JP2001526030A - Cadherin-like polypeptides and related methods and compositions - Google Patents

Abstract

  (57) [Summary] The present invention relates to the discovery of a new family of cadherin-related genes called "onthelins". As described herein, vertebrate onserin proteins exhibit a spatially restricted expression domain, indicating an important role in tissue homeostasis.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Putter formation is the activity by which embryonic cells form an ordered spatial arrangement of differentiated tissues. The physical complexity of higher animals occurs during embryonic development due to the interaction of cell-specific systems and extracellular signaling. Induced interactions are based on body plans (body p
lan) are essential for patterning in spinal development, organ system patterning, and the development of various cell types during tissue differentiation (Davids
on, E., (1990) Development 108: 365-389; Gurdon JB, (1992) Cell 68: 1
85-199; Jessell, TM et al., (1992) Cell 68: 257-270). The effects of developing cell interactions are variable. Typically, responsive cells diversify from one root of cell differentiation to another by inducing cells that differ in both the uninduced and induced states of the responding cells (induction). Sometimes, a cell induces its neighbors to differentiate in a manner similar to itself (homoiogenenetic induction). In other cases, a cell may not be allowed to differentiate into its neighbors as well.
Cell interactions during early development are continuous, so that the initial induction between the two cell types is progressive with increasing diversity. In addition, inducible interactions occur not only in embryos but also in adult cells, which act similarly to establish and maintain morphological patterns and induce further differentiation (JB Gurdon (1992) Cell 68: 185
-199). A diverse system of adhesion molecules and adhesion receptors is involved in the formation of life phenomena such as embryonic development, cell proliferation and differentiation, and wound repair. Over the past decade, the understanding of adhesion molecules and their complexities has been enormous. An overview of the major classes of adhesion receptors is given, with examples of diseases in which they are found and involved. Cadherins establish molecular links between adjacent cells. They form a zipper-like structure at the adhesive junction, the membrane area where the cells come in contact. Via these junctions, actin filament bundles form from cell to cell. Related molecules, such as desmogleins, are components of the desmosome, and cell-cell contacts are common in epithelial cells. Desmosomes serve as anchoring sites for intermediate filaments in the cytoskeleton. When dissociated embryonic cells grow in a dish, they tend to form clusters depending on the tissue of origin. The cadherin homophilic interaction is the basis for this histogenetic segregation and plays an important role in the fractionation of embryonic tissue. Expression of specific adhesion molecules in the embryo is important for cell migration and tissue differentiation. For example,
When neurite cells stop producing N-CAM and N-cadherin and begin presenting integrin receptors, they detach and begin to migrate onto the extracellular matrix. Defective interactions between adhesion molecules play an important role in cancer. The malignant behavior of tumors depends on three characteristics of neoplastic cells. That is, uncontrolled growth,
Local invasiveness and metastatic potential. The presence and absence of adhesion molecules affects each of these properties. As the tumor develops, the adhesion of its constituent cells changes. Cells detach from the tumor, gain the ability to migrate, and invade other organs. If the neoplastic cells reduce their ability to produce matrix proteins (eg, fibronectin), these changes may occur, thus detaching from the tumor. Other cancer cells have lost E-cadherin and become motile and invasive. Highly invasive tumor cells lose their invasiveness when transfected with the normal E-cadherin gene. However, inactivation of the gene leaves an aggressive phenotype.
Induction of the invasive behavior of tumor cells by anti-E-cadherin antibodies also supports the notion that this adhesion molecule suppresses local recruitment and distant metastasis. Cadherins are first discovered in mouse tetratocarcinoma cells (Liaw et al., (1990) EMBO J 9: 2701-2708) and are structurally and functionally similar molecules (Ginsberg et al., (1991) Development 111: 315), which is involved in selective calcium-dependent adhesion interactions between cell surfaces (Walsh et al (1990) J
Neurochem 55: 805). There are many isoforms that are tissue-specific in different organisms. Cells with different cadherins have a propensity to segregate in vitro, whereas cells with the same cadherin tend to aggregate preferentially. This observation is linked to the finding that cadherin expression causes morphological changes, including the positional segregation of cells into layers, and that cadherins sort cell types that differ during morphogenesis, tissue development and regeneration. It is suggested that it plays an important role. Cadherins constitute a family of Ca ++-dependent adhesion molecules that mediate intercellular junctions important for maintenance of tissue structure and morphogenesis. Structurally, cadherin has many domains. These have a signal sequence. There is a propeptide of about 130 residues; an extracellular domain of about 600 residues; a single transmembrane domain; and a well-conserved C-terminal cytoplasmic domain of about 190 residues. Classical cadherins, E- and P-cadherins are large extracellular domains each characterized by a series of five homologous N-terminal repeats, the furthest of which is responsible for binding specificity, membranes Includes a penetrating domain and a C-terminal intracellular domain.
Relatively short intracellular domains interact with various cytoplasmic proteins (eg, β-catenin) to regulate cadherin function. The extracellular domain is further divided into five parts, four of which are repeats of about 110 residues, and the fifth has four conserved cysteines. The calcium-binding region of cadherin is thought to be in the extracellular domain.

[0002] Studies on the tissue expression of various cadherin-related proteins have revealed 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 neurons and muscle cells. Cadherin-related protein expression is also spatially and temporally regulated during development. This is because individual proteins are expressed by specific cells and are expressed in tissues at specific stages of development.
1991)). Ectopic expression of cadherin-related proteins and inhibition of intrinsic expression of cadherin-related proteins prevents the formation of normal tissue structures (Detrick et al.
(1990) Neuron 4: 493-506; Fujimori et al (1990) Development 110: 97-104;
Kintner (1992) Cell 69: 225-236).

[0003] The unique transient expression pattern in tissues of different cadherins and cadherin-related proteins indicates that each subclass of protein has normal in vivo events (eg, maintenance of the intestinal epithelial barrier) and abnormal events (eg, tumors). This is particularly important given its role in metastasis or inflammation. Different subclasses or combinations of subclasses of cadherin-related proteins are responsible for the adhesion events between different cells, for which therapeutic detection and / or intervention may be desirable. For example, autoantibodies from patients with autoimmune dermatosis, which is characterized by blister formation due to melanoma, or reduced cell adhesion, reacts with cadherin-related proteins and provides direct support for cadherin adhesion function in vivo. (Amagai et al (1991) Cell 67: 869-877). Studies have also suggested that cadherin and cadherin-related proteins have a regulatory function in addition to adhesive activity. Matsunaga et al. (Nature 334: 62-64) report that N-cadherin has neurite outgrowth promoting activity. The Drosophila lipid oncogene is E
-Appears to regulate neurite outgrowth and suppress tumor invasion in the same way as cadherin (Mahoney et al., Supra; Frixen et al., (1991) J. Cell. Biol. 113: 1)
73-185; Chen et al., (1991) J. Cell. Biol. 114: 319-327; and Vleminckx
et al. (1991) Cell 66: 107-119). Thus, therapeutic intervention in the modulating activity of cadherin-related proteins expressed in specific tissues is desirable.

SUMMARY OF THE INVENTION The present invention relates to the discovery of a new class of cadherin-related proteins, referred to herein as ontherins. The onserin polypeptides of the present invention, as well as cadherin family proteins, can be used in vertebrates (both adult and embryo).
It is widely involved in the formation and maintenance of ordered spatial arrangements of differentiated tissues of, and could be used to generate and / or maintain different vertebrate tissue sequences both in vitro and in vivo.

[0005] In general, the present invention relates to isolated onserin polypeptides, preferably substantially pure preparations of the present onserin polypeptides. The present invention also relates to recombinantly produced onserin polypeptides. In a preferred embodiment,
The polypeptide has a biological activity that includes one or more of the following activities: the ability to bind to extracellular proteins or matrix components such as cadherins, integrins, desmosome proteins (such as desmoglein and desmocholine);
The ability to bind to intracellular signal transduction proteins such as catenin; and
Binding ability to valent calcium. Onserin polypeptides that specifically antagonize such activity may be provided, for example, by truncation variants, which are also individually contemplated.

[0006] In one embodiment, the polypeptide is an Onserin polypeptide set forth in SEQ ID NO: 2, or a mature polypeptide sequence thereof (eg, residue 13 of SEQ ID NO: 2).
0-889), or the truncated mature form of the extracellular domain (eg, residues 130-708), or the intracellular domain (residues 725-889). Other preferred onserin polypeptides are at least 150, 200 or 25 from the extracellular domain, preferably from the mature extracellular domain.
Includes 0 amino acid residues (eg, residues 130-708). Still other preferred Onserin polypeptides are at least 100, 15 from the intracellular domain.
Contains 0 or 175 amino acid residues.

[0007] Also related members of the Onserin family are contemplated, eg, preferably the Onserin polypeptide is at least 65% relative to the polypeptide set forth in SEQ ID NO: 2.
, 67%, 69%, 70%, 75% or 80% polypeptides having amino acid sequence homology, for example, polypeptides having high homology of 82%, 85%, 90% and 95% Are also contemplated. In a preferred embodiment, the onserin polypeptide may be encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence set forth in SEQ ID NO: 1. Homologs of the onserin of the present invention also include versions of the protein that are resistant to post-translational modifications. The resistance may be, for example, a mutation that changes a modification site (for example, a tyrosine, threonine, serine or asparagine residue), a mutation that prevents glycosylation of a protein, or a protein and an onserin ligand (for example, an extracellular protein or matrix). component)
Due to a mutation that interferes with the interaction.

[0008] The onserin polypeptide may comprise the full-length protein as set forth in SEQ ID NO: 2, or a fragment thereof, eg, a fragment corresponding to one or more specific motifs / domains, or any Fragments corresponding to size, eg, at least 5, 10, 25, 50, 100, 150 or 200 amino acids in length are included. In a preferred embodiment, the Onserin polypeptide comprises a sufficient portion of the extracellular domain that can specifically bind to Ca ²⁺ or some other extracellular ligand. Truncated forms of the protein include, but are not limited to, soluble extracellular fragments.

[0009] In certain preferred embodiments, the invention relates to purified or recombinant onserin polypeptides having a molecular weight of about 95.6 kd (without post-translational modifications). Certain post-translational modifications, such as glycosylation, prenylation, myristylation, etc., increase the apparent molecular weight of the onserin protein as compared to the unemployed polypeptide chain.

[0010] The protein of the present invention may be provided as a chimeric molecule such as a fusion protein. For example,
The Onserin protein may be provided as a recombinant fusion protein comprising a second polypeptide moiety (eg, the second polypeptide moiety has an amino acid sequence that is unrelated (heterologous) to the Onserin polypeptide). For example, glutathione S-
Transferase, for example, may have enzymatic activity such as alkaline phosphatase, for example, may be an epitope tag).

[0011] In yet another embodiment, the invention relates to a nucleic acid encoding an Onserin polypeptide that has the ability to modify, eg, mimic or antagonize, at least a portion of the activity of a wild-type Onserin polypeptide. An example of a nucleic acid sequence encoding onserin is shown in SEQ ID NO: 1.

[0012] In another embodiment, the nucleic acids of the invention include a coding sequence that hybridizes under stringent conditions to all or a portion of one or more of the coding sequences of SEQ ID NO: 1. The coding sequence of the nucleic acid is
It may comprise the same sequence as the coding sequence shown in SEQ ID NO: 1, or may simply be homologous to those sequences. In a preferred embodiment, the nucleic acid is
Encodes a polypeptide that specifically modulates one or more biological activities of a wild-type onserin polypeptide by acting as either an agonist or antagonist.

[0013] Furthermore, in certain preferred embodiments, the Onserin nucleic acids of the invention comprise a transcription regulatory sequence, eg, at least one transcription promoter or enhancer sequence, wherein the regulatory sequence is operably linked to the Onserin gene sequence. .
Such regulatory sequences can be used to render the onserin gene sequence suitable for use as an expression vector. The transcription regulatory sequence may be derived from an onserin gene or may be derived from a heterologous gene.

The present invention also contemplates cells (prokaryotic cells or eukaryotic cells) transfected with the above expression vector and a method for producing an onserin protein by using the expression vector.

[0015] In yet another embodiment, the invention provides a gene activation construct, wherein the construct recombines a heterologous transcriptional regulatory sequence with a genomic onserin, eg, by heterologous recombination, in a cell. Operably linked to a gene coding sequence. Cells having a genomic onserin gene modified by a gene activation construct are also specifically contemplated.

[0016] In yet another embodiment, the present invention provides a nucleic acid that hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 contiguous nucleotides of the sense or antisense sequence of SEQ ID NO: 1. But preferably at least two of the sense or antisense sequences of SEQ ID NO: 1.
5 contiguous nucleotides, more preferably at least 40, 50 or 75
A nucleic acid is provided that hybridizes to a nucleic acid probe corresponding to a number of contiguous nucleotides.

[0017] Yet another aspect of the invention relates to an immunogen comprising an onserin polypeptide in an immunogenic composition, wherein the immunogen is specific to an onserin polypeptide, eg, a humoral response, such as a humoral response. An antibody response, eg, a cell line response, can be induced. In a preferred embodiment, the immunogen contains an antigenic determinant, for example, a unique determinant from the protein shown in SEQ ID NO: 2. A further aspect of the present invention relates to antibodies and antibody formulations that specifically react with epitopes of the onserin immunogen.

The present invention also relates to a transgenic non-human animal having a transgene, for example,
For mice, rats, rabbits, chickens, frogs or pigs, for example, an animal having a variant onserin gene described herein, or an animal mis-expressing an endogenous onserin gene, or one or more onserin proteins of the invention Some animals are deficient in the expression of Such transgenic animals may be useful as animal models for cell and tissue disorders associated with mutated or mis-expressed onserin alleles, or for drug screening applications.

The present invention also provides a probe / primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide is a sense or antisense of SEQ ID NO: 1 or a naturally occurring variant thereof under stringent conditions. Hybridizes to at least 12 contiguous nucleotides of the sequence. In a preferred embodiment, the probes / primers further comprise a label group attached thereto and are detectable. The labeling group can be selected, for example, from the group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme cofactors. The probes of the present invention may be used as part of a diagnostic test kit, for example, to identify a dysfunction associated with misexpression of an onserin protein in a cell sample isolated from a patient, which encodes an onserin protein. Nucleic acid levels may be measured, for example, onserin mR in cells.
The NA level may be measured, or it may be examined whether the genomic onserin gene is mutated or deleted. According to the present invention, these so-called “probes /
A "primer" may be used as part of an "antisense" therapy, which comprises a cell encoding one or more of the onserin proteins of the invention under cellular conditions.
Hybridization (binding) specifically to RNA and / or genomic DNA
The term refers to inhibiting the expression of the protein by administering or in situ generating an oligonucleotide probe or a derivative thereof, for example, by inhibiting transcription and / or translation. Preferably, the oligonucleotide is at least 12
Although nucleotides, primers 25, 40, 50, or 75 nucleotides in length are also contemplated.

In yet another embodiment, the present invention is directed to screening for compounds as inhibitors or activators of the interaction between the Onserin protein and a polypeptide or other factor that interacts with it (Onserin interacting substance). To provide an assay for A typical method comprises: (a) making a reaction mixture comprising (i) an onserin interactor, (ii) an onserin polypeptide, and (iii) a test compound; Examining the interaction with the serine polypeptide. A statistically significant change (activation or inhibition) in the interaction of the Onserin interactor with the Onserin polypeptide in the presence of the test compound, compared to that in the absence of the test compound, indicates that the test compound has Onserin Potential agonists (mimics or activators) or antagonists (inhibitors) for the biological activity of are shown. The reaction mixture may be a cell-free protein formulation, for example, a reconstituted protein mixture or a cell lysate, which may be a cell containing a heterologous nucleic acid that recombinantly expresses an Onserin polypeptide.

[0021] In a preferred embodiment, the step of detecting the interaction between the Onserin interactor and the Onserin polypeptide is a competitive binding assay. In another preferred embodiment, the step of detecting the interaction of the Onserin interactor with the Onserin polypeptide comprises: in a cell-based assay, a second messenger in the cell that responds to signaling mediated by the Onserin polypeptide. Detecting a change in the level of In yet another preferred embodiment, the step of detecting the interaction of the Onserin interactor with the Onserin polypeptide is performed in a cell-based assay by a gene regulated by a transcriptional regulatory sequence responsive to signaling by the Onserin polypeptide. To examine changes in the level of expression.

In a preferred embodiment, the assay step is at least 100, is repeated for more preferably at least 10 ^3, 10 ⁴ or miscellaneous library of 10 ⁵ different test compound. The test compound can be, for example, a peptide, nucleic acid, carbohydrate, small organic molecule, or natural product extract (or fraction thereof).

The invention further contemplates a pharmaceutical formulation of one or more drugs identified in such a drug screening assay. In another embodiment, the present invention provides a molecule, preferably a small organic molecule, that binds to onserin and mimics or antagonizes onserin-induced signaling in a cell.

Yet another embodiment of the present invention relates to the modulation of the biological activity of onserin by, for example, activating or disrupting certain protein-protein interactions, thereby regulating the growth, differentiation or survival of cells. To one or more of them. In general, in vivo, in vitro, or in situ, the method comprises treating cells with an effective amount of an onserin therapeutic and comparing (i) growth rate (ii) differentiation with untreated cells. Or (iii) altering at least one of the cell viability. Thus, the method comprises the use of peptides and peptidomimetics that promote or antagonize the effects of signaling from the Onserin protein by interaction with the Onserin ligand or intracellular signal transduction protein or other molecules identified in the drug screens described above. This can be done using onserin therapeutics. Other onserin therapeutics include antisense constructs that inhibit onserin protein expression, major negative variants of onserin proteins that competitively inhibit onserin interactions upstream of the cell surface, or signal transduction downstream of wild-type onserin proteins. Includes the major negative variants of the onserin protein that competitively inhibits duplication, and gene therapy constructs, including gene activation constructs.

In one embodiment, the method of the present invention for modulating the biological activity of onserin is used to modulate neuronal cell differentiation to maintain neuronal cells at a differentiation stage,
And / or promote neuronal cell differentiation, for example, to prevent apoptosis or other forms of cell death. For example, the present invention may 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 for determining whether a subject, eg, an animal patient, is at risk for a disease characterized by undesirable cell proliferation or differentiation or abnormal control of apoptosis. The method comprises the steps of:
detecting the presence or absence of a genetic disorder characterized by at least one of: i) a mutation in a gene encoding an onserin protein; or (ii) a misexpression of an onserin gene. In a preferred embodiment, detecting the genetic disorder comprises confirming the presence of at least one of the following: a deletion of one or more nucleotides from the onserin gene; Addition of one or more nucleotides; substitution of one or more nucleotides of the gene; translocation of the gene on a chromosome; alteration of the level of a messenger RNA transcript of the gene; The presence of an untyped splicing pattern; a level of protein that is not at the wild-type level; and / or an abnormal level of soluble onserin protein.

For example, detecting a genetic disorder comprises: (i) the sense or antisense sequence of the onserin gene or a naturally occurring variant thereof, or a 5 ′ or 3 ′ that is naturally associated with the onserin gene. Providing a probe / primer containing an oligonucleotide comprising a region of the nucleotide sequence that hybridizes to a flanking sequence; (ii) exposing the probe / primer to tissue nucleic acid; and (iii) It involves detecting the presence or absence of a genetic disorder by hybridization of a primer to a nucleic acid. For example, detecting the disorder involves examining the nucleotide sequence of the onserin gene and optionally flanking nucleic acid sequences using primers / probes. For example, a probe / primer is coupled to a polymerase chain reaction (PCR).
) Or ligation chain reaction (LCR) can also be used. In another embodiment, the level of onserin protein is determined in an immunoassay using an antibody that specifically immunoreacts with onserin protein.

In the practice of the present invention, unless otherwise specified, cell biology, cell culture, molecular biology,
Using conventional methods of transgenic biology, microbiology, recombinant DNA, and immunology, they are within the skill of the art. Such methods are explained fully in the literature. For example, Molecular Cloning A Laboratory Manua
l, 2nd Ed., ed.by Sambrook, Fritsch and Maniatis (Cold Spring Harbor La
boratory Press: 1989); DNA Cloning, Volumes I and II (DN Glover ed.,
1985); Oligonucleotide Synthesis (MJ Gait ed., 1984); Mullis et al. U
.S. Patent No: 4,683,195; Nucleic Acid Hybridization (BD Hames & S. J
Higgins eds. 1984); Transcription And Translation (BD Hames & SJ
Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Li
ss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Per
bal, A Practical Guide To Molecular Cloning (1984); the treatise, Method
s In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For
Mammalian Cells (JH Miller and MP Calos eds., 1987, Cold Spring Ha
rbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. ed
s.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Wal
ker, eds., Academic Press, London, 1987); Handbook Of Experimental Immun.
ology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986); Manipu
lating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spri
ng Harbor, NY, 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 to the development and maintenance of tissues in vertebrates is the type of extracellular communication, called induction, that exists between adjacent cell layers and tissues. In inducible interactions, cell-to-cell contact and / or chemical signals secreted by one cell group affect the developmental fate of a second cell group. Typically, a cell that responds to an inducible signal changes from one cell's fate to another that is not the same as the signaling cell. Whether regulation of extracellular signals results from secreted molecules or cell-cell or cell-substrate interactions is an important mechanism of developmental control. The present invention relates to the discovery of a new family of cadherin-like proteins, referred to herein as "onthelins," which have been shown to have neurotrophic activity. As described below, human once-selling clones were first identified by expression cloning techniques through their ability to protect striatal neurons from cell death. As described herein, onserin gene products express spatially restricted domains that indicate important roles in regulating cell physiology.

[0030] Onserin proteins, through their ability to transmit information from the extracellular to the intracellular matrix, regulate developmental processes, such as those from mesoderm, such as those from dorsal mesoderm, cartilage and tissues involved in spermatogenesis. Modulating the growth, survival, and / or differentiation of tissue, increasing the growth, survival, and / or differentiation of tissue from ectoderm, such as tissue from the epidermis, neural tube, neural crest, or head mesenchyme It can be clearly modulated by modulating, regulating the growth, survival, and / or differentiation of endoderm-derived tissue, such as tissue from the early gastrointestinal tract. Human onserin cDNA was identified by screening for neurotrophic activity using a cDNA library originally cloned in a signal sequence trap assay. Each cDNA was cloned into a plasmid capable of recombinant expression and simultaneously secreted into cultured mammalian cells. A sample of the conditioned medium and a membrane preparation of cells producing the cDNA gene product are applied to the cultured striatal nerve under conditions without neurotrophic activity in the applied sample, and the nerve is timed with defined kinetics . With appropriate neurotrophic activity, the half-life of the cell can be increased. Only one positive corn was identified among the library members screened. First, clones isolated by a nerve survival assay were sequenced and then 3 ′
It was determined to contain the coding sequence fragment. The human cDNA library was screened with its fragments to completely isolate the full-length human cDNA.

The sequence of the human onserin cDNA is presented in SEQ ID NO: 1, and the corresponding full length protein is presented in SEQ ID NO: 2. The first isolated fragment corresponds to the C-terminal fragment of the protein. The sequence of a typical onserin gene has been shown to encode a secretory protein that may anchor to the cell membrane by a transmembrane domain. Apparently, the Onserin protein naturally exists in many different forms, including precursors. The precursor contains an N-terminal signal peptide (N-terminal residue about 130) that directs secretion of at least the N-terminal domain of the protein, but the full-length mature form lacks this signal sequence. In addition, processing of the mature form occurs at several stages, resulting in a biologically active fragment of the protein, such as an extracellular fragment of the mature form of the protein.

Further, the analysis of the protein sequence was carried out at the N-terminal part of the protein, for example, at residues 120-130, 231-241, 339-349, 460-470 and 5
Suggests five cadherin-like motifs, 71-581. Cadherin-like motifs generally have the consensus sequence [LIV] -X- [LIV] -XXDX-
Found by ND- [NH] -XP. 22-25 of SEQ ID NO: 2, 2
66-269, 439-442, 453-456, 504-507, 566-5
69 and 590-593, for example, the consensus sequence N {P}-[S
There are many potential Asn-related glycosylation sites with [T]-{p}. Similarly, the full-length onserin protein can be found, for example, at residue 709-7 of SEQ ID NO: 2.
24 also contains a corresponding transmembrane domain, and thus contains residues 1-708 (
Extracellular domain corresponding to mature form of 131-708) and residues 725-88
Provides the intracellular domain corresponding to 9. For example, Onserin polypeptides that lack transmembrane and intracellular domains due to altered splicing or proteolysis are expected to be completely secreted rather than membrane bound.

The cloned onserin genes described in the appended sequence listing are in addition to being representative of members of an inter-tribal family of related genes (eg, in vertebrates), as well as being members of intra-tribal families. It is contemplated by the present invention that this is the case. That is, it is expected that there is another human paralog of human onserin protein, and the ortholog of each onserin-like gene is conserved among other animals. The expression pattern of the onserin gene has been determined by several different approaches. In one technique, the presence of clones in a signal sequence trap assay from a variety of different embryo and adult tissues was examined. It was observed that the Onserin coding sequence was cloned from several different tissues. The most abundant source by signal sequence assay was brain. Clones were also isolated from testis, kidney and placenta. Accordingly, one aspect of the present invention relates to nucleic acids encoding onserin polypeptides, the onserin polypeptide itself (including various fragments), antibodies immunoreactive with onserin proteins, and the production of such compositions. . In addition, the present invention relates to, for example, abnormal expression (or loss thereof) of onserin,
Diagnostic and therapeutic assays for diseases associated with aberrant processing or splicing of RNA, onserin ligands or signal transducers thereof, and reagents for detecting and treating diseases.

In addition, altering the ability of the onserin protein to transduce intracellular signals by binding the molecule to ions or other extracellular / matrix factors, or by interacting with, for example, cell surfaces and intracellular proteins. Possibly, drug discovery assays are performed to identify reagents that can modulate the biological function of the Onserin protein. Such reagents can be therapeutically useful for altering tissue growth, maintenance and / or differentiation. Other aspects of the invention are described below, or
It will be apparent to those skilled in the art in light of the present disclosure.

For convenience, certain words used in the specification and appended claims are collected here. The term "onthelin" polypeptide refers to a family of polypeptides characterized at least in part by sharing sequences identical or somewhat homologous to all or a portion of the onserin polypeptide set forth in SEQ ID NO: 2. . Onserin polypeptides can be cloned or purified from some of the many eukaryotes, especially vertebrates and especially mammals. In addition, other onserin polypeptides can be produced according to the present invention, which polypeptides are not usually naturally occurring, but rather are produced by artificial mutagenesis techniques. Upon examination, many features of the Onserin protein were observed. In particular, the onserin sequence may encode a secretory protein having a secretory signal sequence corresponding to residues 1-130 of SEQ ID NO: 2 (e.g., a peptide portion that causes at least a portion of the protein to be secreted extracellularly). be pointed out. A "transmembrane" domain refers to, for example, a sequence of amino acids that can retain an onserin polypeptide on the cell surface by crossing the cell membrane surface. An “glycosylated” onserin polypeptide is an onserin polypeptide having a covalent bond to a glycosyl group (eg, diversified with carbohydrates). For example, onserin proteins may be glycosylated at existing residues, mutated to eliminate carbohydrate adducts, or new glycosylation such as N- or O-linked glycosylation. Mutations can be made to provide a site.

As used herein, the term “nucleic acid” refers to deoxyribonucleic acid (DN
A) and polynucleotides such as ribonucleic acid (RNA). The word is
Equivalents include analogs of either RNA or DNA made from nucleotide analogs and, as applied to the described embodiments, include single-stranded (sense or antisense) and double-stranded polynucleotides. It will be understood that

As used herein, the term “gene” or “recombinant gene” comprises an open reading frame encoding an onserin polypeptide, including exons and (optionally) intron sequences. Refers to nucleic acids. "Recombinant gene" refers to a nucleic acid comprising an onserin polypeptide-encoding nucleic acid and an exon sequence encoding onserin, but optionally includes, for example, a chromosomal onserin gene or an intron sequence from an unrelated chromosomal gene. You may. A typical recombinant gene encoding the present Onserin polypeptide is shown in the appended sequence listing. The term "intron" refers to a DNA sequence present in its onserin gene that is not translated into protein and is commonly found between exons. As used herein, the term "transfection" refers to the introduction of a nucleic acid, eg, an expression vector, into a recipient cell by nucleic acid-mediated gene transfer. As used herein, “transformation” refers to the process by which the incorporation of foreign DNA or RNA into a cell results in a change in the cell's genotype, and the transformation of the transformed cell into an onserin polypeptide. The process of developing the
Alternatively, it refers to a process of stopping the expression of the natural form of the Onserin protein when antisense is expressed from the transferred gene.

As used herein, the term “specifically hybridizes” refers to a nucleic acid probe / primer of the invention wherein the nucleic acid probe / primer is at least 15 nucleotides of an onserin gene, such as the onserin sequence shown in SEQ ID NO: 1. A conserved nucleotide, or its complementary sequence, or the ability to hybridize to a naturally occurring variant thereof, as defined herein, a cell encoding a protein other than an onserin protein Nucleic acids (eg, mRNA or genomic DNA)
) With 15% or less, preferably 10% or less, and more preferably 5% or less background hybridization.

As described in detail below, an “onserin therapeutic” is a reagent that can be an agonist (mimic or potentiator) or antagonist of the activity of a native onserin protein. Whether inhibiting or enhancing with respect to modulating the activity of an Onserin protein, the Onserin therapeutic agent is suitably isolated Onserin polypeptide, gene therapy construct, antisense nucleic acid, peptide-like, or herein. It can be a reagent identified in the drug assay provided therein. For a method for treating an animal, an "effective amount" of an onserin therapeutic, when applied as part of a desired dosage regimen, modulates cell growth, differentiation or survival, e.g., modulates neural differentiation. Refers to the amount of an agonist or antagonist in a formulation that provides

As used herein, “phenotype” refers to the overall physical, biological and physiological organization of a cell, for example, having several properties or groups of properties. With respect to the biological activity of an Onserin protein, the terms "induction" or "induce" generally refer to a process or action that specifically effects a cell phenotype. Such an effect may be a phenotypic change, such as a form that causes differentiation to another cell phenotype, or that maintains the cell in a particular cell, such as preventing dedifferentiation or promoting cell survival. It can be. A "patient" or "subject" to be treated means either a human or non-human animal.

The term “vector” according to the present invention refers to a nucleic acid capable of transporting another nucleic acid linked thereto. One preferred type of vector is an episome, ie, a nucleic acid capable of extrachromosomal replication. Preferred vectors are those that allow the autonomous replication and / or expression of the nucleic acid linked thereto. A vector capable of directing the expression of a gene operably linked thereto is referred to herein as an "expression vector". Generally, expression vectors useful in recombinant DNA methods are in the form of "plasmids", commonly referred to as circular double-stranded DNA loops, which do not bind to chromosomes in vector form. As used herein, "plasmid" and "vector"
The plasmid is the most commonly used form of vector. However, the invention also encompasses other forms of expression vectors known in the art that have equivalent functions.

A “transcription regulatory sequence” in the context of the present invention is generally a DNA sequence such as an initiation signal, an enhancer, and a promoter, which refers to a DNA sequence that induces or controls the transcription of an operably linked protein coding sequence. Is a term. In a preferred embodiment, transcription of the recombinant onserin gene is under the control of a promoter sequence (or other transcription regulatory sequence) that controls expression of the recombinant gene in the cell type in which expression is to take place. It will be appreciated that the recombinant gene may be under the control of the same or a different transcriptional regulatory sequence as the sequence controlling transcription of the native onserin gene.

As used herein, the term “tissue-specific promoter” regulates the expression of a DNA sequence that acts as a promoter, ie, a selected DNA sequence operably linked to a promoter, such as a cell derived from a nerve or liver. Selective DN in cells of specific tissues
A sequence that expresses the A sequence is meant. The term refers to the selection DNA in one tissue
So-called "leaky" promoters, which mainly regulate the expression of, and can cause at least low levels of expression in other tissues.

As used herein, the term “transgenic animal” is preferably a non-human mammal, bird or amphibian, in which one or more cells are prepared using methods well known in the art (eg, gene transfer). Method) refers to an animal containing a heterologous nucleic acid artificially introduced by the above method. The nucleic acid is introduced directly or indirectly into the cell by introduction into the precursor of the cell by genetic techniques, for example by microinjection or infection with a recombinant virus. The term genetic approach does not encompass classical crosses or in vitro reproduction, but refers to the introduction of a recombinant DNA molecule. This molecule may be integrated into the chromosome or may be extra-chromosomally replicating DNA. In a typical transgenic animal, the transgene causes cells to express a recombinant form of the onserin protein, eg, in an agonist or antagonist form. However, transgenic animals in which the recombinant onserin gene is silent, such as the FLP or CRE recombinase-deficient constructs described below, are also contemplated. In addition, “transgenic animal” also includes a recombinant animal in which one or more of the onserin genes has been disrupted artificially (including both recombinant methods and antisense methods).

“Non-human animals” of the present invention include vertebrates such as rodents, non-human primates, livestock, birds, amphibians, reptiles, and the like. As used herein, the term "chimeric animal" refers to an animal in which a recombinant gene is present or in which some, but not all cells, express a recombinant product. The term "tissue-specific chimeric animal" refers to an animal in which the onserin gene is present and / or expressed or disrupted in a particular tissue, but not in another tissue.

As used herein, the term “transgene” refers to a nucleic acid sequence that is partially or wholly heterologous, ie, foreign to the introduced animal or cell, or to the introduced animal or cell. Nucleic acid sequences that are homologous or endogenous to the cell, such that they alter the genome of the cell in which they are inserted (eg, are inserted at a different location from the locus of the native gene, or are Are designed to be inserted into the animal's genome). The transgene may include one or more transcription regulatory sequences required for optimal expression of the selected nucleic acid, as well as other nucleic acids, such as introns.

As is well known, the gene for a particular polypeptide may be present as single or multiple copies in the genome of the individual. Such a plurality of genes may be the same, or may have certain modifications (nucleotide substitutions, additions or deletions), but still encode a polypeptide having substantially the same activity. It is. Thus, the term "DNA encoding onserin polypeptide"
"Sequence" refers to one or more genes in a particular individual. Moreover, some differences in nucleotide sequence may be present between individuals of the same species, which are referred to as alleles. Such allelic differences may or may not result in amino acid sequence differences in the encoded polypeptides, but still encode proteins having the same biological activity.

“Homology” and “identity” refer to the similarity between polypeptide sequences, and identity is a more strict comparison. Homology and identity can be determined by comparing specific positions of each sequence that can be aligned for comparison. If the comparison sequence position is occupied by the same amino acid residue, then the polypeptides are said to be identical at that position. If an equivalent site is occupied by the same amino acid (eg, the same amino acid) or is occupied by a similar amino acid (eg, steric and / or electrical properties are similar), then the molecule is homologous at that position. You can say that. The percentage of homology or identity between sequences is a function of the number of matching or homologous positions between the sequences. An "irrelevant" or "heterologous" sequence refers to one having less than 40% identity, preferably less than 25% identity to the Onserin sequence of the invention.

The term “ortholog” refers to a gene or protein that is a homologue by speciation, for example, those that are highly related and those that are considered to have a common lineage based on structure and function. Orthologous proteins are proteins that exert the same recognizable activity in different species. The term “paralog” refers to a gene or protein that is a homologue due to gene duplication, for example, a duplicative variant of a gene in the genome (see also Fritch, WM (1970) Syst. Zool. 19: 99-113).

“Cells”, “host cells” or “recombinant host cells” are used interchangeably herein. It is understood that such terms refer not only to the cell of interest, but also to the progeny or potential progeny of such cell. In practice, such progeny may not be identical to the parental cell, as certain modifications may occur in successive generations due to mutations or the effects of the environment, but are within the scope of the terms herein.

A “chimeric protein” or “fusion protein” defines a first amino acid sequence encoding an onserin polypeptide and a domain that is heterologous and substantially non-homologous to any domain of onserin. And a fusion with the second amino acid sequence. The chimeric protein may have a foreign domain found in an organism that expresses the first protein (which may be in a different protein), or may have a "structure" of a protein structure expressed by a different type of organism. It may be a fusion product such as "between species" and "between genes". Generally, the fusion protein can be represented by the general formula X-onserin-Y,
Onserin is a part of a fusion protein derived from an onserin protein, and X and Y are independently absent or an amino acid sequence unrelated to the onserin sequence in an organism.

As used herein, the term “reporter gene construct” is a nucleic acid comprising an “operable linked reporter gene” of a transcription regulatory sequence. Transcription of the reporter gene is controlled by these sequences. The activity of at least one or more of these regulatory sequences is directly or indirectly regulated by a signal transduction pathway that includes phospholipase, for example, directly or indirectly by a second messenger caused by the phospholipase activity. Has been adjusted. Transcription regulatory sequences are recognized by promoters and enhancer sequences that alter promoter activity, or regulatory sequences that alter the activity or efficiency of RNA polymerase that recognizes the promoter, or effector molecules specifically induced by activation of phospholipase. Other regulatory regions such as regulatory sequences may be included. For example, modulation of promoter activity may be achieved by altering the binding of RNA polymerase to the promoter region or, alternatively, by interfering with the initiation of mRNA transcription or elongation. Such sequences are collectively referred to herein as transcriptional regulatory elements or sequences. In addition, the construct may include a nucleotide sequence that alters the stability or translation rate of the resulting mRNA in response to a second messenger, thereby altering the amount of the reporter gene product.

The term “isolated” as used herein also refers to nucleic acids, such as DNA or RNA, and also refers to molecules that have been separated from DNA or RNA, respectively, which molecules are present in the natural source of the macromolecule. Exists. For example, an isolated nucleic acid encoding an Onserin polypeptide is preferably a nucleic acid sequence of less than 10 kilobases (kb) that is naturally adjacent to the Onserin gene in genomic DNA, more preferably
It contains less than 5 kb of such contiguous sequences, most preferably less than 1.5 kb of such contiguous sequences. As used herein, the term isolated also refers to nucleic acids or peptides that are substantially free of cellular material or media (if produced recombinantly) or chemical precursors or other chemicals (if chemically synthesized). Say. Moreover, "isolated nucleic acid" includes nucleic acid fragments that are not naturally occurring fragments, as well as nucleic acid fragments that are not found in the natural state.

As described below, one aspect of the present invention relates to an isolated nucleic acid comprising a nucleotide sequence encoding an Onserin polypeptide, and / or equivalents of such a nucleic acid. The term "nucleic acid" as used herein includes fragments as equivalents. The term "equivalent" includes a nucleotide sequence that encodes a functionally equivalent onserin polypeptide or a peptide that has the activity of an onserin protein as described herein. Equivalent nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants, and thus are set forth in SEQ ID NO: 1 due to the degeneracy of the genetic code. Includes sequences that differ from the nucleotide sequence of the Onserin coding sequence. The equivalent is that under stringent conditions (ie, in the presence of about 1 M salt, the DN
A) The nucleotide sequence hybridizes with the nucleotide sequence shown in SEQ ID NO: 1 at a melting temperature (Tm) of the A duplex of about 20 to 27 ° C.). In one embodiment, the equivalent further includes a nucleic acid sequence derived from or evolutionarily related to the nucleotide sequence set forth in SEQ ID NO: 1.

Moreover, in some circumstances, either agonists (eg, mimic or promote the biological activity of wild-type onserin protein) or antagonists (eg, inhibit the biological activity of wild-type onserin protein) It is generally understood that a homolog of the onserin polypeptide with limited capacity may be provided as to enhance or inhibit only a portion of the biological activity of the naturally occurring form of the protein. Would. Thus, specific biological effects can be induced by treatment with homologs having limited functions. For example, a truncated form of the onserin protein, eg, a soluble fragment of the extracellular domain, may be provided to competitively inhibit ligand binding to the wild-type onserin protein.

A homologue of the onserin protein of the present invention may be obtained by a mutation method such as point mutation or truncation. For example, the mutation may be one that results in a homolog that retains substantially the same biological activity or a portion of that biological activity as the original Onserin polypeptide. Alternatively, for example, by competitively binding to an onserin ligand and competing with wild-type onserin, or by binding to another onserin protein or adhesion protein (such as cadherin) to form a non-responsive complex. An antagonistic form of the protein that can inhibit the function of the native form of the protein may be obtained. Thus, the onserin proteins and homologs thereof provided by the present invention may positively or negatively regulate cell growth, death and / or differentiation.

In general, a polypeptide of the present invention having the activity of an onserin protein (eg, a biological activity) corresponds to all or a part of the amino acid sequence of an onserin protein shown in SEQ ID NO: 2 ( For example, it is defined as a polypeptide comprising an amino acid sequence (identical or homologous) and agonizing or antagonizing all or a portion of the biological activity of a naturally occurring Onserin protein. Examples of such biological activities include the ability to bind calcium, especially Ca 2+, the ability to bind catenin (α-, β- or γ-catenin) or other proteins containing an “armadillo repeat region”, the desmosome protein Or the ability to bind to receptor-type protein tyrosine phosphatase. The ability of certain embodiments of the Onserin polypeptides of the invention to enhance the biological activity of cells and tissues derived from mesoderm-derived tissues, such as vertebral mesoderm-derived tissues, To be characterized with respect to the ability to promote the differentiation and / or maintenance of tissue of germ layer origin (such as from neural tube, neurite or head mesenchyme), or endoderm-derived tissue (such as gastrally-derived tissue) it can.

The biological activity of the onserin proteins of the invention is described herein or will be reasonably apparent to one of skill in the art. According to the present invention, when the polypeptide is a specific agonist or antagonist of the natural form of the Onserin protein, the polypeptide has biological activity.

[0059] Preferred nucleic acids have at least 60%, 70% or 80% homology to the amino acid sequence of a naturally occurring onserin protein, eg, the amino acid sequence set forth in SEQ ID NO: 2, more preferably at least 85%, most preferably Preferably, it encodes an Onserin polypeptide comprising an amino acid sequence having at least 95% homology. Nucleic acids encoding polypeptides having at least about 98-99% homology to the amino acid sequence shown in SEQ ID NO: 2 are of course within the scope of the present invention, as are the same amino acid sequences as the onserin sequences described in the Sequence Listing. Is within. In one embodiment, the nucleic acid is a cDNA encoding a polypeptide having at least one of the activities of the onserin polypeptide of the invention.

In certain preferred embodiments, the present invention provides a method wherein the range is from 86 kd to 106 kd;
More preferably, it relates to a purified or recombinant onserin polypeptide having a peptide chain having a molecular weight of between 92 kd and 100 kd (for the full length onserin protein), most preferably about 96.5 kd. Certain post-translational modifications, such as glycosylation, phosphorylation, etc., may increase the apparent molecular weight of the onserin protein relative to the unmodified polypeptide, and may result in apparent apparent molecular weight due to cleavage of a particular sequence, such as a prosequence. May decrease. Other preferred onserin polypeptides are mature onserin polypeptides lacking a signal sequence peptide (eg, residues 131-889 of SEQ ID NO: 2), a mature extracellular fragment of the protein (soluble) (eg, SEQ ID NO: 2) Nos. 131 to 708). In a preferred embodiment, the nucleic acid encodes an Onserin polypeptide comprising a Callisum binding domain. "About molecular weight" means in the range of about ± 5 kd.

Another aspect of the invention provides a nucleic acid that hybridizes under high or low stringency conditions to the nucleic acid set forth by SEQ ID NO: 1.
Appropriate stringency conditions to promote DNA hybridization, for example, washing with 6.0 × sodium chloride / sodium citrate (SSC) at about 45 ° C. followed by 2.0 × SSC at 50 ° C. are well known to those skilled in the art. Or Cu
rrent Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6
Found in .3.1-6.3.6. For example, when the salt concentration in the washing step is about 2.0 × SSC, 5
One can choose from a low stringency of 0 ° C. to a high stringency of about 0.2 × SSC, 50 ° C. In addition, the temperature of the washing step can be increased from low stringency conditions, such as room temperature, to high stringency conditions, such as about 65 ° C.

[0062] Nucleic acids that differ from the nucleotide sequence shown in SEQ ID NO: 1 due to the degeneracy of the genetic code are also within the scope of the invention. Such nucleic acids are functionally equivalent peptides (ie,
(A peptide having the biological activity of an onserin polypeptide), but the sequence differs from the sequence in the sequence listing due to the degeneracy of the genetic code. For example, many amino acids are determined by one or more triplets. Codons that specify the same amino acid or analog (eg, CAU and CAC each encode histidine) do not affect the amino acid sequence of the Onserin polypeptide “silent”
Mutations may be caused. However, DNA sequence polymorphisms that cause changes in the amino acid sequence of the onserin polypeptides of the invention may exist, for example, in humans. These changes in one or more nucleotides (up to about 3-5% of the nucleotides) of a nucleic acid encoding a polypeptide having the activity of an onserin polypeptide can be caused by spontaneous allelic variations between individual species. Those skilled in the art will appreciate that

As used herein, the term “onserin gene fragment” refers to a nucleic acid having fewer nucleotides than the nucleotide sequence encoding the entire mature form of the onserin protein, and preferably has the same biological activity as the full-length protein. Encodes the retained polypeptide. Fragment sizes contemplated by the present invention include, for example, those having a length of 5, 10, 25, 50, 75, 100 or 200 amino acids. In a preferred embodiment of the truncated receptor, the polypeptide comprises all or a sufficient portion of the intracellular or extracellular domain to allow the polypeptide to interact with a ligand, ion or other cell surface or intracellular protein. enable.

As shown in the examples below, nucleic acids encoding onserin proteins may be obtained from mRNA present in cells of metazoans. It is also possible to obtain nucleic acids encoding the onserin polypeptides of the invention from genomic DNA of both adults and embryos. For example, a gene encoding an onserin protein may be cloned from a cDNA or genomic library according to the protocols described herein as well as protocols well known to those skilled in the art. If desired, cDNA encoding the onserin protein may be obtained by isolating total mRNA from cells such as mammalian cells (eg, human cells). Using any of a number of known methods, double-stranded cDNA can be prepared from total mRNA and then inserted into a suitable plasmid or bacteriophage vector. The gene encoding the onserin protein can also be cloned using the polymerase chain reaction established according to the nucleotide sequence information provided by the present invention. The nucleic acid of the present invention is DNA
Or it may be RNA. A preferred nucleic acid is a cDNA comprising the nucleotide sequence shown in SEQ ID NO: 1.

[0065] Another aspect of the invention relates to the use of the isolated nucleic acids in "antisense" therapy. As used herein, the term "antisense" therapy refers to the administration of an oligonucleotide probe or a derivative thereof that specifically hybridizes (binds) to cellular mRNA and / or genomic DNA encoding the onserin protein of the present invention under cellular conditions. Or, it refers to inhibiting the expression of the protein by producing it in situ, for example, by inhibiting transcription and / or translation. Binding may be by conventional base pair complementation or by binding to a DNA duplex (specific interaction in the large groove of the duplex helix). In general, "antisense" therapy refers to a range of methods commonly used in the art,
Includes therapies that rely on specific binding to an oligonucleotide sequence.

The antisense constructs of the present invention may, for example, be transcribed in a cell into an R sequence complementary to at least one unique portion of a cellular mRNA encoding an onserin protein.
It may be derivatized to an expression plasmid that produces NA. Alternatively, an antisense construct is an ex vivo generated oligonucleotide probe that, upon introduction into a cell, causes expression inhibition by hybridizing to the mRNA and / or genomic sequence of the onserin gene. Preferably, such oligonucleotide probes are resistant to endogenous nucleases, such as exonucleases and / or endobucleases, and are therefore stable in vivo. Typical nucleic acid molecules used as antisense oligonucleotides are DNA phosphoramidites, phosphothioates and methylphosphonate analogs (see also US Pat. Nos. 5,176,996, 5,264,564 and 5,256,775) or peptide nucleic acids (PNAs). In addition, a general approach to constructing oligomers useful for antisense therapy is described by Van der Krol et al. (1
988) Biotechniques 6: 958-976 and Stein et al. (1988) Cancer Res 48: 26
It is outlined in 59-2668.

Thus, the modified oligomers of the present invention are useful for therapy, diagnosis and research.
For therapeutic use, the oligomers will generally be used in a manner suitable for antisense therapy. For such therapy, the oligomers of the invention can be formulated for various routes of administration, including systemic and local administration. In general, methods and formulations are described in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton,
Found in PA. Injections are preferred for systemic administration and include intramuscular, intravenous, intraperitoneal and subcutaneous injections. For injection, the oligomers of the invention are formulated as solutions, preferably as solutions in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the oligomer may be formulated as a solid formulation and redissolved or suspended immediately before use. Lyophilized forms are also included.

[0068] Systemic administration may be by transmucosal or transdermal means, or the compound may 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. Further, a surfactant may be used to facilitate penetration. Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the oligomer is in the form of a conventional oral dosage form, for example, a capsule, tablet, tonic. For topical administration, the oligomers of the invention are formulated as ointments, salves, gels, or creams as are well known in the art.

In addition to use in therapy, the oligomers of the invention can be used as diagnostic reagents to
The presence or absence of a target DNA or RNA that specifically binds may be detected. Such a diagnostic test is described in further detail below.

Similarly, antisense techniques (eg, microinjection of antisense molecules or transfection with a plasmid whose transcript is antisense to onserin mRNA or gene sequences) can be used to develop and normalize adult tissues. Role of onserin in various cell functions. Such methods are used in cultured cells, but may also be used to create transgenic animals (described above).

The present invention also provides an expression vector comprising a nucleic acid encoding an onserin polypeptide operably linked to at least one transcription regulatory sequence. Operably linked means that the nucleotide sequence is linked to the regulatory sequence in such a way as to allow expression of the nucleotide sequence. Regulatory sequences are recognized in the art and are selected to direct expression of the onserin protein of the invention. Thus, the term "transcription 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 (1
99). For example, a wide variety of expression control sequences and sequences that control the expression of DNA sequences when operably linked may be used in these vectors to express the DNA sequence encoding onthelin. Such useful expression control sequences include, for example, the viral LTR, such as the LTR of Moloney murine leukemia virus, the early and late promoters of SV40, the immediate early promoter of adenovirus or cytomegalovirus, the lac, trp, TAC or TRC.
A T7 promoter whose expression is directed by T7 RNA polymerase, a major operator and promoter region of lambda phage, a control region of fd coat protein, a promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, and an acid phosphatase. Promoters such as Pho5, the promoter of yeast α-mating factor, the baculovirus-based divergent promoter and other sequences known to control the expression of prokaryotic or eukaryotic cells or their viruses, and combinations thereof. Include. It should be understood that the design of the expression vector will depend on factors such as the choice of the host cell to be transformed and / or the type of protein for which expression is desired.

In addition, the ability to control the copy number and expression of other vectors encoded by the vector, such as antibiotic markers, as well as the copy number of the vector, should be considered.
In one embodiment, the expression vector comprises a recombinant gene encoding a polypeptide having agonist activity on the onserin polypeptide of the invention, or a recombinant gene encoding a polypeptide in the form of an antagonist to wild-type onserin protein. . A typical onserin polypeptide of the invention is a soluble truncated form of the protein that has an ion binding domain, eg, retains the ability to bind divalent calcium. Cells are transfected with such an expression vector, whereby a polypeptide, including the fusion protein, encoded by the nucleic acids described herein can be obtained.

In addition, the gene constructs of the present invention can be used as part of a gene therapy protocol, for example, to deliver an agonist or antagonist form of an onserin protein or a nucleic acid encoding the antisense molecule. Thus, another aspect of the invention is the expression vector for transfection in vivo or in vitro, as well as the expression of the onserin protein or antisense molecule in certain cell types, resulting in misexpression of the native form of the protein ( An expression vector for reconstituting or abolishing all or part of the biological function of the transcript induced by onserin in a tissue) or a tissue that has been maintained or differentiated or neoplastic or in excess The present invention relates to an expression vector for delivering a certain form of a protein that inhibits formation and proliferation.

[0074] Expression constructs and antisense constructs of the onserin polypeptides of the invention are
It may be administered in a biologically effective carrier, for example, as a formulation or composition capable of effectively delivering the recombinant gene to cells in vivo. The approach involves the insertion of the gene of the invention into a viral vector or a recombinant bacterial plasmid or a eukaryotic cell plasmid, wherein the viral vector is a recombinant retrovirus,
Includes adenovirus, adeno-associated virus, and herpes simplex virus-1. Viral vectors transfect cells directly. Plasmid D
NA can be delivered, for example, with the aid of cationic liposomes (lipofectin) or derivatized (eg, antibody conjugates).
, Polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers as well as direct injection of gene constructs or Ca in vivo
There is PO ₄ precipitation. Since transduction of appropriate target cells is a critical first step in gene therapy, the choice of a particular gene delivery system will depend on factors such as the target phenotype and route of administration (eg, local or systemic). Would. Furthermore, certain genetic constructs provided for the in vivo transduction of onserin expression are also useful for the in vitro transduction of cells, such as for use in ex vivo tissue culture systems described below.

[0075] A preferred approach for introducing nucleic acids into cells in vivo is by using a viral vector containing the nucleic acid, eg, a cDNA encoding a particular particular onserin polypeptide of interest. Infection of cells with a viral vector has the advantage that most of the target cells will receive the nucleic acid. Furthermore, for example, molecules encoded in the viral vector by the cDNA contained in the viral vector are effectively expressed in cells that have taken up the nucleic acid of the viral vector. Retroviral, adenoviral and adeno-associated viral vectors are typical examples of recombinant gene delivery systems for introducing foreign genes in vivo, particularly into humans. These vectors provide efficient delivery of the gene to the cell, and the introduced nucleic acid is stably integrated into the chromosomal DNA of the host.

In addition to the above-described virus introduction method, the onserin polypeptide of the present invention can also be expressed in animal tissues using a non-viral method. Most non-viral gene transfer methods rely on the usual mechanisms used by mammalian cells for macromolecular uptake and intracellular transport. In a preferred embodiment, a non-viral gene delivery system of the present invention relies on an intracellular pathway for uptake of an onserin polypeptide gene of the present invention by a target cell. Typical gene delivery systems of this type include liposome-based systems, polylysine conjugates, and artificial virus envelopes.

In a clinical setting, a gene delivery system for a therapeutic onserin gene can be introduced into a patient animal by a number of methods common in the art. For example, a pharmaceutical composition for gene delivery can be introduced systemically, for example, by intravenous injection, and the gene delivery carrier, cell or tissue type expression by a transcriptional regulatory sequence that controls the expression of a receptor gene, or a combination thereof. The preferential transfection of the protein in the target cells occurs preferentially due to the specificity of the transfection provided by E. In other embodiments, the initial delivery of the recombinant gene is more restrictive with respect to introduction into the animal, and is entirely local. For example, by catheter (see US Pat. No. 5,328,470) or by stereotactic injection (see, eg, Chen et al.
(1994) PNAS 91: 3054-3057) Gene delivery carriers can be introduced. The onserin gene can be delivered in a gene therapy construct by electroporation using, for example, the method described by Dev et al. (1994) Cancer Treat Rev 20: 105-115.

The pharmaceutical composition of the gene therapy construct may include the gene delivery system in an acceptable diluent, or may include a sustained release matrix in which the gene delivery carrier is embedded. Alternatively, if a complete gene delivery system (eg, a retroviral vector) can be produced intact from recombinant cells, the pharmaceutical composition may contain one or more cells that produce the gene delivery system. .

In yet another embodiment, the invention provides a “gene activation” construct, which alters the transcriptional regulatory sequence of the endogenous onserin gene by homologous recombination of genomic DNA. For example, a gene-activating construct can drive the endogenous promoter of an onserin gene to a heterologous promoter (eg, a promoter that causes constitutive expression of the onserin gene, or gene expression under conditions that are different from the normal expression pattern of onserin). Promoter). A variety of different formats for gene activation constructs are available. For example, PCT publications WO93 / 09222, WO95 of Transkaryotic Therapies, Inc.
/ 31560, WO96 / 29411, WO95 / 31560 and WO94 /
See 12650.

In a preferred embodiment, the nucleotide sequence used as the gene activation construct comprises: (1) DNA from a specific portion of the endogenous onserin gene (exon sequence, intron sequence, promoter sequence, etc.) which directs recombination; (2)
The gene activation construct may include a heterologous transcriptional regulatory sequence operably linked to the sequence encoding the onserin gene of the genome by recombination. For use in culturing onserin-producing cells, the construct may further comprise a reporter gene that detects the presence of the knockout construct in the cell.

The genomic DN of the cell at a location such that the gene activation construct is inserted into the cell and provides a heterologous regulatory sequence operably linked to the native onserin gene.
A. Such insertion occurs by homologous recombination, i.e., the recombination region of the activating construct homologous to the endogenous onserin gene sequence hybridizes to the genomic DNA and recombination with the genomic sequence occurs, and the construct is placed at the corresponding position in the genomic DNA. It is captured.

The term “recombination region” or “target sequence” refers to a segment of a gene activation construct having a sequence that is identical or substantially complementary to a genomic gene sequence (eg, the 5 ′ flanking sequence of a genomic gene) (ie, Part) that can facilitate homologous recombination between the genomic sequence and the target transgene construct.

As used herein, the term “substitution region” refers to the portion of the activation construct that integrates into the endogenous chromosomal location following homologous recombination between the recombination region and the genomic sequence.

A heterologous regulatory sequence, such as that provided in a replacement region, may include one or more various elements, locus control regions, transcription factor binding sites, or combinations thereof. Promoters / enhancers that can be used to control target gene expression in vivo include cytomegalovirus (CMV)
) Promoter / Enhancer (Karasuyama et al., 1989, J. Exp. Med., 169)
: 13), human β-actin promoter (Gunning et al. (1987) PNAS 84: 4831-
4835), a promoter (MMTV LTR) inducible by the glucocorticoid present in the mouse mammary tumor virus long end repeat (Klessig et al. (1984) Mol.
Cell Biol. 4: 1354-1362), Moloney murine leukemia virus long end repeat sequence (MuLV LTR) (Weiss et al. (1985) RNA Tumor Viruses, Cold Spring).
Harbor Laboratory, Cold Spring Harbor, New York), SV40 early or late region promoter (Bernoist et al. (1981) Nature 290: 304-310; Templeton
et al. (1984) Mol. Cell Biol., 4: 817; and Sprague et al. (1983) J. Viro
l., 45: 773), the promoter in the 3 'long end 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) P
NAS 82: 3567-71), as well as the herpes simplex virus LAT promoter (Wolfe
et al. (1992) Nature Genetics, 1: 379-384).

In still other embodiments, the replacement region is native, for example, to activate expression or to dissipate positive control elements, eg, to inhibit expression of a target gene. Are simply deletions of the negative transcription control elements of the gene.

Another aspect of the present invention relates to recombinant forms of the Onserin protein. In addition to the native onserin protein, the recombinant polypeptides of the present invention have at least 60% or 70%, more preferably at least 80%, most preferably at least 85% homology to the amino acid sequence shown in SEQ ID NO: 2. Or a fragment thereof having biological activity. A poly having the activity of an onserin protein (ie, either an agonist or an antagonist) and having at least 90%, more preferably at least 95%, and most preferably at least 98-99% homology to SEQ ID NO: 2. Peptides are also within the scope of the present invention. Such polypeptides as described above include various truncated forms of the protein.

Further, the present invention provides a recombinant form of the invention, which is encoded by a gene from a mammal (eg, a human), amphibian or reptile and has an amino acid sequence evolutionarily related to the onserin protein shown in SEQ ID NO: 2. Related to onserin polypeptides. Preferably, such a recombinant onserin polypeptide is capable of functioning as either an agonist or antagonist for at least one biological activity of the wild-type (authentic) onserin protein set forth in the Sequence Listing. The term "related in evolution" with respect to the amino acid sequence of an Onserin protein refers to both polypeptides having a naturally occurring amino acid sequence as well as variants resulting from mutations of the Onserin polypeptide, for example, induced by recombinant mutagenesis.

The present invention also provides a method for producing the onserin polypeptide of the present invention. For example, a host cell transfected with a nucleic acid vector that directs the expression of a nucleotide sequence encoding a polypeptide of interest can be cultured under suitable conditions to express the peptide. If the recombinant protein does not involve a secretory signal peptide, as in the case of a GST fusion protein, the cells are collected, lysed, and the protein is isolated. Cell culture requires host cells, media and other bioproducts. Suitable media for cell culture are well known in the art. Recombinant methods using methods known in the art also include ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and protein purification, including immunoaffinity purification using antibodies specific for such peptides. Serine polypeptides can be isolated from cell culture media, host cells, or both. In a preferred embodiment, the recombinant onserin peptide is a fusion protein containing a domain that facilitates purification, such as a GST fusion protein or a poly (His) fusion protein.

The present invention also relates to transfected host cells that express a recombinant form of the Onserin polypeptide of the present invention. A host cell can be a eukaryotic or prokaryotic cell. Thus, a recombinant form of the onserin polypeptide can be obtained by microbial or eukaryotic processes using the nucleotide sequence encoding the entire or selected portion of the full-length protein, obtained by cloning the onserin protein.
The polynucleotide sequence is ligated into a genetic construct, such as an expression vector, and transformed or transfected into a host cell that is eukaryotic (yeast, bird, insect or mammal) or prokaryotic (bacterial), and other The protein is produced using standard methods used to produce known proteins, such as cadherin, integrins and the like. Similar methods or modifications thereof may be used to produce recombinant onserin polypeptides according to the present invention by microbiological means or tissue culture methods.

[0090] Recombinant onserin genes can be produced by ligating a nucleic acid encoding an onserin polypeptide into a vector suitable for expression in prokaryotic or eukaryotic cells, or both. Expression vectors used to produce recombinant onserin polypeptides of the present invention include plasmids and other vectors. For example, vectors suitable for expressing the Onserin polypeptide include the following types of plasmids: plasmids derived from pBR322, plasmids derived from pEMBL, plasmids derived from pEX, plasmids derived from pBTac, and those from E. coli. A pUC-derived plasmid for expression in prokaryotic cells, such as.

There are many vectors for expressing recombinant proteins in yeast. For example, YEP24, YIP5, YEP51, YEP52 and YRP17 are useful in introducing genetic constructs into S. cerevisiae (eg, Broach et al. (1983) in Experimental Manipulation of Gene Expres).
sion, ed. M. Inouye Academic Press, p. 83, incorporated herein by reference). These vectors can replicate in E. coli due to the presence of pBR322 ori and in S. cerevisiae due to the replication determinants of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin can be used. In a typical embodiment, the Onserin polypeptide is recombinantly produced using an expression vector obtained by subcloning the coding sequence of the Onserin gene shown in SEQ ID NO: 1.

[0092] Preferred mammalian expression vectors contain both prokaryotic sequences to facilitate propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. . pcDNAI / amp, pcDNAI / ne
o, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr,
pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pH
Yg-derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors have been modified with sequences from bacterial plasmids such as pBR322 to facilitate replication and facilitate drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, bovine papilloma virus (BPV-1) or Epstein-Barr virus (pHEBo, pRE
Transient expression of proteins in eukaryotic cells may be performed using derivatives of viruses such as P-derived and p205). Various methods used for plasmid preparation and transformation of host cells are known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells and general recombinant methods, see Mole.
cular Cloning A Laboratory Manual, 2nd Ed., ed.by Sambrook, Fritsch and
Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17
reference.

In some instances, it may be desirable to express the recombinant onserin polypeptide by using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (pVL1392, pVL13
93 and pVL941), pAcUW-derived vectors (pAcUW1
And pBlueBac-derived vectors (β-gal containing pBlue
eBacIII).

When it is desired to express only a portion of the onserin protein lacking a portion of the N-terminus, ie, a truncated mutant lacking a signal peptide, the oligonucleotide fragment containing the desired sequence to be expressed may be used. Start codon (AT
G) may need to be added. It is known in the art that methionine at the N-terminal position can be enzymatically removed by using methionine aminopeptidase (MAP). MAP is for E. coli (Ben-Bassat et al
(1987) J. Bacteriol. 169: 751-757) and from Salmonella typhimurium, showing its in vitro activity against recombinant proteins (Miller et al. (1987) PNAS 84
: 2718-1722). Therefore, if desired, removal of the N-terminal methionine can be achieved in vivo by expressing onseline-derived polypeptides in a MAP-producing host (eg, E. coli or CM89 or S. cerevisiae), or using purified MAP in vitro. (For example, the above-mentioned method of Miller et al.).

[0095] Alternatively, the polypeptide coding sequence may be included as part of a fusion gene containing a nucleotide sequence encoding another polypeptide. This type of expression system is useful under conditions where it is desirable to obtain an immunogenic fragment of the onserin protein. For example, the VP6 capsid protein of rotavirus can be used as an immunogenic carrier protein for portions of the Onserin polypeptide (in monomeric form or in the form of virus particles). A nucleic acid sequence corresponding to a portion of the onserin protein of the invention (to which an antibody is generated) may be included in the fusion gene construct. The fusion gene construct includes a coding sequence for a late vaccinia virus structural protein that yields a set of recombinant viruses that express a fusion protein containing an onserin epitope as part of a virus particle. The ability to use recombinant hepatitis B virus particles in this role has been shown using immunogenic fusion proteins using hepatitis B surface antigen fusion proteins. Similarly, chimeric constructs encoding fusion proteins containing an onserin protein and a portion of a poliovirus capsid protein can be made to improve the immunogenicity of a set of polypeptide antigens (eg, EP Publication No. 0259149). No .; and Evans et al. (19
89) Nature 339: 385; Huang et al. (1988) J. Virol. 62: 3855; and Schlie
nger et al. (1992) J. Virol. 66: 2).

[0096] Immunogens can also be obtained using multiple antigenic peptide systems for peptide-based immunization. The desired portion of the Onserin polypeptide can be obtained directly by organic synthesis of the peptide on an oligomeric branched lysine core (see, for example, Posnett et al.
(1988) JBC 263: 1719 and Nardelli et al. (1992) J. Immunol. 148: 914). The antigenic determinant for the onserin protein can also be expressed and displayed by bacterial cells.

In addition to using the fusion protein to enhance immunogenicity, the fusion protein can facilitate expression of the protein, and therefore, the Onserin polypeptides of the invention, especially the truncated forms of Onserin It is widely understood that it can be used for protein expression. For example, an onserin polypeptide can be converted to glutathione S-transferase (GS
T fusion) protein. Such GST fusion proteins can facilitate the purification of onserin polypeptides, for example, by using a matrix derived from glutathione (eg, Current Protocols in Molecular).
Biology, eds. Ausubel et al. (NY: John Wiley & Sons, 1991)).

In another embodiment, the N-terminal poly- (His) /
A fusion gene encoding a purified leader sequence, such as an enterokinase cleavage site sequence, allows for purification of the expressed fusion protein by affinity chromatography using a Ni ²⁺ metal resin. The purified leader sequence can then be removed by treatment with enterokinase to obtain a purified protein (see, for example, Hochuli et al. (1987)
) J. Chromatography 411: 177; and Janknecht et al. PNAS 88: 8972).

[0099] Methods for making fusion genes are known to those of skill in the art. In essence, use blunt or staggered ends for ligation, obtain appropriate ends by restriction digestion, fill-in sticky ends as appropriate, alkaline to avoid unwanted ligation Various DNA fragments encoding different polypeptide sequences are ligated together by conventional methods, including phosphatase treatment and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional methods, including automatic DNA synthesizers. Alternatively, PCR amplification of the gene fragment can be performed using an anchor primer that creates a complementary overlap between two consecutive gene fragments (the two fragments are then annealed to yield a chimeric gene sequence) ( For example, Current Protocols in Molecular Biology, eds. Ausubel et
al. John Wiley & Sons: 1992).

The onserin polypeptide is chemically modified by forming a covalent bond or aggregate conjugate with other chemical moieties such as glycosyl groups, lipids, cholesterol, phosphate, acetyl groups, etc. to obtain the onserin derivative Is also good. By attaching a chemical moiety to the amino acid side chain of the protein or the N-terminus or C-terminus of the polypeptide,
A covalently linked Onserin protein may be obtained.

[0101] Where appropriate, multimeric onserin polypeptides are also provided. Soluble forms of the multimers of the onserin polypeptides of the invention may be obtained by methods known in the art. In one embodiment, the onserin multimer is chemically crosslinked using a method that forms a dimer or multimer of the soluble form of the onserin polypeptide. Another method for producing multimers of the soluble form of the Onserin polypeptide is by recombinant methods, for example, by introducing a hinge region. This linker increases the flexibility of the chimeric protein by reducing the steric hindrance between the two fragments, allowing the various onserin monomer subunits to freely and (optionally) simultaneously interact with the onserin ligand, It also allows for proper folding of each part. The linker may be of natural origin, e.g.
It may exist in a random coil between two domains. Alternatively, the linker may be synthetic. For example, the sequence (Gly ₄ Ser) ₃ may be used as a synthetic non-structural linker. This type of linker is described by Huston et.
al. (1988) PNAS 85: 4879; and U.S. Patent Nos. 5,091,513 and 5,258,498. Due to the low risk of immunogenicity, naturally occurring non-structural linkers of human origin are preferred.

Each multimer contains two or more monomers, each containing a soluble form of the Onserin polypeptide or a salt or functional derivative thereof. The upper limit of the number of monomers is not critical, and liposomes having many such monomers may be used. Preferably, such multimers are 2-5 monomers, more preferably 2
It has one or three monomers.

The present invention also relates to onserin polypeptides in isolated form, otherwise other cellular proteins which may ordinarily be bound to the onserin polypeptide, especially adhesion molecules and / or other derivatives. An Onserin polypeptide that is substantially free of a sex polypeptide is utilized. The term "substantially free of other cellular proteins (" contaminating proteins ")" or "substantially pure or purified formulation" refers to less than 20% (dry weight) of contaminating proteins, preferably less than 5%. Onserin polypeptide formulation containing contaminating proteins. Preferred is a functional form of the polypeptide of the invention, which has been purified for the first time by using the cloned genes described herein. "Purified" when referring to a peptide or DNA or RNA sequence means that the molecule is in the substantial absence of other biological macromolecules such as proteins. As used herein, the term "purified" preferably refers to at least 80% by dry weight, more preferably in the range of 95-99% by weight, most preferably at least 99.8% by weight of a biological polymer of the same type. (Water, buffers, and small molecules, especially molecules with a molecular weight of less than 5000 may be present). The term “pure” preferably refers to the above “
It has the same numerical limitations as "purification". “Isolated” and “purified” do not refer to the natural material in its native state as well as to the natural material separated into its components (eg, in an acrylamide gel), but to pure (eg, free of contaminating proteins, or It does not refer to denaturants and polymers, e.g., that do not contain chromatographic reagents such as acrylamide or agarose) or materials that are not available as a solution. In a preferred embodiment, the purified onserin formulation is free of contaminating proteins from the same animal in which onserin is normally produced, and is obtained by recombinant expression, for example, a mammalian onserin protein in yeast or bacterial cells. You may.

As described above for the recombinant polypeptide, the isolated onserin polypeptide includes all or part of the amino acid sequence corresponding to the onserin polypeptide shown in SEQ ID NO: 2, or a sequence homologous thereto. sell.

The isolated peptidyl portion of the onserin protein can also be obtained by screening for a recombinantly produced peptide from the corresponding fragment of the nucleic acid encoding such a peptide. In addition, the fragments are prepared using the conventional Merrifield solid phase f.
Chemical synthesis can also be performed using methods known in the art, such as -Moc or t-Boc chemistry. For example, preferably the Onserin polypeptide of the invention may be arbitrarily split into non-overlapping fragments of a desired length, or preferably split into overlapping fragments of a desired length. Fragments may be produced (by recombinant or chemical synthesis) and tested to identify those peptidyl fragments that may function as agonists or antagonists of the wild-type (eg, authentic) onserin polypeptide. For example, Roma
n et al. (1994) Eur J Biochem 222: 65-73 describes the use of competition assays to examine binding domains using short, overlapping synthetic peptides from large proteins.

The recombinant onserin polypeptide of the present invention is, for example, a protein that is resistant to proteolytic cleavage, obtained by modifying a cleavage recognition site of a protease or the like so as to prevent the release of an extracellular domain by an enzyme. Homologs of authentic proteins such as versions are also included.

Modifications to the structure of the Onserin polypeptides of the invention include, for example, to increase therapeutic or prophylactic efficacy, to enhance stability (eg, to increase lifespan ex vivo and to increase proteolysis resistance in vivo), Alternatively, it may be for post-translational modification. Such a modified peptide is considered a functional equivalent of an onserin polypeptide if it is designed to retain at least one activity of the native protein, or if it is designed to produce a specific antagonist. (However, they may be agonists or antagonists to the biological activity of the authentic protein). Such modified peptides can be obtained, for example, by amino acid substitution, deletion or addition.

For example, replacement of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar amino acids with structurally related amino acids (eg, isosteric and / or isoelectric mutations). ) Will not significantly affect the biological activity of the resulting molecule.
Conservative substitutions are those that take place within a family of amino acids to which the side chains are related. Genetically, the encoded amino acids fall into four families: (1
) Acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine,
Histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polarity
= Glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine; phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. Similarly, the amino acid repertoire can be classified as follows: (1) acidity = aspartic acid, glutamic acid; (2) basicity = chidine, arginine, histidine; (3) aliphatic = glycine, alanine, valine, leucine , Isoleucine, serine, threonine (serine and threonine are aliphatic-
(4) aromatic = phenylalanine, tyrosine, tryptophan; (5) amide = asparagine, glutamine; and (6) sulfur-containing
= Cysteine and methionine. (For example, Biochemistry, 2nd ed., Ed. By L
Stryer, WH Freeman and Co .: 1981). Similar to the wild-type protein, whether the charge in the amino acid sequence of the peptide results in a functional onserin homolog (eg, the resulting polypeptide is functional in mimicking or antagonizing the authentic form) The ability of a variant peptide to produce a response in a cell in a manner described above, or the ability of a variant peptide to competitively inhibit such a response, can be determined. Polypeptides in which one or more substitutions have occurred can be readily tested in the same manner.

The invention further contemplates a method of obtaining a set of combinatorial point and truncation mutants of the onserin proteins of the invention, wherein the method modulates signal transduction and / or ligand binding. It is particularly useful for identifying potential variant sequences that are functional in nature (eg, homologs). The purpose of screening such combinatorial libraries is, for example, to obtain new onserin homologs that can act as agonists or antagonists, or alternatively, new onserin homologs having new activities. By way of example, onserin homologs may be treated according to the invention to provide selective, constitutive activation of catenin-mediated gene transcription, or alternatively, a major negative of catenin-dependent signal transduction. It may be an inhibitor. For example, mutations cause an onserin homolog that interacts with other cell surface proteins and / or binds to extracellular ligands but cannot bind to or signal through intracellular regulatory proteins. Obtainable.

In one embodiment of this method, the amino acid sequences for a population of onserin homologs from different species or other related proteins are aligned, preferably with the greatest possible homology. Such a population of variants may include, for example, onserin homologs from one or more species. Amino acids appearing at each position of the juxtaposed sequence are selected to create a degenerate set of combinatorial sequences. In a preferred embodiment, if a heterogeneous library of Onserin variants is obtained by combinatorial mutation at the nucleic acid level, the library is encoded by a heterogeneous gene library. For example, a synthetic oligonucleotide mixture can be enzymatically linked to a gene sequence, a degenerate set of potential onserin sequences can be expressed as individual polypeptides, or alternatively, a large fusion protein containing a set of onserin sequences. It can be expressed as a set (eg, for phage display).

There are many ways to obtain such a library of potential onserin homologs from degenerate oligonucleotide sequences. Chemical synthesis of the degenerate gene sequence may be performed using a DNA synthesizer, and then the synthetic gene may be ligated into an appropriate expression vector.
The purpose of the degenerate set of genes is to obtain in a mixture all sequences encoding the desired set of potential onserin sequences. The synthesis of degenerate oligonucleotides is well known in the art (eg, Narang, SA (1983) Te
trahedron 39: 3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Clevel
and Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273289;
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 methods have been used for the direct development of other proteins (eg, Scott et al. (19)
90) Science 249: 386390; Roberts et al. (1992) PNAS 89: 24292433; Devlin e
t al. (1990) Science 249: 404406; Cwirla et al. (1990) PNAS 87: 63786382
And US Patent Nos. 5,223,409, 5,198,346 and 5,096,815).

Similarly, a library of fragments of the coding sequence is provided for one onselling clone to provide a heterogeneous population of onserin fragments for screening and subsequent selection of biologically active fragments. You may get it.
Various methods for obtaining such libraries, including chemical synthesis, are known in the art. In one embodiment, the library of fragments of the coding sequence is treated with a nuclease with (i) a double stranded PCR fragment of the onserin coding sequence under conditions where nicking occurs approximately once per molecule;
(Ii) denaturing the double-stranded DNA and (iii) DN from different nicked products.
A is regenerated to form a double-stranded DNA that may contain a sense / antisense pair, and (iv) a single-stranded portion is removed from the regenerated double-strand by treatment with S1 nuclease. The obtained library of fragments can be obtained by ligation into an expression vector. By this exemplary method, an expression library can be derived which encodes N-terminal, C-terminal and internal sequences of various sizes.

Various methods are known in the art for screening gene products of combinatorial libraries created by point mutations or truncations, and for screening cDNA libraries for gene products having particular properties. I have. In general, such methods are suitable for rapid screening of gene libraries obtained by combinatorial mutations of onserin homologs. The most widely used method for screening large gene libraries is to clone the gene library into a replicable expression vector,
Appropriate cells are transformed with the resulting vector library, and then the combinatorial gene is expressed under conditions that facilitate the isolation of the vector encoding the gene whose product was detected by detecting the desired activity. Including

The present invention also provides mimetics that can disrupt the biological activity of the onserin polypeptides of the present invention as inhibitors of protein-protein interactions, such as the interaction between onserin and other cell surface proteins. The present invention provides for the conversion of an onserin protein to obtain an agent, such as a peptide or non-peptide agent. Thus, such mutation methods as described above are also useful for mapping determinants of onserin proteins involved in protein-protein interactions. For example, peptidomimetic compounds that mimic residues that facilitate interaction can be obtained by using scanning mutations to map amino acid residues of the protein involved in binding to other proteins. Then
Such mimetics may be used to interfere with the normal function of the Onserin protein (or its ligand or ion). For example, a non-hydrolyzed peptide analog of such a residue may be substituted with a benzodiazepine (eg, Freidinger et al. In Peptides: Chemistr.
y and Biology, GR Marshall ed., ESCOM Publisher: Leiden, Netherlands,
Azepine) (see Huffman et al. In Peptides: Chemistry and Biol).
ogy, GR Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),
Substituted gamma-lactam ring (Garvey et al. In Peptides: Chemistry and Biology,
GR Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptide (Ewenson et al. (1986) J Med Chem 29: 295; and E
wenson et al. in Peptides: Structure and Function (Proceedings of the 9t
h American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), b
-Turn dipeptide core (Nagai et al. (1985) Tetrahedron Lett 26: 647; and Sato et al. (1986) J Chem Soc Perkin Trans 1: 1231), and b-amino alcohol (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 present invention relates to an antibody that specifically reacts with the onserin protein. For example, by using an immunogen derived from the Onserin protein,
Based on the sequence, anti-protein / anti-peptide antisera or monoclonal antibodies can be obtained by standard protocols (eg, Antibodies: A Laboratory Ma
by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, hamster or rabbit, can be immunized with an immunogenic form of the peptide (eg, an Onserin polypeptide or an antigenic fragment capable of eliciting an antibody response). Methods of conferring immunogenicity on a protein or peptide include conjugation to a carrier. Other methods are well known in the art. The immunogenic portion of the onserin protein can be administered in the presence of an adjuvant. The progress of immunization can be monitored by examining antibody titers in crystals or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess antibody levels. In a preferred embodiment, the subject antibody is immunospecific for an antigenic determinant of an onserin protein of an organism, such as a mammal. The antigenic determinant may be, for example, an antigenic determinant of a protein represented by SEQ ID NO: 2 or a closely related homolog (eg, having at least 70%, preferably at least 80%, more preferably at least 90% homology). It is. In a further preferred embodiment of the invention, for example, to obtain an immunoselective antibody to an individual onserin homolog, the anti-onserin polypeptide antibody is substantially at least, for example, at least 85%, 90
Does not cross-react with proteins having% or 95% homology (ie, does not specifically react). "Substantially non-reactive" means that the antibody has reduced binding affinity, which binding affinity is at least one order of magnitude, more preferably at least two orders, and even more, than the binding affinity of the antibody for the target onserin. Preferably, it is at least three orders of magnitude weaker.

After immunizing an animal with an antigenic formulation of an Onserin polypeptide, anti-Onserin antiserum can be obtained, and if necessary, polyclonal anti-Onserin antibodies can be isolated from the serum. To make monoclonal antibodies, antibody-producing cells (lymphocytes) are collected from the immunized animal and fused with immortalized cells, such as myeloma cells, by standard somatic cell fusion techniques to obtain hybridoma cells. it can. Such techniques are well-known in the art, for example, the hybridoma technique (Koheler and Milstein,
(1975) Nature, 256; originally developed by 495-497), human B cell hybridoma technology (Kozbar et al., (1983) Immunology Today, 4:72) and EBV-hybridoma technology to obtain human monoclonal antibodies. (Cole et al., (19
85) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-
96). Hybridoma cells can be immunochemically screened for the production of antibodies that specifically react with the onserin polypeptide of the invention, and monoclonal antibodies isolated from cultures containing such hybridoma cells.

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

Using both monoclonal and polyclonal antibodies (Abs) raised against authentic onserin polypeptides and antibody fragments such as Fab, F (ab) ₂ , Fv and scFv, the effects of the onserin protein can be measured. It can be blocked, and the role of these proteins, such as tissue differentiation, can be studied. Experiments of this nature can help to decipher the role of onserin proteins that may be involved in controlling proliferation versus differentiation, such as, for example, patterning and tissue formation.

[0118] Antibodies that specifically bind to an Onserin epitope can also be used in immunohistochemical staining of tissue samples to assess the number and pattern of expression of each of the Onserin polypeptides of the invention. Anti-onserin antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and assess onserin protein levels in tissue or biochemical fluid samples as part of a clinical test procedure.
For example, such measurements can be useful in predictive assessment of the onset or progression of a proliferative or differentiated disease. Similarly, the ability to monitor onserin protein levels in an individual will allow one to examine the effect of a given treatment regimen on an individual suffering from such a disease. The level of the Onserin polypeptide may be measured from a body fluid in a sample, such as, for example, serum, cerebrospinal fluid or amniotic fluid, or can be measured, for example, in a tissue created by biopsy. Diagnostic assays using anti-onserin antibodies can include, for example, immunoassays designed to aid in the early diagnosis of disease, particularly those apparent at birth.
Diagnostic assays using anti-onserin polypeptide antibodies can also include immunoassays designed to aid in early diagnosis and phenotyping of neoplastic or hyperplastic diseases.

Other applications of the anti-onserins of the invention include λgt11, λgt18-23, λZ
Immunological screening of a cDNA library constructed in an expression vector such as AP and λORF8. This type of messenger library, with the coding sequence inserted in the correct reading frame and orientation, can produce a fusion protein. For example, λgt11 produces a fusion protein in which the amino terminus comprises a β-galactosidase amino acid sequence and the carboxy terminus comprises a foreign protein. Antibodies can be used to detect antigenic epitopes of onserin proteins, for example, orthologs of onserin proteins from other species, such as by reacting a nitrocellulose filter pulled from the infected plate with an anti-onserin antibody. The positive phage detected by this assay can then be isolated from the infected plate. Thus, the presence of the onserin homolog can be detected and cloned from other animals so that it can replace the human isoform (including splicing variants).

In addition, the nucleotide sequence determined from the cloning of the Onserin gene from an organism can be used to identify and / or clone Onserin homologs in other cell types, eg, from other tissues, and from other organisms. Enables the creation of probes and primers designed for use in For example, the present invention also provides for a nucleotide sequence that hybridizes under stringent conditions to at least 15 contiguous nucleotides of a sense or anti-sense sequence selected from the group consisting of SEQ ID NO: 1 or a natural variant thereof. Also provided is a probe / primer comprising a substantially purified oligonucleotide comprising a region. For example, a primer based on the nucleic acid shown in SEQ ID NO: 1
Used in CR reactions, the onserin homolog can be cloned.
Similarly, transcripts or genomic sequences encoding the same or homologous proteins can be detected using probes based on the onserin sequences of the invention. In a preferred embodiment, the probe further comprises a label group attached thereto and is detectable, for example, the label group is selected from radioisotopes, fluorescent compounds, enzymes and enzyme co-factors.

[0121] Such probes can be used, for example, to measure the level of nucleic acid encoding onserin in a sample of cells from a patient-animal, for example, to detect onserin mRNA levels or to determine whether the genomic onserin gene is mutated. By examining the presence or absence of the deletion, it can be used as a part of a diagnostic test kit for identifying cells or tissues that mis-express the onserin protein. As an example, nucleotide probes can be generated from the onserin gene of the invention, which facilitates histological screening of complete tissues and tissue samples for the presence (or absence) of the transcript encoding onserin. Similar to the diagnostic use of anti-onserin antibodies, probes for onserin messages or genomic onserin sequences can be used, for example, to delete specific cell types, apoptosis, neoplasia and / or hyperplasia (eg, undesirable It can be used for both prophylactic and therapeutic evaluation of allelic variants that would be evident in degenerative diseases characterized by abnormal cell differentiation) or abnormal differentiation of tissues. When used with immunoassays such as those described above, oligonucleotide probes can help determine the molecular basis for developmental diseases that may include some abnormalities involving onserin protein expression (or deletion thereof). be able to. For example, a mutation in polypeptide synthesis can be differentiated from a mutation in the coding sequence.

Thus, the methods of the present invention provide a method for determining whether a subject has a risk for a disease characterized by abnormal apoptosis, cell proliferation and / or differentiation. In a preferred embodiment, the presence or absence of (i) a change affecting the integrity of the gene encoding the onserin protein, or (ii) a genetic damage characterized by misexpression of the onserin gene,
The method can be generally characterized by including detecting in a sample of cells from the subject. By way of example, at least one (i) deletion of one or more nucleotides from the onserin gene, (ii) addition of one or more nucleotides to the onserin gene, (iii) one or more nucleotides of the onserin gene. Onserin genes such as nucleotide substitutions, (iv) significant rearrangement of the gene on the chromosome, (v) significant changes in the level of messenger RNA transcripts of the onserin gene, (vii) methylation patterns of genomic DNA Abnormal modification of (vii) the presence of a non-wild-type splicing pattern of the messenger RNA transcript of the onserin gene, (vii
Such genetic damage can be detected by determining the presence of i) non-wild-type onserin proteins and (ix) inappropriate post-translational modifications of the onserin proteins. As described below, the present invention provides a number of assay techniques for detecting damage in the onserin gene, and importantly, the different underlying underlying onserin-dependent abnormal cell growth, proliferation and / or differentiation. Provides the ability to distinguish between molecular causes.

In a typical embodiment, the onserin gene as set forth in SEQ ID NO: 1 or a natural variant thereof, or a 5 ′ or 3 ′ flanking sequence or intronic sequence that naturally binds to an onserin gene of the invention Or a nucleic acid composition comprising a (purified) oligonucleotide probe comprising a region of a nucleotide sequence capable of hybridizing to a sense or antisense sequence of an onserin gene, such as a natural variant thereof. The nucleic acid of the cell is made available for hybridization, the probe is exposed to the 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 damage, including deletions, substitutions, etc., at either the genomic or mRNA level, and to examine mRNA transcript levels.

[0124] In certain embodiments, the detection of damage is determined by polymerase chain reaction (PCR) such as anchor PCR or RACE PCR (eg, US Pat. No. 4,683,319).
Nos. 5 and 4683202), or alternatively, the ligation chain reaction (see
LCR) (see, eg, Landegran et al., (1988) Science 241: 1077-1080; and Nakazawa et al., (1994) PNAS 91: 360-364). And where the latter can be particularly useful for detecting point mutations in the onserin gene. In merely illustrative embodiments, the method comprises: (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (eg, genomic, mRNA, or both) from the cells of the sample, and (iii)
B.) Contacting the nucleic acid sample with one or more primers that specifically hybridize to the Onserin gene under conditions such that hybridization and amplification of the Onthelin gene (if present) occurs; and To detect the presence or absence of, or the size of the amplification product,
Comparing the length with the control sample.

In yet another embodiment, the level of the onserin protein can be detected by an immunoassay. For example, the cells of a biopsy sample can be lysed and the level of onserin protein present in the cells can be estimated by standard immunoassay techniques. In yet another exemplary embodiment, one that is sensitive to methylation and whose restriction site is present in the onserin gene (including flanking and intronic sequences).
Alternatively, an abnormal methylation pattern of the onserin gene can be detected by digesting genomic DNA from a patient sample using a restriction endonuclease or higher. See, e.g., Buiting et al., (1994) Human Mol. Genet. 3: 893-895. The digested DNA is separated by gel electrophoresis and
Hybridize with a probe from the DNA sequence. The methylation status of the onserin gene can be determined by comparing a restriction pattern generated from sample DNA with a known methylation standard.

In yet another embodiment, the extracellular domain of the onserin protein can be used to quantitatively detect levels of the onserin ligand, whether soluble or membrane-bound. As an example, a soluble form of the Onserin protein can be made. A sample of a bodily fluid, such as plasma, serum, lymph, bone marrow, brain / spinal fluid, urine, etc., or a biopsy cell is contacted with the fragment under conditions in which binding occurs and the level of onserin-containing complex formed is determined. Detection can be by any of a variety of techniques known in the art.
For example, an Onserin polypeptide can be contacted with cells from a biopsy to determine the ability of a sample of Onserin polypeptide to decorate a particular cell. Binding of the sample to the cell population of the Onserin protein can be detected, for example, by using an antibody against the Onserin protein or by detecting a label bound to the Onserin protein. In the latter case, the onserin protein can be labeled, for example, by chemical modification or as a fusion protein. Examples of labels include radioisotopes, fluorescent compounds, enzyme cofactors (which can be added by chemical modification of the protein), and myc, pFL
Epitope tags such as AG, or enzymatic activities such as GST or alkaline phosphatase, which can be added either by chemical modification or creation of fusion proteins, are included.

In addition, the present invention contemplates the detection of soluble forms of the Onserin protein in body fluid samples. For example, Furukawa et al., (1997) Microsc. Res. Tech. 38: 34
As described in 3, soluble form levels of cadherin in serum occur in various disease states. For example, soluble E-cadherin is increased in serum in various skin diseases including pemphigus vulgaris, pemphigus vulgaris and psoriasis, and significantly higher levels of soluble E-cadherin are found in patients with metastatic cancer. To be observed. In various pathological conditions, the production and release of soluble onserin proteins mediates the host response and likely to determine disease progression and outcome, or at least appear to be a detectable diagnostic or prognostic factor. Examining soluble onserin proteins in body fluids is a new tool for obtaining information about various disease states and may be a prognostic assessment for clinicians. For example, the level of soluble onserin protein in body fluids may provide useful information in monitoring, among other things, neurological disease and in treating neoplastic or hyperplastic deformities of ectoderm, mesoderm or endoderm origin. Absent.

The level of soluble onserin present in a given sample can be quantified using known methods and techniques in light of the present disclosure. For example, an antibody that is immunosensitive to the extracellular domain of the Onserin protein can be used to detect and quantify its presence in a sample, for example, by well-known immunoassay techniques. Alternatively, the presence of the receptor in a liquid sample can be detected using a labeled ligand for the protein.

Currently, there are many techniques in the art for identifying extracellular components that bind to onserin, such as ligands for onserin proteins. For example, expression cloning can be performed on cDNA or genomic libraries by isolating cells that have been modified in a labeled form of the extracellular domain of the onserin protein. In a preferred embodiment, the technique uses onserin proteins in in situ assays to detect onserin ligand in tissue samples and whole organisms. Generally, Flanagan and Leder described below: RAP-in situ assay (PCT Publication W O92 / 06220 and Cheng et al, (1994) Cell 79. See 157-168) (R eceptor A ffinity P robe) is Involves the use of an expression cloning system whereby the onserin ligand is obtained based on binding to the onthelin / alkaline phosphatase fusion protein. Generally, the method comprises (i
A) providing a hybrid molecule (affinity probe) comprising an onserin protein covalently linked to an enzymatically active tag, or at least a ligand binding domain thereof, preferably on which a chromogenic substrate is present; Alternatively, contacting the organism with the affinity probe, forming a complex between the probe and the cognate ligand in the sample, removing unbound probe, and (
iii) detecting the affinity complex using a chromogenic substrate for the enzymatic activity binding to the affinity probe.

[0130] Unlike other conventional methods performed only on dispersed cell cultures, this method provides a means for probing non-dispersed and whole tissue and animal samples. In addition to facilitating the cloning of the Onserin ligand, the method may be used to detect the pattern of expression of a specific ligand for the Onserin protein, to measure the affinity of receptor / ligand interaction in a tissue sample, and to It can also be used to generate drug screening assays within. In addition, affinity probes can be used in diagnostic screening to determine if an onserin ligand is mis-expressed.

In still another embodiment of the present invention, wherein other proteins interacting with the onserin protein (
To isolate a sequence encoding an "onserin interacting protein" or "onserin-ip", a "two-hybrid" or "interaction trap" assay can be made using the onserin polypeptides of the invention ( For example, U.S. Pat.No. 5,283,317; Zervous et al., (1993) Cell 72: 223-232; Madura et al.
., (1993) J. Biol. Chem. 268: 12046-12054; Bartel et al., (1993) Biotech.
niques 14: 920-924; Iwabuchi et al., (1993) Oncogene 8: 1693-1696; and Brent WO94 / 10300). This assay is particularly useful for identifying the intracellular components of the onserin-dependent signal transduction pathway.

[0132] Briefly, an interaction trap is by reconstituting a functional transcriptional activator protein from two separate fusion proteins in vivo. In particular, the method uses a chimeric gene that expresses the hybrid protein. By way of example, a first hybrid gene may be a transcriptional activator DNA- fused in reading frame to a sequence encoding an onserin polypeptide, such as the intracellular domain of an onserin protein.
Contains a sequence encoding a binding domain. The second hybrid protein encodes a transcription activation domain fused in frame with the sample gene from the cDNA library. If the “decoy” and the sample hybrid protein are able to interact, eg, form an onserin-dependent complex, they bring the two domains of the transcriptional activator into close proximity. This access is sufficient to cause transcription of a reporter gene operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and to detect and use expression of the reporter gene to interact with onserin and the sample protein. Can be evaluated.

In addition, by making available purified recombinant onserin polypeptides, the present invention provides for the normal cellular function of the onserin proteins of the present invention or the maintenance, differentiation and / or proliferation of cellularity. It facilitates the development of assays that can be used to screen for drugs that are either agonists or antagonists of their role in etiology and their associated diseases. In a general sense,
The assay evaluates the ability of a compound to modulate the binding between an Onserin polypeptide and a molecule, eg, an Onserin interacting protein that interacts with the Onserin polypeptide. Examples of compounds that can be screened for such onserin-mediated interactions include peptides, nucleic acids, carbohydrates, small organic molecules, natural product extraction libraries, such as those isolated from animals, plants, fungi and / or microorganisms. Is done.

In many drug screening programs that test libraries of compounds and natural extracts, high-throughput assays are desirable to maximize the number of compounds examined within a given time period. For example, assays performed in cell-free systems, which may be derived from purified or semi-purified proteins, are often preferred as "primary" screening, in which changes in molecular targets mediated by the test compound are rapidly detected. Develop and allow relatively easy detection. In addition, the effects of cytotoxicity and / or bioavailability of the test compound can be assessed in vitro.
In systems generally negligible, the assay instead focuses primarily on the effect of the drug on the molecular target that will be apparent in the change in binding affinity with the ligand. Thus, in an example of a screening assay of the invention, a reaction mixture is made up to include an Onserin polypeptide, a compound of interest, and a "target molecule", eg, a protein that interacts with an Onserin polypeptide. Examples of target molecules include ligands, intracellular proteins (such as catenin proteins) and onserin itself (or other cadherins with which it interacts), as well as other peptide and non-peptide interacting molecules. Detecting and quantifying the interaction of an Onserin polypeptide with a target molecule provides a means for determining the effect of a compound that inhibits (or enhances) the interaction between Onserin and a target molecule. By constructing dose response curves from data obtained using various concentrations of the test compound, the effects of the compound can be evaluated. In addition, control assays can be performed to obtain a baseline for comparison. In a control assay, the interaction of the Onserin polypeptide and the target molecule is quantified in the absence of the test compound.

[0135] The interaction between the Onserin polypeptide and the target molecule may be detected by various techniques. Modulation of complex formation can be achieved, for example, by using a detectably labeled protein, such as a radiolabeled, fluorescently or enzymatically labeled onserin polypeptide, by immunoassay, by chromatographic detection, or by endogenous acetylase. It can be quantified by detecting sexual activity. In such embodiments with a cell-free assay format, the Onserin polypeptide is preferably a soluble fragment of the full-length protein, such as an extracellular or intracellular domain.

[0136] Typically, either onserin or the target molecule is immobilized to facilitate separation of the complex from the uncomplexed form of one or both proteins, as well as to automate the assay. It is desirable to adapt. Binding of onserin to the target molecule, in the presence and absence of the candidate agent, can be performed in any vessel suitable for containing the reactants. Examples include microtiter plates, test tubes and microcentrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows the protein to be bound to a matrix. For example, glutathione-
The S-transferase / onserin (GST / onserin) fusion protein can be adsorbed onto glutathione sepharose beads (Sigma Chemical. St. Louis. MO) or glutathione-derivatized microtiter plates, which are then, for example, 35S-labeled. The combined cell lysate and test compound are incubated with the mixture under conditions in which complex formation occurs, such as, for example, salts and pH under physiological conditions, although more stringent conditions may be desirable. Following incubation, the beads are washed, unbound label is removed, and the immobilized matrix and radiolabel are examined directly (eg, placing the beads in scintillant) or the complex is subsequently dissociated. After that, examine in the supernatant. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of target molecule found in the bead fraction can be quantified from the gel using standard electrophoresis techniques.

[0137] Other methods for immobilizing proteins and other molecules on matrices are also available for use in the assays of the present invention. For example, either onserin or the target molecule can be immobilized utilizing the binding of biotin and streptavidin.
For example, biotinylated onserin molecules can be prepared using techniques well known in the art (eg, bi
It can be prepared from biotin-NHS (N-hydroxy-succinimide) using an otinylation kit, Pierce Chemicals, Rockford, IL, and immobilized in the wells of a streptavidin-coated 96-well plate (Pierce Chemical). Alternatively, the wells of the plate can be derivatized with an antibody that is reactive with onserin but does not interfere with the interaction between onserin and the target molecule, and the onserin can be trapped in the well by antibody binding. As described above, the preparation of the target molecule and the test compound are incubated in the wells of the plate where onserin is present, and the amount of complex trapped in the wells can be quantified. In addition to the methods described above for GST-immobilized complexes, examples of methods for detecting such complexes include antibodies reactive with the target molecule, or antibodies reactive with the onserin protein and competing with the target molecule. Immunodetection of the complex used; and enzyme-ligation assays by detecting enzyme activity bound to the target molecule, either endogenous or exogenous. In the latter case, the enzyme may be chemically linked to the target molecule or provided as a fusion protein. By way of illustration, a target molecule can be chemically linked to horseradish peroxidase or genetically fused (if it is a polypeptide) and the amount of polypeptide trapped in the complex can be determined, for example, by: It can be evaluated using a chromogenic substrate of an enzyme such as 3,3'-diamino-benzazine tetrahydrochloride or 4-chloro-1-naphthol. Similarly, a fusion protein comprising the polypeptide and glutathione-S-transferase can be provided, and complex formation can be quantified by detecting it with 1-chloro-2,4-dinitrobenzene (Habi
g et al., (1974) J. Biol. Chem. 249: 7130).

For an immunodetection-based method for quantification of proteins captured in a complex,
Antibodies against the protein, such as anti-onserin antibodies, can be used.
Alternatively, the protein to be detected in the complex may be "epitope-tagged," in the form of a fusion protein comprising a second polypeptide (e.g., commercially available) to which the antibody is readily attached (e.g., commercially available) attached to the onserin sequence. (Epitope tagged). For example, the above GS
The T fusion protein can also be used for quantification of binding using an antibody to the GST region. Other useful epitope tags include myc-epitope containing a 10 residue sequence from c-myc (eg, Ellison et al., (1991) J Biol Chem 266: 21150-21).
157), as well as the pFLAG system (International Biotechnologies, Inc) or pEZZ-protein A (Pharamacia, NJ).

For example, the drug screening assay of the present invention comprises the steps of (a) forming a reaction mixture comprising (i) an onserin-interacting polypeptide, (ii) an onserin polypeptide, and (iii) a test compound. (B) detecting the interaction between onserin-ip and the onserin polypeptide. A statistically significant change (enhancement or inhibition) in the interaction of Onserin-ip and Onserin polypeptide in the presence of the test compound, as compared to the interaction in the absence of the test compound, is indicated by the Indicates potential agonists (mimic or activators) or antagonists (inhibitors) of onserin bioactivity. The reaction mixture can be a cell-free protein preparation, such as a reconstituted protein mixture or cell lysate, or can be a recombinant cell containing a heterologous nucleic acid that recombinantly expresses an onserin polypeptide. .

When the onserin polypeptide is involved as part of an oligomeric complex that forms a multimeric cell surface complex, for example, when the complex is homomeric or contains other protein subunits, a cell-free system is For example, it may be a cell membrane preparation or a reconstituted protein mixture. Alternatively, liposome preparations using reconstituted onserin protein can be used. For example, the protein subunit of the onserin-dependent complex can be purified from detergent extracts from both standard and recombinant origins, and reconstituted into artificial lipid vesicles (eg, phosphatidylcholine liposomes) or cell membrane-derived vesicles. (Eg, Bear et al. (1992) Cell 68: 809-818; Newton et al. (1983) B
iochemistry 22: 6110-6117; and Reber et al. (1987) J Biol Chem 262: 11369-1137.
Four). The lamellar structure and size of the resulting liposomes can be characterized using an electron microscope. The external orientation of the onserin protein in the reconstituted membrane can be shown, for example, by immunoelectron microscopy. In the presence of the candidate agent, the interaction of the protein or other cellular components with liposomes having such an onserin complex and liposomes without the protein can be compared.

[0141] In yet another embodiment, a pharmaceutical screening assay is obtained that includes whole cells that express an Onserin polypeptide. Analysis of the recombinant cells can detect the ability of the test agent to alter the activity of the onserin protein. For example, agonists and antagonists of onserin biological activity can be detected by scoring changes in cell proliferation or differentiation (phenotype). The general techniques for detecting each are well known and will vary with the source of the particular reagent cell used in any given assay. For cell-based assays,
The recombinant cells are preferably metazoan cells such as mammalian cells, insect cells, Xenopus cells, and oocytes.

In one embodiment, a cell that expresses an Onserin protein, whether endogenous or heterologous, for example, can induce signal transduction in a Onserin protein ligand, eg, an Onserin-dependent format. Some compounds (including antibodies) can be contacted, and the resulting signal is detected at various points in the pathway or based on a phenotypic change to the reagent cells. By comparison to a control experiment lacking either the protein or the test compound, a test compound that modulates (eg, enhances or inhibits) the pathway can be detected. For example, visual inspection of the morphology of the reagent cells can be used to determine whether the target onserin protein is affected by the added drug.

In addition to morphological studies, changes in the level (s) of intracellular second messengers in response to signaling by the Onserin polypeptide can be detected. For example, in various embodiments, the assay provides for phosphorylation patterns, adenylate cyclase activity (cAMP production),
The ability of a test compound to produce alterations in GTP hydrolysis, calcium mobilization, and / or phospholipid hydrolysis (IP ₃ , DAG production) can be assessed. Detection of changes in intracellular signals, such as changes in second messenger or gene expression, can identify candidate agonists and antagonists for onserin-dependent signaling.

Activation of the Onserin protein can trigger a cascade for activation and inhibition of downstream effectors whose end result is, for example, a detectable change in gene transcription or translation. Potential transcription targets for onserin-dependent signaling include those genes that are controlled by catenin transcription. By selecting transcription control sequences from such target genes that are responsible for up-regulation or down-regulation of these genes, for example, in response to onserin induction, and operably linked to such a promoter for the reporter gene, Provides a transcription-based assay that is sensitive to the ability of a particular test compound to affect the onserin signaling pathway.

In the practice of certain embodiments of the assay, the reporter gene construct is placed in a reagent cell to generate a second messenger-dependent detection signal generated by onserin-dependent induction. Typically, the reporter gene construct is
A reporter gene operably linked to one or more transcriptional regulatory elements responsive to onserin activity at the level of expression of the reporter gene that provides an onserin-dependent detection signal. The amount of transcription from the reporter gene can be measured using any suitable method known to those skilled in the art. For example, mRNA from a reporter gene
Expression can be detected using RNase protection or RNA-based PCR, and the protein product of the reporter gene can be identified by unique staining or unique activity.
The level of expression from the reporter gene can then be compared to the level of expression in either of the same cells in the absence of the test compound, or to the level of transcription in substantially the same cell lacking the target receptor protein. Any difference in the amount of transcript, statistically or otherwise significant, indicates that the test compound has in some way altered the activity of inducing the onserin protein.

As described in further detail below, in a preferred embodiment, the reporter gene product is detected by the intrinsic activity associated with the product. For example, a reporter gene can encode a gene product that produces a detection signal based on color, fluorescence, or luminescence upon enzymatic activity. In another preferred embodiment, the reporter or marker gene confers a selective growth effect. For example, reporter genes enhance cell viability, reduce cell auxotrophy, and / or confer resistance to drugs. Many reporter genes are known to those of skill in the art, and others can be identified or synthesized by methods known to those of skill in the art. Reporter genes include any gene that expresses a detectable gene product, which may be RNA or a protein.

Suitable reporter genes are those that are easily detectable. The reporter gene may include a transcription control sequence of interest, or may be included in a construct in the form of a fusion gene with a gene exhibiting favorable properties. Examples of reporter genes include, but are not limited to, CAT (chloramphenicol acetyltransferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as β-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), B
iochemistry 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).

Thus, yet another embodiment of the pharmaceutical screening assay of the present invention provides a recombinant cell, for example, for performing the specific drug screening method described above, wherein the cell comprises (i) signal transduction An expressible recombinant gene encoding a heterologous onserin polypeptide whose activity regulates gene transcription, and (ii) operably linked to one or more transcriptional regulatory elements responsive to the signal transduction activity of the onserin polypeptide. Reporter gene constructs containing reporter genes. Yet another embodiment of the present invention provides a kit for screening a test compound for identifying an agent that modulates onserin biological activity, including the cells discussed above.

[0149] In yet another embodiment of a pharmaceutical screen, a two-hybrid assay (described above) using an Onserin polypeptide and a target molecule, such as an Onserin interacting protein, is provided. Drug-dependent inhibition or enhancement of the interaction can be scored. After identifying a test compound as a potential modulator of one or more of the biological activities of the onserin protein, the practitioner of the assay of the invention may determine the potency and specificity of the selected compound both in vitro and in vivo. Continue to test. The agents identified in the assays of the invention can be formulated into pharmaceutical preparations for in vivo administration to animals, preferably humans, for subsequent in vivo testing, or for administration to animals as an improved medicament.

Another embodiment of the present invention is directed to inducing and / or maintaining a differentiated state, improving survival, and / or inhibiting cell proliferation by contacting a cell with an agent that modulates an onserin-dependent signal transduction pathway. Or, alternatively, augmentation). The methods of the present invention can be used to generate and / or maintain different groups of tissues both in vitro and in vivo. "Onseline Therapy"
As appropriate, whether inhibitory or potent in modulating the activity of the Onserin protein, the isolated Onserin polypeptide (including both agonistic and antagonistic forms), gene therapy constructs, antisense molecules, It can be any preparation described above, including a peptidomimetic, or an agent identified in a pharmaceutical assay provided herein. In one embodiment, a soluble form of the protein, including the extracellular region of the onserin protein, can be provided as a means for antagonizing the binding of extracellular factors to the cell surface onserin protein.

The therapeutic compounds for onserin of the present invention are believed to play an important role in regulating cell proliferation and maintaining, for example, neuronal, testicular and renal tissues in disease states. It is also clear that transient use of modulators of onserin activity can improve tissue in vivo, for example, in organ development and maintenance, such as ectoderm patterning, and in certain mesoderm and endoderm differentiation processes. . By modulating the proliferation and differentiation potential of different cells, the onserin treatment of the present invention can be used to repair damaged tissue or to improve transplantation and morphology of transplanted tissue. For example, onthelin antagonists and agonists can be used in a difference-based manner to control different stages of tissue repair following a physical, chemical or pathological disorder. The method is also applicable to cell culture techniques.

To further illustrate this aspect of the invention, an in vitro neuronal culture system comprises:
Nerve development and nerve growth factor (NGF), ciliary trophic factor (CNTF)
And has been found to be a fundamental and indispensable tool for the study of the identification of neurotrophic factors such as neurotrophic factor (BDNF) from the brain. Once a neuronal cell has terminally differentiated, it generally does not change to another terminally differentiated cell type. However, neurons nevertheless easily lose their differentiation state. This is commonly observed when they grow in culture from adult tissue and when they form buds during regeneration. The method provides a means to ensure a suitably restrictive environment for maintaining neuronal cells at various stages of differentiation, e.g., to test the specific activity of other trophic factors Can be used in cell cultures designed for In such embodiments of the method of the present invention, the cultured cells are contacted with an onserin therapeutic substance that inhibits the proliferation of neuronal cells, thereby inducing neuronal differentiation (eg, of stem cells) by preventing loss of differentiation, Alternatively, the integrity of terminally differentiated neuronal cells can be maintained. Alternatively, certain onserin therapeutics of the invention induce proliferation and, for example,
It is thought to suppress differentiation to some extent and can be used to prevent differentiation of progenitor cells in culture.

To further illustrate the use of onserin therapeutics, it is noted that endodermal transplantation has emerged as a further approach to central nervous system therapy. For example, one approach to repairing damaged brain tissue involves transplantation of cells from fetal or neonatal animals into the adult brain (Dunnett et al. (1987) J Exp Biol 123: 265-289; and Freund et al.).
(1985) J Nourosci 5: 603-616). Fetal neurons from various brain regions can be successfully incorporated into the adult brain, and such implants can mitigate behavioral deficits. For example, transplantation of fetal dopaminergic neurons can prevent movement disorders caused by impaired dopaminergic release to basal ganglia. Complex cognitive functions that are impaired after neocortical damage can also be partially restored by transplantation of fetal cortical cells.

In addition to transplanting cells cultured in the presence of onserin agonists and antagonists and other in vitro uses, yet another embodiment of the present invention
It relates to the therapeutic application of onserin therapeutics for improving the survival of neurons and other neuronal cells in both the central and peripheral nervous systems. The neurotrophic activity of the Onserin protein depends on the specific Onserin protein and, consequently, the Onserin therapeutic substance that modulates the Onserin biological activity, to maintain normal cells,
Functional capacity and aging; repair and regeneration processes in chemically or mechanically damaged cells; and as a result of loss of differentiation under certain pathological conditions,
It is logical to facilitate the regulation of adult neurons for degeneration and premature death. In light of this understanding, the invention specifically encompasses (i) traumatic injuries, chemical injuries, vascular injuries and deficits (such as ischemia as a result of smoking), as well as infectious / inflammatory and tumor-induced damage. Acute, subacute, or chronic nervous system damage;
) Aging of the nervous system, including Alzheimer's disease; (iii) chronic neurodegenerative diseases of the nervous system, including Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis, and spinocerebellar degeneration; and (iv) ) Treatment (prevention, treatment) of a neurological condition resulting from chronic immunological diseases of the nervous system,
And / or reduced severity) of the onserin therapeutics of the present invention.

Many neurological disorders are associated with degeneration of distinct populations of neuronal elements,
It may be treatable by therapeutic measures, including onserin therapeutics that act as neurotrophic factors. For example, Alzheimer's disease is associated with a deficiency of several neurotransmitters, both released into the neocortex and present with the cortex. For example, a large (75%) loss of neurons has been observed in the basal ganglia of Alzheimer's patients compared to age-matched controls. Alzheimer's disease is among the most common forms of dementia, but some other diseases can result in dementia. Some of these are degenerative diseases characterized by death of neurons in various parts of the central nervous system, especially in the cerebral cortex. However, some forms of dementia are associated with degeneration of the thalamus or white matter below the cortex of the brain. Here, cognitive dysfunction results from the isolation of cortical regions by degeneration of efferent and afferent nerves. Huntington's disease involves intrastriatal and cortical cholinergic neurons and G
Associated with degeneration of ABAergic neurons. Pick's disease is a significant neuronal degeneration of the neocortex of the frontal and anterior temporal lobes, often with the death of striatal neurons. Treatment of patients suffering from such a degenerative condition may be, for example, to modulate the phenomenon of apoptosis resulting in differentiation and loss of neurons (eg, to improve the survival of existing neurons), as well as to precursor the affected area. It may include the application of an Onserin therapeutic to promote differentiation and regrowth by the cells. In addition to degeneration-induced dementia, the pharmaceutical preparations of one or more of the therapeutic agents for onserin of the present invention can be suitably applied in the treatment of neurodegenerative diseases in which tremors and involuntary movements are manifested. Parkinson's disease, for example, primarily affects the subcortical structure and is characterized by degeneration of the nigrostriatal pathway, the raphe nucleus, the locus nucleus, and the motor nucleus of the vagus. Ballism is commonly associated with damage to the hypothalamic nucleus, often due to acute ductal accidents. Also included are neurological and muscular disorders, which ultimately affect somatic division of the peripheral nervous system and manifest as neuromuscular disorders.

Examples include chronic atrophy such as amyotrophic lateral sclerosis, Guillain-Barre syndrome and chronic peripheral neuropathy, and other diseases that can manifest as progressive bulbar palsy or spinal muscular atrophy. is there. The method is amenable to the treatment of cerebellar disorders that result in hypotonia or ataxia, such as those disorders in the cerebellum that result in limb disorders ipsilateral to the injury. For example, a preparation of a therapeutic substance for onserin can be used to treat restricted forms of the cerebellar cortex, including the anterior lobes (worm and leg areas), which are common in alcoholic patients. In a specific embodiment, the method of the invention is used to treat amyotrophic lateral sclerosis. ALS is the name given to a complex disease involving upper and lower motor neurons. The patient may exhibit progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, or a combination of these pathologies. The major pathological abnormalities are characterized by selective, progressive degeneration of lower motor neurons of the spinal cord and upper motor neurons of the cerebral cortex. A neurotrophic onserin therapeutic substance alone or C
Used with other neurotrophic factors such as NTF, BDNF or NGF,
Motor neuron degeneration in LS patients can be prevented or reversed.

The therapeutic substances for onserin of the present invention can also be used for treating autonomic nervous system diseases of the peripheral nervous system, including diseases that affect the nerve response of smooth muscle and endocrine tissues (such as glandular tissues). For example, the methods of the present invention can be used to treat tachycardia or atrial cardiac arrhythmias that can result from degenerative conditions of nerves that stimulate striated muscle of the heart. In addition, the potential role of onserin proteins in dendritic development and maintenance of axonal neurons gives rise to a potential role for certain onserin therapeutics. Thus, potential roles for onserin therapeutics include the induction of axon release and the ability to induce differentiation and / or maintenance of cells that stimulate dendrites. Thus, compositions containing onserin therapeutics can be used to sustain the survival and reprojection of some types of ganglion neuron sympathetic nervous system and sensory and motor neurons. In particular, such therapeutic compositions may be useful, for example, in treatments designed to rescue various neurons from injury-induced death, and in treatments that induce the re-release of these neurons following such injury. . Such diseases include, but are not limited to, CNS trauma infarction, infections (eg, varicella-zoster virus infection), metabolic diseases, nutritional deficiencies, toxic drugs (eg, cisplatin treatment).

In addition, certain onserin therapeutics are useful for selective cleavage of sensory neurons, for example, in the treatment of chronic pain syndromes. Suitably, the onserin therapeutic can be used in neuroprostheses for repair of central and peripheral nerve damage. In particular, the use of prosthetic devices destroys them,
Or when intubating severed axons, certain onserin therapeutics can be added to the prosthetic device to increase the rate of dendritic growth and regeneration. Specific examples of nerve guide tubes are described in U.S. Patent Nos. 5,092,871 and 4,955,892. Thus, the severed axons can be directed by the prosthetic nerve guide to the severed nerve endings.

[0159] In another embodiment, the subject methods can be used in the treatment of neoplastic or proliferative transformation, such as occurs in the central nervous system. For example, some of the onserin therapeutics that induce neuronal cell differentiation can be used to render such transformed cells either post-mitotic or apoptotic. Treatment with onserin therapeutics may promote disruption of autocrine loops, such as the TGF-β or PDGF autostimulation loops, which are thought to be involved in the neoplastic transformation of some neuronal tumors. Thus, onserin therapeutics, for example,
Useful for the treatment of malignant gliomas, medulloblastomas, neuroectodermal and ependymomas.

[0160] Yet another embodiment of the present invention relates to a method wherein the onserin protein is used to form other endodermal patterns (patterns).
ning) and the application of the finding that it is a morphogenetic signal involved in other vertebrate organ development pathways, in addition to the above neuronal differentiation, which has a clear role in both mesodermal and endodermal differentiation processes.

[0161] Another aspect of the invention relates to transgenics that express a heterologous onserin gene of the invention and / or have one or more genomic onserin genes disrupted in at least animal tissues or cell types. Characterized by non-human animals. Accordingly, the invention features animal models of disease pathogenesis having one or more mis-expressed onserin alleles. For example, animals can be produced that have one or more onserin alleles that have been deleted or otherwise inactivated. Such models can then be used to study disorders due to mis-expressed onserin genes, as well as to evaluate the therapeutic potential of similar disorders.

All transgenic animals of the present invention contain a transgene of the present invention in their large number of cells, wherein the transgene is regulated by the onserin protein, eg, a “host” with respect to cell growth, death and / or differentiation. Change the phenotype of cells. Since it is possible to produce the transgenic organisms of the invention using one or more of the transgene constructs described herein, a general description of the production of transgenic organisms by reference to exogenous genetic material will generally be given. Will be given.
This general description uses the methods and materials described herein and those generally known in the art to incorporate a specific transgene sequence into an organism,
Used appropriately by those skilled in the art.

[0163] In one embodiment, the transgene construct is a knockout construct. Such transgene constructs are usually insertional or substitutional constructs (Hasty et al., (1991)
Mol Cell Biol 11: 4509). Transgene constructs for disruption of the onserin gene are designed to facilitate homologous recombination at the genomic onserin gene portion to prevent functional expression of the endogenous onserin gene. In a preferred embodiment, the knockout construct comprises (1) an endogenous onserin gene (
A nucleotide sequence was used that could consist of DNA from several parts of the exon sequence, intron sequence, promoter sequence, etc.) and (2) a marker sequence used to detect the presence of the knockout construct in the cell. The knockout construct is inserted into the cell and integrated into the genomic DNA of the cell at such a location so as to prevent or interrupt transcription of the native onserin gene. Such insertions can occur by homologous recombination, ie, when the knockout construct is genomic DNA
The region of the knockout construct homologous to the endogenous onserin gene sequence is hybridized to genomic DNA such that it is incorporated into the corresponding position of genomic DNA and recombine with the genomic sequence. The knockout construct comprises (1) all or a partial sequence of one or more exons and / or introns of the onserin gene to be disrupted, (2
A) sequences flanking the 5 ′ and 3 ′ ends of the coding sequence of the onthelin gene, or (3) combinations thereof.

[0164] Preferred knockout constructs delete essential structural elements of the endogenous onserin gene by targeted homologous recombination. For example, the targeting construct can recombine with the genomic onserin gene to delete a portion of the coding sequence and / or the essential transcriptional regulatory sequences of the gene.

Alternatively, a knockout construct can be used to interrupt essential structural and / or regulatory elements of the endogenous onserin gene by targeted insertion of a polynucleotide sequence. For example, a knockout construct can recombine with an onserin gene and convert heterologous sequences, such as the neo expression cassette, into structural elements (eg, exons) and / or regulatory elements (eg, enhancers, promoters, intron splice sites, polyadenylation sites, etc.). ) To obtain the target onserin allele with the insertion disruption. Inserted nucleic acids can range in size from one nucleotide (eg, to produce a frameshift) to several kilobases or more, limited only by the efficiency of the targeting technology.

[0166] Depending on the location and characteristics of the disruption, the use of the transgene construct to produce a transgenic animal in which substantially all of the expression of the target onserin gene is inhibited in at least a portion of the animal cells. it can. If only regulatory elements are targeted, somewhat lower levels of expression of the target gene may occur (ie, the target allele is "leaky").

[0167] The nucleotide sequence containing the knockout construct can be obtained using methods well known in the art. Such methods include, for example, screening a genomic library with an Onserin cDNA probe to identify the corresponding genomic Onserin gene and regulatory sequences. Alternatively, if the cDNA sequence can be used as part of a knockout construct, the cDNA may be obtained by screening a cDNA library as described above.

In another embodiment, a “transgenic non-human animal” of the invention is produced by introducing a transgene into the germline of a non-human animal. The transgene can be introduced using embryonic target cells at various stages of development. Various methods are used depending on the stage of development of the embryonic target cell. The particular lineage of any animal used to practice the present invention is generally of good health, good embryo yield,
Selected for good pronuclear visibility and regenerative compatibility in the embryo. In addition, haplotype is a significant factor. For example, when producing transgenic mice, strains such as the C57BL / 6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, ME). A preferred strain is C57BL / 6
Or, it has H-2b, H-2d or H-2q haplotype such as DBA / 1. The lineage used to practice the present invention may itself be transgenic and / or knockout (ie, an animal having one or more partially or completely silenced genes). Obtained from).

[0169] In one embodiment, the transgenic construct is introduced into a single stage embryo.
Conjugates are the best target for microinjection. The use of conjugates as targets for gene transfer has the major advantage that the injected DNA is often incorporated into host genes before the first cleavage (Brinster et al., (1985) PNAS 82: 443).
8-4442). As a result, all cells of the transgenic animal will have the transgene incorporated. This will also be reflected in the effective transfer of the transgene to the offspring of the founder, as generally 50% of the germ cells will carry the transgene.

[0170] Introduction of the transgene nucleotide sequence into the embryo may be accomplished by any method known in the art, for example, microinjection, electroporation or lipofection. Following the introduction of the transgene nucleotide sequence into the embryo, the embryo may be incubated in vitro for various times, or reimplanted into a surrogate host, or both. In vitro incubation to maturity is within the scope of the present invention. One common method is to incubate the embryos in vitro with the species for about 1-7 days, and then reimplant them into a surrogate host.

[0171] Any technique that allows for the addition of exogenous genetic material into the nuclear genetic material is available as long as it does not harm the cell, nuclear envelope or other existing cells or genetic structure. Exogenous genetic material is optionally inserted into the nuclear genetic material by microinjection. Microinjection of cells and cell structures is known and used in the art.

Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized and the embryo is inserted into the fallopian tube. The number of embryos transferred into a particular host varies by species,
Usually, it will be comparable to the number of offspring of a naturally occurring species.

The transgenic progeny of the surrogate host can be screened for the presence and / or expression of the transgene by any suitable method. Screening is often accomplished by Southern or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using antibodies to the protein encoded by the transgene can be used as an alternative or additional method to screen for the presence of the transgene product. Typically, DNA is prepared from excised tissue and analyzed for the transgene by Southern analysis or PCR. Alternatively, tissues or cells suspected of expressing the transgene at high levels may be analyzed by Southern analysis or PC analysis.
R is used to test for the presence and expression of the transgene, but any tissue or cell type can be used for the analysis.

[0174] The transgene can also be introduced into a non-human animal using retroviral infection. Developing non-human embryos can be cultured in vitro to the blastocyst stage. During this time, blastomeres can be targets for retroviral infection (Jaenich, R. (197
6) PNAS 73: 1260-1264). Effective infection of blastomeres can be 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 that carries the transgene (Jahner et al., (1985) PNAS 82: 6927-6931;
Van der Putten et al. (1985) PNAS 82: 6148-6152). Transfection is performed easily and efficiently by culturing blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al., (1987) EMBO J. 6: 383-388). Alternatively, the infection can be made late. Virus or virus-producing cells can be injected into the blastocoel (Jahner et al. (1982) Nature 298: 623-628). Many of the founders will be a transgene mosaic, since uptake occurs only in a subset of the cells that formed the transgenic non-human animal. In addition, the founder has various retroviral insertions of the transgene at different sites in the genome that will generally segregate in progeny. In addition, it is possible to introduce a transgene into the germline by an intrauterine retrovirus in the midgestation embryo (Jahner et al., (1982) supra).
.

A third type of target cells for transgene transfer is embryonic stem cells (ES).
ES cells are obtained from pre-implanted embryos cultured in vitro and fused with the embryos (Evans et al., (1982) Nature 292: 154-156; Bradley et al., (1984) Nature 309: 255.
-258; Grossler et al., (1986) PNAS 83: 9065-9069; and Robertson et al., (1986) Na.
ture 322: 445-448). The transgene can be efficiently introduced into ES cells by DNA transfection or retrovirus-mediated transduction. Thereafter, such transformed ES cells can be combined with blastocysts from a non-human animal. The ES cells then colonize the embryo and contribute to the germline of the resulting chimeric animal. For a review, see Jaenisch, R. (1988) Science 240: 1468-1474.

In one embodiment, changes can be introduced into cultured embryonic stem cells using gene targeting, a method that uses homologous recombination to modify the genome of an animal. By targeting the onserin gene in ES cells, these changes can be introduced into the germline of the animal to produce chimeras. Gene targeting methods include segments in tissue culture cells that are homologous to the onserin locus, and also include sequence modifications (eg, insertions, deletions, point mutations) directed to the onserin genomic sequence. This is achieved by introducing a DNA targeting construct that does The treated cells are then screened for correct targeting to identify and isolate the correctly targeted one.

Gene targeting in embryonic stem cells is indeed contemplated by the present invention as a method of disrupting onserin gene function by using targeted transgene constructs designed to carry out homologous recombination with the onserin genomic sequence. It is a scheme to be performed. The targeting construct can be positioned such that upon recombination with an element of the onserin gene, a positive selectable marker is inserted (or replaced) into the coding sequence of the target onserin gene. The inserted sequence functionally disrupts the onserin gene, but also provides positive selection properties.

In general, the embryonic stem cells (ES cells) used to produce a knockout animal are of the same species as the knockout animal to be produced. Thus, for example, mouse embryonic stem cells are typically used for the production of onserin knockout mice. Embryonic stem cells were obtained from Doetschman et al. ((1985) J. Embryol. Exp. Morphol. 87: 27-4).
Produced and maintained using methods well known to those skilled in the art, as described in 5). Although any lineage of ES cells can be used, the lineage selected is typically incorporated into and becomes part of the germline of the developing embryo to effect germline transmission of the knockout construct. Selected for cell performance. Therefore, any ES cell line that is thought to have the ability is suitable for use in the present invention. Cells are from Robertson in: Teratocarcinomas and Embryonic Stem Cells:
A Practical Approach, E, J. Robertson, ed.IRL Press, Washington, DC
[1987]); by Bradley et al. (1986) Current Topics in Devel. Biol. 20: 357-3
(71); and Hogan et al. (Manipulaying the Mouse Embryo: A Laboratory Ma
nual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1986]
) Are prepared and cultured for knockout construct insertion using methods well known to those skilled in the art as described by

[0179] Insertion of the knockout construct into ES cells can be accomplished using a variety of methods well known to those skilled in the art, including, for example, electroporation, microinjection and calcium phosphate treatment. The preferred method of insertion is electroporation. Each knockout construct to be inserted into a cell must first be in a linear form. Thus, when a knockout construct is inserted into a vector, linearization is accomplished by digesting the DNA with only appropriate restriction endonucleases selected to cleave only within the vector sequence and not within the knockout construction sequence. Is

For insertion, the knockout construct is added to the ES cells under conditions suitable for the chosen insertion method, as known to those skilled in the art. When one or more constructs are introduced into ES cells, each knockout construct can be introduced simultaneously or one by one. When electroporating ES cells, the ES cells and knockout-constructed DNA are exposed to an electric pulse using an electroporation machine according to the manufacturer's usage guidelines. After electroporation, ES cells can typically be recovered under appropriate incubation conditions. The cells are then screened for the presence of the knockout construct.

[0181] Screening can be accomplished using a variety of methods. If the marker gene is an antibiotic resistance gene, the ES cells may otherwise be cultured in the presence of a lethal concentration of the antibiotic. The surviving ES cells have probably incorporated the knockout construct. If the marker gene is other than an antibiotic resistance gene, a Southern blot of ES cell genomic DNA can be probed with a DNA sequence designed to hybridize only to the marker sequence. Alternatively, PCR can be used. Finally, when the marker gene is a gene encoding an enzyme capable of detecting its activity (for example, β-galactosidase), an enzyme substrate can be added to cells under appropriate conditions, and the enzyme activity can be analyzed. Those skilled in the art will be familiar with other useful markers and how to detect their presence in the resulting cells. All such markers are intended to be included within the teachings of the present invention.

The knockout construct may be integrated at several locations in the ES cell genome, or at different locations in the genome of each ES cell due to the occurrence of random insertion events. The desired insertion position is at a position complementary to the DNA sequence to be knocked out, for example, an onserin coding sequence, a transcription regulatory sequence, and the like.
Typically, less than about 1-5 percent of ES cells that take up the knockout construct will, in fact, take up the knockout construct at the desired location. To identify ES cells that have adequately taken up the knockout construct, ES cells can be identified using standard methods.
Total DNA can be extracted from cells. The DNA can then be probed on a Southern blot using a probe or probes designed to hybridize in a specific pattern to genomic DNA that is digested with a particular restriction enzyme. Alternatively, or additionally, by PCR using probes specially designed to amplify DNA fragments of a particular size and sequence,
Genomic DNA can be amplified (ie, only cells that contain the knockout construct at the appropriate location will produce DNA fragments of the appropriate size).

[0183] After identifying the appropriate ES cells containing the knockout construct at the appropriate locations, the cells can be inserted into the embryo. Insertion may be performed by various methods known to those skilled in the art, but the preferred method is microinjection. For microinjection, about 10-30 cells are collected in a micropipette and injected into the embryo at the appropriate stage of development to allow the foreign ES cells containing the knockout construct to integrate into the developing embryo. For example, transformed ES cells can be microinjected into blastocysts.

After the ES cells have been introduced into the embryo, the embryo may be implanted into the uterus of a pseudopregnant foster mother for pregnancy. Although any foster mother may be used, the foster mother is typically selected for its ability to produce and breed children well and for its ability to care for children. Such foster mothers are typically prepared by crossing with a vasectomized male of the same species. The stage of pseudopregnancy foster mother is important for a successful transplant, which is species dependent.

Foster offspring may first be screened for onserin disruption and DNA from progeny tissues may be screened for the presence of the knockout construct using Southern blot and / or PCR as described above. Progeny that appear to be mosaic may then be crossed to each other to produce homozygous knockout animals, provided they are considered to have a knockout construct in the germline. Homozygotes can be identified by Southern blotting of equal amounts of genomic DNA from animals that are the product of the cross and animals that are known heterozygotes and wild-type animals.

Other methods for identifying and characterizing knockout progeny are possible. For example, Northern blots can be used to probe mRNA for the presence or absence of transcription of the onserin gene, marker gene, or both. further,
When Western blots are used to probe antibodies to the onserin protein, or genes, the antibodies to the marker gene product are used to probe the Western blots to determine the expression levels of the knocked out onserin genes in various tissues of offspring. Loss). Finally, in situ analysis (eg, fixation of cells and labeling with antibodies) and / or FACS (fluorescence activated cell sorting) analysis of various cells from the progeny can be performed using appropriate antibodies or onserin ligands. To see for the presence or absence of the knockout construction gene product.

[0187] Animals containing one or more knockout constructs and / or one or more transgene expression constructs are prepared in any of several ways. A preferred preparation method is
To produce a series of animals each containing the desired transgenic phenotype. Such animals are bred together in a series of crosses, backcrosses and selections to ultimately yield a single animal containing all desired knockout and / or expression constructs, where the animal is Other than the presence of the knockout construct and / or the transgene, are otherwise identical to the wild type (genetically identical). Thus, the transgenic avian species was engineered to express a first transgenic bird in which the wild-type onserin gene has been disrupted to express a mutant onserin that retains many other biological functions of the receptor. It can be produced by crossing with a second transgenic bird.

The transformed animals of the present invention, their progeny and cell lines provide some important uses that will be readily apparent to one of ordinary skill in the art. To illustrate, transgenic animals and cell lines have potential as prophylactic or therapeutic treatments for diseases involving abnormal expression or loss of the Onserin gene or abnormal or unwanted activation of receptor signaling. It is particularly useful for screening compounds. Screening for useful drugs involves administering a candidate drug to a transgenic animal over a range of doses and assaying at various time points the effect of the drug on the disease or disorder to be evaluated. Alternatively, or in addition, if appropriate, the drug can be administered before or simultaneously with exposure to induction of the disease.

In one embodiment, the candidate compound is administered to the transgenic animal over a range of doses and the candidate compound is screened by assessing the animal's physiological response to the compound over time. Administration can be oral or by suitable injection, depending on the chemical nature of the compound to be evaluated. In some cases, it is appropriate to administer the compound together with co-factors that enhance the effect of the compound.

When screening cell lines from a subject transgenic animal for compounds useful in treating a variety of disorders, a test compound is added to the cell culture medium at an appropriate time and the cellular response to the compound is determined by the appropriate biochemical And / or assessed over time using histological assays. In some cases, it may be appropriate to provide the relevant compound to the culture medium along with cofactors that enhance the effect of the compound.

The polynucleotides and proteins of the present invention are expected to exhibit one or more uses or biological activities identified below, including those associated with the assays cited herein. You. The uses or activities described for the proteins of the invention may be determined by the administration or use of such proteins, or by the administration or use of polynucleotides encoding such proteins (eg, as gene therapy agents or DNs).
A as a vector suitable for the introduction of A).

Research Uses and Utility The polynucleotides provided by the present invention may be used for various purposes by a research population. For analysis, characterization or therapeutic use, as a marker of tissue in which the corresponding protein is preferentially expressed (constitutively or at a particular stage of tissue differentiation or development or in a disease state), and To identify potential genetic diseases as compared to the patient's endogenous DNA, as chromosomal markers or tags (if labeled) for identification of chromosomes or mapping of relevant gene locations, as molecular weight markers for Southern gels The use of known sequences in the process of discovering other novel polynucleotides as a source for obtaining PCR primers for genetic fingerprinting to find new related DNA sequences to use as hybridization probes As a probe for subtraction, use a "gene chip" or other To select and generate oligomers for binding to carriers, to test expression patterns, to generate anti-protein antibodies using DNA immunization, and to generate anti-DNA antibodies, or Polynucleotides can be used as antigens to induce a response. If encoding a protein that binds or potentially binds to another protein (e.g., as in a receptor-ligand interaction), identify the other protein that binds, and / or an inhibitor of the binding interaction Polynucleotides can be used in an interaction trap assay (e.g., as described in Gyuris et al., Cell 75: 791-803 (1993)) to identify The proteins provided by the present invention may be used in assays to determine biological activity, including a group of multiple proteins for high-throughput screening; to generate antibodies or induce other immune responses; As a reagent (including a labeling reagent) in an assay designed to quantitatively determine the level of a protein (or its receptor) in a biological fluid; the corresponding protein is preferentially expressed (constitutively Or expressed at a particular stage of tissue differentiation or development, or in a disease state) as a marker for tissue; and, of course, may be used to isolate relevant receptors or ligands. Where a protein binds or potentially binds to another protein, the protein can be used to identify other proteins that bind, or to identify inhibitors of the binding interaction. Using proteins involved in these binding interactions, screening for peptides or small molecular inhibitors or agonists for the binding interaction can also be performed. Any or all of these research applications can be evolved into reagent grade or kit formats for commercialization as research products. Methods for performing the above uses are well known to those skilled in the art. A literature disclosing such a method can be found in "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Sp.
ring Harbor Laboratory Press, Sambrook, J., EF Fritsch and T. Maniatis
eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Tec
hniques ", Academic Press, Berger, SL and AR Kimmel eds., 1987.

Use as Nutrition The polynucleotides and proteins of the present invention can be used as nutrient sources or additives. Such uses include, but are not limited to, use as a protein or amino acid additive, use as a carbon source, use as a nitrogen source, and use as a carbohydrate source. In such cases, the proteins or polynucleotides of the present invention may be added as food for a particular organism, or may be administered as a separate solid liquid formulation such as in the form of a powder, pill, solution, suspension or capsule. . In the case of a microorganism, the protein or polynucleotide of the present invention can be added to a medium in which the microorganism is cultured.

Cytokines and Cell Proliferation / Differentiation Activity The proteins of the present invention may exhibit cytokine activity, cell proliferation activity (induction or inhibition) or cell differentiation activity (induction or inhibition), or produce other cytokines in certain cell populations. Can be induced. Many proteinaceous factors (including all known cytokines) that have been found to date have demonstrated activity in one or more factor-dependent cell proliferation assays, thus making the assay a convenient method of confirming cytokine activity. Serve as. The activity of the protein of the present invention is determined in cell lines (32D, DA
2, DA1G, T10, B9, B9 / 11, BaF3, MC9 / G, M + (pr
eB M +), 2E8, RB5, DA1, 123, T1165, HT2, CTL L2, TF-1, Mo7e and CMK). Confirmed by either of The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for T cell or thymocyte proliferation are described in Current Protocols in I.
mmunology, Ed by JE Coligan, AM Kruisbeek, DH Margulies, EM She
vach, W Strober, Pub.Green Publishing Associates and Wiley-Interscienc
e (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Ch
apter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137: 3
494-3500, 1986; Bertagnolli et al., J. Immunol. 145: 1706-1712, 1990; Ber
tagnolli et al., Cellular Immunology 133: 327-341, 1991; Bertagnolli, et
al., J. Immunol. 149: 3778-3783, 1992; Bowman et al., J. Immunol. 152: 17
56-1761, 1994, including, but not limited to. Assays for cytokine production and / or proliferation of spleen cells, lymph node cells or thymocytes are described in Polyclonal T cell stimulation, Kruisbeek, AM
d Shevach, EM In Current Protocols in Immunology. JEea Coligan eds
Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Meas
urement of mouse and human Interferon g, Schreiber, RD In Current Pro
tocols in Immunology. JEea Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John
Includes, but is not limited to, those described in Wiley and Sons, Toronto. 1994. Assays for proliferation and differentiation of hematopoietic and lymphogenic cells are described in the Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottoml
y, K., Davis, LS and Lipsky, PE In Current Protocols in Immunology.
JEea Coligan eds.Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toron
to. 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.
USA 80: 2931-2938, 1983; Measurement of mouse and human interleukin 6
-Nordan, R. In Current Protocols in Immunology. JEea Coligan eds. V
ol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al.,
Proc. Natl. Acad. Sci. USA 83: 1857-1861, 1986; Measurement of human I
nterleukin 11-Bennett, F., Giannotti, J., Clark, SC and Turner, K. J
. In Current Protocols in Immunology. JEea Coligan eds. Vol 1 pp.
6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and hum
an Interleukin 9-Ciarletta, A., Giannotti, J., Clark, SC and Turner,
KJ In Current Protocols in Immunology. JEea Coligan eds. Vol 1 p
p. 6.13.1, including but not limited to those described in John Wiley and Sons, Toronto. 1991. Assays for the response of T cell clones to antigens, specifically identifying APC-T cell interactions as well as direct T cell effects by measuring proliferation and cytokine production, are described in Current Protocols in Immunology, Ed by JE.
Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W Strober, Pub.
Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro
assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their ce
llular receptors; Chapter 7, Immunologic studies in Humans); Weinberger
Natl. Acad. Sci. USA 77: 6091-6095, 1980; Weinberger et al.
, Eur. J. Immun. 11: 405-411, 1981; Takai et al., J. Immunol. 137: 3494-35.
00, 1986; Takai et al., J. Immunol. 140: 508-512, 1988, but are not limited thereto.

Immunostimulatory or Suppressive Activity The protein of the present invention may also exhibit immunostimulatory or immunosuppressive activity, including but not limited to the activity in the assays described herein. Protein is
It may be useful in a variety of deficiencies and disorders, including severe immunodeficiency complications (SCID), eg, in up-regulating or down-regulating the proliferation of T and / or B lymphocytes, and in N
It may be useful in activating the cytolytic activity of K cells and other cell populations. These immunodeficiencies can be genetic and can include viral (
For example, it may be caused by HIV) and bacterial or fungal infections, or may be caused by an autoimmune disease. More particularly, infectious diseases caused by viruses, bacteria, fungi or other infectious agents, such as various fungal infections such as HIV, hepatitis virus, herpes virus, mycobacteria, Leishmania, malaria and Candida species, It can be treated with the protein of the present invention. Of course, in this regard, the proteins of the present invention may be used where a boost to the immune system is needed, ie, in the treatment of cancer. Autoimmune diseases that may be treated using the protein of the present invention include, for example, multiple sclerosis,
Systemic deep elitomades, rheumatoid arthritis, autoimmune pneumonia, Guillain-Barre syndrome, autoimmune thyroiditis, insulin-dependent diabetes mellitus, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory Eye diseases. Such proteins of the invention may be used in the treatment of allergic reactions and conditions, such as asthma or other respiratory diseases. Other conditions for which immunosuppression is desired, including, for example, asthma (particularly allergic asthma) and related respiratory problems may be treated with the proteins of the invention. Other conditions for which immunosuppression is desired (including, for example, organ transplantation)
May be treated using the protein of the present invention. The proteins of the present invention can also be used to enable an immune response 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 interfering with the induction of an immune response. The function of activated T cells may be inhibited by suppressing T cell responses, or by inducing specific resistance in T cells, or both. In general, immunosuppression of a T cell response is an active, non-antigen-specific process that requires continuous exposure of T cells to an inhibitor. Tolerance means non-responsiveness or anergy in T cells, and differs from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent is stopped.
Operationally, resistance may be indicated by a lack of a T cell response when exposed to a specific antigen in the absence of a tolerizing agent. Down-regulating or preventing one or more antigen functions, including but not limited to B-lymphocyte antigen functions (such as, for example, B7), eg, high levels of lymphokines by activated T cells Interference with synthesis may be useful in tissue, skin and organ transplantation and graft versus host disease (GVHD). For example, blocking T cell function should reduce tissue destruction in tissue transplantation. Typically, in tissue transplantation, graft rejection is initiated by the recognition of the tissue piece as foreign by T cells, followed by an immune response that destroys the graft. A monomeric form having the activity of a molecule that inhibits or blocks the interaction of a B7 lymphocyte antigen with its native ligand on immune cells (another B lymphocyte antigen (eg, B7-1, B7-3)) Mixed with peptides,
Alternatively, pre-transplant administration of a soluble, monomeric form of a peptide with B7-2 activity alone, induces binding of the molecule to its native ligand on immune cells without transmitting a responsive costimulatory signal. there is a possibility. Blocking B lymphocyte antigen function in this regard prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may be sufficient to cause a loss of T cell response. The induction of long-term tolerance by B lymphocyte antigen blocking reagents may obviate the need for repeated administration of these blocking reagents. To achieve sufficient immunosuppression or resistance in a subject, it may also be necessary to block the function of the B lymphocyte antigen combination. The efficacy of individual blocking reagents in preventing organ transplant rejection or GVHD can be evaluated using animal models that can predict efficacy in humans. Examples of suitable systems that can be used include allogeneic heart grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which are used to test the immunosuppressive effects of CTLA4Ig fusion proteins in vivo. (Lensch
ow et al., Science 257: 789-792 (1992) and Turka et al., Proc Natl. Acad.
Sci. USA, 89: 11102-11105 (1992)). In addition, a rat model of GVHD (Paul ed., Fundamental Immunology, Ravan Press, New York, 1
989, pp. 846-847) can be used to determine the blocking effect of B lymphocyte antigen function on the progression of the disease in vivo. Also, blocking antigen function may be therapeutically useful for treating autoimmune diseases. Many autoimmune diseases are the result of inappropriate activation of T cells that are reactive to self tissue and promote the production of cytokines and autoantibodies that are involved in the pathology of the disease. Interfering with the activation of autoreactive T cells may reduce or eliminate the symptoms of the disease. Receptors for B lymphocyte antigens: autoantibodies or T cell-derived that inhibit T cell activation using administration of reagents that block T cell costimulation by disrupting ligand interactions and may be involved in the disease process Can interfere with the production of cytokines. In addition, blocking reagents can induce antigen-specific resistance of autoreactive T cells that can lead to long-term remission of the disease. The effectiveness of a blocking reagent in preventing or ameliorating an autoimmune disease can be determined using a number of well-characterized animal models for human autoimmune diseases. Examples are murine experimental autoimmune encephalitis, MRLlpr / lpr mice or N
Systemic deep erythematosus, murine autoimmune collagen arthritis in ZB hybrid mice, diabetes in NOD mice and BB rats, and experimental myasthenia gravis in rats (Paul ed., Fundamental Immunology, Raven Press,
New York, 1989, pp. 840-856). Up-regulation of antigen function (preferably B-lymphocyte antigen function) as a means to up-regulate the immune response is also therapeutically useful.
Up-regulation of the immune response may be in the form of promoting an existing immune response or eliminating the initial immune response. For example, enhancing an immune response by stimulating B lymphocyte antigen function may be useful in the case of a viral infection.
In addition, systemic viral diseases such as influenza, common cold, and encephalitis may be ameliorated by systemic administration of a stimulatory form of B lymphocyte antigen. Alternatively, the T cells are taken from a patient and co-stimulated in vitro with the viral antigen to which the ACPs expressing the peptide of the invention have been added, or combined with a stimulatory form of the soluble peptide of the invention and then in vitro. By reintroducing the T cells activated in the above into the patient, the antiviral immune response in the infected patient may be promoted. Another method of promoting an antiviral immune response is
The infected cells are removed from the patient and transfected with them a nucleic acid encoding a protein of the invention as described herein so that the cells express all or part of the protein on their surface. Will be re-introduced. The infected cells would then be able to transmit the costimulatory signal to the T cells in vivo and thereby activate the T cells. In another application, up-regulation or promotion of antigen function (preferably B lymphocyte antigen function) may be useful in inducing tumor immunity. Administering a tumor cell (eg, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the invention to a tumor-specific tumor in the subject Can overcome resistance. If desired, tumor cells can be transfected to express the peptide combination. For example, an expression vector that directs the expression of tumor cells obtained from a patient in combination with only a peptide having B7-2-like activity or a peptide having B7-1-like activity and / or B7-3-like activity It can be transfected ex vivo. The transfected tumor cells are returned to the patient and the peptide is expressed on the surface of the transfected cells. Alternatively, gene therapy can be used to target tumor cells for transfection in vivo. The presence of a peptide of the invention having the activity of a B lymphocyte antigen on the surface of tumor cells provides the necessary costimulatory signals for T cells to induce an immune response to transfected tumor cells mediated by T cells I do. In addition, MHC
Tumor cells lacking class I or MHC class II molecules, or sufficient MHC
Class I or tumor cells can not be re-expressed MHC class II molecules, MHC class Iα chain protein and beta ₂ microglobulin protein or an MHC class IIα chain Contact and MHC class IIβ chain all or part of the protein (e.g., truncations protein The nucleic acid encoding the cytoplasmic domain can be transfected, thereby allowing the expression of class I or class II MHC proteins on the cell surface. Expression of appropriate Class I or Class II HMC in combination with a peptide having the activity of a B lymphocyte antigen (eg, B7-1, B7-2, B7-3) is transfected by T cells. Induces an immune response against tumor cells. If desired, a gene encoding an antisense construct that blocks the expression of an MHC class II binding protein, such as an invariant chain, can be co-transfected with a DNA encoding a peptide having B lymphocyte antigen activity. It can also enhance the presentation of tumor-associated antigens and induce tumor-specific immunity. Thus, induction of an immune response mediated by T cells in a human subject may be sufficient to overcome tumor-specific resistance in the subject.

The activity of the protein of the invention may be measured, inter alia, by the following method: Suitable assays for thymocyte or spleen cell cytotoxicity are described in Current Protocols
in Immunology, Ed by JE Coligan, AM Kruisbeek, DH Margulies, EM
Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Intersc
ience (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: 2488-2492, 1981; Herrmann et al., J. Immunol. 128:
1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai e
t 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.
Herrmann et al., J. Immunol. 128: 1968-1974, 1982; Handa et al., 1981;
J. Immunol. 135: 1564-1572, 1985; Takai et al., J. Immunol. 137: 3494-3
500, 1986; Bowmanet al., J. Virology 61: 1992-1998; Takai et al., J. Imm
unol. 140: 508-512, 1988; Bertagnolli et al., Cellular Immunology 133: 3
27-341, 1991; including but not limited to the assays described in Brown et al., J. Immunol. 153: 3079-3092, 1994. Assays for T cell-dependent immunoglobulin responses and isotype switching, particularly those that modulate T cell-dependent antibody responses and identify proteins that affect Th1 / Th2 profiles, are described in Maliazewski, J. Immunol. 144: 3028-3033, 1990, including but not limited to assays for B cell function as described in In vitro antibody production, Mond, JJ and Brunswick, M. In Current.
Protocols in Immunology JEColigan eds.Val 1 pp.3.8.1-3.8.16, John Wile
y and Sons, Tronto 1994. Mixed lymphocyte reaction (MLR) assays, which identify proteins that primarily produce Th1 and CTL responses, are described in Current Protocols in Immunology, Ed by
JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W Strober,
Pub.Green Publishing Associates and Wiley-Interscience (Chapter 3, In
Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunol
ogic 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. Im.
munol. 149: 3778-3783, 1992, but is not limited thereto. Dendritic cell-dependent assays, specifically identifying proteins expressed by dendritic cells that activate intact T cells, are described by Guery et al., J. Immunol. 134: 536-544.
, 1995; Inaba et al., Journal of Experimental Medicine 173: 549-559, 199
1; Macatonia et al., Journal of Immunology 154: 5071-5079, 1995; Porgado
r et al., Journal of Experimental Medicine 182: 255-260, 1995; Nair et a
l., 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, including, but not limited to. Assays for lymphocyte survival / apoptosis, specifically identifying proteins that prevent apoptosis following superantibody induction, as well as proteins that regulate lymphocyte homeostasis, are described by Darzynkiewicz et al., Cytometry 13: 795-808, 1992; Gorczyca et al.
, Leukemia 7: 659-670, 1993; Gorczyca et al., Cancer Research 53: 1945-1
951, 1993; Itoh et al., Cell 66: 233-243, 1991; Zacharchuk, Journal of I
mmunology 145: 4037-4045, 1990; Zamai et al., Cytometry 14: 891-897, 199
3; including but not limited to the assays described in Gorczyca et al., International Journal of Oncology 1: 639-648, 1992. Early stage T cell commitment and development assays
Antica et al., Blood 84: 111-117, 1994; Fine et al., Cellular Immunology 15
5: 111-122, 1994; Galy et al., Blood 85: 2770-2778, 1995; Toki et al., Pr
Natl. Acad. Sci. USA 88: 7548-7551, 1991, including, but not limited to.

Hematopoietic modulatory activity The proteins of the present invention are useful in the regulation of hematopoiesis and, therefore, in the treatment of bone marrow and lymphocyte deficiencies. Even minimal biological activity that supports colony forming cells or factor-dependent cell lines has been implicated in regulating hematopoiesis.
For example, supporting the growth of erythroid progenitor cells alone, or in combination with other cytokines, for example, showing utility in treating various anemias, or in combination with radiation therapy / chemotherapy, erythroid progenitor cells And / or stimulating the production of erythroblasts; supporting the proliferation of bone marrow cells such as granulocytes and monocytes / macrophages (ie, traditional CSF activity), eg, in combination with chemotherapy, Useful in preventing myelosuppression that occurs; supporting proliferation of megakaryocytes and then platelets, and thereby preventing or treating various platelet disorders such as thrombocytopenia; It is also commonly used instead of or in complement to platelet infusion; and / or hematopoietic stem cells (mature Becomes all of hematopoietic cells, therefore, a variety of stem cell disease (
It is usually a disease that is treated by transplantation, and is useful in supporting the growth of, for example, aplastic anemia and paroxysmal nocturnal hemoglobinuria), and is also useful in vivo or ex vivo. It is also useful in repopulating the stem cell compartment as normal cells after radiation / chemotherapy with (ie, combined with bone marrow transplantation), or after being genetically engineered for gene therapy. In particular, the activity of the protein of the present invention may be measured by the following method. Assays suitable for the proliferation and differentiation of various hematopoietic cell lines have already been cited. Assays for embryonic stem cell differentiation, particularly those that identify proteins that affect embryonic hematopoiesis, are 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., B
lood 81: 2903-2915, 1993, including, but not limited to. Assays for stem cell survival and differentiation (particularly identifying proteins that regulate lympho-hematopoiesis) are described in Methylcellulose colony forming assays, Freshney, MG In Cultu
re of Hematopoietic Cells.RI 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 ce
lls with high proliferative potential, McNiece, IK and Briddell, RA
In Culture of Hematopoietic Cells.RI Freshney, et al. Eds.Vol pp. 2
3-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental H
ematology 22: 353-359, 1994; Cobblestone area forming cell assay, Ploema
cher, RE In Culture of Hematopoietic Cells.RI Freshney, et al. eds.
Vol pp. 1-21, Wiley-Liss, Inc., New York, NY. 1994; Long term bone ma
rrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M.
and Allen, T. In Culture of Hematopoietic Cells.RI Freshney, et al.
eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term c
ulture initiating cell assay, Sutherland, HJ In Culture of Hematopoiet
ic Cells.RI Freshney, et al. eds.Vol pp. 139-162, Wiley-Liss, Inc.,
Includes, but is not limited to, the assays described in New York, NY. 1994.

Tissue Proliferation Activity The protein of the present invention may be used in compositions used for the growth or regeneration of bone, cartilage, keys, ligaments and / or nervous tissue, as well as wound healing and tissue repair, and in the treatment of burns, lacerations and ulcers Has utility. The protein of the present invention, which induces cartilage and / or bone growth in an environment where bone is not normally formed, has use in healing fractures and cartilage damage or defects in humans and other animals. Such formulations using the protein of the present invention are useful for reducing closed and open fractures and for improving fixation of artificial joints. De novo bone formation induced by osteogenic agents contributes to the repair of congenital, traumatic or oncological resection-induced craniofacial defects, and is also useful in cosmetic surgery. The protein of the present invention may be used in the treatment of periodontal disease and other tooth restoration processes. Such agents provide an environment for attracting osteogenic cells, stimulate the growth of osteogenic cells,
Alternatively, it can induce differentiation of osteogenic cell precursors. The protein of the invention may be osteoporotic or osteoarthritis, for example, by stimulating bone and / or cartilage repair or by blocking the process of tissue destruction mediated by inflammation or inflammatory processes (collagenase activity, osteoclast activity, etc.). May also be useful in the treatment of Another category of tissue development activity that may be due to the proteins of the present invention is key / ligament formation. Proteins of the invention that induce the formation of key / ligament-like or other tissues in an environment in which such tissues are not normally formed can be used to prevent key or ligament laceration, deformation and other key or ligament defects in humans and other animals. Applied to healing, Such formulations using the key / ligament-like tissue inducing protein may be used to prevent damage to the key or ligament tissue as well as to improve the fixation of the key or ligament to bone or other tissue. The de novo key / ligament-like tissue formation induced by the composition of the present invention contributes to the repair of congenital, traumatic, or oncological resection-induced craniofacial defects, and furthermore the key or ligament adhesion Or, it is also useful in cosmetic surgery for repair. The compositions of the present invention provide an environment for attracting key- or ligament-forming cells, stimulate the growth of key- or ligament-forming cells, and induce the differentiation of key- or ligament-forming cell precursors. Or induce the growth of key / ligament cells or progenitors ex vivo so as to effect tissue repair when returned to vivo.
The compositions of the present invention are also useful for keratitis, carpal tunnel syndrome and other key or ligament defects. The compositions of the present invention may include a suitable matrix and / or sequestering agent, such as a carrier well known in the art. The protein of the present invention is useful for the proliferation of nerve cells and the regeneration of nerve and brain tissues, ie, central and peripheral nervous system diseases and neuropathies and mechanical diseases and traumatic diseases (including degeneration, death or trauma to nerve cells or nerve tissues). ) May also be useful. More particularly, diseases of the peripheral nervous system, such as peripheral nerve injury, peripheral and localized neuropathy, and Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral spinal sclerosis and Shy-Drager
The protein may be used in the treatment of diseases of the central nervous system, such as the syndrome. Additional conditions that may be treated according to the present invention include mechanical and traumatic diseases such as spinal cord diseases, cerebrovascular diseases such as head trauma and stroke. Peripheral neuropathy caused by chemotherapy or other medical therapies can be treated with the proteins of the present invention. The protein of the present invention is also useful for promoting more preferable and prompt closure of non-healing wounds including (but not limited to) pressure ulcers, ulcers associated with vascular dysfunction, surgical and trauma wounds, and the like. It is possible. The protein of the present invention may also be used in other tissues such as organs (including pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle or cardiac muscle), and vascular (including vascular endothelium) tissue. Or an activity for promoting the growth of cells constituting such a tissue. Part of the desired effect may be the regeneration of normal tissue by suppressing fibrotic scarring. The proteins of the present invention may also exhibit angiogenic activity. The proteins of the present invention may also be useful in protecting or regenerating the intestine and treating conditions caused by lung or liver fibrosis, reperfusion injury of various tissues, and systemic cytokine damage. The protein of the present invention promotes or suppresses differentiation of the above-mentioned tissue from precursor tissue or cells;
Alternatively, it may be useful for suppressing the growth of the tissue. The activity of the protein of the invention may be measured, inter alia, by the following method: Tissue regeneration activity assay is described in WO 95/16035 (bone, cartilage, key).
International Publication WO95 / 05846 (nerves, neurons); International Publication WO91 / 05
7491 (skin, endothelium), but is not limited thereto. Wound healing activity assays are described in Winter, Epidermal Wound Healing, pps. 71-112.
(Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc.,
Including, but not limited to, those described in Chicago (modified by Eaglstein and Mertz, J. Invest. Dermatol 71: 382-384 (1978)).

Activin / Inhibin Activity The proteins of the present invention may also exhibit activin- or inhibin-related activity. Inhibins are characterized by their ability to inhibit follicle stimulating hormone (FSH) release,
Activins are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, the protein of the present invention, alone or in the form of a heterodimer with a member of the inhibin α family, reduces the fertility of female mammals and reduces the spermatogenesis of male mammals. Can be useful as Administration of a sufficient amount of another inhibin can induce infertility in these mammals. Alternatively, the protein of the present invention, as a homodimer or as a heterodimer with subunits of other proteins of the inhibin-β group, can be used to stimulate fertility based on the ability of activin molecules to stimulate FSH release from anterior pituitary cells. May be useful as therapeutic agents that induce For example, US Pat.
See 98885. The proteins of the invention may also be useful for improving reproduction in sexually immature mammals, resulting in increased fertility during the life of livestock such as cattle, sheep and pigs. The activity of the protein of the invention may be measured, inter alia, by the following method: Activin / inhibin activity assays are described in Vale et al., Endocrinology 91:
562-572, 1972; Ling et al., Nature 321: 779-782, 1986; Vale et al., Nat
ure 321: 776-779, 1986; Mason et al., Nature 318: 659-663,1985; Forage e
Natl. Acad. Sci. USA 83: 3091-3095, 1986, including but not limited to the assays described in US Pat.

Chemotaxis / chemokinetic activity The protein of the present invention can be used for the chemotactic or chemokinetic activity of mammalian cells such as monocytes, neutrophils, T cells, mast cells, eosinophils and / or endothelial cells (for example, (Acting as a chemokine). Chemotactic and chemokinetic proteins can be used to recruit or attract a desired cell population to a desired site of action. Chemotaxis or chemokinesis proteins are particularly advantageous for treating injuries and other trauma to tissues and for treating local infections. For example, attracting lymphocytes, monocytes or neutrophils to a tumor or site of infection can improve the immune response to the tumor or infectious agent. Certain proteins or peptides have chemotactic activity on a particular cell population and, when stimulable, direct or indirectly direct the movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate the directed movement of the cell. Whether a particular protein has chemotactic activity on a cell population can be readily determined by using such a protein or peptide in a known assay for cell chemotaxis. The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for chemotactic activity (assays to identify proteins that induce or interfere with chemotaxis) induce migration across cell membranes. Assays that measure the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for migration and adhesion are described in Current Protocols in Immunology, Ed by JE
Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W. Strober, Pub.
reene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measur
ement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin.
Invest. 95: 1370-1376, 1995; Lind et al. APMIS 103: 140-146, 1995; Mulle
r 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, including, but not limited to.

Hemostatic and thrombolytic activity The proteins of the present invention may also exhibit hemostatic or thrombolytic activity. As a result, such proteins are useful in the treatment of various coagulation disorders, including genetic disorders such as hemophilia, or in the treatment of injuries caused by trauma, surgery or other causes and other hemostasis. Can be promoted. The protein of the present invention may be useful for the treatment and prevention of thrombolysis or inhibition of thrombus formation and conditions resulting therefrom (eg, myocardial infarction and central nervous system infarction (eg, stroke)). The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for haemostatic and thrombolytic activities are described in Linet et al., J. Clin. Pharmacol.
26: 131-140,1986; Burdick et al., Thrombosis Res. 45: 413-419, 1987; Hum
phrey et al., Fibrinolysis 5: 71-79 (1991); Schaub, Prostaglandins 35: 4
67-474, 1988, including, but not limited to.

Receptor / Ligand Activity The protein of the present invention may also exhibit activity as a receptor, receptor ligand or inhibitor or agonist of receptor / ligand interaction. Examples of such receptors and ligands include cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, cells-
Receptors involved in cell interactions and their ligands (such as cell adhesion molecules (selectins, integrins and their ligands)) and receptors / ligands involved in antigen presentation, antigen recognition, development of cellular and humoral immune responses But not limited thereto. Receptors and ligands are also useful for screening potential peptide or small molecule inhibitors for related receptor / ligand interactions. The proteins of the present invention, including but not limited to receptor and ligand fragments, may themselves be useful as inhibitors of receptor / ligand interaction. The activity of the protein of the invention may be measured, inter alia, by the following method: Suitable assays for receptor-ligand activity are described in Current Protocols in Immunolog.
y, Ed by JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach,
W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (C
hapter 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, 1
987; 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. Metho.
ds 175: 59-68, 1994; Stitt et al., Cell 80: 661-670, 1995, including, but not limited to.

Anti-inflammatory activity The protein of the present invention can exhibit anti-inflammatory activity. Anti-inflammatory activity is achieved by stimulating cells involved in the stimulus response, resulting in cell-cell interactions (eg, adhesion).
Inhibits or promotes the chemotaxis of cells involved in the inflammatory process, thereby inhibiting or promoting cell extravasation,
Alternatively, it is exerted by stimulating or suppressing the production of other factors that more directly inhibit or promote the inflammatory response. Symptoms of inflammation (
Includes chronic or acute symptoms, infection-related inflammation (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, lethal endotoxin injury, arthritis, complement mediated Super-acute rejection,
Nephritis, lung injury induced by cytokines or chemokines, inflammatory bowel disease, Crohn's disease, or diseases resulting from overproduction of cytokines such as TNF or IL-1). . The proteins of the invention may also be useful for treating anaphylaxis and hypersensitivity to antigenic substances or materials.

Cadherin / Tumor Inhibition Inhibitory Activity Cadherin is a calcium-dependent adhesion molecule and appears to play a major role during development, particularly in determining specific cell types. Loss or alteration of normal cadherin expression can induce changes to cell adhesion properties associated with tumor growth and metastasis. Cadherin dysfunction has also been implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (autoimmune cutaneous bullous disease), Crohn's disease, and some developmental abnormalities. The cadherin superfamily contains over 40 members, each with a different expression pattern. All members of the superfamily share common conserved extracellular repeats (cadherin domains), but show structural differences in other parts of the molecule. Calcium is required for their adhesion because the cadherin domains bind to calcium and form their tertiary structure. Since the small number of amino acids in the first cadherin domain provides the basis for homophilic adhesion, modification of this recognition site can alter the specificity of cadherin, thereby recognizing only itself Instead, the mutant molecule will be able to bind to different cadherins. In addition, some cadherins are involved in heterophilic adhesion to other cadherins. E-cadherin, a member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in the tumor, the malignant cells become invasive and the cancer metastasizes. Transfecting a polynucleotide expressing E-cadhedrin into a cancer cell line restores the altered cell morphology, restores adhesion between cells and to other substrates, reduces cell growth rates, By dramatically reducing the growth of dependent cells,
The changes associated with cancer were reversed. Thus, reintroducing E-cadhedrin expression returns the carcinoma to a less advanced stage. Other cadherins may have the same inhibitory role in carcinomas from other tissue types. Therefore, cancer can be treated using the protein of the present invention having cadherin activity and the polynucleotide of the present invention encoding such a protein. Introducing such proteins or polynucleotides into cancer cells may reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression. Cancer cells are also known to express cadherins of different tissue types than originally, so these cancer cells can invade different tissues and metastasize. The protein of the present invention having cadherin activity, and the polynucleotide of the present invention encoding such a protein replace cadherin inappropriately expressed in these cells, restore normal cell adhesion properties, and reduce the tendency of cells to metastasize. Or can be removed. Furthermore, an antibody that recognizes and binds to cadherin can be obtained using the protein of the present invention having cadherin activity and the polynucleotide of the present invention encoding such a protein. Such antibodies can also be used to block the adhesion of inappropriately expressed tumor cells cadherin, so that the cells do not form tumors elsewhere. Such anti-cadhedrin antibodies can be used as markers for cancer grade, pathological type, and prognosis. That is, as the cancer progresses, the expression of cadherin decreases, and by using a cadherin-binding antibody, the decrease of cadherin expression can be detected. Using a fragment of a protein of the present invention having cadherin activity, preferably a decapeptide at the cadherin recognition site, and a polynucleotide of the present invention encoding such a protein fragment, to bind to cadherin and prevent cadherin binding that produces undesirable effects Can also block cadherin function. Further, using a fragment of the protein of the present invention having cadherin activity, preferably a truncated soluble cadherin fragment known to be stable in the circulatory system of cancer patients, and a polynucleotide encoding such a protein fragment, Correct cell-cell adhesion can also be disrupted. Assays for cadherin adhesion and entry inhibitory activity are described in Hortsch et al. J Bio
l Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552
, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.

Tumor Inhibitory Activity In addition to the activities described above for the immunological treatment or prevention of tumors, the proteins of the present invention may exhibit other antitumor activities. Certain proteins directly or indirectly affect tumor growth (eg,
(Via ADCC). Certain proteins act on tumor tissue or tumor precursor tissue to inhibit the formation of tissue necessary to support tumor growth (eg, by inhibiting angiogenesis) and to inhibit tumor growth. Tumor inhibitory activity may be exhibited by causing the production of a factor, agent or cell type, or by removing or inhibiting a factor, agent or cell type that promotes tumor growth.

Other Activities The proteins of the present invention may exhibit one or more of the following additional activities or effects: Infectious agents, including but not limited to bacteria, viruses, fungi, and other parasites Killing; height, weight, hair color, eye color, skin or other tissue pigmentation, or the size or form of an organ or body part (eg, breast augmentation or vice versa)
Effects on body characteristics (suppression or promotion); effects on digestion of consumed fat, protein or carbohydrates; appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) ) And effects on behavioral characteristics including, but not limited to, violent behavior; providing analgesic or other pain-relieving effects; promoting differentiation and proliferation of embryonic stem cells in non-hematopoietic lineages; hormones or endocrine Activity; for enzymes,
Correcting enzyme deficiency and treating related disorders; treating hyperproliferative disorders (eg, psoriasis); immunoglobulin-like activity (eg, the ability to bind antibodies or complement);
And the ability to act as an antigen in a vaccine composition to generate an immune response to such proteins or other substances or entities that cross-react with such proteins.

Equivalents Without further than routine experimentation, one of ordinary skill in the art would recognize or identify equivalents for the particular polypeptides, nucleic acids, methods, assays and reagents described herein. Will be able to. Such equivalents are considered to be within the scope of the present invention.

[0208]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/28 C07K 19/00 4C084 19/00 C12N 1/15 4C086 C12N 1/15 1/19 4H045 1 / 19 1/21 1/21 C12P 21/02 C 5/10 21/08 C12P 21/02 C12Q 1/68 A 21/08 G01N 33/50 C12Q 1/68 33/566 G01N 33/50 A61K 31/711 33 / 566 48/00 // A61K 31/711 (C12P 21/08 38/00 C12R 1:93) 48/00 C12N 15/00 ZNAA (C12P 21/08 5/00 A C12R 1:93) A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, C M, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE , ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US , UZ, VN, YU, ZW F term (reference) 2G045 AA29 AA34 AA35 AA40 CB01 CB17 CB26 DA12 DA13 DA14 DA36 D A77 FB02 FB03 4B024 AA01 AA11 BA44 BA80 CA04 CA09 DA01 DA02 DA05 DA11 EA04 GA11 HA14 4B063 QA01 QA13 QA18 QQ02 QQ42 QQ53 QR08 QR55 QR62 QS25 QS34 4B064 AG01 AG27 CA19 CC24 DA01 DA13 4B0AAAAAAAAAX4 BA02 BA44 NA14 ZA021 ZA022 ZA202 ZA812 ZB212 4C086 AA01 EA16 MA01 MA04 ZA02 ZA20 ZA81 ZB21 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA50 DA76 EA20 EA50 FA72 FA74

Claims (71)

[Claims] An onserin polypeptide produced by an isolation and / or recombinant method. 2. A mammalian onserin polypeptide produced by isolation and / or recombinant methods. 3. A human onserin polypeptide produced by isolation and / or recombinant methods. 4. A polypeptide comprising an onserin amino acid sequence and produced by isolation and / or recombinant methods. 5. The polypeptide of claim 4, wherein the onserin amino acid sequence is encoded by a nucleic acid that hybridizes to SEQ ID NO: 1 under stringent conditions. 6. The polypeptide of claim 4, wherein the Onserin amino acid sequence has at least 60% identity to SEQ ID NO: 2 or a portion thereof. 7. The polypeptide of claim 4 or 6, wherein the onserin amino acid sequence is at least 25 amino acid residues in length. 8. An isolated and / or recombinantly produced polypeptide comprising an onserin amino acid sequence sufficient to bind to at least one of Ca ²⁺ , catenin or cadherin, wherein the polypeptide comprises an onserin amino acid. A polypeptide whose sequence has at least 60% identity to residues 130-708 of SEQ ID NO: 2. 9. (i) regulating the differentiation of neural cells, (ii) regulating the survival of differentiated neural cells, (iii) regulating the proliferation of testicular germline cells, and / or (iv) kidney cells The polypeptide according to any one of claims 1 to 8, which regulates the growth of. 10. The polypeptide according to claim 1, which is a fusion protein. 11. The polypeptide according to any one of claims 1 to 8, which promotes differentiation of nerve cells or promotes survival of differentiated nerve cells. 12. The polypeptide of claim 11, wherein the neuron is selected from the group consisting of dopaminergic neurons, motor neurons, gabaergic neurons, serotonergic neurons, and adrenergic neurons. 13. The polypeptide according to claim 11, wherein the nerve cell is a striatal neuron. 14. The polypeptide according to claim 1, which is neurotrophic for nerve cells. 15. The polypeptide according to claim 1, which regulates spermatogenesis. 16. The polypeptide of claim 6, wherein the onserin amino acid sequence has at least 70% identity to SEQ ID NO: 2, or a biologically active fragment thereof. 17. The polypeptide of claim 6, wherein the Onserin amino acid sequence has at least 80% identity to SEQ ID NO: 2, or a biologically active fragment thereof. 18. The polypeptide of claim 6, wherein the Onserin amino acid sequence has at least 90% identity to SEQ ID NO: 2, or a biologically active fragment thereof. 19. The polypeptide of claim 6, wherein the Onserin amino acid sequence has at least 95% identity to SEQ ID NO: 2, or a biologically active fragment thereof. 20. The polypeptide according to claim 6, wherein the amino acid sequence of Onserin is identical to SEQ ID NO: 2, or a biologically active fragment thereof. 21. The polypeptide of claim 4, wherein the onserin amino acid sequence is encoded by a nucleic acid that hybridizes under high stringency conditions to the nucleic acid of SEQ ID NO: 1. 22. The polypeptide of claim 4, wherein the onserin amino acid sequence is encoded by a naturally occurring mammalian onserin gene. 23. The polypeptide of claim 4, wherein the onserin amino acid sequence is encoded by a naturally occurring human onserin gene. 24. The polypeptide of claim 7, wherein the Onserin amino acid sequence corresponds to a fragment consisting of at least 100 amino acid residues of the extracellular or intracellular domain of the Onthelin protein. 25. A single amino acid sequence comprising an onserin amino acid sequence that specifically binds to an onserin protein having the amino acid sequence of SEQ ID NO: 2 and immunologically cross-reacts with an antibody that does not substantially cross-react with cadherin protein. A polypeptide produced by an isolated and / or recombinant method. 26. An antibody or antibody fragment which specifically immunoreacts with an onserin protein and is produced by an isolation and / or recombinant method. 27. A monoclonal antibody that specifically immunoreacts with an onserin protein. 28. A hybridoma that produces the antibody of claim 27. 29. An isolated nucleic acid comprising a coding sequence that encodes an Onserin polypeptide. 30. An isolated nucleic acid comprising an onserin coding sequence that encodes an onserin amino acid sequence that binds to at least one of Ca2 +, catenin, or cadherin. 31. The onserin amino acid sequence hybridizes to (i) an amino acid sequence having at least 60% identity to SEQ ID NO: 2, and (ii) to SEQ ID NO: 1 under stringent conditions. 31. The nucleic acid of claim 30, characterized by one or more of the onserin coding sequences. 32. A nucleic acid comprising (i) the coding sequence of claim 30, and (ii) a heterologous transcriptional regulatory sequence. 33. The nucleic acid of claim 31, wherein the onserin coding sequence is derived from a naturally occurring mammalian onserin gene. 34. The onserin gene is a human onserin gene.
3 nucleic acids. 35. The nucleic acid of claim 30, wherein the onserin amino acid sequence corresponds to an extracellular fragment of an onserin protein. 36. An expression vector which is replicable in at least one of prokaryotic cells and eukaryotic cells and comprises the nucleic acid of claim 30 or 31. 37. A transfection with the expression vector of claim 36,
A host cell that expresses the recombinant polypeptide. 38. A recombinant onserin, comprising culturing the cell of claim 37 in a cell culture medium to express said onserin polypeptide, and then isolating said onserin polypeptide from said subculture. A method for producing a polypeptide. 39. A gene construct comprising the nucleic acid of claim 30 or 31 operably linked to a transcription regulatory sequence to cause expression of an onserin polypeptide in a eukaryotic cell; and (ii) the gene. A recombinant transfection system comprising a gene delivery composition for delivering a construct to a cell and then transfecting the cell with the gene construct. 40. The gene delivery composition is selected from the group consisting of a recombinant virus particle, a liposome, and a polycationic nucleic acid binding agent.
9 recombinant transfection systems. 41. A probe comprising a substantially purified oligonucleotide /
A primer comprising a region of the nucleotide sequence wherein the oligonucleotide hybridizes under stringent conditions to at least 10 contiguous nucleotides of the sense or antisense sequence of SEQ ID NO: 1 or a naturally occurring variant thereof. Probe / primer. 42. The probe / primer of claim 41, wherein the oligonucleotide further comprises a label attached to itself and is detectable. 43. Onserin mR comprising the probe / primer of claim 41
Kit for detecting cells containing NA transcript. 44. A purified preparation of an antisense nucleic acid that hybridizes specifically to the onserin gene under physiological conditions and inhibits its expression,
The nucleic acid is (i) a synthetic oligonucleotide, (ii) a single strand, (iii) a linear strand,
A formulation that is at least one of (iv) 10 to 50 nucleotides in length, and (v) a DNA analog that is resistant to nuclease degradation. 45. A DNA whose antisense nucleic acid is resistant to nuclease degradation
45. The formulation of claim 44 that is analog. 46. A transgenic animal having cells containing a transgene comprising the nucleic acid of claim 29. 47. A transgenic animal wherein the onserin-dependent signal transduction pathway is inhibited in one or more tissues of the animal, either by expression of an antagonistic onserin polypeptide or disruption of the endogenous onserin gene. 48. A method for modulating the proliferation, differentiation or survival of cells in an animal, comprising administering a therapeutically effective amount of an agent that induces, activates or inhibits onserin-dependent signal transduction. . 49. The method of claim 48, comprising administering a nucleic acid construct encoding an onserin polypeptide, wherein said nucleic acid construct is taken up by a cell to be transposed or a cell present in the vicinity thereof and recombined. The method wherein the nucleic acid construct is administered under the conditions being expressed. 50. An onserin protein and an extracellular factor or matrix component,
49. The method of claim 48, comprising administering an agent that inhibits interaction with an intracellular signal transduction molecule. 51. The method of claim 50, wherein said agent is a small organic molecule. 52. The method of claim 50, wherein said agent is a soluble extracellular domain of an onserin protein. 53. A method for determining whether a subject is at risk for a disease characterized by undesired cell proliferation, differentiation or death, comprising: (i) a gene encoding an onserin protein in the subject tissue; And (ii) determining the presence or absence of a genetic disorder characterized by at least one misexpression of the gene. 54. The method of claim 53, wherein the genetic disorder comprises confirming the presence of at least one of the following i-viii: i. Deletion of one or more nucleotides from the gene, ii. The addition of one or more nucleotides to the gene, iii. A substitution of one or more nucleotides of the gene, iv. Significant rearrangement of genes on chromosomes, v. Aberrant methylation of genes, vi. Significant changes in messenger RNA transcript levels of the gene, vii. The presence of a non-wild-type splicing pattern of the messenger RNA transcript of the gene, viii. Protein levels that are not wild-type levels. 55. The method of claim 53 for detecting a genetic disorder, comprising: i. Contains an oligonucleotide comprising a region of the nucleotide sequence that hybridizes to the sense or antisense sequence of SEQ ID NO: 1, or a naturally occurring variant thereof, or the 5 'or 3' flanking sequence naturally associated with the gene. Providing a nucleic acid that: ii. Exposing the nucleic acid to tissue nucleic acid; and iii. A method comprising detecting the presence or absence of a genetic disorder by hybridizing the nucleic acid to a tissue nucleic acid. 56. The method for detecting a genetic disorder comprises detecting the presence or absence of onserin protein on cells of a tissue sample and / or the presence or absence of onserin protein as a soluble protein in a body fluid. 55. The method of claim 54, wherein 57. A method for detecting the presence of an onserin ligand on a cell present in a biological sample, comprising labeling the cell under conditions in which the onserin polypeptide binds to a cognate ligand. And then detecting the presence of the Onserin polypeptide bound to the cells. 58. An assay for identifying a compound that modulates the biological activity of Onserin, comprising: (a) preparing a reaction mixture comprising: (i) an Onserin polypeptide; (ii) A molecule that interacts with the onserin polypeptide (onserin interacting substance); and (iii) a test compound; and (b) detecting an interaction between the onserin polypeptide and the onserin interacting substance, wherein the test compound A change in the interaction of the Onserin interactor with the Onserin polypeptide in the presence of the test compound, as compared to the interaction in the absence of the compound, is indicative of the potential Onserin-modulating activity of the test compound. Assay. 59. The assay of claim 58, wherein said reaction mixture is a cell-free protein formulation. 60. The assay of claim 58, wherein the reaction mixture comprises a recombinant cell containing a heterologous nucleic acid that recombinantly expresses the Onserin polypeptide. 61. The assay of claim 58, wherein detecting the interaction of the Onserin interactor with the Onserin polypeptide comprises a competitive binding assay. 62. The level of a second intracellular messenger responsive to signaling by an interaction between an Onserin interactor and an Onserin polypeptide, wherein the step of detecting an interaction between the Onserin interactor and an Onserin polypeptide 61. The assay of claim 60, wherein the assay detects a change in. 63. The step of detecting an interaction between an Onserin interactor and an Onserin polypeptide comprises detecting a change in the level of gene expression controlled by a transcriptional regulatory sequence responsive to Onserin-dependent signal transduction. 61. The assay of claim 60, comprising: 64. The assay of claim 60, wherein said recombinant cells substantially lack expression of endogenous onserin protein. 65. The assay of claim 58, wherein said reaction mixture is a cell membrane preparation. 66. The assay of claim 58, wherein said reaction mixture is a reconstituted protein mixture. 67. The assay of claim 58, wherein the reaction mixture is a liposome that reconstitutes the onserin polypeptide as a membrane protein. 68. The assay of claim 58, wherein the assay step is repeated on a heterogeneous library of at least 100 different test compounds. 69. The assay of claim 58, wherein said test compound is selected from the group consisting of small organic molecules and natural product extracts. 70. The assay of claim 58, further comprising the step of preparing a pharmaceutical composition of one or more of the identified compounds. 71. A compound that can be identified by the assay of claim 58.