JP2003503056A - Helix polypeptide zalpha29 - Google Patents

Abstract

(57) SUMMARY Disclosed are novel cytokine polypeptides, materials and methods for making them, and methods of use. The polypeptides of the invention comprise at least 15 contiguous amino acid residues of SEQ ID NO: 2 or SEQ ID NO: 4, and are prepared as a polypeptide fusion comprising a heterologous sequence, e.g., an affinity tag. be able to. The polypeptides and polynucleotides encoding them can be used in a variety of therapeutic, diagnostic and research applications.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION Cytokines are polypeptide hormones produced by cells that affect the growth or metabolism of that cell or another cell. In multicellular animals, cytokines control cell proliferation, transfer, differentiation, and maturation. Cytokines play a role in both normal development and pathogenesis including the development of solid tumors. Cytokines are physicochemically diverse, ranging from 5 kDa (TGF-α) to 140 kDa (Muller mimicry inhibitors). They include single polypeptide chains and disulfide-linked homodimers and heterodimers.

Cytokines affect cellular events by binding to cell surface receptors. Binding initiates a series of signaling events within the cell, which ultimately leads to cell division,
It results in phenotypic changes such as protease production, cell transfer, expression of cell surface proteins, and production of additional growth factors. Cell differentiation and maturation are also under the control of cytokines. For example, the hematopoietic factors erythropoietin, thrombopoietin, and G-CSF each stimulate the production of red blood cells, platelets, and neutrophils from precursor cells in the bone marrow. Development of mature cells from pluripotent precursors may require the presence of multiple factors.

The role of cytokines in controlling cellular processes appears to make them candidates or targets for therapeutic intervention; in fact, many cytokines have been approved for clinical use. Interferon-α (IFN-α) is, for example,
Used in the treatment of hairy cell leukemia, chronic myelogenous leukemia, Kaposi's sarcoma, Condyloma acuminata, chronic hepatitis C, and chronic hepatitis B (Aggarwal and Puri, “Common and non-common features of cytokines and cytokine receptors. : Outline ”, Aggarwa
Human cytokines: Diseases and their role in therapy, Blac
kwell Science, Cambridge, MA, 1995, 3-24).

Platelet-derived growth factor (PDGF) is approved in the United States and other countries for the treatment of skin ulcers in diabetic patients. The hematopoietic cytokine erythropoietin has been developed for the treatment of anemia (eg EP 613,683). G-CSF, GM-CSF, IF
N-β, IFN-γ, and IL-2 have also been approved for use in humans (Aggar
wal and Puri, ibid.). Experimental evidence supports further therapeutic use of cytokines and their inhibitors. Inhibition of PDGF receptor activity has been shown to reduce intimal hyperplasia in injured baboon arteries (Giese et al. Restenosis S
ummit VIII, poster session number 23, 1996, US Pat. No. 5,620,687).

Vascular Endothelial Growth Factor (VEGF) has been shown to promote blood vessel growth in ischemic limbs (Isner et al., The Lancet 348 : 370-374, 1996), and for use as a wound healing agent. It has been proposed for the treatment of periodontal disease, for promoting endothelium formation in vascular transplant surgery, and for promoting collateral circulation after myocardial infarction (WIPO Publication No. WO 95/24473; US Pat. No. 5,219,739). Soluble VEGF receptor (Soluble flt-
1) was found to block VEGF binding to cell surface receptors and inhibit vascular tissue proliferation in vitro (Biotechnology News 16 (17): 5-6, 1996).
.

Experimental evidence indicates that inhibition of antagonists can be used to block tumor development (Biotechnology News, 13 November 1997), and angiogenesis is an early indicator of cervical cancer ( Br. J. Cancer 76: 1410-1415, 1997). More recently, thrombopoietin has been shown to stimulate the production of platelets in vivo (Kau
shansky et al., Nature 369 : 568-571, 1994), and the subject of several clinical trials (reviewed by von dem Borne et al., Bailliere's Clin, Haematol. 11: 427-445, 1998).

In view of the demonstrated clinical availability of cytokines, there is a need in the art for additional such molecules for use as both therapeutic agents and research materials and reagents. Cytokines are used in the laboratory to study developmental processes, and as components of cell culture media in the laboratory and industrial settings.

SUMMARY OF THE INVENTION In one aspect of the invention, residues 48-62 of SEQ ID NO: 2, residues 47 of SEQ ID NO: 4.
~ 61, residues 63-104 of SEQ ID NO: 2, residues 62-103 of SEQ ID NO: 4, residues of SEQ ID NO: 2
105-119, residues 104-118 of SEQ ID NO: 4, residues 120-137 of SEQ ID NO: 2, SEQ ID NO: 4
Residues 119-136, SEQ ID NO: 2 residues 138-152, SEQ ID NO: 4 residues 137-151, SEQ ID NO: 2 residues 153-170, SEQ ID NO: 4 residues 152-169, SEQ ID NO: 2 Residues 171-185
, And an isolated polypeptide comprising a sequence of amino acid residues selected from the group consisting of residues 170-184 of SEQ ID NO: 4.

In one embodiment, the isolated polypeptide has 15 to 1500 amino acid residues. In a related embodiment, the sequence of amino acid residues is maltose binding protein, immunoglobulin constant region, polyhistidine tag, and SEQ ID NO: 5.
To a second polypeptide selected from the group consisting of peptides as shown in 1. above, operably linked via a peptide bond or a polypeptide linker.
In another embodiment, the isolated polypeptide contains at least 30 consecutive residues of SEQ ID NO: 2 or SEQ ID NO: 4. In other embodiments, the isolated polypeptide comprises residues 48-185 of SEQ ID NO: 6 or residues 27-190 of SEQ ID NO: 6.
Contains. In a further embodiment, 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 of SEQ ID NO: 4. Contains 26-188.

In a second aspect of the invention there is provided an expression vector containing the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as disclosed above; and a transcription terminator. To be done. In one embodiment, the DNA segment contains nucleotides 79-570 of SEQ ID NO: 7. In another embodiment, the expression vector further contains a secretory signal sequence operably linked to the DNA segment. In a third aspect, the invention provides a cultured cell into which an expression vector as disclosed above has been introduced, wherein the cell expresses a DNA segment. 1
In one embodiment, the expression vector further contains a secretory signal sequence operably linked to the DNA segment, and the polypeptide is secreted by the cell.

In a fourth aspect, the invention provides a method of producing a protein, comprising the steps of: expression as disclosed above under conditions in which a DNA segment is expressed and a polypeptide is produced. Culturing cells into which the vector has been introduced and which produces the polypeptide; and the step of recovering the polypeptide. If the expression vector further contains a secretory signal sequence operably linked to the DNA segment, the polypeptide is secreted by the cell and recovered from the medium in which the cell is cultured. In a fifth aspect, the invention is produced by the method disclosed above,
Provide protein.

In a sixth aspect of the invention there is provided an antibody that specifically binds to the above disclosed polypeptide. In a seventh aspect of the invention there is provided a method of detecting the presence of an antagonist of zalpha29 activity in a test sample. The method comprises the following steps: (a) culturing cells responsive to zalpha29; (b) zal in the presence and absence of the test sample.
exposing the cells to the pha29 polypeptide; (c) comparing the level of response to the zalpha29 polypeptide by a biological or biochemical assay in the presence and absence of a test sample; and (d) The step of determining the presence of an antagonist of zalpha29 activity in the test sample from the comparison is included. These and other aspects of the invention will be apparent upon reference to the following detailed description of the invention and the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION Before describing the present invention in detail, it may be helpful to the understanding of the present invention to define the following terms: The term "affinity tag" as used herein. "Is a polypeptide that can be attached to a second polypeptide to provide purification or detection of the second polypeptide, or to provide a site for attachment of the second polypeptide to a substrate. Indicates a segment. In principle, any polypeptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.

Affinity tags include polyhistidine tracts, protein A (Nilsson et al., EMBO J. 4 : 1075, 1985; Nilsson et al., Methods Enzymol, 198 : 3, 1991), glutamine S transferase (Smith and Johonson, Gene 67). : 31, 1988), maltose binding protein (Kellerman and Ferenc, Methods Enzymol. 90 : 459-463, 1982; Guan et al.
Gene 67 : 21-30, 1987), Glu-Glu affinity tag (Grussenmeyer et al., Proc.Natl.Acad.S.
ci.USA 82 : 7952-4, 1985; see SEQ ID NO: 5), substance P, Flag ™ peptide (Hopp et al., Biotechnology 6 : 1204-10, 1988), streptavidin binding peptide, thioredoxin, ubiquitin. , Cellulose-binding proteins, T7 polymerase, or other antigenic epitopes or binding domains.

See generally Ford et al., Protein Expression and Purification 2 : 95-107, 1991. DNA encoding affinity or other reagents can be obtained from commercial suppliers (e.g., Phar
macia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA; and Eas
tman Kodack, New Haven, CT).

The term “allelic variant” is used herein to indicate any two or more alternative forms of a gene occupying the same chromosomal locus. Allelic modifications can occur spontaneously by unraveling mutations and result in phenotypic polymorphisms within the population.
The genetic mutation can be silent (no change in the encoded polypeptide) or can encode a polypeptide having an altered amino acid sequence. The term allelic variant is also used herein to indicate a protein encoded by an allelic variant of a gene.

The terms “amino terminus” and “carboxy terminus” are used herein to indicate a position within a polypeptide. Where the context allows, these terms are used with reference to a particular sequence or position of a polypeptide to indicate proximal or relative position. For example, a sequence located within the polypeptide at the carboxy terminus of a reference sequence is located proximal to the carboxy terminus of the reference sequence but not necessarily at the carboxy terminus of the complete poly.

“Angiogenesis” refers to the ability of a compound to stimulate the formation of new blood vessels from pre-existing vessels, alone or in concert with one or more additional compounds. Angiogenic activity can be measured as endothelial cell activation, stimulation of protease secretion by endothelial cells, endothelial cell transfer, capillary sprouting, and endothelial cell proliferation. Angiogenesis can also be measured using any of a number of in vivo assays as disclosed herein.

A “complement” of a polynucleotide molecule is a polynucleotide molecule that has a complementary base sequence and is in the opposite orientation as compared to a reference sequence. For example,
The sequence 5 ′ ATGCACGGG 3 ′ is complementary to 5 ′ CCCGTGCAT 3 ′. The term "corresponding", when applied to a position of an amino acid residue in a sequence, means the corresponding position in multiple sequences when the sequences are optimally aligned.

The term “degenerate nucleotide sequence” refers to a nucleotide sequence that contains one or more degenerate codons (as compared to a reference polynucleotide that encodes a polypeptide). Degenerate codons contain triplets of different nucleotides but encode the same amino acid residue (ie, GAU and GAC triplets each encode Asp).

The term “expression vector” is used to refer to a linear or circular DNA molecule, which encodes a polypeptide of interest operably linked to additional segments to provide for its transcription. Contains segments. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

The term “isolated”, when applied to a polynucleotide, has removed the polynucleotide from its natural genetic environment, and thus is free of other extra or undesired coding sequences. , And in a form suitable for use in genetically engineered protein production systems. Such an isolated molecule is
Isolated from their natural environment and includes cDNA and genomic clones.
The isolated DNA molecules of the present invention are free of other genes with which they are normally associated,
It may include naturally occurring 5'and 3'untranslated regions such as promoters and terminators. Identification of associated regions will be apparent to those of skill in the art (e.g., Dyn
See an and Tijan, Nature 316 : 774-78, 1985).

An “isolated” polypeptide or protein is a polypeptide or protein found in circumstances other than its native environment, such as away from blood and animal tissues. An isolated polypeptide can be substantially free of other polypeptides, particularly other polypeptides of animal origin. The polypeptides can be prepared in highly purified form, ie, greater than 95% pure, or greater than 99% pure. As used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimer or alternatively glycosylated or derivatized forms. .

“Operably linked” means that two or more entities are linked together such that they function cooperatively for their intended purpose. DNA
When referring to a segment, the phrase, for example, means that the coding sequence is aligned in the correct reading frame, and transcription is initiated at the promoter and proceeds through the coding segment to the terminator. "Operably linked" when referring to a polypeptide, is covalently (e.g., by a disulfide bond) and non-covalently (e.g., hydrogen bond, hydrophobic bond,
Or both linked sequences (by salt bridge interactions), where the desired function of the sequences is retained. The term "ortholog" refers to a polypeptide or protein obtained from a species that is a functional counterpart of the polypeptide or protein from a different species.
Sequence differences between orthologs are the result of speciation.

A “polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read at the 5 ′ to 3 ′ end. Polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The size of a polynucleotide is expressed as base pairs (abbreviated as "bp"), nucleotides ("nt"), or kilobases ("kb").

Where circumstances permit, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. It is understood that when the term applies to double-stranded molecules, it is used to indicate overall length and is equivalent to the term "base pair". It will be appreciated by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that their ends may be twisted as a result of enzymatic cleavage; thus, a double-stranded polynucleotide molecule. All nucleotides within the range may not be matched. Such unpaired ends generally do not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues joined by peptide bonds that are naturally or synthetically produced. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides." The term "promoter" is used herein in its art-recognized sense to refer to the portion of a gene containing a DNA sequence that provides for the binding of RNA polymerase and initiation of transcription. Promoter sequences are generally genes 5
Found in the'non-code domain, but not always.

A “protein” is a macromolecule containing one or more polypeptide chains. Proteins may also contain non-peptide components such as carbohydrate groups. Carbohydrate and other non-peptide substitutes can be added to the protein by the cell in which it is produced, and will vary with cell type. Proteins are defined herein in terms of their amino acid backbone structure; substitutions such as carbohydrate groups are generally not specified, but may be present nonetheless. A "secretory signal sequence" is a component of a larger polypeptide that directs the larger polypeptide through the secretory pathways of the cell in which it is synthesized ("
Is a DNA sequence encoding a secretory peptide "). Larger polypeptides are generally cleaved to remove the secretory peptide during transit through the secretory pathway.

A “segment” is a portion of a larger molecule (eg, polynucleotide or polypeptide) that has specified properties. For example, a DNA segment encoding a specified polypeptide, when read in the 5'to 3'direction, is one of the longer DNA molecules such as plasmids or plasmid fragments encoding the sequence of amino acids of the specified polypeptide. It is a part. It is understood that the molecular weights and polymer lengths determined by inaccurate analytical methods (eg, gel electrophoresis) are approximate values. When such a value is expressed as "about" X or "approximately" X, it is understood that the stated value of X is accurate to ± 10%. All references cited herein are incorporated by reference in their entirety.

The present invention provides novel cytokine polypeptides and proteins. This novel cytokine is called "zalpha29" and is a poly-protein characteristic of four helix bundle cytokines (eg, erythropoietin, thrombopoietin, G-CSF, IL-2, IL-4, leptin, and growth hormone). It was identified by the presence of peptide and polynucleotide features. Analysis of the human zalpha29 amino acid sequence shown in SEQ ID NO: 2 shows the presence of four amphipathic, alpha helix regions.
These regions contain at least amino acid residues 48-62 (helix A), 105-119 (helix B), 138-152 (helix C), and 171-185 (helix D).

Residues predicted to be within the core of the four helix bundles within these helix regions are positions 48, 51, 52, 55, 58 and 59 of SEQ ID NO: 2. , 62nd, 105th, 108th, 109th, 112th, 115th, 116th, 119th, 138th, 141st, 142nd, 145th, 14th
It occurs at positions 8, 149, 152, 171, 174, 175, 178, 181, 182, and 185. Residues 49, 50, 53, 54, 56, 57, 60, 61, 106, 107, 110, 111, 113, 1
14, 117, 118, 139, 140, 143, 144, 146, 147, 150, 151, 172, 173, 176, 177
, 179, 180, and 184 are expected to be on the exposed surface of the fascicles.

The internal-helix loop contains approximately residues 63-104 (loop AB), 120-137 (loop BC), and 153-170 (loop CD). Human zalpha29 cDNA (SEQ ID NO: 1) encodes a polypeptide of 190 amino acid residues. Without wishing to be bound by theory, this sequence is predicted to include a 26 residue secretory peptide. residue
Cleavage after 26 results in a mature polypeptide (residues 27-190 of SEQ ID NO: 2) with a calculated molecular weight of 18,558 Da (elimination of glycosylation).

However, those skilled in the art will appreciate that some cytokines (eg, endothelial cell growth factor, basic FGF, and IL-1β) do not contain conventional secretory peptides and are secreted by mechanisms that are not understood. Recognize. There is a single consensus N-linked glycosylation site at residues 111-113 in SEQ ID NO: 2. The cDNA also contains a clear polyadenylation signal.

The mouse zalpha29 polypeptide (SEQ ID NO: 4) was predicted to contain helices and loops at similar positions, residues 47-61, 104-118, 137-151, and 170.
A helix at ~ 184; and a loop at residues 62-103, 119-136, and 152-169. See FIG. One of skill in the art will recognize that the predicted domain boundaries are somewhat ambiguous and can vary by ± 5 amino acid residues.

The polypeptide of the invention comprises at least 15 consecutive amino acid residues of SEQ ID NO: 2. In one embodiment of the invention, the polypeptide has a sequence of up to the complete predicted mature polypeptide (residues 27-190 of SEQ ID NO: 2) or primary transcript (residues 1-190 of SEQ ID NO: 2). It comprises 20, 30, 40, 50, 100 or more consecutive residues of number 2. The corresponding mouse zalpha29 polypeptide (SEQ ID NO: 4) is also provided by the present invention. As disclosed in more detail below, these polypeptides may further comprise additional, non-zalpha29, polypeptide sequences.

The polypeptides of the present invention contain an epitope-bearing portion of a protein as set forth 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 8 1: 3998-4002,1984 reference. Epitopes can be linear or conformational, the latter composed of discrete regions of the protein that form the epitope upon protein folding. Linear epitopes are generally at least 6 amino acid residues in length. Relatively short synthetic peptides that mimic portions of protein sequences can elicit antisera that routinely react with partially mimicked proteins. Sutcliff
See e et al., Science 219 : 660-666, 1983.

Antibodies that recognize short, linear epitopes are especially suitable for Western blotting.
(Tobin, Proc. Natl. Acad. Sci. USA 76 : 4350-4356, 1979) in analytical and diagnostic applications with denatured proteins or in the analysis of fixed cell or tissue samples. Is useful in. Antibodies directed against linear epitopes are also useful for detecting fragments of zalpha29 as they may occur in body fluids or the culture medium employed.

The antigenic, epitope-bearing polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies, that specifically bind to the zalpha29 protein. An antigenic, epitope-bearing polypeptide comprises at least 6, often at least 9, generally about 15 to about 30, contiguous amino acid residues of the zalpha29 protein (SEQ ID NO: 2). Included are polypeptides that contain longer portions of the zalpha29 protein, from 30 to 50 residues up to the complete sequence.

The amino acid sequence of the epitope-bearing polypeptide is preferably selected to provide substantial solubility in an aqueous medium, ie the sequence contains relatively hydrophilic residues and is hydrophobic. Residues are substantially avoided. Such residues are za
It contains the inner domain loop of lpha29 and fragments thereof, in particular loop BC (residues 120-137 of SEQ ID NO: 2), which is significantly hydrophilic (see Figure 1C).
In this regard, the polypeptide has residues 99-104, 129-134, and 162 of SEQ ID NO: 2.
Is a polypeptide comprising ~ 167.

Of particular interest within the scope of the present invention is a polypeptide containing the complete four helix bundle of the zalpha29 polypeptide (residues 48-185 of SEQ ID NO: 2). Such a polypeptide further comprises the native zalpha29 amino terminus (residue 27 of SEQ ID NO: 2).
~ 47) and all or part of one or both of the carboxyl-terminal (residues 186-190 of SEQ ID NO: 2) regions, and non-zalpha29 amino acid residues or polypeptide sequences as disclosed in more detail below. .

The polypeptides of the present invention may be prepared with one or more amino acid substitutions, deletions, or additions as compared to SEQ ID NO: 2. These changes are usually of small nature, ie, conservative amino acid substitutions and other changes that do not significantly affect protein or polypeptide folding or activity, as well as amino-terminal methionine residues, maleimide-activated keyhole limpet hemocyanin. An amino or carboxyl-terminal cysteine residue, to facilitate subsequent ligation to
Include amino- or carboxyl-terminal extensions, such as small linker peptides of up to 20-25 residues, or extensions that facilitate purification (affinity tags) as disclosed above. Two or more affinity tags can be used in combination. Polypeptides that include an affinity tag can further include 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.

The present invention further provides a variety of other polypeptide fusions. For example, zalpha
The 29 polypeptide can be prepared as a fusion to a dimerization protein as disclosed in US Pat. Nos. 5,155,027 and 5,567,584. Suitable dimerization proteins in this regard include immunoglobulin constant region domains. Immunoglobulin-zalpha29 polypeptide fusions can be expressed in cells that have been genetically engineered to produce a variety of multimeric zalpha29 analogs. In addition, the zalpha29 polypeptide can be linked to another bioactive molecule such as a cytokine to provide a multifunctional molecule.

One or more helices of the zalpha29 polypeptide may be linked to another cytokine to enhance or otherwise modify its biological properties. Ancillary domains can be fused to zalpha29 polypeptides to target them to specific cells, tissues, or macromolecules such as collagen. For example, zalp
The ha29 polypeptide or protein can be targeted to a predetermined cell type by fusing the zalpha29 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell.

In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. The zalpha29 polypeptide can be attached to more than one moiety, such as an affinity tag, for purification and targeting of the domain. Polypeptide fusions may also include one or more cleavage sites, particularly between domains. Tuan
Et al., Connective Tissue Research 34 : 1-9, 1996.

The polypeptide fusions of the present invention generally comprise no more than about 1,500 amino acid residues, often no more than 1,200 residues, usually no more than 1,000 residues, and in many cases are much smaller. For example, a 164 residue zalpha29 polypeptide (residues 27-190 of SEQ ID NO: 2) is described in E. coli β-galactosidase (residues 1,021; Casadaban et al., J. Bacteriol. 143 : 971-980,1980). , And can be fused to a 10-residue spacer and a 4-residue Factor Xa cleavage site to yield a 1,199-residue polypeptide. In a second example, residues 27-190 of SEQ ID NO: 2 are maltose binding sites (approximately 3
70 residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.

As disclosed above, the polypeptides of the invention comprise at least 15 consecutive residues of SEQ ID NO: 2 or SEQ ID NO: 4. These polypeptides are further
Additional residues may be included as set forth in SEQ ID NO: 2, variants of SEQ ID NO: 2, or another protein as disclosed herein. A "variant of SEQ ID NO: 2" comprises a polypeptide that is at least 85%, at least 90%, or at least 95% identical to the corresponding region of SEQ ID NO: 2. The percent sequence identity is determined by conventional methods.

For example: Altschul et al., Bull. Math. Bio. 48 : 603-616, 1986 and Henikoff and
See Henikoff, Proc. Natl. Acad. Sci. USA 89 : 10915-10919, 1992. Briefly, the two amino acid sequences are shown in Table 1 (where amino acid residues are indicated by the standard one-letter code), with 10 Gip opening penalties and 1 gap extension penalties, and Henikoff and Henikoff ( (Ibid)
Aligned to optimize the alignment score using the "BLOSUM62" scoring matrix of. The percent identity is then calculated as follows:

[0048]

[Equation 1]

[0049]

[Table 1]

The level of identity between amino acid sequences is determined by Pearson and Lipman,
Proc. Natl. Acad. Sci. USA 85: 2444, 1988), but Pearson, Meth
. Enzymol. 183: 63, 1990) disclosed by the "FASTA" similarity search algorithm. Briefly, FASTA first ignores conservative amino acid substitutions, insertions or deletions, and has the highest density of identity (if ktup variable is 1 if the ktup variable is 1) with the region sharing the questionable sequence (eg, SEQ ID NO: 2) ) Or a pair of identities (if k
Sequence similarity is assessed by identifying test sequences with (if tup = 2).

The ten regions with the highest densities of identity were then re-pointed by comparing the similarities of all paired amino acids by the amino acid matrix, with the ends of the regions being the highest score with blunt ends. Try to include only the contributing residues. if"
If there are some regions with scores greater than the "cutoff" value (calculated by a predetermined formula based on the length of the array and the ktup value) then consider the first region with a smooth edge. To determine if the regions can combine to form an appropriate sequence with gaps.Finally, the higher scoring region of the two amino acid sequences is assigned to the Needleman-Bunsch-Sellers algorithm. (Needleman and Wunsch, J. Mo
l. Biol. 48: 444, 1970; Sellers, SIAM J. Appl. Math. 26: 787, 1974), which allows for the insertion and deletion of amino acids.

Suitable parameters for FASTA analysis are as follows: ktup = 1, gap opening penalty = 10, gap extension penalty = 1, and substitution matrix =
BLOSUM62. These parameters can be introduced into the FASTA program by modifying the scoring matrix file (“SMATRIX”) as described in Pearson Addendum 2, 1990 (supra).

FASTA can also be used to determine the sequence identity of nucleic acid molecules using the above ratios. For nucleotide sequence comparisons, ktup values range from 1 to 6, preferably 3 to 6, most preferably 3, with no other parameters.

The present invention includes a polypeptide having one or more conservative amino acid changes when compared to the amino acid sequence of SEQ ID NO: 2. The BLOSUM62 matrix (Table 1) is an amino acid substitution matrix derived from approximately 2,000 locally multiplex sequences of protein sequence segments, representing highly conserved regions of over 500 related proteins (Henikoff and Henikoff, ibid). . In this way, the BLOSUM62 substitution frequency can be used to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the invention.

As used herein, the term “conservative amino acid substitution” refers to BLO greater than −1.
Refers to the substitution represented by the SUM62 value. For example, if an amino acid substitution is 0
It is conservative if it is characterized by a BLOSUM62 value of 1, 2 or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (eg 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (eg 2 or 3).

The protein of the present invention may contain non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, but are not limited to, 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-
Includes azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins.

For example, an in vitro system was employed to chemically suppress the aminoacylated suppressor tR.
NA can be used to suppress nonsense mutations. Methods of amino acid synthesis and tRNA aminoacylation are known in the art. Transcription and translation of the plasmid containing the nonsense mutation is performed in a cell-free system comprising E. coli S30 extract and commercially available systems and other reagents. The protein is purified by chromatography.

See, for example: Robertson et al., J. Am. Chem. Soc. 113: 2722, 1991;
Ellman et al., Methods Enzymol. 202: 301, 1991; Chung et al., Science 25
9: 806-809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90: 10145-1
0149, 1993. In the second method, translation is performed in Xenopus oocytes by microinjecting mutated mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 2
71: 19991-19998, 1996).

In a third method, E. coli cells are used in the absence of the natural amino acid to be replaced (eg, phenylalanine) and the desired non-naturally occurring amino acid (eg, 2-azaphenylalanine, 3-azaphenylalanine, 4- Culture in the presence of azaphenylalanine or 4-fluorophenylalanine). Non-naturally occurring amino acids are incorporated into proteins instead of their natural counterparts. Reference: Koide et al., Biochem. 33: 7470-7476,
1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by chemical modification in vitro. Chemical modifications can be combined with site-directed mutagenesis to further extend the range of substitutions (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

Changes in the amino acid sequence minimize the disruption of higher order structures essential for biological activity in the zalpha29 polypeptide. For example, prevent changes in amino acid residues from disrupting the features of the 4-helix bundle of the protein family. The effects of amino acid sequence changes can be predicted by computer modeling as disclosed above, or determined by crystal structure analysis (see eg Lapthorn et al., Ibid.). The hydrophilicity profile of SEQ ID NO: 2 is shown in Figures 1A-1D. One of skill in the art will consider this hydrophilicity when designing changes in the amino acid sequence of the zalpha29 polypeptide so as not to disrupt the overall profile.

Residues within the 4-helix bundle core can be replaced with other residues as shown in SEQ ID NO: 6. Residues predicted to be on the exposed surface of the 4-helix bundle are relatively resistant to substitution. Other candidate amino acid substitutions in human zalpha29 are suggested by aligning the human sequence (SEQ ID NO: 2) and mouse sequence (SEQ ID NO: 4) shown in Figure 2, but both sequences together represent approximately 85%. It is the same. The cysteine residue at position 160 of SEQ ID NO: 2 (at position 159 of SEQ ID NO: 4) is in loop CD, suggesting that it is involved in interchain disulfide bonds. Therefore, this residue is predicted to be relatively intolerant to substitution.

One skilled in the art will use many well-known techniques, either individually or in combination, to analyze the structural features that affect the folding of the mutant protein or polypeptide and compare them to standard molecules to determine that such modifications are significant. Can be determined. One well-known and accepted method for measuring folding is circular dichroism (CD). Measuring and comparing the CD spectra generated by the modified and standard molecules is a technical routine (Johnson, Proteins 7: 205-214, 1990). Crystallography is another well-accepted 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 (Sch
aanan et al., Science 257: 961-964, 1992).

Essential amino acids of the polypeptides of the present invention can be identified according to techniques 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). In the latter technique, a single alanine mutation is introduced for each residue of the molecule, and the resulting mutant molecule is examined for biological activity as disclosed below, identifying amino acid residues critical for the activity of the molecule. Identify.

Multiple amino acid substitutions may be performed and known mutagenesis and screening, eg,
Reidhaar-Olson and Sauer, Science 241
: 53-57, 1988) or Bowie and Sauer, Proc. Natl. Ac
ad. Sci. USA 86: 2152-2156, 1989). Briefly, these authors randomize two or more positions of a polypeptide simultaneously, select functional peptides, and then sequence the mutated polypeptide to determine possible mutations at each position. A method of determining the spectrum of substitution is disclosed. Other methods that can be used are phage display methods (eg Lowman et al., Biochem. 30: 10832-
10837, 1991; Ladner et al., U.S. Patent 5,223,409; Huse, WIPO Publication WO92 / 06204).
And region-specific mutations (Derbyshire et al., Gene 46: 145, 1986; Ner et al.
., DNA 7: 127, 1988).

The disclosed variants of zalpha29 DNA and polypeptide sequences have already been disclosed D
Produced via NA shuffling (Stemmer, Nature 370: 389-391, 1994 and S
temmer, Proc. Natl. Acad. Sci. USA 91: 10747-10751, 1994). Briefly, mutant genes are generated by in vitro homologous recombination of the parental gene by random fragmentation, followed by PCR reassembly, leading to randomly introduced point mutations. This technique modifies with a gene such as a parental gene such as an allelic variant or a gene from a different species to introduce additional variability into the process.
Selection or screening for the desired activity, followed by further iterations of mutagenesis and assay results in rapid "evolution" of the sequence by selecting for the desired mutation while simultaneously selecting for deleterious changes.

In many cases, the structure of the final polypeptide product will result from processing of the nascent polypeptide chain by the host cell, and the final sequence of the zalpha29 polypeptide produced by the host cell will not necessarily be encoded by the expressed polynucleotide. It does not correspond to all sequences that are processed. For example, expression of the complete zalpha29 sequence in cultured mammalian cells is expected to result in the removal of at least the secretory peptide, whereas the same polypeptide produced in the prokaryotic host is not expected to be cleaved. Differential processing of individual chains results in heterogeneity of the expressed polypeptide.

The zalpha29 proteins of the present invention are characterized by their activity, ie modulation of proliferation, differentiation, migration, adhesion, or metabolism of responsive cell types. The biological activity of the zalpha29 protein is the change in cell proliferation, differentiation, migration or attachment, or changes in cellular metabolism (e.g.,
Assayed using in vitro or in vivo assays designed to detect 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, eg, to determine the effects of amino acid substitutions, deletions or insertions.

However, due to the complexity of developmental processes (eg, angiogenesis, wound healing), in vivo assays are generally employed to confirm and further assess biological activity. Certain in vitro models, such as Pepper et al., Biochem. Bioph
ys. Res. Comm. 189: 824-831, 1992) 3D collagen gel matrix
Models etc. are complex enough to assay for histological effects. The assay may be carried out with a foreign produced protein or may be carried out in vivo or in vitro with cells expressing the polypeptide of interest. Assay is zalpha29
The protein can be performed alone or in combination with other growth factors, eg, in combination with members of the VEGF family or hematopoietic cytokines (eg EPO, TPO, G-CSF, stem cell factor) and the like. Representative assay methods are disclosed below.

The mutagenesis methods described above can be combined with high capacity or high throughput screening methods to detect biological activity of zalpha29 mutant polypeptides. An assay that can be scaled up for high throughput is the mutagenesis assay, which
It can be performed in a 96-well format. Mutagenized DNA molecules that encode active zalpha29 polypeptides can be recovered from host cells and rapidly sequenced by state-of-the-art equipment. These methods allow the importance of individual amino acid residues of the polypeptide of interest to be determined rapidly and can be applied to polypeptides of unknown structure.

The methods discussed above allow one of skill in the art to prepare various polypeptide fragments or variants of SEQ ID NO: 2 or SEQ ID NO: 4 that retain the activity of wild-type zalpha29.

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: 1, and a representative DNA sequence encoding the amino acid sequence of SEQ ID NO: 4 is shown in SEQ ID NO: 3. One of skill in the art will readily recognize that considerable sequence variation is possible within these polynucleotide molecules in view of the degeneracy of the genetic code. SEQ ID NO: 7 is zalpha of SEQ ID NO: 2
A degenerate DNA sequence that includes all of the DNA encoding the 29 polypeptides. Those skilled in the art will appreciate that the degenerate sequence of SEQ ID NO: 7 encodes SEQ ID NO: 2 by replacing T with U.
It will be appreciated that it also provides the entire A sequence.

Thus, a zalpha29 polypeptide-encoding polynucleotide comprising nucleotides 1 to 534 or nucleotides 52 to 534 of SEQ ID NO: 7 is SEQ ID NO: 7 encoding another zalpha29 polypeptide disclosed herein. Segments of the invention are also contemplated by the present invention. Table 2 shows the one-letter code used in SEQ ID NO: 7 and indicates the position of the degenerate nucleotide. "Solution" is the nucleotide indicated by the code letter. "Complement" indicates the code for the complementary nucleotide (s). For example, code Y is C
Or T, the complement R of which means A or G, where A is complementary to T,
G is complementary to C.

[0073]

[Table 2]

The degenerate codons used in SEQ ID NO: 7 include all possible codons for a given amino acid and are as shown in Table 3 below.

[0075]

[Table 3]

One of ordinary skill in the art will recognize that as a representation of all possible codons encoding each amino acid, certain ambiguities are introduced in determining degenerate codons. For example, the degenerate codon of serine (WSN) optionally encodes arginine (AGR) and arginine (MGR
The degenerate codon (GN) may optionally encode serine (AGY). A similar relationship exists between the codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by degenerate sequences can encode variant amino acid sequences, but one of skill in the art can readily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO: 2. Variant sequences can be easily tested for functionality as described herein.

One of ordinary skill in the art will also recognize that different species may use preferential codons. General reference: Grantham et al., Nuc. Acids Res. 8: 1893-912, 1980; Haas et
al., Curr. Biol. 6: 315-24, 1996; Wain-Hobson et al., Gene 13: 355-64, 198.
1; Grosjean and Fiers, Gene 18: 199-209, 1982; Holm, Nuc. Acids Res. 14: 3
075-87, 1986; and Ikemura, J. Mol. Biol. 158: 573-97, 1982. Introduction of suitable codon sequences into recombinant DNA can increase protein production, for example, by making protein translation more efficient within a particular cell type or species. Accordingly, the degenerate codon sequence disclosed in SEQ ID NO: 7 is commonly used in the art and as a template for optimizing expression of polynucleotides in the various cell types and species disclosed herein. To work.

In some aspects of the invention, the isolated polynucleotide hybridizes under stringent conditions to the similarly sized region of SEQ ID NO: 1 or its complementary sequence. In general, stringent conditions are chosen to be about 5 ° C. below the thermal melting point (T _m ) for a specific sequence at constant ionic strength and pH. The T _m is the temperature (under constant 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 0.03M at pH 7 and the temperature is at least about 60 ° C.

As indicated previously, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is a large amount of zalpha2
9 Isolated from tissues or cells that produce RNA. The zalpha29 transcript has been detected in a number of tissues as disclosed below. Total RNA is extracted with guanidine HCl, followed by
It can be prepared by isolation by centrifugation on a CsCl gradient (Chirgwin et al.
, Biochemistry 18: 52-94, 1979). Poly (A) ⁺ RNA is prepared by the method of Aviv and Leder (Aviv and Leder, Proc. Natl. Acad. Sci. USA 69: 1408-1412,
1972). Complementary DNA (cDNA) is prepared from poly (A) ⁺ RNA by known methods. Alternatively, genomic DNA is isolated. The polynucleotide encoding the zalpha29 polypeptide is then identified and isolated, eg, by hybridization or PCR.

The full-length clone encoding zalpha29 can be obtained by a conventional cloning method. Complementary DNA (cDNA) clones are commonly used within protein production systems, but in some applications (e.g., expression in transgenic animals), genomic
CDNA using clones or containing at least one genomic intron
It is preferred to modify the clone. The partial human genome zalpha29 sequence is shown in SEQ ID NO: 14. This sequence consists of the exons from nucleotide 1885 to nucleotide 2112 (corresponding to nucleotides 483-710 of SEQ ID NO: 1).

Partial mouse genomic sequences are shown in SEQ ID NO: 15 and SEQ ID NO: 16. SEQ ID NO: 15
In, nucleotides 6 to 165 are the exons corresponding to nucleotides 40 to 199 of SEQ ID NO: 3. In SEQ ID NO: 16, nucleotides 175 to 295 are exons corresponding to nucleotides 200 to 320 of SEQ ID NO: 3. Methods for preparing cDNA and genomic clones are well known and within the ordinary skill in the art, and use the sequences disclosed herein or portions thereof to probe or prime the library. Expression libraries can be probed with antibodies to zalpha29, receptor fragments or other specific binding partners.

The zalpha29 polypeptide sequences disclosed herein can also be used as probes or primers to clone the 5'non-coding region of the zalpha29 gene. The promoter element from the zalpha29 gene can be used, for example, for the expression of heterologous genes in transgenic animals or patients treated by gene therapy. Cloning of 5'flanking sequences is described in US Pat.
The "gene activation" disclosed in 41,670 also facilitates production of the zalpha29 protein.

Briefly, endogenous zalpha29 gene expression in cells was introduced by introducing into the zalpha29 locus a DNA construct comprising at least a target sequence, a regulatory sequence, an exon, and an unpaired splice donor site. Change. The target array is zalph
The 5'non-coding sequence of a29, which enables homologous recombination of the construct with the exogenous zalpha29 locus, whereby the sequences within the construct are operably linked to the exogenous zalpha29 coding sequence. obtain. In this manner, the endogenous zalpha29 promoter replaces or complements other regulatory sequences, enhancing tissue-specific or otherwise regulated expression.

Those skilled in the art will recognize that the sequences disclosed in SEQ ID NOS: 1-2 and 3-4 represent human and mouse zalpha29 single alleles, respectively. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard techniques.

The present invention further provides corresponding polypeptides and polynucleotides from other species (“Orthologues”). Of particular interest are zalp from mammalian species including mouse, pig, sheep, cow, dog, cat, horse, and other primate polypeptides.
ha29 polypeptide. Orthologs of human zalpha29 can be cloned using the information and compositions provided by the invention, in combination with convenient cloning techniques. For example, cDNA can be cloned using mRNA obtained from a tissue or cell type expressing zalpha29 disclosed above.

A library is then prepared from the mRNA of the positive tissue or cell line. zalpha
The cDNA encoding 29 is then transformed by various methods, such as full or partial human cDNA.
Isolate by probing with NA or with one or more sets of degenerate probes based on the disclosed sequences. The cDNA can also be cloned by the polymerase chain reaction (or PCR) using primers designed from the representative human and mouse zalpha29 sequences disclosed herein (Mullis, US Pat. No. 4,683,202). In a further method, a cDNA library can be used to transform or transfer into a host cell and expression of the cDNA of interest can be detected by an antibody to the zalpha29 polypeptide. Similar techniques can be applied to the isolation of genomic clones.

For zalpha29 polypeptides, including variants and fusion proteins, one of skill in the art can readily use the information provided in Tables 3 and 4 above to generate fully degenerate polynucleotides encoding the polypeptides. it can. Moreover, one of skill in the art can use standard software to devise zalpha29 variants based on the nucleotide and amino acid sequences described herein. Thus, the invention provides a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, sequence No. 6, SEQ ID No. 7 and parts thereof. Suitable forms of computer decryption means are magnetic media and light readable media.

Examples of magnetic media are hard or fixed drives, random access memory
(RAM) chips, floppy disks, digital linear tape (DLT), disk cache, and ZIP disks. Optically readable media include compact discs (eg CD-read only memory (ROM), CD-rewritable (RW), and CD-recordable) and digital versatile / video discs (DVD) (eg DVD-ROM). , DVD-RAM, and DVD + RW).

The zalpha29 polypeptide of the invention is a full-length polypeptide, bioactive fragment,
And a fusion polypeptide, and can be produced according to a conventional technique using a cell into which an expression vector encoding the polypeptide has been introduced. As used herein, "cells into which an expression vector has been introduced" includes both cells that have been directly manipulated by the introduction of foreign DNA molecules and their progeny that contain the introduced DNA.

Suitable host cells are cell types that can be transformed with or transferred to foreign DNA and grown in culture, including bacteria, fungal cells, and cultured higher eukaryotic cells.
Techniques for manipulating cloned DNA molecules and introducing foreign DNA into various host cells have been disclosed by Sambrook et al. (Sambrook et al., Molecular Cloning: A Laboratory.
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor
, NY, 1989, and Ausubel et al., Eds., Current Protocols in Molecular Bio
logy, John Wiley and Sons, Inc., NY, 1987).

Generally, a DNA sequence encoding a zalpha29 polypeptide is operably linked to other genetic elements necessary for its expression in an expression vector, generally elements including transcriptional promoters and terminators. Although the vector commonly contains one or more selectable markers and one or more origins of replication, one of skill in the art would provide the selectable markers within a given system on separate vectors and It will be appreciated that replication may be provided by integrating into the host cell genome. The choice of promoter, terminator, selectable marker, vector and other elements is a matter of routine design within the level of ordinary skill. Many such elements are described in the literature and are available through commercial suppliers.

To direct the zalpha29 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of zalpha29, or may be derived from another secreted protein (eg, t-PA; see US Pat. No. 5,641,655), or may be newly synthesized. The secretory signal sequence is operably linked to the zalpha29 DNA sequence, ie, the two sequences are linked in the correct reading frame, properly positioned, and the newly synthesized polypeptide is directed into the secretory pathway of the host cell. Secretory signal sequences are generally placed 5'to the DNA sequence encoding the polypeptide of interest, although some signal sequences may be placed elsewhere in the DNA sequence of interest (see: Welch et al. , US Patent 5,037,743; Holland et al., US Patent 5,1
43,830).

Expression of zalpha29 polypeptides via the secretory pathway of host cells is expected to lead to the production of multimeric proteins. Such multimers include both homomultimers and heteromultimers, the latter including proteins and zalpha29 comprising only zalpha29 polypeptides.
A heterologous polypeptide (e.g., second 4-helix bundle cytokine polypeptide)
Including proteins containing If the mixture of proteins results from expression, the individual species are isolated by conventional methods. Monomers, dimers, and higher order multimers are separated by, for example, size exclusion chromatography.

Heteromultimers are homomultimers by immunoaffinity chromatography using antibodies specific for individual dimers or by sequential immunoaffinity steps using antibodies specific for individual element polypeptides. Can be separated from. See generally US Pat. No. 5,094,941. Multimers may be constructed in vitro by incubation of the component polypeptides under suitable conditions. Generally, in vitro construction involves incubating the protein mixture under denaturing and reducing conditions, followed by refolding and reoxidation of the polypeptide to form homodimers and heterodimers. The recovery and accumulation of proteins expressed in bacterial cells are as described below.

Cultured mammalian cells may be used as hosts in the present invention. A method for introducing exogenous DNA into a mammalian host cell is transfection with calcium phosphate (
Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Gene
tics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), DEAE-dextran transfection (Ausubel). et al., ibid.), and transfection with liposomes (Hawley-Nelson et al., Focus 15:73, 1993; Cicc
arone et al., Focus 15:80, 1993).

For the production of recombinant polypeptides in cultured mammalian cells, see, for example, Levinson et al. In US Pat. No. 4,713,339 and Hagen et al. In US Pat.
784,950, Palmiter et al. In U.S. Pat.
(Ringold) in U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells are CO
S-1 (ATCC No.CRL1650), COS-7 (ATCC No.CRL1651), BHK (ATCC No.CRL1632), BHK5
70 (ATCC No.CRL10314), 293 (ATCC No.CRL1573; Graham et al., J. Gen. Virol.
36: 59-72, 1977) and Chinese hamster ovaries (e.g. CHO-K1, ATCC).
No. CCL61; or CHO DG44, Chasin et al., Som. Cell. Molec. Genet. 12: 555.
, 1986) Cell line.

Other suitable cell lines are also known in the art and are available from public depository institutions such as the American Type Culture Collection, Manassas, VA. Promoters used in cultured mammalian cells include SV-40 or promoters from cytomegalovirus (reference example: US Pat. No. 4,956,288), metallothionein gene promoters (US Pat. Nos. 4,579,821 and 4,601,978), and adenovirus major late promoter. The expression vector used in mammalian cells is pZP-
1 and pZP-9, which have been deposited under deposit numbers 98669 and 98668 with the American Type Culture Collection, Manassas, VA, USA, respectively, and their derivatives.

Drug selection is generally used to select cultured mammalian cells that have inserted foreign DNA. Such cells are commonly referred to as "transductants." Cultured in the presence of a selective agent,
Cells that can pass the gene of interest to their progeny are called "stable transductants." An exemplary selectable marker is the gene encoding resistance to the antibiotic neomycin. Selection is performed in the presence of a neomycin type drug such as G-418. A selection system can be used to increase the expression level of the gene of interest, a process called "amplification". Amplification is carried out by culturing the transductants in the presence of low levels of selection agent and then increasing the amount of selection agent and selecting for cells which produce high levels of the introduced gene product. An exemplary amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (eg hygromycin resistance, multidrug resistance, puromycin acetyltransferase) can also be used.

The adenovirus system (disclosed in more detail below) can also be used for protein production in vitro. By culturing adenovirus-infected non-293 cells under conditions in which the cells do not divide rapidly, the cells can produce the protein for long periods of time. For example, BHK cells are grown to confluency in cell factories and then exposed to an adenovirus vector encoding a secreted protein of interest. The cells then grow under serum-free conditions, which allow infected cells to survive for several weeks without significant cell division.

In an alternative method, the adenovirus vector-infected 293 cells are adherent cells,
Or grows in suspension culture at relatively high cell densities and produces significant amounts of protein
(Ref: Garnier et al., Cytotechnol. 15: 145-55, 1994). The heterologous protein expressed and secreted by any of the protocols can be repeatedly separated from the cell supernatant, lysate or membrane fraction depending on the situation of the protein expressed in the cell.
Non-secreted proteins can also be effectively obtained within infected 293 cell production protocols.

Insect cells are prepared according to methods known in the art from Autographa californica (Aut.
can be infected with a recombinant baculovirus commonly derived from the nuclear polyhedrosis virus (AcNPV) of Ographa californica). In one method, recombinant baculovirus is produced using the transposon basic system described by Luckow et al., J. Viol. 67: 4566-4579, 1993. This system utilizing transfer vectors is commercially available in kit form (Bac-to-Bac ^™ kit; Life Technologies, Rockville, MD). Transfer vectors (eg pFastBac1 ^™ ; Life Technologies)
Contains the Tn7 transposon, and encodes the DNA encoding the protein of interest as "bacmid
It is transferred to the baculovirus genome retained in E. coli as a large plasmid called "bacmid".

Reference: 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,
J. Biol. Chem. 270: 1543-1549, 1995. In addition, the transfer vector contains an in-frame fusion with DNA encoding the polypeptide extension or an affinity tag disclosed above. Using techniques known in the art, transfer vectors containing the zalpha29 coding sequence are transformed into E. coli host cells, which are screened for bacmids containing an interrupted lacZ gene indicative of recombinant baculovirus. Bacmid DNA containing the recombinant baculovirus genome is isolated by standard techniques and used to transfer Spodoptera frugiperda cells such as Sf9 cells. Recombinant virus expressing the zalpha29 protein is subsequently produced. Recombinant virus strains are created by methods of commonly used techniques.

For protein production, recombinant viruses are used to infect host cells,
A typical example of this is the American procession caterpillar, Pleurotus spp. [Spodoptera frugiperda (Sp
odoptera frugiperda)] (eg, Sf9 or Sf21 cells) or Trichopurcia nai (T
cell lines derived from richoplusia ni) (eg, High Five ^™ ; Invitrogen, Carlsbad, CA). Reference Example: U.S. Patent 5,300,435. Cells are grown and maintained using serum-free medium. Suitable media formulations are known and can be obtained from commercial suppliers. The cells are about 2-5 × 10 ^{5 and} the inoculum density is 1-2 × 1
Grow to a density of 0 ⁶ cells, at which time the recombinant virus strain is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically a MOI of 3. The techniques used are generally known in the art.

Other higher eukaryotic cells, plant cells, avian cells and the like may also be used as hosts. As a vector to express genes in plant cells, Agrobacterium rhizogenes (A
The use of grobacterium rhizogenes has been reviewed by Sinka et al. (Sinka
r et al., J. Biosci. (Bangalore) 11: 47-58, 1987).

Fungal cells, including yeast cells, can be used in the present invention. Yeast species of particular interest in this regard are Saccharomyces cerevisiae, Pichia
Pastoris (Pichia pastoris) and Pichia methanolica
Is. Methods for transforming Saccharomyces cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom include, for example, Kawasaki (US Pat.
4,599,311), Kawasaki et al .; U.S. Pat.
ke; US Pat. No. 4,870,008), Welch et al. (Welch et al. US Pat. No. 5,037,743), and Murray et al. (Murray et al., US Pat. No. 4,845,075). Transformed cells are selected by a phenotype determined by a selectable marker, general drug resistance, or the ability to grow in the absence of a particular nutrient (eg leucine).

Representative examples of vector systems used in Saccharomyces cerevisiae include Kawasaki et al.
awasaki et al .; U.S. Pat. No. 4,931,373), which is a POTI vector system, which allows transformed cells to be selected by growth in glucose-containing medium. Suitable promoters and terminators for use in yeast include glycolytic enzyme genes (see eg Kawasaki; U.S. Patent 4,599,311); Kingsman et al.
et al., U.S. Pat. No. 4,615,974); and Bitter, U.S. Pat. No. 4,977,092)) and alcohol dehydrogenase. See also US patents: 4,990
, 446; 5,063,154; 5,139,936; and 4,661,454.

Hansenula polymorpha, Schizosaccharomyces pombe, Kluyberomys
ces lactis), Kluyberomyces fragillis, Ustilago maydis, Pichia pastoris
, Pichia methanolica, Pichia Guillermon di (Pichia
guillermondii), and other yeast transformation systems are known in the art, including Candida maltosa. Reference example: Gleeson et al., J. Gen. M
icrobiol. 132: 3459-3465, 1986; Cregg, U.S. Patent 4,882,279; and Raymond e.
t al., Yeast 14, 11-23, 1998. Aspergillus cells may be used according to the method of McKnight et al. (US Pat. No. 4,935,349).

Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al. In US Pat. No. 5,162,228. Neurospora (Neur
The method of transforming ospora is described by Lambowitz in U.S. Patent 4,486,533.
It is disclosed in. Recombinant protein production in Pichia methanolica is disclosed in US Patents 5,716,808, 5,736,383, 5,854,039, and 5,888,768.

Prokaryotic host cells include the bacterial Escherichia coli (E. coli)
, Strains of Bacillus and other genera, which are also useful host cells within the scope of the present invention. Aliens transformed into these hosts and cloned there
Techniques for expressing DNA sequences are well known in the art (see eg Sambrook et al., Ibid.
.). When the zalpha29 polypeptide is expressed in bacteria such as E. coli, it is generally retained in the cytoplasm as insoluble granules or is directed into the periplasmic space by bacterial selection sequences.

In the former case, the cells are lysed and the granules are harvested and denatured with, for example, guanidine isothiocyanate or urea. The denatured polypeptide is then renatured and dimerized by diluting the denaturant, eg, dialyzing against a combination of urea solution and redox glutathione, then dialyzing against a buffered salt solution. In the latter case, the polypeptide is recovered from the periplasmic space in a soluble, functional form, in which case the periplasmic contents are disrupted by disrupting the cells (e.g., sonication or osmotic shock). Release and recover protein. Thereby eliminating the need for denaturation and regeneration.

Transformed or transfected host cells are cultured according to conventional techniques in a medium containing nutrients and other components required for the growth of the selected host cells. A variety of suitable media are known in the art, including standardized media and complex media, and generally include carbon sources, nitrogen sources,
Contains essential amino acids, vitamins and minerals. The medium may optionally contain components such as growth factors or serum. Growth medium is generally DNA added from the outside.
, For example, by drug selection or deficiency in essential nutrients that complement the selectable marker carried on the expression vector or co-transfected into the host cell. In liquid culture, air is sufficiently supplied by a simple method such as shaking of a small flask or stirring of a fermentor.

Depending on the intended use, the polypeptides and proteins of the invention may be ≧ 80% pure, ≧ 90% pure, ≧ 95% pure, or in a pharmaceutically pure state, ie macromolecules, especially others. Can be purified to> 99.9% purity with respect to protein and nucleic acid contamination and free of infectious and pyrogenic agents. Purified polypeptides or proteins can be prepared that are substantially free of other polypeptides or proteins, especially those of animal origin.

Expressed recombinant zalpha29 proteins (including chimeric and multimeric proteins) are purified by conventional protein purification methods, typically a combination of chromatographic techniques. General References: Affinity Chromatography: Principles and Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 198.
8; and Scopes, Protein Purification: Principles and Practices, Springer-Verlag, New York, 1994.

A protein comprising a polyhistidine affinity tag (generally about 6 histidine residues) is purified by affinity chromatography on a nickel chelate resin. Reference example: Houchuli et al., Bio / Technol. 6: 1321-1325, 19
88. A protein containing a glu-glu tag can be purified by immunoaffinity chromatography according to a conventional method. Reference example: Grussenmeyer et
al., ibid .. The maltose binding protein fusion is purified on an amylose column according to methods known in the art.

Zalpha29 polypeptides can be prepared via chemical synthesis according to methods known in the art such as exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Reference Example: Merrifield, J. Am. Chem. 85: 2149, 1963; Stewart et
al., Solid Phase Peptide Synthesis (2nd edition), Pier
ce Chemical Co., Rockford, IL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3
: 3, 1986; and Atherton et al., Soli Phase Peptide Synthesis: A Practical
Approach, IRL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for small polypeptide preparation.

The zalpha29 protein may be prepared as a monomer or multimer using methods known in the art; it may be glycosylated or non-glycosylated; it may be pegylated or non-pegylated; The first methionine amino acid residue may or may not be included. Target cells used for zalpha29 activity assay include, but are not limited to, vascular cells (particularly endothelial cells and smooth muscle cells), hematopoietic cells (bone marrow and lymphoid cells), liver cells (hepatic parenchymal cells, Fenestrated endothelial cells, including Kupffer cells, and Ito cells), fibroblasts (including human skin fibroblasts and lung fibroblasts), fetal lung cells, synovial fluid cells, perivascular cells, cartilage Such as cells, osteoblasts, and prostate epithelial cells. Endothelial cells and hematopoietic cells are derived from a common progenitor cell, the hemangioblasts (Choi et al., Development 125: 725-732, 1998).

The activity of the zalpha29 protein can be measured in vitro using cultured cells or in vivo by administering the molecules of the claimed invention to a suitable animal model. Assays that measure cell proliferation or differentiation are well known in the art. For example, assays that measure proliferation include assays such as chemosensitivity to neutral red dye (Cavanaugh et al., Invesigational New Drugs 8: 347-354, 1990), incorporation of radiolabeled nucleotides (e.g., the disclosure below. : Raines and Ross, Methods Enzym
ol. 109: 749-773, 1985; Wahl et al., Mol. Cell Biol. 8: 5016-5025, 1988; a.
nd Cook et al., Analytical Biochem. 179; 1-7, 1989), incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of proliferating cells (Porstmann et al., J. Immunol. Met.
hods 82: 169-179, 1985), and the use of tetrazolium salts (Mosmann, J. Immunol.
Methods 65: 55-63, 1983; Alley et al., Cancer Res. 48: 589-601, 1988; Ma.
rshall et al., Growth Reg. 5: 69-84, 1995; and Scudiero et al., Cancer Re
s. 48: 4827-4833, 1988).

Differentiation can be assayed using appropriate progenitor cells that can be induced to differentiate into a more mature phenotype. Assays for measuring differentiation include, for example, measurement of cell surface markers associated with developmental stage-specific expression of tissues, enzyme activity measurement, functional activity measurement or morphological change measurement (Watt, FASEB, 5: 281). -284, 1991; Franc
is, Differentiation 57: 63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol.
Bioprocesses, 161-171, 1989; all of which are incorporated herein by reference.

The activity of zalpha29 is that of one or more additional growth factors or other macromolecules zalpha29.
It can also be detected by assays designed to measure 29-induced production. Such assay methods include hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF). , An angiogenin, and an assay to quantify the presence of other macromolecules produced by the liver. Suitable assays include mitogenic assays, target binding assays, competitive binding assays, immunological assays (e.g., ELISA) using target cells that respond to macromolecules of interest, and other formats known in the art. is there.

Secretion of metalloproteases is measured from treated primary human dermal fibroblasts, synovial cells and chondrocytes. Relative levels of collagenase, gelatinase and stomalysin produced in response to culture in the presence of zalpha29 protein are measured by zymogram (Loita and Stetler-Stevenson, Cancer Biology 1: 96-106,
1990). Procollagen / collagen by skin fibroblasts and chondrocytes in response to test proteins is measured by incorporating ³ H-proline into nascent secretory collagen.

³ H-labeled collagen was visualized by SDS-PAGE and subjected to autoradiography (
Unemori and Amento, J. Biol. Chem. 265: 10681-10685, 1990). Glycosaminoglycan (GAG) secretion from skin fibroblasts and chondrocytes is 1,9-dimethylmethylene
Measured by the blue dye binding assay (Farndale et al., Biochim. Biophys.
Acta 883: 173-177, 1986). Collagen and GAG assays are also performed in the presence of IL-1β or TGF-β to test the ability of the zalpha29 protein to establish responses to these cytokines.

The monocyte activation assay explored the ability of (1) zalpha29 protein, to further promote monocyte activation, and (2) tested the ability of zalpha29 protein, to induce adhesion or endotoxin induction. To regulate monocyte activation in Fuhlbrigge et al.
, J. Immunol. 138: 3799-3802, 1987). IL-1β and TNFα levels produced in response to activation are measured by ELISA (Biosource, Inc.).
, Camarillo, CA). Monocytes / macrophage cells are activated by CD14 (LPS receptor)
Proteins that are sensitive to endotoxin and have moderate levels of endotoxin-like activity activate these cells.

The hematopoietic activity of the zalpha29 protein can be assayed by culturing on various hematopoietic cells. Suitable assays are the primary bone marrow colony assay and the late lineage restricted colony assay, which are known in the art (eg Holly et al., WIPO Publication WO9.
5/2 1920). Bone marrow cells plated on a suitable semi-solid medium (e.g., 50% methylcellulose with 15% fetal bovine serum, 10% bovine serum albumin and 0.6% PSN antibiotic mixture) were incubated in the presence of the test polypeptide, then Inspect for colony formation under a microscope. Known hematopoietic factors are used as controls. The mitogenic activity of zalpha29 polypeptides on hematopoietic cell lines can be measured as disclosed above.

Cell migration is assayed essentially as disclosed by Kehler et al. (Kehler et al., Ar.
teriosclerosis, Thrombosis, and Vascular Biology 17: 932-939, 1997). A protein is considered to be chemotactic if it induces cell migration from regions of low protein concentration to regions of high protein concentration. A typical assay is performed using a modified Boyden chamber with a polystyrene membrane separating the two chambers (eg, Transwell®; Corning Coaster Corp.). ). Test samples are diluted in medium containing 1% BSA and added to the lower chamber of transwells containing 24-well plates.

Cells are then plated on transwell inserts pretreated with 0.2% gelatin. Cell migration is measured after 4 hours of incubation at 37 ° C. Non-migrated cells are wiped from the top of the transwell membrane, cells attached to the bottom surface of the membrane are fixed and stained with 0.1% crystal violet. The stained cells were then extracted with 10% acetic acid and the absorbance at 600 nm.
To measure. The migration is then calculated from the standard calibration curve. Cell migration can also be measured by the Matrigel method of Grant et al. (Grant et al., "Angiogenesis as a composition of epithelial-mesenchymal interactions", Goldberg and Rosen (Goldberg).
and Rosen), Epithelial-Mesenchymal Interactions in Cancer, Birkhaozel Ferlag (Bir
kheuser Verlag), 1995, 235-248; Baatout, Anticancer Research 17: 451-456.
, 1997).

Cell-adhesive activity is assayed substantially as disclosed by Lafleur et al. (LaFleur et al.
., J. Biol. Chem. 272: 32798-32803, 1997). Briefly, test proteins were applied to microtiter plates, non-specific sites were blocked with BSA, cells (
Smooth muscle cells, white blood cells, or endothelial cells, etc.) are plated at a density of about 10 ⁴ -10 ⁵ cells / well. The wells are incubated at 37 ° C (typically about 60 minutes), then non-adherent cells are removed by gentle washing. Attached cells are quantified by conventional methods (eg, staining with crystal violet, cell lysis, and optical density measurement of lysed cells). Control wells are coated with a known adhesion protein such as fibronectin or vitronectin.

The activity of the zalpha29 protein can be measured by a microphysiometer, a silicon-based biosensor that measures extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiological cellular responses. Typical examples of such devices are Molecular Devices, Sunyvale.
), A Cytosensor ^™ microphysiometer manufactured by CA.
A variety of cellular responses such as cell proliferation, ion transport, energy production, inflammatory response, regulation and receptor activation can be measured by this method. References: McConnell et al., Science 257: 1906-1912, 1992; Pitchford et al., Meth.
Enzymol. 228: 84-108, 1997; Arimilli et al., J. Immunol. Meth. 212: 49.
-59, 1998; and Van Liefde et al., Eur. J. Pharmacol. 346: 87-95, 1998.

The microphysiometer can be used to assay adherent or non-adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in the cell media over time, the microphysiometer directly measures the cellular response to various stimuli, such as the zalpha29 protein, its agonists and antagonists. Using a microphysiometer, zalpha29-responsive eukaryotic cell response
It can be measured relative to control eukaryotic cells that do not respond to the lpha29 polypeptide. Zalpha29-responsive eukaryotic cells consist of cells that have become responsive to zalpha29 by transfecting the zalpha29 receptor, as well as cells that are naturally responsive to zalpha29.

When the responsiveness of cells exposed to zalpha29 peptide was compared to controls not exposed to zalpha29, the difference was measured by changes in extracellular acidification, the difference was zalpha29.
It is a direct measure of the regulatory response. Furthermore, such zalpha29 regulatory response can be assayed under various stimuli. The invention thus provides a method of identifying agonists and antagonists, which method comprises supplying cells responsive to a zalpha29 polypeptide, culturing a first portion of the cells in the absence of the test compound, Culturing a second part of the cell in the presence and detecting an alteration of the cellular response in the second part of the cell as compared to the first part of the cell.

Changes in the cellular response are shown as measurable changes in extracellular acidification rate. zalpha
Culturing the third part of the cells in the presence of 29 protein and in the absence of the test compound resulted in a positive control and test compound agonist activity and z
A control is provided to compare the agonist activity of the lpha29 polypeptide. Antagonists of zalpha29 can be identified by exposing the cells to zalpha29 protein in the presence and absence of the test compound, where a decrease in zalpha29 stimulating activity is an indication of antagonist activity in the test compound. is there.

Expression of zalpha29 polynucleotides in animals provides a model for further study of the biological effects of overproduction or inhibition of protein activity in vivo. Polynucleotides encoding zalpha29 and antisense polynucleotides can be introduced into test animals, such as mice, using viral vectors or naked DNA, and transformed animals can be generated.

One of the in vivo methods of assaying the proteins of the present invention utilizes a viral delivery system. Representative examples of viruses suitable for this purpose are adenovirus, herpesvirus, retroviruses, vaccinia virus, and adenovirus-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the most well-studied gene carrier for foreign nucleic acid delivery. Reference Review: Becker et al., Meth.
Cell Biol. 43: 161-89, 1994; and Douglas and Curiel, Science & Medicine
4: 44-53, 1997.

The adenovirus system offers several advantages. Adenovirus can (i) accommodate relatively large DNA inserts; (ii) can grow to high titers; (iii) can infect a wide range of mammalian cell types; and (iv) many. Different promoters, for example,
It can be used with ubiquitous tissue-specific, regulatable promoters. Adenovirus is stable in the bloodstream and can be injected intravenously.

By removing a portion of the adenovirus genome, large inserts of foreign DNA (up to 7 kb) can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with the cotransfected plasmid. In a typical system, the essential E1 gene was deleted from the viral vector,
The virus does not replicate unless the host cell (eg, human 293 cell line) provides the E1 gene. Adenovirus primarily targets the liver when administered intravenously to naive animals. If the adenovirus delivery system lacks the E1 gene, the virus is unable to replicate in the host cell. However, the host tissue (eg, liver) expresses and processes the heterologous protein (and secretes the signal sequence if present). The secreted proteins enter the circulation in the highly vascularized liver,
The effect on infected animals can be determined.

An alternative to gene delivery consists of removing cells from the body and introducing the vector into the cells as a naked DNA plasmid in the cells. The transformed cells are then reimplanted into the body. Naked DNA vectors can be prepared by methods known in the art, such as transfection, electroporation, microinjection, transduction,
Cell fusion, DEAE dextran, calcium phosphate precipitation, use of gene gun, or
It is introduced into a host cell by using a DNA vector transporter. Reference: Wu et al., J.
Biol. Chem. 263: 14621-14624, 1988; Wu et al., J. Biol. Chem. 267: 963-9.
67, 1992; and Johnston and Tang, Meth. Cell Biol. 43: 353-365, 1994.

Transgenic mice engineered to express the zalpha29 gene, and
Mice that have completely lost zalpha29 gene function, referred to as “knockout mice” (Snouwaert et al., Science 257: 1083, 1992), can also be generated (Lowell et al., Nature 366: 740-742, 1993). These mice can be employed to study the zalpha29 gene and the proteins encoded by it in an in vivo system. Transgenic mice show za during early development
It is particularly useful for studying the role of the lpha29 protein, where it allows the identification of developmental abnormalities or blockages resulting from over or under expression of specific factors. Reference: M
aisonpierre et al., Science 277: 55-60, 1997 and Hanahan, Science 277: 48.
-50, 1997. The promoter for transgenic expression is the promoter from the metallothionein and albumin genes.

Antisense methodology can be used to inhibit zalpha29 gene transcription and study the effects of such inhibition in vivo. Polynucleotide encoding zalpha29
A polynucleotide complementary to a segment (eg, the polynucleotide set forth in SEQ ID NO: 1) is designed to bind to the mRNA encoding zalpha29 and inhibit translation of such mRNA. Such antisense oligonucleotides can also be used to inhibit expression of genes encoding zalpha29 polypeptides in cell culture.

Most 4-helix bundle cytokines, as well as other proteins produced by activated lymphocytes, play important biological roles in cell differentiation, activation, recruitment, and systemic homeostasis throughout the body. Various therapeutic applications are expected for inhibitors of zalpha29 and zalpha29 activity. These applications have applications in the treatment of diseases that require immunomodulation, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis,
Treatment of systemic lupus erythematosus and diabetes.

Zalpha29 is important in the regulation of inflammation and is therefore useful in the treatment of rheumatoid arthritis, asthma and sepsis. There may be a role for zalpha29 in mediating tumorigenesis, in which case zalpha29 antagonists would be useful in treating cancer. zalpha29 is useful in regulating the immune system, in which case zalpha29 and
zalpha29 antagonists reduce graft rejection, prevent graft-host disease, enhance immunity to infectious diseases, immunocompromised patients (e.g. HIV positive patients)
Can be used for the treatment of, or for the improvement of vaccines.

The zalpha29 polypeptide can be administered alone or in combination with other angiogenic or angiogenic agents such as VEGF. When zalpha29 is used in combination with an additional drug, the two compounds may be administered simultaneously or sequentially, depending on the particular condition to be treated.

The zalpha29 protein for pharmaceutical use is formulated according to conventional methods for topical or parenteral delivery, especially for intravenous or subcutaneous administration. In general, pharmaceutical formulations are za
It consists of a combination of the lpha29 polypeptide and a pharmaceutically acceptable vehicle such as saline, buffered saline, 5% dextrose in water and the like. 1 more formulation
It may also include one or more additives, preservatives, solubilizers, buffers, albumin and the like to prevent protein loss on the vial surface. Methods of formulation are well known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed.
., Mack Publishing Co., Easton, PA, 19th ed., 1995.

Zalpha29 is preferably used at a concentration of about 10 to 100 μg / ml total volume, although concentrations ranging from 1 ng / ml to 1000 μg / ml may be used. For local administration, for example for promoting wound healing, the protein is applied in the range of 0.1-10 μg / cm ² trauma area, but the exact dose will depend on the nature and severity of the condition to be treated, the characteristics of the patient. The doctor decides according to the accepted standard, taking into consideration the above. Dose determination is within the level of ordinary skill. Administration is daily or at intervals of treatment. Intravenous administration is accomplished by large volume injection or infusion over a typical period of 1 hour to several hours. Sustained-release preparations may be adopted. Generally, a therapeutically effective amount of zalpha29 is a clinically significant change in the treated condition, such as a clinically significant change in hematopoietic or immune function, a significant decrease in morbidity, or a significantly increased histological The amount is sufficient to give a score and the like.

The zalpha29 proteins, agonists and antagonists are useful in modulating the expansion, proliferation, activation, differentiation, migration, or metabolism of responsive cell types, which cell types affect both primary and cultured cell lines. Including. Of particular interest in this regard are hematopoietic cells (including stem cells and mature bone marrow cells and lymphoid cells), endothelial cells, smooth muscle cells,
Fibroblasts and hepatocytes. The zalpha29 polypeptide is added to the tissue culture medium at a concentration of about 10 pg / ml to about 100 ng / ml for these cell types. Those skilled in the art z
It will be appreciated that the alpha29 protein can be advantageously combined with other growth factors in culture.

In the field of laboratory research, the zalpha29 protein is used as a measure of molecular weight or in assays for measuring circulating levels of the protein, eg diagnosis of diseases or cells characterized by excessive or underproduction of the zalpha29 protein. It can also be used as a reagent in phenotypic analysis.

The zalpha29 protein may also be used to identify inhibitors of its activity.
Test compounds are added to the assay system described above to identify compounds that inhibit the activity of zalpha29 protein. In addition to these assays described above, samples can be tested for inhibition of zalpha29 activity in various assay systems designed to measure the promotion / inhibition of receptor binding or zalpha29 dependent cellular responses. For example, a zalpha29 responsive cell line is transfected with a reporter gene construct that responds to the zalpha29 stimulated cell pathway. This type of reporter gene construct is known in the art and is generally operably linked to a gene encoding an assayable protein such as luciferase.
It comprises the zalpha29 activated serum response element (SRE). Candidate compounds, solutions, mixtures or extracts are tested for their ability to inhibit zalpha29 activity on target cells, as evidenced by the increased stimulation by zalpha29 of reporter gene expression.

This type of assay detects compounds that directly block zalpha29 binding to cell surface receptors, as well as compounds that block the process of the cellular pathway following receptor-ligand binding. In a separate method, a detectable label (e.g., ^{1 25} I, biotin, horseradish peroxidase, FITC, etc.) using zalpha29 labeled with, for compounds or other samples zalpha29 to block binding to the receptor directly Or you can test. In this type of assay, the ability of the test sample to inhibit labeled zalpha29 from binding to the receptor is an indication of inhibitory activity, which activity can be confirmed via secondary assays. The receptor used in the binding assay may be a cellular receptor or a separate solid-phased receptor.

As used herein, “antibody” refers to polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof, such as F (ab ′) ₂ and Fab fragments,
It is a single-chain antibody or the like, and includes an antibody prepared by genetic engineering. Non-human antibodies can be humanized by grafting non-human CDRs into the human skeleton and constant region, or by incorporating whole non-human variable domains (optional,
Substitution of exposed residues "masks" them at the human-like surface, which then results in "coated" antibodies). In some cases, humanized antibodies may preserve non-humanized residues within the human variable region scaffold domain to enhance appropriate binding properties. Through the humanization of antibodies, the biological half-life is extended, reducing the potential for adverse immune reactions upon administration to humans.

Those skilled in the art will appreciate that humanized antibodies with different specific constant domains (ie different Ig
Subclass) can be generated to promote or inhibit various immune functions associated with a particular antibody constant domain. Antibodies are defined as specifically binding if they bind a zalpha29 polypeptide or protein with an affinity that is at least 10 times greater than the binding affinity that controls the (non-zalpha29) polypeptide or protein. The affinity of a monoclonal antibody can be easily determined by those skilled in the art (reference example: Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).

Methods for preparing polyclonal and monoclonal antibodies are well known in the art.
(Reference example: Hurrell, JGR, Ed., Monoclonal Hybridoma Antibodies: Techniqu
es and Applications, CRC Press, Inc., Boca Raton, FL, 1982; incorporated herein by reference. As will be appreciated by those in the art, 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 zalpha29 polypeptides may be increased using 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 peptide or a maltose binding protein. The polypeptide immunogen may be a full length molecule or a portion thereof. When the polypeptide protein is "hapten-like", such moieties are advantageously macromolecular carriers for immunization (keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus. Toxins).

Another technique for generating or selecting antibodies consists of exposing lymphocytes to zalpha29 polypeptides in vitro and selecting antibody display libraries with phage or like vectors (eg, Immobilized or labeled zal
via the use of the pha29 polypeptide). Human antibodies can be produced in transgenic non-human animals, which are made to contain human immunoglobulins, as disclosed in WIPO Publication WO98 / 24893. The endogenous immunoglobulin genes in these animals are preferably inactivated or removed, such as by homologous recombination.

Various assays known to those of skill in the art can be utilized to detect antibodies that specifically bind to zalpha29 polypeptides. An exemplary assay method is Antibodies: A
Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory
See Press, 1988 for details. Representative examples of such assays are: coimmunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, solid phase enzyme immunoassay (ELISA), dot blot assay, western blot assay, inhibition or competition assay, and sandwich. Assay.

Antibodies to zalpha29 can be used in affinity assays for proteins in diagnostic assays that measure circulating levels of the protein; to detect or quantify soluble zalpha29 polypeptides as markers of underlying pathology or disease; immunodiagnosis For immunolocalization within whole animals or within tissue sections, such as for applications in; immunohistochemistry; and as antagonists that block protein activity in vitro and in vivo;
Can be used. Antibodies to zalpha29 for labeling cells expressing zalpha29; for affinity purification of zalpha29 polypeptides and proteins;
It can also be used for analytical methods using FACS; for screening expression libraries; and for producing anti-idiotype antibodies.

Antibodies can be conjugated to other compounds such as therapeutics and diagnostics by known methods of targeting these compounds to cells expressing the zalpha29 receptor. For certain applications, such as in vitro and in vivo diagnostic applications, it is advantageous to employ a labeled antibody. Suitable direct tags or labels are radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, etc .; indirect tags or labels include biotin-avidin or other supplements as intermediates. The use of body / anti-complement pairs may be featured. The antibodies of the invention may be conjugated directly or indirectly to drugs, toxins, radionuclides, etc., and these conjugates may be used for in vivo diagnostic or therapeutic applications (eg, inhibition of cell proliferation). General reference: Ramakrishnan et al., Cancer Res. 56: 1324-1330, 1996.

The polypeptides and proteins of the present invention can be used to identify and isolate receptors. The zalpha29 receptor is involved in the growth regulation of liver, angiogenesis and other developmental processes. For example, zalpha29 proteins and polypeptides can be immobilized on a column and the column can be run with a membrane preparation (Immobilized Affinity Ligand Techniques, Hermanso.
n. et al., Academic Press, San Diego, Calif., 1992, p.195-202).

Proteins and polypeptides have also been radiolabeled (Methods Enzymol., Volume 182, "Guide to Protein Purification", M. Deutsche.
r Edit, Academic Press, San Diego, 1990, 721-737) or photoaffinity labeling (Brunner et al., Ann. Rev. Biochem. 62: 483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33). 1167-1180, 1984), and can be used to tag specific cell surface proteins. Similarly, radiolabeled zalpha29 proteins and polypeptides can be used to clone cognate receptors in a binding assay using cells transfected with an expression cDNA library.

The present invention also provides reagents for use in diagnostic applications. For example, a probe containing the zalpha29 gene, zalpha29 DNA or RNA, or subsequences thereof, can be used to determine the presence of a mutation at or near the zalpha29 locus on chromosome 2p15. Aneuploidy for detectable chromosomal aberrations at the zalpha29 locus,
These include, but are not limited to, gene copy number alterations, insertions, deletions, restriction site alterations and transpositions. These abnormalities can occur within the coding sequence, within introns, or within flanking sequences containing upstream promoters and regulatory regions, and manifest as physical alterations or changes in gene expression levels within the coding sequence. There is.

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 long, often 15 or more nucleotides,
And often 20-30 nucleotides. Short polynucleotides can be used when targeting small regions of a gene for analysis. For global analysis of the gene, the polynucleotide probe can include whole exons or more. The probe will generally include a polynucleotide associated with the signal-generating moiety, such as a radionucleotide.

Generally, these diagnostic methods involve (a) obtaining a genetic sample from a patient; (b) combining this genetic sample with a polynucleotide probe or primer as disclosed above. Incubating under conditions that hybridize to the complementary polynucleotide sequence to produce a first reaction product; and (c)
A step is included that compares this first product to the control reaction product. The difference between the first product and the control reaction product is indicative of a genetic abnormality in the patient. Genetic samples used within the scope of the present invention include genomic DNA, cDNA and RNA.

The polynucleotide probe or primer can be RNA or DNA, and can be a portion of SEQ ID NO: 1, the complement of SEQ ID NO: 1 or RNs thereof.
Would include the A equivalent. Suitable assay methods in this regard include molecular genetic techniques known to those of skill in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis using PCR technology, ligation chain reaction ( Barany, PCR M
ethods and Applications 1: 5-16, 1991), ribonuclease protection assays and other gene binding assay techniques known in the art (Sambrook et al., supra; Ausubel et al., supra; AJ Marian, Chest 108: 255-265, 1995) is included.

Ribonuclease protection assays (see, eg, Ausubel et al., Ibid., Section 4) are based on patient R
Hybridization of the RNA probe to the NA sample, and then exposing the reaction product (RNA-RNA hybrid) to RNase. The hybridizing region of RNA is protected from digestion. In a PCR assay, a patient gene sample is incubated with a pair of polynucleotide primers and the region between these primers is amplified and recovered. Changes in the size or quantity of the recovered product are indicative of patient mutations. Another PCR-based technique that can be used is single-stranded conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applicatio
ns 1: 34-38, 1991).

The polypeptides, nucleic acids and / or antibodies of the present invention can be used in the diagnosis or treatment of diseases associated with cell loss or abnormal cell proliferation (including cancer). Labeled zalph
The a29 polypeptide can be used to image tumors or other sites of abnormal cell growth.

Inhibitors of zalpha29 activity (zalpha29 antagonists) include anti-zalpha29 antibodies and soluble zalpha29 receptors, as well as other peptide and non-peptide agonists (including ribozymes). Such antagonists can be used to block the effects of zalpha29 on cells or tissues. Of particular importance is the use of antagonists of zalpha29 activity in cancer therapy. Improved early detection methods could mediate earlier in tumorigenesis and use growth factor inhibitors to block cell proliferation, angiogenesis, and other events that lead to tumorigenesis and metastasis Can be realized. Inhibitors are also expected to be useful in post-surgical adjunct therapy to inhibit the growth of residual cancer cells. Inhibitors can also be used in combination with other cancer therapeutics.

Zalpha29 inhibitors include, in addition to antibodies, small molecule inhibitors of zalpha29 polypeptides and inactive receptor-binding fragments. The inhibitor is formulated for pharmaceutical use as generally disclosed above, taking into account the exact chemical and physical properties of the inhibitor and the condition to be treated. Relevant decisions are within the level of ordinary skill in the formulation art.

Polynucleotides encoding zalpha29 polypeptides are useful within gene therapy applications where it is desirable to enhance or inhibit zalpha29 activity. When the mammal has a mutated zalpha29 gene or lacks the gene, the zalpha29 gene can be introduced into the cells of the mammal. In one embodiment, the gene encoding the zalpha29 polypeptide is introduced in vivo into a viral vector. Such vectors include attenuated or defective DNA viruses, such as herpes simplex virus (HSV), papilloma virus, Epstein-Barr virus (EBV), adenovirus, adeno-associated virus (AAV), etc. Not limited to.

Defective viruses that completely or almost entirely lack viral genes are preferred. Defective viruses are not infectious after introduction into cells. The use of defective viral vectors allows for administration to cells in a particular localized region without the risk of the vector infecting other cells. Examples of specific vectors include deficient herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2: 320-330,
1991); attenuated adenovirus vectors, such as those described by Stratford-Perricaudet et al., J. Clin. Invest. 90: 626-630, 1992; and defective adeno-associated virus vectors. (Samulski et al., J. Virol. 61: 3096-3101, 1987; Samulski et al., J. Virol. 63: 3822-3888, 1
989), but is not limited to these.

In another embodiment, the zalpha29 gene is cloned into, for example, Anderso (Anderso
n) et al., U.S. Pat.No. 5,399,346; Mann et al., Cell, 33: 153, 1983; Temin (Te
min) et al., U.S. Pat.No. 4,650,764; Temin et al., U.S. Pat.No. 4,980,289; Markowitz et al., J. Virol. 62: 1120, 1988; Temin et al. U.S. Pat.
, 263; Dougherty et al., WIPO published WO 95/07358; and Kuo et al., B
can be introduced into a retroviral vector as described by Lood 82: 845, 1993. Alternatively, the vector can be introduced by liposome-mediated transfection (“lipofection”).

Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner et al., Proc. Na.
tl. Acad. Sci. USA 84: 7413-7417, 1987; Mackey et al., Proc. Natl. Acad. Sci.
USA 85: 8027-8031, 1988). The use of lipofection to introduce foreign genes into specific organs in vivo has certain practical advantages, including molecular targeting of liposomes to specific cells.

Directing transfection to specific cell types is particularly advantageous in tissues with cell heterogeneity, such as pancreas, liver, kidney and brain. Lipids can be chemically linked to other molecules for the purpose of targeting. Peptide and non-peptide molecules can be chemically linked to liposomes. In another embodiment, the cells are removed from the body, the vector is introduced into the cells as a naked DNA plasmid, and the transformed cells are reimplanted into the body as disclosed above. Using the antisense method, patient za as generally disclosed above.
It can inhibit lpha29 gene transcription.

The zalpha29 polypeptides and anti-zalpha29 antibodies can be conjugated directly or indirectly with pharmaceutical agents, toxins, radionuclides, etc., and these conjugates can be used for in vivo diagnostic or therapeutic applications. For example, using a polypeptide or antibody of the invention, the corresponding anti-complementary molecule (e.g., receptor or antigen, respectively)
Can be identified or treated. More specifically, za
a lpha29 polypeptide or anti-zalpha29 antibody, or a biologically active fragment or portion thereof, conjugated to a detectable or cytotoxic molecule, and having a cell, tissue, or organ that expresses the anti-complementary molecule. It can be delivered to an animal.

Suitable detectable molecules can be directly or indirectly bound to the above-described polypeptides or antibodies, and such molecules include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers. , Chemiluminescent markers, magnetic particles and the like. Suitable cytotoxic molecules can be linked directly or indirectly to the polypeptide or antibody, and such molecules include bacterial or plant toxins (e.g., diphtheria toxin, pseudomonas exotoxin, ricin, abrin, Saporin, etc.
), As well as therapeutic radionuclides such as iodine-131, ruthenium-188 or yttrium-90.

These can be linked directly to the above-mentioned polypeptides or antibodies, or indirectly according to known methods, eg by chelating moieties. The polypeptide or antibody can also be conjugated to a cytotoxic drug, such as adreamycin. For indirect binding of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be complexed with one member of a complementary / anti-complementary pair, the other member Binds to the polypeptide or antibody portion. For these purposes, biotin / streptavidin is a typical complementary / anti-complementary pair.

The polypeptide-toxin fusion protein or antibody / fragment-toxin fusion protein can be used to inhibit or eliminate targeted cells or tissues, for example in cancer therapy. Of particular importance in this regard is zalpha
Complexes of 29 polypeptides with cytotoxins and can be used to target cytotoxins to tumors or other tissues undergoing unwanted angiogenesis or angiogenesis. Target cells (ie, those displaying the zalpha29 receptor) bind to the zalpha29-toxin complex, which is then internalized and kills the cell.

The effects of receptor-specific cell killing (target ablation) are revealed by whole animal physiological changes or histological examination. Therefore, ligand-dependent, receptor-directed cytotoxicity can be used to enhance understanding of the physiological importance of protein ligands. One such toxin is saporin. Mammalian cells do not have the receptor for saporin and are therefore non-toxic even when saporin remains extracellularly.

In another embodiment, the zalpha29-cytokine fusion protein or antibody /
Fragment-cytokine fusion proteins are labeled with in vitro cytotoxicity (e.g.,
It can be used to enhance tumor targeting monoclonal antibody mediated) and to enhance in vivo killing of target tissues (eg, blood and bone marrow cancer). In general, Hornick et al., Blood 89: 4437-4447, 1997.
See year. Cytokines are generally toxic when administered systemically. The described fusion proteins can target the cytokine to cells having the desired site of action, eg the binding site for zalpha29, and thereby increase the local concentration of the cytokine.

Cytokines for this purpose include, for example, interleukin-2 and granulocyte-macrophage colony stimulating factor (GM-CSF). Such fusion proteins can be used to cause cytokine-induced killing of tumors and other tissues undergoing angiogenesis or angiogenesis. The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or can be locally introduced at the intended site of action. The present invention is further described by the following non-limiting examples. EXAMPLES Example 1. Northern blot analysis of zalpha29 was performed on commercially produced blots of human RNA (
Human Multiple Tissue Northern Blot I, II and
III; Human Fetal Multiple Tissue Northern Blot
Blot) II; and Human RNA Master Blot; Clontech La
boratories, Inc., Palo Alto, Calif.).

The zalpha29 hybridization probe was generated as a gel-purified PCR amplification product. This growth product was labeled with the oligonucleotide ZC21, as a PCR primer.
720 (SEQ ID NO: 8) and ZC21,721 (SEQ ID NO: 9) were used and made using the cloned zalpha29 cDNA (see SEQ ID NO: 1) as template. PCR amplification was carried out as follows: 1 μl of zalpha29 cDNA (about 2 ng) and 40 pmol each of the oligonucleotide primers ZC21,720 and ZC21,721 were prepared using commercially available reagents (Advan
tage ™ KlenTaq Polymerase Kit, Clontech Laboratories, Inc.) was added according to the manufacturer's recommended protocol.

The reaction was carried out as follows: 94 ° C. for 30 seconds, 94 ° C. for 5 seconds, 55 ° C. for 5 seconds and 68 ° C. for 1 minute for 25 cycles, followed by 68 ° C. for 3 minutes, and Maintain at 4 ° C. 422b
The PCR amplified fragment of p was gel purified and silica gel particles (QIAEX®).
II gel extraction kit; Qiagen, Valencia, Calif.) Was used according to the manufacturer's recommended protocol.

The probe was radiolabeled using a commercially available kit (Rediprime ™ II random-prime labeling system; Amersham Corp., Arlington Heights, IL) according to the manufacturer's protocol. The probe was purified using a commercially available push column (NucTrap® column; Stratagene, La Jolla, Calif .; see US Pat. No. 5,336,412). Hybridization Solution (ExpressHybTM Hybridization Solution; Clontech Laboratories,
Inc.) was used for Northern blot prehybridization and hybridization solutions.

Hybridization was carried out at 65 ° C. overnight. After hybridization, the blots were washed in 2 × SSC, 0.1% SDS at room temperature, then in 0.1 × SSC and 0.1% SDS at 50 ° C.
Washed with. The blots were exposed to film (BIOMAX, Eastman Kodak, New Haven, CT). Overnight exposure showed a band of approximately 870 bases in each lane of all blots. Each RNA sample on the RNA master blot was positive, while the negative control was negative.

Tissues positive by Northern method include heart, ovary, fetal lung, brain, small intestine, fetal liver, placenta, colon (mucosal lining), fetal kidney, lung, peripheral blood leukocytes, liver, stomach, skeletal muscle, thyroid gland. , Kidney, spinal cord, pancreas, lymph node, spleen, trachea, thymus, adrenal gland, prostate, bone marrow, testis, fetal brain. Tissues that were positive by RNA master blot but not Northern were amygdala, aorta, caudate, bladder, cerebellum, uterus, cerebral cortex, pituitary, frontal lobe, salivary gland, hippocampus, mammary gland, medulla oblongata, cecum. , Occipital lobe, trachea, putamen, fetal heart, substantia nigra, fetal spleen, thalamus, fetal thymus and hypothalamic nucleus.

These Northern blots were probed again for the human transferrin receptor. The signal obtained from the transferrin receptor probe was used to normalize the zalpha29 signal. The tissues with the highest ratio of zalpha29 signal to transferrin receptor signal were heart, liver and testis.

Example 2. zalpha29 was added to Stanford G3 Radiation Hybrid Mapping Panel (Research Genetics, Inc.,
Mapped to Chromosome 2 using commercially available ones from Huntsville, AL). This panel contains PCR-capable DNA from each of the 83 irradiated hybrid clones of the entire human genome and two control DNAs (RM donor and A3 recipient). The chromosomal location of the marker can be determined by publicly available WWW servers (http://shgc-www.stanford.edu).

To map zalpha29 using the Stanford G3RH panel, 20 μl of the reaction mixture was placed in a PCR-able 96-well microtiter plate (Stratagene, La Jolla, Calif.) And a thermocycler (RoboCycler® Gradient 96 ; Strata
gene). Each of the 85 PCR reactions was 2 μl of buffer (10 × KlenTaq PCR reaction buffer; Clontech Laboratories, Inc., Palo Alto, Calif.), DNTP mix (2.5 mM each, Perkin-Elmer, Foster City, Calif.) 1.6 μl. ,
Sense primer ZC22,737 (SEQ ID NO: 10) 1 μl, antisense primer ZC2
2,738 (SEQ ID NO: 11) 1 μl, density increasing agent and tracking dye (RediLoad, Res
earch Genetics, Inc., Huntsville, Ariz.) 2 μl, commercially available DNA polymerase / antibody mix (50 × Advantage ™ KlenTaq Polymerase Mix; Clont
ech Laboratories, Inc.) 0.4 μl, 25 ng of DNA from individual hybrid clones or controls, and a total volume of 20 μl of ddH 2 O x μl.

Equal amounts of mineral oil were laminated to these mixtures and sealed. The PCR cycler conditions were as follows: 5 cycles of initial denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 45 seconds, annealing at 64 ° C for 45 seconds and extension at 72 ° C for 75 seconds for 35 cycles; followed by 72 Final extension for 7 minutes at ° C. The reactions were separated by electrophoresis on a 2% agarose gel (obtained from Life Technologies, Gaithersburg, MD). These results show that zalpha29 is> 1 with chromosome 2 framework marker SHGC-30949.
0cR with LOD score of 1 and from the above marker It showed binding at a distance of 10000. Localizes zalpha29 to the 2p16-p15 region of chromosome 2 using a physically mapped peripheral gene.

Example 3. The protein coding region of mouse zalpha29 was amplified by PCR using primers with FseI and AscI restriction sites added to the 5'and 3'ends, respectively. PCR primer
ZC23019 (SEQ ID NO: 12) and ZC23018 (SEQ ID NO: 13) are full-length mouse zalpha
Used in a PCR reaction with a template plasmid containing 29 cDNA (pT7T3D-Pac) as follows: 1 cycle of 95 ° C for 5 minutes; followed by 95 ° C for 0.5 minutes, 58 ° C for 0.5 minutes and 72
15 cycles of 0.5 min at ℃; then 7 min at 72 ℃; then soak at 4 ℃. The PCR reaction product was loaded onto 1.2% (low melting) agarose (SeaPlaque® GTG; FMC Corp., Rockland, ME) gel in TAE buffer (0.04M Tris-acetate, 0.001M EDTA). did. The zalpha29 PCR product was excised from the gel.

The gel slice was melted at 65 ° C. and the DNA was extracted twice with phenol and ethanol precipitated. The PCR product was then digested with FseI + AscI, phenol / chloroform extracted, EtOH precipitated, and TE (Tris / EDTA pH8) 20.
Rehydrated in μl. Next, modify the 567 bp zalpha29 fragment with pAdTrack.
CMV (He et al., Proc. Natl. Acad. Sci. USA 95: 2509-2514, 1998) FseI-AscI
Bound to the site. This construct also contained the green fluorescent protein (GFP) marker gene. 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. In addition, the natural polylinker was replaced with FseI, EcoRV and AscI sites.

This modified form of pAdTrack CMV was named pZyTrack. Binding was performed using a DNA binding and screening kit (Fast-Link ™; Epicentre Technologies, Madison, WI). Clones containing the zalpha29 cDNA were identified by standard mini-prep methods. To linearize the plasmid, pZyTrack zalph
About 5 μg of a29 plasmid was digested with PmeI. Approximately 1 μg of the linearized plasmid was cotransformed into BJ5183 cells with 200 ng of supercoiled pAdEasy (He et al., Ibid.). This cotransformation was performed using a 2.5 kV, 200 ohm and 25 μFa electroporator (Gene Pulser®; Bio-Rad Laboratories, Inc., Hercules, Calif.).

The entire cotransformation mixture was plated on 4LB plates containing 25 μg / ml kanamycin. Minimal colonies were picked, expanded in LB / kanamycin, and recombinant adenovirus DNA was identified by standard DNA miniprep methods. Digestion of recombinant adenovirus DNA with FseI + AscI confirmed the presence of the zalpha29 sequence. Recombinant adenovirus miniprep DNA was added to E. coli host cells (DH10BTM;
Life Technologies, Gaithersburg, Md.) And
DNA is a commercially available plasmid isolation kit (QIAGEN® Plasmid Maxi
Kit; Qiagen, Inc., Valencia, CA) was used as directed by the supplier.

About 5 μg of recombinant adenovirus DNA was digested with PacI enzyme (New England Biolabs) in a reaction volume of 100 μl containing 20-30 U of PacI for 3 hours at 37 ° C. The digested DNA was extracted twice with an equal volume of phenol / chloroform and ethanol precipitated. This DNA pellet was resuspended in 5 μl of distilled water. QBI-293A cells seeded the day before and grown to 60-70% confluence (Quantum Biotechnologies, Inc.
, Montreal, Canada) T25 flasks were transfected with PacI digested DNA. This PacI-digested DNA was prepared using sterile HBS (150 mM NaCl, 20 mM Hepes).
Diluted to a total volume of μl.

In a separate tube, 1 mg / ml of N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethyl-ammonium salt (DOTAP) (Boehringer Mannheim, Indiana, Ind. 25 μl of police) was diluted with HBS to a total volume of 100 μl. To this DOTAP
DNA was added, gently pipetted up and down, and left at room temperature for 15 minutes. Media was removed from 293A cells, and these cells were serum free MEM containing 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids and 25 mM Hepes buffer.
Washed with 5 ml alpha (medium components were obtained from Life Technologies, Gaithersburg, MD).

5 ml of serum-free MEM was added to 293A cells and maintained at 37 ° C. The DNA / lipid mixture was added drop-wise to a T25 flask of 293A cells, mixed gently and incubated at 37 ° C for 4 hours. After 4 hours, aspirate the medium containing the DNA / lipid mixture,
Then replaced with 5 ml of complete MEM containing 5% fetal bovine serum. Transfected cells were monitored for GFP expression and foci (viral plaque) formation.

Seven days after transfection of 293A cells with recombinant adenovirus DNA,
The cells expressed GFP and began to form foci. The crude viral lysate was collected with a cell scraper and transferred to a 50 ml conical tube. Dry ice / ethanol bath and 37 ° C to release most viral particles from these cells
The freeze / thaw cycle was performed 3 times in the water bath.

The above crude lysate was amplified (first (1 °) amplification) to yield an effective “stock” of zalpha29 recombinant adenovirus (rAdV) lysate. Nearly congested (80-90%) 293
A 10 cm plate of A cells was set 20 hours before. 200 ml of crude rAdV lysate was added to each 10 cm plate and these plates were monitored under a white light microscope for CPE (cytopathic effect) and under a fluorescence microscope for expression of GFP for 48-72 hours. 2
When 93A cells all showed CPE, 1 ° stock lysates were collected and freeze / thaw cycles were performed as described above.

In the second round (2 °) amplification, 20 15 cm tissue culture dishes of 293A cells were plated with 80-80 cells.
It was prepared to be 90% confluent. All but 20 ml of 5% MEM medium was removed and each dish was inoculated with 300-500 ml of 1 ° amplified rAdv lysate. After 48 hours, these cells were lysed by virus production. The lysate was collected in a 250 ml polypropylene centrifuge bottle.

To purify rAdV, NP-40 detergent was added to the above crude lysate bottle to a final concentration of 0.5% to lyse all cells. The bottle is on a rotating platform,
The bottle was left for 10 minutes with stirring as fast as possible without tipping. The debris was pelleted by centrifugation at 20,000 x G for 15 minutes. The supernatant was transferred to a 250 ml polycarbonate centrifuge bottle and 0.5 volume of 20% PEG-8000 / 2.5M NaCl solution was added. The bottle was shaken on ice overnight. The bottles were centrifuged at 20,000 x G for 15 minutes and the supernatant was discarded in bleach solution. The precipitate from the two bottles was resuspended in 2.5 ml PBS using a sterile cell scraper.

The virus solution was placed in a 2 ml microcentrifuge tube and centrifuged at 14,000 × G for 10 minutes to remove any additional cell debris. Transfer the supernatant from a 2 ml microcentrifuge tube to a 15 ml polypropylene snap cap tube and 1.34 g / m 2 with CsCl.
The density was adjusted to l. The volume of virus solution was estimated and 0.55 g / ml CsCl was added. CsCl was dissolved, and 1 ml of this solution weighed 1.34 g. Transfer this solution to a polycarbonate thick-wall centrifuge tube (3.2 ml; Beckman # 362305) and TLA-100.
Spin at 348,000 x G for 3-4 hours in a Beckman Optima TLX microcentrifuge using a .4 rotor at 25 ° C. The virus formed a white band. The viral band was collected using a large lumen pipette tip.

The above virus preparations used commercially available columns and cross-linked dextran medium (PD-10 columns pre-packed with Sephadex® G-25M; Pharmacia, Piscataway, NJ). Desalted by gel filtration. The column was equilibrated with 20 ml PBS. Load the virus and allow the virus to flow through the column. 5 ml PBS was added to the column and 8-10 drops of fractions were collected. The optical density of the 1:50 dilution of each fraction was measured spectrophotometrically at 260 nm.

A clear absorbance peak was present between fractions 7-12. These fractions were collected and the optical density (OD) of the 1:25 dilution was measured. Virus concentrations were determined by the following formula: (OD at 250nm) (25) (1.1 × 10 12) = virions / ml. zalpha29 r
The OD of the 1:25 dilution of AdV was 0.059, resulting in a virus concentration of 3.3 × 10 ¹² virions / ml. To preserve the virus, glycerin was added to the purified virus to a final concentration of 15%, gently but effectively mixed and aliquoted and stored at -80 ° C.

Quantum Biotechnologies Ink.
c.) (In Montreal, Canada) The infectivity of the recombinant virus was measured according to the protocol developed. Briefly, two 96-well tissue culture plates were seeded with 1 × 10 ⁴ 293A cells per well in MEM containing 2% fetal bovine serum for each recombinant virus to be assayed. did. After 24 hours, a 10-fold dilution of each virus from 1 × 10 ⁻² to 1 × 10 ⁻¹⁴ was made in MEM containing 2% fetal bovine serum. Each dilution 1
00 μl was placed in each of 20 wells. After 5 days at 37 ° C, the wells were read as positive or negative for CPE and plaque forming unit / ml (PFU) values were calculated.

The TCID ₅₀ used was formulated by Quantum Biotechnology, Inc.
According to. Titers (T) were measured from plates in which the virus used was diluted from 10 ⁻² to 10 ⁻¹⁴ and read 5 days after infection. At each dilution, the ratio (R) of CPE positive wells to the total number of wells was measured. To calculate the titer of undiluted virus sample: Factor, "F" = 1 + d (S-0.5);
Where "S" is the sum of the ratios (R); and "d" is Log10 of the dilution series, eg "d" is equal to 1 in the 10-fold dilution series. T = 1 for undiluted sample
0 ^{(1 + F)} = TCID ₅₀ / ml. To convert TCID ₅₀ / ml to pfu / ml, titer (T)
Subtract 0.7 from the exponent in the calculation of.

The zalpha29 adenovirus had a titer of 1.3 × 10 ¹⁰ pfu / ml. From the foregoing, it will be appreciated that while particular embodiments of the invention have been described herein for purposes of illustration, various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the following claims.

[Sequence list]

[Brief description of drawings]

FIG. 1A is a Hopp / Woods hydrophilicity profile of the amino acid sequence set forth in SEQ ID NO: 2. The profile is based on a sliding 6 residue window. Embedded 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. 1B is the Hopp / Woods hydrophilicity profile of the amino acid sequence set forth in SEQ ID NO: 2. The profile is based on a sliding 6 residue window. Embedded 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. 1C is a Hopp / Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO: 2. The profile is based on a sliding 6 residue window. Embedded 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. 1D is a Hopp / Woods hydrophilicity profile of the amino acid sequence set forth in SEQ ID NO: 2. The profile is based on a sliding 6 residue window. Embedded 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 typical human (SEQ ID NO: 2) and mouse (SEQ ID NO: 4) zalpha29 amino acid sequences.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12P 21/02 C 5/10 C12Q 1/02 C12P 21/02 C12N 15/00 ZNAA C12Q 1 / 02 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD) , SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU , ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72 ) Inventor Gao, Zelen United States, Washington 98052, Redmond, One Hundred Seventeenth Place Northeast 9502 # 3 F Term (reference) 4B024 AA01 AA11 BA21 BA44 CA04 DA02 DA06 EA04 FA02 FA18 GA11 HA01 HA12 HA15 4B063 QA01 QA18 QA19 QA19 QA19 QA19 QA19 QA19 QQ QR77 QR80 QS24 QS28 4B064 AG02 AG27 AG31 CA02 CA10 CA19 CC24 DA01 DA13 4B065 AA93X AA93Y AB01 AC14 BA02 CA24 CA44 CA46 4H045 AA10 AA11 AA20 BA10 BA41 CA40 DA01 DA76 DA86 EA22 EA27 EA28 EA50 FA74

Claims (16)

[Claims] 1. Residues 48 to 62 of SEQ ID NO: 2, residues 47 to 61 of SEQ ID NO: 4, residues 63 to 104 of SEQ ID NO: 2, residues 62 to 103 of SEQ ID NO: 4, the rest of SEQ ID NO: 2. Groups 105-119, residues 104-118 of SEQ ID NO: 4, residues 120-137 of SEQ ID NO: 2, residues 119-1 of SEQ ID NO: 4
36, residues 138 to 152 of SEQ ID NO: 2, residues 137 to 151 of SEQ ID NO: 4, residues of SEQ ID NO: 2
153 to 170, residues 152 to 169 of SEQ ID NO: 4, residues 171-185 of SEQ ID NO: 2, and residues 170 to 184 of SEQ ID NO: 4 containing a sequence of amino acid residues selected from the group consisting of: Separated polypeptide. 2. The isolated polypeptide of claim 1, which is 15 to 1500 amino acid residues long. 3. The isolated polypeptide of claim 2, wherein the sequence of amino acid residues is shown in maltose binding protein, immunoglobulin constant region, polyhistidine tag, and SEQ ID NO: 5. An isolated polypeptide operably linked to a second polypeptide selected from the group consisting of such peptides via a peptide bond or a polypeptide linker. 4. The isolated polypeptide of claim 1, containing at least 30 consecutive residues of SEQ ID NO: 2 or SEQ ID NO: 4. 5. The isolated peptide of claim 1, containing residues 48-185 of SEQ ID NO: 6 or residues 27-190 of SEQ ID NO: 6. 6. Containing 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. Item 2. The isolated peptide according to Item 1. 7. The following operably linked elements: transcription promoter; residues 48-62 of SEQ ID NO: 2, residues 47-61 of SEQ ID NO: 4, residues 63-10 of SEQ ID NO: 2.
4, residues 62-103 of SEQ ID NO: 4, residues 105-119 of SEQ ID NO: 2, residue 1 of SEQ ID NO: 4
04-118, residues 120-137 of SEQ ID NO: 2, residues 119-136 of SEQ ID NO: 4, SEQ ID NO: 2
Residues 138-152, SEQ ID NO: 4 residues 137-151, SEQ ID NO: 2 residues 153-170, SEQ ID NO: 4 residues 152-169, SEQ ID NO: 2 residues 171-185, and SEQ ID NO: Residue 4 of 17
An expression vector comprising a DNA segment encoding a polypeptide containing a sequence of amino acid residues selected from the group consisting of 0 to 184; and a transcription terminator. 8. The expression vector of claim 7, wherein the DNA segment comprises nucleotides 79-570 of SEQ ID NO: 7. 9. An expression vector according to claim 7, wherein the polypeptide comprises residues 48-185 of SEQ ID NO: 6 or residues 27-190 of SEQ ID NO: 6. 10. The expression vector of claim 7, wherein the polypeptide is residues 48-185 of SEQ ID NO: 2, residues 47-184 of SEQ ID NO: 4, residues 27 of SEQ ID NO: 2. ~ 190, or residues 26-188 of SEQ ID NO: 4, an expression vector. 11. An expression vector according to claim 7, which contains a secretory signal sequence operably linked to the DNA segment. 12. A cultured cell into which the expression vector according to any one of claims 7 to 11 has been introduced, wherein the cell expresses a DNA segment. 13. A method of producing a polypeptide comprising the steps of: culturing the cell of claim 12 under conditions in which the DNA segment is expressed and the polypeptide is produced; and Recovering the peptide. 14. A polypeptide produced by the method of claim 13. 15. An antibody that specifically binds to the polypeptide of claim 14. 16. A method of detecting the presence of an antagonist of zalpha29 activity in a test sample comprising the steps of: culturing cells responsive to zalpha29; in the presence and absence of the test sample. exposing the cells to the zalpha29 polypeptide; comparing the level of response to the zalpha29 polypeptide by a biological or biochemical assay in the presence and absence of the test sample; determining the presence of an antagonist of zalpha29 activity.