A diverse system of adhesion molecules and adhesion receptors participates in orchestrating vital biologic phenomena, such as embryogenesis, cell growth and differentiation, and wound repair. Over the past decade there has been immense progress in our understanding of adhesion molecules and their complex interactions. We review the main classes of adhesion receptors and give examples of diseases in which they have been found to be involved.

The cadherins establish molecular links between adjacent cells. They form zipper-like structures at adherens junctions, membrane regions where a cell makes contact with other cells.

Through these junctions, bundles of actin filaments run from cell to cell. Related molecules, such as the desmogleins, are constituents of desmosomes, the intercellular contacts abundant in epithelial cells. Desmosomes serve as anchoring sites for intermediate filaments of the cytoskeleton. When dissociated embryonic cells are grown in a dish, they tend to cluster according to their tissue of origin. The homophilic interaction of cadherins is the basis of this histogenetic separation and has

a key role in segregating embryonic tissues. The expression of specific adhesion molecules in the embryo is crucial for the migration of cells and the differentiation of tissues. For example, when neural-crest cells stop producing N-CAM and N-cadherin and start displaying integrin receptors, they can separate and begin to migrate on the extracellular matrix.

Defective interactions between adhesion molecules have a critical role in cancer. The malignant behavior of a tumor depends on three characteristics of neoplastic cells: uncontrolled growth, local invasiveness, and the ability to metastasize. The presence or absence of adhesion molecules contributes to each of these properties. As the tumor evolves, the adhesive properties of its constituent cells change. The cells detach from the tumor mass and acquire the capacity to migrate and invade other organs. These changes may come about when the neoplastic cells reduce their production of matrix proteins, such as fibronectin, and are thus able to detach from the tumor.

Other cancer cells lose E-cadherin and become motile and invasive. Highly invasive tumor cells lose their invasiveness when transfected with a normal E-cadherin gene, whereas inactivation of the gene restores the aggressive phenotype. The induction of invasive behavior in tumor cells by anti-E-cadherin antibodies also supports the notion that this adhesion molecule suppresses local invasiveness and distant metastasis.

Cadherins, first discovered in mouse teratocarcinoma cells (Liaw et al. (1990) EMBO J.

9: 2701-2708, are structurally and functionally similar molecules [Ginsberg et al. (1991) Development 111: 315) that take part in selective calcium-dependent adhesion interactions between cell surfaces (Walsh et al. (1990) JNeurochem 55: 805. There are a number of different isoforms distributed in a tissue-specific manner in a wide variety of organisms. Cells containing different cadherins tend to segregate in vitro, while those that contain the same cadherins tend to preferentially aggregate together. This observation is linked to the finding that cadherin expression causes morphological changes involving the positional segregation of cells into layers, suggesting they may play an important role in the sorting of different cell types during morphogenesis, histogenesis and regeneration. They may also be involved in the regulation of tight and gap junctions, and in the control of intercellular spacing.

Cadherins comprise a family of Ca++-dependent adhesion molecules that function to mediate cell-cell binding critical to the maintenance of tissue structure and morphogenesis.

Structurally, cadherins comprise a number of domains: these include a signal sequence; a propeptide of around 130 residues; an extracellular domain of around 600 residues; a single

transmembrane domain; and a well-conserved C-terminal cytoplasmic domain of about 150 residues. The classical cadherins, E-,-and P-cadherin, each consist of large extracellular domains characterized by a series of five homologous NH2 terminal repeats, the most distal of which is thought to be responsible for binding specificity, transmembrane domains and carboxy terminal intracellular domains. The relatively short intracellular domains interact with a variety of cytoplasmic proteins, such as P-catenin, to regulate cadherin function. The extracellular domain can be subdivided into 5 parts, 4 of which are repeats of about 110 residues, and the fifth contains 4 conserved cysteines. The calcium-binding region of cadherins is thought to be located in the extracellular domain.

Studies of the tissue expression of the various cadherin-related proteins reveal that each subclass of molecule has a unique tissue distribution pattern. For example, E-cadherin is found in epithelial cells while N-cadherin is found in neural and muscle cells. Expression of cadherin-related proteins also appears to be spatially and temporally regulated during development because individual proteins appear to be expressed by specific cells and tissues at specific developmental stages [for review see Takeichi (1991), supra]. Both the ectopic expression of cadherin-related proteins and the inhibition of native expression of cadherin-related proteins hinders the formation of normal tissue structure (Detrick et al. (1990) Neuron 4: 493-506; Fujimori et al. (1990) Development 110: 97-104; Kintner (1992) Cell 69: 225-236.

The unique temporal and tissue expression pattern of the different cadherins and cadherin- related proteins is particularly significant when the role each subclass of proteins may play in vivo in normal events (e. g., the maintenance of the intestinal epithelial barrier) and in anormal events (e. g., tumor metastasis or inflammation) is considered. Different subclasses or combinations of subclasses of cadherin-related proteins are likely to be responsible for different cell-cell adhesion events in which therapeutic detection and/or intervention may be desirable. For example, auto- antibodies from patients with pemphigus vulgaris, an autoimmune skin disease characterized by blister formation caused by loss of cell adhesion, react with a cadherin-related protein offering direct support for adhesion function of cadherins in vivo (Amagai et al. (1991) Cell 67: 869-877.

Studies have also suggested that cadherins and cadherin-related proteins may have regulatory functions in addition to adhesive activity. Matsunaga et al. (1988) Nature 334: 62-64) reports that N-cadherin has neurite outgrowth promoting activity. The Drosophila fat tumor supressor gene appears to regulate cell growth and supress tumor invasion as does mammalian E-cadherin (see

Mahoney et al., supra; Frixen et al. (1991) J. Cell. Biol., 113: 173-185; Chen et al., (1991) J. Cell<BR> Biol. 114: 319-327; and Vleminckx et al. (1991) Cell 66: 107-119). Thus, therapeutic intervention in the regulatory activities of cadherin-related proteins expressed in specific tissues may be desirable.

Summary of tlre Invention The present invention relates to the discovery of a new class of cadherin-related proteins, referred to herein as ontherins. The ontherin polypeptides of the present invention, like the cadherin family of proteins, are expected to be broadly involved in the formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, both adult and embryonic, and can be used to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.

In general, the invention features isolated ontherin polypeptides, preferably substantially pure preparations of the subject ontherin polypeptides. The invention also provides recombinantly produced ontherin polypeptides. In preferred embodiments the polypeptide has a biological activity including one or more of : the ability to bind extracellular protein or matrix components, such as cadherins, integrins, desmosomal proteins (such as desmoglein and desmocollin); the ability to bind to intracellular signal transduction proteins, such as catenins; and the ability to bind to divalent calcium. Ontherin polypeptides which specifically antagonize such activities, such as may be provided by truncation mutants, are also specifically contemplated.

In one embodiment, the polypeptide is identical with or homologous to an ontherin polypeptide represented in SEQ ID No: 2, or the mature polypeptide sequence thereof (e. g., corresponding to residues 130-889 of SEQ ID. 2), or a truncated mature form of the extracellular domain (e. g., residues 130-708) or intracellular domain (residues 725-889). Other preferred ontherin polypeptides include at least 150,200 or 250 amino acid residues from the extracellular domain, preferably from the mature extracellular domain (e. g., from residues 130-708). Still other preferred ontherin polypeptides include at least 100,150 or 175 amino acid residues from the intracellular domain.

Related members of the ontherin family are also contemplated, for instance, an ontherin polypeptide preferably has an amino acid sequence at least 65%, 67%, 69%, 70%, 75% or 80% homologous to a polypeptide represented by SEQ ID No: 2 though polypeptides with higher

sequence homologies of, for example, 82%, 85%, 90% and 95% or are also contemplated. In a preferred embodiment, the ontherin polypeptide can be encoded by a nucleic acid which hybridizes under stringent conditions with a nucleic acid sequence represented in SEQ ID NO: 1. Homologs of the subject ontherin proteins also include versions of the protein which are resistant to post- translation modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with an ontherin ligand, e. g. an extracellular protein or matrix component.

The ontherin polypeptide can comprise a full length protein, such as represented in SEQ ID No: 2, or it may include a fragment thereof, e. g, corresponding to one or more particular motifs/domains, or to arbitrary sizes, e. g., at least 5,10,25,50,100,150 or 200 amino acids in length. In preferred embodiments, the ontherin polypeptide includes a sufficient portion of the extracellular domain to be able to specifically bind to Ca2+ or some other extracellular ligand.

Truncated forms of the protein include, but are not limited to, soluble extracellular fragments.

In certain preferred embodiments, the invention features a purified or recombinant ontherin polypeptide having a molecular weight (in the absence of post-translational modification) of about 95.6kd. It will be understood that certain post-translational modifications, e. g., glycosylation, prenylation, myristylation and the like, can increase the apparent molecular weight of the ontherin protein relative to the unmodified polypeptide chain.

The subject proteins can also be provided as chimeric molecules, such as in the form of fusion proteins. For instance, the ontherin protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e. g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the ontherin polypeptide, e. g. the second polypeptide portion is glutathione-S-transferase, e. g. the second polypeptide portion is an enzymatic activity such as alkaline phosphatase, e. g. the second polypeptide portion is an epitope tag.

In yet another embodiment, the invention features nucleic acids encoding ontherin polypeptides, which have the ability to modulate, e. g., either mimic or antagonize, at least a portion of the activity of a wild-type ontherin polypeptide. Exemplary ontherin-encoding nucleic acid sequences are represented by SEQ ID No: 1.

In another embodiment, the nucleic acids of the present invention include coding sequences which hybridize under stringent conditions with all or a portion of the coding sequences designated

in one or more of SEQ ID NO: 1. The coding sequences of the nucleic acids can comprise sequences which are identical to the coding sequence represented in SEQ ID No: 1, or it can merely be homologous to those sequences. In preferred embodiments, the nucleic acids encode polypeptides which specifically modulate, by acting as either agonists or antagonists, one or more of the bioactivities of wild-type ontherin polypeptides.

Furthermore, in certain preferred embodiments, the subject ontherin nucleic acids will include a transcriptional regulatory sequence, e. g. at least one of a transcriptional promoter or transcriptional enhancer sequence, which regulatory sequence is operably linked to the ontherin gene sequences. Such regulatory sequences can be used in to render the ontherin gene sequences suitable for use as an expression vector. The transcriptional regulatory sequence can be from an ontherin gene, or from a heterologous gene.

This invention also contemplates the cells transfected with said expression vector whether prokaryotic or eukaryotic and a method for producing ontherin proteins by employing said expression vectors.

In still other embodiments, the subject invention provides a gene activation construct, wherein the gene activation construct is deigned to recombine with a genomic ontherin gene in a cell to provide, e. g., by heterologous recombination, a heterologous transcriptional regulatory sequence operatively linked to a coding sequence of a genomic ontherin gene. Cells having genomic ontherin genes modified by gene activation constructs are also specifically contemplated.

In yet another embodiment, the present invention provides nucleic acids which hybridize under stringent conditions to nucleic acid probes corresponding to at least 12 consecutive nucleotides of either sense or antisense sequences of SEQ ID No: 1; though preferably to at least 25 consecutive nucleotides; and more preferably to at least 40,50 or 75 consecutive nucleotides of either sense or antisense sequence of SEQ ID No: 1.

Yet another aspect of the present invention concerns an immunogen comprising an ontherin polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for an ontherin polypeptide; e. g. a humoral response, e. g. an antibody response; e. g. a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e. g. a unique determinant, from a protein represented by SEQ ID No: 2.

A still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of the ontherin immunogen.

The invention also features transgenic non-human animals, e. g. mice, rats, rabbis, chickens, frogs or pigs, having a transgene, e. g., animals which include (and preferably express) a heterologous form of an ontherin gene described herein, or which misexpress an endogenous ontherin gene, e. g., an animal in which expression of one or more of the subject ontherin proteins is disrupted. Such a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed ontherin alleles or for use in drug screening.

The invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 12 consecutive nucleotides of sense or antisense sequences of SEQ ID NO: 1, or naturally occurring mutants thereof. In preferred embodiments, the probe/primer further includes a label group attached thereto and able to be detected. The label group can be selected, e. g., from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. Probes of the invention can be used as a part of a diagnostic test kit for identifying dysfunctions associated with mis-expression of an ontherin protein, such as for detecting in a sample of cells isolated from a patient, a level of a nucleic acid encoding an ontherin <BR> <BR> <BR> <BR> protein; e. g. measuring an ontherin mRNA level in a cell, or determining whether a genomic ontherin gene has been mutated or deleted. These so-called"probes/primers"of the invention can also be used as a part of"antisense"therapy which refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e. g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding one or more of the subject ontherin proteins so as to inhibit expression of that protein, e. g. by inhibiting transcription and/or translation. Preferably, the oligonucleotide is at least 12 nucleotides in length, though primers of 25,40,50, or 75 nucleotides in length are also contemplated.

In yet another aspect, the invention provides an assay for screening test compounds for inhibitors, or alternatively, potentiators, of an interaction between an ontherin protein and an polypeptide or other factor which interacts therewith (an ontherin interactor). An exemplary method includes the steps of (a) forming a reaction mixture including: (i) an ontherin interactor, (ii) an ontherin polypeptide, and (iii) a test compound ; and (b) detecting interaction of the ontherin interactor and ontherin polypeptides. A statistically significant change (potentiation or inhibition) in the interaction of the ontherin interactor and ontherin polypeptides in the presence of the test compound, relative to the interaction in the absence of the test compound, indicates a potential

agonist (mimetic or potentiator) or antagonist (inhibitor) of ontherin bioactivity for the test compound. The reaction mixture can be a cell-free protein preparation, e. g., a reconsistuted protein mixture or a cell lysate, or it can be a recombinant cell including a heterologous nucleic acid recombinantly expressing the ontherin polypeptide.

In preferred embodiments, the step of detecting interaction of the ontherin interactor and ontherin polypeptides is a competitive binding assay. In other preferred embodiments, the step of detecting interaction of the ontherin interactor and ontherin polypeptides involves detecting, in a cell-based assay, change (s) in the level of an intracellular second messenger responsive to signaling mediated by the ontherin polypeptide. In still another preferred embodiment, the step of detecting interaction of the ontherin interactor and ontherin polypeptides comprises detecting, in a cell-based assay, change (s) in the level of expression of a gene controlled by a transcriptional regulatory sequence responsive to signaling by the ontherin polypeptide.

In preferred embodiments, the steps of the assay are repeated for a variegated library of at least 100 different test compounds, more preferably at least 103,104 or 105 different test compounds. The test compound can be, e. g., a peptide, a nucleic acid, a carbohydrate, a small organic molecule, or natural product extract (or fraction thereof).

The present invention further contemplates the pharmaceutical formulation of one or more agents identified in such drug screening assays.

In other embodiments, the present invention provides a molecule, preferably a small organic molecule, which binds to ontherin and either mimics or antagonizes ontherin-induced signaling in cells.

Yet another aspect of the present invention concerns a method for modulating one or more of growth, differentiation, or survival of a cell by modulating ontherin bioactivity, e. g., by potentiating or disrupting certain protein-protein interactions. In general, whether carried out in vivo, in vitro, or in situ, the method comprises treating the cell with an effective amount of an ontherin therapeutic so as to alter, relative to the cell in the absence of treatment, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of the cell. Accordingly, the method can be carried out with ontherin therapeutics such as peptide and peptidomimetics or other molecules identified in the above-referenced drug screens which agonize or antagonize the effects of signaling from an ontherin protein by interaction with a ligand thereof or an intracellular signal transduction protein. Other ontherin therapeutics include antisense constructs for inhibiting expression of

ontherin proteins, dominant negative mutants of ontherin proteins which competitively inhibit ontherin interactions upstream of the cell surface, or signal transduction downstream of the wild- type ontherin protein, and gene therapy constructs including gene activation constructs.

In one embodiment, the subject method of modulating ontherin bioactivity can be used to modulate the differentiation of a neuronal cell, to maintain a neuronal cell in a differentiated state, and/or to enhance the survival of a neuronal cell, e. g., to prevent apoptosis or other forms of cell death. For instance the present method can be used to affect the differentiation of neuronal cells such as motor neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, and peptidergic neurons.

Another aspect of the present invention provides a method of determining if a subject, e. g. an animal patient, is at risk for a disorder characterized by unwanted cell proliferation or aberrant control of differentiation or apoptosis. The method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding an ontherin protein; or (ii) the mis-expression of an ontherin gene. In preferred embodiments, detecting the genetic lesion includes ascertaining the existence of at least one of : a deletion of one or more nucleotides from an ontherin gene; an addition of one or more nucleotides to the gene, a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; a non-wild type level of the protein; and/or an aberrant level of soluble ontherin protein.

For example, detecting the genetic lesion can include (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of an ontherin gene or naturally occurring mutants thereof, or 5'or 3'flanking sequences naturally associated with the ontherin gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e. g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the ontherin gene and, optionally, of the flanking nucleic acid sequences. For instance, the probe/primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR). In alternate embodiments, the level of an ontherin protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the ontherin protein.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U. S. Patent No: Nucleic Acid Hybridization (B. D. Hames & S. J.

Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. 1. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986).

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Detailed Description of the Invention Of particular importance in the development and maintenance of tissue in vertebrate animals is a type of extracellular communication called induction, which occurs between neighboring cell layers and tissues. In inductive interactions, cell-cell contacts and/or chemical signals secreted by one cell population influence the developmental fate of a second cell population. Typically, cells responding to the inductive signals are diverted from one cell fate to another, neither of which is the same as the fate of the signaling cells.

The regulation of extracellular-originating signals, whether arising from secreted molecules or cell-cell or cell-substrate interactions, is an important mechanism for developmental control.

The present invention concerns the discovery of a new family of cadherin-like proteins, referred to herein as"ontherins", which are demonstrated to have, inter alia, neurotrophic activity. As

described below, a human ontherin clone was first identified by expression cloning techniques though its ability to rescue striatal nerve cells from cell death. As described herein, the ontherin gene product exhibits spatially restricted expression domains indicative of important roles in the regulation of cellular physiology.

The ontherin proteins, through their ability to transduce information from the extracellular to the intracellular matrix, are apparently capable of modulating developmental processes by, for example, regulating proliferation, survival and/or differentiation of mesodermally-derived tissue, such as tissue derived from dorsal mesoderm, cartilage and tissue involved in spermatogenesis; regulating proliferation, survival and/or differentiation of ectodermally-derived tissue, such as tissue derived from the epidermis, neural tube, neural crest, or head mesenchyme; regulating proliferation, survival and/or differentiation of endodermally-derived tissue, such as tissue derived from the primitive gut.

A human ontherin cDNA was identified in a screen for neurotrophic activity using a cDNA library initially cloned with a signal sequence trap assay. The cDNAs were each cloned into a plasmid which allowed recombinant expression, with concomitant secretion into a mammalian cell culture. Samples of conditioned media, as well as membrane preparations of the cells producing the cDNA gene product, were applied to cultured striatal neurons under conditions wherein, absent a neurotrophic activity in the applied sample, the neurons would time with a defined kinetics. In the presence of appropriate neurotrophic activity, the half-life of the cells can be increased. A single positive cone was identified in from amongst the library members screened. The clone initially isolated by the neuron survival assay was sequenced, and determined to include a 3'coding sequence fragment. Screening human cDNA libraries with the fragment, a full length human cDNA was ultimately isolated.

The sequence for a human ontherin cDNA is provided in SEQ ID NO: 1, and the corresponding full length protein is provided in SEQ ID NO: 2. The originally isolated fragment correspond to a C-terminal fragment of the protein.

The sequence of an exemplary ontherin gene indicates it encodes a secreted protein that may be anchored at the cell membrane by a transmembrane domain. The ontherin proteins are apparently present naturally in a number of different forms, including a pro-form. The pro-form includes an N-terminal signal peptide (approximately N-terminal residues 130) for directed secretion of at least the N terminal domain of the protein, while the full-length mature form lacks

this signal sequence. Further processing of the mature form may also occur in some instances to yield biologically active fragments of the protein, such as extracellular fragments of the mature form of the protein.

Moreover, analysis of the protein sequences suggests 5 cadherin-like motifs in the N- terminal portion of the protein, e. g., residues 120-130,231-241,339-349,460-470, and 571-581 of SEQ ID NO: 2. A cadherin-like motif is generally found by the consensus sequence [LIV]-x- [LIV]-x-D-x-N-D- [NH]-x-P. There are also a number of potential Asn-linked glycosylation sites, e. g., having the consensus sequence N- {P}-[ST]-{P}, such as 22-25,266-269,439-442,453-456, 504-507,566-569 and 590-593 of SEQ ID NO: 2.

Likewise, the full-length ontherin protein also includes a transmembrane domain, e. g., corresponding approximately to residues 709-724 of SEQ ID NO: 2, thus providing an extracellular domain corresponding to residues 1-708 (131-708 of the mature form), and an intracellular domain corresponding to residues 725-889. Ontherin polypeptides lacking the transmembrane and intracellular domains, e. g., due to alternate splicing or proteolysis, are expected to be fully secreted rather than membrane bound.

It is contemplated by the present invention that the cloned ontherin gene set out in the appended sequence listing, in addition to representing a member of an inter-species family of related genes (e. g., amongst vertebrates), is also believed to be a part of an intra-species family.

That is, it is anticipated that other human paralogs of the human ontherin proteins exist, and orthologs of each ontherin-like gene are conserved amongst other animals.

The expression pattern for ontherin genes is being determined by several different approaches. In one technique, the occurrence of the clone in signal sequence trap assays from a variety of different embryonic and adult tissues was examined. It was observed that the ontherin coding sequence was cloned from several different tissues. The most abundant source, according to the signal sequence trap assay, was brain. The clone was also isolated from testes, kidney and placenta.

Accordingly, certain aspects of the present invention relate to nucleic acids encoding ontherin polypeptides, the ontherin polypeptides themselves (including various fragments), antibodies immunoreactive with ontherin proteins, and preparations of such compositions.

Moreover, the present invention provides diagnostic and therapeutic assays and reagents for detecting and treating disorders involving, for example, aberrant expression (or loss thereof) of

ontherin, aberrant processing or splicing of ontherin proteins or mRNA, ontherin ligands or signal transducers thereof.

In addition, drug discovery assays are provided for identifying agents which can modulate the biological function of ontherin proteins, such as by altering the binding of ontherin molecules to ions or other extracellular/matrix factors, or the ability of the ontherin protein to transduce intracellular signals, e. g., by interacting with cell surface and intracellular proteins. Such agents can be useful therapeutically to alter the growth, maintenance and/or differentiation of a tissue.

Other aspects of the invention are described below or will be apparent to those skilled in the art in light of the present disclosure.

For convenience, certain terms employed in the specification and appended claims are collected here.

The term"ontherin"polypeptide refers to a family of polypeptides characterized at least in part by being identical or sharing a degree of sequence homology with all or a portion of the ontherin polypeptide represented in SEQ ID No: 2. The ontherin polypeptides can be cloned or purified from any of a number of eukaryotic organisms, especially vertebrates, and particularly mammals. Moreover, other ontherin polypeptides can be generated according to the present invention, which polypeptides do not ordinarily exist in nature, but rather are generated by non- natural mutagenic techniques.

A number of features of the ontherin protein have been observed upon inspection. In particular, it is noted that ontherin sequence encodes a secreted protein having a secretory signal sequence (e. g., a peptidyl portion which causes extracellular secretion of at least a portion of the protein) corresponding to residues 1-130 of SEQ ID NO: 2.

A"transmembrane"domain refers to sequence of amino acids that is capable of retaining the ontherin polypeptide at the cell surface, e. g., by passing through the cell surface membrane.

A"glycosylated"ontherin polypeptide is an ontherin polypeptide having a covalent linkage with a glycosyl group (e. g. a derivatized with a carbohydrate). For instance, the ontherin protein can be glycosylated on an existing residue, or can be mutated to preclude carbohydrate attachment, or can be mutated to provide new glycosylation sites, such as for N-linked or O-linked glycosylation.

As used herein, the term"nucleic acid"refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood

to include, as equivalents. analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.

As used herein, the term"gene"or"recombinant gene"refers to a nucleic acid comprising an open reading frame encoding an ontherin polypeptide, including both exon and (optionally) intron sequences. A"recombinant gene"refers to nucleic acid encoding an ontherin polypeptide and comprising ontherin-encoding exon sequences, though it may optionally include intron sequences which are derived from, for example, a chromosomal ontherin gene or from an unrelated chromosomal gene. Exemplary recombinant genes encoding the subject ontherin polypeptide are represented in the appended Sequence Listing. The term"intron"refers to a DNA sequence present in a given ontherin gene which is not translated into protein and is generally found between exons.

As used herein, the term"transfection"means the introduction of a nucleic acid, e. g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer."Transformation", as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of an ontherin polypeptide or, where anti-sense expression occurs from the transferred gene, the expression of a naturally-occurring form of the ontherin protein is disrupted.

As used herein, the term"specifically hybridizes"refers to the ability of a nucleic acid probe/primer of the invention to hybridize to at least 15 consecutive nucleotides of an ontherin gene, such as an ontherin sequence designated in SEQ ID NO: 1, or a sequence complementary thereto, or naturally occurring mutants thereof, such that it has less than 15%, preferably less than 10%, and more preferably less than 5% background hybridization to a cellular nucleic acid (e. g., mRNA or genomic DNA) encoding a protein other than an ontherin protein, as defined herein.

An"ontherin therapeutic", as described in further detail below, is an agent which is capable of agonizing (mimicing or potentiating) or antagonizing the activity of a native ontherin protein.

Whether inhibitory or potentiating with respect to modulating the activity of an ontherin protein, the ontherin therapeutic can be, as appropriate, an isolated ontherin polypeptide, a gene therapy construct, an antisense nucleic acid, a peptidomimetic, or an agent identified in the drug assays provided herein An"effective amount"of an ontherin therapeutic, with respect to the subject methods for treating animals, refers to an amount of agonist or antagonist in a preparation which, when applied

as part of a desired dosage regimen, provides modulation of growth, differentiation or survival of cells, e. g., modulation of neuronal differentiation.

As used herein,"phenotype"refers to the entire physical, biochemical, and physiological makeup of a cell, e. g., having any one trait or any group of traits.

The terms"induction"or"induce", as relating to the biological activity of an ontherin protein, refers generally to the process or act of causing to occur a specific effect on the phenotype of cell. Such effect can be in the form of causing a change in the phenotype, e. g., differentiation to another cell phenotype, or can be in the form of maintaining the cell in a particular cell, e. g., preventing dedifferentation or promoting survival of a cell.

A"patient"or"subject"to be treated can mean either a human or non-human animal.

As used herein, the term"vector"refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i. e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of"plasmids"which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification,"plasmid"and "vector"are used interchangeably as the plasmid is the most commonly used form of vector.

However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

"Transcriptional regulatory sequence"is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked. In preferred embodiments, transcription of a recombinant ontherin gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally- occurring forms of ontherin genes.

As used herein, the term"tissue-specific promoter"means a DNA sequence that serves as a promoter, i. e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as cells of neuronal or hematopoietic origin. The term also covers so-called"leaky"promoters, which regulate expression of a selected DNA primarily in one tissue, but can cause at least low level expression in other tissues as well.

As used herein, a"transgenic animal"is any animal, preferably a non-human mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.

The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.

This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In an exemplary transgenic animal, the transgene causes cells to express a recombinant form of an ontherin protein, e. g. either agonistic or antagonistic forms. However, transgenic animals in which the recombinant ontherin gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below. Moreover, "transgenic animal"also includes those recombinant animals in which gene disruption of one or more ontherin genes is caused by human intervention, including both recombination and antisense techniques.

The"non-human animals"of the invention include vertebrates such as rodents, non-human primates, livestock, avian species, amphibians, reptiles, etc. The term"chimeric animal"is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant is expressed in some but not all cells of the animal. The term"tissue-specific chimeric animal" indicates that a recombinant ontherin gene is present and/or expressed or disrupted in some tissues but not others.

As used herein, the term"transgene"means a nucleic acid sequence (encoding, e. g., an ontherin polypeptide, or pending an antisense transcript thereto), which is partly or entirely heterologous, i. e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but

which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e. g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.

As is well known, genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same activity. The term"DNA sequence encoding an ontherin polypeptide"may thus refer to one or more genes within a particular individual. Moreover, certain differences in nucleotide sequences may exist between individuals of the same species, which are called alleles. Such allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.

"Homology"and"identity"each refer to sequence similarity between two polypeptide sequences, with identity being a more strict comparison. Homology and identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same amino acid residue, then the polypeptides can be referred to as identical at that position; when the equivalent site is occupied by the same amino acid (e. g., identical) or a similar amino acid (e. g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous at that position. A percentage of homology or identity between sequences is a function of the number of matching or homologous positions shared by the sequences. An"unrelated"or"non-homologous" sequence shares less than 40 percent identity, though preferably less than 25 percent identity, with an ontherin sequence of the present invention.

The term"ortholog"refers to genes or proteins which are homologs via speciation, e. g., closely related and assumed to have common descent based on structural and functional considerations. Orthologous proteins function as recognizably the same activity in different species. The term"paralog"refers to genes or proteins which are homologs via gene duplication, e. g., duplicated variants of a gene within a genome. See also, Fritch, WM (1970) Syst Zool 19: 99- 113.

"Cells,""host cells"or"recombinant host cells"are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A"chimeric protein"or"fusion protein"is a fusion of a first amino acid sequence encoding an ontherin polypeptide with a second amino acid sequence defining a domain (e. g. polypeptide portion) foreign to and not substantially homologous with any domain of an ontherin protein. A chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an"interspecies","intergenic", etc. fusion of protein structures expressed by different kinds of organisms. In general, a fusion protein can be represented by the general formula X-ontherin-Y, wherein ontherin represents a portion of the fusion protein which is derived from an ontherin protein, and X and Y are, independently, absent or represent amino acid sequences which are not related to an ontherin sequences in an organism.

As used herein, a"reporter gene construct"is a nucleic acid that includes a"reporter gene"operatively linked to a transcriptional regulatory sequences. Transcription of the reporter gene is controlled by these sequences. The activity of at least one or more of these control sequences is directly or indirectly regulated by a signal transduction pathway involving a phospholipase, e. g., is directly or indirectly regulated by a second messenger produced by the phospholipase activity. The transcriptional regulatory sequences can include a promoter and other regulatory regions, such as enhancer sequences, that modulate the activity of the promoter, or regulatory sequences that modulate the activity or efficiency of the RNA polymerase that recognizes the promoter, or regulatory sequences that are recognized by effector molecules, including those that are specifically induced upon activation of a phospholipase. For example, modulation of the activity of the promoter may be effected by altering the RNA polymerase binding to the promoter region, or, alternatively, by interfering with initiation of transcription or elongation of the mRNA. Such sequences are herein collectively referred to as transcriptional regulatory elements or sequences. In addition, the construct may include sequences of nucleotides that alter the stability or rate of translation of the resulting mRNA in response to second messages, thereby altering the amount of reporter gene product.

The term"isolated"as also used herein with respect to nucleic acids. such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule. For example, an isolated nucleic acid encoding an ontherin polypeptide preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the ontherin gene in genomic DNA, more preferably no more than 5kb of such naturally occurring flanking sequences, and most preferably less than 1.5kb of such naturally occurring flanking sequence. The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an"isolated nucleic acid"is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.

As described below, one aspect of the invention pertains to isolated nucleic acids comprising nucleotide sequences encoding ontherin polypeptides, and/or equivalents of such nucleic acids. The term nucleic acid as used herein is intended to include fragments as equivalents.

The term equivalent is understood to include nucleotide sequences encoding functionally equivalent ontherin polypeptides or functionally equivalent peptides having an activity of an ontherin protein such as described herein. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will, therefore, include sequences that differ from the nucleotide sequence of the ontherin coding sequence shown in SEQ ID NO: 1 due to the degeneracy of the genetic code. Equivalents will also include nucleotide sequences that hybridize under stringent conditions (i. e., equivalent to about 20-270C below the melting temperature (Tm) of the DNA duplex formed in about 1 M salt) to the nucleotide sequences represented in SEQ ID No: 1. In one embodiment, equivalents will further include nucleic acid sequences derived from and evolutionarily related to, a nucleotide sequence shown in SEQ ID No: l.

Moreover, it will be generally appreciated that, under certain circumstances, it may be advantageous to provide homologs of an ontherin polypeptide which function in a limited capacity as one of either an agonist (e. g., mimics or potentiates a bioactivity of the wild-type ontherin protein) or an antagonist (e. g., inhibits a bioactivity of the wild-type ontherin protein), in order to promote or inhibit only a subset of the biological activities of the naturally-occurring form of the protein. Thus, specific biological effects can be elicited by treatment with a homolog of limited

function. For example, truncated forms of the ontherin protein, e. g., soluble fragments of the extracellular domain, can be provided to competitively inhibit ligand (or ion) binding to the wild- type ontherin protein.

Homologs of the subject ontherin protein can be generated by mutagenesis, such as by discrete point mutation (s), or by truncation. For instance, mutation can give rise to homologs which retain substantially the same. or merely a subset, of the biological activity of the ontherin polypeptide from which it was derived. Alternatively, antagonistic forms of the protein can be generated which are able to inhibit the function of the naturally occurring form of the protein, such as by competitively binding to ontherin ligands and competing with wild-type ontherin, or binding to other ontherin or adhesion proteins (such as cadherins) to form unresponsive complexes. Thus, the ontherin protein and homologs thereof provided by the subject invention may be either positive or negative regulators of cell growth. death and/or differentiation.

In general, polypeptides referred to herein as having an activity of an ontherin protein (e. g., are"bioactive") are defined as polypeptides which include an amino acid sequence corresponding (e. g., identical or homologous) to all or a portion of the amino acid sequences of the ontherin protein shown in SEQ ID NO: 2, and which agonize or antagonize all or a portion of the biological/biochemical activities of a naturally occurring ontherin protein. Examples of such biological activity may include the ability to bind calcium, particularly Ca2+, the ability to bind to a catenin (such as a-, (3-or y-catenin) or other protein containing an"armadillo repeat region", the ability to bind to a desmosomal protein, or the ability to bind to a receptor-type protein tyrosine phosphatase. The bioactivity of certain embodiments of the subject ontherin polypeptides can be characterized in terms of an ability to promote differentiation and/or maintenance of cells and tissue from mesodermally-derived tissue, such as tissue derived from dorsal mesoderm; ectodermally- origin, such as tissue derived from the neural tube, neural crest, or head mesenchyme; or endodermally-derived tissue, such as tissue derived from the primitive gut.

Other biological activities of the subject ontherin proteins are described herein or will be reasonably apparent to those skilled in the art. According to the present invention, a polypeptide has biological activity if it is a specific agonist or antagonist of a naturally-occurring form of an ontherin protein.

Preferred nucleic acids encode an ontherin polypeptide comprising an amino acid sequence at least 60%, 70% or 80% homologous, more preferably at least 85% homologous and most

preferably at least 95% homologous with an amino acid sequence of a naturally occurring ontherin protein, e. g., such as represented in SEQ ID NO: 2. Nucleic acids which encode polypeptides at least about 98-99% homology with an amino acid sequence represented in SEQ ID NO: 2 are of course also within the scope of the invention, as are nucleic acids identical in sequence with the enumerated ontherin sequence of the Sequence listing. In one embodiment, the nucleic acid is a cDNA encoding a polypeptide having at least one activity of the subject ontherin polypeptide.

In certain preferred embodiments, the invention features a purified or recombinant ontherin polypeptide having peptide chain with a molecular weight in the range of 86kd to 106kd, even more preferably in the range of 92kd to I OOkd (for a full-length ontherin protein), an most preferably of about 96.5kd. It will be understood that certain post-translational modifications, e. g., glycosylation, phosphorylation and the like, can increase the apparent molecular weight of the ontherin protein relative to the unmodified polypeptide chain, and cleavage of certain sequences, such as pro- sequences, can likewise decrease the apparent molecular weight. Other preferred ontherin polypeptides include: a mature ontherin polypeptide which lacks the signal sequence peptide, e. g., corresponding to residues 131-889 of SEQ ID No: 2, a mature, extracellular fragment (soluble) of the protein, e. g., corresponding to residues 131-708 of SEQ ID No: 2. In a preferred embodiments, the nucleic acid encodes an ontherin polypeptide which includes a calcium binding domain. By a"molecular weight of about"it is meant with in about 5kd.

Another aspect of the invention provides a nucleic acid which hybridizes under high or low stringency conditions to the nucleic acid represented by SEQ ID NO: 1. Appropriate stringency conditions which promote DNA hybridization, for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C.

Nucleic acids, having a sequence that differs from the nucleotide sequences shown in SEQ ID No: 1 due to degeneracy in the genetic code are also within the scope of the invention. Such nucleic acids encode functionally equivalent peptides (i. e., a peptide having a biological activity of an ontherin polypeptide) but differ in sequence from the sequence shown in the sequence listing

due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC each encode histidine) may result in"silent"mutations which do not affect the amino acid sequence of an ontherin polypeptide. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject ontherin polypeptides will exist among, for example, humans. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acids encoding polypeptides having an activity of an ontherin polypeptide may exist among individuals of a given species due to natural allelic variation.

As used herein, an ontherin gene fragment refers to a nucleic acid having fewer nucleotides than the nucleotide sequence encoding the entire mature form of an ontherin protein yet which (preferably) encodes a polypeptide which retains some biological activity of the full length protein.

Fragment sizes contemplated by the present invention include, for example, 5,10,25,50,75,100, or 200 amino acids in length. In a preferred embodiment of a truncated receptor, the polypeptide will include all or a sufficient portion of an intracellular or extracellular domain to permit the polypeptide to interact with a ligand, and ion, or other cell surface or intracellular proteins.

As indicated by the examples set out below, ontherin protein-encoding nucleic acids can be obtained from mRNA present in cells of metazoan organisms. It should also be possible to obtain nucleic acids encoding ontherin polypeptides of the present invention from genomic DNA from both adults and embryos. For example, a gene encoding an ontherin protein can be cloned from either a cDNA or a genomic library in accordance with protocols described herein, as well as those generally known to persons skilled in the art. A cDNA encoding an ontherin protein can be obtained by isolating total mRNA from a cell, such as a mammalian cell, e. g. a human cell, as desired. Double stranded cDNAs can be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques.

The gene encoding an ontherin protein can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention. The nucleic acid of the invention can be DNA or RNA. A preferred nucleic acid is a cDNA including a nucleotide sequence represented by SEQ ID No: 1.

Another aspect of the invention relates to the use of the isolated nucleic acid in"antisense" therapy. As used herein,"antisense"therapy refers to administration or in situ generation of

oligonucleotide probes or their derivatives which specifically hybridize (e. g. binds) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding a subject ontherin protein so as to inhibit expression of that protein, e. g. by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, "antisense"therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an ontherin protein. Alternatively, the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of an ontherin gene. Such oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e. g. exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U. S. Patents 5,176,996; 5,264,564; and 5,256,775), or peptide nucleic acids (PNAs). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) Biotechniques 6: 958-976; and Stein et al. (1988) Cancer Res 48: 2659-2668.

Accordingly, the modified oligomers of the invention are useful in therapeutic, diagnostic, and research contexts. In therapeutic applications, the oligomers are utilized in a manner appropriate for antisense therapy in general. For such therapy, the oligomers of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the oligomers of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use.

Lyophilized forms are also included.

Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation.

Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics. For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.

In addition to use in therapy, the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind. Such diagnostic tests are described in further detail below.

Likewise, the antisense constructs of the present invention, by antagonizing the normal biological activity of an ontherin protein, e. g., by reducing the level of its expression, can be used in the manipulation of tissue, e. g. tissue maintenance, differentiation or growth, both in vivo and ex vivo.

Furthermore, the anti-sense techniques (e. g. microinjection of antisense molecules, or <BR> <BR> <BR> <BR> <BR> transfection with plasmids whose transcripts are anti-sense with regard to an ontherin mRNA or gene sequence) can be used to investigate the role of ontherin in developmental events, as well as the normal cellular function of ontherin in adult tissue. Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals (described infra).

This invention also provides expression vectors containing a nucleic acid encoding an ontherin polypeptide, operably linked to at least one transcriptional regulatory sequence. Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art- recognized and are selected to direct expression of the subject ontherin proteins. Accordingly, the term transcriptional regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). For instance, any of a wide variety of expression control sequences, sequences that control the expression of a DNA sequence when operatively linked to it, may be used in these vectors to express DNA sequences encoding

ontherin polypeptides of this invention. Such useful expression control sequences, include, for example, a viral LTR. such as the LTR of the Moloney murine leukemia virus, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage X, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e. g., Pho5, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.

Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. In one embodiment, the expression vector includes a recombinant gene encoding a polypeptide having an agonistic activity of a subject ontherin polypeptide, or alternatively, encoding a polypeptide which is an antagonistic form of wild-type ontherin proteins. An exemplary ontherin polypeptide of the present invention is a soluble truncated form of the protein which retains a ion binding domain, e. g., retains the ability to bind to divalent calcium. Such expression vectors can be used to transfect cells and thereby produce polypeptides, including fusion proteins, encoded by nucleic acids as described herein.

Moreover, the gene constructs of the present invention can also be used as a part of a gene therapy protocol to deliver nucleic acids, e. g., encoding either an agonistic or antagonistic form of a subject ontherin proteins or an antisense molecule described above. Thus, another aspect of the <BR> <BR> <BR> <BR> invention features expression vectors for in vivo or in vitro transfection and expression of an ontherin polypeptide or antisense molecule in particular cell types so as to reconstitute the function of, or alternatively, abrogate all or a portion of the biological function of ontherin-induced transcription in a tissue in which the naturally-occurring form of the protein is misexpressed (or has been disrupted); or to deliver a form of the protein which alters maintenance or differentiation of tissue, or which inhibits neoplastic or hyperplastic proliferation.

Expression constructs of the subject ontherin polypeptides, as well as antisense constructs, may be administered in any biologically effective carrier, e. g. any formulation or composition

capable of effectively delivering the recombinant gene to cells in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno- associated virus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e. g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaP04 precipitation carried out in vivo. It will be appreciated that because transduction of appropriate target cells represents the critical first step in gene therapy, choice of the particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, e. g. locally or systemically. Furthermore, it will be recognized that the particular gene construct provided for in vivo transduction of ontherin expression are also useful for in vitro transduction of cells, such as for use in the ex vivo tissue culture systems described below.

A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e. g. a cDNA encoding the particular ontherin polypeptide desired.

Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e. g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid. Retrovirus vectors, adenovirus vectors and adeno-associated virus vectors are exemplary recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.

In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a subject ontherin polypeptide in the tissue of an animal.

Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject ontherin polypeptide gene by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.

In clinical settings, the gene delivery systems for the therapeutic ontherin gene can be introduced into a patient-animal by any of a number of methods, each of which is familiar in the

art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e. g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.

For example, the gene delivery vehicle can be introduced by catheter (see U. S. Patent 5,328,470) or by stereotactic injection (e. g. Chen et al. (1994) PNAS 91: 3054-3057). An ontherin gene can be delivered in a gene therapy construct by electroporation using techniques described, for example, by Dev et al. ( (1994) Cancer Treat Rev 20: 105-115).

The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e. g. retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.

In yet another embodiment, the subject invention provides a"gene activation"construct which, by homologous recombination with a genomic DNA, alters the transcriptional regulatory sequences of an endogenous ontherin gene. For instance, the gene activation construct can replace the endogenous promoter of an ontherin gene with a heterologous promoter, e. g., one which causes constitutive expression of the ontherin gene or which causes inducible expression of the gene under conditions different from the normal expression pattern of ontherin. A variety of different formats for the gene activation constructs are available. See, for example, the Transkaryotic Therapies, Inc PCT publications W093/09222, W095/31560, W096/29411, W095/31560 and W094/12650.

In preferred embodiments, the nucleotide sequence used as the gene activation construct can be comprised of (1) DNA from some portion of the endogenous ontherin gene (exon sequence, intron sequence, promoter sequences, etc.) which direct recombination and (2) heterologous transcriptional regulatory sequence (s) which is to be operably linked to the coding sequence for the genomic ontherin gene upon recombination of the gene activation construct. For use in generating cultures of ontherin producing cells, the construct may further include a reporter gene to detect the presence of the knockout construct in the cell.

The gene activation construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to provide the heterologous regulatory sequences in operative association with the native ontherin gene. Such insertion occurs by homologous recombination, i. e., recombination regions of the activation construct that are homologous to the endogenous ontherin gene sequence hybridize to the genomic DNA and recombine with the genomic sequences so that the construct is incorporated into the corresponding position of the genomic DNA.

The terms"recombination region"or"targeting sequence"refer to a segment (i. e., a portion) of a gene activation construct having a sequence that is substantially identical to or substantially complementary to a genomic gene sequence, e. g., including 5'flanking sequences of the genomic gene, and can facilitate homologous recombination between the genomic sequence and the targeting transgene construct.

As used herein, the term"replacement region"refers to a portion of a activation construct which becomes integrated into an endogenous chromosomal location following homologous recombination between a recombination region and a genomic sequence.

The heterologous regulatory sequences, e. g., which are provided in the replacement region, can include one or more of a variety elements, including: promoters (such as constitutive or inducible promoters), enhancers, negative regulatory elements, locus control regions, transcription factor binding sites, or combinations thereof. Promoters/enhancers which may be used to control the expression of the targeted gene in vivo include, but are not limited to, the cytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., 1989, J. Exp. Med., 169: 13), the human p-actin promoter (Gunning et al. (1987) PNAS 84: 4831-4835), the glucocorticoid-inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig et al.

(1984) Mol. Cell Biol. 4: 1354-1362), the long terminal repeat sequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al. (1985) RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), the SV40 early or late region promoter (Bernoist et <BR> <BR> <BR> <BR> al. (1981) Nature 290: 304-310; Templeton et al. (1984) Mol. Cell Biol., 4: 817; and Sprague et al.

(1983) J. Virol., 45: 773), the promoter contained in the 3'long terminal repeat of Rous sarcoma virus (RSV) (Yamamoto et al., 1980, Cell, 22: 787-797), the herpes simplex virus (HSV) thymidine kinase promoter/enhancer (Wagner et al. (1981) PNAS 82: 3567-71), and the herpes simplex virus LAT promoter (Wolfe et al. (1992) Nature Genetics, 1: 379-384).

In still other embodiments, the replacement region merely deletes a negative transcriptional control element of the native gene, e. g., to activate expression, or ablates a positive control element, e. g., to inhibit expression of the targeted gene.

Another aspect of the present invention concerns recombinant forms of the ontherin proteins. Recombinant polypeptides preferred by the present invention, in addition to native ontherin proteins, are at least 60% or 70% homologous, more preferably at least 80% homologous and most preferably at least 85% homologous with the amino acid sequence represented by SEQ ID No: 2, or a bioactive fragment thereof. Polypeptides which possess an activity of an ontherin protein (i. e. either agonistic or antagonistic), and which are at least 90%, more preferably at least 95%, and most preferably at least about 98-99% homologous with SEQ ID No: 2 are also within the scope of the invention. Such polypeptides, as described above, include various truncated forms of the protein.

The term"recombinant ontherin polypeptide"refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding an ontherin polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. Moreover, the phrase"derived from", with respect to a recombinant ontherin gene, is meant to include within the meaning of"recombinant protein"those proteins having an amino acid sequence of a native ontherin protein, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions (including truncation) of a naturally occurring form of the protein.

The present invention further pertains to recombinant forms of the subject ontherin polypeptides which are encoded by genes derived from a mammal (e. g. a human), reptile or amphibian and which have amino acid sequences evolutionarily related to the ontherin protein represented in SEQ ID No: 2. Such recombinant ontherin polypeptides preferably are capable of functioning in one of either role of an agonist or antagonist of at least one biological activity of a wild-type ("authentic") ontherin protein of the appended sequence listing. The term"evolutionarily related to", with respect to amino acid sequences of ontherin proteins, refers to both polypeptides having amino acid sequences which have arisen naturally, and also to mutational variants of ontherin polypeptides which are derived, for example, by combinatorial mutagenesis.

The present invention also provides methods of producing the subject ontherin polypeptides. For example, a host cell transfected with a nucleic acid vector directing expression

of a nucleotide sequence encoding the subject polypeptides can be cultured under appropriate conditions to allow expression of the peptide to occur. If the recombinant protein is not provided with a secretion signal peptide, such as in the case of a GST fusion protein, the cells may be harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The recombinant ontherin polypeptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such peptide. In a preferred embodiment, the recombinant ontherin polypeptide is a fusion protein containing a domain which facilitates its purification, such as GST fusion protein or poly (His) fusion protein.

This invention also pertains to a host cell transfected to express recombinant forms of the subject ontherin polypeptides. The host cell may be any eukaryotic or prokaryotic cell. Thus, a nucleotide sequence derived from the cloning of ontherin proteins, encoding all or a selected portion of a full-length protein, can be used to produce a recombinant form of an ontherin polypeptide via microbial or eukaryotic cellular processes. Ligating the polynucleotide sequence into a gene construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures used in producing other well-known proteins, e. g. cadherins, integrins and the like.

Similar procedures, or modifications thereof, can be employed to prepare recombinant ontherin polypeptides by microbial means or tissue-culture technology in accord with the subject invention.

The recombinant ontherin genes can be produced by ligating nucleic acid encoding an ontherin polypeptide into a vector suitable for expression in either prokaryotic cells, eukaryotic cells, or both. Expression vectors for production of recombinant forms of the subject ontherin polypeptides include plasmids and other vectors. For instance, suitable vectors for the expression of an ontherin polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL- derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al. (1983) in

Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incorporated by reference herein). These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.

In addition, drug resistance markers such as ampicillin can be used. In an illustrative embodiment, an ontherin polypeptide is produced recombinantly utilizing an expression vector generated by sub- cloning the coding sequence of an ontherin gene represented in SEQ ID No: 1.

The preferred mammalian expression vectors contain both prokaryotic sequences, to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In some instances, it may be desirable to express the recombinant ontherin polypeptide by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the B-gal containing pBlueBac III).

When it is desirable to express only a portion of an ontherin protein, such as a form lacking a portion of the N-terminus, i. e. a truncation mutant which lacks the signal peptide, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed. It is well known in the art that a methionine at the N-terminal position can be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat et al. (1987) J. Bacteriol. 169: 751-757) and Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al.

(1987) PNAS 84: 2718-1722). Therefore, removal of an N-terminal methionine, if desired, can be achieved either in vivo by expressing ontherin-derived polypeptides in a host which produces MAP (e. g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e. g., procedure of Miller et al., supra).

Alternatively, the coding sequences for the polypeptide can be incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide. This type of expression system can be useful under conditions where it is desirable to produce an immunogenic fragment of an ontherin protein. For example, the VP6 capsid protein of rotavirus can be used as an immunologic carrier protein for portions of the ontherin polypeptide, either in the monomeric form or in the form of a viral particle. The nucleic acid sequences corresponding to the portion of a subject ontherin protein to which antibodies are to be raised can be incorporated into a fusion gene construct which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising ontherin epitopes as part of the virion. It has been demonstrated with the use of immunogenic fusion proteins utilizing the Hepatitis B surface antigen fusion proteins that recombinant Hepatitis B virions can be utilized in this role as well. Similarly, chimeric constructs coding for fusion proteins containing a portion of an ontherin protein and the poliovirus capsid protein can be created to enhance immunogenicity of the set of polypeptide antigens (see, for example, EP Publication No: 0259149; and Evans et al.

(1989) Nature 339: 385; Huang et al. (1988) J. Virol. 62: 3855; and Schlienger et al. (1992) J. Virol.

66: 2).

The Multiple Antigen Peptide system for peptide-based immunization can also be utilized to generate an immunogen, wherein a desired portion of an ontherin polypeptide is obtained directly from organo-chemical synthesis of the peptide onto an oligomeric branching lysine core (see, for example, Posnett et al. (1988) JBC 263: 1719 and Nardelli et al. (1992) J. Immunol.

148: 914). Antigenic determinants of ontherin proteins can also be expressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, it is widely appreciated that fusion proteins can also facilitate the expression of proteins, and accordingly, can be used in the expression of the ontherin polypeptides of the present invention, particularly truncated forms of the ontherin protein. For example, ontherin polypeptides can be generated as glutathione-S- transferase (GST-fusion) proteins. Such GST-fusion proteins can enable easy purification of the

ontherin polypeptide. as for example by the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. (N. Y.: John Wiley & Sons, 1991)).

In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly- (His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant protein, can allow purification of the expressed fusion protein by affinity chromatography using a Ni2+ metal resin. The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified protein (e. g., see Hochuli et al. (1987) J. Chromatography 411: 177; and Janknecht et al. PNAS 88: 8972).

Techniques for making fusion genes are known to those skilled in the art. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.

In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

The ontherin polypeptides may also be chemically modified to create ontherin derivatives by forming covalent or aggregate conjugates with other chemical moieties, such as glycosyl groups, lipids, cholesterol, phosphate, acetyl groups and the like. Covalent derivatives of ontherin proteins can be prepared by linking the chemical moieties to functional groups on amino acid sidechains of the protein or at the N-terminus or at the C-terminus of the polypeptide.

As appropriate, formulations of multimeric ontherin polypeptides are also provided. The multimers of the soluble forms of the subject ontherin polypeptides may be produced according to the methods known in the art. In one embodiment, the ontherin multimers are cross-linked chemically by using known methods which will result in the formation of either dimers or higher multimers of the soluble forms of the ontherin polypeptides. Another way of producing the multimers of the soluble forms of the ontherin polypeptides is by recombinant techniques, e. g., by inclusion of hinge regions. This linker can facilitate enhanced flexibility of the chimeric protein

allowing the various ontherin monomeric subunits to freely and (optionally) simultaneously interact with an ontherin ligand by reducing steric hindrance between the two fragments, as well as allowing appropriate folding of each portion to occur. The linker can be of natural origin, such as a sequence determined to exist in random coil between two domains of a protein. Alternatively, the linker can be of synthetic origin. For instance, the sequence (Gly4Ser) 3 can be used as a synthetic unstructured linker. Linkers of this type are described in Huston et al. (1988) PNAS 85: 4879; and U. S. Patent Nos. 5,091,513 and 5,258,498. Naturally occurring unstructured linkers of human origin are preferred as they reduce the risk of immunogenicity.

Each multimer comprises two or more monomers, each comprising the soluble form of an ontherin polypeptide or a salt or functional derivative thereof. The upper limit for the number of monomers in a multimer is not important and liposomes having many such monomers thereon may be used. Such multimers preferably have 2-5 monomers and more preferably 2 or 3.

The present invention also makes available isolated ontherin polypeptides which are isolated from, or otherwise substantially free of other cellular proteins, especially adhesion proteins and/or other inductive polypeptides which may normally be associated with the ontherin polypeptide. The term"substantially free of other cellular proteins" (also referred to herein as "contaminating proteins") or"substantially pure or purified preparations"are defined as encompassing preparations of ontherin polypeptides having less than 20% (by dry weight) contaminating protein, and preferably having less than 5% contaminating protein. Functional forms of the subject polypeptides can be prepared, for the first time, as purified preparations by using a cloned gene as described herein. By"purified", it is meant, when referring to a peptide or DNA or RNA sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other proteins. The term"purified"as used herein preferably means at least 80% by dry weight, more preferably in the range of 95-99% by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present). The term"pure"as used herein preferably has the same numerical limits as "purified"immediately above."Isolated"and"purified"do not encompass either natural materials in their native state or natural materials that have been separated into components (e. g., in an acrylamide gel) but not obtained either as pure (e. g. lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e. g. acrylamide or agarose)

substances or solutions. In preferred embodiments, purified ontherin preparations will lack any contaminating proteins from the same animal from that ontherin is normally produced, as can be accomplished by recombinant expression of, for example, a mammalian ontherin protein in a yeast or bacterial cell.

As described above for recombinant polypeptides, isolated ontherin polypeptides can include all or a portion of an amino acid sequences corresponding to an ontherin polypeptide represented in SEQ ID No: 2 or homologous sequences thereto.

Isolated peptidyl portions of ontherin proteins can also be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, an ontherin polypeptide of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as either agonists or antagonists of a wild-type (e. g., "authentic") ontherin protein. For example, Roman et al. (1994) Eur J Biochem 222: 65-73 describe the use of competitive-binding assays using short, overlapping synthetic peptides from larger proteins to identify binding domains.

The recombinant ontherin polypeptides of the present invention also include homologs of the authentic ontherin proteins, such as versions of those protein which are resistant to proteolytic cleavage, as for example, due to mutations which alter cleavage recongition sequences for protease or the like, e. g., to prevent enzymatic release of the extracellular domain.

Modification of the structure of the subject ontherin polypeptides can be for such purposes as enhancing therapeutic or prophylactic efficacy, stability (e. g., ex vivo shelf life and resistance to proteolytic degradation in vivo), or post-translational modifications. Such modified peptides, when designed to retain at least one activity of the naturally-occurring form of the protein, or to produce specific antagonists thereof, are considered functional equivalents of the ontherin polypeptides (though they may be agonistic or antagonistic of the bioactivities of the authentic protein). Such modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.

For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i. e. isosteric and/or isoelectric mutations) will not have a major effect on the biological activity of the resulting molecule.

Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are can be divided into four families: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine histidine, (3) aliphatic = glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic = phenylalanine, tyrosine, tryptophan; (5) amide = asparagine, glutamine; and (6) sulfur-containing = cysteine and methionine. (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer, WH Freeman and Co.: 1981). Whether a change in the amino acid sequence of a peptide results in a functional ontherin homolog (e. g. functional in the sense that the resulting polypeptide mimics or antagonizes the authentic form) can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the wild-type protein, or competitively inhibit such a response. Polypeptides in which more than one replacement has taken place can readily be tested in the same manner.

This invention further contemplates a method for generating sets of combinatorial point mutants of the subject ontherin proteins as well as truncation mutants, and is especially useful for identifying potential variant sequences (e. g. homologs) that are functional in modulating signal transduction and/or ligand binding. The purpose of screening such combinatorial libraries is to generate, for example, novel ontherin homologs which can act as either agonists or antagonist, or alternatively, possess novel activities all together. To illustrate, ontherin homologs can be engineered by the present method to provide selective, constitutive activation of catenin-mediated gene transcription, or alternatively, to be dominant negative inhibitors of catenin-dependent signal transduction. For instance, mutagenesis can provide ontherin homologs which are able to interact

with other cell surface proteins and/or bind extracellular ligands, yet be unable to bind or signal through intracellular regulatory proteins.

In one aspect of this method, the amino acid sequences for a population of ontherin homologs from different species or other related proteins are aligned, preferably to promote the highest homology possible. Such a population of variants can include, for example, ontherin homologs from one or more species. Amino acids which appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences. In a preferred embodiment, the variegated library of ontherin variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential ontherin sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e. g. for phage display) containing the set of ontherin sequences therein.

There are many ways by which such libraries of potential ontherin homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential ontherin sequences. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos.

Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev.

Biochem. 53: 323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid Res.

11: 477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249: 386-390; Roberts et al. (1992) PNAS 89: 2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U. S. Patents Nos. and 5,096,815).

Likewise, a library of coding sequence fragments can be provided for an ontherin clone in order to generate a variegated population of ontherin fragments for screening and subsequent selection of bioactive fragments. A variety of techniques are known in the art for generating such libraries, including chemical synthesis. In one embodiment, a library of coding sequence fragments can be generated by (i) treating a double stranded PCR fragment of an ontherin coding sequence

with a nuclease under conditions wherein nicking occurs only about once per molecule ; (ii) denaturing the double stranded DNA; (iii) renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products; (iv) removing single stranded portions from reformed duplexes by treatment with S1 nuclease; and (v) ligating the resulting fragment library into an expression vector. By this exemplary method, an expression library can be derived which codes for N-terminal, C-terminal and internal fragments of various sizes.

A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of ontherin homologs.

The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.

The invention also provides for reduction of the ontherin protein to generate mimetics, e. g. peptide or non-peptide agents, which are able to disrupt a biological activity of an ontherin polypeptide of the present invention, e. g. as inhibitors of protein-protein interactions, such as between ontherins and other cell surface proteins. Thus, such mutagenic techniques as described above are also useful to map the determinants of the ontherin proteins which participate in protein- protein interactions. By employing, for example, scanning mutagenesis to map the amino acid residues of a protein which is involved in binding other proteins, peptidomimetic compounds can be generated which mimic those residues which facilitate the interaction. Such mimetics may then be used to interfere with the normal function of an ontherin protein (or its ligands or ions). For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e. g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e. g., see Huffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29: 295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th

American Peptide Symposium) Pierce Chemical Co. Rockland, IL. 1985), b-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26: 647; and Sato et al. (1986) J Chem Soc Perkin Trans 1: 1231), and b-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun 126: 419; and Dann et al. (1986) Biochem Biophys Res Commun 134: 71).

Another aspect of the invention pertains to an antibody specifically reactive with an ontherin protein. For example, by using immunogens derived from an ontherin protein, e. g. based on the cDNA sequences, anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example. Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e. g., an ontherin polypeptide or an antigenic fragment which is capable of eliciting an antibody response). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of an ontherin protein can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies. In a preferred embodiment, the subject antibodies are immunospecific for antigenic determinants of an ontherin protein of a organism, such as a mammal, e. g. antigenic determinants of a protein represented by SEQ ID No: 2 or closely related homologs (e. g. at least 70% homologous, preferably at least 80% homologous, and more preferably at least 90% homologous). In yet a further preferred embodiment of the present invention, in order to provide, for example, antibodies which are immuno-selective for discrete ontherin homologs the anti-ontherin polypeptide antibodies do not substantially cross react (i. e. does not react specifically) with a protein which is, for example, less than 85%, 90% or 95% homologous with the selected ontherin. By"not substantially cross react", it is meant that the antibody has a binding affinity for a non-homologous protein which is at least one order of magnitude, more preferably at least 2 orders of magnitude, and even more preferably at least 3 orders of magnitude less than the binding affinity of the antibody for the intended target ontherin.

Following immunization of an animal with an antigenic preparation of an ontherin polypeptide, anti-ontherin antisera can be obtained and, if desired, polyclonal anti-ontherin antibodies isolated from the serum. To produce monoclonal antibodies, antibody-producing cells

(lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, an include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an ontherin polypeptide of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells.

The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with an ontherin polypeptide. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F (ab) 2 fragments can be generated by treating antibody with pepsin. The resulting F (ab) 2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. The antibody of the present invention is further intended to include bispecific and chimeric molecules having affinity for an ontherin protein conferred by at least one CDR region of the antibody.

Both monoclonal and polyclonal antibodies (Ab) directed against authentic ontherin polypeptides, or ontherin variants, and antibody fragments such as Fab, F (ab) 2, Fv and scFv can be used to block the action of an ontherin protein and allow the study of the role of these proteins in, for example, differentiation of tissue. Experiments of this nature can aid in deciphering the role of ontherin proteins that may be involved in control of proliferation versus differentiation, e. g., in patterning and tissue formation.

Antibodies which specifically bind ontherin epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of each of the subject ontherin polypeptides. Anti-ontherin antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate ontherin protein levels in tissue or biological fluid sample as part of a clinical testing procedure. For instance, such measurements can be useful in predictive valuations of the onset or progression of proliferative or differentiative disorders. Likewise, the ability to monitor ontherin protein levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with

such a disorder. The level of ontherin polypeptides may be measured from bodily fluid, such as in samples of blood serum, cerebral spinal fluid or amniotic fluid, or can be measured in tissue, such as produced by biopsy. Diagnostic assays using anti-ontherin antibodies can include, for example, immunoassays designed to aid in early diagnosis of a disorder, particularly ones which are manifest at birth. Diagnostic assays using anti-ontherin polypeptide antibodies can also include immunoassays designed to aid in early diagnosis and phenotyping neoplastic or hyperplastic disorders.

Another application of anti-ontherin antibodies of the present invention is in the immunological screening of cDNA libraries constructed in expression vectors such askgt 11, Xgt 18- 23, AZAP, and XORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, Agtl 1 will produce fusion proteins whose amino termini consist of ß-galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of an ontherin protein, e. g. orthologs of the ontherin protein from other species, can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-ontherin antibodies.

Positive phage detected by this assay can then be isolated from the infected plate. Thus, the presence of ontherin homologs can be detected and cloned from other animals, as can alternate isoforms (including splicing variants) from humans.

Moreover, the nucleotide sequences determined from the cloning of ontherin genes from organisms will further allow for the generation of probes and primers designed for use in identifying and/or cloning ontherin homologs in other cell types, e. g. from other tissues, as well as ontherin homologs from other organisms. For instance, the present invention also provides a probe/primer comprising a substantially purified oligonucleotide, which oligonucleotide comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least 15 consecutive nucleotides of sense or anti-sense sequence selected from the group consisting of SEQ ID No: 1 or naturally occurring mutants thereof. For instance, primers based on the nucleic acid represented in SEQ ID No: 1, can be used in PCR reactions to clone ontherin homologs. Likewise, probes based on the subject ontherin sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto and able to be detected, e. g. the label group is selected from amongst radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.

Such probes can also be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress an ontherin protein, such as by measuring a level of an ontherin-encoding nucleic acid in a sample of cells from a patient-animal; e. g. detecting ontherin mRNA levels or determining whether a genomic ontherin gene has been mutated or deleted.

To illustrate, nucleotide probes can be generated from the subject ontherin genes which facilitate histological screening of intact tissue and tissue samples for the presence (or absence) of ontherin-encoding transcripts. Similar to the diagnostic uses of anti-ontherin antibodies, the use of probes directed to ontherin messages, or to genomic ontherin sequences, can be used for both predictive and therapeutic evaluation of allelic mutations which might be manifest in, for example, degenerative disorders marked by loss of particular cell-types, apoptosis, neoplastic and/or hyperplastic disorders (e. g. unwanted cell growth) or abnormal differentiation of tissue. Used in conjunction with immunoassays as described above, the oligonucleotide probes can help facilitate the determination of the molecular basis for a developmental disorder which may involve some abnormality associated with expression (or lack thereof) of an ontherin protein. For instance, variation in polypeptide synthesis can be differentiated from a mutation in a coding sequence.

Accordingly, the present method provides a method for determining if a subject is at risk for a disorder characterized by aberrant apoptosis, cell proliferation and/or differentiation. In preferred embodiments, method can be generally characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of (i) an alteration affecting the integrity of a gene encoding an ontherin protein, or (ii) the mis- expression of the ontherin gene. To illustrate, such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from an ontherin gene, (ii) an addition of one or more nucleotides to an ontherin gene, (iii) a substitution of one or more nucleotides of an ontherin gene, (iv) a gross chromosomal rearrangement of an ontherin gene, (v) a gross alteration in the level of a messenger RNA transcript of an ontherin gene, (vii) aberrant modification of an ontherin gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild type splicing pattern of a messenger RNA transcript of an ontherin gene, (viii) a non-wild type level of an ontherin-protein, and (ix) inappropriate post-translational modification of an ontherin-protein. As set out below, the present invention provides a large number of assay techniques for detecting lesions in an ontherin gene, and importantly, provides the

ability to discern between different molecular causes underlying ontherin-dependent aberrant cell growth, proliferation and/or differentiation.

In an exemplary embodiment, there is provided a nucleic acid composition comprising a (purified) oligonucleotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of an ontherin gene, such as represented by SEQ ID NO: 1, or naturally occurring mutants thereof, or 5'or 3'flanking sequences or intronic sequences naturally associated with the subject ontherin genes or naturally occurring mutants thereof. The nucleic acid of a cell is rendered accessible for hybridization, the probe is exposed to nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected. Such techniques can be used to detect lesions at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.

In certain embodiments, detection of the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e. g. U. S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e. g., Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al. (1944) PNAS 91: 360-364), the later of which can be particularly useful for detecting point mutations in the ontherin gene. In a merely illustrative embodiment, the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e. g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to an ontherin gene under conditions such that hybridization and amplification of the ontherin gene (if present) occurs, and (iv) detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.

In still another embodiment, the level of an ontherin-protein can be detected by immunoassay. For instance, the cells of a biopsy sample can be lysed, and the level of an ontherin- protein present in the cell can be quantitated by standard immunoassay techniques. In yet another exemplary embodiment, aberrant methylation patterns of an ontherin gene can be detected by digesting genomic DNA from a patient sample with one or more restriction endonucleases that are sensitive to methylation and for which recognition sites exist in the ontherin gene (including in the flanking and intronic sequences). See, for example, Buiting et al. (1994) Human Mol Genet 3: 893- 895. Digested DNA is separated by gel electrophoresis, and hybridized with probes derived from,

for example, genomic or cDNA sequences. The methylation status of the ontherin gene can be determined by comparison of the restriction pattern generated from the sample DNA with that for a standard of known methylation.

In still other embodiments, the extracellular domain of an ontherin protein can be used to quantitatively detect the level of ontherin ligands, whether soluble or membrane bound. To illustrate, a soluble form of the ontherin protein can be generated. Samples of bodily fluid (s), e. g., plasma, serum, lymph, marrow, cerebral/spinal fluid, urine and the like, or alternatively biopsied cells, can be contacted with the fragment under conditions wherein binding can occur, and the level of ontherin-containing complexes formed can be detected by any of a variety of techniques known in the art. For instance, the ontherin polypeptide can be contacted with cells from a biopsy, and the ability of the ontherin polypeptide to decorate certain cells of the sample is ascertained. The binding of the ontherin protein to cell populations of the sample can be detected, for example, by the use of antibodies against the ontherin protein, or by detection of a label associated with the ontherin protein. In the case of the latter, the ontherin protein can be labeled, for example, by chemical modification or as a fusion protein. Exemplary labels include radioisotopes, fluorescent compounds, enzyme co-factors, which can be added by chemical modification of the protein, and epitope tags such as myc, pFLAG and the like, or enzymatic activities such as GST or alkaline phosphatase which can be added either by chemical modification or by generation of a fusion protein.

Furthermore, the present invention also contemplates the detection of soluble forms of the ontherin proteins in bodily fluid samples. As described in the art, e. g., Furukawa et al. (1997) <BR> <BR> Microsc Res Tech 38: 343, the level of soluble forms of cadherins in sera occurs in various disease states. For instance, soluble E-cadherins are elevated in sera in various skin diseases including bullous pemphigoid, pemphigus vulgaris and psoriasis, and markedly high levels of soluble E- cadherin are observed in patients with metastatic cancers. In various pathologic states, the production and release of soluble ontherin proteins may mediate host response and determine the course and outcome of disease, or at least be a diagnostic or prognostic factor which can be detected. The determination of soluble ontherin proteins in body fluids is a new tool to gain information about various disease states, and may be of prognostic value to a clinician. For example, the level of soluble ontherin protein in a body fluid may give useful information for

monitoring, inter alia, neurological disorders as well as in the treatment of neoplastic or hyperplastic transformations of ectodermal, mesodermal or endodermal origin.

The level of soluble ontherin present in a given sample can be quantitated, in light of the present disclosure, using known procedures and techniques. For example, antibodies immunoselective for the extracellular domain of the ontherin protein can be used to detect and quantify its presence in a sample, e. g., by well-known immunoassay techniques. Alternatively, a labeled ligand of the protein can be used to detect the presence of the receptor in the fluid sample.

A number of techniques exist in the art for now identifying extracellular components which may bind ontherins, e. g., ligands for the ontherin protein. For instance, expression cloning can be carried out on a cDNA or genomic library by isolating cells which are decorated with a labeled form of the extracellular domain of an ontherin protein. In a preferred embodiment, the technique uses the ontherin protein in an in situ assay for detecting ontherin ligands in tissue samples and whole organisms. In general, the RAP-in situ assay described below (for Receptor Affinity Probe) of Flanagan and Leder (see PCT publications WO 92/06220; and also Cheng et al. (1994) Cell 79: 157-168) involves the use of an expression cloning system whereby an ontherin ligand is scored on the basis of binding to an ontherinlalkaline phosphatase fusion protein. In general, the method comprises (i) providing a hybrid molecule (the affinity probe) including the ontherin protein, or at least the ligand binding domain thereof, covalently bonded to an enzymatically active tag, preferably for which chromogenic substrates exist, (ii) contacting the tissue or organism with the affinity probe to form complexes between the probe and a cognate ligand in the sample, removing unbound probe, and (iii) detecting the affinity complex using a chromogenic substrate for the enzymatic activity associated with the affinity probe.

This method, unlike other prior art methods which are carried out only on dispersed cell cultures, provides a means for probing non-dispersed and wholemount tissue and animal samples.

The method can be used, in addition to facilitating the cloning of ontherin ligands, also for detecting patterns of expression for particular ligands of the ontherin protein, for measuring the affinity of receptor/ligand interactions in tissue samples, as well as for generating drug screening assays in tissue samples. Moreover, the affinity probe can also be used in diagnostic screening to determine whether an ontherin ligand is misexpressed.

In yet another aspect of the invention, the subject ontherin polypeptides can be used to generate a"two hybrid"assay or an"interaction trap"assay (see, for example, U. S. Patent No.

Zervos et al. (1993) Cell 72: 223-232; Madura et al. (1993) J Biol Chem 268: 12046- 12054; Bartel et al. (1993) Biotechniques 14: 920-924; Iwabuchi et al. (1993) Oncogene 8: 1693- 1696; and Brent W094/10300), for isolating coding sequences for other proteins which interact with an ontherin protein ("ontherin-interacting proteins"or"ontherin-ip"). This assay can be particularly useful for identifying intracellular components of an ontherin-dependent signal transduction pathway.

Briefly, the interaction trap relies on reconstituting in vivo a functional transcriptional activator protein from two separate fusion proteins. In particular, the method makes use of chimeric genes which express hybrid proteins. To illustrate, a first hybrid gene comprises the coding sequence for a DNA-binding domain of a transcriptional activator fused in frame to the coding sequence for an ontherin polypeptide, e. g., an intracellular domain of an ontherin protein.

The second hybrid protein encodes a transcriptional activation domain fused in frame to a sample gene from a cDNA library. If the bait and sample hybrid proteins are able to interact, e. g., form an ontherin-dependent complex, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the reporter gene can be detected and used to score for the interaction of the ontherin and sample proteins.

Furthermore, by making available purified and recombinant ontherin polypeptides, the present invention facilitates the development of assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function of the subject ontherin proteins, or of their role in the pathogenesis of cellular maintenance, differentiation and/or proliferation and disorders related thereto. In a general sense, the assay evaluates the ability of a compound to modulate binding between an ontherin polypeptide and a molecule, e. g., an ontherin-interacting protein, that interacts with the ontherin polypeptide. Exemplary compounds which can be screened against such ontherin-mediated interactions include peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries, such as isolated from animals, plants, fungus and/or microbes.

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived

with purified or semi-purified proteins, are often preferred as"primary"screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with a ligand. Accordingly, in an exemplary screening assay of the present invention, a reaction mixture is generated to include an ontherin polypeptide, compound (s) of interest, and a"target molecule", e. g., a protein, which interacts with the ontherin polypeptide. Exemplary target molecules include ligands, intracellular proteins (such as catenin proteins), and ontherin itself (or other cadherins with which it may interact), as well as other peptide and non-peptide interacting molecules. Detection and quantification of interaction of the ontherin polypeptide with the target molecule provides a means for determining a compound's efficacy at inhibiting (or potentiating) interaction between the ontherin and the target molecule.

The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. In the control assay, interaction of the ontherin polypeptide and target molecule is quantitated in the absence of the test compound.

Interaction between the ontherin polypeptide and the target molecule may be detected by a variety of techniques. Modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled ontherin polypeptides, by immunoassay, by chromatographic detection, or by detecting the intrinsic activity of the acetylase. In such embodiments which rely on cell-free assay formats, the ontherin polypeptide will preferably be a soluble fragment, e. g. extracellular or intracellular domains, of the full-length protein.

Typically, it will be desirable to immobilize either ontherin or the target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of ontherin to the target molecule, in the presence and absence of a candidate agent, can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes.

In one embodiment, a fusion protein can be provided which adds a domain that allows the protein <BR> <BR> <BR> <BR> to be bound to a matrix. For example, glutathione-S-transferase/ontherin (GST/ontherin) fusion

proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis. MO) or glutathione derivatized microtitre plates, which are then combined with the cell lysates, e. g. an 35S- labeled, and the test compound, and the mixture incubated under conditions conducive to complex formation, e. g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e. g. beads placed in scintillant), or in the supernatant after the complexes are subsequently dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of target molecule found in the bead fraction quantitated from the gel using standard electrophoretic techniques.

Other techniques for immobilizing proteins and other molecules on matrices are also available for use in the subject assay. For instance, either ontherin or target molecule can be immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated ontherin molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e. g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with ontherin, but which do not interfere with the interaction between the ontherin and target molecule, can be derivatized to the wells of the plate, and ontherin trapped in the wells by antibody conjugation. As above, preparations of an target molecule and a test compound are incubated in the ontherin-presenting wells of the plate, and the amount of complex trapped in the well can be quantitated. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the target molecule, or which are reactive with ontherin protein and compete with the target molecule; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule, either intrinsic or extrinsic activity. In the instance of the latter, the enzyme can be chemically conjugated or provided as a fusion protein with the target molecule. To illustrate, the target molecule can be chemically cross-linked or genetically fused (if it is a polypeptide) with horseradish peroxidase, and the amount of polypeptide trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e. g. 3,3'-diamino- benzadine terahydrochloride or 4-chloro-1-napthol. Likewise, a fusion protein comprising the polypeptide and glutathione-S-transferase can be provided, and complex formation quantitated by

detecting the GST activity using l-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249: 7130).

For processes which rely on immunodetection for quantitating proteins trapped in the complex, antibodies against the protein, such as anti-ontherin antibodies, can be used.

Alternatively, the protein to be detected in the complex can be"epitope tagged"in the form of a fusion protein which includes, in addition to the ontherin sequence, a second polypeptide for which antibodies are readily available (e. g. from commercial sources). For instance, the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope tags include myc-epitopes (e. g., see Ellison et al. (1991) J Biol Chem 266: 21150-21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharamacia, NJ).

An exemplary drug screening assay of the present invention includes the steps of (a) forming a reaction mixture including: (i) a ontherin-interacting polypeptide, (ii) an ontherin polypeptide, and (iii) a test compound; and (b) detecting interaction of the ontherin-ip and ontherin polypeptides. A statistically significant change (potentiation or inhibition) in the interaction of the ontherin-ip and ontherin polypeptides in the presence of the test compound, relative to the interaction in the absence of the test compound, indicates a potential agonist (mimetic or potentiator) or antagonist (inhibitor) of ontherin bioactivity for the test compound. The reaction mixture can be a cell-free protein preparation, e. g., a reconsistuted protein mixture or a cell lysate, or it can be a recombinant cell including a heterologous nucleic acid recombinantly expressing the ontherin polypeptide.

Where the ontherin polypeptide participates as part of an oligomeric complex forming a multimeric cell surface complex, e. g., which complex is a homomeric or includes other protein subunits, the cell-free system can be, e. g., a cell membrane preparation or a reconstituted protein mixture. Alternatively, liposomal preparations using reconstituted ontherin protein can be utilized.

For instance, the protein subunits of an ontherin-dependent complex can be purified from detergent extracts from both authentic and recombinant origins can be reconstituted in in artificial lipid vesicles (e. g. phosphatidylcholine liposomes) or in cell membrane-derived vesicles (see, for example, Bear et al. (1992) Cell 68: 809-818; Newton et al. (1983) Biochemistry 22: 6110-6117; and Reber et al. (1987) J Biol Chem 262: 11369-11374). The lamellar structure and size of the resulting liposomes can be characterized using electron microscopy. External orientation of the ontherin

protein in the reconstituted membranes can be demonstrated. for example, by immunoelectron microscopy. The interaction of a protein or other cellular component with liposomes containing such ontherin complexes and liposomes without the protein, in the presence of candidate agents, can be compared.

In yet another embodiment, the drug screening assay is derived to include a whole cell expressing an ontherin polypeptide. The ability of a test agent to alter the activity of the ontherin protein can be detected by analysis of the recombinant cell. For example, agonists and antagonists of the ontherin biological activity can by detected by scoring for alterations in growth or differentiation (phenotype) of the cell. General techniques for detecting each are well known, and will vary with respect to the source of the particular reagent cell utilized in any given assay. For the cell-based assays, the recombinant cell is preferably a metazoan cell, e. g., a mammalian cell, e. g., an insect cell, e. g., a xenopus cell, e. g., an oocyte. In other embodiments, the ontherin protein can be reconstituted in a yeast cell or other lower eukaryote.

In an exemplary embodiment, a cell which expresses the ontherin protein, e. g, whether endogenous or heterologous, can be contacted with a ligand of the ontherin protein, e. g., some compound (including antibodies) which is capable of inducing signal transduction in an ontherin- dependent fashion, and the resulting signaling detected either at various points in the pathway, or on the basis of a phenotypic change to the reagent cell. A test compound which modulates that pathway, e. g., potentiates or inhibits, can be detected by comparison with control experiments which either lack the protein or lack the test compound. For example, visual inspection of the morphology of the reagent cell can be used to determine whether the biological activity of the targeted ontherin protein has been affected by the added agent.

In addition to morphological studies, change (s) in the level of an intracellular second messenger responsive to signaling by the ontherin polypeptide can be detected. For example, in various embodiments the assay may assess the ability of test agent to cause changes in phophorylation patterns, adenylate cyclase activity (cAMP production), GTP hydrolysis, calcium mobilization, and/or phospholipid hydrolysis (IP3, DAG production) upon receptor stimulation.

By detecting changes in intracellular signals, such as alterations in second messengers or gene expression, candidate agonists and antagonists to ontherin-dependent signaling can be identified.

The activation of an ontherin protein may set in motion a cascade involving the activation and inhibition of downstream effectors, the ultimate consequence of which is, in some instances,

a detectable change in the transcription or translation of a gene. Potential transcriptional targets of ontherin-dependent signaling include those genes regulated by catenin transcription. By selecting transcriptional regulatory sequences from such target genes, e. g. that are responsible for the up-or down-regulation of these genes in response to ontherin induction, and operatively linking such promoters to a reporter gene, the present invention provides a transcription based assay which is sensitive to the ability of a specific test compound to influence ontherin signaling pathways.

In practicing one embodiment of the assay, a reporter gene construct is inserted into the reagent cell in order to generate a detection signal dependent on second messengers generated by ontherin-dependent induction. Typically, the reporter gene construct will include a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the ontherin activity, with the level of expression of the reporter gene providing the ontherin-dependent detection signal. The amount of transcription from the reporter gene may be measured using any method known to those of skill in the art to be suitable. For example, mRNA expression from the reporter gene may be detected using RNAse protection or RNA-based PCR, or the protein product of the reporter gene may be identified by a characteristic stain or an intrinsic activity. The amount of expression from the reporter gene is then compared to the amount of expression in either the same cell in the absence of the test compound or it may be compared with the amount of transcription in a substantially identical cell that lacks the target receptor protein. Any statistically or otherwise significant difference in the amount of transcription indicates that the test compound has in some manner altered the inductive activity of the ontherin protein.

As described in further detail below, in preferred embodiments the gene product of the reporter is detected by an intrinsic activity associated with that product. For instance, the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detection signal based on color, fluorescence, or luminescence. In other preferred embodiments, the reporter or marker gene provides a selective growth advantage, e. g., the reporter gene may enhance cell viability, relieve a cell nutritional requirement, and/or provide resistance to a drug. Many reporter genes are known to those of skill in the art and others may be identified or synthesized by methods known to those of skill in the art. A reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein.

Preferred reporter genes are those that are readily detectable. The reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired

transcriptional regulatory sequences or exhibits other desirable properties. Examples of reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta- galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 7: 725-737); bacterial luciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231- 238, Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placental secreted alkaline phosphatase (Cullen and Malim (1992) Methods in Enzymol. 216: 362-368).

Accordingly, yet another embodiment of the subject drug screening assays of the present invention provides a recombinant cell, e. g., for carrying out certain of the drug screening methods above, comprising: (i) an expressible recombinant gene encoding a heterologous ontherin polypeptide whose signal transduction activity modulates gene transcription; and (ii) a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction activity of the ontherin polypeptide. Still another aspect of the present invention provides a kit for screening test compounds to identify agents which modulate ontherin bioactivities, including the above-referenced cell.

In still another embodiment of a drug screening, a two hybrid assay (described supra) can be generated with an ontherin polypeptide and target molecule, e. g., an ontherin interacting protein.

Drug dependent inhibition or potentiation of the interaction can be scored.

After identifying certain test compounds as potential modulators of one or more bioactivities of an ontherin protein, the practioner of the subject assay will continue to test the efficacy and specificity of the selected compounds both in vitro and in vivo. Whether for subsequent in vivo testing, or for administration to an animal as an approved drug, agents identified in the subject assay can be formulated in pharmaceutical preparations for in vivo administration to an animal, preferably a human.

Another aspect of the present invention relates to a method of inducing and/or maintaining a differentiated state, enhancing survival, and/or inhibiting (or alternatively potentiating) proliferation of a cell, by contacting the cells with an agent which modulates ontherin-dependent signal transduction pathways. The subject method could be used to generate and/or maintain an array of different tissue both in vitro and in vivo. A"ontherin therapeutic,"whether inhibitory or potentiating with respect to modulating the activity of an ontherin protein, can be, as appropriate,

any of the preparations described above, including isolated ontherin polypeptides (including both agonist and antagonist forms), gene therapy constructs, antisense molecules, peptidomimetics, or agents identified in the drug assays provided herein. In certain embodiments, soluble forms of the ontherin protein including the extracellular domain of the protein can be provided as a means for antagonizing the binding of extracellular factors to a cell-surface ontherin protein.

The ontherin therapeutic compounds of the present invention are likely to play an important role in the modulation of cellular proliferation and maintenance of, for example, neuronal, testicular and renal tissues during disease states. It will also be apparent that, by transient use of modulators of ontherin activities, in vivo reformation of tissue can be accomplished, e. g. in the development and maintenance of organs such as ectodermal patterning, as well as certain mesodermal and endodermal differentiation processes. By controlling the proliferative and differentiative potential for different cells, the subject ontherin therapeutics can be used to reform injured tissue, or to improve grafting and morphology of transplanted tissue. For instance, ontherin antagonists and agonists can be employed in a differential manner to regulate different stages of organ repair after physical, chemical or pathological insult. The present method is also applicable to cell culture techniques.

To further illustrate this aspect of the invention, in vitro neuronal culture systems have proved to be fundamental and indispensable tools for the study of neural development, as well as the identification of neurotrophic factors such as nerve growth factor (NGF), ciliary trophic factors (CNTF), and brain derived neurotrophic factor (BDNF). Once a neuronal cell has become terminally-differentiated it typically will not change to another terminally differentiated cell-type.

However, neuronal cells can nevertheless readily lose their differentiated state. This is commonly observed when they are grown in culture from adult tissue, and when they form a blastema during regeneration. The present method provides a means for ensuring an adequately restrictive environment in order to maintain neuronal cells at various stages of differentiation, and can be employed, for instance, in cell cultures designed to test the specific activities of other trophic factors. In such embodiments of the subject method, the cultured cells can be contacted with an ontherin therapeutic which inhibits proliferation of neuronal cells, in order to induce neuronal differentiation (e. g. of a stem cell), or to maintain the integrity of a culture of terminally- differentiated neuronal cells by preventing loss of differentiation. Alternatively, certain of the ontherin therapeutics of the present invention are expected to induce proliferation, e. g., and inhibit

differentiation to some degree, and can be used to prevent differentiation of progenitor cells in culture.

To further illustrate uses of ontherin therapeutics, it is noted that intracerebral grafting has emerged as an additional approach to central nervous system therapies. For example, one approach to repairing damaged brain tissues involves the transplantation of cells from fetal or neonatal <BR> <BR> <BR> <BR> animals into the adult brain (Dunnett et al. (1987) JExp Biol 123: 265-289; and Freund et al. (1985)<BR> <BR> <BR> <BR> <BR> <BR> <BR> J Neurosci 5: 603-616). Fetal neurons from a variety of brain regions can be successfully incorporated into the adult brain, and such grafts can alleviate behavioral defects. For example, movement disorder induced by lesions of dopaminergic projections to the basal ganglia can be prevented by grafts of embryonic dopaminergic neurons. Complex cognitive functions that are impaired after lesions of the neocortex can also be partially restored by grafts of embryonic cortical cells. The differential use of ontherin agonists and antagonists in the culture can control the timing and type of differentiation accessible by the culture.

In addition to the implantation of cells cultured in the presence of ontherin agonists and antagonists and other in vitro uses, yet another aspect of the present invention concerns the therapeutic application of an ontherin therapeutics to enhance survival of neurons and other neuronal cells in both the central nervous system and the peripheral nervous system. The neurotrophic activity of ontherin proteins indicates that certain of the ontherin proteins, and accordingly ontherin therapeutic which modulate ontherin bioactivities, can be reasonably expected to facilitate control of adult neurons with regard to maintenance, functional performance, and aging of normal cells; repair and regeneration processes in chemically or mechanically lesioned cells; and prevention of degeneration and premature death which result from loss of differentiation in certain pathological conditions. In light of this understanding, the present invention specifically contemplates applications of the subject ontherin therapeutics to the treatment of (prevention and/or reduction of the severity of) neurological conditions deriving from: (i) acute, subacute, or chronic injury to the nervous system, including traumatic injury, chemical injury, vasal injury and deficits (such as the ischemia resulting from stroke), together with infectious/inflammatory and tumor- induced injury; (ii) aging of the nervous system including Alzheimer's disease; (iii) chronic neurodegenerative diseases of the nervous system, including Parkinson's disease, Huntington's chorea, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations; and

(iv) chronic immunological diseases of the nervous system or affecting the nervous system. including multiple sclerosis.

Many neurological disorders are associated with degeneration of discrete populations of neuronal elements and may be treatable with a therapeutic regimen which includes an ontherin therapeutic that acts as a neurotrophic agent. For example, Alzheimer's disease is associated with deficits in several neurotransmitter systems, both those that project to the neocortex and those that reside with the cortex. For instance, the nucleus basalis in patients with Alzheimer's disease have been observed to have a profound (75%) loss of neurons compared to age-matched controls.

Although Alzheimer's disease is by far the most common form of dementia, several other disorders can produce dementia. Several of these are degenerative diseases characterized by the death of neurons in various parts of the central nervous system, especially the cerebral cortex. However, some forms of dementia are associated with degeneration of the thalmus or the white matter underlying the cerebral cortex. Here, the cognitive dysfunction results from the isolation of cortical areas by the degeneration of efferents and afferents. Huntington's disease involves the degeneration of intrastriatal and cortical cholinergic neurons and GABAergic neurons. Pick's disease is a severe neuronal degeneration in the neocortex of the frontal and anterior temporal lobes, sometimes accompanied by death of neurons in the striatum. Treatment of patients suffering from such degenerative conditions can include the application of ontherin therapeutics in order to control, for example, differentiation and apoptotic events which give rise to loss of neurons (e. g. to enhance survival of existing neurons) as well as promote differentiation and repopulation by progenitor cells in the area affected.

In addition to degenerative-induced dementias, a pharmaceutical preparation of one or more of the subject ontherin therapeutics can be applied opportunely in the treatment of neurodegenerative disorders which have manifestations of tremors and involuntary movements.

Parkinson's disease, for example, primarily affects subcortical structures and is characterized by degeneration of the nigrostriatal pathway, raphe nuclei, locus cereleus, and the motor nucleus of vagus. Ballism is typically associated with damage to the subthalmic nucleus, often due to acute vascular accident. Also included are neurogenic and myopathic diseases which ultimately affect the somatic division of the peripheral nervous system and are manifest as neuromuscular disorders.

Examples include chronic atrophies such as amyotrophic lateral sclerosis, Guillain-Barre syndrome and chronic peripheral neuropathy, as well as other diseases which can be manifest as progressive

bulbar palsies or spinal muscular atrophies. The present method is amenable to the treatment of disorders of the cerebellum which result in hypotonia or ataxia, such as those lesions in the cerebellum which produce disorders in the limbs ipsilateral to the lesion. For instance, a preparation of an ontherin therapeutic can used to treat a restricted form of cerebellar cortical degeneration involving the anterior lobes (vermis and leg areas) such as is common in alcoholic patients.

In an illustrative embodiment, the subject method is used to treat amyotrophic lateral sclerosis. ALS is a name given to a complex of disorders that comprise upper and lower motor neurons. Patients may present with progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, or a combination of these conditions. The major pathological abnormality is characterized by a selective and progressive degeneration of the lower motor neurons in the spinal cord and the upper motor neurons in the cerebral cortex. The therapeutic application of a neurotrophic ontherin therapeutic can be used alone, or in conjunction with other neurotrophic factors such as CNTF, BDNF or NGF to prevent and/or reverse motor neuron degeneration in ALS patients.

Ontherin therapeutics of the present invention can also be used in the treatment of autonomic disorders of the peripheral nervous system, which include disorders affecting the innervation of smooth muscle and endocrine tissue (such as glandular tissue). For instance, the subject method can be used to treat tachycardia or atrial cardiac arrythmias which may arise from a degenerative condition of the nerves innervating the striated muscle of the heart.

Furthermore, a potential role for certain of the ontherin therapeutics derives from the potential role of ontherin proteins in development and maintenance of dendritic processes of axonal neurons. Potential roles for ontherin therapeutics consequently include guidance for axonal projections and the ability to promote differentiation and/or maintenance of the innervating cells to their axonal processes. Accordingly, compositions comprising ontherin therapeutics may be employed to support the survival and reprojection of several types of ganglionic neurons sympathetic and sensory neurons as well as motor neurons. In particular, such therapeutic compositions may be useful in treatments designed to rescue, for example, various neurons from lesion-induced death as well as guiding reprojection of these neurons after such damage. Such diseases include, but are not limited to, CNS trauma infarction, infection (such as viral infection

with varicella-zoster), metabolic disease, nutritional deficiency, toxic agents (such as cisplatin treatment).

Moreover, certain of the ontherin therapeutics may be useful in the selective ablation of sensory neurons, for example, in the treatment of chronic pain syndromes.

As appropriate, ontherin therapeutics can be used in nerve prostheses for the repair of central and peripheral nerve damage. In particular, where a crushed or severed axon is intubulated by use of a prosthetic device, certain of ontherin therapeutics can be added to the prosthetic device to increase the rate of growth and regeneration of the dendritic processes. Exemplary nerve guidance channels are described in U. S. patents 5,092,871 and 4,955,892. Accordingly, a severed axonal process can be directed toward the nerve ending from which it was severed by a prosthesis nerve guide.

In another embodiment, the subject method can be used in the treatment of neoplastic or hyperplastic transformations such as may occur in the central nervous system. For instance, certain of the ontherin therapeutics which induce differentiation of neuronal cells can be utilized to cause such transformed cells to become either post-mitotic or apoptotic. Treatment with an ontherin therapeutic may facilitate disruption of autocrine loops, such as TGF-ß or PDGF autostimulatory loops, which are believed to be involved in the neoplastic transformation of several neuronal tumors. ontherin therapeutics may, therefore, thus be of use in the treatment of, for example, malignant gliomas, medulloblastomas, neuroectodermal tumors, and ependymonas.

Yet another aspect of the present invention concerns the application of the discovery that ontherin proteins are morphogenic signals involved in other vertebrate organogenic pathways in addition to neuronal differentiation as described above, having apparent roles in other endodermal patterning, as well as both mesodermal and endodermal differentiation processes. As described in herein Another aspect of the invention features transgenic non-human animals which express a heterologous ontherin gene of the present invention, and/or which have had one or more genomic ontherin genes disrupted in at least a tissue or cell-types of the animal. Accordingly, the invention features an animal model for developmental diseases, which animal has one or more ontherin allele which is mis-expressed. For example, an animal can be generated which has one or more ontherin alleles deleted or otherwise rendered inactive. Such a model can then be used to study disorders

arising from mis-expressed ontherin genes, as well as for evaluating potential therapies for similar disorders.

The transgenic animals of the present invention all include within a plurality of their cells a transgene of the present invention, which transgene alters the phenotype of the"host cell"with respect to regulation by the ontherin protein, e. g., of cell growth, death and/or differentiation. Since it is possible to produce transgenic organisms of the invention utilizing one or more of the transgene constructs described herein, a general description will be given of the production of transgenic organisms by referring generally to exogenous genetic material. This general description can be adapted by those skilled in the art in order to incorporate specific transgene sequences into organisms utilizing the methods and materials described herein and those generally known in the art.

In one embodiment, the transgene construct is a knockout construct. Such transgene constructs usually are insertion-type or replacement-type constructs (Hasty et al. (1991) Mol Cell Biol 11: 4509). The transgene constructs for disruption of an ontherin gene are designed to facilitate homologous recombination with a portion of the genomic ontherin gene so as to prevent the functional expression of the endogenous ontherin gene. In preferred embodiments, the nucleotide sequence used as the knockout construct can be comprised of (1) DNA from some portion of the endogenous ontherin gene (exon sequence, intron sequence, promoter sequences, etc.) which direct recombination and (2) a marker sequence which is used to detect the presence of the knockout construct in the cell. The knockout construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to prevent or interrupt transcription of the native ontherin gene. Such insertion can occur by homologous recombination, i. e., regions of the knockout construct that are homologous to the endogenous ontherin gene sequence hybridize to the genomic DNA and recombine with the genomic sequences so that the construct is incorporated into the corresponding position of the genomic DNA. The knockout construct can comprise (1) a full or partial sequence of one or more exons and/or introns of the ontherin gene to be disrupted, (2) sequences which flank the 5'and 3'ends of the coding sequence of the ontherin gene, or (3) a combination thereof.

A preferred knockout construct will delete, by targeted homologous recombination, essential structural elements of an endogenous ontherin gene. For example, the targeting construct can

recombine with the genomic ontherin gene can delete a portion of the coding sequence, and/or essential transcriptional regulatory sequences of the gene.

Alternatively, the knockout construct can be used to interrupt essential structural and/or regulatory elements of an endogenous ontherin gene by targeted insertion of a polynucleotide sequence. For instance, a knockout construct can recombine with an ontherin gene and insert a nonhomologous sequence, such as a neo expression cassette, into a structural element (e. g., an exon) and/or regulatory element (e. g., enhancer, promoter, intron splice site, polyadenylation site, etc.) to yield a targeted ontherin allele having an insertional disruption. The inserted nucleic acid can range in size from 1 nucleotide (e. g., to produce a frameshift) to several kilobases or more, and is limited only by the efficiency of the targeting technique.

Depending of the location and characteristics of the disruption, the transgene construct can be used to generate a transgenic animal in which substantially all expression of the targeted ontherin gene is inhibited in at least a portion of the animal's cells. If only regulatory elements are targeted, some low-level expression of the targeted gene may occur (i. e., the targeted allele is "leaky").

The nucleotide sequence (s) comprising the knockout construct (s) can be obtained using methods well known in the art. Such methods include, for example, screening genomic libraries with ontherin cDNA probes in order to identify the corresponding genomic ontherin gene and regulatory sequences. Alternatively, where the cDNA sequence is to be used as part of the knockout construct, the cDNA may be obtained by screening a cDNA library as set out above.

In another embodiment, the"transgenic non-human animals"of the invention are produced by introducing transgenes into the germline of the non-human animal. Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The specific line (s) of any animal used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness. In addition, the haplotype is a significant factor. For example, when transgenic mice are to be produced, strains such as C57BL/6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, ME). Preferred strains are those with H-2b, H-2d or H-2q haplotypes such as C57BL/6 or DBA/1. The line (s) used to practice this invention may themselves be transgenics, and/or may be knockouts (i. e., obtained from animals which have one or more genes partially or completely suppressed).

In one embodiment. the transgene construct is introduced into a single stage embryo. The zygote is the best target for micro-injection. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al. (1985) PNAS 82: 4438-4442). As a consequence, all cells of the transgenic animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.

Introduction of the transgene nucleotide sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.

Following introduction of the transgene nucleotide sequence into the embryo, the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention. One common method in to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.

Any technique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, nuclear membrane or other existing cellular or genetic structures. The exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.

Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.

Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product. Typically, DNA is prepared from excised tissue and analyzed by Southern analysis or PCR for the transgene. Alternatively, the tissues or cells believed to express the

transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.

Retroviral infection can also be used to introduce transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73: 1260-1264).

Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al. (1985) PNAS 82: 6927-6931; Van der Putten et al. (1985) PNAS 82: 6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6: 383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al. (1982) Nature 298: 623-628). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans <BR> <BR> et al. (1981) Nature 292: 154-156; Bradley et al. (1984) Nature 309: 255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322: 445-448). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction.

Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal.

The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, R. (1988) Science 240: 1468-1474.

In one embodiment, gene targeting, which is a method of using homologous recombination to modify an animal's genome, can be used to introduce changes into cultured embryonic stem cells. By targeting the ontherin gene in ES cells, these changes can be introduced into the germlines of animals to generate chimeras. The gene targeting procedure is accomplished by

introducing into tissue culture cells a DNA targeting construct that includes a segment homologous to an ontherin locus. and which also includes an intended sequence modification to the ontherin genomic sequence (e. g.. insertion, deletion, point mutation). The treated cells are then screened for accurate targeting to identify and isolate those which have been properly targeted.

Gene targeting in embryonic stem cells is in fact a scheme contemplated by the present invention as a means for disrupting an ontherin gene function through the use of a targeting transgene construct designed to undergo homologous recombination with ontherin genomic sequences. Targeting construct can be arranged so that, upon recombination with an element of an ontherin gene, a positive selection marker is inserted into (or replaces) coding sequences of the targeted ontherin gene. The inserted sequence functionally disrupts the ontherin gene, while also providing a positive selection trait.

Generally, the embryonic stem cells (ES cells) used to produce the knockout animals will be of the same species as the knockout animal to be generated. Thus for example, mouse embryonic stem cells will usually be used for generation of an ontherin-knockout mice.

Embryonic stem cells are generated and maintained using methods well known to the skilled artisan such as those described by Doetschman et al. (1985) J. Embryol. Exp. Morphol.

87: 27-45). Any line of ES cells can be used, however, the line chosen is typically selected for the ability of the cells to integrate into and become part of the germ line of a developing embryo so as to create germ line transmission of the knockout construct. Thus, any ES cell line that is believed to have this capability is suitable for use herein. The cells are cultured and prepared for knockout construct insertion using methods well known to the skilled artisan, such as those set forth by Robertson in: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, <BR> <BR> <BR> <BR> <BR> <BR> ed. IRL Press, Washington, D. C. [1987]); by Bradley et al. (1986) Current Topics in Devel. Biol.

20: 357-371) ; and by Hogan et al. (Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1986]).

Insertion of the knockout construct into the ES cells can be accomplished using a variety of methods well known in the art including for example, electroporation, microinjection, and calcium phosphate treatment. A preferred method of insertion is electroporation.

Each knockout construct to be inserted into the cell must first be in the linear form.

Therefore, if the knockout construct has been inserted into a vector, linearization is accomplished

by digesting the DNA with a suitable restriction endonuclease selected to cut only within the vector sequence and not within the knockout construct sequence.

For insertion, the knockout construct is added to the ES cells under appropriate conditions for the insertion method chosen, as is known to the skilled artisan. Where more than one construct is to be introduced into the ES cell, each knockout construct can be introduced simultaneously or one at a time.

If the ES cells are to be electroporated, the ES cells and knockout construct DNA are exposed to an electric pulse using an electroporation machine and following the manufacturer's guidelines for use. After electroporation, the ES cells are typically allowed to recover under suitable incubation conditions. The cells are then screened for the presence of the knockout construct.

Screening can be accomplished using a variety of methods. Where the marker gene is an antibiotic resistance gene, the ES cells may be cultured in the presence of an otherwise lethal concentration of antibiotic. Those ES cells that survive have presumably integrated the knockout construct. If the marker gene is other than an antibiotic resistance gene, a Southern blot of the ES cell genomic DNA can be probed with a sequence of DNA designed to hybridize only to the marker sequence Alternatively, PCR can be used. Finally, if the marker gene is a gene that encodes an enzyme whose activity can be detected (e. g. "B-galactosidase), the enzyme substrate can be added to the cells under suitable conditions, and the enzymatic activity can be analyzed. One skilled in the art will be familiar with other useful markers and the means for detecting their presence in a given cell. All such markers are contemplated as being included within the scope of the teaching of this invention.

The knockout construct may integrate into several locations in the ES cell genome, and may integrate into a different location in each ES cell's genome due to the occurrence of random insertion events. The desired location of insertion is in a complementary position to the DNA sequence to be knocked out, e. g., the ontherin coding sequence, transcriptional regulatory sequence, etc. Typically, less than about 1-5 percent of the ES cells that take up the knockout construct will actually integrate the knockout construct in the desired location. To identify those ES cells with proper integration of the knockout construct, total DNA can be extracted from the ES cells using standard methods. The DNA can then be probed on a Southern blot with a probe or probes designed to hybridize in a specific pattern to genomic DNA digested with particular restriction enzyme (s). Alternatively, or additionally, the genomic DNA can be amplified by PCR with probes

specifically designed to amplify DNA fragments of a particular size and sequence (i. e., only those cells containing the knockout construct in the proper position will generate DNA fragments of the proper size).

After suitable ES cells containing the knockout construct in the proper location have been identified, the cells can be inserted into an embryo. Insertion may be accomplished in a variety of ways known to the skilled artisan, however a preferred method is by microinjection. For microinjection, about 10-30 cells are collected into a micropipet and injected into embryos that are at the proper stage of development to permit integration of the foreign ES cell containing the knockout construct into the developing embryo. For instance, the transformed ES cells can be microinjected into blastocytes.

After the ES cell has been introduced into the embryo, the embryo may be implanted into the uterus of a pseudopregnant foster mother for gestation. While any foster mother may be used, the foster mother is typically selected for her ability to breed and reproduce well, and for her ability to care for the young. Such foster mothers are typically prepared by mating with vasectomized males of the same species. The stage of the pseudopregnant foster mother is important for successful implantation, and it is species dependent.

Offspring that are born to the foster mother may be screened initially for ontherin disruptants, DNA from tissue of the offspring may be screened for the presence of the knockout construct using Southern blots and/or PCR as described above. Offspring that appear to be mosaics may then be crossed to each other, if they are believed to carry the knockout construct in their germ line, in order to generate homozygous knockout animals. Homozygotes may be identified by Southern blotting of equivalent amounts of genomic DNA from animals that are the product of this cross, as well as animals that are known heterozygotes and wild type animals.

Other means of identifying and characterizing the knockout offspring are available. For example, Northern blots can be used to probe the mRNA for the presence or absence of transcripts of either the ontherin gene, the marker gene, or both. In addition, Western blots can be used to assess the (loss of) level of expression of the ontherin gene knocked out in various tissues of the offspring by probing the Western blot with an antibody against the ontherin protein, or an antibody against the marker gene product, where this gene is expressed. Finally, in situ analysis (such as fixing the cells and labeling with antibody) and/or FACS (fluorescence activated cell sorting)

analysis of various cells from the offspring can be conducted using suitable antibodies or ontherin ligands, to look for the presence or absence of the knockout construct gene product.

Animals containing more than one knockout construct and/or more than one transgene expression construct are prepared in any of several ways. The preferred manner of preparation is to generate a series of animals, each containing a desired transgenic phenotypes. Such animals are bred together through a series of crosses, backcrosses and selections, to ultimately generate a single animal containing all desired knockout constructs and/or expression constructs, where the animal is otherwise congenic (genetically identical) to the wild type except for the presence of the knockout construct (s) and/or transgene (s). Thus, a transgenic avian species can be generated by breeding a first transgenic bird in which the wild-type ontherin gene is disrupted with a second transgenic bird which has been engineered to express a mutant ontherin which retains most other biological functions of the receptor.

The transformed animals, their progeny, and cell lines of the present invention provide several important uses that will be readily apparent to one of ordinary skill in the art.

To illustrate, the transgenic animals and cell lines are particularly useful in screening compounds that have potential as prophylactic or therapeutic treatments of diseases such as may involve aberrant expression, or loss, of an ontherin gene, or aberrant or unwanted activation of receptor signaling. Screening for a useful drug would involve administering the candidate drug over a range of doses to the transgenic animal, and assaying at various time points for the effect (s) of the drug on the disease or disorder being evaluated. Alternatively, or additionally, the drug could be administered prior to or simultaneously with exposure to induction of the disease, if applicable.

In one embodiment, candidate compounds are screened by being administered to the transgenic animal, over a range of doses, and evaluating the animal's physiological response to the compound (s) over time. Administration may be oral, or by suitable injection, depending on the chemical nature of the compound being evaluated. In some cases, it may be appropriate to administer the compound in conjunction with co-factors that would enhance the efficacy of the compound.

In screening cell lines derived from the subject transgenic animals for compounds useful in treating various disorders, the test compound is added to the cell culture medium at the appropriate time, and the cellular response to the compound is evaluated over time using the appropriate biochemical and/or histological assays. In some cases, it may be appropriate to apply

the compound of interest to the culture medium in conjunction with co-factors that would enhance the efficacy of the compound.

The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).

Research Uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to"subtract-out"known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a"gene chip"or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, <BR> <BR> <BR> <BR> for example, those described in Gyuris et al., 1993, Cell 75: 791-803 and in Rossi et al., 1997, Proc. Natl. Acad. Sci. USA 94: 8405-8410, all of which are incorporated by reference herein) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.

The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological

fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.

Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.

Methods for performing the uses listed above are well known to those skilled in the art.

References disclosing such methods include without limitation"Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and"Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

Nutritional Uses Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.

Cytokine and Cell Proliferation/Differentiation Activity A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G,

T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8. RB5, DA1, 123, Tl 165. HT2, CTLL2, TF-l, Mo7e and CMK.

The activity of a protein of the invention may, among other means. be measured by the following methods: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.

Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137: 3494-3500,1986; Bertagnolli et al., J. Immunol.

145: 1706-1712,1990; Bertagnolli et al., Cellular Immunology 133: 327-341,1991; Bertagnolli, et al., J. Immunol. 149: 3778-3783,1992; Bowman et al., J. Immunol. 152: 1756-1761,1994.

Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon (, Schreiber, R. D. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol 1 pp.

6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology.

J. E. e. a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173: 1205-1211,1991; Moreau et al., Nature 336: 690-692,1988; Greenberger et al., Proc. Natl. Acad. Sci. U. S. A. 80: 2931-2938,1983; Measurement of mouse and human interleukin 6-Nordan, R. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U. S. A. 83: 1857-1861, 1986; Measurement of human Interleukin 11-Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9-Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77: 6091-6095,1980; Weinberger et al., Eur. J. Immun. 11: 405-411,1981; Takai et al., J. Immunol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140: 508-512,1988.

Immune Stimulating or Suppressing Activity A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e. g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e. g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i. e., in the treatment of cancer.

Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired

(including, for example. organ transplantation), may also be treatable using a protein of the present invention.

Using the proteins of the invention it may also be possible to immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.

Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e. g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand (s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e. g., B7-1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand (s) on the immune cells without transmitting the corresponding costimulatory signal.

Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.

The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the <BR> <BR> <BR> <BR> immunosuppressive effects of CTLA41g fusion proteins in vivo as described in Lenschow et al., Science 257: 789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89: 11102-11105 (1992).

In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.

Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block costimulation of T cells by disrupting receptor: ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL11prllpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.

Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.

Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.

In another application, up regulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor cells (e. g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides. For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-1-like activity and/or B7-3-like activity.

The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo.

The presence of the peptide of the present invention having the activity of a B lymphocyte antigen (s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class 11 molecules, can be transfected with nucleic acid encoding <BR> <BR> <BR> <BR> all or a portion of (e. g., a cytoplasmic-domain truncated portion) of an MHC class I a chain protein and B2 microglobulin protein or an MHC class II a chain protein and an MHC class 11 B chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class 11 MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e. g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks

expression of an MHC class 11 associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.

The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.

Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78: Herrmann et al., J. Immunol. 128: 1968-1974,1982; Handa et al., J. Immunol. 135: 1564-1572,1985; Takai et al., J. Immunol. 137: 3494-3500,1986; Takai et al., J. Immunol. 140: 508-512,1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78: 2488-2492,1981; Herrmann et al., J. Immunol. 128: 1968- 1974,1982; Handa et al., J. Immunol. 135: 1564-1572,1985; Takai et al., J. Immunol. 137: 3494- 3500,1986; Bowmanet al., J. Virology 61: 1992-1998; Takai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli et al., Cellular Immunology 133: 327-341,1991; Brown et al., J. Immunol.

153:3079-3092,1994.

Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol.

144: 3028-3033,1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.

Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in

Humans) ; Takai et al., J. Immunol. 137: 3494-3500,1986; Takai et al., J. Immunol. 140: 508-512.

1988; Bertagnolli et al., J. Immunol. 149: 3778-3783.1992.

Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134: 536-544,1995; Inaba et al., Journal of Experimental Medicine 173: 549-559, 1991; Macatonia et al., Journal of Immunology 154: 5071-5079,1995; Porgador et al., Journal of Experimental Medicine 182: 255-260,1995; Nair et al., Journal of Virology 67: 4062-4069,1993; Huang et al., Science 264: 961-965,1994; Macatonia et al., Journal of Experimental Medicine 169: 1255-1264,1989; Bhardwaj et al., Journal of Clinical Investigation 94: 797-807,1994; and Inaba et al., Journal of Experimental Medicine 172: 631-640,1990.

Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13: 795-808,1992; Gorczyca et al., Leukemia 7: 659-670,1993; Gorczyca et al., Cancer Research 53: 1945-1951,1993; Itoh et al., Cell 66: 233-243,1991; Zacharchuk, Journal of Immunology 145: 4037-4045,1990; Zamai et al., Cytometry 14: 891-897,1993; Gorczyca et al., International Journal of Oncology 1:639-648,1992.

Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84: 111-117,1994; Fine et al., Cellular Immunology 155: 111-122,1994; Galy et al., Blood 85: 2770-2778,1995; Toki et al., Proc.

Nat. Acad Sci. USA 88: 7548-7551,1991.

Hematopoiesis Regulating Activitv A protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e. g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i. e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent

myelo-suppression ; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation. including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in <BR> <BR> <BR> <BR> repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i. e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15: 141-151,1995; Keller et al., Molecular and Cellular Biology 13: 473-486,1993; McClanahan et al., Blood 81: 2903-2915,1993.

Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: <BR> <BR> <BR> <BR> Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.

Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89: 5907-5911,1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A. In Culture of Hematopoietic Cells.

R. I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22: 353-359,1994; Cobblestone area forming cell assay, Ploemacher, <BR> <BR> <BR> <BR> R. E. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York, NY. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.

163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay,

Sutherland, H. J. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.

Tissue Growth Activity A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.

A protein of the present invention, which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.

A protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.

Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation. A protein of the present invention, which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of

the present invention may provide an environment to attract tendon-or ligament-forming cells, stimulate growth of tendon-or ligament-forming cells, induce differentiation of progenitors of tendon-or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.

The protein of the present invention may also be useful for proliferation of neural cells and <BR> <BR> <BR> <BR> for regeneration of nerve and brain tissue, i. e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.

Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.

It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity.

A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.

A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.

The activity of a protein of the invention may, among other means, be measured by the following methods: Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. W095/16035 (bone, cartilage, tendon); International Patent Publication No. W095/05846 (nerve, neuronal); International Patent Publication No. W091/07491 (skin, endothelium).

Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71: 382-84 (1978).

Activin/Inhibin Activitv A protein of the present invention may also exhibit activin-or inhibin-related activities.

Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin"family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.

Administration of sufficient amounts of other inhibins can induce infertility in these mammals.

Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-$ group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.

The activity of a protein of the invention may, among other means, be measured by the following methods:

Assays for activin/inhibin activity include, without limitation. those described in: Vale et al., Endocrinology 91: 562-572,1972 ; Ling et al.. Nature 321: 779-782,1986; Vale et al., Nature 321: 776-779.1986; Mason et al., Nature 318: 659-663,1985; Forage et al., Proc. Natl. Acad. Sci.

USA 83: 3091-3095,1986.

Chemotactic/Chemokinetic Activitv A protein of the present invention may have chemotactic or chemokinetic activity (e. g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.

Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.

A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.

Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells.

Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.

The activity of a protein of the invention may, among other means, be measured by the following methods: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.

Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 28; Taub et al. J. Clin.

Invest. 95: 1370-1376,1995; Lind et al. APMIS 103: 140-146,1995; Muller et al Eur. J. Immunol.

25: 1744-1748; Gruber et al. J. of Immunol. 152: 5860-5867,1994; Johnston et al. J. of Immunol.

153: 1762-1768,1994.

Hemostatic and Thrombolytic Activity

A protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e. g., stroke).

The activity of a protein of the invention may, among other means, be measured by the following methods: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26: 131-140,1986; Burdick et al., Thrombosis Res. 45: 413-419, 1987; Humphrey et al., Fibrinolysis 5: 71-79 (1991); Schaub, Prostaglandins 35: 467-474,1988.

Receptor/Ligand Activity A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses). Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.

The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.

Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl.

Acad. Sci. USA 84: 6864-6868,1987; Bierer et al., J. Exp. Med. 168: 1145-1156,1988;

Rosenstein et al., J. Exp. Med. 169: 149-160 1989; Stoltenborg et al., J. Immunol. Methods 175: 59-68,1994; Stitt et al., Cell 80: 661-670,1995.

Anti-Inflammatory Activitv Proteins of the present invention may also exhibit anti-inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.

Cadherin/Tumor Invasion Suppressor Activity Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis.

Cadherin malfunction is also implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities.

The cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule. The cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins.

E-cadherin, one member of the cadherin superfamily. is expressed in epithelial cell types.

Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells'adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage-independent cell growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression.

Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body. Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.

Additionally, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can used to generate antibodies recognizing and binding to cadherins. Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i. e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.

Fragments of proteins of the present invention with cadherin activity, preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and poly-nucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects.

Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of

cancer patients. and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.

Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817,1995; Miyaki et al.

Oncogene 11: 2547-2552,1995; Ozawa et al. Cell 63: 1033-1038,1990.

Tumor Inhibition Activity In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti-tumor activities. A protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.

Other Activities A protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component (s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or

complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.

All of the above-cited references and publications are hereby incorporated by reference.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific polypeptides, nucleic acids, methods, assays and reagents described herein. Such equivalents are considered to be within the scope of this invention.