MXPA98002384A - Citostatine - Google Patents

Abstract

The present invention relates to peptides modulating the growth of cytostatin II, to polynucleotides encoding polypeptides, to methods for producing polypeptides, in particular for the expression of polynucleotides, and to agonists and antagonists of polypeptides. The invention further relates to methods for using such polynucleotides, polypeptides, agonists and antagonists for applications which are related, in part, to clinical, research and diagnostic techniques.

Description

CITOSTATINE II Background of the Invention This invention relates, in part, to newly identified polynucleotides and polypeptides; to variants and derivatives of the polynucleotides and polypeptides; to processes for making the polynucleotides and polypeptides, and to their variants and derivatives; to the agonists and antagonists of the polypeptides; and to the uses of the polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and other aspects, the invention relates to the polynucleotides and polypeptides of human cytostatin II.

BACKGROUND OF THE INVENTION The growth and differentiation of cells and the development of tissues and glands is controlled by autocrine and paracrine factors, such as, such as systemic or systemic factors and hormones that modulate or have a mediating role in the action of hormones, such as growth factors, which in and of themselves can be hormones. Ref.27137 For example, the peptides that cause the growth of the signal locally and that stimulate the differentiation of the cells of the developing epithelium, are very important for the development of the mammary gland. These factors have not been identified or characterized widely, particularly not in humans. A small amount of the factors that play a role in the humoral mediation of the growth and differentiation of cells in tissues and glands, of the mammary glands in particular, has been identified in non-human organisms. One such factor is the growth inhibitor derived from the mammary glands ("MDGI"), which, in mice and cows at least, inhibits the growth of epithelial cells and stimulates the differentiation of epithelial cells. The MDGI was first identified in the milk and mammary glands of the cows. Subsequently, it was identified in the mice. The MDGI is present in at least two forms produced by alternative routes of post-translational processing. The original form is referred to as an MDGI and the second form is called MDGI-2. MDGI is mainly associated with milk fat globule membranes ("MFGM"), as evaluated by immunological assays using anti-MDGI antibodies. Similar time course studies show that MDGI increases dramatically in mammary glands when breastfeeding is initiated, following supply. The MGDI-2 differs from the MDGI in this regard. It was found in the mammary glands during pregnancy but not during lactation. The roles of the two MGDI forms and their action mechanism (s) are not clearly defined. The mouse and bovine MDGI are homologous with each other and with respect to the family of hydrophobic ligand binding proteins of low molecular mass ("low PM HLBP (s)") which include fatty acid binding proteins ( "FABP (s)") of the brain, heart, liver and intestine, the P2 protein of myelin, the differentiation of the associated protein of the adipocytes called gastrotropin p422 and the retinoic acid binding protein ("CRABP" ). These proteins, which bind hydrophobic ligands such as long-chain fatty acids, retinoids and eiconsanoids, are thought to play roles in the transport, sequestration, or metabolism of fatty acids and fatty acid derivatives. However, they are expressed in a specific manner of differentiation, in the cells of the mammary gland, the heart, the liver, the brain and the intestine, and they seem not only to play roles in the basal metabolism but also they have important roles in differentiation and development. The homology of the MDGI with respect to the HHLBPs of low MW raises the possibility that the MDGI, at least as part of its function, binds to the hydrophobic ligand, and is binding to this ligand is important for the mechanisms by which the MDGI inhibits cell growth and stimulates differentiation; although all the other HLBPs of low MW except the gastrotropin, act intracellularly, while the MDGI acts extracellularly, in vitro. Among the low MW HLBPs, the MDGI closely resembles the fatty acid binding proteins. { "FABP"). FABPs have been identified in the brain, heart, liver and intestine. The FABP of the heart, in a similar way to the MDGI, either produced from natural resources or by the expression of a cloned gene in a heterologous host, inhibits the growth of normal mammary epithelial cells ("MEC") of the origin of the mouse . In addition, it stimulates the synthesis of milk protein and stimulates its own expression in these cells. However, unlike the FABP of the bovine heart, the MDGI of the bovine does not bind to the fatty acids, although the two proteins are 95% homologous and it has been suggested that the FABP of the heart can really be of a different form. MDGI. Therefore, even if the MDGI is a HLBP of low MW, its substrate affinities are different from its relative relative members in the family, and therefore probably play a different physiological role. MDGI in vivo is found in capillary endothelial cells and mammary parenchyma, in mice and ls cows. The MDGI appears first in the capillary endothelial cells and finally in the secretory epithelial cells. The location of the MDGI in the mammary capillary endothelium is consistent with a role in the regulation of the proliferation of endothelial cells. Several of the MDGI activities have been demonstrated in vitro. For example, MDGI has been shown to inhibit the supersensitivity induced by L (+) - lactate-, arachidonic acid- and 15-S-hydroxyceicosatetranic acid, from heart cells of the neonatal rat with respect to beta-stimulation. adrenergic Induced hypersensitivity is mediated by a small population of 2-adrenergic receptors and, therefore, it has been suggested that MDGI interferes with the normal function of these receptors. Interaction with these receptors can also be part of the mechanism by which MDGI inhibits the growth of cells.

This activity . it also raises the possibility that the MDGI naturally modulates the beta-adrenergic sensitivity of cardiac myocytes. The effect of MDGI on the differentiation of mammary epithelial cells ("MEC") has been further demonstrated by antisense inhibition experiments using phosphorothioate oligonucleotides. These experiments show that the MDGI antisense molecules reduce the levels of beta-casein and suppress the appearance of the alveolar end buds in organic cultures. In addition, the MDGI suppresses the mitogenic effects of the epidermal growth factor, and the epidermal growth factor antagonizes the activities of the MDGI. The MDGI is the first known growth inhibitor that promotes the differentiation of the mammary glands. The regulatory properties of the MDGI can be completely minimized by a sequence of 11 amino acids, which is represented at the carboxyl terminus of the MDGI and a subfamily of the HLBPs of low MW. Not all mammary epithelial cell lines respond to MDGI in the same way. MDGF inhibits the growth of normal human MEC, which has been passed through for varying periods of time. It also inhibits the growth of mMaCa 20177 from mouse malignant mammary epithelial cell lines, the malignant MaTu and T47D mammary cell lines of humans and inhibits the resumption of the growth of stationary Ehrlich ascites carcinoma cells ("EAC ") in vitro. In contrast, MDGF slightly stimulates the growth of the malignant, human MCF7 mammary epithelial cell line. Finally, the MDGI promotes the differentiation of mouse pluripotent embryonic stem cells. The mechanism of effects of MDGI on cells is not known yet. The resumption of growth of Ehrlich ascites carcinoma cells ("EACs") stationary, in vitro, is effected by a rapid increase in c-fos, c-myc and c-ras cellular mRNAs. The rapid induction of these genes during exposure to MDGI, emphasizes the importance of oncogenic expression with respect to the regulation of growth and reveals a positive correlation between cell growth and the expression of c-fos, c-myc and c-ras. In addition, the effect of MDGI on the expression of these genes indicates that it is a positive effect of cellular proto-oncogene expression, either directly or through one or more signaling pathways, or both. It has also been shown that MDGI can function as a potent tumor suppressor gene.

Human breast cancer cells transfected with an MDGI expression construct exhibited a differentiated morphology, a reduced proliferation rate, a reduced clonogenicity in soft or soft agar, and reduced tumorgenicity in the nude mouse. The human homolog of this gene was assigned coordinates with respect to chromosome lp33-35, a site previously shown to exhibit a frequent loss of heterozygosity in human breast cancer (approximately 40% of tumors). The magnitude of tumor suppressor activity in vivo and in vitro of MDGI is comparable to that previously observed for BRCA1, p53, Rb, and H19. The effects of MDGF on cell growth and differentiation, and on the expression of the cellular proto-oncogene, reiterates the importance of soluble factors in the normal growth and differentiation of cells, tissues, glands and organs, and their roles in the growth of aberrant cells, dysfunction and disease. Clearly, there is a need for factors that regulate the growth and differentiation of normal and abnormal cells. Therefore there is a need for the identification and characterization of factors such as modulate the growth and differentiation of cells, both normally and in disease states. In particular, there is a need to isolate and characterize additional cytostatins that modulate the growth and differentiation of cells such as epithelial cells, particularly mammary epithelial cells, which are essential for the proper development and health of tissues and organs. such as the mammary glands of adult and developing human women.

BRIEF DESCRIPTION OF THE INVENTION For these purposes and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as novel cytostatins, by homology with respect to known cytostatins, such as MDGI, of the amino acid sequence described in the Figure 1. It is a further object of the invention, in addition, to provide polynucleotides that encode cytostatins, particularly polynucleotides that encode the polypeptide designated herein cytostattin II. In a particularly preferred embodiment of this aspect of the invention, the polynucleotide comprises the region encoding human cytostatin II in the sequence described in Figure 1 or in the cDNA in deposit No. 97,287 of the ATCC (referred to herein as the deposited clone). ). According to this aspect of the invention, isolated nucleic acid molecules encoding human cytostatin II are provided, including mRNAs, DNAs, cDNAs, genomic DNAs and, in the additional embodiments of this aspect of the invention, variants, analogues or biological, diagnostic, clinical or therapeutically derived derivatives thereof, including fragments of the variants, analogs and derivatives. Among the preferred embodiments particularly of this aspect of the invention are the allelic variants that are naturally present, of human cytostatin II. It is also an object of the invention to provide cytostatin II polypeptides, particularly the human cytostatin II polypeptides, which modulate the growth activity of epithelial cells. In accordance with this aspect of the invention, novel polypeptides of human origin, referred to herein as cytostatin II as well as fragments, variants, homologs, analogs, and derivatives thereof, are useful biologically, diagnostically or therapeutically.

Among the preferred embodiments particularly of this aspect of the invention are variants of human cytostatin II encoded by the alleles that are naturally present in the human cytostatin II gene. It is another object of the invention to provide a process for producing polypeptides, variants of polypeptide fragments, analogs, derivatives and fragments thereof. In a preferred embodiment of this aspect of the invention, methods are provided for producing the aforementioned cytostatin II polypeptides, which comprise culturing the host cells that are expressly incorporated therein the polynucleotide encoding the exogenously derived human cytostatin II, under conditions for the expression of human cytostatin II in the host and then recovering the expressed polypeptide. It is another object of the invention to provide products, compositions, processes and methods for using the polypeptides and polynucleotides mentioned above for biological, clinical and therapeutic purposes, inter alia. According to certain preferred embodiments of this aspect of the invention, methods are provided for, among other things: modulating the growth of the cells in vitro, ex vivo or in vivo; evaluate the expression of cytostatin II by determining the protein or mRNA; and evaluate variation and genetic aberrations, such as defects, in the genes of cytostatin II. In accordance with certain preferred embodiments of this and other aspects of the invention, probes are provided that specifically hybridize to the human cytostatin II sequences. In certain further preferred embodiments of this aspect of the invention, antibodies are provided against the cytostatin II polypeptides. In certain preferred embodiments particularly in this regard, the antibodies are highly selective for human cytostatin II. According to another aspect of the present invention, cytostatin II agonists are provided, such as those which minimize cytostatin II, bind to the cytostatin II receptors and produce the responses induced by cytostatin II. Also among such agonists are those which interact with cytostatin II, or with other modulators or receptors, and thereby enhance the effects of human cytostatin II. According to yet another aspect of the present invention, cytostatin II antagonists are provided. such as those which minimize cytostatin II, bind to cytostatin II receptors but do not produce the responses induced by cytostatin II, and those that bind to, or interact with human cytostatin II to inhibit its effects. Agonists and antagonists can be used to minimize, increase or inhibit the action of such polypeptides, for example, and they can be used in the treatment of disorders associated with the aberrant growth of cells affected by cytostatin, particularly cytostatin. II. Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description. It should be understood, however, that the following description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.

Brief Description of the Drawings The following drawings show certain embodiments of the invention. They are illustrative only and do not limit the invention in another way described herein. Figure 1 shows the nucleotide and deduced amino acid sequence of human cytostatin II.

GLOSSARY The following illustrative explanations are provided to facilitate the understanding of certain terms frequently used herein, particularly in the examples. The explanations are provided as a convenience and not as a limitation of the invention.

DNA DIGESTION refers to the catalytic cleavage of DNA with a restriction enzyme that only acts on certain sequences in DNA. The various restriction enzymes referred to herein are commercially available and their reaction conditions, cofactors and other requirements for their use are already known and routine for the skilled artisan. For analytical purposes, typically 1 μg of the plasmid or DNA fragment is digested with about 2 units of enzyme in about 20 μl of the buffer. For the purpose of isolating the DNA fragments for the construction of the plasmid, typically 5 to 50 μg of the DNA are deferred with 20 to 250 units of enzyme in a proportionally larger volume. Suitable buffers and substrate amounts for particular restriction enzymes are described in standard laboratory manuals, such as those referred to below, and are specified by commercial suppliers. Incubation times of approximately 1 hour at 37 ° C are commonly used, but conditions may vary according to standard procedures, supplier instructions and particular topics of the reaction. After digestion, the reactions can be subjected to electrophoresis directly on a polyacrylamide gel for analysis or to isolate a desired fragment or for both purposes. "GENETIC ELEMENT" generally means a polynucleotide comprising a region encoding a polypeptide or a region that regulates transcription or translation or other processes important for the expression of the polypeptide in a host cell, or a polynucleotide comprising both a region encoding a palpeptide and a region that regulates expression. The genetic elements may be comprised within a vector that replicates as an episomal element; that is, as a molecule physically independent of the genome of the host cell. The elements may be comprised within minimicrosomes, such as those that arise during the amplification of transfected DNA by the selection of methotrexate in eukaryotic cells. The genetic elements may also be comprised within a genome of the host cell; not in its 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 that the material has been altered from its natural state; For example, if it is present in nature, it has been removed from its original environment. For example, a polynucleotide that is naturally present or a polypeptide that is naturally present in a live animal in its natural state, is not "isolated", but the same polynucleotide or polypeptide separated from some or all of the materials coexisting in the natural system is "isolated" when the term is used here.

As . part of, or following isolation, such polynucleotides can be linked to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for example. Polynucleotides isolated, alone or linked to other polynucleotides such as vectors, can be introduced into host cells, in cultures or in whole organisms. Introduced into host cells in culture or in whole organisms, such DNAs could still be isolated, when the term is used here, because they may not be in their naturally occurring form or in their environment. Similarly, polynucleotides and polypeptides may be present in a composition, such as a medium formulation, solutions for the introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for example, which are compositions that are not naturally present, and in them remained isolated polynucleotides or polypeptides within the meaning of this term when they are employed herein. BINDING or UNION refers to the process of forming phosphodiester linkages between two or more polynucleotides, which are more often double-stranded DNAs., Techniques for ligation or binding are well known in the art and protocols for ligation or binding they are described in standard laboratory manuals and references, such as, for example, Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2 / a. Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) and Maniatis et al., Page 146, as cited below. OLIGONUCLEOTIDE (S) refers to relatively short polynucleotides. Most frequently the terms refer to single-stranded deoxyribonucleotides, but can also refer to single-stranded or double-stranded ribonucleotides, to short DNA RNA hybrids and to short double-stranded DNAs, among others. Oligonucleotides, such as the oligonucleotides of the single-stranded DNA probe, are often synthesized by chemical methods, such as those implemented on automated oligonucleotide synthesizers. However, oligonucleotides can be made by a variety of other methods, including techniques mediated by recombinant DNA in vitro and by the expression of DNAs in cells and organisms. Initially, the chemically synthesized DNAs are obtained without a 5 'phosphate. The 5 'ends of such oligonucleotides are not substrates for the formation of the phosphorodiester linkage by the ligation or binding reactions employing the DNA ligases used to form the recombinant DNA molecules. Where ligation or binding of such oligonucleotides is desired, a phosphate may be added by standard techniques, such as those employing a kinase and ATP. The 3 'ends of the chemically synthesized oligonucleotides generally have a free hydroxyl group and, in the presence of a ligase, such as the T4 DNA ligase, readily form phosphodiester bonds with the 5' phosphate of other polynucleotides. As is well known, this reaction can be prevented, where desired, by the 5 'dephosphorylation of other polynucleotides in a reaction. PLASMIDS are generally designated herein by a subscript p preceded and / or followed by capital letters and / or numbers, in accordance with standard naming conventions that are familiar to those skilled in the art. Starting from the fact that the described plasmids are either commercially available, publicly available on an unrestricted basis, or can be constructed from the plasmids available by the routine application of well-known published methods. 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 skilled in the art. In addition, those skilled in the art can easily construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention, will be readily apparent to those skilled in the art from the present disclosure. The POLYUCLEOTIDE (s) generally refer to any polyribonucleotide or polydeoxyribonucleotide, which may be an unmodified RNA or DNA, or modified DNA or RNA. Accordingly, for example, polynucleotides are used herein to refer to, inter alia, single-stranded and double-stranded DNA, DNA is a mixture of the single-stranded and double-stranded regions, single-stranded DNA strand and double strand, and RNA which is a mixture of single-strand and double-strand regions, hybrid molecules comprising DNA and RNA that can be single strand or, more typically, double strand or mix of single-strand and double-strand regions. In addition, the polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions can be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically they involve only one region of any of the molecules. One of the molecules of a triple helical region is often an oligonucleotide. When used here, the term polynucleotide includes the DNAs or RNAs described above, which contain one or more modified bases. Accordingly, DNAs or RNAs with modified skeletons for reasons of stability or for other reasons, are "polynucleotides" as this term is proposed herein. In addition, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name but two examples, are polynucleotides when the term is used herein. It will be appreciated that a wide variety of modifications have been made to DNA or RNA that serve many useful purposes known to those skilled in the art. The term "polynucleotide" when used herein encompasses both chemically, enzymatically or metabolically modified forms of the polynucleotides, as well as the chemical forms of DNA or RNA characteristic of viruses and cells, including simple and complex cells, inter alia.

Description of the invention The present invention relates to novel cytostatin II polypeptides and polynucleotides, inter alia, as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a new human cytostatin II, which is related by an homology of the amino acid sequence to the growth inhibitor derived from the mammary gland ("MDGF") found in cows and the rats. The invention relates especially to the amino acid and polynucleotide sequences of cytostatin II described in Figure 1.

Polynucleotides In accordance with one aspect of the present invention, there are provided isolated polynucleotides which encode the mature polypeptide having the deduced amino acid sequence of Figure 1 or the mature polypeptide encoded by the human cDNA in deposit No. 97287 of the ATCC referred to herein as the "deposited clone". Using the information provided herein, such as the polynucleotide sequence described in Figure 1, a polynucleotide of the present invention encoding the human cytostatin II polypeptide can be obtained using standard cloning and selection methods, such as those for cloning. of the cDNAs that use the mRNA of the epithelial cells as the starting material. Illustrative of the invention, the polynucleotide described in Figure 1 was discovered in a cDNA library derived from mRNA of human fetal brain tissue. The human cytostatin II of the invention is closely related to other proteins of the cytostatin family of growth modulating factors, as shown by the sequencing results of the cDNA encoding human cytostatin II in Deposit No. 97287 of the ATCC . This cDNA sequence, described in Figure 1, contains an open reading frame that encodes a protein of approximately 132 amino acid residues with a molecular weight of about 14.8 kDa. The protein exhibits the highest degree of homology to the growth inhibitor derived from the mouse mammary gland (also called "MDGI"), with which it shares a 64% identity and a similarity of 79% over a length of 132 amino acids. The polynucleotides of the present invention may be in the form of RNA, such as mRNA, or in the form of the APN, including, for example, cDNA and genomic DNA obtained by cloning or produced by synthetic chemical techniques or by a combination of them. The DNA can be single-stranded or double-stranded, and if it is single-stranded, it can be the coding strand or the non-coding strand (antisense). The polynucleotides may have sequences that are naturally present, such as those of the allelic variants that are naturally present, they may have the sequences that have been altered, for example, 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 or that of the deposited clone. It can also 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 or of the deposited cDNA. The polynucleotides of the present invention which encode the polypeptide of Figure 1 or the polypeptide encoded by the deposited cDNA may include, but are not limited to, the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and the sequences. of additional coding, such as those encoding a forward or secretory sequence, such as a pre-, or pro- or preproprotein sequence; the coding sequence of the mature polypeptide, with or without the additional coding sequences mentioned above, together with additional, non-coding sequences, including but not limited to introns and 5 'and 3' non-coding sequences, such as non-translated or translated sequences , transcribed, which play a role in transcription, mRNA processing - including separation and polyadenylation signals, for example - ribosome binding and mRNA stability. In accordance with the foregoing, the term "polynucleotide encoding a polypeptide" when used herein, encompasses polynucleotides which include a sequence encoding a polypeptide of the present invention, particularly human cytostatin II having the amino acid sequence described in Figure 1 or the amino acid sequence of human cytostatin II encoded by the cDNA in ATCC No. 97287. The term encompasses polynucleotides that include a single continuous region or the discontinuous regions encoding the polypeptide, together with additional regions that may also contain coding or ns sequences. coding. The present invention further relates to variants of the polynucleotides described hereinabove, which code for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. A variant of the polynucleotide can be a variant that is naturally present, such as an allelic variant that is naturally present, or it can be a variant that is not known to be naturally present. Such variants that are not naturally present in the polynucleotides can be made by mutagenesis techniques, including those applied to the polynucleotides, cells or organisms. The present invention includes polynucleotides that encode the same mature polypeptide shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone. In addition, the invention includes variants of such polynucleotides that encode a fragment, derivative or analogue of the polypeptide of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. Among the variants in this regard are variants that differ from the polynucleotides mentioned above by substitutions, deletions or additions of nucleotides. Substitutions, deletions or additions may involve one or more nucleotides. The variants can be altered in the coding or non-coding regions or in both. Alterations in the coding regions can produce substitutions, deletions or additions of amino acids, conservative or non-conservative. Variants of the invention may have a sequence that is present in nature or may have a sequence that is not naturally present. As indicated hereinabove, the polynucleotide may have a coding sequence which is an allelic variant that is naturally present, of the coding sequence shown in Figure 1 or of the coding sequence of the deposited clone. As is known in the art, an allelic variant is an alternative form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides. Among the preferred embodiments particularly of the invention in this regard are the polynucleotides that encode the polypeptides having the amino acid sequence of cytostatin II described in Figure 1 or the amino acid sequence of cytostatin II of the cDNA of the deposited clone; the variants, analogs, derivatives and fragments thereof, and the -fragments of the variants, analogs and derivatives. Additionally, polynucleotides encoding the variants, analogs, derivatives and fragments of cytostatin II, and the variants, analogs and derivatives of the fragments, which have the amino acid sequence of the cytostatin II polypeptide are particularly preferred in this respect. Figure 1 or of the reservoir in which several, a small value of 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residue are substituted, deleted or added, in any combination. Especially preferred among these are substitutions, additions and inactive deletions, which do not alter the properties and activities of cytostatin II. In this respect, conservative substitutions are also particularly preferred. Polypeptides having the amino acid sequence of Figure 1 or the deposit are more preferred., without substitutions. Preferred embodiments further of the invention are polynucleotides that are over 85% identical to a polynucleotide encoding the polypeptide of cytostatin II having the amino acid sequence described in Figure 1, or variants, close homologues, derivatives and analogs of them, as described above. Alternatively, polynucleotides comprising a region that is more than 85% identical to a polynucleotide encoding the cytostatin II polypeptide of the deposited clone cDNA are more preferred. In this regard, polynucleotides that are more than 90% identical thereto are particularly preferred, and among these particularly preferred polynucleotides, those with 95% or more identity are especially preferred. In addition, those with 97% or more of identity are highly preferred among those with 95% or more of identity, and among these, those with 98% or more and 99% or more of identity are highly preferred in particular, with 99% or more that is more preferred. Also particularly preferred in this regard are polynucleotides that encode a polypeptide having the amino acid sequence of the cytostatin described in Figure 1 or the deposited clone. As described elsewhere herein, the polynucleotide may encode the polypeptide in a continuous region or a plurality of two or more discontinuous exons, and it may also comprise additional regions, which are not related to the region or regions coding. Are most highly preferred in this regard are polynucleotides that comprise a region that is more than 85% identical to the portion encoding the cytostatin II polirucleótido described in Figure 1. Alternatively, most highly preferred are polynucleotides that comprise a region that is more than 85% identical to the portion encoding cytostatin II of the cDNA of the deposited clone. Among such polynucleotides, those more than 90% identical thereto are particularly preferred, and, among these particularly preferred polynucleotides, those with 95% or more identity are especially preferred. In addition, those with 97% or more of identity are highly preferred among those with 95% or more of identity, and among those those with 98% or more and 99% or more of identity are highly preferred in particular, with 99% or more that is the most preferred of these. The present invention also includes polynucleotides in which the sequence encoding the mature polypeptide is fused in the same reading frame with respect to the additional sequences. Such sequences include signal sequences, which facilitate the transport of the nascent protein in the endoplasmic reticulum, prosequences are associated with inactive precursor forms of the polypeptide, which can facilitate protein trafficking in a cell or outside a cell or can improve the persistence of the protein in a cell or in an extracellular compartment. Such sequences can also be added to facilitate production and purification, or to add additional functional domains, as described elsewhere herein. Accordingly, the polynucleotides of the invention may, in addition to a mature citostina encode, particularly cytostatin II, for example, a leader sequence or manager, such as a signal peptide which functions as a secretory sequence for controlling transport polypeptide to the lumen of the endoplasmic reticulum. The forward or leader sequence can be removed from the host cell, as is generally the case for the signal peptides, producing another precursor protein or the mature polypeptide. A precursor protein that has a forward sequence is often called a preprotein. The polynucleotides can also encode a polypeptide which is the mature protein plus the additional amino or carboxyl terminal amino acids, or the amino acids inside the mature polypeptide (when the mature form has more than one polypeptide chain, for example). Such sequences may play a role in processing a protein from the precursor to a mature form, they may facilitate trafficking or transport of the protein, they may prolong or shorten the half-life of the protein or they may facilitate the manipulation * of a protein for the protein. evaluation or production, among other things. As is generally the case in situ, additional amino acids can be processed separately 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. When the prosequences are removed, such inactive precursors are usually activated. Some or all of the prosequences can be removed before activation. In general, such precursors are called proproteins. In summary, a polynucleotide of the present invention can encode a mature protein, a mature protein plus a leader or leader sequence (which can be referred to as a preprotein), a precursor of a mature protein having one or more prosequences which do not are the forward or directing sequences of a preprotein, or a preprotein, which is a precursor to a proprotein, which has a forward or direct sequence and one or more prosequences, which are generally removed during the processing steps that produce the forms active and mature polypeptides.

A polynucleotide of the present invention can encode a mature pre-, or prepropolypeptide or precursor as described above, inter alia, fused to additional amino acids, such as those which provide additional functionalities. Thus, for example, the polypeptide can be fused to a marker sequence, such as a peptide, which facilitates the purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as the label provided in the pQE-9 vector, among others, many of which are commercially available. As described in Gentz et al., Proc. Nati Acad. Sci., USA 86: 821-824 (1989), for example, hexa-histidine provides convenient purification of the fusion protein. Typically, it does not adversely affect the structure or function of the protein, and binds efficiently, selectively and closely to the metal chelate resins, particularly the nickel chelate resins. For example, as is well known, hexa-histidine tags often bind particularly well to nickel-NTA resin, which is well known and readily available, and can be obtained commercially, for example, from Qiagen. In addition, the metal-histidine interaction is not only stable for a variety of conditions useful for removing non-specifically bound material, but also the fusion polypeptide can be bound and removed under non-denaturing, mild conditions. The hexa-histidine tag may be more conveniently fused to the amino or carboxyl terminus of the cytostatin polypeptide. A label of the hexa-histidine type is particularly useful for bacterial expression. Another marker sequence useful in certain other preferred embodiments is a hemagglutinin ("HA") tag, particularly when a mammalian cell is used for expression; for example, COS-7 cells. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by ilson et al., Cell 37: 767 (1984), for example.

Probes The present invention further relates to polynucleotides that hybridize to the cytostatin sequences described hereinabove, particularly the cytostatin 2 sequences. Polynucleotides having at least 50% identity to the described sequences are preferred in this regard. here before Particularly preferred are sequences that. They have at least 70% identity. In this regard, the present invention relates especially to polynucleotides which hybridize under stringent conditions to the polynucleotides described hereinbefore. When used herein, the term "stringent conditions" means that hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. Particularly preferred embodiments in this regard, furthermore, are the polynucleotides which hybridize to the polynucleotides described above and encode the polypeptides which retain substantially the same function or biological activity as the mature polypeptide encoded by the cDNA of Figure 1 or the CDNA of the deposited clone.

Deposited Materials A deposit containing a human cytostatin II cDNA was deposited with the American Type Culture Collection ("ATCC") on September 26, 1995. The deposit, which has been given the number 97287, is referred to herein as "the deposited clone. " The deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the purposes of the Patent Procedure. This deposit is provided only as a convenience to those skilled in the art and is not an indication or an admission that a deposit is required for authorization or authorization., such as that required under 35 U.S.C. § 112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded herein, without being incorporated herein by reference and being controlling in the case of any conflict with any description of the sequences herein. . A license may be required to take, use or sell the deposited materials, and none of these licenses is granted hereby.

Polypeptides The present invention further relates to a human cytostatin II polypeptide which has the deduced amino acid sequence of Figure 1 or which has the amino acid sequence encoded by the deposited clone. The invention also relates to fragments, analogs and derivatives of these polypeptides.

The terms "fragment", "derivative" and "analogue" when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide that retains essentially the same function or biological activity of such a polypeptide. Accordingly, 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 can be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In certain preferred embodiments it is a recombinant polypeptide. The fragment, derivative or analogue of the polypeptide of Figure 1 or that encoded by the cDNA in the deposited clone can 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 a substituted amino acid residue may or may not be 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 to another compound, such as a compound to increase the half-life of the polypeptide (eg, 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 the purification of the mature polypeptide or a sequence of proprotein. Such fragments, derivatives and the like are considered to be within the reach of those skilled in the art from the teachings herein. Among the preferred embodiments particularly of the invention in this regard are polypeptides having the amino acid sequence of cytostatin II described in Figure 1, variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments. Alternatively, the preferred embodiments particularly of the invention in this regard are polypeptides having the amino acid sequence of cytostatin II of the cDNA in the deposited clone, variants, analogs, derivatives and fragments thereof, and variants , the analogues and the derivatives of the fragments. Particularly preferred in this regard are variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments, having the amino acid sequence of the cytostatin II polypeptide of Figure 1 or CDNA in the deposited clone, in which several, from a small number, from 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are substitutions, additions and inactive deletions, which do not alter the properties and activities of cytostatin II. Also, conservative substitutions are especially preferred in this respect. Polypeptides having the amino acid sequence of Figure 1 or the clone deposited without the substitutions are more highly preferred. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. The term "isolated" means that the material has been altered from its natural state; for example, that, if it is present in nature, then it has been removed from its original environment. For example, a polynucleotide that is present naturally or polypeptide that is naturally present in a living animal in its natural state, is not "isolated", but rather the same polynucleotide or polypeptide separated from some or all of the materials coexisting in the natural system is "isolated", when the term is used here.

As part of, or following isolation, such polynucleotides can be linked to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for example. Isolated polynucleotides, alone or linked to the other polynucleotides such as vectors, can be introduced into host cells, in cultures or in whole organisms. Introduced into host cells in the culture or in whole organisms, such cDNAs would still be isolated, when the term is used here, because they may not be in their naturally occurring form or in their environment . Similarly, polynucleotides and polypeptides may be present in a composition, such as a medium formulation, a solution for introduction into cells, a composition or solution for the chemical or enzymatic reaction, and the like, which are not compositions that are naturally present, and there remain isolated polynucleotides or polypeptides within the meaning of this term when it is employed herein.

Vectors, host cells, expression The present invention also relates to vectors which include the polynucleotides of the present invention, the host cells which are genetically engineered with the vectors of the invention and the production of the polypeptides of the invention by recombinant techniques. The host cells can be genetically engineered to incorporate the polynucleotides and to express the polypeptides of the present invention. For example, polynucleotides can be introduced into host cells using well known techniques of infection, transduction, transfection, transvection and transformation. The polynucleotides can be introduced alone or with other polynucleotides. Such other polynucleotides may be introduced independently, co-introduced or introduced attached to the polynucleotides of the invention. Thus, for example, the polynucleotides of the invention can be transfected into host cells with another separate polynucleotide encoding a selectable marker, using standard techniques for cotransfection and selection in, for example, mammalian cells. In this case the polynucleotides will generally be stably incorporated into the genome of the host cell. Alternatively, the polynucleotides can be linked to a vector that contains a selectable marker for propagation in a host. The construction of the vector can be introduced into the host cells by the techniques mentioned above. In general, a plasmid vector is introduced as the DNA in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. Electroporation can also be used to introduce the polynucleotides into a host. If the vector is a virus, it can be packed in vitro or introduced into a packaging cell and the packaged virus can be transduced into the cells. A wide variety of techniques suitable for making polynucleotides and for introducing polynucleotides into cells according to this aspect of the invention are well known and routine to those skilled in the art. Such techniques are reviewed extensively in Sambrook et al., Cited elsewhere, which are illustrative of many of the laboratory manuals that detail these techniques. According to this aspect of the invention the vector can be, for example, a plasmid vector, a single-strand or double-stranded phage vector, a viral vector of. DNA or single-stranded or double-stranded RNA. Such vectors can be introduced into cells as polynucleotides, preferably DNA, by well-known techniques for introducing DNA and RNA into cells. The vectors, in the case of phage and viral vectors, can also be and preferably are introduced into cells as a 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 will generally only occur in complement host cells. Preferred among the vectors, in certain aspects, are those for the expression of the polynucleotides and polypeptides of the present invention. In general, such vectors comprise regions of cis-acting control for expression in a host operably linked to the polynucleotide to be expressed. Appropriate trans-drive factors are either supplied by the host, supplied by a complementary vector or supplied by the vector itself during introduction into the host. In certain preferred embodiments in this regard, the vectors provide specific expression. Such a specific expression may be the expression or the inducible expression only in certain cell types or both inducible and specifically for the cells. 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 for this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and routinely employed by those skilled in the art. The engineered host cells can be cultured in the conventional nutrient medium, which can be modified where appropriate to, inter alia, activate the promoters, select the transformants or amplify the genes. The culture conditions, such as temperature, pH and the like, previously used with the host cell selected for expression will generally be suitable for the expression of the polypeptides of the present invention as will be apparent to those skilled in the art. A wide variety of expression vectors can be used to express a polypeptide of the invention. Such vectors include chromosomal, episomal and virus derivatives, for example, vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from chromosomal elements from yeast, from viruses such as baculoviruses, papoviruses such as SV40, vaccinia virus, adenovirus, contagious epithelioma virus, pseudorabies virus and retrovirus, and vectors derived from combinations thereof, such as those derived from the genetic elements of the plasmid and the bacteriophage, such as cosmids and phagemids, all can be used for expression according to this aspect of the present invention. In general, any vector suitable for maintaining, propagating or expressing the polynucleotides to express a polypeptide in a host can be used for expression in this regard. The appropriate DNA sequence can be inserted into the vector by any of a variety of routine and well-known techniques. In general, a DNA sequence for expression is linked to an expression vector by cleavage or separation of the DNA sequence and the expression vector with one or more restriction endonucleases and then joining the restriction fragments together using the T4 DNA ligase. The methods for restriction and linkage that can be used for this purpose are well known and routine for those skilled in the art. Suitable methods in this regard, and for the construction of expression vectors using alternative techniques, which are also well known and routine by those skilled in the art, are described in greater detail in Sambrook et al., Cited elsewhere. part here. The DNA sequence in the expression vector is linked or operably linked to the appropriate expression control sequence (s), including, for example, a promoter to direct transcription of the mRNA. Representative elements of such promoters include the phage lambda PL promoter, the E. coli lac, the trp and tac promoters, the SV40 initial and final promoters and the retroviral LTR promoters, to name just a few of the well-known promoters. It will be understood that numerous promoters not mentioned, which are suitable for use in this aspect of the invention, are well known and can be readily employed by those skilled in the art in the manner illustrated by the description and examples herein. In general, expression constructs will contain sites for the initiation and termination of transcription and, in the transcribed region, a ribosome binding site for translation or translation. The coding portion of the mature transcribed elements expressed by the constructs will include an AUG of initiation of translation or translation at the start and end codon appropriately positioned at the end of the polypeptide to be translated or translated. In addition, the constructions may contain the control regions that regulate as well as engender the expression. In general, according to. Many commonly practiced procedures, such regions will operate by controlling transcription, such as sites and union enhancers of repressors, among others. Vectors for propagation and expression will generally include selectable markers. Such markers may also be suitable for amplification or the vectors may contain additional markers for this purpose. In this regard, expression vectors preferably contain one or more selectable marker genes to provide a phenotypic character for the selection of transformed host cells. Preferred markers include the dihydrofolate reductase or neomycin resistance genes for the culture of eukaryotic cells, and the tetracycline or ampicillin resistance genes for the culture of 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, can be introduced into an appropriate host using a variety of well-known techniques, suitable for expression in the same of a desired polypeptide. Representative examples of the 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. Guests for a wide variety of expression constructs are already known, and those skilled in the art will be enabled by the present disclosure to readily select a host for the expression of a polypeptide in accordance with this aspect of the present invention. More particularly, the present invention also includes recombinant constructs, such as expression constructs, that comprise one or more of the sequences described above. The constructs comprise a vector, such as a plasmid or a viral vector, within which such a sequence of the invention has been inserted. The sequence can be inserted in a forward or inverse orientation. In certain preferred embodiments in this regard, the construct further comprises the regulatory sequences, which include, for example, a promoter, operably linked to the sequence. Large numbers of 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. The following vectors, which are commercially available, are provided by way of example. Among the preferred vectors for use in bacteria are pQE70, pQE60 and PQE-9, available from Qiagen; pBs, pDlO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and? trc99a, pKK223-3, pKK233-3,? DR540, pRIT5 available from Pharmacia. Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed only by way of illustration of the many commercially available and well known vectors that are available to those skilled 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, the introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host can be used in this aspect of the invention. Promoter regions can be selected from any desired gene that utilizes vectors that contain a reporter transcription unit that lacks a promoter region, such as a transcription unit of chloramphenicol acetyl transferase ("cat"), downstream of a site or restriction sites for the introduction of a candidate promoter fragment; that is, a fragment that may contain a promoter. As is well known, the introduction into the vector of a fragment containing the promoter at the restriction site upstream of the cat gene engenders the production of CAT activity, which can be detected by standard CAT assays. Vectors suitable for this purpose are well known and readily available. Two such vectors are pKK232-8 and pCM7. Accordingly, promoters for the expression of the polynucleotides of the present invention include not only well-known and readily available promoters, but also promoters that can be readily obtained by the prior art, using a reporter gene. Among the known bacterial promoters suitable for the expression of the polynucleotides and the polypeptides according to the present invention are the lacZ and lacZ promoters of E. coli, the promoters of T3 and T7, the gpt promoter, the lamba promoters. PR, PL and the trp promoter. Among the known eukaryotic promoters, suitable in this regard are the immediate initial promoter of CMV, the HSV thymidine kinase promoter., the initial and final SV40 promoters, the promoters of the retroviral LTRs, and the metallothionein promoters such as the mouse I-metallothionein promoter. The selection of appropriate vectors and promoters for expression in a host cell is a well-known procedure and the techniques required for the construction of the expression vector, the introduction of the vector into the host and expression in the host are of the Routine experience in the technique. The present invention also relates to host cells containing the constructions described 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. The introduction of the construction into the host cell can be effected by calcium phosphate transfection, dextran-DEAE mediated transfection, 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 the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be produced synthetically 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 or translation systems can also be employed to produce such proteins using the RNAs derived from the DNA constructs of the present invention. Expression and cloning vectors suitable for use with prokaryotic and eukaryotic hosts are described by Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2 / a. Ed. Cold Spring Harbor Laboratory Dam, Cold Spring Harbor, N. Y. (1989). In general, the recombinant expression vectors will include the origins of replication, a promoter derived from a gene highly expressed for the direct transcription of a downstream structural sequence, and a selectable marker to allow isolation of the vector containing the cells after the exposure to the vector. Suitable promoters include those derived from genes encoding glycolytic enzymes such as 3-phosphoglycerate kinase ("PGK"), factor a, acid phosphatase, and proteins for heat shock, among others. Selectable markers include the penicillin resistance gene of E. coli and the trpl gene of S. cerevisiae. The transcription of the DNA encoding the polypeptides of the present invention by the higher eukaryotes can be increased by the insertion of an enhancer sequence into the vector. Enhancers are cis-acting or driving elements of DNA, usually around 10 to 300 bp which act to increase the transcriptional activity of a promoter in a given host cell type. Examples of the enhancers include the SV40 enhancer, which is located on the final side of the replication origin in base pairs (bp) 100 to 270, the cytomegalovirus initial promoter enhancer, the polyoma enhancer on the final side of the origin of the replication, and the adenovirus breeders. The polynucleotides of the invention, which encode the heterologous structural sequence of a polypeptide of the invention, will generally be inserted into the vector using standard techniques so that it is operably linked or linked to the promoter for expression. The polynucleotide will be positioned so that the transcription initiation site is located appropriately 5 'to the ribosome binding site. The ribosome binding site will be 5 'to the AUG that initiates the translation or translation of the polypeptide to be expressed. In general, there will be no other open reading frames that start with the initiation codon, usually UAG, and are between the ribosome binding site and the initiation AUG. Also in general, there will be a stop codon for translation or translation to. end of the polypeptide and there will be a polyadenylation signal and a transcription termination signal properly positioned at the 3 'end of the transcribed region. For the secretion of the translated or translated protein in the lumen of the endoplasmic reticulum, in the periplasmic space or in the extracellular environment, the appropriate secretion signals can be incorporated into the expressed polypeptide. The signals can be endogenous with respect to the polypeptide and they can be heterologous signals. The polypeptide can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals but also additional heterologous functional regions. Thus, for example, 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. Also, the region can be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final preparation of the polypeptide. The addition of portions of the peptide to the polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among other things, are familiar and routine techniques in the art. Prokaryotic hosts suitable for propagation, maintenance or expression of the polynucleotides and polypeptides according to the invention, include Escherischia coli, Bacillus subtilis and Salmonella typhimurium. Several species of Pseudomonas, Streptomyces, and Staphylococcus are suitable hosts in this regard. In addition, many other guests also known to those skilled in the art can be employed in this regard. As a representative but non-limiting example, expression vectors useful for bacterial use may comprise a selectable marker and the bacterial origin of replication derived from commercially available plasmids comprising the genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMI (Promega Biotec, Madison, Wl, USA). These "skeleton" sections of pBR322 are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of an appropriate host strain and growth of the host strain to an appropriate cell density, wherein the selectable promoter that is inducible is induced by the appropriate means (e.g., temperature shift or exposure to the host). chemical inducer) and the cells are cultured for an additional period. The cells are then typically collected by centrifugation, broken or altered by physical or chemical means, and the resulting crude extract retained for further purification. The microbial cells used in the expression of proteins can be broken or altered by any convenient method, including freeze-thaw cyclization, sound application, mechanical alteration or rupture, or the use of agents for the lysis of cells, such methods are well known to those skilled in the art. Various culture systems of mammalian cells can also be used for expression. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described in Gluzman, Cell 2_3: 175 (1981). Other cell lines capable of the expression of a compatible vector include for example, cell lines C127, 3T3, CHO, HeLa and BHK. The expression vectors of mammals will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, acceptor and donor sites of the splices, transcriptional termination sequences, and the non-transcribed flanking sequences of 5 ', which are necessary for expression. In certain preferred embodiments for this purpose, the DNA sequences derived from the SV40 splice sites, and the SV40 polyadenylation sites are used for the required non-transcribed genetic elements of these types. The cytostatin II polypeptide can be recovered and purified from the cultures of the recombinant cells by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anionic or cation exchange chromatography, chromatography of phosphocellulose, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. High-performance liquid chromatography ("HPLC") can also be used especially for the final steps of purification. Well-known techniques for refolding the protein can be employed to regenerate the active conformation when the polypeptide is denatured during isolation and / or purification. The polypeptides of the present invention include naturally purified products, products of synthetic chemical processes, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast cells. , of higher plants, insects and mammals. Depending on the host employed in a recombinant production process, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, the polypeptides of the invention may also include a modified methionine residue, in some cases as a result of the processes mediated by the host.

Additional illustrative applications The cytostatin II polynucleotides and polypeptides can be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of cytostatin II. Among these applications in the characterization of cells and organisms and in the growth or development of cells and organisms. Additional applications refer to the diagnosis or treatment or disorders of cells, tissues and organisms. Accordingly, inter alia, the cytostatin II growth inhibitory and growth differentiating activity is useful for inhibiting growth and stimulating the differentiation of tumorigenic cells, such as tumorigenic cells in vitro, as well as for treatment purposes. The same activities can be applied for the treatment of the growth of aberrant cells in an organism, such as the cells of a tumor. In this regard, the cytostatin II polypeptides are preferred, particularly cytostatin II having the amino acid sequence described in Figure 1 or the amino acid sequence of cytostatin II of the cDNA of the deposited clone. Similarly, the ability of cytostatin II to inhibit the growth of endothelial cells, such as endothelial cells of venules, can be used to prevent, alter or slow angiogenesis in culture or in situ. In a related vein, since tumorigenic cells at sites of metastasis, as well as those at an original site, must attract new blood vessels for growth, inhibition of cytostatin II from endothelial cells of venules may be useful to reduce the metastatic potential or to slow the progression of metastatic disease. In addition, the activity of cytostatin II that inhibits the growth of mammary epithelial cells and the differentiation of mammary gland modulation can also be used. to promote the formation of alveolar buds, help the development of differentiated lobuloalveoli, and stimulate the production of milk protein and the accumulation of fat droplets. Such lactation-stimulating activity can aid milk production in mammals that produce commercial milk, and may be useful in aiding the production of milk by human mothers, for example. In a related application, the modulating activity of cytostatin II that affects breast size may be useful in helping to return from an enlarged breast to normal size after parturition or delivery. Inhibition of cytostatin activity II, for example, by antisense phosphorothioates or antibodies, may be useful for the selective inhibition of endogenous cytostatin II activity in mammary epithelial cells to suppress the appearance of the buds of the extremities or alveolar terminals and to reduce the level of beta-casein. As described below, these and other activities and properties of the cytostatin II polynucleotides and polypeptides of the invention have various applications and uses in numerous fields including applications involving the growth of cells in vitro, commercial production of milk and the products of milk, and the diagnosis and treatments that are related to the fields of oncology, cardiology, immunology, endocrinology, hematology, metabolic disorders, musculoskeletal problems and gynecology and obstetrics, to name just a few. Full-length cytostatin II cDNA can be used partially or completely as a hybridization probe for cDNA and genomic DNA to isolate full-length cDNAs and genomic clones that encode cytostatin II and to isolate the cDNA and genomic clones of other genes that have a high sequence similarity to the human cytostatin II gene. Such probes generally have at least 20-bases. Preferably, however, the probes have at least 30 bases and do not exceed 50 bases. Such probes can also be used to identify additional cDNA clones corresponding to a full-length transcript and a clone or genomic clones containing the complete human cytostatin II gene, including the regulatory and promoter regions, the exons, and the introns. For example, the coding region of the cytostatin II gene can be isolated by screening or screening using the known DNA sequence to synthesize an oligonucleotide probe. Once an oligonucleotide having a sequence complementary to that of a gene of the present invention has been labeled, it is then used to select a library of human cDNA, genomic DNA or genomic mRNA to determine for which members of the library hybridizes the probe. The polynucleotides and polypeptides of the present invention can be used as reagents and search materials to discover treatments and diagnostics for human diseases.

Binding molecules of Cytostatine II This invention also provides a method for the identification of molecules, such as receptor molecules, that bind to cytostatin II. The proteins encoding the genes, which bind to cytostatin II, such as the receptor proteins, can be identified by numerous methods known to those skilled in the art, for example, the frame or arrangement of the ligands and the classification of FACS. Such methods are described in many laboratory manuals such as, for example, Coligan et al., Current Protocols in Immunology 1_ (2): Chapter 5 (1991).

For example, the cloning of the expression can be used for this purpose. For this purpose the polyadenylated RNA is prepared from a cytostatin II response cell, a cDNA library is created from this RNA, the library is divided into groups or sets and the groups or sets are transfected individually into cells that do not work in response to cytostatin II. The transfected cells are then exposed to a labeled cytostatin II. (Cytostatin II can be labeled by a variety of well known techniques including standard methods of radioiodination or the inclusion of a site recognition site-specific protein kinase). Following the exposure, the cells are fixed and the binding of the cytostatin is determined. These procedures are conveniently carried out on glass slides. The groups or sets are identified from the cDNA that produced the binding cells of the cytostatin II produced. The subgroups are prepared from those that are positive, transfected into the host cells and selected as described above. Using a process of reselection and iterative reassortment, one or more unique clones encoding putative or putative binding molecules, such as a receptor, can be isolated.

Alternatively, a labeled ligand can be linked or linked by photoaffinity to a cell extract, such as a membrane or a membrane extract, prepared from cells expressing a molecule to which they bind, such as a receptor molecule. The crosslinked material is resolved by electrophoresis of the polyacrylamide gel ("PAGE") and exposed to an X-ray film. The tagged complex containing the ligand-receptor can be cleaved, resolved into fragments of the peptide, and subjected to the microsequencing of the protein. The amino acid sequence obtained from microsequencing can be used to design single or degenerate oligonucleotide probes to select cDNA libraries to identify the genes encoding the putative receptor. The polypeptides of the invention can also be used to evaluate the binding capacity of cytostatin II of the cytostatin II binding molecules, such as the receptors, in the cell preparations or in the free preparations of the cells.

Agonists and antagonists and tests or evaluations for them The invention provides a method of selecting the compounds to identify those which enhance or block the action of cytostatin II on cells, such as their interaction with the cytostatin II binding molecules as the receptors. An agonist is a compound which increases the natural biological functions of cytostatin II, although antagonists reduce or eliminate such functions. For example, a cell compartment, such as a membrane or a preparation thereof, such as a membrane preparation, can be prepared from a cell that expresses a molecule that binds cytostatin II, such as a molecule of a regulatory or signaling pathway modulated by cytostatin II. The preparation is incubated with cytostatin II labeled in the absence or presence of a candidate molecule which may be an agonist or antagonist of cytostatin II. The ability of the candidate molecule to bind to the binding molecule is reflected in the reduced binding of the tagged ligand. Molecules that come together for free, that is, without inducing the effects of cytostatin II during the binding of the cytostatin II binding molecule, are more likely to be good antagonists. Molecules bond well and produce effects that are the same or that closely resemble each other. The cytostatin II-like effects of potential agonists and antagonists can be measured, for example, by determining the activity of a second messenger system following the interaction of the candidate molecule with an appropriate cell or cell preparation, and comparing the effect with that of cytostatin II or molecules that produce the same effects as cytostatin II. The second messenger systems that may be useful in this regard include but are not limited to the second messenger systems of the hydrolysis of the AMP guanylate cyclase, the ion channel or the phosphoinositide. Another example of an assay or evaluation for cytostatin II antagonists is a competitive assay that combines cytostatin II and a potential antagonist with cytostatin II receptors bound to the membrane or recombinant cytostatin II receptors under appropriate conditions. a competitive inhibition assay. Cytostatin II can be labeled, such as by radioactivity, such that the number of cytostatin II molecules bound to the receptor can be accurately determined to assess the effectiveness of the potential antagonist. Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention and whereby their activity is inhibited or extinguished. Potential antagonists may also be small organic molecules, a peptide, a polypeptide such as a protein or closely related antibody that binds to the same sites on the binding molecule, such as a receptor, without inducing the activities induced by cytostatin II. , whereby the action of cytostatin II is prevented by excluding cytostatin II from the union. Potential antagonists include a small molecule which binds to and occupies the polypeptide binding site whereby binding to cell binding or agglutination molecules, such as receptors, is prevented so that normal biological activity is prevented . Examples of the small molecules include but are not limited to small organic molecules, peptides or molecules similar to peptides. Other potential antagonists include antisense molecules. The antisense technology can be used to control the expression of the gene through antisense DNA or RNA or through the formation of a triple helix. Antisense techniques are described, for example, in-Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXINUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988). The formation of the triple helix is described, for example, in Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). The methods are based on the binding of a polynucleotide to a complementary DNA or RNA. For example, the 5 'coding portion of a polynucleotide encoding the mature polypeptide of the present invention can be used to design an antisense RNA oligonucleotide 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, whereby the transcription and production of cytostatin II is prevented. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks the translation or translation of the mRNA molecule into the cytostatin II polypeptide. The oligonucleotide described above can also be delivered to the cells in such a way that the antisense RNA or DNA can be expressed in vivo to inhibit the production of cytostatin II.

Antagonists can be employed in a composition with a pharmaceutically acceptable carrier, for example, as described hereinafter. Antagonists can be used, for example, to treat hypertrophy of cardiac myocytes or leukemia.

Compositions The invention also relates to compositions comprising the polynucleotide or the polypeptides described above or the agonists or antagonists. Accordingly, the polypeptides of the present invention can be used in combination with a sterile or non-sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject or patient. Such compositions comprise, for example, an additive for the medium 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, a saline solution, a buffered saline solution, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation must be adapted to the mode of administration.

Games or Sets The invention further relates to pharmaceutical kits or packages 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), there may be a notice in the form prescribed by a governmental agency that regulates the manufacture, use or sale of pharmaceutical or biological products, which reflect the approval by the agency for manufacturing, the use or sale of the product for human administration.

Administration The polypeptides of the present invention can be used alone or in conjunction with other compounds, such as the therapeutic compounds. The pharmaceutical compositions can be administered in any effective, convenient manner, including, for example, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes, among others.

The pharmaceutical compositions are generally administered in an amount effective for the treatment or prophylaxis of a specific indication or indications. In general, the compositions are administered in an amount of at least about 10 μg / kg body weight. In most cases they will be administered in an amount that does not exceed approximately 8 mg / kg of body weight per day. Preferably, in most cases, the dose is from about 10 μg / kg to about 1 mg / kg of body weight, daily. It will be appreciated that the optimal dosage will be determined by standard methods for each modality and indication of the treatment, taking into account the indication, its severity, the route of administration, the conditions of complication and the like.

Gene Therapy The cytostatin II polynucleotides, polypeptides, agonists and antagonists which are polypeptides, can be used according to the present invention for the expression of such polypeptides in vivo, in the treatment modalities frequently referred to as "gene therapy". .

Thus, for example, the cells of a patient can be designed with a polynucleotide, such as a DNA or RNA, which encodes an ex vivo polypeptide, and the designed cells can then be provided to a patient to be treated with the polypeptide . For example, the cells can be designed ex vivo by the use of a retroviral plasmid vector containing the RNA encoding a polypeptide of the present invention. Such methods are well known in the art and their use in the present invention will be apparent from the teachings herein. Similarly, cells can be designed in vivo for the expression of a polypeptide in vivo by methods known in the art. For example, a polynucleotide of the invention can be designed for expression in a defective retroviral vector of replication, as described above. The construction of the retroviral expression can be isolated and then introduced into a packaging cell that is transduced with a retroviral plasmid vector containing the RNA encoding a polypeptide of the present invention such that the packaging cell now produces viral particles. infectious that contain the gene of interest. These producer cells can be administered to a patient to design cells in vivo and the expression of the polypeptide in vivo. These and other methods of administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. Retroviruses from which the vectors of the retroviral plasmids mentioned hereinabove can be derived include, but are not limited to, Moloney Murine Leukemia Virus, the spleen necrosis virus, retroviruses such as the Virus of Rous sarcoma, Harvey's sarcoma virus, bird leukosis virus, gibbon monkey leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from the Moloney Murine Leukemia Virus. Such vectors also include one or more promoters for the expression of the polypeptide. Suitable promoters which may be employed include, but are not limited to, the retroviral ITR; 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 cell promoters include, but are not limited to , histone, polymerase III of RNA, and promoters of β-actin). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and parvovirus B19 promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein. The nucleic acid sequence encoding the polypeptide of the present invention will be placed under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major final 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; the promoters of thermal shocks, the promoter of albumin; the ApoAI promoter; the promoters of human globin; the promoters of viral thymidine kinase, such as the thymidine kinase promoter of Herpes Simplex; the retroviral LTRs (including the modified retroviral LTRs described herein above); the β-actin promoter; and the promoters of human growth hormones. The promoter may also be the natural promoter which controls the gene encoding the polypeptides. The retroviral plasmid vector is used to transduce the packaging cell lines to form the producer cell lines. Examples of packaging cells that can be transfected include, but are not limited to, the cell lines of PE501, PA317,? -2,? -AM, PA12, T19-14X, VT-19-17-H2,? CRE, CRIP, GP + E-86, Gp + envAml2, and DAN as described in Miller, A., Human Gene Therapy 1: 5-14 (1990). The vector can be transduced into the packaging cells by any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and precipitation with CaP0. In an alternative, the retroviral plasmid vector can be encapsulated in 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 can then be used to transduce the eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells express the sequence (s) of nucleic acids encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as haematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells , and bronchial epithelial cells.

Polynucleotide assays This invention is also related to the use of cytostatin II polynucleotides to detect complementary polynucleotides such as, for example, a diagnostic reagent. Detection of the mutated form of cytostatin II provides a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to a disease resulting from under-expression, overexpression or altered expression of cytostatin II, such as, for example, breast cancer. Individuals that carry mutations in the human cytostatin II gene can be detected at the DNA level by a variety of techniques. The nucleic acids for diagnosis can be obtained from the cells of a patient, such as blood, urine, saliva, tissue biopsies and autopsy material. Genomic DNA can be used directly for detection or can be amplified enzymatically using PCR (Saiki et al., Nature, 324: 163-166 (1986)) prior to analysis. RNA or cDNA can also be used in the same routes. As an example, PCR primers complementary to the nucleic acid encoding cytostatin II can be used to identify and analyze the expression and mutations of cytostatin II. For example, deletions and insertions can be detected by a change in the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing the amplified DNA to the radiolabeled cytostatin II RNA or alternatively, the antisense DNA sequences of the radiolabeled cytostatin II. The paired or perfectly corresponding sequences can be distinguished from the duplicates not corresponding or not adjusted by the RNase A digestion or by differences in the melting temperatures. The sequence differences between a reference gene and the genes that have the mutations can also be relieved by direct DNA sequencing. In addition, the cloned DNA segments can be used as probes to detect specific DNA segments. The sensitivity of such methods can be greatly improved by the appropriate use of PCR or other amplification method. For example, a sequencing primer is used with the double-stranded PCR product or a model molecule or single-stranded matrix generated by the modified PCR. The determination of the sequence is carried out by conventional procedures with the radiolabelled nucleotide or by automatic sequencing procedures with fluorescent labels. Genetic testing based on differences in DNA sequences can be achieved by detecting the alteration in the electrophoretic mobility of the DNA fragments in the gels, with or without denaturing agents. Deletions and small insertions of the sequences can be visualized by electrophoresis of the high resolution gel. DNA fragments from different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are delayed in the gel at different positions according to their specific melting or partial melting temperatures (see , for example, Myers et al., Science, 230: 1242 (1985)). Sequence changes at specific sites can also be revealed by nuclease protection assays, such as the RNase and SI protection or the chemical cleavage method (eg, Cotton et al., Proc. Nati. Acad. Sci., USA, 85 4397-4401 (1985)). Accordingly, detection of the specific DNA sequence can be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes (for example, Restriction fragment length polymorphisms ("RFLP") and Southern blotting of genomic DNA In addition to more conventional DNA sequencing and gel electrophoresis, mutations can also be detected by in situ analysis.

Polypeptide assays The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of the cytostatin II protein in cells and tissues, including the determination of normal and abnormal levels. Accordingly, for example, a diagnostic assay according to the invention for detecting overexpression of the cytostatin II protein compared to normal control tissue samples can be used to detect the presence of myocardial infarction, for example . The assay techniques that can be used to determine the levels of a protein, such as a cytostatin II protein of the present invention, in a sample derived from a host, are well known to those skilled in the art. Such assay methods include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays. Among t, ELISA assays are frequently preferred. An ELISA assay comprises initially preparing an antibody specific for cytostatin II, preferably a monoclonal antibody. In addition, usually a reporter antibody is prepared, which binds to the monoclonal antibody. The reporter antibody is attached to a detectable reagent such as a radioactive, fluorescent or enzymatic reagent, in this example the enzyme horseradish peroxidase. In order to carry out an ELISA test, a sample is removed from a host and incubated on a solid support, for example a polystyrene dish or disk, which binds the proteins in the sample. Any of the protein-free sites on the disk or dish are then covered by incubation with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the disc or dish, during which time the monoclonal antibodies bind to any of the cytostatin II proteins fixed to the polystyrene disc or dish. The unbound monoclonal antibody is removed by washing with a buffer solution. The reporter antibody bound or linked to the horseradish peroxidase is placed on the plate or disk, leading to the binding of the reporter antibody with any monoclonal antibody bound to cytostatin II. The antibody from the unbound reporter is then removed by washing. The reagents to verify the activity of the peroxidase, including a colorimetric substrate, are then added to the dish or disk. The immobilized peroxidase, bound or linked to cytostatin II through the primary or secondary antibodies, produces a colored reaction product. The amount of the color developed in a given period of time indicates the amount of the cytostatin II protein present in the sample. Quantitative results are typically obtained by reference to a standard curve. A competition assay can be employed where antibodies specific for cytostatin II bound or bound to a solid support and labeled cytostatin II and a sample derived from the host are passed over the solid support and the amount of the label detected fixed to the Solid support can be correlated with an amount of cytostatin II in the sample.

Chromosome assays The sequences of the present invention are also valuable for the identification of chromosomes. The sequence is located as a target or target specifically for and can be hybridized to a particular location on an individual human chromosome. In addition, there is a current need to identify particular sites on the chromosome. Few chromosome labeling reagents, based on the actual sequence data (repeated polymorphisms) are currently available for chromosome localization labeling. The assignment of chromosomes of the DNAs to the chromosomes according to the present invention, is an important first step in the correlation of these sequences with the genes associated with the disease. Briefly, the sequences can be assigned coordinates with respect to the chromosomes by preparing the PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3 'untranslated or translated region of the gene is used to rapidly select primers that do not extend more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for the selection by PCR of the hybrids of the somatic cells containing the individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will produce an amplified fragment. The assignment of coordinates by PCR of the somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same primers of the oligonucleotides, sublocalization with the panels of the fragments can be achieved from the chromosomes or specific groups of the large genomic clones in an analogous manner. Other coordinate assignment strategies that can be used similarly for the assignment of coordinates with respect to their chromosome include in situ hybridization, preselection with the chromosomes classified by the flow, labeled and pre-selection by hybridization to construct the cDNA libraries specific for chromosomes.

Fluorescence in situ hybridization ("FISH") of a cDNA clone for chromosomal diffusion of the metaphase can be used to provide a precise chromosomal location in one step. This technique can be used with a cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a single chromosomal location with sufficient signal strength for simple detection. FISH requires the use of the clones from which the label of the expression sequence (EST) is 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 give good results in a reasonable percentage of time. For a review of this technique, see Verma et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York (1988). Once a sequence has been assigned coordinates with respect to an accurate chromosomal location, the physical position of the sequence on the chromosome can be correlated with the data from the genetic map. Such data are found, for example, in V. McKusick, MENDELIAN INHERITANCE IN MAN, available online through Johns Hopkins University, Welch Medical Library. The relationship between the genes and the diseases to which they have been assigned coordinates with respect to the same chromosomal region, are identified through the analysis of binding or binding (co-descendence of physically adjacent genes). Next, it is necessary to determine the differences in the cDNA or the genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any of the normal individuals, then the mutation is likely to be the causative agent of the disease. With the common resolution of the techniques of assignment of physical coordinates and the assignment of genetic coordinates, a cDNA located precisely with respect to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes a ratio of the coordinate assignment of 1 megabase and one gene per 20 kb).

Immunological Applications The polypeptides, their fragments or other derivatives, or analogs thereof, or the cells expressing them, can be used as an immunogen to produce the antibodies therefor. These antibodies can be, for example, monoclonal or polyclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of a Fab expression library. Various methods known in the art can be used for the production of such antibodies and fragments. Antibodies raised against polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administration of the polypeptides to an animal, preferably different from a human. The antibody thus obtained will then bind to the polypeptides themselves. In this way, even a sequence encoding only a fragment of the polypeptides can be used to generate the antibodies that bind to the complete natural polypeptides. Such antibodies can then be used to isolate the polypeptide from the tissue expressing this polypeptide. For the expression of the monoclonal antibodies, any technique which provides the antibodies produced by the cultures of the continuous cell lines 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 _ : 72 (1983) and the hybridoma-EBV technique to produce human monoclonal antibodies (Colé et al., pages 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss. Inc. (1985). The techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single chain antibodies for the immunogenic polypeptide products of this invention. Also, transgenic mice, or other organisms such as other mammals, can be used to express the humanized antibodies to the products of the immunogenic polypeptides of this invention.

EXAMPLES The present invention is further described by the following examples. The examples are provided only to illustrate the invention by reference to the specific embodiments. These amplifications, while illustrating certain specific aspects of the invention, do not represent the limitations or circumscribe the scope of the invention described. Certain terms used here are explained in the following glossary.

All parts or amounts described in the following examples are by weight, unless otherwise specified. Unless otherwise specified, the preparation of the size of the fragments in the examples below was carried out using standard polyacrylamide gel electrophoresis ("PAGE") techniques in 8 percent gels, as shown in FIG. described, for example, by Goeddel et al., Nucleic Acids Res. 8_: 4057 (1980). Unless otherwise described, the linkages or linkages were made using buffer solutions, incubation temperatures and standard times, approximately equimolar amounts of the DNA fragments to be bound or bound and about 10 units of the DNA ligase. of T4 ("ligase") per 0.5 μg of DNA. All examples were carried out using standard techniques, which are well known and routine for those skilled in the art, except where otherwise described in detail.

EXAMPLE 1 Expression and purification of human cytostatin II using bacteria The DNA sequence encoding human cytostatin II in the deposited polynucleotide was amplified using the PCR oligonucleotide primers specific for the carboxyl terminal sequence of the amino acids of the human cytostatin II protein and for the 3 'sequences of the vector with regarding the gene. Additional nucleotides containing the restriction sites to facilitate cloning were added to the 5 'and 3' sequences respectively. The 5 'oligonucleotide primer had the 5' CGC GGA TCC GTG GAG GCT TTC TG 3 'sequence containing the underlined BamHl restriction site, followed by 14 nucleotides of the human cytostatin II coding sequence starting from the second codon; that is, the codon that follows from the AUG for the presumed or probable N-terminal methionine. The 3 'primer has the sequence 5' CGC AAG CTT TTA TGC CTT CTC ATA GTG 3 'containing the underlined Hind III restriction site, followed by 18 nucleotides complementary to the last 6 codons of cytostatin II including the stop codon.

The restriction sites were convenient for the restriction enzyme sites in the pQE-70 vectors of bacterial expression, which were used for bacterial expression in these examples. . { Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311). PQE-70 encodes antibiotic resistance to ampicillin ("Ampr") and contains a bacterial origin of replication ("ori"), an inducible IPTG promoter, a ribosome binding site ("RBS"), an 6-His tag and the restriction enzyme sites. PQE-70 was digested with BamHI and HindIII and the amplified human cytostatin II DNA was ligated or ligated into the digested vector DNA of BamHI / HindIII. The insertion in the restricted BamHl / HindIII vector placed the coding region of cytostatin II downstream of the IPTG-inducible promoter and in frame with an initiation AUG for translation or translation. The ligation or binding mixture was transformed into competent E. coli cells using standard procedures. Such procedures are described in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2ND Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Strain M15 / re? 4 of E. coli, which contains multiple copies of the plasmid pREP4, which expresses the lac repressor and confers resistance to kanamycin ("Kanr), was used to carry out the illustrative example described herein. This strain, which is only one of many that are suitable for the expression of cytostatin II, is commercially available from Qiagen. Transformants were identified by their ability to grow on LB plates in the presence of ampicillin. The DNA plasmid was isolated from the resistant colonies and the identity of the cloned DNA was confirmed by the restriction analysis. Clones containing the desired constructs were grown overnight ("0 / N") in a liquid culture in an LB medium supplemented with both ampicillin (110 ug / ml) and kanamycin (25 ug / ml). The O / N culture was used to inoculate a large culture, at a dilution of about 1: 100 to 1: 250. The cells were grown at an optical density of 600 hm (O.D.600) between 0.4 and 0.6. The isopropyl-B-D-thiogalactopyranoside ("IPTG") was then added to a final concentration of 1 mM to induce transcription from the lac repressor responsive promoters, inactivating the lacl repressor. The cells were subsequently incubated additionally for 3 to 4 hours. The cells were then collected by centrifugation and broken or altered, by standard methods. The inclusion bodies were purified from the broken or altered cells using the routine collection techniques, and the protein was solubilized from the inclusion bodies in the urea 8. The 8M urea was exchanged in the salt solution buffered with 2X phosphate ("PBS") and the protein was then refolded in a standard PD-10 solution. The protein was further purified by size exclusion chromatography and then by additional chromatography step to remove the endotoxin. The preparation of the sterile protein was stored in PBS 2X at a concentration of 95 micrograms per ml. Analysis of the preparation by standard methods of electrophoresis of the polyacrylamide gel revealed that the preparation contained approximately 80% of monomeric cytostatin II having the expected molecular weight of approximately 14 kDa.

Example 2 Cloning and expression of human cytostatin II in a baculovirus expression system The cDNA sequence encoding the full-length human cytostatin II protein in the deposited region is amplified using the PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene: The 5 'primer has the sequence GC GGA TCC CGT GGA GGC TTT CTG TGC containing the restriction enzyme site of BamIIl followed by codons 2-5 and 2 bases of codon 6 of the sequence of cytostatin II of Figure 1. Inserted in a vector of expression, as described below, the 5 'end of the amplified fragment encoding human cytostatin II, provides an efficient signal for the initiation of translation or translation in eukaryotic cells, as described by Kozac, M. , J. Mol. Biol., 196: 947-950 (1987), among others. The 3 'primer has the sequence 5' GC GGT ACC TTA TGC CTT CTC ATA GTG '3' containing the restriction to Asp718 underlined followed by the complementary nucleotides for the coding cod and the codices for the latter. five amino acids of the human cytostatin II cDNA of Figure 1. The amplified fragment is isolated from a 1% agarose gel using a commercially available kit or set ("Geneclean", BIO 101 Inc., La Jolla, CA. .). The fragment is then digested with BamHl and Asp718 and again purified on a 1% agarose gei. This IraymenLo was designated here as F2.

The pA2-GP vector is used to express the cytostatin II protein in the bacuiovirus expression system, using standard methods, such as those described in Summer et al., A MANUAL OF PROCEDURES, Texas Agricultural Experimental Station Rull No. 1 55 (1987). This expression vector contains the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites. The signal peptide of the AcMNPV gi67, including the N-terminal methylanin, is located just upstream of a BamHI site. The polyadenylation site of simian virus 40 ("SV40") is used for efficient polyadenylation. For a facilitated selection of the recombinant virus, the beta-galactosidase gene of E. coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene. Polyhedrin sequences are flanked on both sides by the viral sequences for cell-mediated homologous recombination, with wild-type viral DNA to generate the viable or available virus expressing the cloned polynucleotide. Many other baculovirus vectors could be used in place of pA2-GP, such as pAc373, pVL941 and pAcIMl. Such vectors are described in Luckow et al., Virology 170: 31-39, among others. The plasmid is digested with the restriction enzymes BamHl and Asp7i8 and then it is def? Rrilad? u Lij. ±? auau xa l? oiaLd ia luLco t iiidi Uc icl iicl ü, u? ? ? ? -aüu? routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit or set ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated here "V2". The fragment F2 and the dephospho- phoidal plasmid V2 are together with the liyasa of the T-DNA. The HB101 cells of E. coli were transformed with the ligation or binding mixture and dispersed on the culture bins. The barferias containing the plasmid with the human cytostatin II gene are digested by the digestion of DNA from the individual colonies using the BamHI and the Asp718 and then the product of the digestion is analyzed by electrophoresis of the gel. The sequence of the cloned fragment is confirmed by the sequencing of the DNA. This plasmid is designated here pBacCytostatin II. 5 μg of plasmid pBacCytostatin II is co-transfected with 1.0 μg of commercially available linearized baculovirus DNA (baculovirus DNA "BaculoGold ™", Pharmingen, San Diego, CA. ), using the lipofection method described by Felgner et al., Proc. Nati Acad. Sci. USA 84: 7413-7417 (1987). 1 μg of BaculoGold ™ virus DNA and 5 μg of plasmid pBacCytostatin II are mixed in a sterile cavity of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD). After this, 10 μl of Lipofectin plus 90 μl of Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added dropwise to the cells of the Sf9 insects (ATCC CRL 1711) seeded on a 35 mm tissue culture plate with 1 ml of Grace's medium without serum. The plate is shaken back and forth to mix the recently added solution. 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 bovine serum is added. The plate is placed again in an incubator and the culture is continued at 27 ° C for four days. After four days the supernatant is collected and plaque assayed, as described by Summers and Smith (supra). An agar gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used to allow the identification and facilitated isolation of the gal expression clone, which produces the blue-stained plates. (A detailed description of a "plaque assay" of this type can also be found in the user guide for the cultivation of insect cells and baculovirology distributed by Life Technologies Inc., Gaithersburg, pages 9-10). Four days after serial dilution, the virus is added to the cells. The plates stained blue are collected with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar is removed by a short centrifugation and the supernatant containing the recombinant baculovirus is used to infect the Sf9 cells seeded on 35 mm discs or dishes. Four days later the supernatants of these disks or culture dishes are collected and then stored at 4 ° C. A clone containing the appropriately inserted cytostatin II is identified by the DNA analysis which includes the sequencing and the assignment of the restriction coordinates. This is designated here as V-cytostatin II. The Sf9 cells are grown in Grace's medium supplemented with 10% heat inactivated FBS. The cells are infected with the V-Cytostatin II of the recombinant baculovirus at a multiplicity of sites of infection ("MOI") of 2. Six hours later the medium is removed and replaced with the SF900 medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 μCi of 35S-methionine and 5 μCi of 35S cysteine (Amersham) are added. The cells are further incubated for 16 hours and then collected by centrifugation, used and labeled proteins are visualized by SDS-PAGE and autoradiography.

EXAMPLE 3 Expression of human cytostatin II in COS cells The plasmid of expression, Cytostatin II HA, is derived from the pcDNAI / Amp vector (Invitrogen) which contains: 1) the SV40 origin of the replication, 2 the ampicillin resistance gene, 3) the origin of E. coli replication, 4) the promoter of CMV followed by the polylinker region, an intron of SV40 and the polyadenylation site. A DNA fragment encoding the complete cytostatin II precursor and an HA tag fused in frame to its 3 'end is cloned into the polylinker region of the vector so that the expression of the recombinant protein is driven by the promoter of CMV. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al., Cell 37_: 7 ^ 7 (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 strategy for the construction of the plasmid is as follows. The DNA sequence encoding the cytostatin II of the deposited clone was constructed by PCR on the original cloned EST using two primers. The 5 'primer is GCGC GGATCC GCC ACC ATG GTG GAG GCT TTC TGT, which contains the underlined BamHl site followed by 8 nucleotides of the cytostatin II coding sequence that starts from the initiation codon. The 3 'sequence is GCGC TCTAGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA TGC CTT ATA GTG containing the Bbal underlined site, a stop codon of translation or translation, an HA tag and the last 12 nucleotides of the cytostatin II coding sequence (which does not include the stop codon). Therefore, the PCR product contains a BamHI site, the cytostatin II coding sequence followed by an HA tag fused to cytostatin II in the frame, a stop codon for translational termination or translation then of the HA tag, and a Xbal site. The DNA fragment amplified by PCR and the vector, pcDNAI / Amp, are deferred with BamHl and Xbal and then ligated or ligated. The binding mixture is transformed into the SURE strain of E. coli (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture is plated on the plates of the ampicillin medium and selected the resistant colonies. The plasmid DNA is isolated from the transformants and examined by restriction analysis to verify the presence of the correct fragment. For expression of recombinant cytostatin II, COS cells are transfected with the expression vector using the methods described, for example, in DEAE-DEXTRAN, as described for example in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). Expression of the HA-cytostatin II fusion protein was detected by radiolabelling and immunoprecipitation, using the methods described in, for example, Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2 / a. Ed .; Laboratory Press, Cold Spring Harbor, New York (1988). Cells are labeled for 8 hours with 35S-cysteine two days after transfection. The culture medium is then collected and the cells are used with detergent (RIPA buffer solution (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) ( Wilson et al., Id.) Both the cell lysate and the culture medium are precipitated with an HA-specific monoclonal antibody.The precipitated proteins are analyzed on 15% SDS-PAGE gels, which show an expression product of the expected size.

Example 4 Tissue distribution of cytostatin II expression Northern blot analysis is carried out to examine the levels of cytostatin II expression in human tissues, using the methods described, inter alia, by Sambrook et al, cited above. The whole cellular RNA samples are isolated with the RNAzol ™ system (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, Tx. 77033). Approximately 10 μg of the total RNA isolated from each specified human tissue are separated on a 1% agarose gel. The gel is impregnated or stained on a full-length cytostatin II gene from a nylon filter and hybridized to a labeled polynucleotide probe. The labeling reaction is done according to the Stratagene Prime-It set or set with the 50 ng DNA fragment. The labeled DNA is purified with a Select-G-50 column (5 Primer-3 Primer, Inc. 5603 Araphos Road, Boulder, CO 80303). The filter is then hybridized with the full length, labeled, radioactive cytostatin II gene at 1,000,000 cpm / ml in 0.5 M NaP04, pH 7.4 and 7% SDS overnight at 65 ° C. After washing twice at room temperature and twice at 60 ° C with 0.5 x SSC, 0.1% SDS, the filter is dried and then exposed to a film at -70 ° C overnight with an intensifying screen or grid. The mRNA for cytostatin II is abundant in the brain.

EXAMPLE 5 Therapeutic Expression of the Human Cytostatin II Gene Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in a tissue-culture medium and separated into small pieces. Small pieces of tissue are placed on a wet surface of a tissue culture bottle, approximately ten pieces are placed in each bottle or container. The bottle is turned upside down, closed tightly and left at room temperature overnight. After 24 hours at room temperature, the container is inverted - the pieces of the tissue remain fixed in the bottom of the container - and fresh medium is added (for example, the F12 medium of Ham, with 10% FBS, penicillin and streptomycin) . The tissue is then incubated at 37 ° C for about a week. At this time, fresh media is added and subsequently changed every few days. After an additional two weeks in culture, suggest a monolayer of fibroblasts. The monolayer is trypsinized and scaled in larger flasks. A vector for gene therapy is deferred with restriction enzymes for the cloning of a fragment to be expressed. The digested vector is treated with bovine intestinal phosphatase to prevent self-union or self-adherence. The dephosphorylated linear vector is fractionated on an agarose gel and purified. The cytostatin cDNA capable of the expression of active cytostatin II is isolated. The ends of the fragment are modified, if necessary, for cloning into the vector. For example, the 5"overlay can be treated with the DNA polymerase to create disheveled ends.The 3 'overhanging ends can be removed using the nuclease SI.The linkers can be attached to the blunt ends with the DNA ligase of T4 Equal amounts of the linear skeleton of Moloney murine leukemia virus and the fragment of cytostatin II are mixed together and bound using T4 DNA ligase. The binding mixture is used to transform the E. coli and the bacteria are then placed on plates, on the kanamycin containing the agar. The restriction and phenotype analysis of kanamycin confirm that the vector has the gene inserted properly. The packing cells were grown in the tissue culture at a confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% bovine serum (CS), penicillin and streptomycin. The vector containing the cytostatin II gene is introduced into the packaging cells by standard techniques. Infectious viral particles that contain the cytostatin II gene are collected from the packaging or packaging cells, which are now called the producer cells. The fresh medium is added to the producer cells, and after an appropriate incubation period the medium is collected from the plates of the confluent producer cells. The middle, which contains the infectious viral particles, is filtered through a Millipore filter to remove the disunited producer cells. The filtered medium is then used to infect the cells of the fibroblasts. The medium is removed from a subconfluent plate of fibroblasts and rapidly replaced with the filtered medium. The polybrene (Aldrich) can be included in the medium to facilitate transduction. After the appropriate incubation, the medium is removed and replaced with a fresh medium. If the virus concentration is high, then virtually all fibroblasts will be infected and no selection is required. If the concentration is low, then it is necessary to use a retroviral vector having a selectable marker, such as neo or his, to select the cells transduced for expansion. The designed fibroblasts can be injected into rats, either alone or after they have been grown to converge on beads or microcarrier beads, such as 3 beads or cytodex beads. Injected fibroblasts produce the product of cytostatin II, and the biological actions of the protein are transported to the host. It will be clear that the invention can be practiced in a different manner than that described particularly in the description and preceding examples. Numerous modifications and variations of the present invention are possible in the light of the foregoing teachings and, therefore, are within the scope of the appended claims.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (21)

1. An isolated polynucleotide, characterized in that it comprises a region at least 95% identical in sequence to an RNA or DNA encoding amino acids 1-132 in Figure 1.

2. An isolated polynucleotide according to claim 1, characterized in that the region is continuous or is formed by a plurality of non-contiguous exons.

3. An isolated polynucleotide according to claim 2, characterized in that the region is a genomic DNA or a cDNA.

4. An isolated polynucleotide according to claim 3 characterized in that the sequence of the region is that of nucleotides 16-411 in Figure 1.

5. An isolated polynucleotide according to claim 1, characterized in that the region is at least 95% identical in sequence to an RNA or DNA encoding amino acids 1-132 in Figure 1.

6. An isolated polynucleotide according to claim 5, characterized in that the sequence of the region is that of nucleotides 16-396 in Figure 1.

7. An isolated polynucleotide, characterized in that it comprises a region at least 95% identical in sequence to an RNA or DNA encoding the cytostatin II polypeptide of the human cDNA insert of Deposit No.: 97287 of the ATCC.

8. An isolated polynucleotide according to claim 7, characterized in that the RNA or DNA encodes the mature polypeptide of the human cDNA insert of Deposit No. 97287 of the ATCC.

9. An isolated polynucleotide according to claim 7, characterized in that the region is the coding region of the cDNA insert of Deposit No.:97287 of the ATCC.

10. An isolated polynucleotide according to claim 7, characterized in that the region is the cDNA insert of Deposit No. 97287 of the ATCC.

11. An expression vector, characterized in that it comprises cis-functioning control elements, effective for the expression in a host cell of an operably linked polynucleotide, wherein the polynucleotide is a polynucleotide according to claim 1.

12. An expression vector according to claim 11, characterized in that the control elements are effective for inducible expression of the polynucleotide in the host cell.

13. An expression vector, characterized in that it comprises the performance control elements, effective for the expression in a host cell of an operably linked polynucleotide, wherein the polynucleotide is a polynucleotide according to claim 7.

14. A host cell, characterized in that it has stably incorporated therein a polynucleotide according to claim 1.

15. A host cell, characterized in that it has stably incorporated into it a cDNA insert according to claim 7.

16. A host cell, characterized in that it has expressly incorporated therein an expression vector according to claim 11.

17. A host cell, characterized in that it has expressively incorporated therein an expression vector according to claim 13.

18. A process for manufacturing a polypeptide, characterized in that it comprises the step of expressing in a host cell a polynucleotide according to claim 1.

19. A process for making a polypeptide, characterized in that it comprises the step of expressing in a host cell a polynucleotide according to claim 7.

20. A polypeptide, characterized in that it is encoded by a polynucleotide according to claim 1.

21. A polypeptide, characterized in that it is encoded by a polynucleotide according to claim 7.