AU728798B2 - Vertebrate deltex proteins, nucleic acids, and antibodies, and related methods and compositions - Google Patents

Description

WO 97/18822 PCT/US96/18675 VERTEBRATE DELTEX PROTEINS, NUCLEIC ACIDS, AND ANTIBODIES, AND RELATED METHODS AND COMPOSITIONS 1. INTRODUCTION The present invention relates to vertebrate deltex genes and their encoded protein products, as well as derivatives and analogs thereof. The invention further relates to production of vertebrate Deltex proteins, derivatives and antibodies. Related therapeutic compositions and methods of therapy and diagnosis are also provided.

2. BACKGROUND OF THE INVENTION In Drosophila melanogaster, the so called "Notch group" of genes has been implicated in events crucial for the correct developmental choices of a wide variety of precursor cells (for review, see Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245-1247; Artavanis-Tsakonas and Simpson, 1991, Trends Genet. 7:403-408). The accumulated genetic and molecular studies suggest that these genes encode elements of a cell communication mechanism which includes cell surface, cytoplasmic, and nuclear -components.

Very little is known about the mechanisms underlying cell fate choices in higher organisms such as vertebrates; a knowledge of such mechanisms could provide insights into pathologies associated with abnormal differentiation events. Thus, a need exists in the art to obtain and characterize the human members of the "Notch group" of genes, including deltex, since these genes appear to play crucial roles in the determination of cell fate.

Numerous developmental genetic studies in recent years have shown that the Notch locus plays a central role in regulative events influencing cell fate decisions in Drosophila in a very broad spectrum of developing tissues (reviewed in Artavanis-Tsakonas and Simpson, 1991, Trends Genet. 7:403-408; and in Artavanis-Tsakonas et al., 1991, Ann.

Rev. Cell Biol. 7:427-452). This pleiotropy of Notch function is revealed by mutations affecting all stages of development and a variety of tissues Welshons, 1965. Science 150:1122-1229; Welshons, 1971, Genetics 68:259-268; Shellenbarger and Mohler. 1978, 3 Dev Biol. 62:432-446). A dramatic illustration of Notch function is seen in the development of the embryonic nervous system, whereby loss of function mutations cause the misrouting of epithelial precursor cells into a neural developmental pathway and result in what has been -1- WO 97/18822 PCT/US96/18675 termed a 'neurogenic' phenotype (Poulson, 1937, Proc. Natl. Acad. Sci. USA, 23:133-137; Lehman et al., 1983, Roux's Arch. Dev. Biol. 192:62-74).

In attempts to understand the molecular contexts by which the Notch protein communicates signals from the cell surface to the nucleus to effect changes in cell fate, genetic means have been used to identify loci that interact phenotypically with various Notch alleles. These genetic studies led to the definition of a small group of interacting loci, which has been operationally termed the 'Notch group' (Artavanis-Tsakonas and Simpson, 1991, Trends Genet. 7:403-408). The other members of the Notch group are deltex (Xu and Artavanis-Tsakonas, 1991, Genetics 126:665-677), Enhancer of (split) [E(spl)] (Knust et al., 1987, EMBO J. 6:4113-4123; Hartley et al., 1988, Cell 55:785-795; Preiss et al., 1988, EMBO J. 7:3917-3927: Klambt et al., 1989, EMBO J. 8:203-210), and mastermind (mam) (Smoller et al., 1990, Genes Dev. 4:1688-1700). mastermind, Hairless the Enhancer of (split), and Suppressor of Hairless encode nuclear proteins (Smoller et al., 1990, Genes Dev. 4:1688-1700; Bang et al., 1992 Genes Dev. 6:1752-1769; Maier et al., 1992, Mech Dev. 38:143-156; Delidakis et al., 1991, Genetics 129:803-823; Schrons et al., 1992, Genetics 132:481-503; Furukawa et al., 1991, J. Biol Chem. 266:23334-23340; Furukawa et al, 1992, Cell 69:1191-1197; Schweisguth et al., 1992, Cell 69:, 1199-1212). deltex mutations suppress the pupal lethality conferred by certain heteroallelic combinations of the Abruptex class of Notch alleles (Xu et al., 1990, Genes Dev. 4:464-475). From this same genetic screen, the genes Delta and mastermind were also identified, both of which belong to the same 'neurogenic' class of genes as Notch because of the similar mutant phenotypes they produce. Moreover. subsequent analysis has shown that alleles of deltex exhibit genetic interactions with those of Delta, mastermind, Hairless, and Su(H), a further suggestion of functional links among these loci (Xu and Artavanis-Tsakonas, 1990, Genetics 126:665- 677).

The manner by which Notch is thought to influence determinative events is indirect, that is, not through the direct specification of cellular fates. Instead, recent experimental studies (Coffman et al, 1993, Cell 73:659-671; Fortini et al, Nature, in press) indicate that Notch activity delays differentiation, and in this manner renders precursor cells competent to receive and/or interpret any number of specific developmental cues (Cagan and Ready, 1989, Genes Dev. 3:1099-1112). In loss of function mutants, this inhibition is lost and cells assume default pathways of differentiation. For example, during the development -2- WO 97/18822 PCT/US96/18675 of the Drosophila nervous system, cells that normally would become epidermis instead adopt a neural fate in the absence of Notch function. However, a salient feature of Notch activity is its pleiotropy. Notch is required for the proper specification of many other cell types, including those of the compound eye (Cagan and Ready, 1989, Genes Dev. 3:1099-1112), ovary (Ruohola et al., 1991, Cell 66:433-449; Xu et al., 1992, Development 115:913-922), and mesoderm (Corbin et al., 1991, Cell 67:311-323). Similarly, the widespread expression patterns exhibited by vertebrate Notch cognates suggest also a broad-based functional role in these species (Coffman et al, 1993, Cell 73:659-671; Coffman et al., 1990, Science 249:1438-1441; Weinmaster et al., 1991, Development 113:199-205; Weinmaster et al., 1992, Development 116:931-941; Kopan and Weintraub, 1993, J. Cell Biol. 121:631-641; Franco del Amo et al., 1992, Development 115:737-744; Ellisen et al., 1991, Cell 66:649- 661; Stifani et al., 1992, Nature Genetics 2:119-127).

Notch homologs have been isolated from a variety of vertebrate species and have been shown to be remarkably similar to their Drosophila counterpart in terms of structure, expression pattern and ligand binding properties (Rebay et al., 1991, Cell 67:687-699; Coffman et al., 1990, Science 249:1438-1441; Ellisen et al, 1991, Cell 66:649-661; Weinmaster et al., 1991, Development 113:199-205). Two human Notch homologs have been isolated (PCT Publication No. WO 92/19737 dated November 12, 1992), termed hN and TAN-1. A human Notch (TAN-I) malfunction has been associated with a lymphatic cancer (Ellisen et al., 1991, Cell 66:649-661).

Notch encodes a large, structurally-complex transmembrane protein, consistent with an involvement in cell-cell communication (Wharton et al., 1985, Cell 43:567-581; Kidd et al., 1986, Mol. Cell. Biol. 6:3094-3108). Notch has an extracellular domain containing 36 tandem EGF-like repeats and 3 Notch/linl2 repeats. The intracellular domain bears several common structural motifs including 6 cdcl0/SW16/ankyrin repeats ("ANK" repeats) Lux et al., 1990, Nature 344:36-42; Breeden and Nasmyth, 1987, Nature 329:651-654; Michaely and Bennett, 1992, Trends Cell Biol. 2:127-129; Blank et al., 1992, Trends Biochem. Sci. 17:135-140; Bennett, 1992, J. Biol. Chem. 267:8703-8706)), a polyglutamine stretch known as 'opa', and a PEST motif (Stifani et al., 1992, Nature Genetics 2:119-127). The remarkable degree to which these motifs have been conserved in 3 5 homologs isolated from mice (Weinmaster et al., 1991, Development 113:199-205; Weinmaster et al., 1992, Development 116:931-941; Kopan and Weintraub, 1993, J. Cell WO 97/18822 PCT/US96/18675 Biol. 121:631-641), rats (Kopan and Weintraub. 1993, J. Cell Biol. 121:631-641; Franco del Amo et al., 1993, Genomics 15:259-264), humans (Ellisen et al., 1991, Cell 66:649- 661; Stifani et al., 1992, Nature Genetics 2:119-127; PCT Publication No. WO 92/19737 dated November 12, 1992), and Xenopus (Coffman et al, 1993, Cell 73:659-671; Coffman et al., 1990, Science 249:1438-1441) implies that they will have a common biochemical mode of action. In particular, ANK repeats, which constitute the most conserved region amino acid identity) between Notch and its vertebrate counterparts (Stifani et al., 1992, Nature Genetics 2:119-127), are thought to mediate protein-protein interactions among diverse groups of proteins, including those involved in signal transduction processes and cytoskeletal interactions (Lux et al., 1990, Nature 344:36-42; Breeden and Nasmyth, 1987, Nature 329:651-654; Michaely and Bennett, 1992, Trends Cell Biol. 2:127-129; Blank et al., 1992, Trends Biochem. Sci. 17:135-140; Bennett, 1992, J. Biol. Chem. 267:8703- 8706). Indeed, Rebay et al. (1993, Cell 74:319-329) have recently demonstrated that the ANK repeats are crucial for Notch-mediated signaling events. Both EGF-like repeats and ankyrin motifs are found in a variety of proteins known to interact with other protein molecules. Indeed, evidence has shown a direct interaction between Notch and the products of the Delta and Serrate loci, which also encode transmembrane proteins containing EGFlike repeats (Fehon et al., 1990, Cell 61:523-534; Rebay et al., 1991, Cell 67:687-699).

In Drosophila, it has been demonstrated that dominant 'activated' phenotypes result from in vivo overexpression of a Notch protein lacking most extracellular, ligandbinding sequences, while 'dominant-negative' phenotypes result from overexpression of a protein lacking most intracellular sequences (Rebay et al., 1993, Cell 74:319-329).

In Drosophila, Deltex has been demonstrated to play a critical role in development and other physiological processes, in particular, in the signaling pathway of Notch which is involved in cell fate (differentiation) determination. We have demonstrated through expression studies conducted in cultured Drosophila cells, in yeast, and in the 0 imaginal wing disc that Drosophila Deltex mediates the intracellular portion of the signal transduction cascade involved in Notch function (Diederich et al., 1994, Development 120:473-481). These studies show that Drosophila Deltex is localized within the cytoplasm, that it is a protein of unique sequence, that it displays homotypic interactions, and that it directly physically interacts with the Drosophila Notch intracellular ANK repeats.

Additionally, we have demonstrated that Drosophila Deltex directly interacts with the ANK repeats of human Notch.

The ANK repeat motif is shared by many proteins and has been implicated in protein-protein interactions (Lux et al.. 1990, Nature 344:36-42, Thompson et al., 1991, Science 253:762-768, reviewed in Bennett, 1992, J. Biol. Chem. 267:8703-8706, Blank et al., 1992, Trends Biochem. Sci. 17:135-140, Rebay et al., 1993, Cell 74:319-329).

Moreover, an in vivo functional analysis of various truncated forms of Notch has implicated these ANK repeats in downstream signaling events and that dominant 'activated' phenotypes result from in vivo overexpression of a Notch protein lacking most extracellular, ligand binding sequences, while 'dominant negative' phenotypes result from overexpression of a protein lacking most intracellular sequences (Rebay et al., 1993, Cell, 74:319-329).

Furthermore, deltex displays genetic interactions with Notch and Delta. both transmembrane proteins, and with mastermind, a nuclear localized protein (Smoller et al., 1990, Genes Dev. 4:1688-1700). This makes deltex the first identified cytoplasmic component of the S" Notch group of interacting loci.

We have also subsequently demonstrated that a fragment mostly composed of S the ankyrin reats of the tch protein mndiates mdlelar ieractis bebel Nd)tch and the Su(H) protein (Fortini et al., 1994, Cell 79:273-282). The Drosophila Su(H) gene encodes a protein of 594 amino acids and binds to the promoters of several viral and cellular genes and interacts directly with a viral transactivator protein termed Epstein-Barr virus Snuclear antigen 2 (EBNA2), which enables a virus to subvert the normal program of B cell 25 differentiation (Schweisguth, et al, 1992. Cell 69: 1199; Furukawa et al.. 1991, J.

Biol. Chem. 266:23334). Genetic and molecular studies suggest that Deltex and Delta may act in concert to multimerize Notch proteins and to interfere with the cytoplasmic retention of Su(H) by Notch, thus activating the Notch signaling pathway (Diederich, et al., 1994, Development 120:473; Fortini, et al., 1994, Cell, 79:273; Matsuno, et al., unpublished, Artavanis-Tsakonas et al., 1995, Science, 268:225-232). This pathway is believed to control nuclear events in order to influence the progression of uncommitted cells to a more differentiated state. Three loci encoding putative nuclear proteins Hairless, Enhancer of split, and mastermind, have been implicated in these nuclear events.

6 Despite the cloning of a Drosophila deltex gene (See PCT Publication WO 95/19770 published July 27, 1995), no vertebrate deltex gene had been obtained prior to the present invention.

Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.

SUMMARY OF THE INVENTION The present invention relates to nucleotide sequences of vertebrate deltex genes, and amino acid sequences of the encoded vertebrate Deltex proteins. The invention further relates to fragments and other derivatives, and analogs, of vertebrate Deltex proteins as well as antibodies thereto. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. Production of the foregoing proteins and derivatives, by recombinant methods, is provided.

The present invention provides a purified vertebrate Deltex protein, which protein is encoded by a first nucleic acid which hybridizes under low stringency conditions to a second nucleic acid consisting of the double stranded vertebrate Deltex nucleic acid sequence contained in plasmid pBS hdx as deposited with the ATCC and assigned Accession No. 97341 or (b) hybridizes under low stringency conditions to a second nucleic acid consisting of the nucleotide sequence depicted in Figure 2A-C (SEQ ID NO:11) or a sequence complementary thereto, and which protein is able to be bound by an antibody to a Deltex protein consisting of the amino acid sequence depicted in Figure 2A-C (SEQ ID NO:12), said low stringency conditions comprising hybridization in a buffer consisting of 35% formamide, 5X SSC, 50 mM Tris-HCI (pH 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate, for 18-20 hours at 40 0 C, washing in a buffer consisting of 2X SSC, 25 mM Tris-HCI (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 55°C, and washing in a buffer consisting of 2X SSC, 25 mM Tris-HCI (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 3 :EisabehJSPEC11614-97.DOC In a'specific embodiment, the invention relates to human deltex nucleic acids and proteins.

In another specific embodiment, the invention relates to mammalian deltex nucleic acids and proteins.

In specific embodiments, the invention relates to vertebrate Deltex protein derivatives and analogs of the invention which are functionally active, or which comprise one or more domains of a vertebrate Deltex protein, including but not limited to the SH3binding domains, ring-H2-Zinc fingers, domains which mediate binding'to Notch or to a Notch derivative containing Notch cdcl0/SW16/ankyrin repeats, or any combination of the foregoing.

The present invention also relates to therapeutic and diagnostic methods and compositions based on vertebrate Deltex proteins and nucleic acids. The invention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic 15 compound of the invention. Such therapeutic compounds (termed herein "Therapeutics') include: vertebrate Deltex proteins and analogs and derivatives (including fragments) thereof; antibodies thereto: nucleic acids encoding the vertebrate Deltex proteins, analogs, or derivatives: and vertebrate deltex antisense nucleic acids. In a preferred embodiment. a 0 Therapeutic of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant 6a WO 97/18822 PCT/US96/18675 state. In other specific embodiments, a Therapeutic of the invention is administered to treat a nervous system disorder or to promote tissue regeneration and repair.

In one embodiment, Therapeutics which antagonize, or inhibit, vertebrate Notch and/or Deltex function (hereinafter "Antagonist Therapeutics") are administered for therapeutic effect. In another embodiment, Therapeutics which promote vertebrate Notch and/or Deltex function (hereinafter "Agonist Therapeutics") are administered for therapeutic effect.

Disorders of cell fate, in particular hyperproliferative cancer) or hypoproliferative disorders, involving aberrant or undesirable levels of expression or activity or localization of vertebrate Notch and/or Deltex protein can be diagnosed by detecting such levels, as described more fully infra.

In a preferred aspect, a Therapeutic of the invention is a protein consisting of Sat least a fragment (termed herein "adhesive fragment") of vertebrate Deltex which mediates binding to a Notch protein or a fragment thereof.

The invention also provides methods of inactivating Notch function in a cell, methods of identifying a compound that inhibits or reduces the binding of a vertebrate Deltex protein to a Notch protein, and methods of expanding non-terminally differentiated cells.

4. DESCRIPTION OF THE FIGURES Figure 1A-F. Nucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) of Drosophila deltex cDNA.

Figure 2A-C. Composite nucleotide sequence (SEQ ID NO: 11) derived from the cDNA (nucleotide 1 to 2547), and deduced amino acid sequence (SEQ ID NO:12) of the human deltex locus. The predicted amino acid sequence is depicted below the DNA sequence. The symbol: designates the start of T05200 and the end of T05200. Core H and C residues in Ring-H2-zinc finger are shown by underlining. PCR primers hdx-1 to 4 (SEQ ID NO:26), (SEQ ID NO:27), (SEQ ID NO:28), and (SEQ ID NO:29), respectively, are indicated in bold. X and N represent amino acid residues and nucleotides, respectively, not yet determined.

Figure 3. Aligned amino acid sequences of human Deltex WO 97/18822 PCT/US96/18675 (SEQ ID NO:12) and Drosophila Deltex (SEQ ID NO:2) proteins. Those positions at which residues are identical are shaded. Sites in which amino acids are chemically similar are boxed.

4A-B. Amino acid sequence of Drosophila Deltex (SEQ ID NO:2) and designated fragments implicated in protein-protein interactions. Fragments A-D (SEQ ID NOS: 13-16, respectively) are shown.

Figure 5. Schematic diagram of Deltex fragments mediating Deltex-Deltex interactions.

Figure 6. Schematic diagram of the Deltex and Notch fragments mediating Deltex-Notch interactions.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to nucleotide sequences of vertebrate deltex genes, and amino acid sequences of their encoded Deltex proteins. The invention further relates to fragments and other derivatives, and analogs, of vertebrate Deltex proteins.

Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. Production of the foregoing proteins and derivatives, by recombinant methods, is provided.

In a specific embodiment, the invention relates to a human deltex gene and protein.

In a another specific embodiment, the invention relates to a mammalian deltex gene and protein.

The invention also relates to vertebrate Deltex protein derivatives and analogs of the invention which are functionally active, they are capable of displaying one or more-known functional activities associated with a full-length (wild-type) vertebrate Deltex protein. Such functional activities include but are not limited to antigenicity [ability to bind (or compete with a vertebrate Deltex protein for binding) to an anti-vertebrate Deltex protein antibody], immunogenicity (ability to generate antibody which binds to a vertebrate Deltex protein), ability to bind (or compete with a vertebrate Deltex protein for binding) to Notch or a second Deltex protein or other proteins or fragments thereof, ability to bind (or compete with a vertebrate Deltex protein for binding) to a receptor or ligand for a vertebrate Deltex protein.

-8- WO 97/18822 PCT/US96/18675 The invention further relates to fragments (and derivatives and analogs thereof) of a vertebrate Deltex protein which comprise one or more domains of a vertebrate Deltex protein (see infra), including but not limited to the SH3-binding domains, ring-H2fingers, domains which mediate binding to Notch (or a derivative thereof containing the Notch ANK repeats) or to a second Deltex molecule or fragment thereof, or any combination of the foregoing.

Antibodies to vertebrate Deltex proteins, their derivatives and analogs, are additionally provided.

Our prior attempts to clone human, zebrafish, and Xenopus deltex using Drosophila deltex as a probe were unsuccessful. In contrast to such prior failures, the present invention is based on the successful cloning of human deltex. As described by way of example below, we have used an innovative methodology to clone the transcription unit corresponding to human deltex. As described therein (see Section our results show a significant structural conservation of Deltex in humans, indicative of functional conservation. Moreover, we demonstrate that human Deltex displays direct molecular interaction with both human and Drosophila Notch intracellular ANK repeats (see Section Knowledge of the sequence of human deltex allows the identification of regions strongly conserved between Drosophila and human deltex, and provides a method for readily isolating other vertebrate deltex genes by use of such strongly conserved regions (see Sections 5.6 and 8 infra).

The vertebrate deltex nucleic acid and amino acid sequences and antibodies thereto of the invention can be used for the detection and quantitation of vertebrate deltex mRNA and protein, to study expression thereof, to produce vertebrate Deltex proteins, fragments and other derivatives, and analogs thereof, in the study, assay, and manipulation of differentiation and other physiological processes, and are of therapeutic and diagnostic use, as described infra. The agonists and antagonists of Deltex function can be used to alter the ability of a cell to differentiate. The vertebrate deltex nucleic acids and antibodies can also be used to clone vertebrate deltex homologs of other species, as described infra. Such vertebrate deltex homologs are expected to exhibit significant homology to each other, and encode proteins which exhibit the ability to bind to a Notch protein.

The present invention also relates to therapeutic and diagnostic methods and compositions based on vertebrate Deltex proteins and nucleic acids. The invention provides -9- WO 97/18822 PCT/US96/18675 for treatment of disorders of cell fate or differentiation by administration of a therapeutic compound of the invention. Such therapeutic compounds (termed herein "Therapeutics") include: vertebrate Deltex proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the vertebrate Deltex proteins, analogs, or derivatives; and vertebrate deltex antisense nucleic acids. In a preferred embodiment, a Therapeutic of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant state.. In other specific embodiments, a Therapeutic of the invention is administered to treat a nervous system disorder or to promote tissue regeneration and repair.

In one embodiment, Therapeutics which antagonize, or inhibit, Notch and/or vertebrate Deltex function (hereinafter "Antagonist Therapeutics") are administered for therapeutic effect. In another embodiment, Therapeutics which promote Notch and/or vertebrate Deltex function (hereinafter "Agonist Therapeutics") are administered for therapeutic effect.

Disorders of cell fate, in particular hyperproliferative cancer) or hypoproliferative disorders, involving aberrant or undesirable levels of expression or activity or localization of Notch and/or vertebrate Deltex protein can be diagnosed by detecting such levels, as described more fully infra.

In a preferred aspect, a Therapeutic of the invention is a protein consisting of at least a fragment (termed herein "adhesive fragment") of vertebrate Deltex that mediates binding to a Notch protein, a second Deltex protein, or a fragment of Notch or Deltex.

The invention is illustrated by way of examples infra which disclose, inter alia, the cloning and sequencing of human deltex, and the identification of regions of human Deltex which are predicted to bind to the ANK repeats of Notch, or which are predicted to bind to regions of human Deltex.

For clarity of disclosure, and not by way of limitation, the detailed 0description of the invention is divided into the subsections set forth below.

5.1. ISOLATION OF THE VERTEBRATE DELTEX NUCLEIC ACIDS The invention relates to the nucleotide sequences of vertebrate deltex nucleic acids. In specific embodiments, human deltex nucleic acids comprise the cDNA sequence shown in Figure 2A-C (SEQ ID NO:11), or the coding region thereof (nucleotide numbers 504-2363), or nucleic acids encoding a human Deltex protein having the sequence of SEQ ID NO:12). The invention provides nucleic acids consisting of at least 8 nucleotides a hybridizable portion) of a vertebrate deltex sequence; in other embodiments, the nucleic acids consist of at least 25 (continuous) nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of a vertebrate deltex sequence, or a full-length vertebrate deltex coding sequence. The invention also relates to nucleic acids hybridizable to or complementary to the foregoing sequences. In specific aspects, nucleic acids are provided which comprise a sequence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a vertebrate deltex gene. In a. specific embodiment, the sequence is naturally occurring.

In other specific embodiments, the invention provides nucleic acids comprising at least 110, 150, or 200 continuous nucleotides of the sequence of 1 SEQ ID NO:11. In other embodiments, the invention provides a nucleic acid'%ncoding the first 25, 50, 100, 150, 200, or 230 amino acids of SEQ ID NO:12.

In a specific embodiment, vertebrate deltex nucleic acids comprise those nucleic acids which are substantially homologous to the nucleic acids encoding the amino terminal 180 amino acids (encoded, by nucleotide numbers 504-1044 of SEQ ID:11) 20 of human deltex, or fragments thereof. In one embodiment, the vertebrate deltex nucleic acid has at least 50% identity over the corresponding nucleotide sequence of an identically sized human deltex. In another embodiment this identity is greater than 55%. In a preferred embodiment, the nucleotide sequence identity of the vertebrate deltex is greater 25 than 60%. In a more preferred embodiment this identity is greater than 65%. In a most preferred embodiment, the nucleotide sequence identity of the vertebrate deltex is greater than 70% over that of the corresponding nucleotide sequence of identically sized human deltex.

In another specific embodiment, vertebrate deltex nucleic acids comprise 3 0those nucleic acids which are substantially homologous to the nucleic acids encoding the central region amino acids of human deltex nucleotide numbers 1045-1821 of SEQ ID NO:11) or fragments thereof. In one embodiment, the nucleic acids encoding the central amino acids of the vertebrate Deltex protein has at least 50% nucleotide sequence identity with the corresponding human deltex sequence of identical size. In another embodiment this identity is greater than 55%. In a preferred embodiment, this nucleotide sequence identity is 11 WO 97/18822 PCT/US96/18675 greater than 60%. In a more preferred embodiment this identity is greater than 65%. In a most preferred embodiment, the homology of the nucleic acids encoding the central region amino acids of the vertebrate deltex has a nucleotide sequence identity that is greater than over that of the corresponding nucleotide sequence of identically sized human deltex.

In another specific embodiment, vertebrate deltex nucleic acids comprise those nucleic acids which are substantially homologous to the nucleic acids encoding the 180 carboxy terminal amino acids of human deltex (nucleotide numbers 1822-2366), or fragments thereof. In one embodiment, the nucleic acids encoding the carboxy terminal region of the vertebrate Deltex protein has at least 50% nucleotide sequence identity over the corresponding human deltex sequence of identical size. In another embodiment this identity is greater than 55%. In a preferred embodiment, this identity is greater than In a more preferred embodiment this identity is greater than 65%. In a most preferred embodiment, the identity of the nucleotides encoding the amino terminal amino acids of the vertebrate deltex is greater than 70% over that of the corresponding nucleotide sequence of identically sized human deltex.

In a specific embodiment, a nucleic acid which is hybridizable to a vertebrate deltex nucleic acid having sequence SEQ ID NO:11), or to a nucleic acid encoding a vertebrate deltex derivative, under conditions of low stringency is provided. By way of example and not limitation, procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792): Filters containing DNA are pretreated for 6 h at 40*C in a solution containing formamide, 5X SSC, 50 mM Tris-HCl (pH 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 jg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10 6 cpm 32 P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40*C, and then washed for 1.5 h at 55C in a solution containing 2X SSC, 25 mM Tris-HCI (pH 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60*C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68*C and reexposed to film. Other conditions of low stringency which may be used are well known in the art as employed for cross-species hybridizations).

12- WO 97/18822 PCT/US96/18675 In another specific embodiment, a nucleic acid which is hybridizable to a vertebrate deltex nucleic acid under conditions of high stringency is provided. By way of example and not limitation, procedures using such conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 jg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65*C in prehybridization mixture containing 100 jig/ml denatured salmon sperm DNA and 5-20 X 106 cpm of "P-labeled probe. Washing of filters is done at 37*C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.

This is followed by a wash in 0.1X SSC at 50*C for 45 min before autoradiography. Other conditions of high stringency which may be used are well known in the art.

Nucleic acids encoding derivatives fragments) of vertebrate Deltex 1 proteins (see Section and vertebrate deltex antisense nucleic acids (see Section 5.11) are additionally provided. As is readily apparent, as used herein, a "nucleic acid encoding a fragment or portion of a vertebrate Deltex protein" shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of the vertebrate Deltex protein and not the other contiguous portions of the vertebrate Deltex protein as a continuous sequence.

Specific embodiments for the cloning of a vertebrate deltex gene, a human deltex gene, presented as a particular example but not by way of limitation, follows: For expression cloning (a technique commonly known in the art), an expression library is obtained or is constructed by methods known in the art. For example, mRNA human) is isolated, cDNA is made and ligated into an expression vector a bacteriophage derivative) such that it is capable of being expressed by the host cell into which it is then introduced. Various screening assays can then be used to select for the expressed vertebrate Deltex product. In a preferred aspect, anti-human Deltex antibodies can be used to select the recombinant host cell expressing a cloned vertebrate deltex gene.

In a specific embodiment, PCR is used to amplify the desired vertebrate deltex sequence in a genomic or cDNA library, prior to selection (see, by way of example Section 8, infra). Oligonucleotide primers representing known vertebrate deltex sequences, preferably regions known to be conserved between Drosophila and human, can be used as primers in PCR. The synthetic oligonucleotides may be utilized as primers to amplify by 13- WO 97/18822 PCT/US96/18675 PCR sequences from a source (RNA or DNA), preferably a cDNA library, of potential interest. PCR can be carried out, by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp"). The DNA being amplified can include human mRNA or cDNA or genomic DNA. One can choose to synthesize several different degenerate primers, for use in the PCR reactions. It is also possible to vary the stringency of hybridization conditions used in priming the PCR reactions, to allow for greater or lesser degrees of nucleotide sequence similarity between the known vertebrate deltex nucleotide sequence and the nucleic acid homolog being isolated. For cross species hybridization, low stringency conditions are preferred (see supra). For same species hybridization, moderately stringent or highly stringent conditions are preferred (see supra). After successful amplification of a segment of a vertebrate deltex gene homolog, that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone. This, in turn, will permit the determination of the gene's complete nucleotide sequence, the analysis of its expression, and the production of its protein product for functional analysis, as described infra. In a preferred aspect, human genes encoding Deltex proteins may be identified in this fashion. Alternatively to selection by hybridization, the PCR-amplified DNA can be inserted into an expression vector for expression cloning as 2 0 described above.

In the event that it is desired to isolate a vertebrate deltex gene by crossspecies hybridization (either by direct hybridization to a vertebrate deltex probe representing all or a part of a vertebrate deltex gene of a different species, or by PCR using oligonucleotide primers derived from the sequence of a vertebrate deltex gene of a different species), the desired vertebrate deltex gene can be isolated as set forth in Example 8, by screening with a probe, or priming for PCR with an oligonucleotide, containing deltex sequences encoding regions highly conserved between human and Drosophila. For example, the human Deltex amino acid stretches 414-419 (SEQ ID NO:30), 475-480 (SEQ 3 ID NO:31), 504-511 (SEQ ID NO: 32), 531-539 (SEQ ID NO:33) and 557-564 (SEQ ID NO:34) are conserved in Drosophila Deltex amino acid stretches 549-555 (SEQ ID 603-608 (SEQ ID NO:36), 632-639 (SEQ ID NO:37), 659-667 (SEQ ID NO:38) and 685- 692 (SEQ ID NO:39), respectively. In a preferred embodiment, a pair of oligonucleotides comprising sequences separated by a length in the range from 50-500 nucleotides is used as primers in PCR. The invention envisions the use of nucleic acids encoding conserved 14- WO 97/18822 PCT/US96/18675 regions of the Deltex protein in combination to isolate the Deltex encoding nucleic acids of other organisms, by use in PCR to amplify the desired sequence or less preferably, without PCR, as a probe in selection by virtue of direct colony hybridization Grunstein, M.

and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 3961).

In the event that it is desired to isolate a deltex gene by cross-species hybridization (either by direct hybridization to a deltex probe representing all or a part of a deltex gene of an evolutionarily distant, different species, or by PCR using oligonucleotide primers derived from the sequence of a deltex gene of a different, evolutionarily distant species), the desired deltex gene can be isolated by a more gradual method of evolutionary walking via first isolating a deltex gene from a more closely related species, identifying the portions of deltex which are conserved cross-species, and then screening with a probe or priming for PCR with a nucleic acid containing the conserved sequence. This method, while more cumbersome, is straightforward and can be readily carried out by routine methods. For example, if it is desired to proceed further down the evolutionary tree, one may first isolate a murine deltex gene using nucleic acids encoding human Deltex as a probe. A conserved portion of the murine deltex sequence is then used to screen or amplify deltex in an avian library; a conserved portion of the avian clone is used to screen an amphibian library, a conserved portion of the amphibian clone is used to screen a fish library, etc. If desired, the species to be selected in the next round of screening can be selected from among various species by hybridizing the deltex probe to a Southern blot containing genomic DNA from each species, and selecting a species to which hybridization occurs.

The above-methods are not meant to limit the following general description of methods by which clones of vertebrate deltex may be obtained.

Any eukaryotic cell can potentially serve as the nucleic acid source for the molecular cloning of the vertebrate deltex gene. The DNA may be obtained by standard procedures known in the art from cloned DNA a DNA "library"), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired human cell (see, for example Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 2d. Ed., Cold Spring Harbor, New York; Glover, D.M. 1985, DNA Cloning: A Practical Approach, MRL 15 WO 97/18822 PCT/US96/18675 Press, Ltd., Oxford, U.K. Vol. I, II.) Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will contain only exon sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.

"In the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired gene may be accomplished in a number of ways. For example, if an amount of a portion of a vertebrate deltex (of any species) gene or its specific RNA, or a fragment thereof the adhesive domain, is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton, W. and Davis, 1977, Science 196, 180; Grunstein, M. And Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 3961). Those DNA fragments with substantial homology to the probe will hybridize. For cross species hybridization, low stringency conditions are preferred (see supra). For same species hybridization, moderately stringent conditions are preferred (see supra). It is also possible to identify the appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected according to a known restriction map if such is available. Further selection can be carried out on the basis of the properties of the gene. Alternatively, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones, or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein that, has similar or identical electrophoretic migration, isoelectric focusing behavior, proteolytic digestion maps, binding to Notch, or antigenic properties as known for vertebrate Deltex. If an antibody to vertebrate Deltex is available, the vertebrate Deltex protein may be identified by binding of labeled antibody to the putatively vertebrate Deltex synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay)-type procedure.

16- WO 97/18822 PCT/US96/18675 The vertebrate deltex gene can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization. Such DNA fragments may available, purified vertebrate deltex DNA of another species human).

Immunoprecipitation analysis or functional assays ability to bind Notch) of the in vitro translation products of the isolated products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments that contain the desired sequences. In addition, specific mRNAs may be selected by adsorption of polysomes isolated from cells to immobilized antibodies specifically directed against vertebrate Deltex protein. A radiolabelled vertebrate deltex cDNA can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template. The radiolabelled mRNA or cDNA may then be used as a probe to identify the vertebrate deltex DNA fragments from among other genomic DNA Sfragments.

Alternatives to isolating the vertebrate deltex genomic DNA include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence or making cDNA to the mRNA which encodes the vertebrate deltex gene. For example, RNA for cDNA cloning of the vertebrate deltex gene can be isolated from cells which express vertebrate Deltex. Other methods are possible and within the scope of the invention.

The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used.

Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as PBR322, pUC, or Bluescript (Stratagene) plasmid derivatives. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to 0fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and vertebrate deltex gene may be modified by horopolymeric tailing. Recombinant molecules can be introduced 17- WO 97/18822 PCTUS96/1 8675 18 into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.

In an alternative method, the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionization, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinant DNA molecules that incorporate the isolated vertebrate deltex gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated Srecombinant DNA.

The vertebrate deltex sequences provided by the instant invention include those nucleotide sequences encoding substantially the same amino acid sequences as found in native vertebrate Deltex protein, and those encoded amino acid sequences with functionally equivalent amino acids, all as described in Section 5.6 infra for vertebrate Deltex derivatives.

Throughout the description and claims of the specification the word "comprise" and Svariations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

5.2. EXPRESSION OF VERTEBRATE DELTEX NUCLEIC ACIDS The nucleic acid coding for a vertebrate Deltex protein or a functionally active fragment or other derivative thereof can be inserted into an appropriate expression vector, a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. The necessary transcriptional and translational signals can also be supplied by the native vertebrate deltex gene and/or its flanking regions. A variety of host-vector systems may be utilized to express the proteincoding sequence. These include but are not limited to vertebrate cell systems infected with virus vaccinia virus, adenovirus, etc.); insect cell systems infected with virus baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.

In a specific embodiment, a molecule comprising a portion of a vertebrate deltex gene which WO 97/18822 PCT/US96/18675 encodes a protein that binds to Notch or to a molecule comprising the Notch ANK repeats is expressed. In another embodiment, a molecule comprising a portion of a vertebrate deltex gene which encodes a protein that binds to a fragment of a Deltex protein is expressed. In specific embodiments, mammalian deltex gene is expressed, or a sequence encoding a functionally active portion of mammalian Deltex. In other specific embodiments, the human deltex gene is expressed, or a sequence encoding a functionally active portion of human Deltex. In a specific embodiment, a chimeric protein comprising a Notch-binding domain of a vertebrate Deltex protein is expressed. In other specific embodiments, a full-length vertebrate deltex cDNA is expressed, or a sequence encoding a functionally active portion of a vertebrate Deltex protein. In yet another embodiment, a fragment of a vertebrate Deltex protein comprising a domain of the protein, or other derivative, or analog of a vertebrate Deltex protein is expressed.

Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of a nucleic acid sequence encoding a vertebrate Deltex protein or peptide fragment may be regulated by a second nucleic acid sequence so that the vertebrate Deltex protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a vertebrate Deltex protein may be controlled by any promoter/enhancer element known in the art.

Promoters which may be used to control vertebrate deltex gene expression include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A..78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the -lactamase (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.

75:3727-3731), tac (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25), XPL, or trc promoters; see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase promoter region or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl.

19- WO 97/18822 PCT/US96/18675 Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor.Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89- 94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

Expression vectors containing vertebrate deltex gene inserts can be identified by three general approaches: nucleic acid hybridization, presence or absence of "marker" gene functions, and expression of inserted sequences. In the first approach, the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted vertebrate deltex gene. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions thymidine kinase activity, resistance to antibiotics, transformation WO 97/18822 PCT/US96/18675 phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. For example, if the vertebrate deltex gene is inserted within the marker gene sequence of the vector, recombinants containing the vertebrate deltex insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the vertebrate deltex gene product in in vitro assay systems, binding to Notch, binding with antibody.

Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered vertebrate Deltex protein may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification phosphorylation, cleavage) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.

In other specific embodiments, the vertebrate Deltex protein, fragment, analog, or derivative may be expressed as a fusion, or chimeric protein product (comprising the protein, fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, by use of a peptide synthesizer.

-21 WO 97/18822 PCT/US96/18675 Both cDNA and genomic sequences can be cloned and expressed.

In other embodiments, a vertebrate deltex cDNA sequence may be chromosomally integrated and expressed. Homologous recombination procedures known in art may be used.

5.3. IDENTIFICATION AND PURIFICATION

OF

THE VERTEBRATE DELTEX GENE PRODUCTS In particular aspects, the invention provides amino acid sequences of vertebrate Deltex, preferably human Deltex, and fragments and derivatives thereof which comprise an antigenic determinant can be recognized by an antibody) or which are functionally active, as well as nucleic acid sequences encoding the foregoing. "Functionally active" material as used herein refers to that material displaying one or more known functional activities associated with the full-length (wild-type) vertebrate Deltex protein product, binding to Notch or a portion thereof, binding to another Deltex molecule or portion thereof, binding to any other Deltex ligand, antigenicity (binding to an antivertebrate Deltex antibody), immunogenicity (generating anti-Deltex antibody), Notch intracellular signal transduction, etc.

In specific embodiments, the invention provides fragments of a vertebrate Deltex protein consisting of at least 6 amino acids, 10 amino acids, 50 amino acids, or of at least 75 amino acids. In other embodiments, the proteins comprise, or alternatively, consist essentially of; one or more of the SH3-binding domains SEQ ID NOS: 17-21 of Table SIII); one or more ring-H2-zinc finger domains SEQ ID NO:25), or a portion which binds to Notch comprising the first approximately 230 amino acids of vertebrate Deltex), or any combination of the foregoing, of a vertebrate Deltex protein. Fragments, or proteins comprising fragments, lacking some or all of the foregoing regions of vertebrate Deltex are also provided. Molecules comprising more than one copy of the foregoing regions are also provided. Nucleic acids encoding the foregoing are provided.

Once a recombinant which expresses a vertebrate deltex gene sequence is identified, the gene product can be analyzed. This is achieved by assays based on the physical or functional properties of the product, including radioactive labelling of the product followed by analysis by gel electrophoresis, immunoassay, etc. Chemically synthesized proteins, derivatives, and analogs can be similarly analyzed.

-22- WO 97/18822 PCT/US96/18675 Once a vertebrate Deltex protein is identified, it may be isolated and purified by standard methods including chromatography ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. The functional properties may be evaluated using any suitable assay (see Section 5.7).

Alternatively, the amino acid sequence of a vertebrate Deltex protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant.

Once the amino acid sequence is thus known, the protein can be synthesized by standard chemical methods known in the art see Hunkapiller et al., 1984, Nature 310:105- 111).

By way of example, the deduced amino acid sequence (SEQ ID NO:12) of a human Deltex protein is presented in Figure 2A-C.

5.4. STRUCTURE OF THE VERTEBRATE DELTEX GENES AND PROTEINS The structure of the vertebrate deltex genes and proteins can be analyzed by various methods known in the art.

5.4.1. GENETIC ANALYSIS The cloned DNA or cDNA corresponding to the vertebrate deltex gene can be analyzed by methods including but not limited to Southern hybridization (Southern, 1975, J. Mol. Biol. 98:503-517), Northern hybridization (see Freeman et al., 1983, Proc.

2 Natl. Acad. Sci. U.S.A. 80:4094-4098), restriction endonuclease mapping (Maniatis, 1982, Molecular Cloning, A Laboratory, Cold Spring Harbor, New York), and DNA sequence analysis. Polymerase chain reaction (PCR; U.S. Patent Nos. 4,683,202, 4,683,195 and 4,889,818; Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7652-7656; Ochman et al., 1988, Genetics 120:621-623; Loh et al., 1989, Science 243:217-220) followed by Southern hybridization with a vertebrate deltex-specific probe can allow the detection of the vertebrate deltex genes in DNA from various cell types. In one embodiment, Southern hybridization can be used to determine the genetic linkage of vertebrate deltex. Northern 3 hybridization analysis can be used to determine the expression of the vertebrate deltex genes.

Various cell types, at various states of development or activity can be tested for vertebrate deltex gene expression. The stringency of the hybridization conditions for both Southern -23- WO 97/18822 PCT/US96/18675 and Northern hybridization can be manipulated to ensure detection of nucleic acids with the desired degree of relatedness to the specific vertebrate deltex probe used.

Restriction endonuclease mapping can be used to roughly determine the genetic structure of the vertebrate deltex gene. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis. Alternatively, restriction maps can be deduced, once the nucleotide sequence is known.

DNA sequence analysis can be performed by any techniques known in the art, including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol.

65:499-560), the Sanger dideoxy method (Sanger et al., 1977, Proc. Natl. Acad. Sci.

U.S.A. 74:5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Patent No.

4,795,699; Sequenase, U.S. Biochemical Corp.), or Taq polymerase, or use of an automated DNA sequenator Applied Biosystems, Foster City, CA). The cDNA sequence of a human deltex gene is shown in Figure 2A-C (SEQ ID NO: 11) and is described in Section 6, infra.

5.4.2. PROTEIN ANALYSIS The amino acid sequence of a vertebrate Deltex protein can be derived by deduction from the DNA sequence, or alternatively, by direct sequencing of the protein, with an automated amino acid sequencer. The amino acid sequence of a representative vertebrate Deltex protein comprises the sequence substantially as depicted in Figure 2A-C (SEQ ID NO:12), and detailed in Section 6, infra.

The vertebrate Deltex protein sequence can be further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824).

A hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of a vertebrate Deltex protein and the corresponding regions of the gene sequence which encode such regions. Hydrophilic regions are predicted to be immunogenic.

Secondary, structural analysis (Chou and Fasman, 1974, Biochemistry 13:222) can also be done, to identify regions of a vertebrate Deltex protein that assume specific secondary structures.

Manipulation, translation, and secondary structure prediction, as well as open reading frame prediction and plotting, can also be accomplished using computer software programs available in the art.

-24- WO 97/18822 PCTIUS96/18675 Other methods of structural analysis can also be employed. These include but are not limited to X-ray crystallography (Engstom, 1974, Biochem. Exp. Biol. 11:7-13) and computer modeling (Fletterick and Zoller 1986, Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

GENERATION OF ANTIBODIES TO VERTEBRATE DELTEX PROTEINS AND DERIVATIVES THEREOF According to the invention, a vertebrate Deltex protein, its fragments or other derivatives, or analogs thereof, may be used as an immunogen to generate antibodies which recognize such an immunogen. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. In a preferred embodiment, antibodies which specifically bind to vertebrate Deltex proteins are produced. In a more preferred embodiment, an antibody which binds to a vertebrate Deltex protein mammalian, preferably human) but does not bind to (full length) Drosophila Deltex protein, is produced. In a preferred embodiment, such an antibody is produced by using as immunogen, regions least conserved between Drosophila melanogaster and the vertebrate Deltex protein.

In another embodiment, antibodies to a particular domain of a vertebrate Deltex protein are produced. In a specific embodiment, an antibody is produced which binds to a fragment of vertebrate Deltex which binds to Notch; in another embodiment, an antibody binds to a molecule comprising the first 230 amino-terminal amino acids of vertebrate Deltex. In another embodiment the antibody binds to an amino-terminal fragment of vertebrate Deltex containing not more than the first 200 amino acids of vertebrate Deltex.

In yet another embodiment, an antibody binds to a fragment of vertebrate Deltex which binds to a second Deltex molecule.

Various procedures known in the art may be used for the production of polyclonal antibodies to a vertebrate Deltex protein or derivative or analog. In a particular embodiment, rabbit polyclonal antibodies to an epitope of the vertebrate Deltex protein having a sequence depicted in Figure 2A-C or a subsequence thereof, can be obtained. For 3 the production of antibody, various host animals can be immunized by injection with a native vertebrate Deltex protein, or a synthetic version, or derivative fragment) thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used WO 97/18822 PCT/US96/18675 to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.

In a preferred embodiment, polyclonal or monoclonal antibodies are produced by use of a hydrophilic portion of a vertebrate Deltex peptide identified by the procedure of Hopp and Woods (1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824)).

For preparation of monoclonal antibodies directed toward a vertebrate Deltex protein sequence or analog thereof, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495- 497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) can be used. In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (PCT Publication No.

WO 89/12690 dated December 28, 1989). According to the invention, human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc.

Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96), or by other methods known in the art. In fact, according to the invention, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule specific for a vertebrate Deltex protein together with genes from a human antibody 0molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention. Non-human antibodies can be humanized by the method of Winter (see U.S. Patent No. 5,225,539).

According to the invention, techniques described for the production of single chain antibodies Patent 4,946,778) can be adapted to produce vertebrate Deltex protein-specific single chain antibodies. An additional embodiment of the invention utilizes -26- WO 97/18822 PCT/US96/18675 the techniques described for the construction of Fab expression libraries (Huse et al., 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for vertebrate Deltex proteins, derivatives, or analogs.

Antibody fragments and other derivatives which contain the idiotype (binding domain) of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay). For example, to select antibodies which recognize a specific domain of a vertebrate 1 Deltex protein, one may assay generated hybridomas for a product which binds to a vertebrate Deltex fragment containing such domain. For selection of an antibody specific to human Deltex protein(s), one can select on the basis of positive binding to a human Deltex protein and a lack of binding to Drosophila Deltex protein.

In a specific embodiment, antibodies specific to a phosphorylated epitope of vertebrate Deltex are produced.

The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the protein sequences of the invention for imaging these proteins, measuring levels thereof in appropriate physiological samples, etc. Antibodies to vertebrate Deltex (since it normally colocalizes with Notch) can be used to determine the intracellular distribution of Notch and/or vertebrate Deltex, in diagnostic methods such as described infra. The antibodies also have use in immunoassays. In another embodiment of the invention (see infra), anti-vertebrate Deltex antibodies and fragments thereof containing the binding domain are Therapeutics.

5.6. VERTEBRATE DELTEX PROTEINS, DERIVATIVES AND ANALOGS The invention further provides vertebrate Deltex proteins, and derivatives (including but not limited to fragments) and analogs of vertebrate Deltex proteins. Nucleic acids encoding vertebrate Deitex protein derivatives and protein analogs are also provided.

In one embodiment, the vertebrate Deltex proteins are encoded by me vertebrate deltex -27- WO 97/18822 PCT/US96/18675 nucleic acids described in Section 5.1 supra. In particular aspects, the proteins, derivatives, or analogs are of mouse or rat; agricultural stock such as cow, sheep, horse, goat, pig and the like; pets such as cats, dogs; or other domesticated mammals, or primate Deltex proteins.

The production and use of derivatives and analogs related to vertebrate Deltex are within the scope of the present invention. In a specific embodiment, the derivative or analog is functionally active, capable of exhibiting one or more functional activities associated with a full-length, wild-type vertebrate Deltex protein.

In particular, vertebrate Deltex derivatives can be made by altering vertebrate deltex.sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as a vertebrate deltex gene may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of vertebrate deltex genes which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. Likewise, the vertebrate Deltex derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a vertebrate Deltex protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.

Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In a specific embodiment of the invention, proteins consisting of or comprising a fragment of a vertebrate Deltex protein consisting of at least 10 (continuous) amino acids of the vertebrate Deltex protein is provided. In other embodiments, the -28 WO 97/18822 PCT/US96/18675 fragment consists of at least 20 or 50 amino acids of the vertebrate Deltex protein. In specific embodiments, such fragments are not larger than 35, 100 or 200 amino acids.

Derivatives or analogs of vertebrate Deltex include but are not limited to those peptides which are substantially homologous to human Deltex or fragments thereof.

In a specific embodiment, derivatives or analogs of vertebrate Deltex include those peptides which are substantially homologous to the amino terminal 180 amino acids (1- 180) of human Deltex. In one embodiment, the amino terminal region of the vertebrate Deltex protein has at least 30% identity over the amino terminal amino acid sequence of identically sized human Deltex. In another embodiment this identity is greater than In a preferred embodiment, the amino terminal amino acid identity of the vertebrate Deltex is greater than 45%. In a more preferred embodiment this identity is greater than 55%. In a most preferred embodiment, the homology of the amino terminal amino acids of the vertebrate Deltex is greater than 65% over the corresponding human Deltex amino terminal amino acid sequence of identical size.

In another specific embodiment, derivatives or analogs of vertebrate Deltex include those peptides which are substantially homologous to the central region (amino acids 181-441) of human Deltex, or fragments thereof. In one embodiment, the central region of the vertebrate Deltex protein has at least 30% identity with the corresponding human Deltex sequence of identical size. In another embodiment this identity is greater than 35%. In a preferred embodiment, the amino acid identity of the central region of vertebrate Deltex and human Deltex is greater than 45%. In a more preferred embodiment this identity is greater than 55%. In a most preferred embodiment, the homology of the central amino acids of the vertebrate Deltex to corresponding human Deltex amino acids of identical size is greater than Additionally, derivatives or analogs of vertebrate Deltex include but are not limited to those peptides which are substantially homologous to the carboxy terminal amino acids of human Deltex or fragments thereof. In one embodiment, the carboxy terminal region of the vertebrate Deltex protein (the carboxy terminal 180 amino acids) has at least identity over the amino acid sequence of identical size. In another embodiment this identity is greater than 50%. In a preferred embodiment, the amino terminal amino acid identity of the vertebrate Deltex is greater than 55%. In a more preferred embodiment this identity is greater than 60%. In a most preferred embodiment, the homology of the amino terminal amino acids of the vertebrate Deltex is greater than -29- WO 97/18822 PCT/US96/18675 In another preferred embodiment, derivatives or analogs of vertebrate Deltex comprise regions conserved between Drosophila and human Deltex (see Section 8).

The vertebrate Deltex protein derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned vertebrate deltex gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding a derivative or analog of a vertebrate Deltex protein, care should be taken to ensure that the modified gene remains within the same translational reading frame as the vertebrate deltex gene, uninterrupted by translational stop signals, in the gene region where the desired vertebrate Deltex activity is encoded.

Additionally, the vertebrate Deltex-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting, ones, to facilitate further in vitro modification.

Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem 253:6551), use of TAB® linkers (Pharmacia), etc.

Manipulations of the vertebrate deltex sequence may also be made at the protein level. Included within the scope of the invention are vertebrate Deltex protein fragments or other derivatives or analogs which are differentially modified during or after translation, by acetylation, phosphorylation, carboxylation, amidation, derivatization by Sknown protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 acetylation, formylation, oxidation, reduction, etc.

In a preferred aspect, phosphorylation or, alternatively, dephosphorylation is carried out, which can be to various extents, on the purified vertebrate Deltex protein or WO 97/18822 PCTIUS96/18675 derivative thereof. The phosphorylation state of the molecule may be important to its role in intracellular signal transduction of Notch function. Phosphorylation can be carried out by reaction with an appropriate kinase possibly cdc2 or CK II). Dephosphorylation can be carried out by reaction with an appropriate phosphatase.

In addition, analogs and derivatives of vertebrate Deltex proteins can be chemically synthesized. For example, a peptide corresponding to a portion of a vertebrate Deltex protein which comprises the desired domain, or which mediates the desired activity in vitro, can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the vertebrate Deltex protein sequence. Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, a-amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, tbutylalanine, phenylglycine, cyclohexylalanine, 13-alanine, designer amino acids such as 3methyl amino acids, Ca-methyl amino acids, and Na-methyl amino acids.

In a specific embodiment, the vertebrate Deltex derivative is a chimeric, or fusion, protein comprising a vertebrate Deltex protein or fragment thereof (preferably consisting of at least a domain or motif of the vertebrate Deltex protein, or at least 10 amino acids of the vertebrate Deltex protein) joined at its amino or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a vertebrate Deltex-coding sequence joined in-frame to a coding sequence for a different protein). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, by use of a peptide synthesizer. A specific embodiment relates to a chimeric protein comprising a fragment of a vertebrate Deltex protein which comprises a domain or motif of the vertebrate Deltex protein, a portion which binds to a Notch protein or to a second Deltex protein, an SH-3 binding domain, a ring-H2-zinc finger domain, etc. In a particular embodiment, a chimeric nucleic acid can be constructed, encoding a fusion protein consisting of a vertebrate Deltex Notch-binding fragment joined to a non-Deltex protein. As another example, and not by way of limitation, a recombinant -31 WO 97/18822 PCT/US96/18675 molecule can be constructed according to the invention, comprising coding portions of both a vertebrate deltex gene and another gene which is a member of the "Notch group." Another specific embodiment relates to a chimeric protein comprising a fragment of a vertebrate Deltex protein of at least six amino acids. Particular examples of the construction and expression of fusion proteins comprising human Deltex or various Notch fragments, are described in Section 7.

Other specific embodiments of derivatives and analogs are described in the subsection below and examples sections infra.

5.6.1. DERIVATIVES OF VERTEBRATE DELTEX CONTAINING ONE OR MORE DOMAINS OF THE PROTEIN In a specific embodiment, the invention provides vertebrate Deltex derivatives and analogs, in particular vertebrate Deltex fragments and derivatives of such fragments, that comprise or consist of one or more domains of the vertebrate Deltex protein, including but not limited to a region which binds to a Notch protein (or a molecule comprising the ANK repeats thereof), a region which binds to a second Deltex protein or portion thereof, an SH3-binding domain, or a ring-H2-zinc finger domain. In specific embodiments, the vertebrate Deltex derivative may lack all or a portion of one or more of the foregoing domains.

In specific embodiments directed to the domains of the human Deltex protein, the aforesaid domains consist of approximately the following amino-acid sequences (see Section 6.1.1 infra): SH3 binding domains: SEQ ID NOS: 17-21 Ring-H2-zinc finger domain: SEQ ID Other binding fragments, smaller than those set forth above, can be identified by routine methods, by construction of nucleic acids encoding such fragments and assays for binding via the interaction trap method described in Section 7 infra.

In a specific embodiment, relating to a vertebrate Deltex protein of a species Sother than human, fragments comprising specific domains of vertebrate Deltex are those comprising domains in the respective vertebrate Deltex protein most homologous to the specific domain of the human Deltex protein.

-32 WO 97/18822 PCT/US96/18675 We have demonstrated that Drosophila Deltex binds to human Notch-1 and 2, suggesting evolutionary conservation of biochemical activity between human and Drosophila Deltex. We have also demonstrated that human Deltex binds to human Notch-1 and 2, and that human Deltex binds to Drosophila Notch. Using the interaction trap system (described infra) as our assay we systematically examined, by deletion analysis, the domains of Notch and Deltex which are responsible for protein-protein interactions. Both Deltex- Deltex as well as Deltex-Notch interactions were detected. Deletion constructs encoding various fragments (described below) of Drosophila Deltex, Drosophila Notch and human Notch were expressed as fusion constructs (LexA or ACT fusions), and assayed.

The sequences of fragments A-D (SEQ ID NOS:13-16, respectively) of Drosophila Deltex which were expressed are shown in Fig. 4A-B.

Figure 5 summarizes the Deltex-Deltex interactions we have detected.

Fragment A interacts with Fragment A (homotypic interactions). Fragment B interacts with Fragment B (homotypic interactions). Fragment C interacts with Fragment C (homotypic interactions). In addition, we detected interactions between fragments C and B. However, we can only detect the fragment C-B interaction if fragment C is tested as the "bait" as the LexA fusion). If Fragment B is the bait, this interaction is not detected. All the other aforesaid interactions occur irrespective of which fragment is used as the bait. Fragment A consists of amino acids 1-303. Fragment B consists of amino acids 306-486. Fragment C consists of amino acids 514-737.

The heterotypic interaction between Notch and Deltex is occurred between the ANK repeat region of Notch and fragment D of Deltex (which is part of fragment A and includes amino acids 1-204). Drosophila Notch ANK repeats as well as the ANK repeats of both human Notch proteins (encoded by TAN-1 and hN) were tested in this interaction assay and showed positive binding to fragment D. The following fragments containing the ANK repeat region were used: Drosophila Notch amino acids: 1889-2076 (numbering per Wharton et al., 1985, Cell 43:567-581); Human Notch TAN-1 amino acids: 1826-2146; Human Notch hN amino acids: 1772-2093. All displayed interactions with fragment D.

Figure 6 summarizes schematically this interaction.

In specific embodiments, vertebrate Deltex regions are provided that are most homologous to Drosophila fragment A (SEQ ID NO:13), fragment B (SEQ ID NO: 14), fragment C (SEQ ID NO:15), and fragment D (SEQ ID NO:16), shown in Figure 4A-B.

-33 WO 97/18822 PCT/US96/18675 Binding interactions between fragments are indicated by arrows in Figures 5 and 6. Such regions homologous to A-D are predicted also to display the binding interactions shown in Figures 5 and 6. Thus, amino acids 1-237, 238-391, 392-620, and 1-175 of SEQ ID NO:12 correspond to Drosophila fragments A-D, respectively. Molecules comprising one or more of the foregoing regions are provided. Accordingly, by way of example, a molecule comprising amino acid numbers 1-237 of SEQ ID NO: 12 is predicted to bind the Notch ankyrin repeats.

Also provided are inhibitors peptide inhibitors) of the foregoing protein interactions with Notch or with a second Deltex protein.

The ability to bind to a Notch protein or a Deltex protein (or derivative thereof) can be demonstrated by in vitro assays such as the interaction trap technique (Section 7, infra).

The nucleic acid sequences encoding Notch or vertebrate Deltex proteins or fragments thereof, for use in such assays, can be isolated from porcine, bovine, equine, feline, canine, as well as primate sources and any other mammals in which homologs of known genes can be identified. For example, the Notch protein or portion thereof comprising the ANK repeats which can be expressed and assayed for binding to Deltex or a Deltex derivative can be derived from any of the Notch homologs: human hN, human TAN-i, Xenopus, and Drosophila.

Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as the aforesaid domains may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of the vertebrate deltex genes which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. Likewise, the vertebrate Deltex proteins, fragments or derivatives thereof, of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of the domains including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence ("conservative" changes).

The derivatives, analogs, and peptides of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level.

-34- WO 97/18822 PCT/US96/18675 Additionally, the nucleic acid sequence can be mutated in vitro or in vivo; and manipulations of the sequence may also be made at the protein level.

In addition, analogs and peptides can be chemically synthesized.

5.7. IN VITRO ASSAYS OF VERTEBRATE DELTEX PROTEINS, DERIVATIVES AND ANALOGS The functional activity of vertebrate Deltex proteins, derivatives and analogs, can be assayed in vitro by various methods.

For example, in one embodiment, where one is assaying for the ability to bind or compete with wild-type vertebrate Deltex for binding to anti-vertebrate Deltex antibody, various immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labelled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

In another embodiment, where one is assaying for the ability to mediate binding to Notch or portion thereof Notch ankyrin repeats) to a second Deltex protein or portion thereof, one can carry out assays such as that described infra in Section 7.

Other methods will be known to the skilled artisan and are within the scope of the invention.

In another embodiment, a method of identifying a molecule that inhibits or reduces the binding of a vertebrate Deltex protein to a Notch protein is provided. In this 3 manner, agonists and antagonists of Deltex can be identified. Such a method comprises the steps of contacting a Notch protein and a vertebrate Delta protein such that binding between the Notch protein and the Deltex protein can occur, in the presence of one or more WO 97/18822 PCT/US96/18675 molecules which are desired to be tested for the ability to inhibit or reduce binding between the Notch protein and the Deltex protein, and identifying the molecules that inhibit or reduce the binding of the Deltex protein to the Notch protein. Any of various binding assays known in the art can be used to carry out such a method, including but not limited to yeast interaction trap assays, cell culture in vitro aggregation assays, and soluble binding assays using purified Notch and Deltex proteins. A specific embodiment is as follows: Cultured cells are cotransfected with plasmid expression constructs that place Notch and deltex under distinct, inducible promoters. Notch expression in these cells is first induced to ensure proper cell surface localization; Deltex expression is then induced. These cells are then aggregated with cells expressing Delta, to produce mutual capping of Notch and Delta at the point of mutual contact (see Singer 1992, Science, 255:1671-1677; Fehon et al., 1990, Cell, 61:523-534; Heitzler, et al., 1991, Cell, 64:1083-1092). Under these conditions, Deltex colocalizes with the capped Notch by virtue of its binding to Notch. The cells are then incubated in the presence of one or more molecules (preferably, purified molecules) which are desired to be tested for the ability to inhibit binding between Notch and Deltex.

Molecules which inhibit or reduce the binding of Deltex to Notch will result in an increased localization of Deltex throughout the cell cytoplasm. This increased localization can be determined according to methods known in the art immunofluorescent staining with antibody to Deltex). The method can also be carried out using derivatives of Notch and Deltex that mediate binding to Deltex and to Notch, respectively.

5.8. THERAPEUTIC USES The invention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic compound of the invention. Such therapeutic compounds (termed herein "Therapeutics") include: vertebrate Deltex proteins and analogs and derivatives (including fragments) thereof as described hereinabove); antibodies thereto (as described hereinabove); nucleic acids encoding the vertebrate Deltex proteins, analogs, or derivatives as described hereinabove); and vertebrate deltex antisense nucleic acids. As stated supra, the Antagonist Therapeutics of the invention are those Therapeutics which antagonize, or inhibit, a vertebrate Deltex function and/or Notch function. Such Antagonist Therapeutics are most preferably identified by use of known convenient in vitro assays, based on their ability to inhibit binding of vertebrate Deltex -36- WO 97/18822 PCT/US96/18675 to another protein a Notch protein), or inhibit any known Notch or vertebrate Deltex function as preferably assayed in vitro or in cell culture, although genetic assays in Drosophila or mouse) may also be employed. In a preferred embodiment, the Antagonist Therapeutic is a protein or derivative thereof comprising a functionally active fragment such as a fragment of vertebrate Deltex which mediates binding to Notch, or an antibody thereto.

In other specific embodiments, such an Antagonist Therapeutic is a nucleic acid capable of expressing a molecule comprising a fragment of vertebrate Deltex which binds to Notch, or a vertebrate deltex antisense nucleic acid (see Section 5.11 herein). It should be noted that preferably, suitable in vitro or in vivo assays, as described infra, should be utilized to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue, since the developmental history of the tissue may determine whether an Antagonist or Agonist Therapeutic is desired.

In another embodiment of the invention, a nucleic acid containing a portion of a vertebrate deltex gene is used, as an Antagonist Therapeutic, to promote vertebrate deltex inactivation by homologous recombination (Koller and Smithies, 1989, Proc. Natl.

Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

The Agonist Therapeutics of the invention, as described supra, promote vertebrate Deltex function. Such Agonist Therapeutics include but are not limited to proteins and derivatives comprising the portions of Notch that mediate binding to vertebrate Deltex, the ANK repeats, and nucleic acids encoding the foregoing (which can be administered to express their encoded products in vivo).

Further descriptions and sources of Therapeutics of the inventions are found in Sections 5.1 through 5.7 herein.

Molecules which retain, or alternatively inhibit, a desired vertebrate Deltex property, binding to Notch, binding to an intracellular ligand, can be used therapeutically as inducers, or inhibitors, respectively, of such property and its physiological 3 correlates. In a specific embodiment, a peptide in the range of 6-50 or 15-25 amino acids; and particularly of about 10, 15, 20 or 25 amino acids) containing the sequence of a portion of vertebrate Deltex which binds to Notch is used to antagonize Notch function. In a specific embodiment, such an Antagonist Therapeutic is used to treat or prevent human or other malignancies associated with increased Notch expression cervical cancer, colon cancer, breast cancer, squamous adenocarcinomas (see infra)). Derivatives or analogs of -37 WO 97/18822 PCT/US96/18675 vertebrate Deltex can be tested for the desired activity by procedures known in the art, including but not limited to the assays described in the examples infra. For example, molecules comprising Deltex fragments which bind to Notch ANK repeats (see Section 7), can be obtained and selected by expressing deletion mutants of human Deltex (or of a nucleotide sequence of another species and assaying for binding of the expressed product to Notch by any of several methods, such as the interaction trap system described in the Examples Sections infra. In one specific embodiment, peptide libraries can be screened to select a peptide with the desired activity; such screening can be carried out by assaying, for binding to Notch or a molecule containing the Notch ANK repeats.

The Agonist and Antagonist Therapeutics of the invention have therapeutic utility for disorders of cell fate. The Agonist Therapeutics are administered therapeutically (including prophylactically): in diseases or disorders involving an absence or decreased (relative to normal, or desired) levels of Notch or vertebrate Deltex function, for example, in patients where Notch or vertebrate Deltex protein is lacking, genetically defective, biologically inactive or underactive, or underexpressed; and in diseases or disorders wherein in vitro (or in vivo) assays (see infra) indicate the utility of vertebrate Deltex agonist administration. The absence or decreased levels in Notch or vertebrate Deltex function can be readily detected, by obtaining a patient tissue sample from biopsy tissue) and assaying it in vitro for protein levels, structure and/or activity of the expressed Notch or vertebrate Deltex protein. Many methods standard in the art can be thus employed, including but not limited to immunoassays to detect and/or visualize Notch or vertebrate Deltex protein Western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect Notch or vertebrate Deltex expression by detecting and/or visualizing respectively Notch or vertebrate deltex mRNA Northern assays, dot blots, in situ hybridization, etc.) 30 In vitro assays which can be used to determine whether administration of a specific Agonist Therapeutic or Antagonist Therapeutic is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a Therapeutic, and the effect of such Therapeutic upon the tissue sample is observed. In one embodiment, where the patient has a malignancy, a sample of cells from such malignancy is plated out or grown in culture, and the cells are then exposed 38 WO 97/18822 PCT/US96/18675 to a Therapeutic. A Therapeutic which inhibits survival or growth of the malignant cells by promoting terminal differentiation) is selected for therapeutic use in vivo. Many assays standard in the art can be used to assess such survival and/or growth; for example, cell proliferation can be assayed by measuring 'H-thymidine incorporation, by direct cell count, by detecting changes in transcriptional activity of known genes such as protooncogenes fos, myc) or cell cycle markers; cell viability can be assessed by trypan blue staining, differentiation can be assessed visually based on changes in morphology, etc.

In a specific aspect, the malignant cell cultures are separately exposed to an Agonist Therapeutic, and an Antagonist Therapeutic; the result of the assay can indicate which type of Therapeutic has therapeutic efficacy.

In another embodiment, a Therapeutic is indicated for rse which exhibits the desired effect, inhibition or promotion of cell growth, upon a patient cell sample from tissue 1 having or suspected of having a hyper- or hypoproliferative disorder, respectively. Such hyperor hypoproliferative disorders include but are not limited to those described in Sections 5.8.1 through 5.8.3 infra.

In another specific embodiment, a Therapeutic is indicated for use in treating nerve injury or a nervous system degenerative disorder (see Section 5.8.2) which exhibits in 2 0 vitro promotion of nerve regeneration/neurite extension from nerve cells of the affected patient type.

In addition, administration of an Antagonist Therapeutic of the invention is also indicated in diseases or disorders determined or known to involve a Notch or Deltex dominant activated phenotype ("gain of function" mutations.) Administration of an Agonist Therapeutic is indicated in diseases or disorders determined or known to involve a Notch or Deltex dominant negative phenotype ("loss of function" mutations). The functions of various structural domains of the Notch protein have been investigated in vivo, by ectopically expressing a series of Drosophila Notch deletion mutants under the hsp70 heatshock promoter, as well as eye-specific promoters (see Rebay et al., 1993, Cell 74:319-329). Two classes of dominant phenotypes were observed, one suggestive of Notch loss-of function mutations and the other of Notch gain-of-function mutations. Dominant "activated" phenotypes resulted from overexpression of a protein lacking most extracellular sequences, while dominant "negative" phenotypes resulted from overexpression of a protein lacking most intracellular sequences. The results indicated that Notch functions as a -39 WO 97/18822 PCT/US96/18675 receptor whose extracellular domain mediates ligand-binding, resulting in the transmission of developmental signals by the cytoplasmic domain. The phenotypes observed also suggested that the ANK repeat region within the intracellular domain plays an essential role in Notch mediated signal transduction events (intracellular function). We have shown that Drosophila Deltex binds to the Notch ANK repeat region.

In various specific embodiments, in vitro assays can be carried out with representative cells of cell types involved in a patient's disorder, to determine if a Therapeutic has a desired effect upon such cell types.

In another embodiment, cells of a patient tissue sample suspected of being pre-neoplastic are similarly plated out or grown in vitro, and exposed to a Therapeutic. The Therapeutic which results in a cell phenotype that is more normal less representative of a pre-neoplastic state, neoplastic state, malignant state, or transformed phenotype) is selected for therapeutic use. Many assays standard in the art can be used to assess whether a pre-neoplastic state, neoplastic state, or a transformed or malignant phenotype, is present.

For example, characteristics associated with a transformed phenotype (a set of in vitro characteristics associated with a tumorigenic ability in vivo) include a more rounded cell morphology, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, release of proteases such as plasminogen activator, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton surface protein, etc. (see Luria et al., 1978, General Virology, 3d Ed., John Wiley Sons, New York pp. 436-446).

In other specific embodiments, the in vitro assays described supra can be carried out using a cell line, rather than a cell sample derived from the specific patient to be treated, in which the cell line is derived from or displays characteristic(s) associated with the malignant, neoplastic or pre-neoplastic disorder desired to be treated or prevented, or is derived from the neural or other cell type upon which an effect is desired, according to the 3 present invention.

In a specific embodiment, an antagonist of Notch and/or Deltex function that can be used as an Antagonist Therapeutic is a molecule comprising a Deltex protein or portion thereof that mediates binding to Notch, covalently bound to a protease or proteolytically active fragment thereof. Such protease preferably is able to cleave a Notch protein. The molecule is preferably a fusion protein the covalent bond is a peptide WO 97/18822 PCT/US96/18675 bond). The Deltex protein is preferably a vertebrate protein, most preferably human.

Accordingly, the invention provides a method of targeting or inactivating proteins to which Deltex binds Notch) in a cell. According to this method, the molecule comprising the Deltex protein or portion thereof and the protease sequences is produced through chemical or via molecular biological techniques. This molecule fusion protein) is introduced into the cell by techniques known in the art transfection of the cell with a nucleic acid encoding the molecule such that its expression occurs intracellularly). Inside the cell, the molecule can bind to Notch and/or other Deltex binding partners. Upon such binding, the protease portion of the molecule cleaves the protein to which the molecule is bound, thus inactivating it. For example, a fusion protein containing domain I of human Deltex and the protease thermolysin, when introduced into the cell would bind to and cleave Notch, thereby inactivating the Notch signaling pathway. Molecules which would inactivate protein function by binding thereto, can be used as an alternative to proteases.

The Antagonist Therapeutics are administered therapeutically (including prophylactically): in diseases or disorders involving increased (relative to normal, or desired) levels of Notch or vertebrate Deltex function, for example, where the Notch or vertebrate Deltex protein is overexpressed or overactive; and in diseases or disorders wherein in vitro (or in vivo) assays indicate the utility of vertebrate Deltex antagonist administration. The increased levels of Notch or vertebrate Deltex function can be readily detected by methods such as those described above, by quantifying protein and/or RNA. In vitro assays with cells of patient tissue sample or the appropriate cell line or cell type, to determine therapeutic utility, can be carried out as described above.

5.8.1. MALIGNANCIES Malignant and pre-neoplastic conditions which can be tested as described supra for efficacy of intervention with Antagonist or Agonist Therapeutics, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to those described below in Sections 5.8.1 and 5.9.1.

Malignancies and related disorders, cells of which type can be tested in vitro (and/or in vivo), and upon observing the appropriate assay result, treated according to the 3 5 present invention, include but are not limited to those listed in Table 1 (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia): -41 WO 97/18822 PCTIUS96/I 8675 TABLE 1 MALIGNANCIES AND RELATED DISORDERS acute leukemia acute lymphocytic leukemia acute myelocytic leukemia myeloblastic promyelocytic myelomonocytic monocytic erythroleukemia.

chronic leukemia chronic myelocytic; (granulocytic) leukemia chronic lymphocytic leukemia Polycythemia vera Lymphoma Hodgkin's disease non-Hodgkin's disease Multiple myeloma Waldenstrbm's macroglobulinemia Heavy chain disease Solid tumors sarcomas and carcinomas fibrosarcoma myxosarcoma liposarcoma, chondrosarcoma osteogenic, sarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcoma synovioma, mesothelioma Ewing's tumor leiomyosarcoma rhabdomyosarcoma colon carcinoma pancreatic cancer breast cancer ovarian cancer prostate cancer squamous cell carcinoma basal cell carcinoma -42 WO 97/18822 PCT/US96/18675 adenocarcinoma sweat gland carcinoma sebaceous gland carcinoma papillary carcinoma papillary adenocarcinomas cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma choriocarcinoma seminoma embryonal carcinoma Wilms' tumor cervical cancer testicular tumor lung carcinoma small cell lung carcinoma bladder carcinoma epithelial carcinoma glioma astrocytoma medulloblastoma craniopharyngioma ependymoma pinealoma hemangioblastoma acoustic neuroma oligodendroglioma menangioma melanoma neuroblastoma retinoblastoma In specific embodiments, malignancy or dysproliferative changes (such as metaplasias and dysplasias) are treated or prevented in epithelial tissues such as those in the cervix, esophagus, and lung.

Malignancies of the colon and cervix can exhibit increased expression of human Notch relative to such non-malignant tissue (see PCT Publication WO 94/07474 published April 14, 1994, incorporated by reference herein in its entirety). Thus, in specific embodiments, malignancies of the colon or cervix are treated or prevented by administering an effective amount of an Antagonist Therapeutic of the invention. The presence of -43 WO 97/18822 PCT/US96/18675 increased Notch expression in colon, and cervical cancer suggests that many more cancerous and hyperproliferative conditions exhibit upregulated Notch. Thus, in specific embodiments, various cancers, breast cancer, squamous adenocarcinoma, seminoma, melanoma, and lung cancer, as well as other hyperproliferative disorders, can be treated or prevented by administration of an Antagonist Therapeutic.

5.8.2. NERVOUS SYSTEM DISORDERS Nervous system disorders, involving cell types which can be tested as described supra for efficacy of intervention with Antagonist or Agonist Therapeutics, and which can be treated upon thus observing an indication of therapeutic utility, include but are notlimited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelinatidn.

SNervous system lesions which may be treated in a patient (including human and non-human vertebrate patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (iii) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from nonnervous system tissue; (iv) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including -44- WO 97/18822 PCT/US96/18675 but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis; (vi) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (vii) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (viii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (ix) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virusassociated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons (see also Section For example, and not by way of limitation, Therapeutics which elicit any of the following effects may be useful according to the invention: 0 increased survival time of neurons in culture; (ii) increased sprouting of neurons in culture or in vivo; (iii) increased production of a neuron-associated molecule in culture or in vivo, choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.

WO 97/18822 PCT/US96/18675 Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci. 4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy Sthat may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

5.8.3. TISSUE REPAIR AND REGENERATION In another embodiment of the invention, a Therapeutic of the invention is used for promotion of tissue regeneration and repair, including but not limited to treatment of benign dysproliferative disorders. Specific embodiments are directed to treatment of cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes), treatment of keloid (hypertrophic scar) formation (disfiguring of the skin in which the scarring process interferes with normal renewal), psoriasis (a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination), and baldness (a condition in which terminally differentiated hair follicles (a tissue rich in Notch) fail to function properly).

Deltex agonists and antagonists can also be used to manipulate the differentiation state of non-terminally differentiated stem and progenitor) cells. For example, a stem cell can be exposed to such an agonist to inhibit its differentiation and -46- WO 97/18822 PCT/US96/18675 achieve expansion of the stem cell population by incubation in vitro under growth conditions. Such stem cells have use in transplantation for in vivo repopulation of their progeny cells and tissue regeneration. (For methods that can be used in the foregoing, see United States patent application Serial No. 08/537,210 filed September 29, 1995 by Artavanis-Tsakonas et al., entitled "Manipulation of Non-Terminally Differentiated Cells Using the Notch Pathway," which is incorporated by reference herein in its entirety.) For example, a method for the expansion of a precursor cell a human stem or progenitor cell) comprises contacting the cell with an amount of a vertebrate human) Deltex protein or functionally active portion thereof effective to inhibit differentiation of the cell, and exposing the cell to cell growth conditions such that the cell proliferates. In various embodiments, the precursor cell can be but is not limited.to a hematopoietic precursor cell, epithelial precursor cell, kidney precursor cell, neural precursor cell, skin precursor cell, o setoblast precursor cell, chondrocyte precursor cell, liver precursor cell, and muscle cell.

5.9. PROPHYLACTIC USES 5.9.1. MALIGNANCIES The Therapeutics of the invention can be administered to prevent progression to a neoplastic or malignant state, including but not limited to those disorders listed in Table 1. Such administration is indicated where the Therapeutic is shown in assays, as described supra, to have utility for treatment or prevention of such disorder. Such prophylactic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium.

Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell -47- WO 97/18822 PCT/US96/18675 uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.

Alternatively or in addition to the presence of abnormal cell growth characterized as hyperplasia, metaplasia, or dysplasia, the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a patient, can indicate the desirability of prophylactic/therapeutic administration of a Therapeutic of the invention. As mentioned supra, such characteristics of a transformed phenotype include morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton cell surface protein, etc. (see also id., at pp.

84-90 for characteristics associated with a transformed or malignant phenotype).

In a specific embodiment, leukoplakia, a benign-appearing hyperplastic or dysplastic lesion of the epithelium, or Bowen's disease, a carcinoma in situ, are preneoplastic lesions indicative of the desirability of prophylactic intervention.

In another embodiment, fibrocystic disease (cystic hyperplasia, mammary dysplasia, particularly adenosis (benign epithelial hyperplasia)) is indicative of the desirability of prophylactic intervention.

In other embodiments, a patient which exhibits one or more of the following predisposing factors for malignancy is treated by administration of an effective amount of a Therapeutic: a chromosomal translocation associated with a malignancy the Philadelphia chromosome for chronic myelogenous leukemia, t(14; 18) for follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy (a possible forerunner of multiple myeloma), and a first degree kinship with persons having a cancer or precancerous disease showing a Mendelian (genetic) inheritance pattern familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, 3 5 neurofibromatosis of Von Recklinghausen, retinoblastoma, carotid body tumor, cutaneous melanocarcinoma, intraocular melanocarcinoma, xeroderma pigmentosum, ataxia -48- WO 97/18822 PCT/US96/18675 telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplastic anemia, and Bloom's syndrome; see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 112-113) etc.) In another specific embodiment, an Antagonist Therapeutic of the invention is administered to a human patient to prevent progression to breast, colon, or cervical cancer.

5.9.2. OTHER DISORDERS In other embodiments, a Therapeutic of the invention can be administered to prevent a nervous system disorder described in Section 5.8.2, or other disorder liver cirrhosis, psoriasis, keloids, baldness) described in Section 5.8.3.

5.10. DEMONSTRATION OF THERAPEUTIC OR PROPHYLACTIC

UTILITY

The Therapeutics of the invention can be tested in vivo for the desired therapeutic or prophylactic activity. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, prior to administration to humans, any animal model system known in the art may be used.

5.11. ANTISENSE REGULATION

OF

VERTEBRATE DELTEX EXPRESSION The present invention provides the therapeutic or prophylactic use of nucleic acids of at least six nucleotides that are antisense to a gene or cDNA encoding vertebrate Deltex or a portion thereof. "Antisense" as used herein refers to a nucleic acid capable of hybridizing to a portion of a vertebrate deltex RNA (preferably mRNA) by virtue of some sequence complementarity. Such antisense nucleic acids have utility as Antagonist Therapeutics of the invention, and can be used in the treatment or prevention of disorders as described supra in Section 5.8 and its subsections.

The antisense nucleic acids of the invention can be oligonucleotides that are double-stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced sequences.

-49- WO 97/18822 PCT/US96/18675 In a specific embodiment, the vertebrate deltex antisense nucleic acids provided by the instant invention can be used for the treatment of tumors or other disorders, the cells of which tumor type or disorder can be demonstrated (in vitro or in vivo) to a vertebrate deltex gene or a Notch gene. Such demonstration can be by detection of RNA or of protein.

The invention further provides pharmaceutical compositions comprising an effective amount of the vertebrate deltex antisense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described infra in Section 5.12. Methods for treatment and prevention of disorders (such as those described in Sections 5.8 and 5.9) comprising administering the pharmaceutical compositions of the invention are also provided.

In another embodiment, the invention is directed to methods for inhibiting the expression of a vertebrate deltex nucleic acid sequence in a prokaryotic or eukaryotic cell comprising providing the cell with an effective amount of a composition comprising an antisense vertebrate deltex nucleic acid of the invention.

Vertebrate deltex antisense nucleic acids and their uses are described in detail below.

5.11.1. VERTEBRATE DELTEX ANTISENSE NUCLEIC ACIDS The vertebrate deltex antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides (ranging from 6 to about 50 oligonucleotides). In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100-nucleotides, or at least 200 nucleotides. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or doublestranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl.

Acad. Sci. 84:648-652; PCT Publication No. WO 88/09810, published December 15, 1988) or blood-brain barrier (see, PCT Publication No. WO 89/10134, published April 1988), hybridization-triggered cleavage agents (see, Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents (see, Zon, 1988, Pharm. Res. 5:539-549).

WO 97/18822 WO 97/ 8822PCT/US96/1 8675 In a preferred aspect of the invention, a vertebrate deltex antisense oligonucleotide is provided, preferably of single-stranded DNA. In a most preferred aspect, such an oligonucleotide comprises a sequence antisense to the sequence encoding an SH3binding domain or a Notch-binding domain of vertebrate deltex or zinc finger domain, most preferably, of human detex. The oligonucleotide may be modified at any position on its structure with substituerns generally known in the art.

The vertebrate deltex antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5 '-methoxycarboxymethyluradil, 5-methoxyuracil, 2-methylthio-N6isopentenyladenine, uradil-5-oxyacetic acid wybutoxosine, pseudouradil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil- 2 0 5-oxyacetic acid methylester, uracil-5-oxyacetic acid 5-inethyl-2-thiouracil, 3-(3-amino- 3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

In another embodiment, the oligonucleotide comprises at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the oligonucleotide comprises at least one modified phosphate backbone selected from the group. consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methyiphosphonate, an ailkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the oligonucleotide is an ca-anomeric olig onucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual (3-units, the strands run parallel to each other (Gautier et al., 1987, Nuci. Acids Res. 15:6625-6641).

51 WO 97/18822 PCT/US96/18675 The oligonucleotide may be conjugated to another molecule, a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

Oligonucleotides of the invention may be synthesized by standard methods known in the art, by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res.

16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.

In a specific embodiment, the vertebrate deltex antisense oligonucleotide comprises catalytic RNA, or a ribozyme (see, PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al., 1990, Science 247:1222-1225). In another embodiment, the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

In an alternative embodiment, the vertebrate deltex antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector can be introduced in vivo such that it is taken up by a cell, within which cell the vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the vertebrate deltex antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art.

Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding the vertebrate deltex antisense RNA can be by any promoter known in the art to act in vertebrate, preferably human, cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner -52 WO 97/18822 SPCT/US96/18675 et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), etc.

The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a vertebrate deltex gene, preferably a human deltex gene. However, absolute complementarity, although preferred, is not required. A sequence "complementary to at least a portion of an RNA," as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA,'forming a stable duplex; in the case of double-stranded vertebrate deltex antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with a vertebrate deltex RNA it may Scontain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

5.11.2. THERAPEUTIC UTILITY OF VERTEBRATE DELTEX ANTISENSE NUCLEIC ACIDS The vertebrate deltex antisense nucleic acids can be used to treat (or prevent) malignancies or other disorders, of a cell type which has been shown to express vertebrate deltex or Notch. In specific embodiments, the malignancy is cervical, breast, or colon cancer, or squamous adenocarcinoma. Malignant, neoplastic, and pre-neoplastic cells which can be tested for such expression include but are not limited to those described supra in Sections 5.8.1 and 5.9.1. In a preferred embodiment, a single-stranded DNA antisense vertebrate deltex oligonucleotide is used.

Malignant (particularly, tumor) cell types which express vertebrate deltex or Notch RNA can be identified by various methods known in the art. Such methods include but are not limited to hybridization with a vertebrate deltex or Notch-specific nucleic acid by Northern hybridization, dot blot hybridization, in situ hybridization), observing the ability of RNA from the cell type to be translated in vitro into Notch or vertebrate Deltex, immunoassay, etc. In a preferred aspect, primary tumor tissue from a patient can be assayed for Notch or vertebrate Deltex expression prior to treatment, by immunocytochemistry or in situ hybridization.

-53 WO 97/18822 PCT/US96/18675 Pharmaceutical compositions of the invention (see Section 5.12), comprising an effective amount of a vertebrate deltex antisense nucleic acid in a pharmaceutically acceptable carrier, can be administered to a patient having a malignancy which is of a type that expresses Notch or vertebrate deltex RNA or protein.

The amount of vertebrate deltex antisense nucleic acid which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Where possible, it is desirable to determine the antisense cytotoxicity of the tumor type to be treated in vitro, and then in useful animal model systems prior to testing and use in humans.

In a specific embodiment, pharmaceutical compositions comprising vertebrate deltex antisense nucleic acids are administered via liposomes, microparticles, or microcapsules. In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the vertebrate deltex antisense nucleic acids. In a specific embodiment, it may be desirable to utilize liposomes targeted via antibodies to specific identifiable tumor antigens (Leonetti et al., 1990, Proc. Natl. Acad. Sci. U.S.A.

87:2448-2451; Renneisen et al., 1990, J. Biol. Chem. 265:16337-16342).

5.12. THERAPEUTIC/PROPHYLACTIC ADMINISTRATION AND COMPOSITIONS The invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a Therapeutic of the invention. In a preferred aspect, the Therapeutic is substantially purified. The subject is preferably an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.

Various delivery systems are known and can be used to administer a Therapeutic of the invention, encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a Therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings oral mucosa, rectal and intestinal mucosa, etc.) and may be -54- WO 97/18822 PCT/US96/18675 administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, s aid implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.

In another embodiment, the Therapeutic can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.) In yet another embodiment, the Therapeutic can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball Wiley, New York (1984); Ranger and Peppas, J.

Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg.

71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, the brain, thus requiring only a fraction of the WO 97/18822 PCT/US96/18675 systemic dose (see, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

In a specific embodiment, administration of a Therapeutic into a Notchexpressing cell is accomplished by linkage of the Therapeutic to a Delta (or other toporythmic) protein or portion thereof capable of mediating binding to Notch. Contact of a Notch-expressing cell with the linked Therapeutic results in binding of the linked Therapeutic via its Delta portion to Notch on the surface of the cell, followed by uptake of the.linked Therapeutic into the Notch-expressing cell.

In a specific embodiment, the Therapeutic is delivered intracellularly by expression from a nucleic acid vector, or by linkage to a Delta protein capable of binding to Notch followed by binding and internalization, or by receptor-mediated or diffusion mechanisms).

In a specific embodiment where the Therapeutic is a nucleic acid encoding a protein Therapeutic, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see Joliot et al., 1991, Proc. Natl. Acad. Sci.

USA 88:1864-1868), etc. Alternatively, a nucleic acid Therapeutic can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

In specific embodiments directed to treatment or prevention of particular 0disorders, preferably the following forms of administration are used: Disorder Preferred Forms of Administration Cervical cancer Topical Gastrointestinal cancer Oral; intravenous Lung cancer Inhaled; intravenous -56 WO 97/18822 PCT/US96/18675 Leukemia Intravenous; extracorporeal Metastatic carcinomas Intravenous; oral Brain cancer Targeted; intravenous; intrathecal Liver cirrhosis Oral; intravenous Psoriasis Topical Keloids Topical Baldness Topical Spinal cord injury Targeted; intravenous; intrathecal Parkinson's disease Targeted; intravenous; intrathecal Motor neuron disease Targeted; intravenous; intrathecal Alzheimer's disease Targeted; intravenous; intrathecal The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, 57 WO 97/18822 PCT/US96/18675 etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. In another preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to mammals. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The Therapeutics of the invention can be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2 -ethylamino ethanol, histidine, procaine, etc.

The amount of the Therapeutic of the invention which will be effective in the 0treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for 58 WO 97/18822 PCT/US96/18675 intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5% to by weight; oral formulations preferably contain 10% to 95% active ingredient.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

5.13. DIAGNOSTIC UTILITY Vertebrate Deltex proteins, analogues, derivatives, and subsequences thereof, vertebrate deltex nucleic acids (and sequences complementary thereto), anti-vertebrate Deltex antibodies, have uses in diagnostics. Such molecules can be used in assays, such as immunoassays, to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting vertebrate Deltex expression, or monitor the treatment thereof. In particular, such an immunoassay is carried out by a method comprising contacting a sample derived from a patient with an anti-vertebrate Deltex antibody under conditions such that immunospecific binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody. In a specific aspect, such binding of antibody, in tissue sections, preferably in conjunction with binding of anti-Notch can be used to detect aberrant Notch and/or vertebrate Deltex localization or aberrant levels of Notch-vertebrate Deltex colocalization in a disease state. In a specific embodiment, antibody to vertebrate Deltex can be used to assay in a patient tissue or serum sample for the presence of vertebrate Deltex where an aberrant level of vertebrate Deltex is an indication of a diseased condition. Aberrant levels of vertebrate Deltex binding ability in an endogenous Notch protein, or aberrant levels of binding ability to Notch (or other vertebrate Deltex ligand) in an endogenous vertebrate Deltex protein may be indicative of a disorder of cell fate cancer, etc.) By "aberrant levels," is meant increased or decreased levels relative to that 59- WO 97/18822 PCT/US96/18675 present, or a standard level representing that present, in an analogous sample from a subject not having the disorder.

The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.

Vertebrate deltex genes and related nucleic acid sequences and subsequences, including complementary sequences, and other toporythmic gene sequences, can also be used in hybridization assays. Vertebrate deltex nucleic acid sequences, or subsequences thereof comprising about at least 8 nucleotides, can be used as hybridization probes.

Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with aberrant changes in vertebrate Deltex expression and/or activity as described supra. In particular, such a hybridization assay is carried out by a method comprising contacting a sample containing nucleic acid with a nucleic acid probe capable of hybridizing to vertebrate deltex DNA or RNA, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization.

6. EXAMPLE: CLONING AND CHARACTERIZATION OF HUMAN DELTEX As described herein, we have accomplished the isolation and molecular characterization of human deltex. We report the cloning and sequencing of human deltex.

Human deltex encodes five putative SH3 domain binding sites and a ring-H2-zinc finger in similar locations to the corresponding motifs found in Drosophila Deltex.

6.1. RESULTS 6.1.1. MOLECULAR CLONING OF THE HUMAN DELTEX LOCUS Human deltex was isolated through a combination of computer and biochemical screens. Initially, a human expressed sequence tag database was screened for homology against the amino acid sequence of Drosophila Deltex. The critical part of this search involved the assumption that stop codons in a particular reading frame of the database WO 97/18822 PCT/US96/18675 are the result of sequencing mistakes. Accordingly, stop codons were ignored and the open reading frame was extended in a different frame. The predicted amino acid sequence encoded by the hypothetical open reading frames were then compared with the protein product of the Drosophila deltex transcription unit.

We previously identified the Drosophila deltex transcription unit by showing via germline-mediated transformation experiments that a genomic fragment containing this transcription unit is capable of complementing most deltex mutant defects. Moreover, this genomic fragment rescues the normally lethal genetic interaction that results when flies are doubly mutant for deltex and nd. Finally, Northern analysis indicates a maternal loading of deltex transcripts into the developing oocyte, a finding that is consistent with the maternal effect observed upon embryogenesis in eggs laid by homozygous mutant mothers (Xu and Artavanis-Tsakonas, 1990 Genetics 126:665-677). cDNA clones homologous to the transcription unit were isolated from an embryonic cDNA library, the complete nucleotide sequence (SEQ ID NO: 1) and predicted protein product were then determined (SEQ ID NO:2).

Comparison of the amino acid sequence of Drosophila Deltex with that predicted for what we deduced to be hypothetical open reading frames in the database identified a sequence:gnl I dbest I 24254 T05200 with significant homology to Drosophila Deltex. Within T05200, five conserved stretches of amino acids were found in different reading frames, at residues 7-39 (SEQ ID NO:4), 102-149 (SEQ ID NO:6), 138-245 (SEQ ID NO:8), and 200-310 (SEQ ID NO:10) corresponding to Drosophila Deltex residues 545- 555 (SEQ ID NO:3), 565-580 (SEQ ID NO:5), 581-616 (SEQ ID NO:7) and 602-638 (SEQ ID NO:9) respectively. These sequences are shown in Table II, identical amino acids are shown in bold.

-61 WO 97/18822 PCT/US96/18675 TABLE II

AMINO

ACID

NOS.

Drosophila 602-638 VYGEKVGVQPIGSMSWSIISKNLPGHEGQNTIQIVYD Deltex T05200 200-310 IYGEKTGTQPPGKMEFHLIPHSLXFGPDTQTXRIVYD Drosophila 565-580 LSRCQHLMHLQCLNGM Deltex T05200 102-149 LGRCGHMYHLLCLVAM Drosophila 581-616 IIAQQNEMNKNLFIECPVCGIVYGEKVGNQPIGSMS Deltex T05200 138-245 LVAMYSNGNKDGSLQCPTCKPSMGRRRVRSRLGRWS Drosophila 545-555 QPCPMCMEELV Deltex T05200 7-39 EDCTICMERLV A series of two 5' primers (hdx-1 (SEQ ID NO:26) and hdx-2 (SEQ ID NO:27)) and two 3' primers (hdx-3 (SEQ ID NO:28) and hdx-4 (SEQ ID NO:29 were synthesized based on the DNA sequence of gnl I dbest I 24254 T05200.

PCR reactions were performed using the four different primer combinations and a human fetal brain cDNA library (Invitrogene) as the template. The PCR product -was sequenced and found to have the same DNA sequence as gnl I dbest I 24254 T05200.

The PCR product generated using the hdx-1 and hdx-4 primers was then labeled and used to screen another human fetal brain cDNA library. The isolate was sequenced (SEQ ID NO:11) and the predicted protein determined (SEQ ID NO:12) (Figure 2A-C). Not greater than 107 continuous nucleotides of SEQ ID NO:11 were present in T05200. Applying standard techniques, the cDNA isolate obtained using the PCR product as probe was then labeled and itself used as a probe to screen a northern blot containing poly(A)I mRNA isolated from various human tissue samples. This probe was observed to hybridize to a 5.4-kb RNA in -62- WO 97/18822 PCT/US96/18675 heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. When this probe was used to screen a zoo blot (a blot containing genomic EcoRI-digested DNA of various species, obtained from Clontech) by Southern hybridization, hybridization was observed in genomic human, monkey, rat, mice, dog, cow, and yeast DNA. Hybridization was not observed in the rabbit and chicken genomic

DNA.

Structural analysis of the human Deltex protein: The predicted human Deltex product has 720 amino acids and an estimated molecular mass of approximately 80 kDa. The 180 amino terminal residues of human Deltex have an approximate identity of 33% with the corresponding amino acid residues of Drosophila Deltex and the nucleic acids encoding these amino acids have an approximate 52% identity. The 180 carboxy terminal amino acids of human Deltex have an approximate 48% identity with the corresponding amino acid residues of Drosophila Deltex and the nucleic acids encoding these carboxy terminal amino acids have an approximate 49% identity.

A structural analysis of human Deltex protein revealed a conserved structure among Deltex proteins (see Figure Like Drosophila Deltex, human Deltex has both ring-H2-zinc finger (amino acids 411-471) (SEQ ID NO:25) and putative SH3-binding domains. Noticeably absent from the human Deltex are the two opa repeats that subdivide the primary structure of the Drosophila Deltex into three domains. Each of the Drosophila Deltex domains I, II, and III, has been found using the yeast "interaction trap assay" to be capable of mediating homotypic interactions (see infra).

i) Domain I: Domain I corresponds to.the N-terminal 303 amino acids of 3 Drosophila and the first 237 amino acids of human Deltex. In Drosophila, we have demonstrated that the region of human Deltex corresponding to the first 175 amino acids of domain I is essential and sufficient to bind the Notch ANK repeats and overexpression of this domain can rescue loss-of-function phenotypes of Deltex. Since Drosophila Deltex can bind to human Notch-1 and 2, conservation -63- WO 97/18822 PCT/US96/18675 of binding activity between human Deltex and Drosophila domain I is suggested.

Furthermore, this has been demonstrated. See infra.

ii) Putative SH3 binding domains: Domain II of Drosophila Deltex contains a putative SH3-binding site (amino acids 476-484) (Matsuno et al., 1995, Development 121(8):2633-2644).

Five putative SH3-binding sites (SEQ ID NOS: 17-21) are found in human Deltex (within amino acids 226-377) in a position corresponding to the SH3-binding site in domain I of Drosophila Deltex (Table lI).

SH2 and SH3 domains are conserved protein modules so named based on their homology to the oncogene Src (Src Homology). These motifs have -been implicated in mediating protein-protein interactions in a number of signal transduction pathways (reviewed in Cell 71:359-362;Science 252:668-674; Trends S Cell Biol. 3:8-13; FEBS 307:55-61). Recently, a complementary motif that binds to the SH3 domain has been identified and called simply an 'SH3-binding domain' ("SH3-BD") (Science 259:1157-1161). The core binding region of SH3-BD is proline-rich and approximately ten residues in length. As shown in Table III, this motif, as defined from a mouse protein that experimentally bound an SH3 domain (SEQ ID NO:23), is shown aligned to the putative SH3-binding site in Drosophila Deltex (SEQ ID NO:22) and the five regions (SEQ ID NOS: 17-21) that may represent human versions of this motif. These regions are located centrally in the Deltex protein. For reference, regions of the protein encoded by the Drosophila Son ofsevenless (SOS) protein (SEQ ID NO:24), which may also contain SH3-BD, is shown. The Son of sevenless encoded protein, a putative guanine nucleotide exchange factor (GNEF), has been shown to bind to an 'adaptor' protein (drk) containing only SH2 and SH3 modules, although the actual residues that mediate binding have not been accurately defined (Simon et al., 1993, Cell73:169-177 and Olivier et al., 1993, Cell 73:179-191).

-64- WO 97/18822 PCT/US96/18675 TABLE III Putative SH-3 Domain Binding Sites in Deltex Proteins SEQ ID NO: Fly Deltex RAP-VPPPLPLHPRQQ 22 Mouse 3BP-1 RAPTMPPPLPPVPPQP 23 .Fly Son of sevenless RA--VPPPLPPRRKER 24 Human Deltex I 226-244 VXPAPPLSXPXXPGGPPGA 17 II 271-285 XSPGXPPRSPGAPGG 18 m 311-322 SIPPGVPALPVK 19 IV 353-365 RAPKPILHPPPVS V 368-377 VKPVPGVPGV 21 There are currently only six SH3-containing proteins identified in Drosophila, any one of which may be a direct binding partner of Deltex, and thus an indirect partner of Notch.

The functional requirement of the Deltex domain II-III in Drosophila which contains the putative SH3 domain binding site and ring-H2-zinc finger has been suggested from deletion analyses, in which the deletion of these domains resulted in a significant reduction of the ability to activate the Notch signaling pathway. These results indicate that domains II and III are not redundant.

iii) Ring-H2-zinc finger: Human deltex (nucleotides 1734-1916 of SEQ ID NO: 11) encodes a ring-H2-zinc finger (SEQ ID NO: 25), appearing as amino acids 411-471 of SEQ ID NO: 12 in that part of human Deltex which corresponds to domain III of Drosophila Deltex. This type of zinc finger is believed to be involved in protein/protein interactions.

WO 97/18822 PCT/US96/18675 6.2. MATERIAL AND METHODS 6.2.1. SEQUENCE DETERMINATION AND ANALYSIS The EcoRI-cDNA insert was subcloned directly in both orientations into Bluescript KS. Overlapping deletions were produced on the insert using the DNAse I method to generate bidirectional deletions (Eberle et al., 1993, Biotechniques 14:408). The resulting deletions were analyzed using an IBI automatic sequencer.

DNA sequence manipulations were performed using Intelligenetic's PC-GENE software. Open reading frame prediction and plotting were performed using the University of Wisconsin program CODONPREFERENCE (Gribshov et al., 1984, Nucl. Acids Res. 12:539-549). The GenPept and SWISS-PROT databases were searched with all or part of the deduced amino acid sequence using the FASTA program (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444-2448) available by the GenBank FASTA server through BITNET.

7. EXAMPLE: HUMAN DELTEX BINDS HUMAN AND DROSOPHILA NOTCH We have demonstrated in Drosophila a specific and direct physical interaction between Deltex and Notch ANK repeats (Diederich et al., 1994, Development 120:473-481). To study protein-protein interactions between human Deltex and various cytoplasmic domains of human and Drosophila Notch receptors, we conducted expression studies in yeast using the so-called 'interaction trap' assay technique (Zervos et al., 1993, Cell 72:223-232).

In this assay, one protein segment is fused to the DNA-binding domain of the LexA protein, which in turn binds to the promoter of a LexAop-lacZ reporter construct without activating transcription. These constructs are referred as pEG.

A second foreign protein segment is fused to an acidic transcriptional activation domain that does not bind DNA on its own. These constructs are referred to as pJG. Coexpression of these two proteins in yeast cells results in the functional reconstruction of an active LexA "hybrid" transcription factor if the foreign proteins physically interact with one another. Activity of the hybrid transcription factor is monitored by transcription of the 3-galactosidase reporter gene.

Expression of fusion proteins from the pJG construct is induceu when yeast cells -66- WO 97/18822 PCT/US96/18675 are cultured in the galactose media but not in the glucose media. Therefore, positive interaction should be observed only in galactose media.

The constructs examined using the yeast interaction were as follows: the pEGhDeltex construct contains the entire coding region of human Deltex; pJGhNotch-1 encodes the ankyrin repeats region of human Notch-1 from am acids 1826-2147; pJGhNotch-2 encodes the ankyrin repeats region of human Notch- from amino acids 1826-2147; pJGhNotch-2 encodes the ankyrin repeats region of human Notch- 2 from amino acids 1772-2084; pJGhNotch encodes the ankyrin repeats of Drosophila Notch from amino acids 1827-2259; and JGfHairless contains the entire coding region of Drosophila Hairless.

As presented in Table IV, significant induction of (-galactosidase activity was observed when yeast cells cotransfected with pEGhDeltex and pJGhNotch-1, pJGHNotch-2 or pJGHfNotch, were cultured in galactose media (Table IV). These results indicate that human Deltex binds to the ankyrin repeats human Notch-1, human Notch-2 as well as that of Drosophila Notch. Standard deviation is presented in the parentheses.

TABLE IV Media Coexpressed Constructs Galactose Glucose pEGhDeltex/pJGhNotch-1 732(35) 4(1) pEGhDeltex/pJGhNotch-2 195(25) 11(5) pEGhDeltex/pJGfNotch 892(184) 14(4) pEGhDeltex/pJGHairless 61(13) 20(2) pEGhDeltex/pJG 38(1) 21(5) pEG/pJGhNotch-1 47(13) 13(5) pEG/pJGhNotch-2 48(7) 19(8) pEG/pJGfNotch 69(18) 20(5) pEG/pJGHairless 59(4) 39(14) pEG/pJG 60(10) 32(4) -67 WO 97/18822 PCT/US96/18675 8. EXAMPLE: CLONING OF VERTEBRATE DELTEX GENES The evolution of humans and Drosophila diverged about 600 million years ago. As discussed supra, Deltex protein demonstrates a conserved structure in these two evolutionary distant species. Knowledge of the conserved regions of the protein allows one to design synthetic degenerate primers for use in hybridization and PCR reactions which enable the cloning of Deltex encoding nucleic acids in other organisms.

Five regions of high conservation between human and Drosophila are found in amino acid stretches of human Deltex amino acid numbers 414-419 (SEQ ID NO:30), 475-480 (SEQ ID NO:31), 504-511 (SEQ ID NO:32), 531-539 (SEQ ID NO:33) and 557-564 (SEQ ID NO:34). These sequences are conserved in Drosophila Deltex amino acid stretches 549-555 (SEQ ID NO:35), 603-608 (SEQ ID NO:36), 632-639 (SEQ ID NO:37), 659-667 (SEQ ID NO:38) and 685- 692 (SEQ ID NO:39), respectively. Conserved amino acid stretches may be used alone or in combination to isolate the deltex encoding nucleic acids of other organisms.

By way of example, a murine deltex gene is obtained as follows: Standard techniques are utilized to synthesize a series of degenerate primers encoding amino acids 414-419 in Drosophila (SEQ ID NO:30) and 549-555 in human (SEQ ID NO:35) in a 5' to 3' orientation. A second series of degenerate primers corresponding to the antisense strand of the nucleic acids encoding amino acids 475-480 in Drosophila (SEQ ID NO:31) and 603-608 in human (SEQ ID NO:36) is also synthesized. The two series of primers are added to a mixture containing mouse embryonic cDNA as template for the PCR amplification.. PCR is carried out at a range of stringencies, according to methods commonly known, to allow for varying degrees of nucleotide similarity between the known deltex sequences and the mouse nucleic acid homolog being isolated.

After successful PCR amplification, the segment of mouse deltex gene is molecularly cloned and sequenced through techniques known in the art. This segment is used as a probe to isolate a complete cDNA and genomic clone. The complete nucleotide sequence of the mouse deltex homolog is determined by sequence analysis.

-68 9. DEPOSIT OF MICROORGANISMS Plasmid pBS hdx containing a cDNA insert encoding a full-length human deltex as a EcoRI insert in Bluescript vector (Stratagene) was deposited by S. Leslie Misrock, of Pennie Edmonds, 1155 Avenue of the Americas, New York, New York 10036 on behalf of Yale University on November 17, 1995, with the American Type Culture Collection, 1 0801 University Boulevard, Manassas, Virginia 20110-2209, under the provisions of the Budapest Treaty of the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures, and assigned accession number 97341.

The present invention is not to be limited in scope by the microorganism deposited or the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those S* 15 skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which are incorporated in their entireties.

2 '2 69l '-t 1 li r WO 97/18822 PCT/US96/18675 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Artavanis-Tsakonas, Spyridon Matsuno, Kenji (ii) TITLE OF INVENTION: VERTEBRATE DELTEX PROTEINS, NUCLEIC ACIDS, AND ANTIBODIES, AND RELATED METHODS AND

COMPOSITIONS

(iii) NUMBER OF SEQUENCES: 39 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Pennie Edmonds STREET: 1155 Avenue of the Americas CITY: New York STATE: New York COUNTRY: U.S.A.

ZIP: 10036-2711 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION

DATA:

APPLICATION NUMBER: To Be Assigned FILING DATE: 22-NOV-1995

CLASSIFICATION:

(viii) ATTORNEY/AGENT

INFORMATION:

NAME: Misrock, S. Leslie REGISTRATION NUMBER: 18,872 REFERENCE/DOCKET NUMBER: 7326-036 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (212) 790-9090 TELEFAX: (212) 869-9741/8864 TELEX: 66141 PENNIE INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 3771 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 345..2555 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AAATGCTAGA AAAACCGTTT TTACCATCAA ACGTGAATTC TTAAGCTGCG CCTAAACGAA ACCGAGTGAC TAAAGAACCA GAACGAAAAC TTCGGGAAAA TGGAAGCCAG GGAAAATCAG 120 GGATAACTAA CGCTGGCAGC GGGTCCACCA TTTTTAATTT CTTTGTTTAT TTTGTGCCCA 180 TCTTCGCGAG CGAGCGAGAT AGCGCGACAG CAACAGCAAG AGAGAGCGAG AGAGAGAGTG 240 WO 97/1 8822 PCT/US96/18675 AGTGAGTGAG AGCTAGTGMA GAGAGCGCAG GAGGAGTTGG ATATGGAAAT GGGCATGGAT 300 ATGGCAATGG GCTCACTCCA CGGATAACGG ATCAACTGCA AGCA ATG GCC AGC AGC 356 Met Ala Ser Ser 1 GCC GGP.

Ala Gi GCC GCC Ala Ala GGG CCG Gly Pro TCG CGC Ser Arg GAA CGC Glu Arg GAT CCC Asp Pro GAA TCG Glu Ser CCC ATG Arg Met GAG TGG Glu Trp AAC ATG Asn Met 150 CAA ACC Gin Thr 165 AAT TTT Asn Phe AGC ATT Ser Ile CCA CAA Pro Gin CAG TAC Gin Tyr 230 GTG CCC Val Pro 245 LAGT GCG *Ser Ala TCC ACT *Ser Ser CCC GTG Pro Val GGC AAG Gly Lys GCC CAC Ala His AGC CTG Ser Leu GAG GCG Ciu Ala TTA TAC Leu Tyr 120 TCG GGC Ser Gly 135 CAC GTC His Val TTG GAC Leu Asp TCC AAT Cys Asn CGG CGT Arg Arg 200 CAG GCC Gin Ala 215 AAC ACT Asn Thr ATG ACC Met Thr

GCA

Ala

TGI

Cys

AAC

Asn

TGG

Trp,

CC

Ala

GAG

Glu

GAA

Clii 105

GCA

Ala

GC

Gly

CAG

Gin

CTG

Leu

CTC

185

ACC

Thr

AAC

Asn

CTA

Leu

AGG

Arg

TCC

Ser 10

CCC

Ala

CAC

His

CTG

Leu

AAG

Lys

CAG

Gin 90

ACG

Thr

CCC

Pro

AGT

Ser

TGC

Cys

TGC

Cys 170

ACC

Thr

CAA

Gin

CAA

Gin

CCC

Pro

CAA

Gin 250

GGA

Gly

ACC

Thr

GCC

Ala

CCC

Pro

AAA

Lys 75

TAC

Tyr

CC

Arg

AGC

Ser

GCC

Ala

ATC

Ile 155

AAC

Asn

CAC

His

CAG

Gin

CTC

Leu

AAA

Lys 235 CAG Gin TCC GTT GTT CCC Ser Vai Vai Pro 15 ATG GCC CTG TCC Met Ala Leu Ser 30 CAC GCC GTC TGC His Ala Val Cys 45 TAT TCG CCG GCG Tyr Ser Pro Ala 60 CTG ACG CGC GTC Leu Thr Arg Val TAC GTC AAC GTG Tyr Val Asn Val 95 TCC GCC CTG CTG Ser Cly Leu Leu 110 TCG CCG CCG GGC Ser Pro Ala Gly 125 GAT AGC AAC AAC Asp Ser Asn Asn 140 ATC GAG GAC GCC Ile Ciu Asp Ala ACC CAC ATC CGC Thr His Ile Gly 175 GTG CCC CAA CCC Vai Arg Gin Pro 190 CCG CCG TAT CCC Ala Pro Tyr Pro 205 AAG TCG AAT TCC Lys Ser Asn Ser 220 CTG GGC GAC ACC Leu Cly Asp Thr CAC CCA TTG CCC His Pro Leu Pro 255 OCT CGC GGA GGT AGC Gly Cly Gly Gly Ser

ACC

Thr

GTG

Val1

GTG

Val

ATG

Met

CC

Arg

ACC

Thr

AAG

Lys

GAC

Asp

TGG

Trp 160

CTG

Leu

AGC

Ser

TTG

Leu

CC

Al a

AAG

Lys 240

ACC

Thr GCC GGA Ala Cly TGG GAG Trp Gu TCG CAG Ser Gin CTG AGC Leu Ser ACA ATG Thr Met ATC GOT Ile Oiy GGC ACC Gly Thr .130 TGG CGG Trp Arg 145 GCG AGG Ala Arg CCG TAC Pro Tyr GGA CCC Ciy Pro GTG AAA Val Lys 210 AGC GTO.

Ser Val 225 AGC CTC Ser Leu ACC CAT Ser His TCC GGT Ser Gly TTC GAG Phe Giu

CAC

His

GAT

Asp

ACC

Thr

GTT

Vai 115

AAO

Lys

CCC

Pro

GGC

Oly

ACC

Thr

ATG

Met 195

CTA

Leu

AGC

Se r

CAC

His

CAA

Gin

TTG

Leu

GCG

Ala

CAG

Gin 100

CGG

Arg

TOG

Trp

TAC

Tyr

CAA

Glu

ATT

Ile 180

CGC

Arg

ACG

Thr

AGC

Se r

AGA

Arg

GTG

Val1 260 404 452 500 548 596 644 692 740 788 836 884 932 980 1028 1076 1124 -71- WO 97/18822 PCTIUS96/1 8675 GAG GAG GAG GAG CAT GAG CTC GAG CAT CAA CAG CAG GAG GAG GAG CAA 1172 Gin Gin Gin Gin His Gin Leu Gin His Gin Gin Gin Gin Gin Gin Gin 265 270 275 CAT CAT CAC CAG CAT GAG CAA CAA GAG CAT GAG CAA GAG GAG CAA CAT 1220 His His His Gin His Gin Gin Gin Gin His Gin Gin Gin Gin Gin His 280 285 290 GAG ATG GAG CAC CAT GAG ATC CAT CAT GAG ACG GCG CCC AGG AAG CCG 1268 Gin Met Gin His His Gin Ile His His Gin Thr Ala Pro Arg Lys Pro 295 300 305 CCC AAG AAG CAC AGC GAG ATC TCC ACC ACC AAT CTA CGC GAG ATA GTC 1316 Pro Lys Lys His Ser Giu Ile Ser Thr Thr Asn Leu Arg Gin Ile Leu 310 315 320 AAC AAC CTA AAC ATC TTC AGG AGC AGC ACT AAG GAG CAA TCG AAC ATG 1364 Asn Asn Leu Asn Ile Phe Ser Ser Ser Thr Lys His Gin Ser Asn Met 325 330 335 340 TCG ACG GCG GCC AGT GCC ACT TGA TCC TCC TGA TCG CCC TCG CTG CAC 1412 Ser Thr Aia Ala Ser Aia Ser Ser Ser Ser Ser Ser Ala Ser Leu His 345 350 355 CAT GCC AAC CAT CTG TCG CAT GCG CAC TTT TGG CAC GCG AAG AAG ATG 1460 His Ala Asn His Leu Ser His Ala His Phe Ser His Ala Lys Asn Met 360 365 370 CTG ACT CCC TCG ATG AAC AGT CAT CAT AGT CGC TGC TCG GAG GGA TCG 1508 Leu Thr Ala Ser Met Asn Ser His His Ser Arg Cys Ser Glu Gly Ser 375 380 385 CTG GAG TCG CAA AGG AGC AGC CGG ATG GGC TCC CAT CGC TCG AGA TCG 1556 Leu Gin Ser Gin Arg Ser Ser Arg Met Gly Ser His Arg Ser Arg Ser 390 395 400 CGA AGG CGG ACC TCG GAG ACG GAG ACG AAC AGT GTG AAA TCG CAT CGG 1604 Arg Thr Arg Thr Ser Asp Thr Asp Thr Asn Ser Val Lys Ser His Arg 405 410 415 420 CGG AGA CCC AGT GTG GAG ACC GTG TCC ACT TAC CTG AGC CAC GAG AGC 1652 Arg Arg Pro Ser Val Asp Thr Val Ser Thr Tyr Leu Ser His Giu Ser 425 430 435 AAG GAG AGC CTG CCC AGC AGG AAC TTT CCC ATT TCG GTC AAT GAT CTG 1700 Lys Glu Ser Leu Arg Ser Arg Asn Phe Ala Ile Ser Val Asn Asp Leu 440 445 450 CTG GAG TCC TCG GTT GGC AGG GAT GAA GTT TTT GTG CCC TCC GTG GCG 1748 Leu Asp Cys Ser Leu Gly Ser Asp Giu Val Phe Val Pro Ser Val Pro 455 460 465 CCA'-.TCG TGG CTG GGG GAA AGG GGG CCG GTG CCG CCG CCA TTA CCA CT 1796 Pro Ser Ser-Leu Gly Giu Arg Ala Pro Vai Pro Pro Pro Leu Pro Leu 470 475 480 CAT CCG CGA GAG CAA GAG GAG GAG CAA CAA GAG GAG CAA GAG CTG GAG 1844 His Pro Arg Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Leu Gin 485 490 495 500 ATC CAA GAG GAG CAA GAG GCG GAG GAG GAG GAG GAG CAA TCA ATC CCC 1892 Met Gin Gin Gin Gin Gin Ala Gin Gin Gin Gin Gin Gin Ser Ile Ala 505 510 515 GGT TGG ATT GTG GGC GTG GAG CCG GCC ACC GAT ATG ATA TCG CGT TTT 1940 Gly Ser Ile Val Gly Val Asp Pro Ala Ser Asp Met Ile Ser Arg Phe 520 525 530 GTC AAG GTG CTG GAG CCA CCC CTG TGG CCC AAT GCC GAG CCC TGT CCC 1988 Val Lys Val Val Glu Pro Pro Leu Trp Pro Asn Ala Gin Pro Cys Pro -72- WO 97/18822 WO 97/ 8822PCT/US96/1 8675 ATG TGC ATG Met Cys Met 550 CTG AGT CGC Leu Ser Arg 565 ATA ATT GCC Ile Ile Ala CCT GTA TGC Pro Val Cys GGC AGC ATG Gly Ser Met 615 GGT CAG AAC Gly Gin Asn 630 GAG GAG CTG GTG CAC TCC GCC Giu Glu Leu Val His Ser Ala TGC CAG Cys Gin

CAT

His 570 ATG CAT TTG Met His Leu CAG AAT Gin Asn 560 CAG TGC Gin Cys 575 AAC CTT Asn Leu 545 CCG GCC ATT TCG Pro Ala Ile Ser CTC AAT GGG Leu Asn Gly

CAG

Gin CAA AAC Gin Asn 585 ATC GTT Ile Val GAA ATG AAC Giu Met Asn TAC GGC GAG Tyr Gly Giu 605 ATA ATT AGC Ile Ile Ser AAG TTC ATC Phe Ile 590 AAG GTC Lys Val AAG AAT Lys Asn GAG TGC Giu Cys 595 GGC AAT CAG CCC ATT Gly Asn Gin Pro Ile 610 CTG CCA GGA CAC GAG Leu Pro Gly His Giu TCG TGG AGC Ser Trp Ser ACC ATA CAG Thr Ile Gin TAC GAC ATT GCA Tyr Asp Ile Ala 640 GGA CTG CAG Gly Leu Gin GAG GAG CAT CCG Giu Giu His Pro

CAT

His 650 GGT CGT GCC Gly Arg Ala

TTC

Phe 655

CTG

Leu TTC GCC GTG GGA Phe Ala Val Gly GGG CGA AAG GTT Gly Arg Lys Val 675 CGG ATC TGC Arg Ile Cys TTG CCG GAC TGC Leu Pro Asp Cys CGC TTC CTC AAG Arg Phe Leu Lys 680 CGA TCG GTG ACC Arg Ser Val Thr 695 GAT CAC AAG, ACG Asp His Lys Thr ATT GCA TTC GAT Ile Ala Phe Asp CGG CTG CTT Arg Leu Leu ACC GGA CGC Thr Gly Arg

GAG

Giu 700

ATG

Met GAT GTG GTG ATC Asp Val Val Ile TTC TCG ATC GGA Phe Ser Ile Gly 690 TGG AAC AGT GTG Trp Asn Ser Val 705 ACC TAT TTG CAG Thr Tyr Leu Gin 2036 2084 2132 2180 2228 2276 2324 2372 2420 2468 2516 2565 2625 2685 2745 2805 2865 2925 2985 3045 3105 3165 CAG TTC Gin Phe TTT CCG GAT Phe Pro Asp

CCC

Pro 720 710 CGA ACC Arg Thr 725 ATG CAA CAG Met Gin Gin GTG CAC CTG Val His Leu GGC GTG Giy Val 735 ACG GAT TAAGGATTAG Thr Asp TTCCCTGTCC CCAAGTAGAA CTACCAACCA ACCAATCAAC

CTCGATCATT

AATCCTCGTT

TCGAACCCTA

ATTCGTATTT

GTTAGTTAGT

CTAGTATTAG

CGCCATTCGA

TGTTGATATA

TCTCTTTCAT

CTCTTCCATT

TACTATGATA

AAACAACAGC

GTACGCACAT

TAGTTAGTTG

GCATTATCCT

GTGCAAGCTG

GCTAGCTATA

CAGATCCATG

CGTCGTTAAG

TATTTTTTTT

AAACCACAAT

ACGCAATAAG

TAGCTGTAGT

GATTCTTGAT

TGCCAAAATC

ACCATTGCCC

TGAATTTTCT

TTACTTTCTA

ATAGATATAT

TGCAATTGTA

TTGGCGTACA

TCCCAAGAGA

TCCTGATTCG

GTAGCGCTCC

ATCTCTCCAT

TTATATCGGA

CACCCACCCA

CATAATCTCA

TGTAATAGCG

GCTTCCTTTC

TCATATGTAT

ATCTTGACCC

ATTCAAGCCA

CGTTTATAGG

CTCTCTCGGT

TTTATATAGG

CCGAAGTCCC

GTGTGTGTGC

TTCGAGCTGC

CGCTCTTCCA

TAGCTAGTTA

AAGACACCTA

AGCCAAGCCA

ATATGTATAT

TTCGAATTTG

ATT~iAATAG -73- WO 97/18822 PCT/UJS96/1 8675 TATTTTGAGA GAGGAAATGG AGATGGGTAA ATTCGATAGA CTTGTCTCAC

TTGTCTTGGC

CATTTAATCT

CTTTCATTC-

AACGGAGCAT

TTAGGAGC

ATACATATAC ATATAcATAC CATAACAATA

ATTTTTTTTA

ATTGATATCA

TCGAGCATCG

GCATATAGTA

TGTAGAGATC

GATATGATAT

GATATGATTT

TTTGACGAAT

ATTCCACAAC

GCGAATTTGA

TAGTTGTAAC

ATACATAAAC

TCGAATCCCT

AACGAACTAT

GTACGGACAG

TACTAAGTTG

AAATTCCACA

TGTGATTTTA

GCAGCCAGAT

ATATTTTAAC

TGCATACATT

CGTATACATC

CTAGCGGCTA

TATTTAGCAC

ATTTGAATTA

ATTCCATTAC

ATAGCCCCAT

TGATGAATTG

GCCAATATAT

CTGACCGCGC

TGATTAGTTA

TTCATTATTA

GCATATACAT

AGCCATACGA

TTGCTTTCAT

AGCATATATA

CACCATATTT

TTAAAGTTCA

3225 3285 3345 3405 3465 3525 3585 3645 3705 3765 3771 ATACAGTACA TTTATATATA GTTCAAATAA AGTAACTTCA TTCATGTTCA.

AAAAAAAAAA

AAAAAA

INFORMATION FOR SEQ ID NO:2: SEQUENCE

CHARACTERISTICS:

LENGTH: 737 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE (xi) SEQUENCE Ala Ser Ser Ala TYPE: protein DESCRIPTION: SEQ ID NO:2: Gly Ser Ala Ala Ser Gly Ser Val Val Pro Gly Gly Gly Ser Ala Ala Ser Ser Cys Ala 25 Ala Gly Ser Gly Gly Thr Met Ala Ala His Ala Leu Ser Thr Val Cys Val Pro Pro Val 40 Gly Lys Asn His Trp Glu Ser Gin Phe Glu Ser Arg His Leu Glu Arq Trp Leu Pro Tyr Ser Pro Ala Val His Ala Lys Leu Thr Arg Val Leu Ser Asp Ala Pro Ser Leu Glu Tyr Thr Met Thr Ile Gly Val 115 Gly Thr Lys Glu Ser Glu Ala Thr Arg Ser Pro'Ser Ser Val Asn Val Arg Gly Leu Leu Thr 110 Pro Ala Gly Lys 125 Ser Asn Asn Asp Arg Arg Met Leu Trp Glu Trp 130 Trp Arg Pro Tyr Asn Ser 135 His Leu Gly Gly Ser Ala Val Gin Cys Ile 155 Asp Leu Cys Asn 170 Asn Leu Thr His 185 Asp 140 Ile Glu Asp Ala Ala Arg Gly Pro Tyr Thr Glu Gin Thr 165 Thr His Ile Gly Leu 175 Val Arg Gin Pro Ser 190 Ile Asn Phe Cys 180 -74- WO 97/18822 WO 9718822PCTIUS96/18675 Gly Val Ser 225 Ser Ser Gin Gin Pro 305 Arg Gin Al a Ala Ser 385 Arg Lys Ser Val Pro 465 Pro Gin Gin 545 Pro Prc Lys 210 Val Leu His Gin Gin 290 Arg Gin Ser Ser Lys 370 Giu Ser Ser HIis ksn 450 :3er eu ;ln e r er ~ro lia Met 195 *Leu *Ser *His Gin Gin 275 Gin Lys Ile Asn Leu 355 Asn Gly Arg His Giu 435 Asp Val Pro Leu Ile 515 Arg Cys Ile Arg Thr Ser Arg Val 260 Gin His Pro Leu Met 340 His Met Ser Ser Arg 420 Ser Leu Pro Leu 3 in 500 Al a Phe Pro Ser Ser *Pro *Gin Val 245 Gin His Gin Pro Asn 325 Ser His Leu Leu Arg 405 Arg Lys Leu Pro His 485 Met Giy Vai Met Leu Ile Gin Tyr 230 Pro Gin His Met Lys 310 Asn Thr Ala Thr Gin 390 Thr Arg Giu Asp Ser 470 Pro Gin Ser Lys Cys 550 Ser Arg Arg 200 Gin Ala 215 Asn Thr Met Thr Gin Gin His Gin 280 Gin His 295 Lys His Leu Asn Ala Ala Asn His 360 Aia Ser 375 Ser Gin Arg Thr Pro Ser Ser Leu 440 Cys Ser 455 Ser Leu Arg Gin Gin. Gin Ile Val 520 Val Val 535 Met- Giu Arg Cys Thr Asn Leu Arg His 265 His His Ser Ile Ser 345 Leu Met Arg Ser Val 425 Arg Leu Gly Gin Gin 505' Gly Giu Giu Gin Gin Gin Gin Leu Pro Lys 235 Gin Gin 250 Gin Leu Gin Gin Gin Ile Giu Ile 315 Phe Ser 330 Ala Ser Ser His Asn Ser Ser Ser 395 Asp Thr 410 Asp Thr Ser Arg Gly Ser Glu Arg 475 Gin Gin 490 Gin Ala Val Asp Pro Pro Leu Val 555 His Leu *Ala Pro 205 Lys Ser 220 Leu Gly His Pro Gin His Gin Gin 285 His His 300 Ser Thr Ser Ser Ser Ser Ala His 365 His His 380 Arg Met Asp Thr Val Ser Asn Phe 445 Asp Glu 460 Ala Pro Gin Gin Gin Gin Pro Ala 525 Leu Trp 540 His Ser Met His Tyr Asn Asp Leu Gin 270 His Gin Thr Thr Ser 350 Phe Ser Gly Asn Thr 430 Al a Val Val Glm Gln 510 Se r Pro Ala Leu *Pro Ser Thr Pro 255 Gin Gin Thr Asn Lys 335 Ser Ser Arg Ser Ser 415 Tyr Ile Phe Pro Gin 495 Gin Asp Asn Gin Gin Leu Aia Lys 240 Thr Gin Gin Ala Leu 320 His Ser His Cys His 400 Val Leu Ser Val Pro 480 Gln Glm Me t Ala Asn 560 -ys WO 97/18822 WO 9718822PCTIUS96/18675 Leu Asn Phe Ile Asn Gin 610 Pro Gly 625 Ser Gly Ala Val Arg Lys Phe Ser 690 Trp Asn 705 Thr Tyr Asp

INFO

Gly Giu 595 Pro His Leu Gly Val1 675 Ile Ser Leu Met 580 Cys Ile Giu Gin Phe 660 Leu Gly Vai Gln 565 Ile Pro Gly Gly Thr 645 Pro Arg Arg Asp Arg 725 Ile Vai Ser Gin 630 Giu Arg Phe Ser His 710 Thr Ala Gin Gin Cys Giy Ile 600 Met Ser Trp 615 Asn Thr Ile Giu His Pro Ile Cys Tyr 665 Leu Lys Ile 680 Val Thr Thr 69S Lys Thr Gin Met Gin Gin 570 Asn Glu Met Val Tyr Gly Ser Ile Ile 620 Gin Ile Val 635 His Pro Gly 650 Leu Pro Asp Aia Phe Asp Gly Arg Giu 700 Phe Asn met 715 Leu Vai His 730 Lys 590 Lys Lys Asp Ala Pro 670 Arg Val Pro Giy 575 Asn Val Asn Ile Phe 655 Leu Leu Val Asp Val 735 Leu Gly Leu Aia 640 Phe Gly Leu Ile Pro 720 Thr RMATION~ FO Tl M^ I SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Gin Pro Cys Pro Met Cys Met Giu Glu Leu Val 1 5 INFORMATION FOR SEQ ID NO:4: Wi SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Giu Asp Cys Thr Ile Cys Met Giu Arg Leu Val 1 5 INFORMATION FOR SEQ ID -76- WO 97/18822 PCT/US96/18675 SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Ser Arg Cys Gin His Leu Met His Leu Gin Cys Leu Asn Gly Met 1 5 10 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Leu Gly Arg Cys Gly His Met Tyr His Leu Leu Cys Leu Val Ala Met 1 5 10 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 36 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ile Ile Ala Gin Gin Asn Glu Met Asn Lys Asn Leu Phe Ile Glu Cys 1 5 10 Pro Val Cys Gly Ile Val Tyr Gly Glu Lys Val Gly Asn Gin Pro Ile 25 Gly Ser Met Ser INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 36 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: -77- WO 97/18822 PCT/US96/186 7 Leu Val Ala Met Tyr Ser Asn Gly Asn Lys Asp Gly Ser Leu Gin Cys 1 5 10 Pro Thr Cys Lys Pro Ser Met Gly Arg Arg Arg Val Arg Ser Arg Leu 25 Gly Arg Trp Ser INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 37 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Val Tyr Gly Glu Lys Val Gly Val Gin Pro Ile Gly Ser Met Ser Trp 1 5 10 Ser Ile Ile Ser Lys Asn Leu Pro Gly His Glu Gly Gin Asn Thr Ile 25 Gin Ile Val Tyr Asp INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 37 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Ile Tyr Gly Glu Lys Thr Gly Thr Gin Pro Pro Gly Lys Met Glu Phe 1 5 10 His Leu Ile Pro His Ser Leu Xaa Phe Gly Pro Asp Thr Gin Thr Xaa 25 Arg Ile Val Tyr Asp INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 2547 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 504..2363 -78- WO 97/18822 WO 9718822PCT/US96/18675 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:il: GCGAGAAGCC CCACTGAAGC

TGGCTGAGAG

CTGGCGGGGA

CTGCCTCACT

GGCATAAGAG

GAAGGGGTCr

TCAGAGAGAA

GAGGAAGTTG

TGGCCCCTGA

CCGCAGGAGc

ACAGGGACCA

CCCTACCTGA

AGACACTTGC

TTGCAGCCTG

CCCAGAGTTA

CCGCCTGCTG

GGCAGTGGCG

CGGGCGCAGG GTCTCGGACG CAGTTGGGAG TGCAAAGGGC AGCAGGCTGT GGCCCAGGCC TCCTGGGTGA CAGGCCCTGT AGAGACAACA CAGAAGAGGC TGGACCTCGA ACAGGGGCGG GCCAGCCGAG GGGGCCAAGG ACTTTAGAGC TGTTTCCTCC TTTCCAGGGC AGCACCCTTT ATCGGAGAAG GCTCTACAGG GATGGCCATC CCACATTCCT TTAACGGAGG TCTCTAGGCC GAAAGGAGGC CAGACGGTCC TTGCTGTCCC CCTGGCGAGA CCAGGCCCAG GAGGAGCTGG GCCTGCA.ATA GTGGGGGACC GCC ATG TCA CGG CCA GGC CAC GGT GGG CTG, Met Ser Arg Pro Gly His Gly Gly Leu 120 180 240 300 360 420 480 530 CCT GTG AAT Pro Val Asn GTG GTG TGG GAG Val Val Trp Giu GOT CTG GGC Gly Leu Gly TGT CTG AAT Cys Leu Asn CAC CAC ATT His His Ile TTC CCA CCG Phe Pro Pro GAG CAC AGC Glu His Ser .35 GAG AAC GTG Giu Asn Val CAG AAC Gin Asn CGC TGG Arg Trp, CTG AAG Leu Lys GTG CCC CGG Val Ala Arg CGG CCC TAC ACG Arg Pro Tyr Thr GAG GAC GCT CC Glu Asp Ala Arg GCC ACC Ala Thr GGT TCC Gly Ser ATC GAC Ile Asp CGC CCC Arg Pro

GTG

Val1

GTG

Val1 CTC CTC GOG CAG GTG GAC GCC CAG CTT GTG-CCC TAC ATO Val Leu Gly Gin Val Asp Ala Gin Leu Val Pro Tyr Ile 65 CTG CAG TCC ATG CAC CAG TTT CGC Leu Gin Ser Met His Gin Phe Arg 80 GTG CGG CCC AAC TTC TAC GAC CCG Vai Arg Arg Asn Phe Tyr Asp Pro CAG CAC Gin Asp TCC TCG Ser Ser 100 GGC GCA Gly Ala ACA CCC ACC ATG Thr Gly Thr Met GCG .CCG GGC Ala Pro Gly 578 626 674 722 770 818 866 914 962 1010 1058 1106 ATC GTG TGG Ile Val Trp.

TGG GAG AAC GAC Trp Giu Asn Asp TCG ACG Trp Thr GCC TAC Ala Tyr 120 GAT ATG GAC Asp Met Asp CCG TOG CTC Pro Trp Leu 140 AAC AGC ATG Asn Ser Met

ATC

Ile 125

GAC

Asp ATC ACC ATC Ile Thr Ile GCC TAC GAG Ala Tyr Giu AAG CAG CAC Lys Gin His 135 ATC TAC TTC Ile Tyr Phe CTC TCA TOG Leu Ser Ser

CTA

Leu 145 Ccc Arg TTC TGC TAC Phe Cys Tyr

CTC

Leu 150 TCG CAG ATG Ser Gin Met CAC ACG CC Gin Thr Arg CCC CCC CGT CTG Arg Arg Arg Leu CGC CTG GAO Arg Leu Asp TAO CCC CTC Tyr Pro Leu GTG GGC TCC ATO Val Gly Ser Ile GGH CAG CCC TGC Gly Gin Pro Cys AAG TCG CAG TCG Lys Ser Gin Ser GTG- GGT GBC AGC Val Gly Xaa Ser .195 -79- WO 97/1 8822 Xaa Gin Gin .PCTIUS96/1 8675 CTG YTG GTC AAC Leu Leu Val Asn AGC ACG CGC CCC GTC TCC AAC GTC Ser Thr Arg Ala Vai Ser Asn Val 210 215 ATC CTG Ile Leu YCG CCG Xaa Pro 235 AGY GTC Ser Val 250 CCC GTC Pro Vai CC CCC Ala Pro GGG CCC Giy Pro GGG GTC Giy Vai 315 CAT CCG His Pro 330 CTG CCC Leu Pro CCC GTG Pro Val TGC CC Cys Arg GAT GTG Asp Val 395 GAC TGC2 Asp Cys 410 GGC GTG Cly Val GGC CC Giy Arg TCC AAT C Ser Asn C 4 ATC TAC C Ile Tyr C

GYC

Xaz 22(

YCC

Xaa

ACC

Thr

GAG

Giu

GGC

Giy

CAG

Gin 300

CCC

Pro

GCC

Aia

GTG

Vai

PAGC

Ser

AAG

Lys 380

GTT

Ia 1

%CC

r'hr

'TT

.eu

[GT

~ys

;GC

;iy k60

;GG

fly

TCG

Ser

MCA

Xaa

TTC

*Phe

ICCC

*Pro

GCA

Gly 285

CC

Arg

GCA

Aia

CTG

Leu

TGC

Cys

AAG

Lys 365

ACC

Thr CGA3 Arg

ATC

Ile Arg I Gly I 445

AAC

AsnI

GAG

Glu

CAC

Gir

CCT

Pro

ACA~

Thr

GMG

Xaa 270

C

Ala

ACC

Thr

CTC

Leu

CA

Alia

CTG

Leu 350 PkGC Ser Lys k.GA k.rg "ys

:AC

{is 130

:AC

is LaG ~ys

LAG

~ys

CGT

Arg

CGA

GlCy

GCC

Gly 255

YCC

Xaa

CC

Arg

ACC

Thr

CCG

Pro

GGC

Cly 335

ACC

Thr

GAC

Asp

AAC

Lys

TAC

Tyr

ATC

Met 415

AAG,

Lys

ATC

Met

CAT

Asp

ACC

Thr

CG]

Arg Gly 240C

CNC

Xaa

TCT

Ser

ACC

Thr

ACH

-Xaa

CTG

Vai 320

ATC

Met

CCC

Arg

GTG

Val 1.AG Lys

ATG

M4et 400

GAG

Giu

GC

Giy

TAG

Tyr 3C 31 y

;GT

3;iy

AAC

Lys 225

ICCT

*Pro

GNG

*Xaa

CCC

Pro

CCC

Pro

GTC

Val 305

AAC

Lys

ACC

Thr

CC

Aia

AAG

Lys

CAC

His 385

GAG

Gin

CGA

Arg

CTC

Val

CAC

His

AC

Ser 465

AC

Thr

GTG

Val C CA Pro

OTO

Leu

CC

Gly

CCC

Gly 290

AGO

Ser

AAC

Asn

CCC

Cly

CCC

Pro

CCC

Pro 370

CTT

Leu

AAG

Lys

CTC

Leu

CCC

Arg

CG

Leu 450

CTG

LeuC

GAG

Gin I

MCC

Xaa

GC

Gly

TCN

Xaa

CYC

Xaa 275

GAG

Gin

CC

Ala

TTG

Leu

ATA

Ile

PACG

Lys 355 G TG

AA

Lys

GTG

lYal 3TC lal ?ro 135

:TG

jeu

:AG

ln

CG

~ro CCC CC *Pro Ala

CCTT

*Ala Leu 245 GAA GTG Ciu Val 260 CCC CCA Pro Pro AAC AAC Asn Asn CCC CC Arg Ala AAT CCT Asn Gly 325 CTG CTC Leu Leu 340 CCC ATC Pro Ile OCT GC Pro Gly AAG ACT Lys Ser AAA AAC Lys Asn 405 ACA GCA Thr Ala GAG CTC Glu Leu TC CTC Cys Leu TGC CCC Gys Pro COT CCC ccC Prc 23(

GC

NI~h Xaa

CGG

Arg

CTC

Leu

TCC

Ser 310

ACT

Thr

TC

Gys

CTG

Leu

GTG

Val1

AC

Lys 390

CCA

Pro rCA Ser

GTC

la 1

;TG

Ia 1

CC

r'hr 170 Prc G TG Val

TTC

Phe

AGO

Ser

AAC

*Asn 295

*ATC

Ile Gly

CC

Ala

CAC

His

CCC

Pro 375

AAT

Asn

CCT

Pro

GCC

Gly

GC

Cly

GCC

Ala 455

TC

Cys ICTC TOG ILeu Ser CCC CCC Arg Pro NAC OCT Xaa Cly 265 CCC GC Pro Cly 280 CCC BCC Arg Xaa CCC CCC Pro Pro CCC CTC Pro Val CCC CCC Ala Cly 345 CCC CCC Pro Pro 360 CCC CTC Cly Val CCC GAG Pro Ciu CAT GAG Asp Glu TAC GAG Tyr Ciu 425 CC COT Arg Leu 440 ATG TAC Met Tyr AAC CC Lys Ala GA,- TTC Giu Phe 1154 1202 1250 1298 1346 1394 1442 1490 1538 1586 1634 1682 1730 1778 1826 1874 1922 1970 Pro Gly Lys Met WO 97/18822 PCT/US96/18675 480 485 CAC CTC ATC CCC CAC TCG CTG CCC GGC TTC CCT GAT ACC CAG ACC ATC 2018 His Leu Ile Pro His Ser Leu Pro Gly Phe Pro Asp Thr Gin Thr Ile 490 495 500 505 CGC ATC GTC TAT GAC ATC CCC ACA GGC ATC CAG GGC CCT GAG CAC CCC 2066 Arg Ile Val Tyr Asp Ile Pro Thr Gly Ile Gin Gly Pro Glu His Pro 510 515 520 AAC CCC GGG AAG AAG TTC ACC GCA AGA GGA TTC CCT CGC CAC TGC TAT 2114 Asn Pro Gly Lys Lys Phe Thr Ala Arg Gly Phe Pro Arg His Cys Tyr 525 530 535 CTA CCC AAC AAC GAG AAA GGC CGG AAG GTG CTG CGG CTG CTC ATC ACG 2162 Leu Pro Asn Asn Glu Lys Gly Arg Lys Val Leu Arg Leu Leu Ile Thr 540 545 550 GCC TGG GAG AGA AGA CTC ATC TTC ACT ATC GGC ACG TCC AAC ACC ACG 2210 Ala Trp Giu Arg Arg Leu Ile Phe Thr Ile Gly Thr Ser Asn Thr Thr 555 560 565 GGC GAG TCG GAC ACC GTG GTG TGG AAC GAG ATC CAC CAC AAG ACC GAG 2258 Gly Glu Ser Asp Thr Val Val Trp Asn Glu Ile His His Lys Thr Glu 570 575 580 585 TTT GGA TCC AAC CTC ACG GGA CAC GGC TAC CCG GAC GCT AGC TAC CTA 2306 Phe Gly Ser Asn Leu Thr Gly His Gly Tyr Pro Asp Ala Ser Tyr Leu 590 595 600 GAC AAC GTG CTG GCT GAG CTC ACA GSC CAG GGC GTA TCC GAG GCT GCA 2354 Asp Asn Val Leu Ala Glu Leu Thr Xaa Gin Gly Val Ser Glu Ala Ala 605 610 615 GGC AAG GCT TGAGGSCCAA GGCTGCCCAC CTTCCCTCCT GSTTTGGCCC 2403 Gly Lys Ala 620 TGGTCCGGCA AATGCCTCCT TCGCCAGGTG TGTCCTGGTA GCCCAGGTTC AGGGCTGGGG 2463 AGGAGCCTGC GGAAGGGGCC GCAGCCATTC AGGGGACTGN CTGGNGGAAG TTGGATGAGG 2523 AGAGNTGGAT TTNAGGTTGG CCCC 2547 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 620 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Ser Arg Pro Gly His Gly Gly Leu Met Pro Val Asn Gly Leu Gly 1 5 10 Phe Pro Pro Gin Asn Val Ala Arg Val Val Val Trp Glu Cys Leu Asn 25 Glu His Ser Arg Trp Arg Pro Tyr Thr Ala Thr Val Cys His His Ile 40 Glu Asn Val Leu Lys Glu Asp Ala Arg Gly Ser Val Val Leu Gly Gin 55 Val Asp Ala Gin Leu Val Pro Tyr Ile Ile Asp Leu Gin Ser Met His 70 75 -81- WO 97/18822 PCT/US96/18675 Gin Phe Tyr Asp Asn Asp Ile Gin 130 Leu Gly 145 Arg Gin Tyr Pro Gly Xaa Asn Ser 210 Lys Val 225 Pro Pro Xaa Leu Pro Gly Pro Gly 290 Val Ser 305 Lys Asn I Thr Gly I Ala Pro I 3 Lys Pro 1 370 His Leu L 385 Gin Lys V Arg Leu V Val Arg P 4 His Leu L Ar Pr

GI'

1i! As Phf Thi Let Ser 195 Thr Xaa Gly Xaa Kaa 275 Gln kla eu le ~ys 155 ral lys 'al 'a1 ro 35 eu g Gin Asp o Ser Ser 100 y Gly Aia 5 I Ala Tyr I Cys Tyr Arg Arg 165 Thr Val 180 Ser Gly *Arg Aia Pro Ala Ala Leu 245 Glu Val 260 Pro Pro Asn Asn Arg Ala Asn Gly 325 Leu Leu 340 Pro Ile I Pro Gly I Lys Ser I Lys Asn I 405 Thr Ala S 420 Glu Leu V Cys Leu V Th Al Tr Gli Lei 151 Ar Glj Glr Val Pro 230 Gly Xaa Arg Leu Ser 310 rhr -ys .eu tal ys 190 'ro er 'al 'al r Giy Thr a Pro Gly p Thr Ala 120 Lys Gin 135 .1 Ile Tyr 0 T Arg Arg r Ser Ile Pro Cys 200 Ser Asn 215 Pro Leu *Val Arg Phe Xaa 4 Ser Pro 280 Asn Arg 295 Ile Pro I Gly Pro A Ala Ala C His Pro I 360 Pro Gly 375 Asn Pro G Pro Asp G Gly Tyr G 4 Glyr Arg L 440 Ala Met T Mel Lyi 10! Ty~ Hif Phe Leu Pro 185 Ser Val Ser Pro ly 265 3ly (aa ?ro lal ;ly 145 ,ro ral hlu hlu lu 25 eu yr t s Arg Pro 90 Gly Ile Arg Asn Glu Trp 110 Asp Met As Pro Trp Asn Ser 155 Arg Arg 170 I Lys Ser Xaa Gin Ile Leu Xaa Pro 235 Ser Val 250 Pro Val Ala Pro Gly Pro Gly Val 315 His Pro 330 Leu Pro Pro Val Cys Arg Asp Val 395 Asp Cys 410 Gly Val I Gly Arg C Ser Asn c -82- Leu 140 Met Arg Gin Gin Xaa 220 Xaa Thr Glu Gly i Gin 300 Pro Ala I Val Ser I 3y Lays 380 lal A rhr I ,eu A :ys G 4 ;iy

A

Ile 125 Asp Ser Leu Ser Cys 205 Ser Xaa Phe Pro Gly 285 krg kla eu .ys .ys 165 'hr Lrg :le irg ly 45 sn Cys Ile Thr Leu Ser Ser Gin Met Xaa 160 Asp Leu Ala 175 Trp Pro Val 190 Leu Leu Val Gin Arg Arg Pro Gly Gly 240 Thr Gly Xaa 255 Xaa Xaa Ser 270 -Ala Arg Thr Thr Thr Xaa Leu Pro Val 320 Ala Gly Met 335 Leu Thr Arg 350 Ser Asp Val Lys Lys Lys Arg Tyr Met 400 Cys Met Glu 415 His Lys Gly 430 His Met Tyr Lys Asp Gly WO 97/18822 WO 97/ 8822PCTIUS96/1 8675 Ser 465 Thr Pro Thr Ala Arg 545 Phe Trp His Thr Leu Gin Cys Pro Cys Lys Ala Ile Gin Gly Gly Arg 530 Lys Thr Asn Gly Xaa 610 Pro Pro 500 Gin Phe Leu Gly Ile 580 Pro Gly Gly 485 Asp Gly Pro Arg Thr 565 His Asp Val Met Gin Giu His 535 Leu Asn Lys Ser Giu Thr His 520 Cys Ile Thr Thr Tyr 600 His 490 Arg Asn Leu Ala Gly 570 Phe Asp Tyr Giy 475 Leu Ile Ile Val Pro Gly Pro Asn 540 Trp Giu 555 Giu Ser Gly Ser Asn'Vai Glu Lys Thr Pro His Ser 495 Tyr Asp Ile 510 Lys Lys Phe 525 Asn Giu Lys Arg Arg Leu Asp Thr Val 575 Asn Leu Thr 590 Leu Aia Giu 605 Gly 480 Leu Pro Thr Gly Ile 560 Val1 Gly Leu Giu Aia Ala Gly 615 Lys Ala 620 INFORMATION FOR SEQ ID NO:i3: SEQUENCE CHARACTERISTICS: LENGTH: 303 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) Met 1 Gly Ala Trp, Ser Leu Thr Ile SEQUENCE DESCRIPTION: SEQ ID NO:13: Ala Ser Ser Ala Gly Ser Ala Ala Ser S Gly Gly Ser Ala Ala Ser Ser Cys Ala Gly Ser Gly Gly Pro Pro Val Asn His Glu Phe Glu Ser Arg Gly Lys Trp Leu 55 Gin His Leu Giu Arg Ala His Ala Lys 70 Ser Asp Ala Asp Pro Ser Leu Giu Gin Met Thr Gin Glu Ser Giu Ala Giu Thr 100 105 Gly Val Arg Arg Met Leu Tyr Ala Pro 115 120 Gly Ser Thr Met Ala His Pro Tyr Lys Leu Tyr Tyr Arg Ser Ser Ser -83- WO 97/18822 PCTIUS96/1 8675 Gly Thr Lys Trp Giu Trp 130 Ser Gly Gly Ser Ala 135 Ser Asn Asn Asp Trp, 145 Ala Pro Gly Val Ser 225 Ser Ser Gin Arg Arg Tyr Pro Lys 210 Val Leu His Gin Pro Gly Thr Met 195 Leu Ser His Gin Gin 275 Tyr Giu Ile Arg Thr Ser Arg Val 260 Gin Met His Vai 150 Thr Leu Asp Phe Cys Asn Ile Arg Arg 200 Gin Gin Ala 215 Tyr Asn Thr 230 Pro Met Thr Gin Gin Gin His His Gin 280 Gin Cys Leu Cys 170 Leu Thr Thr Gin Asn Gin Leu Pro Arg Gin 250 His Gin 265 His Gin Ile Asn His Gin Leu Lys 235 Gin Leu Gin Ile Thr Val Ala Lys 220 Leu His Gin Gin His 300 Giu Asp His Ile Arg Gin 190 Pro Tyr 205 Ser Ash Gly Asp Pro Leu His Gin 270 Gin His 285 Ala Gly 175 Pro Pro Ser Thr Pro 255 Gin Gin Trp, 160 Leu Ser Leu Ala Lys 240 Thr Gin Gin Gin Gin 290 Gin His Gin Met Gin His His Gin Ile 295 His Gin Thr INFORMATION FOR SEQ ID NO:i4: Wi SEQUENCE CHARACTERISTICS: CA) LENGTH: 181 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Arg Lys Pro Pro Lys Lys His Ser Giu Ile 4. 5 10 Gin Ile Leu Asn Asn Leu Asn Ilie Phe Ser 25 Ser Asn Met Ser Thr Ala Ala Ser Ala Ser 40 Ser Leu His His Ala Asn His Leu Ser His 55 Lys Asn Met Leu Thr Ala Ser Met Asn Ser 70 Giu Gly Ser Leu Gin Ser Gin Arg Ser Ser 90 Ser Arg Ser Arg Thr Arg Thr Ser Asp Thr 100 105 Ser His Arg Arg Arg Pro Ser Val Asp Thr 115 120 Ser Thr Thr Ser Ser Thr Ser Ser Ser Ala His Phe His His Ser Arg Met Gly Asp Thr Asn Val Ser Thr 125 Asn Leu is Lys His Ser Ser Ser His Arg Cys Ser His Ser Val 110 Tyr~ Leu -84- WO 97/18822 WO 97/ 8822PCT/US96/1 8675 His Glu Ser Lys Glu Ser Leu Arg Ser Arg Asn Phe Ala Ile Ser Val 130 135 140 Asn Asp Leu Leu Asp Cys Ser Leu Giy Ser Asp Giu Val Phe Val Pro 145 150 155 160 Ser Val Pro Pro Ser Ser Leu Gly Glu Arg Ala Pro Val Pro Pro Pro 165 170 175 Leu Pro Leu His Pro 180 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 224 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Ile Ala Gly Ser Ile Val Gly Val Asp Pro Ala Ser Asp Met Ile Ser Pro Ala Asn Ile Gin Gly Gly Val1 145 Lys Ser Asn Tyr Arg Cys Ile Gly Giu Pro His Leu 130 Gly Val1 Ile Ser Leu 210 Val Met Arg Ala 70 Cys Met Asn Glu Ile 150 Leu Val Lys Met Pro Val Leu Giu Tyr Ile Ile Pro Pro Phe 170 Arg Asn Val Leu Trp His Ser Met His Met Asn 75 Gly Giu Ile Ser Val Tyr Gly Arg 140 Asp Cys 155 Asp Arg Giu Asp Met Phe His Leu 220 Pro Asn Ala Gin Leu Gin Lys Asn Lys Val Lys Asn 110 Asp le 125 Ala Phe Pro Leu Arg Leu Val Val 190 Pro Asp 205 Gly Val Gin Pro Leu Phe Asn Pro Se r Ala Arg 160 Phe Trp Thr Asp INFORMATION FOR SEQ ID NO:16: WO 97/18812 WO 9718822PCT/US96/18675 Wi SEQUENCE CHARACTERISTICS: LENGTH: 204 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Ala Ser Ser 1 Ala Gly 5 Ser Ala Ala Ser Gly Ser Val Val Pro Gly 10 Gly Ala Trp Ser Leu Thr Ile Gly Trp 145 Ala Pro Gly Gly Glu Gin Ser Met Gly Thr 130 Arg Arg Tyr Gly Ser Phe His Asp Thr Val Lys Pro Gly Thr Ser Ala Gly Gly Glu Ser Leu Glu Ala Asp Gin Glu 100 Arg Arg Trp Giu Tyr Asn Glu Gin 165 Ile Asn 180 Ala Pro Axg Arg 70 Pro Ser Met Trp Met 150 Thr Phe Ser Ser Cys 25 Pro Val Asn 40 Gly Lys Trp, 55 Ala His Ala Ser Leu Glu Glu Ala Giu 105 Leu Tyr Ala 120 Ser Gly Gly 135 His Val Gin Leu Asp Leu Cys Asn Leu 185 Ala His Leu Lys Gin 90 Thr Pro Ser Cys Cys 170 Thr Thr Ala Pro Lys 75 Tyr Arg Ser Ala Ile 155 Asn His Ala Ala Ser Thr Val1 Gly Pro 125 Ser Glu His Arg Ser Thr Cys Val Ala Val Val Met Val Arg Leu Thr Gly Lys Asn Asp Ala Trp 160 Gly Leu 175 Pro Ser Gly Pro Met 195 Arg Ser Ile Arg Thr Gin Gin Ala INFORMATION FOR SEQ ID NO:17: Wi SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Val Xaa Pro Ala Pro Pro Leu Ser Xaa Pro Xaa Xaa Pro Gly Gly Pro 1 5 10 Pro Gly Ala -86- WO 97/18822 PCT/US96/18675 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Xaa Ser Pro Gly Xaa Pro Pro Arg Ser Pro Gly Ala Pro Gly Gly 1 5 10 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Ser Ile Pro Pro Gly Val Pro Ala Leu Pro Val Lys 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 13 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Arg Ala Pro Lys Pro Ile Leu His Pro Pro Pro Val Ser 1 5 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Val Lys Pro Val Pro Gly Val Pro Gly Val 1 5 INFORMATION FOR SEQ ID NO:22: -87- WO 97/18822 PCT/US96/18675 SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Arg Ala Pro Val Pro Pro Pro Leu Pro Leu His Pro Arg Gin Gin 1 5 10 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Arg Ala Pro Thr Met Pro Pro Pro Leu Pro Pro Val Pro Pro Gin Pro 1 5 10 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Arg Ala Val Pro Pro Pro Leu Pro Pro Arg Arg Lys Glu Arg INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 61 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Thr Ile Cys Met Glu Arg Leu Val Thr Ala Ser Gly Tyr Glu Gly 1 5 10 Val Leu Arg His Lys Gly Val Arg Pro Glu Leu Val Gly Arg Leu Gly 25 -88- WO 97/18822 .PCT/US96/18675 Arg Cys Gly His Met Tyr His Leu Leu Cys Leu Val Ala Met Tyr Ser 40 Asn Gly Asn Lys Asp Gly Ser Leu Gin Cys Pro Thr Cys 55 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO26: CACCATCTGC ATGGAGCGAC TGGT 24 INFORMATION FOR SEQ ID NO:27:.

SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: TGTGGCCACA TGTACCACCT GCTG 24 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: TACGGGGAGA AGACGGGTAC GCAG 24 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA -89- WO 97/18822 PCT/US96/18675 Xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: AAGATGGAGT TCCACCTCAT

CCC

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Met Glu Arg Leu Val 1 INFORMATION FOR SEQ ID NO:31: SEQUENCE

CHARACTERISTICS:

LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY:.unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Tyr Gly Glu Lys Thr Gly 1 INFORMATION FOR SEQ ID NO:32: SEQUENCE

CHARACTERISTICS:

LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Thr Ile Arg Ile Val Tyr Asp Ile 1 INFORMATION FOR SEQ ID NO:33: SEQUENCE

CHARACTERISTICS:

LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Gly Phe Pro Arg His Cys Tyr Leu Pro WO 97/18822 PCT/US96/18675 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) Arg 1 SEQUENCE DESCRIPTION: SEQ ID NO:34: Arg Leu Ile Phe Thr Ile Gly INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Met Cys Met Glu Glu Leu Val 1 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) Tyr SEQUENCE DESCRIPTION: SEQ ID NO:36: Gly Glu Lys Val Gly INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 8amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) Thr 1 SEQUENCE DESCRIPTION: SEQ ID NO:37: Ile Gin Ile Val Tyr Asp Ile -91- WO 97/18822 PCT/US96/18675 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Gly Phe Pro Arg Ile Cys Tyr Leu Pro 1 INFORMATION FOR SEQ ID NO:39: SEQUENCE

CHARACTERISTICS:

LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Arg Arg Leu Leu Phe Ser Ile Gly 1 -92- WO 97/18822 PCT/US96/1 8675 International Application No: PCT/ Addrtessa depositare institidon aincludiinptal ode adcuty 12301 Parkiawn Drive Rockville, MD 20852 us Date of deposit INovember 17. 1995 Accession Number'* 97341 B. ADDITIONAL INDICATIONS leave blank if rot applicable). This iaformation is ommnuedi on' a separate attached sheet C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE'dtb 21 D. SEPARATE FURNISHING OF INDICATIONS (leave blank if we pplicatle The inldications listed below will be submittedl to the International Bureau later Specify the general nature of the indications e.g..

'Accession Number of Deposit") E.-enThs sheet was received with the International application when filed (to be checked by the receiving Office) (Authonze~d Officer) 0 Ile date of receipt (from the applicant) by the Interaional Burneau was (Authorized Officer) Form PCT/RO/1 34 (January 198 1) -92.1-

Claims (89)

1. A purified vertebrate Deltex protein, which protein is encoded by a first nucleic acid which hybridizes under low stringency conditions to a second nucleic acid consisting of the double stranded vertebrate Deltex nucleic acid sequence contained in plasmid pBS hdx as deposited with the ATCC and assigned Accession No. 97341 or (b) hybridizes under low stringency conditions to a second nucleic acid consisting of the nucleotide sequence depicted in Figure 2A-C (SEQ ID NO: 11) or a sequence complementary thereto, and which protein is able to be bound by an antibody to a Deltex protein consisting of the amino acid sequence depicted in Figure 2A-C (SEQ ID NO: 12), said low stringency conditions comprising hybridization in a buffer consisting of 35% formamide, 5X SSC, mM Tris-HCl (pH 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 tg/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate, for 18-20 hours at 40 0 C, washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 55 0 C, and washing in a buffer consisting of 2X SSC, 25 mM Tris- HCI (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 60 0 C.

2. The protein of claim 1 which is a mammalian protein.

3. The protein according to any one of claims 1-2 which is a human protein.

4. The protein according to any one of claims 1-3 consisting of the amino acid sequence depicted in Figure 2A-C (SEQ ID NO: 12). A purified protein comprising a fragment of a vertebrate Deltex protein, said fragment consisting of at least 10 continuous amino acids of the vertebrate Deltex protein of claim 1 which fragment displays one or more functional activities associated with a full- length vertebrate Deltex protein. NY2 1142893.1 94

6. A purified protein comprising a fragment of a vertebrate Deltex protein, said fragment consisting of at least 20 continuous amino acids of the vertebrate Deltex protein of claim 1 which fragment displays one or more functional activities associated with a full- length vertebrate Deltex protein.

7. A purified fragment of a vertebrate Deltex protein consisting of at least continuous amino acids of the vertebrate Deltex protein of claim 1, which fragment displays one or more functional activities associated with a full-length vertebrate Deltex protein.

8. The fragment of claim 7 which consists of at least 20 continuous amino acids of the vertebrate Deltex protein.

9. The protein of claim 5 in which the protein is able to be bound by an antibody to a Deltex protein. A purified protein comprising a fragment consisting of at least continuous amino acids of the vertebrate Deltex protein of claim 1, which fragment binds to a Notch protein or to a molecule comprising the cdc10/SW16/ankyrin repeats of a Notch protein.

11. A purified protein comprising a fragment of a first vertebrate Deltex protein, which fragment consists of at least 10 continuous amino acids and binds to a second Deltex protein or to a molecule comprising a fragment of a second Deltex protein, which fragment of the first vertebrate Deltex protein is encoded by a first nucleic acid which (a) hybridizes under low stringency conditions to a second nucleic acid consisting of the double stranded vertebrate Deltex nucleic acid sequence contained in plasmid pBS hdx as deposited with the ATCC and assigned Accession No. 97341 or hybridizes under low stringency conditions to a second nucleic acid consisting of the nucleotide sequence depicted in Figure 2A-C (SEQ ID NO:11) or a sequence complementary thereto, said low stringency conditions comprising hybridization in a buffer consisting of 35% formamide, 5X SSC, 50 mM Tris- HCI (pH 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg/ml denatured NY2- 1142893.1 L r salmon sperm DNA, and 10% (wt/vol) dextran sulfate, for 18-20 hours at 40 0 C, washing in a buffer consisting of2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for hours at 55C, and washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH mM EDTA, and 0.1% SDS, for 1.5 hours at 60 0 C.

12. A purified protein comprising a fragment of at least 10 continuous amino acids of the vertebrate Deltex protein of claim 1, which fragment comprises a SH3- binding domain of the vertebrate Deltex protein.

13. A purified chimeric protein comprising a functionally active fragment of at least 10 continuous amino acids of the vertebrate Deltex protein of claim 1 joined via a peptide bond to an amino acid sequence of a protein other than the vertebrate Deltex protein.

14. The protein of claim 13 in which the fragment binds to a Notch protein or to a molecule including the cdc 10/SW 16/ankyrin repeats of a Notch protein. The protein of claim 13 in which the fragment includes an SH3-binding domain.

16. The protein of claim 13 in which the fragment includes a zinc finger domain.

17. A purified derivative of the protein of claim 4, which derivative is able to display one or more functional activities of the protein of claim 4, and which derivative has one or more insertions, deletions, or substitutions relative to the protein.

18. A purified peptide consisting of an amino acid sequence in the range of 10-35 amino acids, said sequence being a portion of the vertebrate Deltex protein sequence of claim 1. SNY2- 1142893.1 96

19. The derivative of claim 17 which is able to be bound by an antibody to the protein of claim 4, which antibody is unable to bind to Drosophila Deltex protein. A purified molecule including the amino acid sequence of the human Deltex protein of claim 3.

21. A purified antibody which binds to the vertebrate Deltex protein of claim 1, and which does not bind to a Drosophila Deltex protein.

22. The antibody of claim 21 which binds to a human Deltex protein.

23. The antibody according to any one of claims 21 or 22 which is monoclonal.

24. A fragment or derivative of the antibody according to any one of claims 21-23 containing the binding domain of the antibody. A purified nucleic acid encoding a vertebrate Deltex protein, which nucleic acid hybridizes under low stringency conditions to a second nucleic acid consisting of the double stranded vertebrate Deltex nucleic acid sequence contained in plasmid pBS hdx as deposited with the ATCC and assigned Accession No. 97341 or (b) hybridizes under low stringency conditions to a second nucleic acid consisting of the nucleotide sequence depicted in Figure 2A-C (SEQ ID NO:11) or a sequence complementary thereto, and which encoded vertebrate Deltex protein is able to be bound by an antibody to a Deltex protein consisting of the amino acid sequence depicted in Figure 2A-C (SEQ ID NO:12), said low stringency conditions comprising hybridization in a buffer consisting of formamide, 5X SSC, 50 mM Tris-HCl (pH 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 jpg/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate, for 18-20 hours at 40 0 C, washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 55 0 C, and washing in a buffer NY2- 1142893.1 'I. r- i 97 consisting of 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 60 0 C.

26. The nucleic acid of claim 25 which lacks introns.

27. The nucleic acid according to any one of claims 25 or 26 which encodes a protein consisting of the amino acid sequence depicted in Figure 2A-C (SEQ ID NO: 12).

28. The nucleic acid of claim 25 which comprises the coding region of the nucleotide sequence depicted in Figure 2A-C (part of SEQ ID NO: 11).

29. A purified nucleic acid complementary to the nucleic acid according to any one of claims 25-28. The nucleic acid of claim 25 which encodes a human Deltex protein.

31. A purified first nucleic acid which hybridizes to a second nucleic acid under conditions of low stringency, said second nucleic acid comprising the nucleotide sequence depicted in Figure 2A-C (SEQ ID NO: 11) or a sequence complementary thereto, said first nucleic acid comprising at least 110 continuous nucleotides of SEQ ID NO:11, said conditions of low stringency comprising hybridization in a buffer consisting of formamide, 5X SSC, 50 mM Tris-HCl (pH 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 jpg/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate, for 18-20 hours at 40 0 C, washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH mM EDTA, and 0.1% SDS, for 1.5 hours at 55 0 C, and washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 60 0 C.

32. A purified first nucleic acid which hybridizes to a second nucleic acid under conditions of high stringency, said second nucleic acid comprising the nucleotide sequence depicted in Figure 2A-C (SEQ ID NO: 11) or a sequence complementary thereto, said first nucleic acid comprising at least 110 continuous nucleotides of SEQ ID NO:11, said NY2 1142893.1 98 conditions of high stringency comprising hybridization in a buffer consisting of 6X SSC, mM Tris-HCl (pH 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 100 jg/ml denatured salmon sperm DNA, for 48 hours at 65 0 C, and wash in a buffer consisting of 2X SSC, 0.01% PVP, 0.01% Ficoll and 0.01% BSA for 1 hour at 37 0 C.

33. A purified first nucleic acid which hybridizes to a second nucleic acid, or a sequence complementary thereto, under conditions of low stringency, said second nucleic acid encoding a protein comprising the amino acid sequence depicted in Figure 2A-C (SEQ ID NO: 12), said first nucleic acid encoding a protein comprising the first 50 amino acids of SEQ ID NO: 12, said conditions of low stringency comprising hybridization in a buffer consisting of 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate, for 18-20 hours at 40 0 C, washing in a buffer consisting of 2X SSC, 25 mM Tris-HC1 (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 55 0 C, and washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for hours at 60 0 C.

34. A purified first nucleic acid which hybridizes to a second nucleic acid, or a sequence complementary thereto, under conditions of high stringency, said second nucleic acid encoding a protein comprising the amino acid sequence depicted in Figure 2A-C (SEQ ID NO: 12), said first nucleic acid encoding a protein comprising the first 50 amino acids of SEQ ID NO: 12, said conditions of high stringency comprising hybridization in a buffer consisting of 6X SSC, 50 mM Tris-HCl (pH 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 100 jg/ml denatured salmon sperm DNA, for 48 hours at 65 0 C, and wash in a buffer consisting of 2X SSC, 0.01% PVP, 0.01% Ficoll and 0.01% BSA for 1 hour at 37 0 C. A purified first nucleic acid which hybridizes to a second nucleic acid, or a sequence complementary thereto, under conditions of high stringency, said second nucleic acid comprising nucleotide numbers 500-1044, 1045-1821, or 1822-2380 of SEQ ID NO: 11, said conditions of high stringency comprising hybridization in a buffer consisting of 6X SSC, 50 mM Tris-HCl (pH 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and NY2 1142893.1 99 100 gg/ml denatured salmon sperm DNA, for 48 hours at 65 0 C, and wash in a buffer consisting of 2X SSC, 0.01% PVP, 0.01% Ficoll and 0.01% BSA for 1 hour at 37 0 C.

36. A purified first nucleic acid which hybridizes to a second nucleic acid, or a sequence complementary thereto, under conditions of low stringency, said second nucleic acid comprising nucleic acids 500-1044, 1045-1821, or 1822-2370 of SEQ ID NO: 11, said conditions of low stringency comprising hybridization in a buffer consisting of formamide, 5X SSC, 50 mM Tris-HCl (pH 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate, for 18-20 hours at 40 0 C, washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH mM EDTA, and 0.1% SDS, for 1.5 hours at 55°C, and washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 60 0 C.

37. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 25 amino acids of SEQ ID NO: 12, or a sequence complementary thereto, which protein displays one or more functional activities associated with the vertebrate Deltex protein of claim 1.

38. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 50 amino acids of SEQ ID NO: 12, or a sequence complementary thereto, which protein displays one or more functional activities associated with the vertebrate Deltex protein of claim 1.

39. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 100 amino acids of SEQ ID NO: 12, or a sequence complementary thereto, which protein displays one or more functional activities associated with the vertebrate Deltex protein of claim 1. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 150 amino acids of SEQ ID NO:12, or a sequence complementary NY2 1142893.1 100 thereto, which protein displays one or more functional activities associated with the vertebrate Deltex protein of claim 1.

41. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 230 amino acids of SEQ ID NO: 12, or a sequence complementary thereto, which protein displays one or more functional activities associated with the vertebrate Deltex protein of claim 1.

42. A purified nucleic acid comprising 110 continuous nucleotides of SEQ ID NO: 11, or a sequence complementary thereto, which nucleic acid encodes an amino acid sequence that displays one or more functional activities associated with the vertebrate Deltex protein of claim 1.

43. A purified nucleic acid encoding the protein of claim

44. A purified nucleic acid encoding the protein of claim A purified nucleic acid encoding the protein of claim 11.

46. A purified nucleic acid encoding the protein of claim 12.

47. A purified nucleic acid encoding the chimeric protein of claim 13.

48. The nucleic acid of claim 25 in which the nucleic acid sequence consists of the human Deltex coding sequence contained in plasmid pBS hdx as deposited with the ATCC and assigned accession number 97341.

49. A nucleic acid vector comprising the nucleic acid of claim A nucleic acid vector comprising the nucleic acid of claim 26. NY2 1142893.1 \i 101

51. A recombinant cell transformed with the nucleic acid vector of claim 49.

52. A recombinant cell transformed with the nucleic acid vector of claim

53. A method for producing a vertebrate Deltex protein comprising growing the recombinant cell of claim 51, such that the encoded vertebrate Deltex protein is expressed by the cell; and recovering the expressed vertebrate Deltex protein.

54. A method for producing a protein comprising growing a cell containing a recombinant nucleic acid comprising the nucleic acid of claim 33, such that the protein is expressed by the cell; and recovering the expressed protein. A pharmaceutical composition comprising a therapeutically effective amount of the vertebrate Deltex protein of claim 1; and a pharmaceutically acceptable carrier.

56. The composition of claim 55 in which the vertebrate Deltex protein is a human Deltex protein.

57. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 7; and a pharmaceutically acceptable carrier.

58. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 10; and a pharmaceutically acceptable carrier.

59. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 11; and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 12; and a pharmaceutically acceptable carrier. NY2 1142893.1 102

61. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 13; and a pharmaceutically acceptable carrier.

62. A pharmaceutical composition comprising a therapeutically effective amount of a purified derivative of the vertebrate Deltex protein of claim 1, which derivative is characterized by the ability to bind to a Notch protein or to a molecule comprising the cdc 10/SW 16/ankyrin repeats of a Notch protein.

63. A pharmaceutical composition comprising a therapeutically effective amount of the nucleic acid of claim 25; and a pharmaceutically acceptable carrier.

64. The pharmaceutical composition of claim 63 in which the vertebrate Deltex protein is a human Deltex protein. A pharmaceutical composition comprising a therapeutically effective amount of the antibody of claim 21, or a purified fragment .or derivative of the antibody containing the binding domain thereof; and a pharmaceutically acceptable carrier.

66. A method of treating or preventing a disease or disorder in a subject comprising administering to a subject in need of such treatment or prevention a therapeutically effective amount of a purified molecule which antagonizes the function of the vertebrate Deltex protein of claim 1, in which the disease or disorder is a disease or disorder of cell fate or differentiation.

67. The method according to claim 66 in which the disease or disorder is amalignancy characterized by increased Notch activity or increased expression of a Notch protein or of a Notch derivative capable of being bound by an anti-Notch antibody, relative to said Notch activity or expression in an analogous non-malignant sample.

68. The method according to claim 66 in which the disease or disorder is cervical cancer. NY2 1142893.1

69. The method according to claim 66 in which the disease or disorder is breast cancer. The method according to claim 66 in which the disease or disorder is colon cancer.

71. The method according to claim 66 in which the malignancy is selected from the group consisting of melanoma, seminoma, and lung cancer.

72. The method according to claim 67 in which the subject is a human.

73. The method according to claim 66 in which the molecule is an antibody to vertebrate Deltex or a derivative of said antibody containing the binding domain thereof, which antibody does not bind to Drosophila Deltex.

74. The method according to claim 66 in which the molecule is a protein comprising a portion of a vertebrate Deltex protein capable of binding to a Notch protein or to a second molecule comprising the cdc10/SW16/ankyrin repeats of a Notch protein. The method according to claim 66 in which the molecule is a protein comprising the SH3 binding domain of a vertebrate Deltex protein.

76. The method according to claim 66 in which the molecule is a protein comprising the zinc finger domain of a vertebrate Deltex protein.

77. The method according to claim 66 in which the molecule is an oligonucleotide which consists of at least six nucleotides; comprises a sequence complementary to at least a portion of an RNA transcript of a vertebrate deltex gene; and (c) hybridizes to the RNA transcript under conditions of low stringency, said conditions comprising hybridization in a buffer consisting of 35% formamide, 5X SSC, 50 mM Tris- HC1 (pH 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 gg/ml denatured NY2 1142893.1 104 salmon sperm DNA, and 10% (wt/vol) dextran sulfate, for 18-20 hours at 40 0 C, washing in a buffer consisting of 2X SSC, 25 mM Tris-HC1 (pH 5 mM EDTA, and 0.1% SDS, for hours at 55C, and washing in a buffer consisting of 2X SSC, 25 mM Tris-HC1 (pH mM EDTA, and 0.1% SDS, for 1.5 hours at 60 0 C.

78. A method of treating or preventing a disease or disorder in a subject in need of such treatment or prevention comprising administering to the subject a therapeutically effective amount of a purified molecule which promotes the function of the vertebrate Deltex protein of claim 1, in which the disease or disorder is a disease or disorder of cell fate or differentiation.

79. A method of treating or preventing a malignancy in a subject comprising administering to a subject in need of such treatment or prevention an effective amount of the vertebrate Deltex protein of claim 1. The method according to claim 79 in which the Deltex protein is a human Deltex protein.

81. A method of treating or preventing a malignancy in a subject comprising administering to a subject in need of such treatment or prevention an effective amount of the nucleic acid of claim

82. A method of treating or preventing a malignancy in a subject comprising administering to a subject in need of such treatment or prevention an effective amount of the antibody of claim 21.

83. A method for treating a patient with a tumor, of a tumor type characterized by expression of a Notch or vertebrate deltex gene, comprising administering to the patient an effective amount of an isolated oligonucleotide, which oligonucleotide (a) consists of at least six nucleotides; comprises a sequence complementary to at least a portion of an RNA transcript of the vertebrate deltex gene; and hybridizes to the RNA NY2 1142893.1 105 transcript under conditions of low stringency, said conditions comprising hybridization in a buffer consisting of 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ug/ml denatured salmon sperm DNA, and (wt/vol) dextran sulfate, for 18-20 hours at 40 0 C, washing in a buffer consisting of 2X SSC, mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 55 0 C, and washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 60 0 C.

84. An isolated oligonucleotide consisting of at least six nucleotides, and comprising a sequence complementary to at least a portion of an RNA transcript of a vertebrate deltex gene, which oligonucleotide hybridizes to the RNA transcript under conditions of low stringency, said conditions comprising hybridization in a buffer consisting of 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 -tg/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate, for 18-20 hours at 40 0 C, washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 55 0 C, and washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 60 0 C. A pharmaceutical composition comprising the oligonucleotide of claim 84; and a pharmaceutically acceptable carrier.

86. A method of inhibiting the expression of a nucleic acid sequence encoding a vertebrate Deltex protein in a cell comprising providing the cell with an effective amount of the oligonucleotide of claim 84..

87. A method of diagnosing a disease or disorder characterized by an aberrant level of Notch-vertebrate Deltex protein binding activity in a patient, comprising measuring the ability of a Notch protein in a sample derived from the patient to bind to the vertebrate Deltex protein of claim 1, in which an increase or decrease in the ability of the Notch protein NY2- 1142893.1 106 to bind to the vertebrate Deltex protein, relative to the ability found in an analogous sample from a normal individual, indicates the presence of the disease or disorder in the patient.

88. A method of identifying a molecule that inhibits or reduces the binding of a vertebrate Deltex protein to a Notch protein, comprising: contacting a Notch protein or fragment thereof that mediates binding to a Deltex protein, and (ii) the vertebrate Deltex protein of claim 1 or fragment thereof that mediates binding to a Notch protein, such that binding between the Notch protein or fragment and the Deltex protein or fragment can occur, in the presence of one or more molecules which are desired to be tested for the ability to inhibit or reduce binding between the Notch protein or fragment and the Deltex protein or fragment; and identifying the one or more molecules that inhibit or reduce the binding of the Deltex protein or fragment to the Notch protein or fragment.

89. The method of claim 88 in which the Deltex protein is a human Deltex protein. A method of inactivating Notch function in a cell comprising introducing into the cell a molecule, said molecule comprising a Deltex protein of claim 1 or portion thereof that mediates binding to a Notch protein; and a protease or proteolytically active portion thereof.

91. A method for the expansion of a precursor cell comprising contacting the cell with an amount of a vertebrate Deltex protein of claim 1 or functionally active portion thereof effective to inhibit differentiation of the cell, and exposing the cell to cell growth conditions such that the cell proliferates.

92. The protein of claim 1, which protein has the amino acid sequence depicted in Figure 2A-C (SEQ ID NO:12); or is able to be bound by an antibody to a NY2 1142893.1 107 human Deltex protein, and is encoded by a first nucleic acid that hybridizes under low stringency conditions to a second nucleic acid consisting of the double stranded human Deltex nucleotide sequence contained in plasmid pBS hdx as deposited with the ATCC and assigned accession number 97341, said low stringency conditions comprising hybridization in a buffer consisting of 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 [g/ml denatured salmon sperm DNA, and (wt/vol) dextran sulfate, for 18-20 hours at 40 0 C, washing in a buffer consisting of 2X SSC, mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 55 0 C, and washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 60 0 C.

93. A purified fragment of the protein of claim 3, said fragment consisting of at least 10, at least 20, or at least 50 continuous amino acids of the human Deltex sequence depicted in Figure 2A-C (SEQ ID NO:12), and able to be bound by an antibody to a human Deltex protein consisting of said human Deltex sequence.

94. A purified fragment of the protein of claim 3, in which said fragment (a) consists of at least 10, at least 20, or at least 50 continuous amino acids of the human Deltex sequence depicted in Figure 2A-C (SEQ ID NO: 12), binds to a Notch protein, a molecule comprising the cdc10/SW16/ankyrin repeats of a Notch protein, a second Deltex protein, or to a molecule comprising a fragment of a second Deltex protein, and is able to be bound by an antibody to said human Deltex protein. A purified fragment of the protein of claim 3, said protein consisting of the amino acid sequence depicted in Figure 2A-C (SEQ ID NO: 12), which fragment (a) comprises an SH3-binding domain or the ring-H2-zinc finger domain of said protein, is able to be bound by an antibody to said human Deltex protein and consists of at least continuous amino acids of the human Deltex sequence depicted in Figure 2A-C (SEQ ID NO:12). NY2 1142893.1 108

96. A chimeric protein comprising a functionally active fragment of the protein of claim 3, in which said fragment consists of at least 10, at least 20, or at least continuous amino acids of the human Deltex sequence depicted in Figure 2A-C (SEQ ID NO: 12), and is joined via a peptide bond to an amino acid sequence of a protein other than said human Deltex protein.

97. A purified fragment of the protein of claim 3, said protein consisting of the amino acid sequence depicted in Figure 2A-C (SEQ ID NO: 12), in which the fragment (a) lacks an SH3 binding domain of the protein, the ring-H-2-zinc finger of the protein, the cdc1O/SW16/ankyrin repeat binding domain of the protein, or the Deltex binding domain of the protein, is able to bind to an antibody to said human Deltex protein, and consists of at least 10 continuous amino acids of the human Deltex sequence depicted in Figure 2A-C (SEQ ID NO:12).

98. A molecule comprising a fragment of the protein of claim 3, said fragment consisting of at least 10, at least 20, or at least 50 continuous amino acids of the human Deltex sequence depicted in Figure 2A-C (SEQ ID NO: 12); or is able to be bound by an antibody to a human Deltex protein, and is encoded by a first nucleic acid that hybridizes under low stringency conditions to a second nucleic acid consisting of the double stranded human Deltex nucleotide sequence contained in plasmid pBS hdx as deposited with the ATCC and assigned accession number 97341, said low stringency conditions comprising hybridization in a buffer consisting of 35% formamide, 5X SSC, 50 mM Tris-HCl (pH mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate, for 18-20 hours at 40 0 C, washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 55 0 C, and washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA, and 0.1% SDS, for 1.5 hours at 60 0 C.

99. The protein or molecule of claim 1, 11, 92 or 98 which nucleic acid hybridizes to plasmid pBS hdx under highly stringent conditions, said highly stringent conditions comprising hybridization in a buffer consisting of 6X SSC, 50 mM Tris-HCl (pH NY2 1142893.1 109 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 100 g/ml denatured salmon sperm DNA, for 48 hours at 65 0 C, and wash in a buffer consisting of 2X SSC, 0.01% PVP, 0.01% Ficoll and 0.01% BSA for 1 hour at 37 0 C.

100. The protein, fragment, derivative, or molecule of any one of claims 9, 92-95 or 97-100 in which the antibody does not bind to Drosophila Deltex protein.

101. A pharmaceutical composition comprising a therapeutically effective amount of the protein, fragment, or molecule of any one of claims 92-101; and a pharmaceutically acceptable carrier.

102. A use of a composition according to any one of claims 55-65, 85 or 102 in the preparation of a medicament for treating or preventing a disease or disorder of cell fate or differentiation in a subject in need of such treatment or prevention.

103. A use according to claim 103 wherein said disease or disorder is cervical cancer, colon cancer, melanoma, seminoma or lung cancer.

104. A purified vertebrate Deltex protein substantially as hereinbefore described with reference to any one of the examples.

105. A purified nucleic acid encoding a vertebrate Deltex protein substantially as hereinbefore described with reference to the examples regarding cloning of the Deltex gene.

106. A purified molecule comprising the vertebrate Deltex protein of claim 1 or 2.

107. A purified nucleic acid comprising the human Deltex nucleic acid contained in plasmid pBS hdx as deposited with the ATCC and assigned Accession No.

97341. NY2 1142893.1 110 DATED 16 November, 2000 PHILLIPS ORMONDE FITZPATRICK Attorneys for YALE UNIVERSITY NY2 1142893.1