WO1999046276A1 - Zap-1 tumor associated genes and their uses - Google Patents

Abstract

Methods for isolating ZAP-1 genes are provided. Deletion of the ZAP-1 locus is associated with human tumors, particularly carcinomas. The ZAP-1 nucleic acid compositions find use in identifying homologous or related proteins and the DNA sequences encoding such proteins; in producing compositions that modulate the expression or function of the protein; and in studying associated physiological pathways. In addition, modulation of the gene activity in vivo is used for prophylactic and therapeutic purposes, such as treatment of cancer, identification of cell type based on expression, and the like.

Description

ZAP-1 TUMOR ASSOCIATED GENES AND THEIR USES

INTRODUCTION Background Control of cellular proliferation is of great importance for many normal and abnormal biological processes; including development, wound healing, programmed cell death, angiogenesis and tumorigenesis. A myriad of components involved in the regulation of cell proliferation have been identified, including growth factors, cell cycle regulators, oncogenes and tumor suppressor genes. In turn, a growing body of evidence suggests that the nature of the local cellular environment can dramatically mediate a cell's response to regulatory components. By providing the architecture whereby cells form attachments, localize factors and migrate to particular positions, matrix proteins may direct the outcome of exposure to regulatory molecules. Molecules involved in cell attachment, the extracellular matrix, intercellular interactions, the internal cytoskeleton, and overall tissue architecture may ultimately dictate the cell's proiiferative status. Such molecules include the collagens, integrins, laminins, fibronectins, receptor tyrosine kinases, etc.

Nineteen types of collagen have been identified, encoded by 33 genes. Types I, II, III, V, and XI constitute the fibrillar collagens, whereas types IV, VI to X, and XII to XIX represent the structurally diverse, nonfibrillar members. The XV and XVIII collagens are characterized by a collagenous sequence with frequent interruptions and large amino and carboxyterminal noncollagenous domains. These are widely found in basement membrane zones, and have a pronounced vascular association.

Rehn et al. (1994) P.N.A.S. 91(10):4234-4238; and Oh et al. (1994) P.N.A.S. 91(10):4229-4233 describe the characterization of the mouse and human cDNAs encoding alpha 1 (XVIII) collagen. Subsequently, O'Reilly et al. (1997) Cell 88(2):277-285 identified endostatin, an angiogenesis inhibitor that is a 20 kDa C-terminal fragment of collagen XVIII. Endostatin specifically inhibits endothelial

-1- proliferation and potently inhibits angiogenesis and tumor growth (See International patent application WO97/15666).

Collagens have also been implicated in regulation of cell proliferation through the activation of tyrosine kinases. Takeuchi et al. (1997) J Biol Chem 272(46):29309-29316 demonstrated that the attachment of cells to collagen stimulated tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK), and enhanced MAPK activity. Collagen also activates platelets through a pathway dependent on the Fc receptor gamma-chain and the tyrosine kinase Syk (Gibbins et al. (1997) FEBS Lett 413(2):255-259).

Cell adhesion to extracellular matrices is fundamental for maintaining normal tissue architecture and function. Changes in adhesion can occur as a result of modifications of the composition or integrity of the extracellular matrix or as a result of disease associated changes in the expression and/or function of adhesion receptors. Such alterations in cell adhesion can have profound effects on the phenotypic traits of cells, and as a result, can be of primary importance in facilitating disease-associated breakdown of normal tissue function. This is apparent in cancer, where neoplastic transformation can lead to alterations in tumor cell growth, changes in the composition or integrity of tissue proteins, tumor cell migration, invasion, and ultimately metastasis formation.

Metastatic dissemination of tumor cells includes several steps: detachment of tumor cells from the primary tumor, traversing of the basement membrane, and migration into the extracellular matrix. In order to migrate from their original site, tumor cells have to cross several barriers, such as basement membranes, interstitial tissues and extracellular matrices, which are composed primarily of collagen, proteoglycans, elastin, laminin and other glycoproteins.

Tumor cells over express and secrete proteases that are capable of degrading the components of these barriers and thus facilitate their migration. The classes of proteases which have been implicated in the process of tumor invasion and metastasis include metalloproteases, serine proteases and cathepsins. The tissue inhibitors of metalloproteinases (TIMPs) are naturally occurring proteins that specifically inhibit matrix metalloproteinases, thus maintaining balance between matrix destruction and formation. An imbalance between MMPs and the associated TIMPs may play a significant role in the invasive phenotype of malignant tumors. However, a better understanding of the process of metastasis and tumor invasion is required before proteases can be used as therapeutic targets.

The identification of proteins involved with tumor growth and metastasis is of great interest for clinical and research purposes. Understanding the involvement of structural proteins may provide new therapeutic approaches.

Relevant Literature

Roberts et al. (1998) Cancer Genet Cvtoαenet 100(1): 10-20 constructed YAC contig extending approximately 6 Mbp in the chromosome 1p22 region that spans the D1S435 and D1S236 loci. Following the establishment of the contig a physical map of the region was constructed, which allowed this contig to be joined with another reported previously, thereby generating a well characterized 15 Mbp YAC contig in the 1p22-31 region. Cytogenetic mapping of chromosome 1 is reported by Ariyama et al. (1995) Genomics 25(1): 114-123; and Vernole et al. (1995) Cvtoqenet Cell Genet 70(1-2):23-25. Microsatellite mapping and allelic imbalance on chromosome 1 in human breast cancer is disclosed by Hoggard et al. (1995) Genes Chromosomes Cancer 12(11:24-31.

Mertens et al. (1997) Cancer Res 57(13):2765-2780 studied cytogenetic information from a number of tumors. All tumor types displayed unique combinations of chromosomal imbalances, but also showed overlap among the profiles of the different diagnostic entities, indicating that similar molecular mechanisms may be operative in the development of many types of neoplasia. Chromosome segment 1 p22-pter, among others, was commonly deleted. Lee et al. (1996) Cancer Res 56(19):4297-4301 demonstrated a loss of heterozygosity defines a critical region in chromosome 1p22 commonly deleted in human malignant mesothelioma.

Aburatani et al. (1996) The American Journal of Human Genetics 59(4):A61 demonstrated that the DNA marker 7-14 was homozygously deleted in a gastric carcinoma cell line.

-3- SUMMARY OF THE INVENTION

Isolated nucleotide compositions and sequences are provided for ZAP-1 genes. Loss of heterozygosity at the ZAP-1 locus is associated with the oncogenesis of human cancers. The ZAP-1 nucleic acid compositions find use in identifying homologous or related genes; in producing compositions that modulate the expression or function of its encoded protein, ZAP-1; for gene therapy; mapping functional regions of the protein; and in studying associated physiological pathways.

In addition, modulation of the gene activity in vivo is used for prophylactic and therapeutic purposes, such as treatment of cancer, identification of cell type based on expression, and the like. ZAP-1 , anti-ZAP-1 antibodies and ZAP-1 nucleic acid sequences are useful as diagnostics, and to identify cancers having mutations in this gene.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS ZAP-1 gene compositions and methods for their isolation are provided.

Certain human cancers, particularly epithelial cell tumors, e.g. carcinomas of the breast, colon, cervix and lung; mesotheliomas, neuroblastomas, efc, show loss of heterozygosity of ZAP-1, indicating that the gene product functions as a tumor suppressor. The ZAP-1 genes and fragments thereof, encoded protein, and anti-ZAP-1 antibodies are useful in characterizing the phenotype of tumors that are associated with this gene. The characterization is useful for determining further treatment of the patient. Tumors may be typed or staged as to the ZAP-1 status, e.g. by detection of mutated or deleted sequences, antibody quantitation of the protein products, and functional assays for altered ZAP-1 activity levels. Tumors associated with loss of ZAP-1 include a number of carcinomas known to have deletions in the region of chromosome 1 P22, particularly the region between D1S2627 and D1S2889. The ZAP-1 protein or biologically active fragments thereof is therapeutically useful for inhibition of tumor development. CHARACTERIZATION OF ZAP-1 Comparative sequence alignments indicate that ZAP-1 represents a novel member of the collagen gene family. In addition to the presence of an extensive GZZ domain, which commonly makes up the majority of most collagens, ZAP-1 has a genomic structure similar to most members of the collagen gene family. Homology with known human collagens are between 40-50% at the amino acid level. For the most part, significant alignments occur solely at the glycine residues of the GZZ repeat units. The association of ZAP-1 loss of heterozygosity (LOH) with tumors and its extensive collagen-like domain predicts involvement in control of tumorigenesis or angiogenesis at the level of cell attachment, proteolytic activation as a soluble growth factor, or activation/deactivation of cell surface receptors such as ligand-induced receptor tyrosine kinases.

ZAP-1 is alternatively spliced to form two mRNAs variant forms, ZAP-1A and ZAP-1B. The mRNAs are approximately 8.5 and 4.5 kb in length, respectively. Both forms are expressed at low levels in a wide variety of adult and fetal tissues. The chromosomal location of the human gene has been localized to 1P22. The ZAP-1A nucleic acid sequence is provided as SEQ ID NO:1 , where the coding sequence extends from nt. 40 to 5118, and the encoded polypeptide sequence as SEQ ID NO:2. The ZAP-1 B nucleic acid sequence is provided as SEQ ID NO:3, and the encoded polypeptide as SEQ ID NO:4. The term "ZAP-1 genes" is herein used generically to designate the two alternative forms, unless otherwise indicated.

Members of the nonfibrillar collagen subgroups, particularly those that are associated with or integral to extracellular matrix, have been found to release biologically active peptides. The central collagenous domain is cleaved away from the noncollagenous flanking domains, releasing soluble factors. In particular, the carboxy-terminal fragment, of approximately 150 to 200 amino acids, is released as an inhibitor of cellular proliferation, e.g. endothelial cells involved in angiogenesis.

The collagenous domain of ZAP-1A extends roughly from the region of amino acid 489 to 1456, where the domain boundaries may be within around about 10 amino acids before or after those positions. The polypeptide regions flanking the

-5- collagenous domain may be synthesized as separate molecules, or may be released from the intact protein by cleavage with protease, chemical treatment, etc.

IDENTIFICATION OF ZAP-1 SEQUENCES Homologs of ZAP-1 are identified by any of a number of methods. A fragment of the provided cDNA may be used as a hybridization probe against a cDNA library from the target organism of interest, where low stringency conditions are used. The probe may be a large fragment, or one or more short degenerate primers.

Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 6XSSC (0.9 M sodium chloride/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in 1XSSC (0.15 M sodium chloride/0.015 M sodium citrate). Sequence identity may be determined by hybridization under stringent conditions, for example, at 50°C or higher and 0.1XSSC (15 mM sodium chloride/01.5 mM sodium citrate). Nucleic acids having a region of substantial identity to the provided ZAP-1 sequences, e.g. allelic variants, genetically altered versions of the gene, etc., bind to the provided ZAP-1 sequences under stringent hybridization conditions. By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes may be any species, e.g. primate species, particularly human; rodents, such as rats and mice, canines, felines, bovines, ovines, equines, yeast, nematodes, etc.

Between mammalian species, e.g. human and mouse, homologs have substantial sequence similarity, i.e. at least 75% sequence identity between nucleotide sequences. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al. (1990) J Mol Biol 215:403-10. The sequences provided herein are essential for recognizing ZAP-1 related and homologous proteins in database searches. in general, variants of the invention have a sequence identity greater than at least about 75%, preferably at least about 85%, more preferably at least about 90%, and can be greater than at least about 95% or more as determined by the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular). For the purposes of this invention, a preferred method of calculating percent identity is the Smith-Waterman algorithm, using the following. Global DNA sequence identity must be greater than 65% as determined by the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular) using an affine gap search with the following search parameters: gap open penalty, 12; and gap extension penalty, 1.

ZAP-1 NUCLEIC ACID COMPOSITIONS

Nucleic acids encoding ZAP-1 may be cDNA or genomic DNA or a fragment thereof. The term "ZAP-1 gene" shall be intended to mean the open reading frame encoding specific ZAP-1 polypeptides, introns, as well as adjacent 5' and 3' non- coding nucleotide sequences involved in the regulation of expression, up to about 20 kb beyond the coding region, but possibly further in either direction. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into a host genome. The term "cDNA" as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3' and 5' non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns, when present, removed by nuclear RNA splicing, to create a continuous open reading frame encoding a ZAP-1 protein.

A genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. It may further include the 3' and 5' untranslated regions found in the mature mRNA. It may further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking

-7- genomic DNA at either the 5' or 3' end of the transcribed region. The genomic DNA may be isolated as a fragment of approximately 120 kbp; and substantially free of flanking chromosomal sequence. The genomic DNA flanking the coding region, either 3' or 5', or internal regulatory sequences as sometimes found in introns, contains sequences required for proper tissue and stage specific expression.

The sequence of the 5' flanking region may be utilized for promoter elements, including enhancer binding sites, that provide for developmental regulation in tissues where ZAP-1 is expressed. The tissue specific expression is useful for determining the pattern of expression, and for providing promoters that mimic the native pattern of expression. Naturally occurring polymorphisms in the promoter region are useful for determining natural variations in expression, particularly those that may be associated with disease.

Alternatively, mutations may be introduced into the promoter region to determine the effect of altering expression in experimentally defined systems. Methods for the identification of specific DNA motifs involved in the binding of transcriptional factors are known in the art, e.g. sequence similarity to known binding motifs, gel retardation studies, etc. For examples, see Blackwell et al. (1995) Mol Med 1: 194-205; Mortlock et al. (1996) Genome Res. 6: 327-33; and Joulin and Richard-Foy (1995) Eur J Biochem 232: 620-626. The regulatory sequences may be used to identify cis acting sequences required for transcriptional or translational regulation of ZAP-1 expression, especially in different tissues or stages of development, and to identify cis acting sequences and trans acting factors that regulate or mediate ZAP-1 expression. Such transcription or translational control regions may be operably linked to a ZAP-1 gene in order to promote expression of wild type or altered ZAP-1 or other proteins of interest in cultured cells, or in embryonic, fetal or adult tissues, and for gene therapy.

The nucleic acid compositions of the subject invention may encode all or a part of the subject polypeptides. Double or single stranded fragments may be obtained of the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. For the most part, DNA fragments will be of at least 15 nt, usually

-8- at least 18 nt or 25 nt, and may be at least about 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening probes, etc. Larger DNA fragments, i.e. greater than 100 to 250 nt are useful for production of the encoded polypeptide. For use in amplification reactions, such as PCR, a pair of primers will be used. The exact composition of the primer sequences is not critical to the invention, but for most applications the primers will hybridize to the subject sequence under stringent conditions, as known in the art. It is preferable to choose a pair of primers that will generate an amplification product of at least about 50 nt, preferably at least about 100 nt. Algorithms for the selection of primer sequences are generally known, and are available in commercial software packages. Amplification primers hybridize to complementary strands of DNA, and will prime towards each other.

The ZAP-1 genes are isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the DNA will be obtained substantially free of other nucleic acid sequences that do not include a ZAP-1 sequence or fragment thereof, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant", i.e. flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.

The DNA may also be used to identify expression of the gene in a biological specimen. Methods of probing samples for the presence of particular nucleotide sequences, as genomic DNA or RNA, is well established in the literature. DNA or mRNA is isolated from a cell sample. The mRNA may be amplified by RT-PCR, using reverse transcriptase to form a complementary DNA strand, followed by polymerase chain reaction amplification using primers specific for the subject DNA sequences. Alternatively, the mRNA sample is separated by gel electrophoresis, transferred to a suitable support, e.g. nitrocellulose, nylon, etc., and then probed with a fragment of the subject DNA as a probe. Other techniques, such as oligonucleotide ligation assays, in situ hybridizations, and hybridization to DNA probes arrayed on a solid chip may also find use. Detection of mRNA hybridizing to the subject sequence is indicative of ZAP- 7 gene expression in the sample. The sequence of a ZAP-1 gene, including flanking promoter regions and coding regions, may be mutated in various ways known in the art to generate

-9- targeted changes in promoter strength, sequence of the encoded protein, etc. The DNA sequence or protein product of such a mutation will usually be substantially similar to the sequences provided herein, i.e. will differ by at least one nucleotide or amino acid, respectively, and may differ by at least two but not more than about ten nucleotides or amino acids. The sequence changes may be substitutions, insertions or deletions. Deletions may further include larger changes, such as deletions of a domain or exon. Other modifications of interest include epitope tagging, e.g. with the FLAG system, HA, etc. For studies of subcellular localization, fusion proteins with green fluorescent proteins (GFP) may be used. Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for site specific mutagenesis may be found in Gustin et al., Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985); Colicelli et al, Mol Gen Genet 199:537-9 (1985); and Prentki et al, Gene 29:303-13 (1984). Methods for site specific mutagenesis can be found in Sambrook et al, Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al, Gene 126:35- 41 (1993); Sayers et al, Biotechniques 13:592-6 (1992); Jones and Winistorfer, Biotechniques 12:528-30 (1992); Barton et al, Nucleic Acids Res 18:7349-55 (1990); Marotti and Tomich, Gene Anal Tech 6:67-70 (1989); and Zhu, Anal Biochem 177:120-4 (1989). Such mutated genes may be used to study structure-function relationships of ZAP-1, or to alter properties of the protein that affect its function or regulation.

ZAP-1 POLYPEPTIDES The subject gene may be employed for producing all or portions of ZAP-1 polypeptides. Fragments of interest include the carboxy and amino terminal, noncollagenous domains. Such domains will usually include at least about 50 amino acids of the provided sequence, more usually at least about 100 amino acids, and may include 150 amino acids or more, up to the complete domain. The sequence of such fragments may be modified through manipulation of the coding sequence, as described above. Truncations may be performed at the carboxy or amino

-10- terminus of the fragment, e.g. to determine the minimum sequence required for biological activity.

Assays for the biological activity of the protein or fragments thereof may be determined as described in the art. Inhibition of cellular proliferation, as may be associated with oncogenesis and/or angiogenesis, is determined through in vivo or in vitro models. Animal models for tumor formation are well known, and may be used to determine the effect of a polypeptide on the overall process of tumor associated morbidity and mortality. In vitro systems, such as inhibition of proliferation of defined cell types, may be performed to better define the target of the peptide. The effect of ZAP-1 polypeptides on activation of tyrosine kinases may be determined through analyzing a direct effect on isolated kinase proteins, on cultured cells, etc.

For expression, an expression cassette may be employed. The expression vector will provide a transcriptional and translational initiation region, which may be inducible or constitutive, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. These control regions may be native to a ZAP-1 gene, or may be derived from exogenous sources.

Peptides may be expressed in prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression. For large scale production, a unicellular organism, such as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates, particularly mammals, e.g. COS 7 cells, may be used as the expression host cells. In some situations, it is desirable to express the ZAP-1 gene in eukaryotic cells, where the ZAP-1 protein will benefit from native folding and post-translational modifications. Small peptides can also be synthesized in the laboratory. Peptides that are subsets of the complete ZAP-1 sequence may be used to identify and investigate parts of the protein important for function, or to raise antibodies directed against these regions.

Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression

-1 1- host may be present. Expression vectors may be used for the production of fusion proteins, where the exogenous fusion peptide provides additional functionality, i.e. increased protein synthesis, stability, reactivity with defined antisera, an enzyme marker, e.g. β-galactosidase, etc. Expression cassettes may be prepared comprising a transcription initiation region, the ZAP-1 gene or fragment thereof, and a transcriptional termination region. Of particular interest is the use of sequences that allow for the expression of functional epitopes or domains, usually at least about 8 amino acids in length, more usually at least about 15 amino acids in length, to about 25 amino acids, and up to the complete open reading frame of the gene. After introduction of the DNA, the cells containing the construct may be selected by means of a selectable marker, the cells expanded and then used for expression.

When the ZAP-1 is to be secreted, the coding sequence for an extracellular domain will be fused, in frame, with sequences that permit secretion, including a signal peptide. Signal peptides may be exogenous or native. The subject peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream. The subject peptides may also be combined with other proteins, such as the Fc of an IgG isotype, which may be complement binding, with a toxin, such as ricin, abrin, diphtheria toxin, or the like, or with specific binding agents that allow targeting to specific moieties on a target cell. When the ZAP-1 is to be expressed on the surface of the cell, the coding sequence for the extracellular domain will be fused, in frame, with sequences encoding a peptide that anchors the extracellular domain into the membrane and a signal sequence. Such anchor sequences include transmembrane domains from cell surface proteins, e.g. CD4, CD8, slg, etc.

Where targeting is desired, the active domain of ZAP-1 may be produced as a fusion protein with an antibody that is specific for a target cell of interest, thereby providing for an antitumor antibody composition. The antibody may be produced as a single chain, instead of the normal multimeric structure. Single chain antibodies are described in Jost et al. (1994) J.B.C. 269:26267-73, and others. DNA sequences encoding the variable region of the heavy chain and the variable region

-12- of the light chain are ligated to a spacer encoding at least about 4 amino acids of small neutral amino acids, including glycine and/or serine. The protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original antibody. With the availability of the protein or fragments thereof in large amounts, by employing an expression host, the protein may be isolated and purified in accordance with conventional ways. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. The purified protein will generally be at least about 80% pure, preferably at least about 90% pure, and may be up to and including 100% pure. Pure is intended to mean free of other proteins, as well as cellular debris.

Formulations Formulations of ZAP-1 or ZAP-1 fragments are administered to a host affected by tumor growth or metastasis. The compounds of the present invention are administered at a dosage that reduces tumor growth while minimizing any side-effects. It is contemplated that the composition will be obtained and used under the guidance of a physician for in vivo use. Various methods for administration may be employed. The polypeptide formulation may be given orally, or may be injected intravascularly, intratumor, subcutaneously, peritoneally, inhalation, etc. The dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc. to maintain an effective dosage level. In many cases, oral administration will require a higher dose than if administered intravenously. The amide bonds, as well as the amino and carboxy termini, may be modified for greater stability on oral administration.

-13- Administration may be performed over several cycles, as described in Boehm et al, supra.

The compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration. The ZAP-1 may be systemic after administration or may be localized by the use of an implant that acts to retain the active dose at the site of implantation.

In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

For oral preparations, the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives

-14- such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

The compounds can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention. Similarly, unit dosage forms for injection or intravenous administration may comprise the compound of the present invention in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. Implants for sustained release formulations are well-known in the art.

Implants are formulated as microspheres, slabs, etc. with biodegradable or non- biodegradable polymers. For example, polymers of lactic acid and/or glycolic acid form an erodible polymer that is well-tolerated by the host. The implant containing ZAP-1 is placed in proximity to the site of the lesion, so that the local concentration of active agent is increased relative to the rest of the body.

The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the

-15- effect to be achieved, and the pharmacodynamics associated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Typical dosages for systemic administration range from 0.1 μg to 100 milligrams per kg weight of subject per administration. A typical dosage may be one tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release. Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific compounds are more potent than others. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound.

The use of liposomes as a delivery vehicle is one method of interest. The liposomes fuse with the cells of the target site and deliver the contents of the lumen intracellularly. The liposomes are maintained in contact with the cells for sufficient time for fusion, using various means to maintain contact, such as isolation, binding agents, and the like. In one aspect of the invention, liposomes are designed to be aerosolized for pulmonary administration. Liposomes may be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus, etc. The lipids may be any useful combination of known liposome forming lipids, including cationic lipids, such as phosphatidylcholine. The remaining lipid will normally be neutral lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.

-16- For preparing the liposomes, the procedure described by Kato et al. (1991) J. Biol. Chem. 266:3361 may be used. Briefly, the lipids and lumen composition containing the nucleic acids are combined in an appropriate aqueous medium, conveniently a saline medium where the total solids will be in the range of about 1-10 weight percent. After intense agitation for short periods of time, from about 5-60 sec, the tube is placed in a warm water bath, from about 25-40° C and this cycle repeated from about 5-10 times. The composition is then sonicated for a convenient period of time, generally from about 1-10 sec. and may be further agitated by vortexing. The volume is then expanded by adding aqueous medium, generally increasing the volume by about from 1-2 fold, followed by shaking and cooling. This method allows for the incorporation into the lumen of high molecular weight molecules.

For use in the subject methods, ZAP-1 may be formulated with other pharmaceutically active agents, particularly other anti-metastatic, anti-tumor or anti- angiogenic agents. Angiostatic compounds of interest include angiostatin, endostatin, carboxy terminal peptides of collagen alpha (XV), etc.

Cytotoxic and cytostatic agents of interest include adriamycin, alkeran, Ara-C,

BICNU, busulfan, CNNU, cisplatinum, cytoxan, daunorubicin, DTIC, 5-FU, hydrea, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, velban, vincristine, vinblastine, VP-16, carboplatinum, fludarabine, gemcitabine, idarubicin, irinotecan, leustatin, navelbine, taxol, taxotere, topotecan, etc.

Antibodies specific for ZAP-1

The expressed ZAP-1 polypeptides are useful for the production of antibodies, where short fragments provide for antibodies specific for the particular polypeptide, and larger fragments or the entire protein allow for the production of antibodies over the surface of the polypeptide. Antibodies may be raised to the wild-type or variant forms of ZAP-1. Antibodies may be raised to isolated peptides corresponding to these domains, or to the native protein. Antibodies are prepared in accordance with conventional ways, where the expressed polypeptide or protein is used as an immunogen, by itself or conjugated

-17- to known immunogenic carriers, e.g. KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the like. Various adjuvants may be employed, with a series of injections, as appropriate. For monoclonal antibodies, after one or more booster injections, the spleen is isolated, the lymphocytes immortalized by cell fusion, and then screened for high affinity antibody binding. The immortalized cells, i.e. hybridomas, producing the desired antibodies may then be expanded. For further description, see Monoclonal Antibodies: A Laboratory Manual, Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1988. If desired, the mRNA encoding the heavy and light chains may be isolated and mutagenized by cloning in E. coli, and the heavy and light chains mixed to further enhance the affinity of the antibody. Alternatives to in vivo immunization as a method of raising antibodies include binding to phage "display" libraries, usually in conjunction with in vitro affinity maturation.

DIAGNOSTIC USES

The subject nucleic acid and/or polypeptide compositions may be used to analyze a patient sample for deletions or mutations in ZAP-1. Biochemical studies may be performed to determine whether mutations in a ZAP-1 coding region or control regions is associated with cancers, particularly carcinomas, e.g. prostate, breast, lung, mesothelioma, neuroblastoma, etc. Disease associated polymorphisms may include deletion or truncation of the gene, mutations that alter expression level, that affect the protein structure, etc.

A number of methods are available for analyzing nucleic acids for the presence or absence of a specific sequence. Where large amounts of DNA are available, genomic DNA is used directly. Analysis of genomic DNA may use whole chromosomes or fractionated DNA, e.g. Southern blots, etc. Comparative Genomic Hybridization (CGH), as described in U.S. Patent no. 5,665,549, provides methods for determining the relative number of copies of a genomic sequence. The intensity of the signals from each labeled subject nucleic acid and/or the differences in the ratios between different signals from the labeled subject nucleic acid sequences are compared to determine the relative copy numbers of the nucleic acid sequences as

-18- a function of position along the reference chromosome spread. Other methods for fluorescence in situ hybridization are known in the art, for a review, see Fox et al. (1995) Clin Chem 41(11):1554-1559.

Alternatively, the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis. Cells that express ZAP-1 may be used as a source of mRNA, which may be assayed directly or reverse transcribed into cDNA for analysis. The nucleic acid may be amplified by conventional techniques, such as the polymerase chain reaction (PCR), to provide sufficient amounts for analysis. The use of the polymerase chain reaction is described in Saiki, et al. (1985) Science 239:487, and a review of techniques may be found in Sambrook, et al. Molecular Cloning: A Laboratory Manual. CSH Press 1989, pp.14.2-14.33. Alternatively, various methods are known in the art that utilize oligonucleotide ligation as a means of detecting polymorphisms, for examples see Riley et al (1990) N.A.R. 18:2887- 2890; and Delahunty et al. (1996) Am. J. Hum. Genet. 58:1239-1246. A detectable label may be included in an amplification reaction. Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, efc., where the binding partner is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.

The sample nucleic acid, e.g. genomic DNA, amplification product or cloned fragment, is analyzed by one of a number of methods known in the art. The nucleic acid may be sequenced by dideoxy or other methods, and the sequence of bases compared to a wild-type ZAP-1 sequence. Hybridization with the variant sequence may also be used to determine its presence, by Southern blots, dot blots, etc. The

-19- hybridization pattern of a control and variant sequence to an array of oligonucleotide probes immobilised on a solid support, as described in US 5,445,934, or in WO95/35505, may also be used as a means of detecting the presence of variant sequences. Single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), and heteroduplex analysis in gel matrices are used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility. Alternatively, where a polymorphism creates or destroys a recognition site for a restriction endonuclease, the sample is digested with that endonuclease, and the products size fractionated to determine whether the fragment was digested. Fractionation is performed by gel or capillary electrophoresis, particularly acrylamide or agarose gels.

Screening for mutations in ZAP-1 may be based on the functional or antigenic characteristics of the protein. Protein truncation assays are useful in detecting deletions that may affect the biological activity of the protein. Various immunoassays designed to detect polymorphisms in ZAP-1 proteins may be used in screening. Where many diverse genetic mutations lead to a particular disease phenotype, functional protein assays have proven to be effective screening tools.

Changes in the promoter or enhancer sequence that may affect expression levels oiZAP-1 can be compared to expression levels of the normal allele by various methods known in the art. Methods for determining promoter or enhancer strength include quantitation of the expressed natural protein; insertion of the variant control element into a vector with a reporter gene such as β-galactosidase, luciferase, chloramphenicol acetyltransferase, etc. that provides for convenient quantitation; and the like. Antibodies specific for a ZAP-1 may be used in staining or in immunoassays.

Samples, as used herein, include cells, e.g. biopsy samples, biological fluids such as semen, blood, cerebrospinal fluid, tears, saliva, lymph, dialysis fluid and the like; organ or tissue culture derived fluids; and fluids extracted from physiological tissues.

Also included in the term are derivatives and fractions of such fluids. The cells may be dissociated, in the case of solid tissues, or tissue sections may be analyzed. Alternatively a lysate of the cells may be prepared.

-20- Diagnosis may be performed by a number of methods to determine the absence or presence or altered amounts of normal or abnormal ZAP-1 in patient cells. For example, detection may utilize staining of cells or histological sections, performed in accordance with conventional methods. Cells are permeabilized to stain cytoplasmic molecules. The antibodies of interest are added to the cell sample, and incubated for a period of time sufficient to allow binding to the epitope, usually at least about 10 minutes. The antibody may be labeled with radioisotopes, enzymes, fluorescers, chemiluminescers, or other labels for direct detection. Alternatively, a second stage antibody or reagent is used to amplify the signal. Such reagents are well known in the art. For example, the primary antibody may be conjugated to biotin, with horseradish peroxidase-conjugated avidin added as a second stage reagent. Alternatively, the secondary antibody conjugated to a flourescent compound, e.g. flourescein, rhodamine, Texas red, etc. Final detection uses a substrate that undergoes a color change in the presence of the peroxidase. The absence or presence of antibody binding may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc.

An alternative method for diagnosis depends on the in vitro detection of binding between antibodies and ZAP-1 in a lysate. Measuring the concentration of ZAP-1 binding in a sample or fraction thereof may be accomplished by a variety of specific assays. A conventional sandwich type assay may be used. For example, a sandwich assay may first attach ZAP- 1 -specific antibodies to an insoluble surface or support. The particular manner of binding is not crucial so long as it is compatible with the reagents and overall methods of the invention. They may be bound to the plates covalently or non-covalently, preferably non-covalently.

The insoluble supports may be any compositions to which polypeptides can be bound, which is readily separated from soluble material, and which is otherwise compatible with the overall method. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports to which the receptor is bound include beads, e.g. magnetic beads, membranes and microtiter plates. These are typically made of glass, plastic (e.g. polystyrene),

-21- polysaccharides, nylon or nitrocellulose. Microtiter plates are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.

Patient sample lysates are then added to separately assayable supports (for example, separate wells of a microtiter plate) containing antibodies. Preferably, a series of standards, containing known concentrations of normal and/or abnormal

ZAP-1 is assayed in parallel with the samples or aliquots thereof to serve as controls.

Preferably, each sample and standard will be added to multiple wells so that mean values can be obtained for each. The incubation time should be sufficient for binding, generally, from about 0.1 to 3 hr is sufficient. After incubation, the insoluble support is generally washed of non-bound components. Generally, a dilute non-ionic detergent medium at an appropriate pH, generally 7-8, is used as a wash medium.

From one to six washes may be employed, with sufficient volume to thoroughly wash non-specifically bound proteins present in the sample. After washing, a solution containing a second antibody is applied. The antibody will bind ZAP-1 with sufficient specificity such that it can be distinguished from other components present. The second antibodies may be labeled to facilitate direct, or indirect quantification of binding. Examples of labels that permit direct

3 125 measurement of second receptor binding include radiolabels, such as H or I, fluorescers, dyes, beads, chemilumninescers, colloidal particles, and the like. Examples of labels which permit indirect measurement of binding include enzymes where the substrate may provide for a colored or fluorescent product. In a preferred embodiment, the antibodies are labeled with a covalently bound enzyme capable of providing a detectable product signal after addition of suitable substrate. Examples of suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art. The incubation time should be sufficient for the labeled ligand to bind available molecules. Generally, from about 0.1 to 3 hr is sufficient, usually 1 hr sufficing.

-22- After the second binding step, the insoluble support is again washed free of non-specifically bound material. The signal produced by the bound conjugate is detected by conventional means. Where an enzyme conjugate is used, an appropriate enzyme substrate is provided so a detectable product is formed.

Other immunoassays are known in the art and may find use as diagnostics.

Ouchterlony plates provide a simple determination of antibody binding. Western blots may be performed on protein gels or protein spots on filters, using a detection system specific for ZAP-1 as desired, conveniently using a labeling method as described for the sandwich assay.

MODULATION OF GENE EXPRESSION The ZAP-1 genes, gene fragments, or the encoded protein or protein fragments are useful in gene therapy to treat disorders associated with ZAP-1 defects. Expression vectors may be used to introduce the ZAP-1 gene into a cell. Such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks.

The gene orZAP-1 protein may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intramuscular administration, as described by Furth et al. (1992) Anal Biochem 205:365-368. The DNA may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun" as described in the literature (see, for example, Tang et al. (1992) Nature 356:152-154), where gold microprojectiles are coated with the ZAP-1 or DNA, then bombarded into skin cells.

-23- Antisense molecules can be used to down-regulate expression of ZAP-1 in cells. The anti-sense reagent may be antisense oligonucleotides (ODN), particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA. The antisense sequence is complementary to the mRNA of the targeted gene, and inhibits expression of the targeted gene products. Antisense molecules inhibit gene expression through various mechanisms, e.g. by reducing the amount of mRNA available for translation, through activation of RNAse H, or steric hindrance. One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the transcriptional initiation is oriented such that an antisense strand is produced as an RNA molecule. Alternatively, the antisense molecule is a synthetic oligonucleotide. Antisense oligonucleotides will generally be at least about 7, usually at least about 12, more usually at least about 20 nucleotides in length, and not more than about 500, usually not more than about 50, more usually not more than about 35 nucleotides in length, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like. It has been found that short oligonucleotides, of from 7 to 8 bases in length, can be strong and selective inhibitors of gene expression (see Wagner ef al. (1996) Nature Biotechnology 14:840-844).

A specific region or regions of the endogenous sense strand mRNA sequence is chosen to be complemented by the antisense sequence. Selection of a specific sequence for the oligonucleotide may use an empirical method, where several candidate sequences are assayed for inhibition of expression of the target gene in an in vitro or animal model. A combination of sequences may also be used, where several regions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methods known in the art (see Wagner et al. (1993) supra, and Milligan ef al, supra.) Preferred oligonucleotides are chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such

-24- modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O- phosphorothioate, 3'-CH2-5'-O-phosphonate and 3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity. The α-anomer of deoxyribose may be used, where the base is inverted with respect to the natural β-anomer. The 2'-OH of the ribose sugar may be altered to form 2'-O- methyl or 2'-O-allyl sugars, which provides resistance to degradation without comprising affinity. Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. 5- propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.

As an alternative to anti-sense inhibitors, catalytic nucleic acid compounds, e.g. ribozymes, anti-sense conjugates, efc. may be used to inhibit gene expression.

Ribozymes may be synthesized in vitro and administered to the patient, or may be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell (for example, see International patent application WO 9523225, and

Beigelman et al. (1995) Nucl. Acids Res 23:4434-42). Examples of oligonucleotides with catalytic activity are described in WO 9506764. Conjugates of anti-sense ODN with a metal complex, e.g. terpyridylCu(ll), capable of mediating mRNA hydrolysis are described in Bashkin et al. (1995) Appl Biochem Biotechnol 54:43-56.

-25- GENETICALLY ALTERED CELL OR ANIMAL MODELS FOR ZAP-1 FUNCTION The subject nucleic acids can be used to generate transgenic animals or site specific gene modifications in cell lines. Transgenic animals may be made through homologous recombination, where the normal ZAP-1 locus is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like.

The modified cells or animals are useful in the study of ZAP-1 function and regulation. For example, a series of small deletions and/or substitutions may be made in the ZAP-1 gene to determine the role of different exons in oncogenesis, protein-protein interactions, efc. Of interest are the use of ZAP-1 to construct transgenic animal models for cancer or metastasis, where expression of ZAP-1 is specifically reduced or absent, e.g. in epithelial tissue, efc. Conditional knock-outs are of interest, where the gene is inactivated only after exposure to a defined signal, for example by introduction of lox sites flanking the ZAP-1 gene, in combination with an inducible Cre expression construct. Specific constructs of interest include antisense ZAP-1, which will block ZAP-1 expression, expression of dominant negative ZAP-1 mutations, and conditional expression of ZAP-1 genes.

DNA constructs for homologous recombination will comprise at least a portion of the ZAP-1 gene with the desired genetic modification, and will include regions of homology to the target locus. DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection are included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al. (1990) Methods in EnzvmoloQV 185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, efc.

Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF). When ES or embryonic cells have been transformed, they may be used to produce transgenic animals. After transformation,

-26- the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo manipulation and blastocyst injection. Blastocysts are obtained from 4 to 6 week old superovulated females. The ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting offspring screened for the construct. By providing for a different phenotype of the blastocyst and the genetically modified cells, chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogeneic or congenic grafts or transplants, or in in vitro culture. The transgenic animals may be any non-human mammal, such as laboratory animals, domestic animals, etc. The transgenic animals may be used in functional studies, drug screening, efc., e.g. to determine the effect of a candidate drug on oncogenesis, metastasis, efc.

IN VITRO MODELS FOR ZAP-1 FUNCTION

Drug screening may be performed using a genetically altered cell or animal, purified ZAP-1 protein, or ZAP-1 protein in combination with other cellular proteins, such as collagens, fibronectins, cell surface receptors, efc. One can identify ligands or substrates that bind to, modulate or mimic the action of ZAP-1. Areas of investigation include the development of cancer treatments, metastasis, efc.

Drug screening identifies agents that provide a replacement for ZAP-1 function, that modulate ZAP-1 expression, or that inhibit agents such as proteases that may disrupt ZAP-1 during tumorigenesis. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays

-27- may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like. The purified protein may also be used for determination of three- dimensional crystal structure, which can be used for modeling intermolecular interactions, efc.

ZAP-1 is also of interest for modeling extracellular matrix and other cellular architecture that may be compromised during metastasis. For example, Batimastat (BB-94) and marimastat (BB-2516) are synthetic, low-molecular weight matrix metalloprotease inhibitors with a collagen-mimicking hydroxamate structure. Analogs of these drugs may be developed based on ZAP-1 protein structure.

The term "agent" as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of ZAP-1. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and

-28- oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, efc. to produce structural analogs.

Where the screening assay is a binding assay, one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, efc. may be used. The mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and

40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hours will be sufficient.

The compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host for treatment of cancer, efc. The therapeutic agents may be administered in a variety of ways, orally, topically, parenterally e.g. subcutaneously, intraperitoneally, by viral infection, intravasculariy, efc. Depending upon the manner of introduction, the compounds

-29- may be formulated in a variety of ways. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%.

The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.

The following examples are offered by illustration not by way of limitation.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, efc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

Example 1 Characterization of ZAP-1 Materials and Methods

PCR primers. (SEQ ID NO:5) 8700-011 : 5'-CCCACTTCTGCACTGTTTGC-3'; (SEQ ID NO:6) 8700-012: 5'-GTTGGAGGCAAGTTCATTCTG-3'.

LQH analysis. Primer sequences flanking polymorphic microsatellite loci (D1S2889, D1S2766, D1S1618, D1S1673, and D1S435) were obtained from the

Whitehead Genome Center Database (http://www-genome.wi.mit.edu/cgi-

-30- bin/contig/phys_map). PCR amplification, PCR product analysis, and calculation of the allelic ratios of heterozygous loci was performed essentially as described (Larson et al. (1997) Cancer Research 57: 4082-4090).

Tumor specimens and cell lines. Tumor specimens and matched non-involved tissue were provided by Memorial Sloan-Kettering Cancer Center. Cell Lines were provided by Memorial Sloan-Kettering Cancer Center and University of Tokyo. DNAs were extracted as previously described (Hampton et al. (1994) PNAS 91 : 6953- 6957).

cDNA selection. cDNA selection was performed as previously described (Lovett et al. (1991) PNAS 88: 9628-9632, Morgan et al. (1992) Nuc. Acids Res. 20: 5173-5179) and cDNA selection products were cloned into pCR2.1 as directed by the manufacturer (Invitrogen, Carlsbad, CA).

cDNA libraries. Fetal brain (Clontech, Palo Alto, CA) and adult frontal cortex (Stratagene, La Jolla, CA) cDNA libraries were screened as directed by the manufacturer.

Northern analysis. Human adult and fetal multiple tissue Northern blots were purchased from Clontech (Palo Alto, CA).

BAC sequencing. DNA from BAC clones was prepared using Qiagen DNA prep kits and further purified by CsCI gradient. DNA was sonicated and DNA fragments were repaired using nuclease BAL-31 and T4 DNA polymerase. DNA fragments of 0.8-2.2 kb were size-fractionated by agarose gel electrophoresis and ligated into pUC9 vector. Inserts of the plasmid clones were amplified by PCR and sequenced using standard ABI dye-primer chemistry (ABI, Foster City, CA). Preliminary sequence analysis and assembly were performed using the InnerPeace(IP) system developed at Sequana Theraputics Inc. (La Jolla, CA).

-31- Results

The 'address' of publicly available yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) clones which contained marker 7-14 was identified, and PCR primers 8700-011 and 8700-012, which are specific for 7-14 were made.

A search of the Whitehead Genome Center and Cooperative Human Linkage Center data bases revealed that the YAC clones positive for marker 7-14 mapped to chromosome 1p22 in an ~5cM region defined by markers D1S2627 and D1S2889. This area of the genome had been previously reported to exhibit a significant loss of heterozygosity (LOH) in a wide variety of tumor types including breast, colon, mesothelioma, cervical carcinoma and lung.

Genotypic analysis of a panel of 48 breast tumor/normal DNA pairs was performed. It was found that 40% of informative tumors show a LOH in this region. In addition, a screen of 96 tumor cell lines showed that 5 cervical carcinoma, 2 colon, 5 lung, 1 mesothelioma, and 1 neuroblastoma cell lines have homozygous deletions that include marker 7-14. These data suggest that marker 7-14 is located within close proximity of a tumor suppresser gene involved in the development of a wide variety of epithelial tumor types.

To identify transcription units encoded within the vicinity of 7-14, BAC clones 91.e.21 and 123.b.18 were subjected to cDNA selection using fetal brain, breast, and prostate cDNA libraries. In addition, BAC 91.e.21 DNA was randomly sheared and subcloned as a lambda phage library which was in turn sequenced resulting in

~10-fold coverage of the original BAC insert. Sequence analysis of one of the selected cDNA products (p1f5) indicated that it encoded a peptide containing a repetitive GZZ (G=glycine-Z=any amino acid) motif, which is commonly found in members of the collagen gene family. Analysis of the 91.e.21 BAC sequence indicated that the p1f5 sequence is interrupted, consistent with p1f5 representing 2 adjacent exons within the same transcription unit. A subsequent directed search of the 91.e.21 sequence for GZZ domains revealed the presence of several additional collagen-repeat like exons.

-32- Northern analysis using p1f5 as a probe indicate that this gene is expressed at low levels as both an 8.5 and a 4.5 kbp transcript in a wide variety of adult and fetal tissues. Several overlapping cDNA clones have been identified which correspond to the original p1f5 probe and/or map to the 91. e.21 and 123.b.18 BACs. Consistent with the Northern data, there are two splice variants (A and B) encoded by the same gene.

Mapping experiments indicate that both splice variants extend in either direction beyond the area represented by each of the two BAC clones. As such, the 2M homozygous deletion is certain to involve this transcript. Detailed analysis of the genomic sequence represented by BAC 91.e.21 gave no evidence for the presence of an additional transcription unit. These data demonstrate that ZAP-1 is the only gene directly affected by the 2M homozygous deletion, and therefore indicate its role as a tumor suppressor gene.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

-33-

Claims

WHAT IS CLAIMED IS:

1. An isolated nucleic acid molecule other than a naturally occurring chromosome comprising a sequence encoding a mammalian ZAP-1 protein, or fragment thereof of at least about 18 nucleotides.

2. An isolated nucleic acid according to Claim 1 , wherein said ZAP-1 protein is human.

3. An isolated nucleic acid molecule according to Claim 2, wherein said mammalian ZAP-1 protein is selected from the group consisting of SEQ ID NO:2 and

SEQ ID NO:4.

4. An isolated nucleic acid molecule according to Claim 3, wherein said nucleic acid comprises the nucleotide sequence of SEQ ID NO:1 or a fragment thereof.

5. An isolated nucleic acid molecule according to Claim 3, wherein said nucleic acid comprises the nucleotide sequence of SEQ ID NO:3 or a fragment thereof.

6. A cell comprising a nucleic acid composition according to Claim 1.

7. A purified polypeptide composition comprising at least 50 weight % of the protein present as a mammalian ZAP-1 protein

8. A purified polypeptide composition according to Claim 7, wherein said ZAP-1 protein is a human ZAP-1 protein.

9. A purified polypeptide composition according to Claim 8, wherein said protein comprises the amino acid sequence of SEQ ID NO:2.

-34-

10. A purified polypeptide according to Claim 8, wherein said protein comprises the amino acid sequence of SEQ ID NO:4.

11. A purified polypeptide according to Claim 8, wherein said ZAP-1 protein is a fusion protein comprising an exogenous fusion peptide.

12. A purified polypeptide according to Claim 8, wherein said human ZAP- 1 protein is substantially similar to the protein comprising the amino acid sequence of SEQ ID NO:2.

13. A purified polypeptide according to Claim 8, wherein said human ZAP- 1 protein is substantially similar to the protein comprising the amino acid sequence of SEQ ID NO:4.

14. An antibody, characterized as specifically reacting with a human ZAP-1 polypeptide.

15. An antibody according to Claim 14, wherein said antibody is a monoclonal antibody.

16. A purified polypeptide composition comprising a fragment of at least 10 amino acids of a human ZAP-1 protein.

17. A purified polypeptide composition according to Claim 16, wherein said fragment comprises a functional domain.

18. A purified polypeptide fragment according to Claim 17, wherein said fragment comprises an epitope.

19. A method for determining the ZAP-1 status of a tumor, the method comprising:

-35- analyzing the genomic DNA or mRNA of said tumor for the presence of ZAP- 1 alleles, wherein the deletion of one or both alleles is indicative that said tumor is ZAP-1 associated.

20. A method according to Claim 19, wherein said analyzing step comprises fluorescence in situ hybridization with a ZAP-1 specific probe.

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