  • Inter-helix loops comprise approximately residues 63 through 104 (loop A-B), 120 through 137 (loop B-C), and 153 through 170 (loop C-D).
  • the human zalpha29 cDNA (SEQ ID NO:l) encodes a polypeptide of 190 amino acid residues.
  • this sequence is predicted to include a secretory peptide of 26 residues. Cleavage after residue 26 will result in a mature polypeptide (residues 27-190 of SEQ ID NO:2) having a calculated molecular weight (exclusive of glycosylation) of 18,558 Da.
  • cytokines e.g., endothelial cell growth factor, basic FGF, and IL-l ⁇
  • the cDNA also includes a clear polyadenylation signal.
  • the mouse zalpha29 polypeptide (SEQ JO NO:4) is predicted to include helices and loops at analogous positions, including helices at residues 47-61, 104-118, 137-151, and 170-184; and loops at residues 62-103, 119-136, and 152-169. See Fig. 2.
  • Polypeptides of the present invention comprise at least 15 contiguous amino acid residues of SEQ ID NO:2. Within certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of SEQ ID NO:2, up to the entire predicted mature polypeptide (residues 27 to 190 of SEQ ID NO:2) or the primary translation product (residues 1 to 190 of SEQ ID NO:2).
  • Corresponding mouse zalpha29 polypeptides are also provided by the invention. As disclosed in more detail below, these polypeptides can further comprise additional, non-zalpha29, polypeptide sequence(s).
  • polypeptides of the present invention are polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ ID NO:2 or SEQ ID NO:4.
  • An "epitope” is a region of a protein to which an antibody can bind. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002, 1984.
  • Epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least 6 amino acid residues in length.
  • Relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, Sutcliffe et al., Science 219:660-666. 1983.
  • Antibodies that recognize short, linear epitopes are particularly useful in analytic and diagnostic applications that employ denatured protein, such as Western blotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979), or in the analysis of fixed cells or tissue samples.
  • Antibodies to linear epitopes are also useful for detecting fragments of zalpha29, such as might occur in body fluids or cell culture media.
  • Antigenic, epitope-bearing polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies, that specifically bind to a zalpha29 protein.
  • Antigenic, epitope-bearing polypeptides contain a sequence of at least six, often at least nine, commonly from 15 to about 30 contiguous amino acid residues of a zalpha29 protein (e.g., SEQ ID NO:2).
  • Polypeptides comprising a larger portion of a zalpha29 protein, i.e. from 30 to 50 residues up to the entire sequence, are included.
  • amino acid sequence of the epitope-bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided.
  • Such regions include the interdomain loops of zalpha29 and fragments thereof, in particular loop B-C (residues 120-137 of SEQ ID NO:2), which is markedly hydrophilic (see Fig. IC).
  • Polypeptides in this regard include those comprising residues 99-104, 129-134, and 162-167 of SEQ ID NO:2.
  • polypeptides that comprise the entire four-helix bundle of a zalpha29 polypeptide (e.g., residues 48-185 of SEQ ID NO:2).
  • Such polypeptides may further comprise all or part of one or both of the native zalpha29 amino-terminal (residues 27-47 of SEQ ID NO:2) and carboxyl- terminal (residues 186-190 of SEQ ID NO:2) regions, as well as non-zalpha29 amino acid residues or polypeptide sequences as disclosed in more detail below.
  • Polypeptides of the present invention can be prepared with one or more amino acid substitutions, deletions or additions as compared to SEQ ID NO:2. These changes will usually be of a minor nature, that is conservative amino acid substitutions and other changes that do not significantly affect the folding or activity of the protein or polypeptide, and include amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, an amino or carboxyl-terminal cysteine residue to facilitate subsequent linking to maleimide-activated keyhole limpet hemocyanin, a small linker peptide of up to about 20-25 residues, or an extension that facilitates purification (an affinity tag) as disclosed above. Two or more affinity tags may be used in combination.
  • Polypeptides comprising affinity tags can further comprise a polypeptide linker and/or a proteolytic cleavage site between the zalpha29 polypeptide and the affinity tag.
  • exemplary cleavage sites include thrombin cleavage sites and factor Xa cleavage sites.
  • a zalpha29 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Suitable dimerizing proteins in this regard include immunoglobulin constant region domains. Immunoglobulin-zalpha29 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zalpha29 analogs. In addition, a zalpha29 polypeptide can be joined to another bioactive molecule, such as a cytokine, to provide a multi-functional molecule.
  • a bioactive molecule such as a cytokine
  • One or more helices of a zalpha29 polypeptide can be joined to another cytokine to enhance or otherwise modify its biological properties.
  • Auxiliary domains can be fused to zalpha29 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen).
  • a zalpha29 polypeptide or protein can be targeted to a predetermined cell type by fusing a zalpha29 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell.
  • a zalpha29 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain.
  • Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9, 1996.
  • Polypeptide fusions of the present invention will generally contain not more than about 1,500 amino acid residues, often not more than about 1,200 residues, usually not more than about 1,000 residues, and will in many cases be considerably smaller.
  • a zalpha29 polypeptide of 164 residues can be fused to E. coli -galactosidase (1,021 residues; see Casadaban et al., I. Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a 4-residue factor Xa cleavage site to yield a polypeptide of 1,199 residues.
  • residues 27-190 of SEQ ID NO:2 can be fused to maltose binding protein (approximately 370 residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.
  • the polypeptides of the present invention comprise at least 15 contiguous residues of SEQ ID NO:2 or SEQ ID NO:4. These polypeptides may further comprise additional residues as shown in SEQ ID NO:2, a variant of SEQ ID NO:2, or another protein as disclosed herein.
  • "Variants of SEQ ID NO:2" includes polypeptides that are at least 85%, at least 90%, or at least 95% identical to the corresponding region of SEQ ID NO:2.
  • Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as:
  • 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. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, I. Mol. Biol. 48:444, 1970; Sellers, SIAM I. Appl Math.
  • 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 from one to six, preferably from three to six, most preferably three, with other parameters set as default.
  • the present invention includes polypeptides having one or more conservative amino acid changes as compared with the amino acid sequence of SEQ ID NO:2.
  • the BLOSUM62 matrix (Table 1) 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, ibid.). Thus, 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.
  • the term "conservative amino acid substitution” refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, 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 one 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).
  • the proteins of the present invention can also comprise non-naturally occuring amino acid residues.
  • Non-naturally occuring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine.
  • Several methods are known in the art for incorporating non-naturally occuring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tR ⁇ As. Methods for synthesizing amino acids and aminoacylating tR ⁇ A are known in the art.
  • E. 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 occuring amino acid(s) (e.g., 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • the desired non-naturally occuring amino acid(s) e.g., 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine.
  • non-naturally occuring amino acid is inco ⁇ orated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994. Naturally occuring amino acid residues can be converted to non-naturally occuring 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).
  • Amino acid sequence changes are made in zalpha29 polypeptides so as to minimize disruption of higher order structure essential to biological activity. For example, changes in amino acid residues will be made so as not to disrupt the four- helix bundle characteristic of the protein family.
  • the effects of amino acid sequence changes can be predicted by computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al., ibid.).
  • a hydrophilicity profile of SEQ ID NO:2 is shown in Figs. 1A-1D. Those skilled in the art will recognize that this hydrophilicity will be taken into account when designing alterations in the amino acid sequence of a zalpha29 polypeptide, so as not to disrupt the overall profile.
  • Residues within the core of the four-helix bundle can be replaced with other residues as shown in SEQ ID NO: 6.
  • the residues predicted to be on the exposed surface of the four-helix bundle will be relatively intolerant of substitution.
  • Other candidate amino acid substitutions within human zalpha29 are suggested by alignment of the human (SEQ ID NO:2) and mouse (SEQ ID NO:4) sequences as shown in Fig. 2, which sequences are approximately 85% identical overall.
  • the cysteine residue at position 160 of SEQ ID NO:2 (position 159 of SEQ ID NO:4) lies in loop C-D, suggesting its participation in an interchain disulfide bond. This residue is thus expected to be relatively intolerant of substitution.
  • One skilled in the art may employ many well known techniques, independently or in combination, to analyze and compare the structural features that affect folding of a variant protein or polypeptide to a standard molecule to determine whether such modifications would be significant.
  • One well known and accepted method for measuring folding is circular dichroism (CD). Measuring and comparing the CD spectra generated by a modified molecule and standard molecule are routine in the art (Johnson, Proteins 7:205-214, 1990). Crystallography is another well known and accepted method for analyzing folding and structure. Nuclear magnetic resonance (NMR), digestive peptide mapping and epitope mapping are other known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al., Science 257:961-964, 1992).
  • NMR nuclear magnetic resonance
  • digestive peptide mapping and epitope mapping are other known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al., Science 257:961-964, 1992).
  • 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, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991).
  • site-directed mutagenesis or alanine-scanning mutagenesis Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991.
  • single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule.
  • variant genes are generated by in vitro homologous recombination by random fragmentation of a parent gene followed by reassembly using PCR, resulting in randomly introduced point mutations.
  • This technique can be modified by using a family of parent genes, such as allelic variants or genes from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes. In many cases, the structure of the final polypeptide product will result from processing of the nascent polypeptide chain by the host cell, thus the final sequence of a zalpha29 polypeptide produced by a host cell will not always correspond to the full sequence encoded by the expressed polynucleotide.
  • expressing the complete zalpha29 sequence in a cultured mammalian cell is expected to result in removal of at least the secretory peptide, while the same polypeptide produced in a prokaryotic host would not be expected to be cleaved.
  • Differential processing of individual chains may result in heterogeneity of expressed polypeptides.
  • Zalpha29 proteins of the present invention are characterized by their activity, that is, modulation of the proliferation, differentiation, migration, adhesion, or metabolism of responsive cell types.
  • Biological activity of zalpha29 proteins is assayed using in vitro or in vivo assays designed to detect cell proliferation, differentiation, migration or adhesion; or changes in cellular metabolism (e.g., production of other growth factors or other macromolecules).
  • Many suitable assays are known in the art, and representative assays are disclosed herein. Assays using cultured cells are most convenient for screening, such as for determining the effects of amino acid substitutions, deletions, or insertions.
  • in vivo assays will generally be employed to confirm and further characterize biological activity.
  • Certain in vitro models such as the three-dimensional collagen gel matrix model of Pepper et al. (Biochem. Biophys. Res. Comm. 189:824-831. 1992), are sufficiently complex to assay histological effects.
  • Assays can be performed using exogenously produced proteins, or may be carried out in vivo or in vitro using cells expressing the polypeptide(s) of interest.
  • Assays can be conducted using zalpha29 proteins alone or in combination with other growth factors, such as members of the VEGF family or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stem cell factor). Representative assays are disclosed below. Mutagenesis methods as disclosed above can be combined with high volume or high-throughput screening methods to detect biological activity of zalpha29 variant polypeptides. Assays that can be scaled up for high throughput include mitogenesis assays, which can be run in a 96-well format. Mutagenized DNA molecules that encode active zalpha29 polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • the present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zalpha29 polypeptides disclosed above.
  • a representative DNA sequence encoding the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:l
  • a representative DNA sequence encoding the amino acid sequence of SEQ ID NO:4 is shown in SEQ ID NO:3.
  • SEQ ID NO:7 is a degenerate DNA sequence that encompasses all DNAs that encode the zalpha29 polypeptide of SEQ ID NO: 2.
  • SEQ ID NO:7 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T.
  • zalpha29 polypeptide-encoding polynucleotides comprising nucleotides 1-534 or nucleotides 52-534 of SEQ ID NO:7, and their RNA equivalents are contemplated by the present invention, as are segments of SEQ ID NO:7 encoding other zalpha29 polypeptides disclosed herein.
  • Table 2 sets forth the one-letter codes used within SEQ ED NO:7 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C. TABLE 2
  • degenerate codons used in SEQ ID NO:7 encompassing all possible codons for a given amino acid, are set forth in Table 3, below.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ ED NO:l, or a sequence complementary thereto, under stringent conditions.
  • 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.
  • Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.
  • cDNA Complementary DNA
  • genomic DNA can be isolated.
  • Polynucleotides encoding zalpha29 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • cDNA Complementary DNA
  • SEQ ED NO: 14 This sequence comprises an exon from nucleotide 1885 to nucleotide 2112 (corresponding to nucleotides 483-710 of SEQ ED NO:l).
  • Partial mouse genomic sequences are shown in SEQ ED NO: 15 and SEQ ED NO: 16.
  • nucleotides 6-165 are an exon corresponding to nucleotides 40-199 of SEQ ED NO:3.
  • nucleotides 175-295 are an exon corresponding to nucleotides 200-320 of SEQ ID NO:3.
  • DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
  • the targeting sequence is a zalpha29 5' non- coding sequence that permits homologous recombination of the construct with the endogenous zalpha29 locus, whereby the sequences within the construct become operably linked with the endogenous zalpha29 coding sequence.
  • an endogenous zalpha29 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
  • the present invention further provides counterpart polypeptides and polynucleotides from other species ("orthologs").
  • zalpha29 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides.
  • Orthologs of human zalpha29 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zalpha29 as disclosed above.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a zalpha29-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequence.
  • a cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human and mouse zalpha29 sequences 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 a zalpha29 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • any zalpha29 polypeptide including variants and fusion proteins
  • one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that polypeptide using the information set forth in Tables 3 and 4, above.
  • those of skill in the art can use standard software to devise zalpha29 variants based upon the nucleotide and amino acid sequences described herein.
  • the present invention thus provides a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ D NO:l, SEQ ED NO:2, SEQ ED NO:3, SEQ ED NO:4, SEQ ED NO:6, SEQ ED NO:7, and portions thereof.
  • Suitable forms of computer-readable media include magnetic media and optically-readable media.
  • Examples of magnetic media include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZEP disk.
  • Optically readable media are exemplified by compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD-recordable), and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).
  • compact discs e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD-recordable
  • DVD digital versatile/video discs
  • the zalpha29 polypeptides of the present invention can be produced according to conventional techniques using cells into which have been introduced an expression vector encoding the polypeptide.
  • a "cell into which has been introduced an expression vector” includes both cells that have been directly manipulated by the introduction of exogenous DNA molecules and progeny thereof that contain the introduced DNA.
  • 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.
  • a DNA sequence encoding a zalpha29 polypeptide 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 zalpha29, or may be derived from another secreted protein (e.g., t-PA; see, U.S. Patent No. 5,641,655) or synthesized de novo.
  • the secretory signal sequence is operably linked to the zalpha29 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly sythesized polypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
  • zalpha29 polypeptides via a host cell secretory pathway are expected to result in the production of multimeric proteins.
  • Such multimers include both homomultimers and heteromultimers, the latter including proteins comprising only zalpha29 polypeptides and proteins including zalpha29 and heterologous polypeptides (e.g., a second four-helix-bundle cytokine polypeptide). If a mixture of proteins results from expression, individual species are isolated by conventional methods. Monomers, dimers, and higher order multimers are separated by, for example, size exclusion chromatography. Heteromultimers can be separated from homomultimers by immunoaffinity chromatography using antibodies specific for individual dimers or by sequential immunoaffinity steps using antibodies specific for individual component polypeptides.
  • Multimers may also be assembled in vitro upon incubation of component polypeptides under suitable conditions.
  • in vitro assembly will include incubating the protein mixture under denaturing and reducing conditions followed by refolding and reoxidation of the polypeptides to from homodimers and heterodimers. Recovery and assembly of proteins expressed in bacterial cells is disclosed below.
  • Cultured mammalian cells can be used as 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 I.
  • 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.,
  • Expression vectors for use in mammalian cells include pZP-1 and pZP-9, which have been deposited with the American Type Culture Collection, Manassas, VA USA under accession numbers 98669 and 98668, respectively, and derivatives thereof.
  • 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.”
  • An exemplary 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.
  • An exemplary amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • the adenovirus system can also be used for protein production in vitro.
  • 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.
  • 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 can also be effectively obtained.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) according to methods known in the art.
  • recombinant baculovirus is produced through the use of a transposon-based system described by Luckow et al. (I. Virol. 67:4566-4579, 1993). This system, which utilizes transfer vectors, is commercially available in kit form (Bac-to-BacTM kit; Life Technologies, Rockville, MD).
  • the transfer vector (e.g., pFastBaclTM; Life Technologies) contains a Tn7 transposon to move the DNA encoding the protein of interest into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551- 1556, 1994; and Chazenbalk and Rapoport, I. Biol. Chem. 270:1543-1549, 1995.
  • transfer vectors can include an in-frame fusion with DNA encoding a polypeptide extension or affinity tag as disclosed above.
  • a transfer vector containing a zalpha29-encoding sequence is transformed into E. coli host cells, and the cells are 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, such as Sf9 cells.
  • Recombinant virus that expresses zalpha29 protein is subsequently produced.
  • Recombinant viral stocks are made by methods commonly used the art.
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., High FiveTM cells; Invitrogen, Carlsbad, CA). See, for example, U.S. Patent No. 5,300,435. Serum-free media are used to grow and maintain the cells. Suitable media formulations are known in the art and can be obtained from commercial suppliers.
  • the cells are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells, at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • eukaryotic cells can also be used as hosts, including plant cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., I. Biosci. (Bangalore) 11:47- 58, 1987.
  • 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 5. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No.
  • Transformation systems for other yeasts including Hansenula polymorpha, 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., I. Gen. Microbiol. 132:3459-3465, 1986; Cregg, U.S. Patent No. 4,882,279; and Raymond et al., Yeast 14, 11-23, 1998. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No.
  • 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.).
  • the polypeptide When expressing a zalpha29 polypeptide in bacteria such as E. coli, the polypeptide 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 polypeptide 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 polypeptide 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.
  • Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
  • polypeptides and proteins of the present invention can be purified to >80% purity, >90% purity, >95% purity, or to 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 polypeptide or protein can be prepared substantially free of other polypeptides or proteins, particularly those of animal origin.
  • Expressed recombinant zalpha29 proteins are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general,
  • Proteins comprising a polyhistidine affinity tag are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol 6: 1321-1325,
  • Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Gmssenmeyer et al., ibid. Maltose binding protein fusions are purified on an amylose column according to methods known in the art.
  • zalpha29 proteins can be prepared as monomers or multimers; glycosylated or non-glycosylated; pegylated or non- pegylated; and may or may not include an initial methionine amino acid residue.
  • Target cells for use in zalpha29 activity assays include, without limitation, vascular cells (especially endothelial cells and smooth muscle cells), hematopoietic (myeloid and lymphoid) cells, liver cells (including hepatocytes, fenestrated endothelial cells, Kupffer cells, and Ito cells), fibroblasts (including human dermal fibroblasts and lung fibroblasts), fetal lung cells, articular synoviocytes, pericytes, chondrocytes, osteoblasts, and prostate epithelial cells. Endothelial cells and hematopoietic cells are derived from a common ancestral cell, the hemangioblast (Choi et al., Development 125:725-732, 1998).
  • Activity of zalpha29 proteins can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to an appropriate animal model.
  • Assays measuring cell proliferation or differentiation are well known in the art.
  • assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990), incorporation of radiolabelled nucleotides (as disclosed by, e.g., Raines and Ross, Methods Enzymol 109:749-773, 1985; Wahl et al., Mol. Cell Biol. 8:5016-5025, 1988; and Cook et al., Analytical Biochem.
  • Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporated herein by reference).
  • Zalpha29 activity may also be detected using assays designed to measure zalpha29-induced production of one or more additional growth factors or other macromolecules.
  • assays include those for determining the presence of hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF ⁇ ), interleukin-6 (EL-6), VEGF, acidic fibroblast growth factor (aFGF), angiogenin, and other macromolecules produced by the liver.
  • Suitable assays include mitogenesis assays using target cells responsive to the macromolecule of interest, receptor-binding assays, competition binding assays, immunological assays (e.g., ELISA), and other formats known in the art.
  • Metalloprotease secretion is measured from treated primary human dermal fibroblasts, synoviocytes and chondrocytes.
  • the relative levels of collagenase, gelatinase and stromalysin produced in response to culturing in the presence of a zalpha29 protein is measured using zymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-106, 1990).
  • Procollagen/collagen synthesis by dermal fibroblasts and chondrocytes in response to a test protein is measured using 3 H-proline incorporation into nascent secreted collagen.
  • 3 H-labeled collagen is visualized by SDS-PAGE followed by autoradiography (Unemori and Amento, J. Biol.
  • GAG Glycosaminoglycan secretion from dermal fibroblasts and chondrocytes is measured using a 1,9- dimethylmethylene blue dye binding assay (Farndale et al., Biochim. Biophys. Acta 883:173-177, 1986). Collagen and GAG assays are also carried out in the presence of EL-l ⁇ or TGF- ⁇ to examine the ability of zalpha29 protein to modify the established responses to these cytokines.
  • Monocyte activation assays are carried out (1) to look for the ability of zalpha29 proteins to further stimulate monocyte activation, and (2) to examine the ability of zalpha29 proteins to modulate attachment-induced or endotoxin-induced monocyte activation (Fuhlbrigge et al., J. Immunol. E38: 3799-3802, 1987).
  • EL-l ⁇ and TNF ⁇ levels produced in response to activation are measured by ELISA (Biosource, Inc. Camarillo, CA).
  • Monocyte/macrophage cells by virtue of CD14 (LPS receptor), areakily sensitive to endotoxin, and proteins with moderate levels of endotoxin- like activity will activate these cells.
  • Hematopoietic activity of zalpha29 proteins can be assayed on various hematopoietic cells in culture. Suitable assays include primary bone marrow colony assays and later stage lineage-restricted colony assays, which are known in the art (e.g., Holly et al., WEPO Publication WO 95/21920). Marrow cells plated on a suitable semi-solid medium (e.g., 50% methylcellulose containing 15% fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN antibiotic mix) are incubated in the presence of test polypeptide, then examined microscopically for colony formation. Known hematopoietic factors are used as controls. Mitogenic activity of zalpha29 polypeptides on hematopoietic cell lines can be measured as disclosed above. Cell migration is assayed essentially as disclosed by Kahler et al.
  • a protein is considered to be chemotactic if it induces migration of cells from an area of low protein concentration to an area of high protein concentration.
  • a typical assay is performed using modified Boyden chambers with a polystryrene membrane separating the two chambers (e.g., Transwell®; Coming Costar Corp.). The test sample, diluted in medium containing 1% BSA, is added to the lower chamber of a 24-well plate containing Transwells. Cells are then placed on the Transwell insert that has been pretreated with 0.2% gelatin. Cell migration is measured after 4 hours of incubation at 37°C.
  • Non-migrating cells are wiped off the top of the Transwell membrane, and cells attached to the lower face of the membrane are fixed and stained with 0.1% crystal violet. Stained cells are then extracted with 10% acetic acid and absorbance is measured at 600 nm. Migration is then calculated from a standard calibration curve. Cell migration can also be measured using the matrigel method of Grant et al. ("Angiogenesis as a component of epithelial-mesenchymal interactions" in Goldberg and Rosen, Epithelial-Mesenchymal Interaction in Cancer, Birkhauser Verlag, 1995, 235-248; Baatout, Anticancer Research 17:451-456, 1997).
  • Cell adhesion activity is assayed essentially as disclosed by LaFleur et al. (I. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter plates are coated with the test protein, non-specific sites are blocked with BSA, and cells (such as smooth muscle cells, leukocytes, or endothelial cells) are plated at a density of approximately 10 4 - 10 5 cells/well. The wells are incubated at 37°C (typically for about 60 minutes), then non-adherent cells are removed by gentle washing. Adhered cells are quantitated by conventional methods (e.g., by staining with crystal violet, lysing the cells, and determining the optical density of the lysate).
  • cells such as smooth muscle cells, leukocytes, or endothelial cells
  • Control wells are coated with a known adhesive protein, such as fibronectin or vitronectin.
  • a known adhesive protein such as fibronectin or vitronectin.
  • the activity of zalpha29 proteins can be measured with a silicon-based biosensor microphysiometer that measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses.
  • An exemplary such device is the 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 et al., Science 257:1906-1912, 1992; Pitchford et al., Meth. Enzymol.
  • the microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zalpha29 proteins, their agonists, and antagonists.
  • the microphysiometer can be used to measure responses of a zalpha29-responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zalpha29 polypeptide.
  • Zalpha29-responsive eukaryotic cells comprise cells into which a receptor for zalpha29 has been transfected creating a cell that is responsive to zalpha29, as well as cells naturally responsive to zalpha29. Differences, measured by a change in extracellular acidification, in the response of cells exposed to zalpha29 polypeptide relative to a control not exposed to zalpha29, are a direct measurement of zalpha29-modulated cellular responses.
  • the present invention thus provides methods of identifying agonists and antagonists of zalpha29 proteins, comprising providing cells responsive to a zalpha29 polypeptide, 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 change in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • the change in cellular response is shown as a measurable change in extracellular acidification rate.
  • Culturing a third portion of the cells in the presence of a zalpha29 protein and the absence of a test compound provides a positive control for the zalpha29-responsive cells and a control to compare the agonist activity of a test compound with that of the zalpha29 polypeptide.
  • Antagonists of zalpha29 can be identified by exposing the cells to zalpha29 protein in the presence and absence of the test compound, whereby a reduction in zalpha29- stimulated activity is indicative of antagonist activity in the test compound.
  • Zalpha29-encoding polynucleotides and antisense polynucleotides can be introduced into test animals, such as mice, using viral vectors or naked DNA, or transgenic animals can be produced.
  • viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia vims, and adeno-associated vims (AAV).
  • Adenovims a double-stranded DNA vims, is currently the best studied gene transfer vector for delivery of heterologous nucleic acids. For review, see Becker et al., Meth. Cell Biol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine 4:44-53, 1997.
  • the adenovims system offers several advantages.
  • Adenovims can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Because adenovimses are stable in the bloodstream, they can be administered by intravenous injection. By deleting portions of the adenovims genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co- transfected plasmid.
  • the essential El gene is deleted from the viral vector, and the vims will not replicate unless the El gene is provided by the host cell (e.g., the human 293 cell line).
  • the host cell e.g., the human 293 cell line.
  • adenovims primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the vims cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a 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.
  • An alternative method of gene delivery comprises removing cells from the body and introducing a vector into the cells as a naked DNA plasmid. The transformed cells are then re-implanted in the body. Naked DNA vectors are introduced into host cells by methods known in the art, including transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol. Chem. 263:14621-14624, 1988; Wu et al., I. Biol. Chem. 267:963- 967, 1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.
  • mice engineered to express a zalpha29 gene, and mice that exhibit a complete absence of zalpha29 gene function, referred to as "knockout mice"
  • mice can be employed to study the zalpha29 gene and the protein encoded thereby in an in vivo system.
  • Transgenic mice are particularly useful for investigating the role of zalpha29 proteins in early development in that they allow the identification of developmental abnormalities or blocks resulting from the over- or underexpression of a specific factor. See also, Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science 277:48-50, 1997. Promoters for transgenic expression include promoters from metallothionein and albumin genes.
  • Antisense methodology can be used to inhibit zalpha29 gene transcription to examine the effects of such inhibition in vivo.
  • Polynucleotides that are complementary to a segment of a zalpha29-encoding polynucleotide e.g., a polynucleotide as set forth in SEQ YD NO:l
  • Such antisense oligonucleotides can also be used to inhibit expression of zalpha29 polypeptide - encoding genes in cell culture.
  • Zalpha29 and inhibitors of zalpha29 activity are expected to have a variety of therapeutic applications. These therapeutic applications include treatment of diseases which require immune regulation, including autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, and diabetes. Zalpha29 may be important in the regulation of inflammation, and therefore would be useful in treating rheumatoid arthritis, asthma and sepsis.
  • Zalpha29 may be useful in modulating the immune system, whereby zalpha29 and zalpha29 antagonists may be used for reducing graft rejection, preventing graft-vs-host disease, boosting immunity to infectious diseases, treating immunocompromised patients (e.g., HEV + patients), or in improving vaccines.
  • Zalpha29 polypeptides can be administered alone or in combination with other vasculogenic or angiogenic agents, including VEGF.
  • VEGF vasculogenic or angiogenic agents
  • the two compounds can be administered simultaneously or sequentially as appropriate for the specific condition being treated.
  • zalpha29 proteins are formulated for topical or parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.
  • pharmaceutical formulations will include a zalpha29 polypeptide in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, or the like.
  • a pharmaceutically acceptable vehicle such as saline, buffered saline, 5% dextrose in water, or the like.
  • Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
  • Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995.
  • Zalpha29 will preferably be used in a concentration of about 10 to 100 ⁇ g/ml of total volume, although concentrations in the range of 1 ng/ml to 1000 ⁇ g/ml may be used.
  • the protein will be applied in the range of 0.1-10 ⁇ g/cm 2 of wound area, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. Dosing is daily or intermittently over the period of treatment. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. Sustained release formulations can also be employed.
  • a therapeutically effective amount of zalpha29 is an amount sufficient to produce a clinically significant change in the treated condition, such as a clinically significant change in hematopoietic or immune function, a significant reduction in morbidity, or a significantly increased histological score.
  • Zalpha29 proteins, agonists, and antagonists are useful for modulating the expansion, proliferation, activation, differentiation, migration, or metabolism of responsive cell types, which include both primary cells and cultured cell lines.
  • hematopoietic cells including stem cells and mature myeloid and lymphoid cells
  • endothelial cells smooth muscle cells
  • fibroblasts and hepatocytes.
  • Zalpha29 polypeptides are added to tissue culture media for these cell types at a concentration of about 10 pg/ml to about 100 ng/ml. Those skilled in the art will recognize that zalpha29 proteins can be advantageously combined with other growth factors in culture media.
  • zalpha29 proteins can also be used as molecular weight standards or as reagents in assays for determining circulating levels of the protein, such as in the diagnosis of disorders characterized by over- or under-production of zalpha29 protein or in the analysis of cell phenotype.
  • Zalpha29 proteins can also be used to identify inhibitors of their activity. Test compounds are added to the assays disclosed above to identify compounds that inhibit the activity of zalpha29 protein. In addition to those assays disclosed above, samples can be tested for inhibition of zalpha29 activity within a variety of assays designed to measure receptor binding or the stimulation inhibition of zalpha29-dependent cellular responses. For example, zalpha29-responsive cell lines can be transfected with a reporter gene constmct that is responsive to a zalpha29- stimulated cellular pathway.
  • Reporter gene constmcts of this type are known in the art, and will generally comprise a zalpha29-activated semm response element (SRE) operably linked to a gene encoding an assayable protein, such as luciferase.
  • SRE semm response element
  • Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zalpha29 on the target cells as evidenced by a decrease in zalpha29 stimulation of reporter gene expression.
  • Assays of this type will detect compounds that directly block zalpha29 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding.
  • zalpha29 tagged with a detectable label e.g., 125 I, biotin, horseradish peroxidase, FEEC, and the like.
  • a detectable label e.g., 125 I, biotin, horseradish peroxidase, FEEC, and the like.
  • Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
  • antibodies includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies.
  • 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.
  • Antibodies are defined to be specifically binding if they bind to a zalpha29 polypeptide or protein with an affinity at least 10-fold greater than the binding affinity to control (non- zalpha29) polypeptide or protein.
  • the affinity of a monoclonal antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).
  • polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats.
  • the immunogenicity of a zalpha29 polypeptide may be increased through the use of 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 a zalpha29 polypeptide or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine semm albumin (BSA) or tetanus toxoid) for immunization.
  • KLH keyhole limpet hemocyanin
  • BSA bovine semm albumin
  • tetanus toxoid tetanus toxoid
  • Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to zalpha29 polypeptides, and selection of antibody display libraries in phage or similar vectors (e.g., through the use of immobilized or labeled zalpha29 polypeptide).
  • Human antibodies can be produced in transgenic, non- human animals that have been engineered to contain human immunoglobulin genes as disclosed in WEPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
  • a variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to zalpha29 polypeptides.
  • 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, radio-immunoassays, radio- immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays.
  • Antibodies to zalpha29 may be used for affinity purification of the protein, within diagnostic assays for determining circulating levels of the protein; for detecting or quantitating soluble zalpha29 polypeptide as a marker of underlying pathology or disease; for immunolocalization within whole animals or tissue sections, including immunodiagnostic applications; for immunohistochemistry; and as antagonists to block protein activity in vitro and in vivo.
  • Antibodies to zalpha29 may also be used for tagging cells that express zalpha29; for affinity purification of zalpha29 polypeptides and proteins; in analytical methods employing FACS; for screening expression libraries; and for generating anti-idiotypic antibodies.
  • Antibodies can be linked to other compounds, including therapeutic and diagnostic agents, using known methods to provide for targetting of those compounds to cells expressing receptors for zalpha29.
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, 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 of the present invention may also be directly or indirectly conjugated to dmgs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications (e.g., inhibition of cell proliferation). See, in general, Ramakrishnan et al., Cancer Res. 56:1324-1330, 1996.
  • Polypeptides and proteins of the present invention can be used to identify and isolate receptors.
  • Zalpha29 receptors may be involved in growth regulation in the liver, blood vessel formation, and other developmental processes.
  • zalpha29 proteins and polypeptides can be immobilized on a column, and membrane preparations mn over the column (as generally disclosed in Immobilized Affinity Ligand Techniques, Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202). Proteins and polypeptides can also be radiolabeled (Methods Enzymol, vol. 182, "Guide to Protein Purification", M.
  • radiolabeled zalpha29 proteins and polypeptides can be used to clone the cognate receptor in binding assays using cells transfected with an expression cDNA library.
  • the present invention also provides reagents for use in diagnostic applications.
  • the zalpha29 gene, a probe comprising zalpha29 DNA or RNA, or a subsequence thereof can be used to determine the presence of mutations at or near the zalpha29 locus at chromosome 2pl5.
  • Detectable chromosomal aberrations at the zalpha29 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes, and rearrangements. These aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level.
  • Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used.
  • PCR primers are at least 5 nucleotides in length, often 15 or more nt, and frequently 20-30 nt. Short polynucleotides can be used when a small region of the gene is targetted for analysis.
  • a polynucleotide probe may comprise an entire exon or more. Probes will generally comprise a polynucleotide linked to a signal-generating moiety such as a radionucleotide.
  • these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (c) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient.
  • Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA.
  • the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ED NO: l, the complement of SEQ ED NO:l, or an RNA equivalent thereof.
  • Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymo ⁇ hism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications 1:5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; A.J.
  • Ribonuclease protection assays comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase. Hybridized regions of the RNA are protected from digestion.
  • RNA-RNA hybrid reaction product
  • PCR assays a patient genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient.
  • Another PCR-based technique that can be employed is single strand conformational polymo ⁇ hism (SSCP) analysis (Hayashi, PCR Methods and Applications L34-38, 1991).
  • the polypeptides, nucleic acids and/or antibodies of the present invention may be used in diagnosis or treatment of disorders associated with cell loss or abnormal cell proliferation (including cancer).
  • Labeled zalpha29 polypeptides may be used for imaging tumors or other sites of abnormal cell proliferation.
  • Inhibitors of zalpha29 activity include anti- zalpha29 antibodies and soluble zalpha29 receptors, as well as other peptidic and non- peptidic agents (including ribozymes). Such antagonists can be used to block the effects of zalpha29 on cells or tissues. Of particular interest is the use of antagonists of zalpha29 activity in cancer therapy.
  • Inhibitors are also expected to be useful in adjunct therapy after surgery to prevent the growth of residual cancer cells. Inhibitors can also be used in combination with other cancer therapeutic agents.
  • zalpha29 inhibitors include small molecule inhibitors and inactive receptor-binding fragments of zalpha29 polypeptides.
  • Inhibitors are formulated for pharmaceutical use as generally disclosed above, taking into account the precise chemical and physical nature of the inhibitor and the condition to be treated. The relevant determinations are within the level of ordinary skill in the formulation art.
  • Polynucleotides encoding zalpha29 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zalpha29 activity. If a mammal has a mutated or absent zalpha29 gene, a zalpha29 gene can be introduced into the cells of the mammal.
  • a gene encoding a zalpha29 polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA vims, such as, but not limited to, he ⁇ es simplex vims (HSV), papillomavirus, Epstein Barr vims (EBV), adenovims, adeno-associated vims (AAV), and the like.
  • HSV he ⁇ es simplex vims
  • EBV Epstein Barr vims
  • AAV adeno-associated vims
  • Defective vimses which entirely or almost entirely lack viral genes, are preferred.
  • a defective vims is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • vectors examples include, but are not limited to, a defective he ⁇ es simplex vims 1 (HSVl) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-330, 1991); an attenuated adenovims vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-630, 1992; and a defective adeno-associated vims vector (Samulski et al., J. Virol. 61:3096-3101, 1987; Samulski et al., J. Virol. 63:3822-3888, 1989).
  • HSVl defective he ⁇ es simplex vims 1
  • a zal ⁇ ha29 gene can be introduced in a retroviral vector as described, for example, by 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., I. Virol. 62:1120, 1988; Temin et al., U.S. Patent No. 5,124,263; Dougherty et al., WEPO Publication WO 95/07358; and Kuo et al., Blood 82:845, 1993.
  • the vector can be introduced by liposome-mediated transfection ("lipofection").
  • lipofection 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 84:7413-7417, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027- 8031, 1988).
  • lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages, including molecular targeting of liposomes to specific cells.
  • Directing transfection to particular cell types is 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 pu ⁇ ose of targeting.
  • Peptidic and non-peptidic molecules can be coupled to liposomes chemically.
  • cells are removed from the body, a vector is introduced into the cells as a naked DNA plasmid, and the transformed cells are re-implanted into the body as disclosed above.
  • Antisense methodology can be used to inhibit zalpha29 gene transcription in a patient as generally disclosed above.
  • Zalpha29 polypeptides and anti-zalpha29 antibodies can be directly or indirectly conjugated to dmgs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • polypeptides or antibodies of the present invention may be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance).
  • zalpha29 polypeptides or anti-zalpha29 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 anti-complementary molecule.
  • Suitable detectable molecules can be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like.
  • Suitable cytotoxic molecules can be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, saporin, and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium- 188 or yttrium-90.
  • polypeptides or antibodies can also be conjugated to cytotoxic dmgs, such as adriamycin.
  • cytotoxic dmgs such as adriamycin.
  • the detectable or cytotoxic molecule may be conjugated with a member of a complementary/anticomplementary pair, where the other member is bound to the polypeptide or antibody portion.
  • biotin streptavidin is an exemplary complementary/anticomplementary pair.
  • Polypeptide-toxin fusion proteins or antibody/fragment-toxin fusion proteins may be used for targeted cell or tissue inhibition or ablation, such as in cancer therapy.
  • conjugates of a zalpha29 polypeptide and a cytotoxin which can be used to target the cytotoxin to a tumor or other tissue that is undergoing undesired angiogenesis or neovascularization.
  • Target cells i.e., those displaying the zalpha29 receptor
  • bind the zalpha29-toxin conjugate which is then internalized, killing the cell.
  • the effects of receptor-specific cell killing (target ablation) are revealed by changes in whole animal physiology or through histological examination.
  • ligand-dependent, receptor-directed cyotoxicity can be used to enhance understanding of the physiological significance of a protein ligand.
  • One such toxin is saporin.
  • Mammalian cells have no receptor for saporin, which is non-toxic when it remains extracellular.
  • zalpha29-cytokine fusion proteins or antibody/fragment-cytokine fusion proteins may be used for enhancing in vitro cytotoxicity (for instance, that mediated by monoclonal antibodies against tumor targets) and for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers).
  • target tissues for example, blood and bone marrow cancers.
  • cytokines are toxic if administered systemically.
  • the described fusion proteins enable targeting of a cytokine to a desired site of action, such as a cell having binding sites for zalpha29, thereby providing an elevated local concentration of cytokine.
  • Suitable cytokines for this pu ⁇ ose include, for example, interleukin-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Such fusion proteins may be used to cause cytokine-induced killing of tumors and other tissues undergoing angiogenesis or neovascularization.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intra-arterially or intraductally, or may be introduced locally at the intended site of action.
  • Zalpha29 Northern blot analysis was performed using commercially prepared blots of human RNA (Human Multiple Tissue Northern Blots I, I , and Ed; Human Fetal Multiple Tissue Northern Blot II; and Human RNA Master Blot; Clontech Laboratories, Inc., Palo Alto, CA).
  • the zalpha29 hybridization probe was generated as a gel purified PCR amplification product.
  • the amplification product was made using oligonucleotides ZC21,720 (SEQ ID NO:8) and ZC21,721 (SEQ ED NO:9) as PCR primers and a cloned zalpha29 cDNA (see SEQ ED NO: l) as template.
  • PCR amplification was performed as follows: 1 ⁇ l of zalpha29 cDNA ( ⁇ 2 ng) and 40 pmoles each of oligonucleotide primers ZC21,720 and ZC21,721 were added to a reaction mixture containing commercially available reagents (AdvantageTM KlenTaq Polymerase Kit, Clontech Laboratories, Inc.) following the manufacturer's recommended protocol.
  • the reaction was mn as follows: 94°C for 30 seconds, 25 cycles of 94°C for 5 seconds, 55°C for 5 seconds, and 68°C for 1 minute, followed by 68°C for 3 minutes and a hold at 4°C.
  • the 422 bp PCR amplified fragment was gel purified and recovered using silica gel particles (QIAEX® II gel extraction kit; Qiagen, Valencia, CA) according to the manufacturer's recommended protocol.
  • the probe was a radioactively labeled using a commercially available kit (RediprimeTM II random-prime labeling system; Amersham Co ⁇ ., Arlington Heights, EL) according to the manufacturer's protocol.
  • the probe was purified using a a commercially available push column (NucTrap® column; Stratagene, La Jolla, CA; see U.S. Patent No. 5,336,412).
  • a hybridization solution (ExpressHybTM
  • Hybridization Solution Clontech Laboratories, Inc.
  • Hybridization Solution Clontech Laboratories, Inc.
  • Hybridization took place overnight at 65 °C. Following hybridization, the blots were washed in 2X
  • the positive tissues on the Northerns included heart, ovary, fetal lung, brain, small intestine, fetal liver, placenta, colon (mucosal lining), fetal kidney, lung, peripheral blood leukocyte, liver, stomach, skeletal muscle, thyroid, kidney, spinal cord, pancreas, lymph node, spleen, trachea, thymus, adrenal gland, prostate, bone marrow, testis, fetal brain.
  • Northerns included amygdala, aorta, caudate nucleus, bladder, cerebellum, utems, cerebral cortex, pituitary gland, frontal lobe, salivary gland, hippocampus, mammary gland, medulla oblongata, appendix, occipital lobe, trachea, putamen, fetal heart, substantia nigra, fetal spleen, thalamus, fetal thymus, and subthalamic nucleus.
  • the Northern blots were reprobed for human transferrin receptor.
  • the resulting signal generated from the transferrin receptor probe was used to normalize the zalpha29 signal.
  • the tissues with the greatest ratio of zalpha29 signal to transferrin receptor signal were heart, liver, and testis.
  • This panel contains PCRable DNAs from each of 83 radiation hybrid clones of the whole human genome, plus two control DNAs (the RM donor and the A3 recipient).
  • a publicly available WWW server http://shgc- www.stanford.edu) allows chromosomal localization of markers.
  • Each of the 85 PCR reactions consisted of 2 ⁇ l buffer (10X KlenTaq PCR reaction buffer; (Clontech Laboratories, Inc., Palo Alto, CA), 1.6 ⁇ l dNTPs mix (2.5 mM each, Perkin- Elmer, Foster City, CA), 1 ⁇ l sense primer ZC22,737 (SEQ ED NO: 10), 1 ⁇ l antisense primer ZC22,738 (SEQ ED NO: 11), 2 ⁇ l of a density increasing agent and tracking dye (RediLoad, Research Genetics, Inc., Huntsville, AL), 0.4 ⁇ l of a commercially available DNA polymerase/antibody mix (50X AdvantageTM KlenTaq Polymerase Mix; Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and x ⁇ l ddH2O for a total volume of 20 ⁇ l.
  • 2 ⁇ l buffer 10X KlenTaq PCR reaction buffer; (
  • the mixtures were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows: an initial 5-minute denaturation at 94°C, 35 cycles of a 45-second denaturation at 94°C, 45 seconds annealing at 64°C and 75 seconds extension at 72°C; followed by a final extension of 7 minutes at 72°C.
  • the reactions were separated by electrophoresis on a 2% agarose gel (obained from Life Technologies, Gaithersburg, MD).
  • PCR primers ZC23019 (SEQ ED NO: 12) and ZC23018 (SEQ ED NO: 13) were used with a template plasmid (pT7T3D-Pac) containing the full-length murine zalpha29 cDNA in a PCR reaction as follows: one cycle at 95 °C for 5 minutes; followed by 15 cycles at 95°C for 0.5 min., 58°C for 0.5 min., and 72°C for 0.5 min.; followed by 72°C for 7 min.; followed by a 4°C soak.
  • the PCR reaction product was loaded onto a 1.2% (low melt) agarose (SeaPlaque® GTG; FMC Co ⁇ ., Rockland, ME) gel in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA).
  • the zalpha29 PCR product was excised from the gel.
  • the gel slice was melted at 65°, and the DNA was extracted twice with phenol and precipitated with ethanol.
  • the PCR product was then digested with Fsel + Ascl, phenol/chloroform extracted, EtOH precipitated, and rehydrated in 20 ⁇ l TE (Tris/EDTA pH 8).
  • the 567-bp zalpha29 fragment was then ligated into the Fsel-Ascl sites of a modified pAdTrack CMV (He et al., Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998).
  • This constmct also contained the green fluorescent protein (GFP) marker gene.
  • GFP green fluorescent protein
  • the CMV promoter driving GFP expression was replaced with the SV40 promoter and the SV40 polyadenylation signal was replaced with the human growth hormone polyadenylation signal.
  • the native polylinker was replaced with Fsel, EcoRV, and Ascl sites.
  • This modified form of pAdTrack CMV was named pZyTrack.
  • Ligation was performed using a DNA ligation and screening kit (Fast-LinkTM; Epicentre Technologies, Madison, WI). Clones containing the zalpha29 cDNA were identified by standard mini-prep procedures.
  • Fast-LinkTM Epicentre Technologies, Madison, WI.
  • Clones containing the zalpha29 cDNA were identified by standard mini-prep procedures.
  • To linearize the plasmid approximately 5 ⁇ g of the pZyTrack zalpha29 plasmid was digested with Pmel. Approximately 1 ⁇ g of the linearized plasmid was cotransformed with 200ng of supercoiled pAdEasy (He et al., ibid.) into BJ5183 cells.
  • the co-transformation was done using an electroporator (Gene Pulser®; Bio-Rad Laboratories, Inc., Hercules, CA) at 2.5 kV, 200 ohms, and 25 ⁇ Fa.
  • the entire co- transformation mixture was plated on 4 LB plates containing 25 ⁇ g/ml kanamycin. The smallest colonies were picked and expanded in LB/kanamycin, and recombinant adenovims DNA was identified by standard DNA miniprep procedures. Digestion of the recombinant adenovims DNA with Fsel + Ascl confirmed the presence of the zalpha29 sequence.
  • the recombinant adenovims miniprep DNA was transformed into E.
  • coli host cells DH10BTM; Life Technologies, Gaithersburg, MD
  • DNA was prepared using a commercially available plasmid isolation kit (QIAGEN® Plasmid Maxi Kit; Qiagen, Inc., Valencia, CA) as directed by the supplier.
  • plasmid isolation kit QIAGEN® Plasmid Maxi Kit; Qiagen, Inc., Valencia, CA
  • Approximately 5 ⁇ g of recombinant adenoviral DNA was digested with PacI enzyme (New England Biolabs) for 3 hours at 37°C in a reaction volume of 100 ⁇ l containing 20-30U of Pad.
  • the digested DNA was extracted twice with an equal volume of phenol/chloroform and precipitated with ethanol.
  • the DNA pellet was resuspended in 5 ⁇ l distilled water.
  • a T25 flask of QBI-293A cells Quantantum Biotechnologies, Inc.
  • the Pad-digested DNA was diluted to a total volume of 50 ⁇ l with sterile HBS (150 mM NaCl, 20 mM HEPES).
  • HBS sterile HBS
  • 25 ⁇ l of lmg/ml N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethyl-ammonium salts (DOTAP) was diluted to a total volume of 100 ⁇ l with HBS.
  • the DNA was added to the DOTAP, mixed gently by pipeting up and down, and left at room temperature for 15 minutes.
  • the media was removed from the 293A cells, and the cells were washed with 5 ml semm-free MEMalpha containing 1 mM sodium pyruvate, 0.1 mM MEM non- essential amino acids, and 25 mM HEPES buffer (media components obtained from Life Technologies, Gaithersburg, MD).
  • 5 ml of semm-free MEM was added to the 293 A cells and held at 37°C.
  • the DNA lipid mixture was added drop-wise to the T25 flask of 293A cells, mixed gently and incubated at 37°C for 4 hours. After 4 hours the media containing the DNA lipid mixture was aspirated off and replaced with 5 ml complete MEM containing 5% fetal bovine semm.
  • the transfected cells were monitored for GFP expression and formation of foci (viral plaques).
  • the cmde lysate was amplified (primary (1°) amplification) to obtain a working "stock" of zalpha29 recombinant adenovims (rAdV) lysate.
  • Ten 10-cm plates of nearly confluent (80-90%) 293A cells were set up 20 hours in advance.
  • 200 ml of cmde rAdV lysate was added to each 10-cm plate, and the plates were monitored for 48 to 72 hours for CPE (cytopathic effect) under the white light microscope and expression of GFP under the fluorescent microscope.
  • CPE cytopathic effect
  • This lysate was collected into 250-ml polypropylene centrifuge bottles.
  • NP-40 detergent was added to a final concentration of 0.5% to the bottles of cmde lysate to lyse all cells. Bottles were placed on a rotating platform for 10 minutes, agitating as fast as possible without the bottles falling over. The debris was pelleted by centrifugation at 20,000 X G for 15 minutes. The supernatants were transferred to 250-ml polycarbonate centrifuge bottles, and 0.5 volume of 20% PEG-8000/2.5 M NaCl solution was added. The bottles were shaken overnight on ice. The bottles were centrifuged at 20,000 X G for 15 minutes, and supernatants were discarded into a bleach solution.
  • the precipitate from 2 bottles was resuspended in 2.5 ml PBS.
  • the vims solution was placed in 2-ml microcentrifuge tubes and centrifuged at 14,000 X G for 10 minutes to remove any additional cell debris.
  • the supernatant from the 2-ml microcentrifuge tubes was transferred to a 15-ml polypropylene snapcap tube and adjusted to a density of 1.34 g/ml with CsCl.
  • the volume of the vims solution was estimated, and 0.55 g/ml of CsCl was added.
  • the CsCl was dissolved, and 1 ml of this solution weighed 1.34 g.
  • the solution was transferred to polycarbonate thick-walled centrifuge tubes (3.2ml; Beckman #362305) and spun at 348,000 X G for 3-4 hours at 25°C in a Beckman Optima TLX micro-ultracentrifuge with a TLA- 100.4 rotor.
  • the vims formed a white band. Using wide-bore pipette tips, the vims band was collected.
  • the vims preparation was desalted by gel filtration using commercially available columns and cross-linked dextran media (PD-10 columns prepacked with Sephadex® G-25M; Pharmacia, Piscataway, NJ). The column was equilibrated with 20 ml of PBS. The vims was loaded and allow it to mn into the column. 5 ml of PBS was added to the column, and fractions of 8-10 drops were collected. The optical densities of 1:50 dilutions of each fraction was determined at 260 nm on a spectrophotometer. A clear absorbance peak was present between fractions 7-12. These fractions were pooled, and the optical density (OD) of a 1:25 dilution determined.
  • PD-10 columns prepacked with Sephadex® G-25M; Pharmacia, Piscataway, NJ The column was equilibrated with 20 ml of PBS. The vims was loaded and allow it to mn into the column. 5 ml of PBS was added to the column
  • the OD of a 1:25 dilution of the zalpha29 rAdV was 0.059, giving a vims concentration of 3.3 X 10 12 virions/ml.
  • glycerol was added to the purified vims to a final concentration of 15%, mixed gently but effectively, and stored in aliquots at -80°C.
  • TCED 50 formulation used was as per Quantum Biotechnologies, Inc., above.
  • the titer (T) was determined from a plate where vims used was diluted from 10 -2 to 10 -14 , and read 5 days after the infection. At each dilution a ratio (R) of positive wells for CPE per the total number of wells was determined.
  • 0.7 is subtracted from the exponent in the calculation for titer (T).
  • the zalpha29 adenovims had a titer of 1.3 X 10 1 pfu/ml.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Cette invention a trait à de nouveaux polypeptides de cytokine, à des substances et à des techniques permettant de les produire ainsi qu'à des méthodes d'utilisation. Ces polypeptides, qui comprennent au moins 15 restes d'acides aminés contigus appartenant à la SEQ ID N° 2 ou à la SEQ ID N° 4, peuvent être préparés en tant que fusions polypeptidiques, lesquelles comprennent des séquences hétérologues, telles que des marqueurs d'affinité. Les polypeptides et les nucléotides les codant peuvent être utilisés dans une large variété de domaines, thérapeutiques, diagnostiques et de recherche.
EP00941509A 1999-06-28 2000-06-16 Polypeptide spirale zalpha29 Withdrawn EP1192254A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US343163 1994-11-22
US34316399A 1999-06-28 1999-06-28
PCT/US2000/016736 WO2001000831A1 (fr) 1999-06-28 2000-06-16 Polypeptide spirale zalpha29

Publications (1)

Publication Number Publication Date
EP1192254A1 true EP1192254A1 (fr) 2002-04-03

Family

ID=23344968

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00941509A Withdrawn EP1192254A1 (fr) 1999-06-28 2000-06-16 Polypeptide spirale zalpha29

Country Status (6)

Country Link
EP (1) EP1192254A1 (fr)
JP (1) JP2003503056A (fr)
AU (1) AU5621000A (fr)
CA (1) CA2377580A1 (fr)
MX (1) MXPA02000138A (fr)
WO (1) WO2001000831A1 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999053051A2 (fr) * 1998-04-09 1999-10-21 Genset Etiquettes de sequences exprimees (est) des 5' et proteines humaines codees

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0100831A1 *

Also Published As

Publication number Publication date
CA2377580A1 (fr) 2001-01-04
WO2001000831A1 (fr) 2001-01-04
AU5621000A (en) 2001-01-31
JP2003503056A (ja) 2003-01-28
MXPA02000138A (es) 2002-07-02

Similar Documents

Publication Publication Date Title
US7691981B2 (en) Growth factor homolog zvegf3
CA2413258C (fr) Proteine zcyto21de type interferon
EP1988102B1 (fr) Facteur de croissance homologue ZVEGF3
EP2194130A1 (fr) La cytokine ZCYTO18
WO1999055869A1 (fr) Nouveaux facteurs et materiels de croissance polypeptidiques et leurs procedes de production
WO1999032515A2 (fr) Homologue de l'angiopoietine, adn le codant et procede de production dudit homologue
US6406888B1 (en) Helical cytokine zalpha33
US20020147328A1 (en) Connective tissue growth factor homologs
WO2001012665A2 (fr) Cytokine zalpha48 a enroulement en helice
US6531576B1 (en) Four-helical bundle protein zsig81
US20030153050A1 (en) Helical polypeptide zalpha29
CA2387371A1 (fr) Cytokine helicoidale zalpha33
WO2001000831A1 (fr) Polypeptide spirale zalpha29
EP1268804A2 (fr) Proteine helicoidale zalpha51
US6756214B2 (en) Protein zlmda33
EP1181369B1 (fr) Proteine en faisceau a quatre helices zsig81
US20030022316A1 (en) CUB domain protein zcub3 and materials and methods for making it
US20020009775A1 (en) Helical protein zalpha51
US20020192750A1 (en) Neuropilin homolog zcub5
WO2001018205A1 (fr) Polypeptide secrete zalpha30
WO2001029223A2 (fr) Proteine de membrane ztsl1 de type 1
CA2321176A1 (fr) Homologues du facteur de croissance pour le tissu conjonctif
WO1998044117A1 (fr) Chimiokine zsig-35 d'origine humaine
WO2002012334A2 (fr) Zsnki, polypeptide de venin de serpent

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020111

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17Q First examination report despatched

Effective date: 20031106

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040517