+

WO1997035028A1 - Cytostatine iii - Google Patents

Cytostatine iii Download PDF

Info

Publication number
WO1997035028A1
WO1997035028A1 PCT/US1996/003697 US9603697W WO9735028A1 WO 1997035028 A1 WO1997035028 A1 WO 1997035028A1 US 9603697 W US9603697 W US 9603697W WO 9735028 A1 WO9735028 A1 WO 9735028A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
cells
dna
iii
Prior art date
Application number
PCT/US1996/003697
Other languages
English (en)
Inventor
Jian Ni
Guo-Liang Yu
Reiner L. Gentz
Original Assignee
Human Genome Sciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to PCT/US1996/003697 priority Critical patent/WO1997035028A1/fr
Priority to AU53659/96A priority patent/AU5365996A/en
Publication of WO1997035028A1 publication Critical patent/WO1997035028A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • CYTOSTATIN III This invention relates, in part, to newly identified polynucleotides and polypeptides; varxants and derxvatives of the polynucleotxdes and polypeptides,- processes for making the polynucleotides and the polypeptxdes, and thexr varxants and derxvatxves; agonxsts and antagonists of the polypeptides; and uses of the polynucleotides, polypeptides, varxants, derxvatives, agonxsts and antagonxsts.
  • the invention relates to polynucleotides and polypeptides of human Cytostatxn III.
  • the growth and differentia ion of cells and the development of tissues and glands is controlled by autocnne and paracrxne actors, such as systemic hormones and factors that modulate or mediate the action of hormones, such as growth factors, which themselves may be hormones.
  • peptides that locally signal growth cessation and stimulate differentiatxon of cells of the developxng epithelium are very important to mammary gland development. These factors largely have not been identified or characterized, particularly not in humans.
  • mammary-derived growth inhibitor CMDGI mammary-derived growth inhibitor
  • GENES GENES
  • ONCOGENES GENES
  • HORMONES ADVANCES IN CELLULAR AND MOLECULAR BIOLOGY OF BREAST CANCER, Dickson et al., Eds., Kluwer Academic Publishers, Boston (1991)
  • MDGI was first identified in milk and mammary glands of cows. Subsequently, it was identified in mice. In mice and cows, at least, MDGI has been shown to inhibit epithelial cell growth and stimulate epithelial cell differentiatio .
  • MDGI occurs in at least two forms produced by alternative routes of post-translational processing.
  • the original form is referred to as MDGI and the second form is called MDGI-2.
  • MDGI is associated primarily with milk fat globule membranes ("MFGM”) , as assessed by immunological assays using anti-MDGI antibodies. Similar time course studies show that MDGI increases dramatically in mammary glands when lactation begins, following delivery. MDGI-2 differs from MDGI in this respect. It is found in mammary glands during pregnancy but not during lactation. (Grosse et al. cited above and Kurtz et al. J. Cell. Biol. 110: 1779-1789 (1990))
  • Mouse and bovine MDGI are homologous to one another and to a family of low molecular mass hydrophobic ligand-binding proteins ("low MW HLBP(s) n ), which includes fatty acid-binding proteins (n FABP(s) n ) from brain, heart, liver and intestine, myelin P2 protein, the differentiation associated protein of adipocytes called p422 gastrotropin and cellular retinoic acid-binding protein (“CRABP”) .
  • CRABP fatty acid-binding proteins
  • the homology of MDGI to the low MW HLBPs raises the possibility that MDGI, at least as part of its function, binds a hydrophobic ligand, and that binding to this ligand is important to the mechanism(s) by which MDGI inhibits cell growth and stimulates differentiation. It should be noted, however, that the other low MW HLBPs, except gastrotropin, act intracellularly, whereas MDGI acts extracellularly, at least in vitro. (Yang et al., J. Cell Biol. 127: 1097-1109 (1994).
  • MDGI fatty acid binding proteins
  • FBP fatty acid binding proteins
  • Heart FABP like MDGI, whether produced from natural sources or by expression of a cloned gene in a heterologous host, inhibits growth of normal mammary epithelial cells ("MEC") of mouse origin. In addition, it stimulates milk protein synthesis and it stimulates its own expression in these cells.
  • MEC mammary epithelial cells
  • bovine MDGI does not bind fatty acids, although the two proteins are 95% homologous and it has been suggested that heart FABP actually may be a form of MDGI.
  • MDGI In vivo MDGI is found in capillary endothelial cells and in the mammary parenchyma, in mice and cows. (See, for instance, Grosse et al. cited above. ) MDGI appears first in the capillary endothelial cells and later in the secretory epithelial cell ⁇ . The location of MDGI in the mar ⁇ mary capillary endothelium is consistent with a role in regulating endothelial cell proliferation.
  • MDGI inhibits L(+) -lactate-, arachidonic acid- and 15-S-hydroxyeicosatetraenoic acid-induced supersensitivity of neonatal rat heart cell ⁇ to beta-adrenergic stimulation.
  • a ⁇ reported by Burton et al., BBRC 205: 1822-1828 (1994) the induced hypersensitivity is mediated by a small population of beta 2-adrenergic receptors and, therefore, it has been suggested that MDGI interferes with the normal function of these receptors. Interaction with these receptors might also be part of the mechanism by which MDGI inhibits cells growth. This activity also raises the possibility that MDGI naturally modulates the beta-adrenergic sensitivity of cardiac myocytes.
  • H-FABP can be a potent inducer of cardiac myotrophy, capable of stimulating protein synthesis and c-jun expression in myocytes, and increasing their surface area.
  • MDGI mammary epithelial cells
  • MDGI The regulatory properties of MDGI can be fully mimicked by an 11-amino acid sequence, which is represented in the carboxyl terminus of MDGI and a subfamily of the low MW HLBPs.
  • MDGF inhibits growth of normal human MEC, passaged for varying lengths of time. (Yang et al. cited above.) It also inhibits growth of the mouse rnammary malignant epithelial cell lines MaCa 20177, the human malignant mammary cell lines MaTu and T47D and it inhibits the resumption of growth of stationary Ehrlich ascites carcinoma cells ("EAC") in vitro. In contrast, MDGF slightly stimulates growth of the human malignant mammary epithelial cell line MCF7. Finally, MDGI promotes differentiation of mouse pluripotent embryonic stem cells.
  • MDGI can function as a potent tumor suppressor gene.
  • Human breast cancer cells transfected with an MDGI expression construct exhibited differentiated morphology, reduced proliferation rate, reduced clonogenicity in soft agar, and reduced tumorgenicity in nude mice.
  • the human homologue of this gene was mapped to chromosome lp33-35, a locus previously shown to exhibit frequent loss of heterozygosity in human breast cancer (about 40% of tumors) .
  • the magnitude of the in vivo and in vitro tumor suppressor activity of MDGI is comparable to that previously observed for BRCA1, p53, Rb, and H19.
  • cytostatins akin to MDGI that modulate growth and differentiation of cells, such as epithelial cells, particularly mammary epithelial cells, are essential to the proper development and health of tissue and organs, such as mammary glands of developing and adult females, particularly human females, and, among other things, can play a role in preventing, ameliorating or correcting dysfunctions or diseases.
  • polypeptides inter alia, that have been identified as novel cytostatins by homology between the amino acid sequence set out in Figure l (SBQ ID NO:2) and known amino acid sequences of other proteins such as MDGI proteins.
  • the polynucleotide comprises the region encoding human Cytostatin III in the sequence set out in Figure l (SEQ ID NO:2) .
  • nucleic acid molecule encoding a mature polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97332.
  • isolated nucleic acid molecules encoding human Cytostatin III, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically, diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives.
  • Cytostatin III polypeptides particularly human Cytostatin III polypeptides, that modulate growth activity of epithelial cells, stimulate milk production in both humans and cows and promote involution of the breast.
  • Cytostatin III novel polypeptides of human origin referred to herein as Cytostatin III as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing.
  • products, compositions and methods for, among other things: assessing Cytostatin III expression in cells by determining Cytostatin III polypeptides or Cytostatin III-encoding mRNA; modulating cell growth in vitro, ex vivo or in vivo by exposing cells to Cytostatin III polypeptides or polynucleotides as disclosed herein; assaying genetic variation and aberrations, such as defects, in Cytostatin III genes; and administering a Cytostatin III polypeptide or polynucleotide to an organism to augment Cytostatin III function or remediate Cytostatin III dysfunction.
  • probes that hybridize to human Cytostatin III sequences.
  • antibodies against Cytostatin III polypeptides there are provided antibodies against Cytostatin III polypeptides.
  • the antibodies are highly selective for human Cytostatin III.
  • Cytostatin III agonists are provided.
  • preferred agonists are molecules that mimic Cytostatin III, that bind to Cytostatin III-binding molecules or receptor molecules, and that elicit or augment Cytostatin III-induced responses.
  • Cytostatin III antagonists are those which mimic Cytostatin III so as to bind to Cytostatin III receptor or binding molecules but not elicit a Cytostatin III-induced response or more than one Cytostatin III-induced response. Also among preferred antagonists are molecules that bind to or interact with Cytostatin III so as to inhibit an effect of Cytostatin III or more than one effect of Cytostatin III or which prevent expression of Cytostatin III.
  • the agonists and antagonists may be used to mimic, augment or inhibit the action of Cytostatin III polypeptides. They may be used, for instance, for purposes relating to growth of cells in vitro or for purposes relating to treatment of disorders associated with aberrant growth of cells a fected by cytostatins, particularly Cytostatin III.
  • compositions comprising a Cytostatin III polynucleotide or a Cytostatin III polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism.
  • the compositions comprise a Cytostatin III polynucleotide for expres ⁇ ion of a Cytostatin III polypeptide in a host organism for treatment of disease.
  • Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of Cytostatin III.
  • Figure 1 shows the nucleotide and deduced amino acid ⁇ equence of human Cytostatin III.
  • Figure 2 shows the regions of similarity between amino acid sequences of cytostatin and MDGI polypeptides (SEQ ID NO:11-15) .
  • Figure 3 shows structural and functional features of Cytostatin III deduced by the indicated techniques, as a function of amino acid sequence.
  • DIGESTION of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes referred to herein are commercially available and their reaction conditions, cofactors and other requirements for use are known and routine to the skilled artisan.
  • plasmid or DNA fragment is digested with about 2 units of enzyme in about 20 ⁇ l of reaction buffer.
  • isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in proportionately larger volumes.
  • Incubation times of about 1 hour at 37"C are ordinarily used, but conditions may vary in accordance with standard procedures, the supplier's instructions and the particulars of the reaction.
  • reactions may be analyzed, and fragments may be purified by electrophoresis through an agarose or polyacrylamide gel, using well known methods that are routine for those skilled in the art.
  • GENETIC ELEMENT generally means a polynucleotide comprising a region that encodes a polypeptide or a region that regulates transcription or translation or other processes important to expression of the polypeptide in a host cell, or a polynucleotide comprising both a region that encodes a polypeptide and a region operably linked thereto that regulates expression.
  • Genetic elements may be comprised within a vector that replicates as an episomal element; that is, as a molecule physically independent of the host cell genome. They may be comprised within mini-chromosomes, such as those that arise during amplification of transfected DNA by methotrexate selection in eukaryotic cells. Genetic element ⁇ al ⁇ o may be compri ⁇ ed within a ho ⁇ t cell genome,- not in their natural state but, rather, following manipulation such as isolation, cloning and introduction into a host cell in the form of purified DNA or in a vector, among others.
  • ISOLATED means altered from "by the hand of man” from its natural state,- i.e., that, if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a naturally occurring polynucleotide or a polypeptide naturally present in a living animal in its natural state is not “isolated, " but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.
  • isolated means that it is separated from the chromosome and cell in which it naturally occurs.
  • polynucleotides can be joined to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance.
  • the isolated polynucleotides, alone or joined to other polynucleotides such as vectors, can be introduced into ho ⁇ t cell ⁇ , in culture or in whole organisms. Introduced into host cells in culture or in whole organisms, such DNAs still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment.
  • polynucleotides and polypeptides may occur in a composition, such as a media formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein.
  • LIGATION refers to the process of forming phosphodiester bonds between two or more polynucleotides, which most often are double stranded DNAs.
  • OLIGONUCLEOTIDE(S) refers to relatively short polynucleotides. Often the term refers to single-stranded deoxyribonucleotides, but it can refer as well to single-or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs, among others.
  • Oligonucleotides such as single-stranded DNA probe oligonucleotides, often are synthesized by chemical methods, such as those implemented on automated oligonucleotide synthesizers. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
  • oligonucleotides typically are obtained without a 5' phosphate.
  • the 5' ends of such oligonucleotides are not substrates for phosphodiester bond formation by ligation reactions that employ DNA ligases typically used to form recombinant DNA molecules.
  • a phosphate can be added by standard techniques, such as those that employ a kinase and ATP.
  • the 3' end of a chemically synthesized oligonucleotide generally has a free hydroxyl group and, in the presence of a ligase, such as T4 DNA ligase, readily will form a phosphodiester bond with a 5' phosphate of another polynucleotide, such as another oligonucleotide.
  • a ligase such as T4 DNA ligase
  • this reaction can be prevented selectively, where desired, by removing the 5' phosphates of the' other polynucleotide(s) prior to ligation.
  • PLASMIDS generally are designated herein by a lower case p preceded and/or followed by capital letters and/or numbers, in accordance with standard naming conventions that are familiar to those of skill in the art.
  • plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well known, published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of ⁇ uch plasmids, as well a ⁇ other vectors, in the present invention will be readily apparent to those of skill from the present disclo ⁇ ure.
  • POLYNUCLEOTIDE(S) generally refer ⁇ to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thu ⁇ , for instance, polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as ino ⁇ ine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein.
  • polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.
  • POLYPEPTIDES as used herein, includes all polypeptides a ⁇ described below. The basic structure of polypeptides is well known and has been described in innumerable textbooks and other publications in the art.
  • the term is used herein to refer to any peptide or protein comprising two or more amino acids joined to each other in a linear chain by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • polypeptides as is well known and as the term is used herein, generally are formed of the 20 naturally occurring amino acids, and that the amino acids in a polypeptide generally are joined to one another in a linear chain by peptide bonds between the alpha carboxyl and the alpha amino groups of adjacent, succeeding amino acids.
  • the sequence of amino acids in a chain usually, but not always, is written beginning (on the left and at the top) with the amino acid having a free alpha amino group. This amino acid is taken as the amino terminus of the polypeptide, also referred to as the N-terminus.
  • Bach successive amino acid then is listed in turn, ending with the amino acid having a free carboxyl group (at bottom and right) , which is taken as the carboxyl terminus of the polypeptide, also called the C-terminus.
  • amino acid residues Individual amino acids in a polypeptide commonly are referred to as amino acid residues, and as residues. Generally, the amino acids in a polypeptide are numbered beginning with the amino terminus and proceeding integer by integer and residue by residue to the carboxyl terminus. However, for polypeptides that first are synthesized in cells as precursors to a mature form, it also is common to begin numbering amino acids with the first residue of the mature form. Then, the upstream residues (i.e., those closer to the N-terminus) are assigned negative numbers counting back from residue one (the N-terminus of the mature form) to the N-terminus of the earliest precursor form. Other numbering scheme ⁇ also have been employed, but less commonly.
  • polypeptides often contain amino acid ⁇ other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acid ⁇ , and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural proceases, such as processing and other post ⁇ translational modifications, but also by chemical modification techniques which are well known to the art. Even the common modifications that occur naturally in polypeptides are too numerous to list exhaustively here, but they are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.
  • polypeptides of the present are, to name an illustrative few, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylino ⁇ itol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as
  • polypeptides are not always entirely linear.
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural proce ⁇ sing event and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification is common in naturally occurring and synthetic polypeptides and such modifications may be pre ⁇ ent in polypeptides of the present invention, as well.
  • the amino terminal residue of polypeptides made in B. coli, prior to proteolytic processing almost invariably will be N-formylmethionine.
  • polypeptides made by expressing a cloned gene in a host for instance, the nature and extent of the modifications in large part will be determined by the host cell posttranslational modification capacity and the modification signals present in the polypeptide amino acid sequence.
  • glycosylation often does not occur in bacterial hosts such as B. coli. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell.
  • Insect cell often carry out the same posttranslational glycosylations as mammalian cells and, for' this reason, insect cell expression systems have been developed to express efficiently ma ⁇ malian proteins having native patterns of glycosylation, inter alia. Similar considerations apply to other modifications.
  • polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized by expres ⁇ ing a polynucleotide in a host cell.
  • VARIANT(S) of polynucleotides or polypeptides are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively. Variants in this sense are described below and elsewhere in the present disclosure in greater detail.
  • changes in the nucleotide sequence of the variant may be silent. That is, they may not alter the amino acids encoded by the polynucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptide with the same amino acid sequence as the reference. Also as noted below, changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleotide changes may re ⁇ ult in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination.
  • substitutions are those that vary from a reference by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative sub ⁇ titutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and lie; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
  • RECEPTOR MOLECULE refers to molecules which bind or interact specifically with Cytostatin III polypeptides of the present invention, including not only classic receptors, which are preferred, but also other molecules that specifically bind to or interact with polypeptides of the invention (which al ⁇ o may be referred to as “binding molecules” and “interaction molecules,” respectively and as “Cytostatin III binding molecules” and “Cytostatin III interaction molecules.” Binding between polypeptides of the invention and such molecules, including receptor or binding or interaction molecules may be exclusive to polypeptides of the invention, which is very highly preferred, or it may be highly specific for polypeptides of the invention, which is highly preferred, or it may be highly specific to a group of proteins that includes polypeptides of the invention, which is preferred, or it may be ⁇ pecific to several groups of proteins at least one of which includes polypeptides of the invention.
  • Such molecules generally are proteins, which may be single or multichain proteins and multisubunit or multiprotein complexe ⁇ , such as those of classic cell surface receptors, which are highly preferred in the invention.
  • Receptor molecules also may be non ⁇ protein molecules that bind to or interact specifically with polypeptides of the invention.
  • Such molecules may occur in membranes, such as classic cell surface receptors, or they may occur intracellularly, in the cytosol, inside organelles, or in the surface of organelles, for instance.
  • membrane bound receptors particularly cell membrane receptors, especially cell surface receptors.
  • preferred receptors are those that occur in the membranes of organelles, particularly nuclear membrane receptors and mitochondrial membrane receptors.
  • Receptors also may be non-naturally occurring, such as antibodies and antibody-derived reagents that bind specifically to polypeptides of the invention. DESCRIPTION OF THE INVENTION
  • the present invention relates to novel Cytostatin III polypeptides and polynucleotides, among other things, as described in greater detail below.
  • the invention relates to polypeptides and polynucleotides of a novel human Cytostatin III, which is related by amino acid sequence homology to the mammary derived growth inhibitor ("MDGF") found in cows and mice.
  • MDGF mammary derived growth inhibitor
  • the invention relates especially to Cytostatin III having the nucleotide and amino acid sequences set out in Figure 1 (SEQ ID NO:l and 2), and to the Cytostatin III nucleotide and amino acid sequences of the cDNA in ATCC Deposit No.
  • a polynucleotide of the present invention encoding human Cytostatin III polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA from cell ⁇ of a breast lymph node a ⁇ starting material.
  • standard cloning and screening procedures such as those for cloning cDNAs using mRNA from cell ⁇ of a breast lymph node a ⁇ starting material.
  • the polynucleotide set out in Figure 1 (SEQ ID NO:l) was discovered in a cDNA library derived from cells of a human breast lymph node.
  • Human Cytostatin III of the invention is structurally related to other proteins of the cytostatin family of growth modulating factors, as shown by the results of sequencing the cDNA encoding human Cytostatin III in the deposited clone.
  • the human cDNA sequence thu ⁇ obtained is set out in Figure 1 (SEQ ID NO:l) . It contains an open reading frame encoding a protein of about 135 amino acid residues with a deduced molecular weight of about 15.9 kDa. The protein exhibits greatest homology to mouse mammary- derived growth inhibitor CMDGI") , among known proteins.
  • the first 133 residues of the Cytostatin III of Figure l (SEQ ID NO:2) have about 33% identity and about 62% similarity with the amino acid sequence of mouse MDGI.
  • Polynucleotides of the present invention may be in the form of RNA, such a ⁇ mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA may be the coding strand, also known as the sen ⁇ e strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
  • a polynucleotide of the present invention may a naturally occurring sequence, such as that of a naturally occurring allelic variant, or it may have a sequence that does not occur in nature, such as a sequence that has been produced, for instance, by in vitro mutagenesis techniques.
  • the coding sequence which encodes the polypeptide may be identical to the coding sequence of the polynucleotide shown in Figure 1 (SEQ ID NO:l) . It also may be a polynucleotide with a different sequence, which, as a result of the redundancy (degeneracy) of the genetic code, encodes the polypeptide of the DNA of Figure 1 (SEQ ID NO:l) .
  • Polynucleotides of the present invention which encode the polypeptide of Figure 1 may include, but are not limited to the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequence ⁇ , together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing - including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA.
  • polynucleotide encoding a polypeptide encompasses polynucleotides which include a sequence encoding a polypeptide of the present invention, particularly the human Cytostatin III having the amino acid ⁇ equence ⁇ et out in Figure 1 (SEQ ID NO:2) .
  • the term encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide, together with additional regions, that also may contain coding and/or non-coding sequences.
  • the present invention further relates to variants of the herein above described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) .
  • a variant of the polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.
  • Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.
  • the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NO:2) . Further, the invention includes variants of such polynucleotides that encode a fragment, derivative or analog of the polypeptide of Figure l (SEQ ID NO:2) . Among variants in this regard are variants that differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non- conservative amino acid substitutions, deletions or additions.
  • Variants of the invention may have a sequence that occurs in nature or they may have a sequence that does not occur naturally.
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID NO:l) .
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides.
  • polypeptides having the amino acid sequence of Cytostatin III set out in Figure l SEQ ID NO:2
  • polynucleotides encoding Cytostatin III variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments which have the amino acid sequence of the Cytostatin III polypeptide of Figure 1 (SEQ ID NO:2) in which several, a few, 5 to 10, l to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination.
  • silent substitutions, additions and deletions which do not alter the propertie ⁇ and activities of the Cytostatin III.
  • conservative substitutions are also especially preferred in this regard.
  • polynucleotides encoding a polypeptide having the amino acid sequence of the Cytostatin III set out in Figure 1 (SEQ ID NO:2) .
  • the polynucleotide may encode the polypeptide in a continuous region or in a plurality of two or more discontinuous exons, and it may comprise additional regions ae well, which are unrelated to the coding region or region ⁇ .
  • polynucleotides that comprise a region that is more than 85% identical to the Cytostatin III-encoding portion of the polynucleotide set out in Figure 1 (SEQ ID NO:l) .
  • polynucleotides that comprise a region that is more than 85% identical to the Cytostatin Ill-encoding portion of the cDNA the deposited clone.
  • those more than 90% identical to the same are particularly preferred, and, among these particularly preferred polynucleotides, those with 95% or more identity are especially preferred.
  • those with 97% or more identity are highly preferred among those with 95% or more identity, and among these those with 98% or more and 99% or more identity are particularly highly preferred, with 99% or more being the more preferred of these.
  • the present invention also includes polynucleotides in which the sequence encoding the mature polypeptide is fused in the same reading frame to additional sequences.
  • sequences include signal sequences, which facilitate transport of the nascent protein into the endoplasmic reticulum, pro-sequences that are associated with inactive precursor forms of the polypeptide, which may facilitate trafficking of the protein in a cell or out of a cell or may improve persistence of the protein in a cell or in an extracellular compartment.
  • sequences also may be added to facilitate production and purification, or to add additional functional domains, as discussed elsewhere herein.
  • polynucleotides of the invention may encode, in addition to a mature cyto ⁇ tatin, particularly Cytostatin III, for example, a leader ⁇ equence, such as a signal peptide which functions as a secretory sequence for controlling transport of the polypeptide into the lumen of the endoplasmic reticulum.
  • a leader ⁇ equence such as a signal peptide which functions as a secretory sequence for controlling transport of the polypeptide into the lumen of the endoplasmic reticulum.
  • the leader sequence may be removed by the host cell, as is generally the case for signal peptides, yielding another precursor protein or the mature polypeptide.
  • a precursor protein having a leader sequence often is called a preprotein.
  • a polynucleotide of the present invention may encode a mature or precursor pre-, pro- or prepropolypeptide as discus ⁇ ed above, among others, fused to additional amino acids, such as those which provide additional functionalities.
  • the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide.
  • the marker sequence is a hexa-histidine peptide, such as the tag provided in the vector pQB-9, among others, many of which are commercially available. As described in Gentz et al. , Proc. Natl. Acad.
  • hexa-histidine provides for convenient purification of the fusion protein. Typically, it does not adversely affect protein structure or function, and it binds efficiently, selectively and tightly to metal chelate resins, particularly nickel chelate resins.
  • metal chelate resins particularly nickel chelate resins.
  • hexa-histidine tags often bind especially well to nickel-NTA resin, which is well known and readily available and can be obtained commercially from, for instance, Qiagen.
  • the histidine-metal interaction not only is stable to a variety of conditions useful to remove non- specifically bound material, but also the fusion polypeptide can be bound and removed under mild, non-denaturing conditions.
  • the hexa-histidine tag can be fused most conveniently to the amino or the carboxyl terminus of the cyto ⁇ tatin polypeptide.
  • a tag of the hexa-histidine type is particularly useful for bacterial expression.
  • HA hemagglutinin
  • Another useful marker sequence in certain other preferred embodiments is a hemagglutinin ("HA") tag, particularly when a mammalian cell is used for expression; e.g., COS-7 cells.
  • the HA tag corresponds to an epitope derived of influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37: 767 (1984) , for instance.
  • the present invention further relates to polynucleotides that hybridize to the herein above-described cytostatin sequences, particularly Cytostatin III sequences.
  • polynucleotides that have at least 50% identity to the sequences described herein above.
  • sequences that have at least 70% identity.
  • the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • a probe as discu ⁇ ed above derived from the full length Cytostatin III cDNA, including the entire Cytostatin III cDNA of Figure 1 (SEQ ID NO:l) or of the deposited clone, or the coding region of thereof, or any part thereof useful as a probe, may be used as a hybridization probe for cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding Cytostatin III and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the human Cytostatin III gene.
  • Such probes generally will comprise at least 20 bases.
  • such probes will have at least 30 bases.
  • Particularly preferred probes will have at least 30 bases and will have 50 bases or less.
  • the coding region of the Cytostatin III gene may be isolated by screening using the known DNA sequence to synthesize an oligonucleotide probe. Labeled an oligonucleotide having a sequence complementary to that of a gene of the present invention then is used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease, as further discussed herein relating to polynucleotide assays, inter alia.
  • the polynucleotides also may encode a polypeptide which is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance) .
  • sequence ⁇ may play a role in processing of a protein from precursor to a mature form, may facilitate protein trafficking, may prolong or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things.
  • the additional amino acids may be processed away from the mature protein by cellular enzymes.
  • a precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide.
  • prosequence ⁇ When prosequence ⁇ are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
  • a polynucleotide of the present invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein) , a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
  • a leader sequence which may be referred to as a preprotein
  • a preproprotein which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
  • the cDNA deposit is referred to herein as "the deposited clone” or as "the cDNA of the depo ⁇ ited clone.”
  • the deposited clone was deposited with the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, USA, on November 6, 1995 and assigned ATCC Deposit No. 97332.
  • the deposited material is a pBluescript SK (-) plasmid (Stratagene, La Jolla, CA) that contains the full length human Cytostatin III cDNA.
  • sequence of the polynucleotides contained in the deposited material, as well as the amino acid sequence of the polypeptide encoded thereby, are controlling in the event of any conflict with any description of sequences herein.
  • a license may be required to make, use or sell the deposited materials, and no such license is hereby granted.
  • the present invention further relates to a human Cytostatin III polypeptide which has the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or which has the amino acid sequence encoded by the depo ⁇ ited clone.
  • the invention also relates to fragments, analogs and derivatives of these polypeptides.
  • fragment when referring to the polypeptide of Figure 1 (SEQ ID NO:2) or that encoded by the deposited cDNA, means a polypeptide which retains essentially the same biological function or activity as such polypeptide.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In certain preferred embodiments it is a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO:2) or that encoded by the cDNA in the deposited clone may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the
  • particularly preferred embodiments of the invention in this regard are polypeptides having the amino acid sequence of Cytostatin III set out in Figure 1 (SEQ ID NO:2), variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments.
  • particularly preferred embodiments of the invention in this regard are polypeptides having the amino acid sequence of the Cytostatin III of the cDNA in the deposited clone, variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments.
  • variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments having the amino acid sequence of the Cytostatin III polypeptide of Figure 1 (SEQ ID NO:2) or of the cDNA in the deposited clone, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are sub ⁇ tituted, deleted or added, in any combination.
  • silent substitutions, additions and deletions which do not alter the properties and activities of the Cytostatin III.
  • conservative substitutions are also especially preferred in this regard.
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material has been altered from its natural state; e.g., that, if it occurs in nature, it has been removed from its original environment.
  • a naturally occurring polynucleotide or polypeptide naturally present in a living animal in its natural state is not “isolated, " but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural ⁇ y ⁇ tem is "isolated", as the term is employed herein.
  • polynucleotides can be joined to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expres ⁇ ion in a host, for instance.
  • the isolated polynucleotides, alone or joined to other polynucleotides such as vectors, can be introduced into ho ⁇ t cells, in culture or in whole organisms. Introduced into host cells in culture or in whole organisms, such DNAs still would be i ⁇ olated, as the term is used herein, because they would not be in their naturally occurring form or environment.
  • polynucleotides and polypeptides may occur in a composition, such as a media formulation, a solution for introduction into cells, a composition or solution for chemical or enzymatic reaction, and the like, which are not naturally compositions, and therein remain isolated polynucleotides or polypeptides within the meaning of that term as it i ⁇ employed herein.
  • polypeptides comprising fragments of Cyto ⁇ tatin III, mo ⁇ t particularly fragments of the Cytostatin III having the amino acid set out in Figure 1 (SEQ ID NO:2) , or having the amino acid sequence of the Cytostatin III of the deposited clone, and fragments of variants and derivatives of the Cytostatin III of Figure 1 (SEQ ID NO:2) or of the deposited clone.
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned Cytostatin III polypeptides and variants or derivatives thereof.
  • fragments may be "free-standing,” i.e., not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region.
  • the presently discussed fragments mo ⁇ t preferably form a single continuous region.
  • several fragments may be comprised within a single larger polypeptide.
  • certain preferred embodiments relate to a fragment of a Cytostatin III polypeptide of the present comprised within a precursor polypeptide designed for expression in a host and having heterologous pre and pro- polypeptide regions fused to the amino terminus of the Cytostatin III fragment and an additional region fused to the carboxyl terminus of the fragment. Therefore, fragments in one aspect of the meaning intended herein, refers to the portion or portions of a fusion polypeptide or fusion protein derived from Cytostatin III.
  • Cytostatin III preferred fragments of Cytostatin III are fragments about 5-15, 10-20, 15-40, 25-50, 35-60, 50-75, 65-80, 65-90, 65- 100, 50-100, 75-100, 90-115, 80-135, 90-130, 100-125 and 110-135 amino acids long.
  • 65-90 amino acids in this context means a polypeptide fragment of 65, 65 plus or minus several, a few, 5, 4, 3, 2 or l amino acid to 90 or 90 plus or minus several a few, 5, 4, 3, 2 or 1 amino acid residues, i.e., ranges a broad as 65 minus several amino acids to 90 plus several amino a"ids to as narrow as 65 plus several amino acids to 90 minus several amino acids.
  • the recited ranges plus or minus as many as 5 amino acids at either or at both extremes are particularly highly preferred.
  • the recited ranges means plus or minus as many a ⁇ 3 amino acids at either or at both extremes.
  • ranges plus or minus 1 amino acid at either or at both extremes are fragments 5-15, 10-20, 15-40, 25-50, 35- 60, 50-75, 65-80, 65-90, 65-100, 50-100, 75-100, 90-115, 80-135, 90-130, 100-125 and 110-135 amino acids in length are preferred.
  • Truncation mutants include Cytostatin III polypeptides having the amino acid sequence of Figure l (SEQ ID NO:2) , or of the deposited clone, or of variants or derivatives thereof, except for deletion of a continuous series of residues (that is, a continuous region, part or portion) that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or, as in double truncation mutants, deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Fragments having the size ranges set out about also are preferred embodiments of truncation fragments, which are especially preferred among fragments generally.
  • fragments characterized by structural or functional attributes of Cytostatin III are fragments characterized by structural or functional attributes of Cytostatin III.
  • Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions") , beta-sheet and beta-sheet-forming regions ("beta-regions”) , turn and turn-forming regions (“turn-regions”) , coil and coil-forming regions ("coil-regions”) , hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic region ⁇ , flexible regions, surface-forming regions and high antigenic index regions of Cytostatin III.
  • Certain preferred regions in these regards are set out in Figure 3, and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figure 1 (SEQ ID NO:2) .
  • such preferred regions include Gamier-Robson alpha-regions, beta-regions, turn-regions and coil-regions, Chou-Fasman alpha- regions, beta-regions and turn-regions, Kyte-Doolittle hydrophilic regions and hydrophilic regions, Eisenberg alpha and beta amphipathic regions, Karplus-Schulz flexible regions, Emini surface-forming region ⁇ and Jameson-Wolf high antigenic index regions.
  • fragments in this regard are those that comprise regions of Cytostatin III that combine several structural features, such as several of the features set out above.
  • the regions defined by the residues about 10 to about 20, about 40 to about 50, about 70 to about 90 and about 100 to about 110 of Figure 1 (SEQ ID NO:2) which all are characterized by amino acid compositions highly characteristic of turn-regions, hydrophilic regions, flexible-regions, surface-forming regions, and high antigenic index-regions, are especially highly preferred regions.
  • Such regions may be comprised within a larger polypeptide or may be by themselves a preferred fragment of the present invention, as discussed above. It will be appreciated that the term "about” as used in this paragraph ha ⁇ the meaning ⁇ et out above regarding fragment ⁇ in general.
  • Further preferred regions are those that mediate activities of Cytostatin III.
  • Mo ⁇ t highly preferred in this regard are fragments that have a chemical, biological or other activity of Cytostatin III, including those with a similar activity or an improved activity, or with a decreased undesirable activity.
  • Highly preferred in this regard are fragments that contain regions that are homologs in sequence, or in position, or in both sequence and to active regions of related polypeptides, such as the related polypeptides set out in Figure 2 (SEQ ID NO: - ) , which include Mouse MDGI, Bovine MDGI, Human MDGI, Cytostatin I and Cytostatin II.
  • truncation mutants as discussed above.
  • the invention also relates to, among others, polynucleotides encoding the aforementioned fragments, polynucleotides that hybridize to polynucleotides encoding the fragments, particularly those that hybridize under stringent conditions, and polynucleotides, such as PCR primers, for amplifying polynucleotides that encode the fragments.
  • preferred polynucleotides are those that correspondent to the preferred fragments, as discussed above.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells can be genetically engineered to incorporate polynucleotides and express polypeptides of the present invention.
  • polynucleotides may be introduced into ho ⁇ t cells using well known techniques of infection, transduction, transfection, transvection and transformation.
  • the polynucleotides may be introduced alone or with other polynucleotides.
  • Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention.
  • polynucleotides of the invention may be transfected into host cells with another, separate, polynucleotide encoding a selectable marker, using standard techniques for co- tran ⁇ fection and ⁇ election in, for instance, mammalian cells.
  • the polynucleotides generally will be stably incorporated into the host cell genome.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • the vector construct may be introduced into ho ⁇ t cells by the aforementioned techniques.
  • a plasmid vector is introduced as DNA in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid.
  • Electroporation also may be used to introduce polynucleotides into a ho ⁇ t. If the vector i ⁇ a viru ⁇ , it may be packaged in vitro or introduced into a packaging cell and the packaged virus may be transduced into cell ⁇ .
  • the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector.
  • Such vectors may be introduced into cells as polynucleotides, preferably RNA, by well known techniques for introducing DNA and RNA into cell ⁇ .
  • the vectors, in the case of phage and viral vectors also may be and preferably are introduced into cells as packaged or encapsidated virus by well known techniques for infection and transduction.
  • Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells.
  • vectors are those for expression of polynucleotides and polypeptides of the present invention.
  • such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed.
  • Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the ho ⁇ t.
  • the vectors provide for specific expres ⁇ ion.
  • Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell-specific.
  • Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
  • a variety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art.
  • the engineered host cells can be cultured in conventional nutrient media, which may be modified as appropriate for, inter alia, activating promoters, selecting transformant ⁇ or amplifying genes.
  • Culture conditions such as temperature, pH and the like, previously used with the host cell selected for expression generally will be suitable for expression of polypeptides of the present invention as will be apparent to those of skill in the art.
  • vectors can be used to express a polypeptide of the invention.
  • Such vectors include chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, ⁇ uch as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention.
  • any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host may be used for expression in this regard.
  • the appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and routine techniques.
  • a DNA sequence for expression is joined to cin expression vector by cleaving the DNA sequence and the expression vector with one or more restriction endonucleases and then joining the restriction fragments together using T4 DNA ligase.
  • Procedures for restriction and ligation that can be used to this end are well known and routine to those of skill. Suitable procedures in this regard, and for constructing expression vectors using alternative techniques, which also are well known and routine to those skill, are set forth in great detail in Sambrook et al. cited elsewhere herein.
  • the DNA sequence in the expression vector is operatively linked to appropriate expres ⁇ ion control sequence(s), including, for instance, a promoter to direct mRNA transcription.
  • a promoter to direct mRNA transcription include the phage lambda PL promoter, the E. coli lac, trp and tae promoters, the SV40 early and late promoters and promoter ⁇ of retroviral LTR ⁇ , to name ju ⁇ t a few of the well-known promoter ⁇ .
  • promoters include the phage lambda PL promoter, the E. coli lac, trp and tae promoters, the SV40 early and late promoters and promoter ⁇ of retroviral LTR ⁇ , to name ju ⁇ t a few of the well-known promoter ⁇ .
  • numerous promoters not mentioned are suitable for use in thi ⁇ aspect of the invention are well known and readily may be employed by those of skill in the manner illustrated by the discussion and the examples herein.
  • expression constructs will contain site ⁇ for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding' portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
  • constructs may contain control regions that regulate as well as engender expression.
  • control regions that regulate as well as engender expression.
  • such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.
  • Vectors for propagation and expression generally will include selectable markers. Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.
  • Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing E. coli and other bacteria.
  • the vector containing the appropriate DNA sequence as described elsewhere herein, as well as an appropriate promoter, and other appropriate control sequences, may be introduced into an appropriate ho ⁇ t using a variety of well known techniques suitable to expression therein of a desired polypeptide.
  • appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells,- and plant cells. Hosts for of a great variety of expres ⁇ ion constructs are well known, and those of skill will be enabled by the present disclosure readily to select a host for expressing a polypeptides in accordance with this aspect of the present invention.
  • the present invention also includes recombinant constructs, such as expression constructs, comprising one or more of the sequences described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which such a sequence of the invention has been inserted.
  • the sequence may be inserted in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • suitable vectors and promoters are known to those of skill in the art, and there are many commercially available vectors suitable for use in the present invention.
  • vectors which are commercially available, are provided by way of example.
  • vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen,- pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with this aspect of the present invention. It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention.
  • Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase ("cat") transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment,- i.e., a fragment that may contain a promoter.
  • a reporter transcription unit lacking a promoter region such as a chloramphenicol acetyl transferase ("cat") transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment,- i.e., a fragment that may contain a promoter.
  • a promoter-containing fragment at the restriction site upstream of the cat gene engenders production of CAT activity, which can be detected by standard CAT assays.
  • Vectors suitable to this end are well known and readily available. Two such vector ⁇ are pKK232-8 and pCW7. Thu ⁇ , promoters for expres ⁇ ion of polynucleotides of the present invention include not
  • bacterial promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E. coli lad and lacZ and promoters, the T3 and T7 promoter ⁇ , the TS tae promoter, the lambda PR, PL promoters and the trp promoter.
  • eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV”), and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • RSV Rous sarcoma virus
  • metallothionein promoters such as the mouse metallothionein-I promoter.
  • the present invention also relates to host cells containing the above-described constructs discussed above.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al. BASIC METHODS IN MOLECULAR BIOLOGY, (1986) .
  • Constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expres ⁇ ion vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) .
  • recombinant expressionvectors will include origins of replication, a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.
  • Enhancers are cis- acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • Polynucleotides of the invention encoding the heterologous structural sequence of a polypeptide of the invention generally will be inserted into the vector using standard techniques so that it is operably linked to the promoter for expres ⁇ ion.
  • the polynucleotide will be po ⁇ itioned ⁇ o that the transcription start site is located appropriately 5 ' to a ribosome binding site.
  • the ribosome binding site will be 5 ' to the AUG that initiates translation of the polypeptide to be expressed.
  • secretion signals may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
  • a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
  • region also may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
  • the addition of peptide moieties to polypeptxdes to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) .
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA) .
  • pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • Cells typically then are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those ⁇ killed in the art.
  • Variou ⁇ mammalian cell culture systems cam be employed for expres ⁇ ion, as well.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblast, described in Gluzman et al., Cell 23:175 (1981).
  • Other cell lines capable of expressing a compatible vector include for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines.
  • the Cytostatin III polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well k n techniques for refolding protein may be employed to regenerate tive conformation when the polypeptide is denatured during isolation and or purification.
  • HPLC high performance liquid chromatography
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non- glyco ⁇ ylated. In addition, polypeptide ⁇ of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • Cytostatin III polynucleotides and polypeptides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties Cytostatin III. Among these are applications in characterizing cells and organisms and in growing cells and organisms. Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms. These aspects of the invention are illustrated further by the following discussion.
  • This invention is also related to the use of the Cytostatin III polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of Cytostatin III associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or su ⁇ ceptibility to a disease which results from under-expression over-expre ⁇ ion or altered expre ⁇ ion of Cyto ⁇ tatin III, ⁇ uch a ⁇ , for example, breast cancer.
  • Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysis. PCR (Saiki et al., Nature, 324: 163-166 (1986)) .
  • RNA or cDNA may also be used in the same ways.
  • PCR primers complementary to the nucleic acid encoding Cytostatin III can be used to identify and analyze Cytostatin III expression and mutations.
  • deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled Cyto ⁇ tatin III RNA or alternatively, radiolabeled Cytostatin III antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
  • Sequence differences between a re erence gene and genes having mutations also may be revealed by direct DNA sequencing.
  • cloned DNA segments may be employed as probes to detect specific DNA segments.
  • the sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method.
  • a sequencing primer is used with double- stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
  • DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230: 1242 (1985)).
  • Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985)).
  • nuclease protection assays such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985)).
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., restriction fragment length polymorphisms ("RFLP”) and Southern blotting of genomic DNA.
  • restriction enzymes e.g., restriction fragment length polymorphisms ("RFLP") and Southern blotting of genomic DNA.
  • mutations also can be detected by in situ analysis.
  • sequences of the present invention are also valuable for chromosome identification.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosome ⁇ according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • Thi ⁇ can be accomplished using a variety of well known techniques and libraries, which generally are available commercially.
  • some trial and error may be necessary to identify a genomic probe that gives a good in situ hybridization signal.
  • sequence ⁇ can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primer ⁇ are then u ⁇ ed for PCR ⁇ creening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping ⁇ trategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA librarie ⁇ .
  • Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • Thi ⁇ technique can be u ⁇ ed with cDNA as short as 500 or 600 bases,- however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • FISH requires use of the clones from which the expres ⁇ sequence tag (EST) was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time.
  • EST expres ⁇ sequence tag
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb) .
  • the present invention also relates to a diagnostic assays such as quantitative and diagnostic assays for detecting levels of Cytostatin III protein in cells and tissues, including determination of normal and abnormal levels.
  • a diagnostic assay in accordance with the invention for detecting over-expression of Cytostatin III protein compared to normal control tissue samples may be used to detect the presence of myocardial infarction, for example.
  • Assay techniques that can be used to determine levels of a protein, such as an Cytostatin III protein of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioiramunoassays, competitive-binding assays, Western Blot analysi ⁇ and ELISA a ⁇ ays.
  • An ELISA assay initially comprises preparing an antibody specific to Cytostatin III, preferably a monoclonal antibody.
  • a reporter antibody generally is prepared which binds to the monoclonal antibody.
  • the reporter antibody is attached a detectable reagent such as radioactive, fluorescent or enzymatic reagent, in this example horseradish peroxidase enzyme.
  • a sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin.
  • a non-specific protein such as bovine serum albumin.
  • the monoclonal antibody i ⁇ incubated in the dish during which time the monoclonal antibodies attach to any Cytostatin III protein ⁇ attached to the poly ⁇ tyrene di ⁇ h. Unbound monoclonal antibody i ⁇ wa ⁇ hed out with buffer.
  • a competition as ⁇ ay may be employed wherein antibodie ⁇ specific to Cytostatin III attached to a solid support and labeled Cyto ⁇ tatin III and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of Cytostatin III in the sample.
  • the polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodie ⁇ .
  • the pre ⁇ ent invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature 256: 495-497 (1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4: 72 (1983) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. LiSS, Inc. (1985) .
  • the above-described antibodies may be employed to isolate or to identify clone ⁇ expressing the polypeptide or purify the polypeptide of the present invention by attachment of the antibody to a solid support for isolation and/or purification by affinity chromatography.
  • the growth inhibitory and differentiation stimulating activity of Cytostatin III is useful to inhibit growth and stimulate differentiation of tumor cells, for instance in vitro, as for growth of cells for research, industrial or commercial purposes, for example.
  • the same activities may be applied to treatment of aberrant cell growth in an organism, such as growth of cells of a tumor.
  • Cytostatin III polypeptides are preferred, particularly the Cyto ⁇ tatin III having the amino acid ⁇ equence ⁇ et out in Figure 1 (SEQ ID NO:2) or the amino acid ⁇ equence of the Cytostatin III of the cDNA of the deposited clone.
  • Cytostatin III to inhibit growth of Cytostatin Ill-sen ⁇ itive cell ⁇ , such as Cytostatin III-sen ⁇ itive endothelial cell ⁇ , including, for instance, venus endothelial cella, may be u ⁇ ed to prevent, ⁇ low or alter cell growth in culture or in ⁇ itu.
  • Cytostatin III may be used to inhibit Cytostatin III- sensitive cells involved in tumor vascularization, such as Cyto ⁇ tatin III-sensitive venus endothelial cells for instance, and may be useful slow tumor growth, or reduce metastatic potential of tumors or slow progression of metastatic disease.
  • Cytostatin III also may be useful to modulate b-adrenergic activity of certain Cytostatin Ill-sensitive cells, such as Cytostatin Ill-sensitive cardiac myocytes.
  • activity of Cytostatin III such a ⁇ activity that modulates mammary gland differentiation or affects the growth of mammary epithelial cells may be used to promote formation of alveolar buds, aid development of differentiated lobuloalveoli, and stimulate the production of milk protein and the accumulation of fat droplets.
  • lactation-stimulating activity may aid milk production in commercial milk-producing mammals. It also may be useful to aid milk-production by human mothers, for instance.
  • modulating activity of Cytostatin III that affects breast size may be useful to aid return of an enlarged brea ⁇ t to normal size after parturition.
  • Inhibition of Cytostatin III activity for instance, by antisense phosphorothioates or by antibodies, may be useful for selective inhibition of endogenous Cytostatin III activity in mammary epithelial cells to suppress the appearance of alveolar end buds and to lower the beta-casein level.
  • Cytostatin III polynucleotides and polypeptides of the invention have various applications and use ⁇ in numerous fields including applications involving growth of cells in vitro, commercial production of milk and milk products, and diagnosis and treatments relating to the fields of oncology, cardiology, immunology, endocrinology, hematology, metabolic disorder ⁇ , musculoskelatal problems and gynecology and obstetrics, to name a few.
  • This invention also provides a method for identification of molecules, such as receptor molecules, that bind Cyto ⁇ tatin III.
  • Genes encoding proteins that bind Cytostatin III, such as receptor proteins can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Such methods are described in many laboratory manuals such as, for instance, Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991) .
  • RNA polyadenylated RNA is prepared from a cell responsive to Cytostatin III
  • a cDNA library is created from this RNA, the library i ⁇ divided into pool ⁇ and the pool ⁇ are transfected individually into cells that are not responsive to Cytostatin III.
  • the transfected cells then are exposed to labeled Cytostatin III.
  • Cytostatin III can be labeled by a variety of well-known techniques including standard methods of radio- iodination or inclusion of a recognition site for a site-specific protein kinase.
  • the cells are fixed and binding of cytostatin is determined. These procedures conveniently are carried out on glass slides.
  • Pools are identified of cDNA that produced Cytostatin III- binding cells. Sub-pools are prepared from these positives, transfected into host cells and screened as described above. Using an iterative sub-pooling and re-screening proces ⁇ , one or more single clones that encode the putative binding molecule, such as a receptor molecule, can be isolated.
  • a labeled ligand can be photoaffinity linked to a cell extract, such as a membrane or a membrane extract, prepared from cells that express a molecule that it binds, such as a receptor molecule.
  • Cross-linked material is resolved by polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray film.
  • PAGE polyacrylamide gel electrophoresis
  • the labeled complex containing the ligand-receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing.
  • the amino acid sequence obtained from microsequencing can be used to design unique or degenerate oligonucleotide probes to screen cDNA libraries to identify genes encoding the putative receptor molecule.
  • Polypeptides of the invention also can be used to assess Cytostatin III binding capacity of Cytostatin III binding molecules, such as receptor molecules, in cells or in cell-free preparations.
  • the invention also provides a method of screening compounds to identify those which enhance or block the action of Cytostatin III on cells, such as its interaction with Cytostatin Ill-binding molecules such as receptor molecules.
  • An agonist i ⁇ a compound which increa ⁇ e ⁇ the natural biological function ⁇ of Cyto ⁇ tatin III or which function ⁇ in a manner ⁇ imilar to Cyto ⁇ tatin III, while antagonis s decrease or eliminate such functions.
  • a cellular compartment such as a membrane or a preparation thereof, such as a membrane-preparation, may be prepared from a cell that expresses a molecule that binds Cytostatin III, such as a molecule of a signaling or regulatory pathway modulated by Cytostatin III.
  • the preparation is incubated with labeled Cytostatin III in the absence or the pre ⁇ ence of a candidate molecule which may be a Cytostatin III agonist or antagonist.
  • the ability of the candidate molecule to bind the binding molecule is reflected in decreased binding of the labeled ligand.
  • Molecules which bind gratuitously, i.e., without inducing the effects of Cytostatin III on binding the Cytostatin III binding molecule, are most likely to be good antagonists. Molecules that bind well and elicit effects that are the same a ⁇ or closely
  • Cytostatin Ill-like effects of potential agonists and antagonists may by measured, for instance, by determining activity of a second messenger system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of Cytostatin III or molecules that elicit the same effects as Cytostatin III.
  • Second mes ⁇ enger systems that may be useful in this regard include but are not limited to AMP guanylate cyclase, ion channel or phosphoino ⁇ itide hydrolysis second messenger systems.
  • Cytostatin III antagonists are a competitive assay that combines Cytostatin III and a potential antagonist with membrane-bound Cytostatin III receptor molecules or recombinant Cytostatin III receptor molecules under appropriate conditions for a competitive inhibition assay.
  • Cytostatin III can be labeled, such as by radioactivity, ⁇ uch that the number of Cyto ⁇ tatin III molecules bound to a receptor molecule can be determined accurately to assess the effectiveness of the potential antagonis .
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a receptor molecule, without inducing Cytostatin Ill-induced activities, thereby preventing the action of Cytostatin III by excluding Cytostatin III from binding.
  • Potential antagonists include a small molecule which binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such as receptor molecules, such that normal biological activity is prevented.
  • small molecules include but are not limited to small organic molecules, peptides or peptide-like molecule ⁇ .
  • Antisen ⁇ e technology can be used to control gene expres ⁇ ion through antisense DNA or RNA or through triple-helix formation. Antisense techniques are discussed, for example, in - Okano, J. Neurochem. 56: 560 (1991); OLIGODBOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988) . Triple helix formation is discussed in, for instance Lee et al. , Nucleic Aci. s Research 6: 3073 (1979); Cooney et al., Science 241: 456 (19881 and Dervan et al. , Science 251: 1360 (1991) .
  • the methods are based on binding of a polynucleotide to a complementary DNA or RNA.
  • the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of Cytostatin III.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into Cytostatin III polypeptide.
  • the oligonucleotides described above can also be delivered to cells such that the antisen ⁇ e RNA or DNA may be expressed in vivo to inhibit production of Cytostatin III.
  • the antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
  • the antagonists may be employed for instance to treat cardiac myocte hypertrophy or leukemia
  • composition ⁇ comprising the polynucleotide or the polypeptides discussed above or the agonist ⁇ or antagoniat ⁇ .
  • the polypeptides of the present invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject.
  • a pharmaceutical carrier suitable for administration to a subject such as a pharmaceutical carrier suitable for administration to a subject.
  • Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient.
  • Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration.
  • the invention further relates to pharmaceutical packs and kit ⁇ comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.
  • Polypeptides of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
  • compositions generally are administered in an amount effective for treatment or prophylaxis of a specific indication or indications.
  • the compositions are administered in an amount of at least about 10 ⁇ g/kg body weight. In most cases they will be administered in an amount not in exces ⁇ of about 8 mg/kg body weight per day.
  • dose is from about 10 ⁇ g/kg to about 1 mg/kg body weight, daily. It will be appreciated that optimum dosage will be determined by standardmethods for each treatment modality and indication, taking into account the indication, its severity, route of administration, complicating conditions and the like.
  • Cytostatin III polynucleotides, polypeptides, agonists and antagonists that are polypeptides may be employed in accordance with the present invention by expression of such polypeptides in vivo, in treatment modalities often referred to as "gene therapy.”
  • cells from a patient may be engineered with a polynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo, and the engineered cells then can be provided to a patient to be treated with the polypeptide.
  • a polynucleotide such as a DNA or RNA
  • cells may be engineered ex vivo by the use of a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by procedures known in the art.
  • a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above.
  • the retroviral expression construct then may be isolated and introduced into a packaging cell is tran ⁇ duced with a retroviral pla ⁇ mid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a patient for engineering cells in vivo and expres ⁇ ion of the polypeptide in vivo.
  • Retroviruse ⁇ from which the retroviral plasmid vectors herein above mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma irus, and mammary tumor virus.
  • the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
  • Such vectors well include one or more promoters for expressing the polypeptide.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter,- and the human cytomegalovirus (CMV) promoter described in Miller et al., Biotechniques 7: 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, RNA polymerase III, and ⁇ -actin promoters) .
  • CMV cytomegalovirus
  • viral promoter ⁇ which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoter ⁇ , and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter,- or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter,- heat shock promoters,- the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoter ⁇ , such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs herein above described) ,- the ⁇ -actin promoter; and human growth hormone promoters.
  • the promoter also may be the native promoter which controls the gene
  • the retroviral plasmid vector is employed to tran ⁇ duce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, Y-2, Y-AM, PA12, T19-14X, VT-19-17- H2, YCRE, YCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, A., Human Gene Therapy l: 5-14 (1990).
  • the vector may be transduced into the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP04 precipitation.
  • the retroviral pla ⁇ mid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
  • the producer cell line will generate infectious retroviral vector particles, which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed to transduce eukaryotic cell ⁇ , either in vitro or in vivo.
  • the transduced eukaryotic cells will express the nucleic acid sequence( ⁇ ) encoding the polypeptide.
  • Bukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblast ⁇ , myobla ⁇ t ⁇ , keratinocytes, endothelial cells, and bronchial epithelial cells.
  • ligations were accomplished using standard buffers, incubation temperatures and times, approximately equimolar amounts of the DNA fragments to be ligated and approximately 10 units of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of DNA.
  • ligase T4 DNA ligase
  • the DNA sequence encoding human Cytostatin III in the deposited polynucleotide was amplified using PCR oligonucleotide primers specific to the amino acid carboxyl terminal sequence of the human Cyto ⁇ tatin III protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning were added to the 5' and 3' sequences respectively.
  • the 5' oligonucleotide primer had the sequence 5' CGC GCA TGC CTC CCA ACC TCA C 3' (SBQ ID NO:3) containing the underlined SphI restriction site, which encodes a start AUG, followed by 13 nucleotides of the human Cytostatin III coding sequence set out in Figure l (SEQ ID NO:l) beginning with the second base of the second codon.
  • the 3' primer had the sequence 5' GCG AAG CTT CTA TCT GAC CTT CCT G 3' (SEQ ID NO:4) containing the underlined Hind III restriction site followed by 16 nucleotides complementary to the last 16 nucleotides of the Cytostatin III coding sequence set out in Figure 1 (SEQ ID NO:l), including the stop codon.
  • the restrictions sites were convenient to restriction enzyme sites in the bacterial expression vectors pQE-7, which were used for bacterial expression in these examples. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311) .
  • pQE-7 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin of replication ("ori") , an IPTG inducible promoter, a ribosome binding site (“RBS”) , a 6-His tag and restriction enzyme sites.
  • E. coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expre ⁇ es lac repressor and confers kanamycin resistance (“Kanr") , was used in carrying out the illustrative example described here.
  • This strain which is only one of many that are suitable for expressing Cytostatin III, is available commercially from Qiagen.
  • Tran ⁇ formant ⁇ were identified by their ability to grow on LB plates in the presence of ampicillin. Pla ⁇ mid DNA was isolated from resistant colonies and the identity of the cloned DNA was confirmed by restriction analysis.
  • Clones containing the desired constructs were grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ug/ml) and kanamycin (25 ug/ml) .
  • the O/N culture was used to inoculate a large culture, at a dilution of approximately 1:100 to 1:250.
  • the cells were grown to an optical density at 600nm ("OD600”) of between 0.4 and 0.6.
  • Isopropyl-B-D-thiogalactopyranoside (“IPTG”) wa ⁇ then added to a final concentration of 1 mM to induce tran ⁇ cription from lac repre ⁇ or sensitive promoters, by inactivating the lad repressor.
  • IPTG Isopropyl-B-D-thiogalactopyranoside
  • Cells subsequently were incubated further for 3 to 4 hours. Cells then were harvested by centrifugation and disrupted, by standard methods.
  • Inclusion bodies were purified from the disrupted cells using routine collection techniques, and protein was solubilized from the inclusion bodies into 8M urea.
  • the 8M urea solution containing the solubilized protein was pas ⁇ ed over a PD-10 column in 2X pho ⁇ phate buffered ⁇ aline ("PBS") , thereby removing the urea, exchanging the buffer and refolding the protein.
  • PBS 2X pho ⁇ phate buffered ⁇ aline
  • the protein was purified by a further step of chromatography to remove endotoxin. Then, it was sterile filtered.
  • the sterile filtered protein preparation was stored in 2X PBS at a concentration of 95 micrograms per mL.
  • the cDNA sequence encoding the full length human Cytostatin III protein, in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
  • the 5' primer has the sequence 5' GC GGA TCC TCC CAA CCT CAC TGG CTA C 3' (SEQ ID NO:5) containing the underlined BamHI restriction enzyme site followed by 19 bases of the sequence of Cytostatin III of Figure 1 (SEQ ID NO:l) . Inserted into an expre ⁇ ion vector, as described below, the 5' end of the amplified fragment encoding human Cytostatin III provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196: 947-950 (1987) is appropriately located in the vector portion of the construct.
  • the 3' primer has the sequence 5' GC GGT ACC CTA TCT GAC CTT CCT G 3' (SEQ ID NO:6) containing the Asp718 restriction followed by nucleotides complementary to the last 16 nucleotides of the Cytostatin III coding sequence set out in Figure 1 (SEQ ID NO:l) , including the stop codon.
  • the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with BamHI and Asp7l8 and again is purified on a 1% agarose gel. This fragment is designated herein F2.
  • the vector pA2-GP is used to express the Cytostatin III protein in the baculovirus expression system, using standard methods, such as those described in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station Bulletin No. 1555 (1987) .
  • This expression vector contains the strong polyhedrin promoter of the Autographa califo ica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites.
  • the signal peptide of AcMNPV gp67, including the N-terminal methionine, is located just upstream of a BamHI site.
  • the polyadenylation site of the simian virus 40 (“SV40") is used for efficient polyadenylation.
  • SV40 simian virus 40
  • the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene.
  • the polyhedrin sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.
  • baculoviru ⁇ vectors could be u ⁇ ed in place of pA2- GP, such as pAc373, pVL941 and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required.
  • Such vectors are de ⁇ cribed in Luckow et al., Virology 170: 31-39, among other ⁇ .
  • the plasmid is digested with the restriction enzymes BamHI and Asp718 and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
  • the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated herein "V2".
  • Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase.
  • E.coli HB101 cells are transformed with ligation mix and spread on culture plates.
  • Bacteria are identified that contain the plasmid with the human Cytostatin III gene by digesting DNA from individual colonies using BamHI and Asp718 and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This pla ⁇ mid is designated herein pBacCyto ⁇ tatin III.
  • plasmid pBacCytostatin III 5 ⁇ g of the plasmid pBacCytostatin III is co-tran ⁇ fected with 1.0 ⁇ g of a commercially available linearized baculovirus DNA ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA.) , using the lipofection method described by Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987) .
  • BaculoGoldTM baculovirus DNA linearized baculovirus DNA
  • the plate is then incubated for 5 hours at 27"C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27"C for four days.
  • plaque as ⁇ ay After four days the supernatant is collected and a plaque as ⁇ ay is performed, a ⁇ described by Summers and Smith, cited above.
  • An agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expres ⁇ ing clones, which produce blue-stained plaques. (A detailed description of a "plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc. , Gaithersburg, page 9-10) .
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS.
  • the cells are infected with the recombinant baculovirus V-Cytostatin III at a multiplicity of infection ("MOI") of about 2 (about l to about 3) .
  • MOI multiplicity of infection
  • the medium is removed and is replaced with SF900 II medium minu ⁇ methionine and cy ⁇ teine (available from Life TECH ⁇ Inc., Gaither ⁇ burg) .
  • 5 ⁇ Ci of 35S-methionine and 5 ⁇ Ci 35S cy ⁇ teine available from Amersham
  • the cells are further incubated for 16 hours and then they are harvested by centrifugation, lysed and the labeled proteins are visualized by SDS-PAGE and autoradiography. Active proteins are then produced by dialysis with PBS.
  • the expression plasmid, Cytostatin III HA is made by cloning a cDNA encoding Cytostatin III into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.) .
  • the expres ⁇ ion vector pcDNAI/amp contains: (1) an E.coli origin of replication effective for propagation in E. coli and other prokaryotic cell; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
  • a DNA fragment encoding the entire Cytostatin III precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector so that recombinant protein expres ⁇ ion i ⁇ directed by the CMV promoter.
  • the HA tag corre ⁇ ponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al., Cell 37: 767 (1984) .
  • the fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the plasmid con ⁇ truction strategy is as follows.
  • the Cytostatin III cDNA of the depo ⁇ it clone of the deposited clone is amplified using primers that contained convenient restriction sites, much as described above regarding the construction of expres ⁇ ion vectors for expres ⁇ ion of Cytostatin III in E. coli and S. fugiperda.
  • one of the primers contains a hemagglutinin tag ("HA tag") as described above.
  • Suitable primers include that following, which are used in this example.
  • the 5' primer containing the underlined BamHI site, an AUG start codon and 5 codons thereafter, forming the hexapeptide haemaglutinin tag, has the following sequence: 5' GC GGA TCC ACC ATG CCT CCC AAC CTC ACT 3' (SEQ ID NO:7) .
  • the 3' primer containing the underlined Xbal site and 15 bp of 3' coding sequence (at the 3' end) has the following sequence: 5' GC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA TCT GAC CTT CCT GAA 3' (SEQ ID NO:8) .
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with and then ligated.
  • the ligation mixture is transformed into B. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the Cytostatin III-encoding fragment.
  • COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989) . Cells are incubated under conditions for expres ⁇ ion of Cyto ⁇ tatin III by the vector.
  • Expre ⁇ sion of the Cytostatin III HA fusion protein is detected by radiolabelling and immunoprecipitation, using methods described in, for example Harlow et al. , ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.,- Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988) . To this end, two days after transfection, the cells are labeled by incubation in media containing 35S-cysteine for 8 hours.
  • the cells and the media are collected, and the cells are wa ⁇ hed and the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above.
  • Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expres ⁇ ion product of the expected size is seen in the cell lysate, which is not seen in negative controls.
  • RNAzolTM B sy ⁇ tem Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033
  • RNA is isolated from tissue samples. The RNA is size resolved by electrophoresis through a 1% agarose gel under strongly denaturing conditions. RNA is blotted from the gel onto a nylon filter, and the filter then is prepared for hybridization to a detectably labeled polynucleotide probe.
  • the antisen ⁇ e strand of the coding region of the cDNA insert in the deposited clone is labeled to a high specific activity.
  • the cDNA is labeled by primer extension, u ⁇ ing the Prime-It kit, available from Stratagene.
  • the reaction i ⁇ carried out using 50 ng of the cDNA, following the standard reaction protocol as recommended by the supplier.
  • the labeled polynucleotide is purified away from other labeled reaction components by column chromatography using a Select-G-50 column, obtained from 5-Prime - 3-Prime, Inc. of 5603 Arapahoe Road, Boulder, CO 80303.
  • the labeled probe is hybridized to the filter, at a concentration of 1,000,000 cpm/ml, in a small volume of 7% SDS, 0.5 M NaP04, pH 7.4 at 65'C, overnight.
  • the probe solution is drained and the filter is wa ⁇ hed twice at room temperature and twice at 60"C with 0.5 x SSC, 0.1% SDS.
  • the filter then is dried and exposed to film at -70'C overnight with an intensifying screen.
  • Fibroblasts are obtained from a subject by skin biopsy.
  • the resulting tissue i ⁇ placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask.
  • the flask i ⁇ turned upside down, closed tight and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted - the chunks of tissue remain fixed to the bottom of the flask - and fre ⁇ h media i ⁇ added (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) .
  • the tis ⁇ ue i ⁇ then incubated at 37"C for approximately one week. At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblast ⁇ emerge ⁇ . The monolayer i ⁇ trypsinized and scaled into larger flask ⁇ .
  • a vector for gene therapy is digested with restriction enzymes for cloning a fragment to be expres ⁇ ed.
  • the dige ⁇ ted vector i ⁇ treated with calf inte ⁇ tinal phosphatase to prevent self-ligation.
  • the dephosphorylated, linear vector is fractionated on an agarose gel and purified.
  • Cytostatin cDNA capable of expressing active Cytostatin III, i ⁇ isolated.
  • the ends of the fragment are modified, if necessary, for cloning into the vector. For instance, 5" overhanging may be treated with DNA polymerase to create blunt ends. 3 ' overhanging ends may be removed using SI nuclease.
  • Linkers may be ligated to blunt ends with T4 DNA ligase. Equal quantities of the Moloney murine leukemia virus linear backbone and the Cytostatin III fragment are mixed together and joined using T4 DNA ligase. The ligation mixture is used to transform E. Coli and the bacteria are then plated onto agar- containing kanamycin. Kanamycin phenotype and restriction analysis confirm that the vector has the properly inserted gene.
  • Packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS) , penicillin and streptomycin.
  • DMEM Dulbecco's Modified Eagles Medium
  • CS calf serum
  • the vector containing the Cytostatin III gene is introduced into the packaging cells by standard techniques. Infectious viral particles containing the Cytostatin III gene are collected from the packaging cells, which now are called producer cells.
  • Fresh media is added to the producer cells, and after an appropriate incubation period media is harvested from the plates of confluent producer cells.
  • the media containing the infectious viral particles, i ⁇ filtered through a Millipore filter to remove detached producer cells.
  • the filtered media then is used to infect fibroblast cells.
  • Media is removed from a sub-confluent plate of fibroblast ⁇ and quickly replaced with the filtered media.
  • Polybrene Aldrich
  • the media is removed and replaced with fre ⁇ h media. If the titer of virus is high, then virtually all fibroblast ⁇ will be infected and no selection is required. If the titer is low, then it is neces ⁇ ary to use a retroviral vector that has a selectable marker, such as neo or his, to select out transduced cell ⁇ for expansion.
  • Engineered fibroblast ⁇ then may be injected into rat ⁇ , either alone or after having been grown to confluence on microcarrier beads, such as cytodex 3 beads.
  • the injected fibroblast ⁇ produce Cyto ⁇ tatin III product, and the biological actions of the protein are conveyed to the ho ⁇ t.
  • Example 6 Expression of recombinant Cytostatin III in CHO cells
  • the vector pCl is used for the expression of Cytostatin III protein.
  • Plasmid pCl is a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 371461. Both plasmids contain the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Lift Technologies) ⁇ upplemented with the chemotherapeutic agent methotrexate.
  • a selective medium alpha minus MEM, Lift Technologies
  • MTX methotrexate
  • DHFR target enzyme
  • a second gene is linked to the DHFR gene it is usually co-amplified and over-expres ⁇ ed. It i ⁇ state of the art to develop cell lines carrying more than 1,000 copies of the genes. Subsequently, when the methotrexate i ⁇ withdrawn, cell line ⁇ contain the amplified gene integrated into the chromosome(s) .
  • Plasmid pCl contains for the expres ⁇ ion of the gene of interest a strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al., Molecular and Cellular Biology, March 1985, 438-4470) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530, 1985) .
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • Down ⁇ tream of the promoter are the following single restriction enzyme cleavage sites that allow the integration of the genes: BamHI, Pvull, and Nrul.
  • the pla ⁇ mid contains translational stop codons in all three reading frames followed by the 3' intron and the polyadenylation site of the rat preproinsulin gene.
  • Other high efficient promoters can also be used for the expression, e.g., the human ⁇ -actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
  • the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well.
  • Stable cell line ⁇ carrying a gene of intere ⁇ t integrated into the chromo ⁇ ome can al ⁇ o be ⁇ elected upon co-transfection with a selectable marker ⁇ uch a ⁇ gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g. G418 plus methotrexate.
  • the plasmid pCl is digested with the restriction enzyme BamHI and then dephosphorylated using calf inte ⁇ tinal phosphatase by procedures known in the art.
  • the vector is then isolated from a 1% agarose gel.
  • cytostatin III ATCC # 97332
  • the DNA sequence encoding cytostatin III, ATCC # 97332 is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
  • the 5' primer has the sequence 5' GC GGA TCC TCC CAA CCT CAC TGG CTA C 3' (SEQ ID NO:9) containing the underlined BamHI re ⁇ triction enzyme site followed by 19 bases of the sequence of Cytostatin III of Figure 1 (SEQ ID NO:l) .
  • An efficient signal for initiation of translation in eukaryotic cells as described by Kozak, M. , J. Mol. Biol. 196: 947-950 (1987) is appropriately located in the vector portion of the construct.
  • the 3' primer has the ⁇ equence 5' GC GGT ACC CTA TCT GAC CTT CCT G 3' (SEQ ID NO:10) containing the A ⁇ p718 re ⁇ triction followed by nucleotides complementary to the last 16 nucleotides of the Cytostatin III coding sequence set out in Figure 1 (SEQ ID NO:l) , including the stop codon.
  • amplified fragments are isolated from a 1% agarose gel as described above and then digested with the endonuclease BamHI and then purified again on a 1% agarose gel.
  • the isolated fragment and the dephosphorylated vector aare then ligated with T4 DNA ligase.
  • E.coli HB101 cells are then transformed and bacteria identified that contained the plasmid pCl inserted in the correct orientation using the restriction enzyme BamHI.
  • the sequence of the inserted gene is confirmed by DNA sequencing.
  • Chinese hamster ovary cells lacking an active DHFR enzyme are used for transfection.
  • 5 ⁇ g of the expression plasmid Cl are cotransfected with 0.5 ⁇ g of the plasmid pSVneo using the lipofectin method (Feigner et al., supra) .
  • the plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in alpha minus MEM supplemented with l mg/ml G418.
  • the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 days. After this period, single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25, 50 nm, 100 nm, 200 nm, 400 nm) . Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 ⁇ M, 2 ⁇ M, 5 ⁇ M) . The same procedure is repeated until clones grow at a concentration of 100 ⁇ M.
  • the expression of the desired gene product is analyzed by Western blot analysis and SDS-PAGE.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI,
  • MOLECULE TYPE DNA (genomic)
  • CTCTGGGTCC CTCCTCACCC CTCCCCGTGT TAATCTGTAA CTTGGAGCCC CCAGGACAAA 678
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention a trait aux polypeptides Cytostatine III, à des polynucléotides codant pour ces polypeptides et à des procédés de production de tels polypeptides, notamment par expression des polynucléotides ainsi qu'aux agonistes et antagonistes desdits polypeptides. L'invention concerne également des procédés d'utilisation de tels polynucléotides, polypeptides, agonistes et antagonistes pour certaines applications se rapportant, entre autres, à la recherche et à des techniques diagnostiques et cliniques.
PCT/US1996/003697 1996-03-19 1996-03-19 Cytostatine iii WO1997035028A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1996/003697 WO1997035028A1 (fr) 1996-03-19 1996-03-19 Cytostatine iii
AU53659/96A AU5365996A (en) 1996-03-19 1996-03-19 Cytostatin iii

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1996/003697 WO1997035028A1 (fr) 1996-03-19 1996-03-19 Cytostatine iii

Publications (1)

Publication Number Publication Date
WO1997035028A1 true WO1997035028A1 (fr) 1997-09-25

Family

ID=22254865

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/003697 WO1997035028A1 (fr) 1996-03-19 1996-03-19 Cytostatine iii

Country Status (2)

Country Link
AU (1) AU5365996A (fr)
WO (1) WO1997035028A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5977309A (en) * 1995-03-24 1999-11-02 Human Genome Sciences, Inc. Cytostatin I
US6046027A (en) * 1997-07-22 2000-04-04 Incyte Pharmaceuticals, Inc. Human retinoid binding protein

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, Vol. 55, issued 01 June 1995, HUYNH et al., "Tumor Suppressor Activity of the Gene Encoding Mammary-derived Growth Inhibitor", pages 2225-2231. *
GENOMICS, Vol. 34, No. 1, issued April 1996, PHELAN et al., "The Human Mammary-Derived Growth Inhibitor (MDG1) Gene: Genomic Structure and Mutation Analysis in Human Breast Tumors", pages 63-68. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5977309A (en) * 1995-03-24 1999-11-02 Human Genome Sciences, Inc. Cytostatin I
US6287812B1 (en) 1995-03-24 2001-09-11 Human Genome Sciences, Inc. Nucleic acid molecules encoding cytostatin I
US6046027A (en) * 1997-07-22 2000-04-04 Incyte Pharmaceuticals, Inc. Human retinoid binding protein

Also Published As

Publication number Publication date
AU5365996A (en) 1997-10-10

Similar Documents

Publication Publication Date Title
US6232291B1 (en) Cytostatin III
US6066724A (en) Human endometrial specific steroid-binding factor I, II and III
WO1997033899A1 (fr) Molecule i induisant l'apoptose
US6576745B1 (en) Human cystatin F antibodies
US6174992B1 (en) Human endometrial specific steroid-binding factor I, II and III
US6008022A (en) Human cytokine polypeptide
US20080096821A1 (en) Cytostatin II
US5945303A (en) Human hematopoietic-specific protein
WO1997034013A1 (fr) Cytokine ii chimiotactique
US7309780B2 (en) Antibodies to natural killer cell activating factor II
US5919658A (en) Human cystatin F
US5977309A (en) Cytostatin I
WO1997035028A1 (fr) Cytostatine iii
US20060171918A1 (en) Apoptosis inducing molecule I
WO1997036915A1 (fr) Cystatine f humain
EP0856006A1 (fr) Cytostatine ii
WO1997037022A1 (fr) Facteur ii d'activation de cellules tueuses naturelles chez l'homme
WO1997027747A1 (fr) Cytostatine i
US20040197874A1 (en) Human cytokine polypeptide
EP0896614A1 (fr) Facteur i, ii et iii de liaison de steroides specifique a l'endometre humain
CA2233361A1 (fr) Cytostatine ii
WO1998044109A1 (fr) Cytostatine ii

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AU BG BR BY CA CN CZ EE FI GE HU JP KG KP KR KZ LT LV MD MN MX NO NZ PL RO RU SG SI SK TJ TM UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

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

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97533420

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载