CA2238404A1 - Vertebrate deltex proteins, nucleic acids, and antibodies, and related methods and compositions - Google Patents

Abstract

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, e.g., by recombinant methods is provided. In a specific embodiment, the invention relates to human deltex nucleic acids and proteins. The present invention also relates to therapeutic and diagnostic methods and compositions based on vertebrate Deltex proteins, nucleic acids, and antibodies. 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.

Description

W O 97/18822 PCTrUS96/18675 ~E~TEBR~TE DELTEX PROTEINS,rRUCL~IC ACrDS, A~D
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 TE~E INVENTION
In Drosophila melanogaster, the so called "Notch group" of genes has been implicated in events crucial for the correct develol~",el~l 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 ~ccurn~ t~ dgenetic and molecular studies suggest that these genes encode elements of a cellcf~mm~lni~tion m~rh~ni.cm which inrl~ 5 cell surface, cytoplasmic, and nuclear components.
2 0 Very little is known about the m~c.h~ni.cmc underlying cell fate choices in higher organisms such as vertebrates; a knowledge of such mpçh~ni~m~ 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 delt~x. since these genes appear ~o play crucial roles in the de~ermination of 2 5 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 30 and Simpson, 1991, Trends Genet. 7:403-408; and in Artavanis-Tsakonas et al., 1991, Ann.
~ev. Cell Biol. 7:427-452). This pleiotropy of Notch function is revealed by mutations affecting all stages of development and a variety of tissues (e.g., Welshons, 1965. Science 150:1122-1229; Welshons, 1971, Genetics 68:259-268; Shellenbarger and Mohler. 1978, Dev Biol. 62:432-446). A dramatic illustration of Notch function is seen in the de~elopment 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 I

CA 02238404 1998-0~-22 O97/18822 PCT~US96/18675 termed a 'neurogenic' phenotvpe (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 c~"",~",lirA~os 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; ~artley 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 (H), the Enhancer f (split), and Suppressorof Hairless (S~(H)) encode nuclear proteins (Smoller et al., 1990, Genes ~:)ev. 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, ~. Biol Chem. 266:23334-23340; Furukawa èt 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 delte~ 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 int~ t 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 W O97/18822 PCT~US96/18675 of the Drosophik.~ nervous system, cells that normally would become epidermis inslead adopt a neural fate in the absence of Notch function. However, a salient ~eanlre of Notch activity is its pleiotropy. Notch is required for the proper ~ecirlc~Lion of many other cell types, inrl~ in~ those of the compound eye (Cagan and Ready, 1989, Genes Dev. 3:1099-1112), ovary (Ruohola et al., 1991, Cell 66:433~49; Xu et al., l992, Development 115:913-922), and mesoderm (Corbin et al., 1991, Cell 67:311-323). Sirnilarly, 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 10 249:1438-1441; Wei~ a~ et al., 1991, Development 113:199-205; Weh.~ l 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 (~Pn~oti~s 2:119-127).
Notch homologs have been isolated from a variety of vertebrate species and have been shown to be lc~llldlk~bly similar to their Drosophila counL~ a-L in terms of structure, expression pattern and ligand binding pro~)c,Lies (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: 199-20~). Two hùman Notch 20 homologs have been isolated (PCT Publication No. WO 92/19737 dated November 12, 19923, termed hN and TAN-l. A human Notch (TAN-l) malfunction has been associated with a lymphatic cancer (Ellisen et al., 1991, Cell 66:649-661).
Notch encodes a large, structurally-complex tr~n~m~mhrane protein.
25 consis~ent with an involvement in cell-cell co~ tion (Wharton et al., 1985, Cell 43:567-581; Kidd et al., 1986, Mol. Cell. Biol. 6:3094-3108). Notch has an ex~racellular domain containing 36 tandem EG~-like repeats and 3 Notch/linl2 repeats. The intracellular domain bears several cornmon structural motifs tnr~ in~ 6 cdclO/SWl6/ankyrin repeats ("ANK" repeats) Lux et al., 1990, Nature 344:36-42; Breeden and Nasmyth, 1987, Nature 30 329:651-654; Michaely and Bennett, 1992, Trends Cell Biol. 2:127-129; Blank et al., 1992, Trends Biochem. Sci. 17:13~-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 lc:,llalk;~ble degree to which these motifs have been conserved in

3~ homologs isolated from mice (Weil~l~a~,Ler et al., 1991, Development 113:199-205;
Weinmaster et al., 1992, Development 116:931-941; Kopan and Weintraub, 1993, J. Cell CA 02238404 1998-0~-22 Biol. 121:631-641), rats (Kopan and Weintraub. 1993, J. Cell Biol. 121:631-641; Franco del Amo et al., 1993, Genomics 15:259-26~, humans (Ellisen e~ al., 1991, Cell 66:649-661; Stifani et al., 1992, Nature t~en~ti~s 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., l990, Science 249:1438-1441) implies that they will have a common biot~hPrnir~l mode of action. In particular, ANK repeats, which cDllsLiLuLe the most conserved region (--70% amino acid identity) between Notch and its ve~L~b.dte c~unLe~a~L~ (Stifani et al., 1992, Nature Gen~oti~s 2:119-127), are thought to mediate protein-protein interactions among 10 diverse groups of proteins, inrl~l~ling 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 Rçrln~tt 1992, Trends Cell Biol. 2:127-129; Blank et al., 1992, Trends Biochem. Sci. 17:135-140; Bennett, 1992, J. Biol. Chem. 267:8703-87~6). Indeed, Rebay et al. (1993, Cell 74:319-329) have recently demonstrated that the ANK repeats are crucial for Notch-mPAi~ted ~i~n~lin~ events. Both EGF-like repeats and ankyrin motifs are found in a variey of proteins known to interact with other protein molecules. Indeed, evidence has shown a direct interaction between Notch and the products of the Delfa and Serrate loci, which also encode transmembrane proteins containin, EGF-20 like repeats (Fehon et al., 1990, Cell 61:523-534; Rebay et al., l991, Cell 67:687-699).
In Drosophila, it has been demonstrated that dominant 'activated' phenoypes result from in vivo overexpression of a Notch protein lackin~ most extracellular, ligand-- binding sequences. while 'dominant-negative' phenoypes result from ove~ ssion of a 25 protein lacking most intrace~ r sequences (Rebay et al., 1993, Cell 74:31g-329) In Drosophila, Deltex has been demonstrated to play a critical role in development and other physiological processes, in particular, in the .~ign~lin~ pathway of Notch which is involved in cell fate (differentiation) determination. We have demonstrated throu~h expression studies conducted in cultured Drosophila cells, in yeast, and in the 3 ~ im~in~l wing disc that Drosophila Deltex mP~ t~s the intracellular portion of the signal transduction cascade involved in Notch func~ion (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 homoypic interactions, and that it 35 directly physically inLeld~ . with the Drosophila Notch intracellular ANK repeats.

CA 02238404 1998-0~-22 W O 97/1882Z PCTnJS96/18675 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., 19911 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 si~n~lin~ events and that dominant 'activated' phenotypes 10 result from in vivo ove~e~ ession of a Notch protein lacking most extracellular, ligand binding sequences, while 'rlomin~nt negative' phenotypes result from overexpression of a protein lacking most intracellular se~lenres (Rebay et al., 1993, Cell, 74:319-329).
Furthermore, deltex displays genetic interactions with Notch and Delta, both tr~nsm~mhrane 15 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 Notch group of i~lLeld. ~illg loci.
We have also subsequently demonstrated that a fragment mostly composed of the ankyrin repeats of the Notch protein mediate molecular interactions between Notch and 20 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 i~ .d~ directly with a viral transactivator protein termed Epstein-Barr virus -~ nuclear antigen 2 (EBNA2), which enables a virus to subvert the normal program of B cell 25 differentiation (Schweisguth, F., et al, 1992, Cell 69: l l99; Furukawa T., et al.. 1991, J.
Biol. Chem. 266:23334). Genetic and molecular studies suggest that Deltex and Delta may act in concert to m--lrimf~rize Notch proteins and to il~L~ere with the cytoplasmic retention of Su(H) by Notch, thus activating the Notch .si~n~3lin~ pathway (Diederich, ~., et al., 1994, Development 120:473; Fortini, M., et al., 1994, Cell, 79:273; Matsuno, K., et al., 3 ~ unpublished, Artavanis-Tsakonas et al., 1995, Science, 268:225-232). l'his pathway is believed to control nuclear events in order to inflllen~e the progression of uncomrnitted cells to a more dir~~ iated state. Three loci encoding putative nuclear proleins Hairless, Enhancer of split, and mastermind, have been implicated in these nuclear events.

CA 02238404 1998-0~-22 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.

3. S~}3~A~Y OF THE INVENI ION
The present invention relates to nucleotide sequences of vertebrate deltex 10 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 fr~m~ntc or derivatives are also within the scope of the invention. Production of the foregoing proteins and derivatives, 15 e.g., by recombinant methods, is provided.
In a specific embodiment, the invention relates to human deltex nucleic acids and proteins.
In another specific embodiment, the invention relates to m~mm~ n deltex nucleic acids and proteins.
2 0In specific embodirnents, 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 SH3 - binding domains, ring-H2-Z;inc fmgers, ~iom~in~ which mediate binding to Notch or to a 25 Notch derivative containing Notch cdclO/SW16/ankyrin ("ANK") repeals, 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 dir~erenliation by administration of a therapeutic 3 ~ compound of the invention. Such therapeutic compounds (termed herein "Therapeutics") include: ~ertebrate Deltex proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the vertebrate Deltex proteins. analogs, or derivatives: and vertebrate deltex ~nti.~erl~e nucleic acids. In a preferred embodimen~. a 3~ Therapeutic of the invention is administered to treat a cancelous condition, or to prevent ~urc~les~ion from a pre-neoplastic or non-m~iign~nt state into a neoplastic or a m~lign~nt CA 02238404 l998-05-22 , state. In other specific embodiments, a Th~ uLi~, of the invention is ~ cd to treat a nervous system disorder or to promote tissue .c,~e~ dLion and repair.
In one embodiment, Thcld~tulics which aIltagonize, or inhibit, vertebrate Notch and/or Deltex function (he~eillisrL~l ".Ant~goni.~t The,~ulics") are a-l",i~ ,ed for theld~euLic effect. In another embodiment, Th~_.~t;uLics which promote velLtl,~ Notch and/or Deltex function (hel~ drl~l "Agonist Theld~tuLics") are ~ lcd for Lhe~l tulic effect.
Disordel~ of cell fate, in particular lly~elyioliferative ~e.g., cancer~ or 10 hypoprolirtlaLi./e disorders, involving aberrant or ulldeshable levels of e~lession or activity or ~ i7~tiC~Il of v~ Notch and/or Deltex protein can be ~i~gnnsed by detecting such levels, as described more fully infra.
In a preferred aspect, a Therapeutic of the invention is a protein consisting ofat least a fragment ~termed herein "adhesive fragment") of vertebrate Deltex which m~di~tPs binding to a Notch protein or a fragment thereof.
The invention also provides mPtho-i~ of inactivating Notch function in a cell, methods of id~,llliÇyillg a c0lll~3uulld ~at inhibits or reduces the binding of a vertebrate Deltex protein to a Notch protein, and methods of r~r~nrling non-terminally dirrtlc;l~Li~d 2 0 cells.

4. D~SCRIPTION OF T~ FIC;:URES
~ Figure lA-F. Nucleotide sequence (SEQ ID NO:l) and cleclllred amino acid 2 5 sequence (SEQ ID NO:2~ of Drosophila delt~x cDNA.
Figure 2A-C. Composite nucleotide sequence (SEQ ID NO: I l) derived from the cDNA (nucleotide 1 to 2547), and dedllred 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: * ~i~si~n~trs the start of T05200 and $ the end of T05200. Core H
3 ~ and C residues in Ring-H2-zinc finger are shown by underlining. PCR primers hdx-l to 4 (SEQ ID NO:26), (SEQ ID NO:27), (SEQ ID NO:28), and (SEQ ID NO:29), respectively, are in~lir~tfd in bold. X and N l~.csenL amino acid residues and nucleotides, le~c~,Lively, not yet deL~ lilled.
3 5 Fi~ure 3. Aligned amino acid sequences of human Deltex (SEQ ID NO:12) and Drosophila Deltex ~SEQ ID NO:2) plOt~ S. Those positions at which residues are itlrntir~l are shaded. Sites in which arnino acids are rhPmir~Tly similar are boxed.
Figure 4A-B. Amino acid sequ~nre of Drosophila Deltex (SF.Q ID NO:2) and d~cign~h~d fr~gmPnt~ irnplicated in protein-protein interactions. Fr~gmrntc A-D
(SEQ ID NOS:13-16, respectively) are shown.
Figure 5. Srh -.~ diagrarn of Deltex r~;-g...r ~i m~ ting Deltex-Deltex interactions.
Figure 6. Schrm~tir. rli~r~m of the Deltex and Notch rld~".~ c m~ t;n~
Deltex-Notch ~lLt,ld~;liO~

5. DETAILED DESCRIPrION OF THE INVENTION
The present invention relates to nucleotide sequences of vertebrate deltex genes, and arnino acid sequences of their enrodP(l Deltex proteins. The invention further relates to fia~".r~ and other del;vdliv~;s, and analogs, of vGlLcbldL~ Deltex proteins.
Nucleic acids erlr.o-ling such r~ r~ or derivatives are also within the scope of the invention. Production of the fol~goillg proteins and d~livd~iYes, e.g., by recombinant 2 ~ methods, is provided.
In a specific emborlimrnt the invention relates to a hurnan deltex gene and protein.
- In a another specific embodiment, the invention relates to a m~mm~ n 2 5 deltex gene and protein.
The invention also relates to vertebrate Deltex protein derivatives and analogs of the invention which are fimrtion~lly active, i.e., they are capable of displaying one or more known filnr.tinn~l activities associated with a fi~ length (wild-type) vertebrate Deltex protein. Such r~...- ~i.",~l a.,liviLics include but are not lirnited to ~ntigeniriy [ability to bind 30 (or cr~mretf~ with a vt;lLt;bldte Deltex protein for binding) to an anti-vertebrate Deltex protein antibody], immnnogenicity (ability to genel~LL~ antibody which binds to a vertebrate Deltex protein), ability to bind (or colLl~eLe with a veLl~;lJldL~ Deltex protein for binding) to Notch or a second Deltex protein or other proteins or fr~gm~ont~ thereof, ability to bind (or 35 compete with a ve.l~lJldL~ Deltex protein for binding) to a r~eptor or ligand for a v.,.l~biaLe Deltex protein.

The invention further relates to fr7gmpntc (and delivaLives and analogs thereof) of a ~e-Lbldt~ Deltex protein which comprise one or more ~lom~in~ of a vertebrate Deltex protein (see infra), inrl~ nE but not limited to ~e SH3-binding dom~inc, ring-H2-zinc fingers, dom~in~ which mediate binding to Notch (or a d~livdLi~e thereof cont~inin~ the Notch ANK repeats) or to a second Deltex molecule or fragment thereof, or any co~ ation of the forcg(,illg.
Antibodies to Velk;~ Deltex pruL~-ILs, their derivatives and analogs, are a~lrlition~ily provided.
Our prior ~LIC,n~,uL~. 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 sl~rcecc~ul cloning of human deltex. As described by way of example below, we have used an innovative methodology to clone the transcription unit 15 corresponding to human deltex. As described therein (see Section 6), our results show a ~i~"ili-~nl structural conservation of Deltex in humans, indicative of functional conservation. Moreover, we demonstrate that human Deltex displays direct molPcul~r interaction with both human and Drosophila Notch intrare~ r ANK repeats (see Section 7). Knowledge of the sequpnre of human deltex allows the i~lP,~liri.~1linn of regions strongly 2 ~ conserved between Drosophila and human deltex, and provides a method for readily i~ol,.ting other vertebrate deltex genes by use of such strongly cons~ilved regions (see Sections 5.6 and 8 infra).
~- The vertebrate deltex nucleic acid and amino acid seqller~res and antibodies 2 5 thereto of the invention can be used for the detection and 4uallLiL~tion of vertebrate deltex rnRNA and protein, to study expression tnereof, 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 thelayc~lLic and tii~gnnstic use, as described infra. The agonists and antagonists of Deltex function can be used to alter 3 ~ the ability of a cell to dir~~ Lial~. 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 signihr~nt 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 g for Llcatlllent of disorders of cell fate or dirr~ tinn by ~ i"i~l,aLion of a therapeutic compound of the invention. Such tht;ldy~ulic compounds (termed herein "TheldL~uLiL,sn) include: vertebrate Deltex ~lo~il~s and analogs and dc.ivdtives (inrlntl;n~ fr~rnPnte) thereof; antibodies thereto; nucleic acids Pnrorlin~ the v~...,I~ldte Deltex proteins, analogs, or deli~aliv~s; and ~/t;lLt;lJldlt: deltex ~nti~e.r~e nucleic acids. In a ~refell~d embodiment, a Therapeutic of the invention is ~rl~ r.~ed to treat a c~lcer.~us condition, or to prevent progression from a pre-neoplastic or non-m~ n~nt state into a neoplastic or a m~ n~nt state. In other specific embodiments, a Theld~)euLi~; of the invention is ~ d to treat 10 a nervous system disorder or to promote tissue regeneration and repair.
In one embodiment, Therapeutics which antagonize, or inhibit, Notch and/or ~e.~ dt~ Deltex function ~ereinafter "Antagonist Therapeutics") are a~imini~tPred for therapeutic effect. In another embodiment, Therapeutics which promote Notch and/or vertebrate Deltex function (heleillaf~t:r "Agonist Theldl,euLics") are administered for therapeutic effect.
Disorders of cell fate, in particular hyperprolir~ld~ive(e.g.~ cancer) or hypoproliferative disorders, involving aberrant or undesirable levels of expression or activity or loc~li7~tion of Notch and/or vertebrate Deltex protein can be diagnosed by detectin~ such 2 0 levels, as described more fully infra.
In a preferred aspect, a Therapeutic of ~e invention is a protein con~i.ct;n~ ofat least a fragment (termed herein "adhesive fragment") of \~1 L~late Deltex that mediates binding to a Notch protein, a second Deltex protein, or a fragment of Notch or Deltex.
2~; 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 fli~clnsl-re~ and not by way of limit~tion, the detailed 30 description of the invention is divided into the subsectinnc set forth below.
.1. ISOLATION O~ T~ VERTEBRATE DE:LTEX NUCLEIC ACIDS
The invention relates to the nucleotide sequences of vertebrate deltex nucleic 35 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 -O 97/18822 PCT~US96/18675 504-2363), or nucleic acids encoding a hurnan Deltex protein (e.g., having the sequenre of SEQ ID NO: 12). The illve-~Liun provides nucleic acids Co~ .g of at least 8 nucleotides (i.e., a hybridizable portion) of a ~,~,LC~ deltex seqU~llre; in other embotlimPnt~, the nucleic acids consist of at least 25 (ccntimlouc) ~ oti~i~s, 50 mlrl~otirles, 100 nucleotides, 150 nucleotides, or 200 nucleotides of a Velt~l~ldLt~ deltex sequence, or a full-length ~e~l~biaLe de~tex coding sequence. The iulv~ idn also relates to nucleic acids hybridizable to or complem~nt~ry to the f~ g se4~ es. In specific acpects, nucleic acids are provided which comprise a sequt?nre complr~ y to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a vertebrate deltex gene. In a specific em~odirnent, the sequence is naturally oC~;ullLIlg.
In other specific embodim~ts, the invention provides nucleic acids cu~ g at least 110, 150, or 200 contim-rn~c nucleotides of the sequence of SEQ ID NO: 11. In other emborlimtont~. the invention provides a nucleic acid colllylisillg the first 25, 50, 100, 150, 200, or 230 amino acids of SEQ ID NO: 12.
In a specific emboflim~nt, v~ aL~ deltex nucleic acids comprise those nucleic acids which are substantially hom--logous to the nucleic acids encoding the amino terrninal 180 amino acids (encoded, e.g., by nucleotide numbers 504-1044 of SEQ ID:11) Of human deltex, or fr~gm~ntc thereof. In one embodiment, the vr~t;bldl~ deltex nucleic acid has at least 50% identity over the corresponding nucleotide sequence of an identically sized human deltex. In another embodiment ~is identity is greater than 55 % . In a preferred embodiment, the nucleotide seqnPnre identity of the vertebrate deltex is greater than 60%. In a more ,~lefell~d embodiment this identity is greater than 65%. In a most preferred embodiment, the nucleotide seque~re identity of the vertebrate deltex is greater than 70% over that of the corresponding nucleotide seqn~nre of icientir~Tly sized human deltex.
In another specific embodiment, ~ biate deltex nucleic acids co~ ise those nucleic acids which are srlbst~nti~lly homologous to the nucleic acids encoding the central region arnino acids of hurnan deltex (e.g., nucleotide numbers 1045-1821 of SE~Q ID
NO: 11) or fr~gmPnt~ thereof. In one embodirnent, the nucleic acids encoding the central arnino acids of the vell~bld~ Deltex protein has at least 50~ nucleotide seqn~n-e identity with the corresponding human deltex sequence of i~lentir~l size. In another embodirnent this identity is greater than 55%. In a ~l~r~lled embodiment, this nucleotide sequence identity is W O 97/18822 PCT~US96/18675 greater than 60%. In a more pr~r~lled embodiment this identity is greater than 65%. In a most preferred embodiment, the homology of the nucleic acids tonro(lin~ the central region amino acids of the ~el~ JIat~: deltex has a ~ ol;Ae seqllenre identity that is greater than 65% over that of the corresponding nucleotide sequenre of irlent;r~lly sized human deltex.
In another specific embo~lim~ont~ v~ Le deltex nucleic acids Co~ e those nucleic acids whlch are ~ s~ ly homologous to the nucleic acids çnrorlin~ the 180 carboxy terrninal amino acids of human deltex (--~-- lcoliAe numbers 1822-2366), or fr~mlonr~ thereof. In one embodiment, the nucleic acids encoding the carboxy terminal 10 region of the ~/e.L~blàle Deltex protein has at least 50% nucleotide sequence identity over the corresponding human deltex sequence of i-l~ontir~l size. In another embodiment this identity is greater than 55%. In a ~ler~ ,d embodiment, this identity is greater than 60%.
In a more preferred embodiment this identity is greater than 65%. In a most p~er~ d 15 embo~l;m-o~t the identity of the nucleotides encoding the amino terminal amino acids of the v~ blate deltex is greater than 70% over that of the corresponding nucleotide seql~nre of i~lentir~11y sized human deltex.
In a specific embodiment, a nucleic acid which is hybridizable to a velLel, delt~x nucleic acid (e.g., having seq~l~nre SEQ ID NO: 11), or to a nucleic acid encoding a 20 ~/elL~bl~te deltex derivative, under conditions of low stringency is provided. By way of example and not lim;t~t;~n, procedures using such cnn-liti~ n~ 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 35%
2 5 formarnide, 5X SSC, 50 mM Tris-HC1 (pH 7.5), 5 mM EDTA, 0.1 % PVP, 0.1 % Ficoll, 1 % BSA, and 500 ,~lg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following morlifi~tion~: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ~Lg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 106 cpm 32P-labeled probe is used. Filters are inrllb~t~(l in llyb~ t~ mixture for 18-20 h at 3 ~ 40~C, and then washed for 1.5 h at 55~C in a soltttion containing 2X SSC, 25 mM
Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1 % SDS. The wash solution is replaced with fresh solution and inçllhat.od an additional 1.5 h at 60~C. Filters are blotted dry and exposed for autoradiography. If nPct~s~y, filters are washed for a third tirne at 65-68~C and reexposed 35 to f~lm. Other conditions of low ~Llillgell~;y which may be used are well known in the art (e.g., as employed ~or cross-species hybridizations).

W O 97/18822 PCT~US96/18675 In another specific embodiment, a nucleic acid which is hybridizable to a vertebrate deltex nucleic acid under co~ c of high ~ ;e~ is provided. By way of example and not limit~tion, proce~lules using such conditions of high slL~ ,y are as follows: Plchybl;.l;~tiQn of filters c~ g DNA is carried out for 8 h to OVC~ lt at 65~C in buffer composed of 6X SSC, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.0Z%
PVP, 0.02% Ficoll, 0.02% BSA, and 500 ~Lg/rnl denatured salmon sperrn DNA. Filters are hybridized for 48 h at 65~C in plehybli~ mixture c~ .i,.i"g 100 ~g/ml denatured salmon sperm DNA and 5-20 X 106 cpm of 32P-labeled probe. Washing of filters is done at 10 37~C for 1 h in a solution co.~ g 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.~1% BSA.
This is followed by a wash in 0. lX SSC at ~0~C for 45 min before autoradiography. Other con~itionc of high ~Llill~,ell-;y which may be used are well known in the art.
Nucleic acids encoding derivatives (e.g., fr~m~ntc) of vertebrate Deltex proteins (see Section 5.6), and vertebrate deltex antisense nuc}eic acids (see Section 5.11) are ~rlition~lly provided. As is readily a~palellL, as used herein, a "nucleic acid encoding a fragment or portion of a vertebrate Deltex protein" shall be construed as lt:r~llillg to a nucleic acid enrotling only the recited fragment or portion of the Vtllt~bl~ Deltex protein and not the other contiguous portions of the ve~ Deltex protein as a con~in~louc 2 0 seq~l~onre.
Specific embo-~imPntc for the cloning of a vertebrate deltex gene, e.g., a human deltex gene, picse.lLed as a particular example but not by way of limitation, follows:
-- For expression cloning (a technique commonly known in the art), an 25 expression library is obtained or is constructed by methods known in the art. For example7 mRNA (e.g., human) is isolated, cDNA is made and ligated into an expression vector (e.g., a bacteriophage derivative) such that it is capable of being expressed by the host cell into which it is then introduced. Various sclee~ lg assays can then be used to select for the expressed VC~ Deltex product. In a p.erell~d aspect, anti-human Deltex antibodies 3 ~ can be used to select the recombinant host cell ekyressillg a cloned vertebrate deltex gene.
In a specific emborlimf~nt. 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 l~pr~se~ known vertebrate deltex sequences, 3 5 preferably regions known to be conserved between Drosophila and human, can be used as primers in PCR. The ~yllllle~ic oligonucleotides may be utilized as primers to amplify by W O 97/18822 PCT~US96/18675 PCR sequences from a source (RNA or DNA), ~,lert.~ly a cDNA library, of potential interest. PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taq polyl"c.ase tGene Amp~). The DNA being amplified can include human mRNA or cDNA or genomic DNA. One can choose to synth~si7P several dirre.~l degenerate primers, for use in the PCR reactions. Ie is also possible to vary the sL~ ,tl,.,y of hybr;-li7~ion conditions used in prirning the PCR reactions, to allow for greater or lesser degrees of nucleotide se~ e-nf4 sirnilarity between the lcnowrl vc.~braL deltex nucleotide sequence and the nucleic acid homolog being i~ol~tPA For cross species hybri-li7~tirn low 10 ..~ ency con~itiQn~ are ~-er~ d (see supra). For same species 11Y7L71;~ moderately ~,L~ ent or highly sll"lgGlll con~Tition~ are ~l~r~.led (see supra). After sllcce~sf~ll amp'lifir~titln of a segment of a vertebrate deltex gene homolog, that se~ P~,( may be molecularly cloned and sequPnre~l, and utili_ed as a probe to isolate a complete cDNA or 15 genomic clone. This, in turn, will perrnit the ~L~I ~--i-~,-lion of the gene's complete nucleotide sequ~nre, t'ne analysis of its ~ ,;.;.ion, and the production of its protein product for functional analysis, as desclil,e1 infra. In a plGr~..ed aspect, human genes eAlrorlin~
Deltex proteins may be i(1entifipd in this fashion. Al~ll~LivGly to selection by hy'ori-ii7~tion t'ae PCR-amplified DNA can be inserted into an G~ ,sion vector for G~yre;.sioll clorLing as 20 described above.
In tne event that it is desired to isolate a ~ LGLiate deltex gene by cross-species hybridization (eit'ner by direct hybridization to a vclL~blak deltex probe lc:plt:sell~ g all or a part of a VelLebld~ deltex gene of a different species, or by PCR using2~; oligonucleotide primers derived from the sequence of a vertebrate deltex gene of a different species), the desired Vt;lkblaL~ deltex gene can be isolated as set forth in Exarnple 8, by screening with a probe, or prirning for PCR with an oligonucleotide, CO~ deltex sequ,onrçs encoding regions highly conserved bt;l~ccll hurnan and Drosophila. For ple, the human Deltex amino acid stretches 414419 (SEQ ID NO:30), 475480 (SEQ
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 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. In a ~.~r~ d embodiment, a pair of oligonucleotides 3~; Cu~ liSillg sequences s~pdlaL~d by a length in the range from 50-500 nucleotides is used as primers in PCR The invention ellvi~ions the use of nucleic acids encoding conselved W O 97/18822 PCT~US96/18675 regions of the Deltex protein in combination to isolate the Deltex ~ ~ro(l;,.g nucleic acids of other o~ ..c, by use in PCR to amplify the desired seql~Pnt~e or less l)leL;-~al)ly~ without PCR, as a probe in sel~c*cn by virtue of direct colony hybri-li7~ti-~n (e.g., C~U~l~iL~, M.
and Hogness, D., 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 r~.,se .l;..~ all or a part of a del~ex gene of an evolutionarily distant, dirf~ species, or by PCR using oligo~llcl.-oti~.o primers derived from the sequence of a deltex gene of a ~irr~ , evolutionarily distant 10 species), the desired del~ex gene can be isolated by a more gradual method of evolutionary walking via first isolating a deltex gene from a more closely related species, idellliryillg the portions of deltex which are conserved cross-species, and then sclcc.liLIg with a probe or prirning for PCR with a nucleic acid containing the cons~;~ vtd sequence. This method, 15 while more cumbersome, is straigllLro~ d and can be readily carried out by routine mPthorlc. For exarnple, 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 cons~,ved 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 2 ~ amphibian library, a con erved 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 sc.eel"l~g can be selected from among various species by hybridizing the deltex probe to a Southern blot - cont~ining genomic DNA from each species, and selecting a species to which hybridization 2 5 OCCUrS.

The above-methods are not meant to limit the following general description of me~ods by which clones of v~ l;,late deltex may be obtained.
Any eukaryotic cell can potentially serve as ~e nucleic acid source for the 3 ~ molecular cloning of the vertebrate de~tex gene. The DNA may be obtained by standard procedlnes known in the art from cloned DNA (e.g., a DNA "library"), by ~ .omir~~yll~esis, by cDNA cloning, or by the cloning of genomic DNA, or fr~gm~ntc thereof, purified from the desired human cell (see, for example Sambrook et al., 1989, Molecular 35 Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 2d. Ed., Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL

. - 15 -O 97/18822 PCT~US96/18675 Press, Ltd., Oxford, U.K. Vol. I, II.) Clones derived from genomic DNA rnay contain regulatory and intron DNA regions in ~itirn to coding regions; clones derived from cDNA will contain only exon se~,P~-r~s. Whatever the source, the gene should be mrlec~ rly cloned into a suitable vector for prop~tinn of the gene.
In the molecular clor~ing of the gene from genomic DNA, DNA fr~gTnPIlt~
are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using various restriction ~l~ymes. Al~l~ ely, one may use DNAse in the ~lcSI;llce of ~ ng~"~c to fid~ the DNA, or the DNA can be physically sheared, as for example, by sol~irAIi~n The linear DNA fiA~ ; can then be separated accol~ g to size by s~1dald terhniql~,Ps, inr~ in~ but not lirnited to, agarose and polydc~yl~l~ide gel electrophoresis and colurnn chromatography.
Once the DNA rlA~ are gellelat~.d, idPntifir~tion of the specific DNA
fragment cont~inin~ the desired gene may be accomplished in a number of ways. For example, if an amount of a portion of a i/t~b~dle deltex (of any species) gene or its specific RNA, or a fragment thereof e.g., the a&esive domain, is available and can be purified and labeled, the gene.dL~d DNA fiA~ may be screened by nucleic acid hybridization to the labeled probe (Benton, W. and Davis, R., 1977, Science 196, 180; GlullsL~ , M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 3961). Those DNA fragments with suhst~nti~l homology to the probe will l~yblidi~. For cross species hybri~ Ati- n, low stringency conditions are plc~.led (see supra). For same species hyl,l;~ /ion, moderately stringent conditions are ~c~.led (see s~pra). It is also possible to identify the appl~,pliate 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 ~let~?ct~-l by assays based on the physical, chPminAI, or immunological properties of its e*,lessed product. For example, cDNA clones, or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein that~ e.g., has similar or i(iPnti~AI elecL~hol~Lic migration, isoelectric focusing behavior, proteolytic digestion maps, binding to Notch, or Antigenir plopellies as known for vertebrate Deltex. If an antibody to vertebrate Deltex is available, the v~ ldl~ Deltex protein may be i~ tified by binding of labeled antibody to the putatively vel~bia~e Deltex synth~.ci7.ing clones, in an ELISA (enzyme-linked i""--""~sorbent assay)-type procedure.

The ~cllcbl~L~ deltex gene can also be i~iPntifipd by rnRNA s~i~ction by nucleic acid hybririi7~tion followed by in vitro tr~n~l~*on In this pioc~lu.e, fragmPntc are used to isoiate compl~ "~.,y mRNAs by llyi~ ol~ Such DNA r~ ." .~lx may re~.~se.ll avaiiable, purified ~kl~ld~ deltex DNA of another species (e.g., human).

Tmmnnn~lcL;i~iL~tion analysis or filnrtion~l assays (e.g., ability to bind Notch) of the in vitro tr~ncl~tion products of the isolated products of the isolated mRNAs i~iPntifiP5 the mRNA
and, therefore, the compl~"~ .y DNA fr~mPntc that contain the desire~ seql~enrp-c- In litir~rl, specffic rnRNAs may be selected by adsorption of polysomes isolated from celis to 10 im~nobiiized antibodies specifir~lly directed against vcllc~iatc Deltex protein. A
radiolabelled ve~lc~ tc deltex cDNA can be synth-osi7.Pd using the selected mRNA (from the adsorbed polysomes) as a tprnpl~tp. The radiolabelled mRNA or cDNA may then be used as a probe to identify the vc.L~b.dte deltex DNA fragments from arnong other genornic DNA
fr~gmpnt~ .
AlLelllatives to isolating the vt;lLcbrd~c deltex genornic DNA rnclude, but are not limited to, chpmir~lly ~ylllllr~s;~.;llg the gene sequence itself from a known seqUpnrp or making cDNA to the mRNA which encodes the ~ L~ deltex gene. For example, RNA
for cDNA cloning of the vertebrate deltex gene can be isolated from cells which express 20 vertebrate Deltex. Other methods are possible and within the scope of the invention.
The i~7~?ntifi~i and isolated gene can then be inserted into an dppl~pli~
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 2~i system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda d~livativt;s, or plasmids such as PBR322, pUC, or Bluescript (Stratagene) plasrnid deliv~tives. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complelnell~ly- cohesive termini. However, if the complem~n~ry restriction sites used to 3 ~ fragrnent the DNA are not present in the cloning vector, the ends of the DNA moi~c~ s may be el,,.ylllalically morlifiPri Al~lLativ~ly, any site desired may be produced by ligatirg nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific ~h~mir~liy s5~ rd oligonucleotides encoding restriction endon--c~e~e35 recognition seq~lenres~ In an all~ll~LiY~ method, the cleaved vector and v~"lt;bldLe del~ex gene may be moclifi~-d by hornopolymeric tailing. Recombinant molecules can be introduced O 97/18822 PCT~US96/18675 into host cells via lla~r ., ~ inn, Ll~iLion, infection, electroporation, etc., so that rnany copies of the gene s~q~lenre are gene~dt~,d.
ID an ~ ive m~th~d, the desired gene may be i-1~ntifi~ and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Elllich llcl.L for ~e desired gene, for example, by size fr~rtn7~tn~ can be done before insertion into the cloning vector.
In specific embodiments, LLo~f ..I~ l of host cells witb. lecolllbil~ll DNA
molecules that i,lcolpolat~ the isolated -v~lL~ L~ deltex gene, cDNA, or synth~si7ed DNA
sequenre euables generation of multiple copies of the gene. Thus, the gene may be obtained in large q~ lies by growing ~.aulsÇolm~-L~, isolating the recoml,il.alll DNA molecules from the Ll~rO~ lL~ and, when n~ce~s~y, r~ lg the inserted gene from the isolated recombinant DNA.
The vertebrate deltex sequences provided by the instant invention include those nucleotide sequences ~nro~ling suhst~nti~lly the same amino acid sequences as found in native vertebrate Deltex protein, and those encoded arn~no acid sequences with functionally equivalent amino acids, all as described in Section 5.6 infra for vertebrate Deltex del iV~LiY'~,S .
~i.2. EXPRESSION OF VERTEBRATE DELTEX ~YUCLE:IC ACIDS
The nucleic acid coding for a vertebrate Deltex protein or a functionally active fragment or other de-ivalive thereof can be inserted into an a,u~lopliate expression vector, i.e., a vector which contains the n~ceS~ y elements for the ~anSc.i~Lion and translation of the inserted protein-coding seq~-~nre. The necessary Lldnsc.i~tiollal and translational signals can also be supplied by the native velleb~d~ deltex gene and/or its fl~nking regions. A variety of host-vector systems may be utilized to express the protein-coding sequen~e. These include but are not lirnited to vertebrate cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microOrga~ s such as yeast containing yeast vectors, or bacteria Ll~iro.l~led with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in ~eir strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable Lldns~ tion and tr~n~l~tion elements may be used.
In a specific embodirnent, a molecule con~ il-g a portion of a v~lL~blate delte~; gene which W O 97/18822 PCT~US96/~8675 encodes a protein that binds to Notch or to a mn~ cu~ g the Notch ANK repeats is ~;Ayl~,ss~d. In another embollim~nt~ a m~lf~cul~co~li"illg a portion of a VL,l~ lr~l~ del~ex gene which encodes a protein that binds to a r~ of a Deltex protein is e~ ,ssed. In othêr specific embo lim-o-ntc, m~mm~ n deltex gene is eA~,essed, or a sequence f~nro lin~ a f~lnrtion~lly active portion of m~mm~ n Deltex. In other specific embo-lim~ntc, the hu nan a'eltex gene is eA~l~esscd, or a se~ e~e c;~rO~ a fimrtirln~lly active portion of human Deltex. In a specific embodiment, a chimeric protein Co~ Li~.iulg a Notch-binding do_ain of a v~l~bldL~ Deltex protein is eA~lessc~. In ot'ner specific embo~lim~ntc, a full-iength 10 vertebrate deltex cDNA is expressed, or a sequence encoding a Çull~,Liollally active portion of a ~clLtbl,dL~ Deltex protein. In yet another embo(limPnt, a r~ lellL of a ~ L~bl~Lr Deltex protein collllulisillg a domain of the protein, or other delivdLiv~, or analog of a vertebrate Deltex protein is ~ re~sed.
Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors COllld~ lg a chirneric gene Co~ Lillg of app~ lidte Lld~ Lional/tr~n~ tir~n~l control signals and the protein coding se~ n~es. These methods may inc}ude in vitro l~colllbil~lL DNA and synthetic techniques and in vivo recc,~llbh~lL~ (genetic ltcollll)illdlion). Expression of a nucleic acid sequence 2 0 encoding a ~/~l L~ldL~; Deltex protein or peptide fragment may be regulated by a second nucleic acid sequence so that the ~,~lLt;l~-dL~ Deltex protein or peptide is expressed in a host LldlL~rollned with the lecclllbi~ DNA mnlf~c~ o For example, expression of a ~ertebrate - Deltex protein may be controlled by any promoter/enl1ancel element known in the art.
25 Promoters which may be used to control vertebrate deltex gene expression include, but are not limited to, the SV40 early promoter region (Bernoist and Charnbon, 1981, NaNre 290:304-310), the promoter cont~;n~oci in the 3' long terminal repeat of Rous sarcoma virus (Yarnamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Aead. Sci. U.S.A. ,78:1441-1445), the regulatory sequences of the 3 ~ metallothionein gene (Brinster et al., 1982, Nature 296:3942); prokaryotic expression vectors such as the ~ r~ ce (Villa-Kamaroff et al., 1978, Proc. Natl. Aead. Sci. U.S.A.
75:3727-3731), tac (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25), ~PL. or trc promoters; see also "Useful ~ Leil.~. from recombinant baeteria" in Scientific American, 3--, 1980, 242:74-94; plant e,~ ssion vectors colllp.~ lg the nopaline synthetase promoter region or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl.

Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosph~t~
c~l~o~ylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter el~m~ont~ from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehy~lLog~.lasc) l~rO111O~C~, PGK (phosphoglycerol kinase) promoter, alkaline phosl)h~. se promoter, and the following ar~irnal .l~ls~;lipLional control regions, which exhibit tissue sl,ec;.r~i~y and have been utilized in ,l,.,.cge".~ animals: elastase I gene control region which is active in ~Cl~,dLic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, I987, Hepatology 7:425-515); insulin 10 gene control region which is active in pa,lc-~lic beta cells (~T~n~h~n, 1985, Nature 3}5:115-122), immlm~ obulin gene control region which is active in lymphoid cells (Gr~.scrhPrll et al., 1984, Cell 38:647-658; Adarnes et al., 1985, Nature 318:533-538;
~lrlr~n-lPr et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse "l~,lll.l~ly tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et ah, 1986, C:ell 45:485-495~, albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-l~ u~in gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; ~T~mmPr et al., 1987, Science 235:53-58; alpha l-antitrypsin gene control region which is active in the liver (Kelsey et al., 20 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 oligodt;~ldlo-;ylt: cells in the brain ~ he~-l et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region 25 which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypoth~l~mll~ (Mason et al., 1986, Science 234:1372-1378).
Expression vectors co~ ,l.g vertebrate deltex gene inserts can be i-iPrltiftPd by three general approaches: (a) nucleic acid hybritli7~tion, (b) pl~sellce or absence of 30 "marker" gene functions, and (c) e~ essiol1 of inserted seqllenres. In the first approach, ~e presence of a foreign gene inserted in an expression vector can be ~i~tectPd by nucleic acid hybridization using probes comprising seqnPnres that are homologous to an inserted vertebrate deltex gene. In the second approach, the recombinant vectorthost system can be 35 jrlentifiPd and selected based upon the ~l~sence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, l~C;cl1nre to antibiotics, transformation W O g7~18822 PCT~US96/18675 pheno~pe, oc~ cinn body form~ti- n in baculovirus, etc.) caused by the insertion of foreign genes in the vector. For exarnple, if the vertebrate deltex gene is inserted within the rnarker gene seql-~nre of the vector, recom~inants cu,.l~;,.i,.g the vcll~b~aL~ deltex insert can be i(lPntifiP(l by the absence of the marker gene r~""~;O" In the third approach, lecoll,bi~
expression vectors can be i(lentifiecl by assdyillg the foreign gene product e~l1ssed by ~e recombinant. Such assays can be based, for example, on the physical or fim~tion~l properties of the vertebrate deltex gene product in in vitro assay systems, e.g., binding to Notch, binding with antibody.
Once a particular l~colllbillall~ DNA molecule is j~1~ntifi~d 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 e~Lessiu,l 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 (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.
In addition, a host cell strain may be chosen which mofin]~ft~s the expression 20 of the inserted seql-~nres, or modifies and processes the gene product in the specif1c fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the g~n~L~ y engil-~.o,.ed vertebrate Deltex protein may be controlled. Furthermore, different host cells have cha~acLt:lislic and specific mPrh~nicm~ for 25 the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Applu~ te cell lines or host systerns can be chosen to ensure the desired mo~ifir~tion and process,illg of the foreign protein expressed.
In other specific embodirnents, the vertebrate Deltex protein, fragment, analog, or de~ivdliv~ may be expressed as a fusion, or chimeric protein product (CO~ liSillg 3 ~ the protein, rldgl~le~ll, 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 a~.u~.iaL~ nucleic acid seq~-en~f c encoding the desired amino acid sequences tO each other by methods known in the art, in the proper coding frame, and e,i~iesshlg the chirneric 3 5 product by methods commo~ly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques. e.g., by use of a peptide synth,-~i7tor.

Bot'n cDNA and g~nnmir seql1enr~C can be cloned and e~.re.sed.
In other emborlim~ntc, a v lle'lJlaL~ deltex cDNA se.lu~"lce may be chrnm- som~lly integrated and expresse. Homologous recombination plocedu,~s known in the art may 'oe used.

5.3. IDENTl~lCATION AND PURIFICATION OF
'1~; Vl~RTEBRATE DELTEX GENE PRODUCTS
In particular aspects, the invention provides armino acid seq~ res of 10 vertebrate Deltex, preferably human Deltex, and fr~gm~ntc and derivatives thereof which coll,~lise an antigenic d~ (i.e., can be recognized by an antibody) or which are functionally active, as well as nucleic acid sequences ~nroriin~ the folcgo"~g. rFnnrtirln~lly 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 15 product, e.g., 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 anti-vertebrate Deltex antibody), immlmogenicity (geneldtil.g anti-Deltex antibody), Notch intracçll~ r signal Ll,~n.cd~ ion, etc.
2 0 In specific embo(limP,nts, the invention provides fr~gm~ntc of a v~ bldL~
Deltex protein consisting of at least 6 amino acids, 10 arnirlo acids, 50 amino acids, or of at least 75 amino acids. In other embotlim~ntc7 t'ne proteins comprise, or alternatively, consist eCce lti~lly of; one or more of tne SH3-binding ~iom~in.C (e.g., SEQ ID NOS: 17-21 of Table III); one or more ring-H2-zinc finger clom~inc (e.g., SEQ ID NO:25), or a portion which binds to Notch (e.g., comprising the first approximately 230 amino acids of vertebrate Deltex), or any combination of the Çol~goillg, of a vertebrate Deltex protein. Fr~gm~ontc, or proteins COlll~ illg fr~gm~ntc~ lacking some or all of the foregoing regions of vertebrate Deltex are also provided. Molecules Cu~ liSillg more than one copy of the foregoing 3 o regions are also provided. Nucleic acids encoding the foregoing are provided.
Once a recombinant which ~ ,sses a vel ~ Lt; deltex gene sequen~e is identified, the gene product can be analyzed. This is achieved by assays based on the physical or functional properties of the product, inrll-rling radioactive labelling of the product followed by analysis by gel electrophoresis, immnn~cc~y, etc. Ch~mi~ y synthPsi7ed pro~n~s, derivatives, and analogs can be similarly analy_ed.

W 097~g8Z2 PCTnUS96/18675 Once a vclL~,dL~ Deltex protein is iriPntifiP~i, it may be isolated and purifiedby ~l~ldcud TnPth~c inrll-tiin~ ch~o~ .hy (e.g., ion PY~ n~e~ affinity, and sizing colurnn chLo~ d~hy), ce-.l . ir~ irJn, dirrt.e.,Lial solubility, or by any other ~Li~d~d terhnirl~le for the ~ ;ri~lir~n of proteins. The fimrtion~l p.ope.lies rnay be evaluated using any suitable assay (see Section 5.7).
AlLc~ ive:ly, the arnino acid seqll~onre of a ~elLcbldte Deltex protein can be tiPAl-red from the mlrl~PotirlP se(ll~p-nre of the C]~ lcliC gene conL~ ed in the recr,mhin~nt Once the arnino acid spq~npnre is thus known, the protein can be 5y~ P~ d by s~dard 10 rhPmir~1 mPtht~tlc known in the art (e.g., see Hunkapiller et al., 1984, Nature 310:105-111).
By way of exarnple, the ~hP(l~-red amino acid sequence (SEQ ID NO:123 of a human Deltex protein is ~l~sellLcd in Figure 2A-C.

5.4. STRUCTURE OF THE VERTEBRATE
~ELTEX GENES AND PROTEINS
The aL~u~lule 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 vcll~blalt: deltex gene can be analyzed by methods inr.lll-ling but not limited to Southern hybridization (Southern, 1975, J. Mol. Biol. 98:503-517), Northern hybridization (see e.g., Freeman et al., 1983, Proc.
Natl. Acad. Sci. U.S.A. 80:4094-4098), restriction endomlri~e mapping (Mar~iatis, 1982, Molecular Cloning, A Laboratory, Cold Spring Harbor, New York), and DNA sequenceanalysis. 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 30 et al., 1988, G~nt~tirs 120:621-623; Loh et al., 1989, Science 243:217-220) followed by Southern hybrirli7~ti~ with a vcllc~ldte deltex-specific probe can allow the detection of the Vc~lt;bldLc~ deltex genes in DNA from various cell types. In one embodiment, Southern hyhri~i7~tit n can be used to deL~,llfille the genetic linkage of vertebrate deltex. Northern llyl~ irn analysis can be used to cle~ t~ the expression of the verlebl~Lc deltex genes.
Various cell types, at various states of development or activity can be tested for vertebrate - deltex gene ~ ,ssion. The stringency of the hybri~li7~ticn c-)nriit-on~ for both Southern WO 97/18822 PCT~US96/18675 and Northern hybr;tli7~tinn can be m~nirulAte(~ to ensure ~letectirn of nucleic acids with the desired degree of re~ c~ to the speci~lc ~l~bldlt deltex probe used.
Restriction ~lldo~ r~ e ,~ g can be used to roughly ~i~L~ k the genetic ~LIU~IU1G of the ~ bldLt delte~c gene. Restriction rnaps derived by restriction erl-io~-lrlP~e cleavage can be confirmP~ by DNA seql-~nre analysis. AlL."llaliv~ly, restriction maps can be ~eAllce~l, once the nucieotide sequence is known.
DNA sequence analysis can be pc.~ll-ed by any L~cl~ ues known in the art, inri-l~ing but not lirnited to the method of Maxarn and Gilbert (1980, Meth. Enzymol.
10 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. Bi-~rh~mir~l Corp.), or Taq polymerase, or use of an d DNA seq~lçn~tor (e.g., Applied Bio~y~lt...~, 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 AN~LYSIS
The arnino acid seq lPnre of a vertebrate Deltex protein can be derived by 2 0 deduction from the DNA sequence, or all~ dLiv~ly, by direct sequencing of the protein, e.g., with an automated amino acid seq Irnrer~ The amino acid sequence of a representative ve,L~b.dL~ Deltex protein c~ es the sçql~nre sllh~ct~nti~lly 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 irn~nunogenic.
Secontl~.y, ~LluLLul~l analysis (Chou and Fasman, 1974, Biochemistry 13:222) can also be done, to identify regions of a v~ ,bidte Deltex protein that assume specific secondary structures.
Manipulation, translation, and secorl~ry structure prediction, as well as open 35 reading frarne pre-iiction and plotting, can also be accomplished using co~ u~r software programs available in the art.

W O 97~18822 . PCT~US96~8675 Other methods of structural analysis can also be employed. These include but are not limited to X-ray crystallography (Ellg~lUL~ 74, Biochem. Exp. Biol. 11:7-13) and cu.,.~uL~r mf drling (Fletterick and Zoller (eds.), 1986, Computer Graphics and ~ol~c~ r Mûdeling, in Current Co~ lionc in Molecular Biology, C~old Spring Harbor Labo,dLoly, Cold Spring Harbor, New York).

5.5. GENERATION OF ~NIIBODIES TO V~;;~l~;~RATE
D~LTli~X PROTEINS AND DERIVATIVES THEREOF
According to the invention, a vertebrate Deltex protein, its fr~Emrnt.c 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 e~rcssion library. In a plerellcd embo-limr~t antibodies which sperifir~l~y bind to velL~bldt~ Deltex 15 proteins are produced. In a more preferred embodiment, an antibody which binds to a velLt:bldL~ Deltex protein (e.g., m~mm~ n, preferably human) but does not bind to ~full length) Drosophila Deltex protein, is produced. In a ~ler~llt;d embodiment, such an antibody is produced by using as immlm~gen, regions least conserved between Drosophila 2 o melanogaster and the vertebrate Deltex protein.
In another embodiment, antibodies to a particular domain of a v~lkb.dlt:
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 co~ g the first 230 amino-terminal amino acids of vertebrate Deltex. In another embodirnent the antibody binds to an amino-terminal fragment of vel~ te Deltex co~t~ining 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.
3 o Various procedures known in the art may be used for the production of polyclonal antibodies to a vertebrate Deltex protein or deliv~tiv~ or analog. In a particular embo~limPnt, 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 ~e production of antibody, various host animals can be ill,..,,~..;,~d by injection with a 3~
native VGll~ Deltex protein, or a synthetic version, or derivative (e.g., fragment) thereof, inrl~ ing but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to ~crease the immunological response, depending on th~- host species, and in~ ing but not limited to Freund's (complete and incomplete), mineral gels such as ~11llll;.l~..
hydroxide, surface active s--hs~ es such as Iysolecitbin, pluronic polyols, polyanions, peptides, oil ~mnlcion~7 keyhole Iimpet hemo-;ydu lS, diniLIo~llenol, and potentially useful human adjuvants such as BCG (bacille CalmPttP-Guerin) and co~ b~ Itliull~ parvum.
In a l)lcf~lled embor1im~nt polyclonal or monoclr)n~1 antibodies are produced by use of a hydrophilic portion of a Vel~ )ldtt: Deltex peptide (e.g., irl~ntifi~d by the proce.lule of Hopp and Woods (1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824)).For ~l~a~ion of monoclonal antibodies directed toward a Vt~l~ebldL~; Deltex protein sequence or analog thereof, any l~ch~ ue which provides for the production of antibody molecules by contin--ol-~ cell lines in culture may be used. For example, the hybridoma t~rhnit~ 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, Imrnunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer ~herapy, Alan R. Liss, Inc., pp. 77-96) can be used. In an additional embodiment of the invention, monoclonal antibodies carl be produced in germ-free animals (PCT Publication No.20 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 ~ ~fo~ ulg human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 2 5 PP 77-96), or by other methods known in the art. In fact, according to the invention, techniqll~s 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 vt;l~ll.dl~ Deltex protein together with genes from a human antibody 30 molecule of ~J~llO~)lidt~ biological activity can be used; such antibodies are within the scope of this invention. Non-human antibodies can be h~ d by the method of Winter (see U.S. Patent No. 5,225,539).
According to the invention, techniques described for the production of single 3~; chain antibodies (U.S. Patent 4,946,778) can be adapted to produce Yertebrate Deltex protein-specific single chain antibodies. An ~tll1i~jon~ embodiment of the inYention utilizes W 0971~88~Z P ~ nUS96/I8675 the t~rhni~ described for the construction of Fab expression libraries (Huse et al., 1989, Science 246:1275-1281) to allow rapid and easy iclt~ lion of mnnoclon~1 Fab fragTn~ntc with the desired specificity for v~Lebl~Le Deltex proteins, dclivdLi~es, or analogs.
Antibody fra~mPnt~ and other de.-v~liYes which contain the idiotype (binding domain) of the molecule can be ~;f.~r,~"t d by known tPrhniques For example, such fragments include but are not limited to: the P(ab')2 fragment which can be produced by pepsin digestion of the allliboly molecule; the Fab' fr~gmPntc which can be 8enerated by reducing the ~liclllfirlP bridges of the F(ab')2 fragment, and the Fab fr~m~ntc which can be 10 ~,eneldt~d by treating the antibody molecule with papain and a reducing agent.
In the pro~lllction of antibodies, scle.,.~lg 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 Deltex protein, one may assay ge~ d~d hybridomas for a product which binds to a vertebrate Deltex fragment cont~;ning 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 Drosop~ila Deltex protein.
In a specific embodi-m--ent~ antibodies specific to a phosphorylated epitope of 2 0 vertebrate Deltex are produced.
The foregoing antibodies can be used in methods known in the art relating to the loc~li7~tion and activity of the protein sequences of the i~v~--Lion e.g., for imaging these proteins, measuring levels thereof in ~plo~iate physiological samples, etc. Antibodies to 25 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 imm-lnr ~ ys. In another embodiment of the invention (see infra), anti-vertebrate Deltex antibodies and fr~gm.ontc thereof containing the binding domain are Th~.dp~ lics.

5.6. VERTEBRATli~ DI:LTEX PROTEINS, DERIVATIVES AND ANALOGS
The invention further provides vertebrate Deltex proteins, and derivatives (including but not limited to fr~gm~nt~) and analogs of vertebrate Deltex proteins. Nucleic 3~
acids encoding vertebrate De~tex protein derivatives and protein analogs are also provided.
In one embodiment, the vertebrate Deltex proteins are en~o~le-l by ~ne vertebrate deltex W O 97/18822 PCT~US96/18675 nueleic aeids described in Seetion 5. l supra. In partieular aspeets, the proteins, de.ivdLives, or analogs are of mouse or rat; agrir~tltnr~l stock sueh as eow, sheep, horse, goat, pig and the like; pets such as eats, dogs; or other dC)lllp~l;r~ d m~mm~ls. or prirnate Deltex proteins.
The produetion and use of delivaLiv~s and analogs related to vertebrate Deltex are within the scope of the present ~ .,nlion. In a specific embodiment, the dc.ivdlive or analog is functionally active, i.e., capable of e~lliliLing one or more f~
a.,LiviLies associated with a full-length, wild-type v~lL~bldt~ Deltex protein.
In partieular, vertebrate Deltex dc.ivdLiv~s ean be made by altering vc.L~blate deltex seqnPn~es by sub~ .ll.c, a~itinnc or deletions that provide for fillll-L;o~ y equivalent moleeules. Due to the degt;ncla~;y of nucleotide coding seqllenres~ other DNA
sequenees whieh eneode substantially the same amino aeid sequPnre as a vertebrate deltex 15 gene may be used in the practice of the present invention. These inelude but are not limited to m~lPoti~P sequenees COl~ iulg all or portions of vertebrate deltex genes whieh are altered by the snhstitl-tion of different eodons that encode a fimrtinn~lly equivalent arnino acid residue within the sequenee, thus plu.luculg a silent ehange. Likewise, the vertebrate Deltex derivatives of the invention include, but are not limited to, those Co..Ldillil.g, as a 2~ ~illl~y amino aeid sequenee, all or part of the amino aeid seql~er~re of a v~ b.dte Deltex protein inrln~ing altered sequenees in which funetionally equivalent amino aeid residues are substin-t~d for residues within the sequence resulting in a silent change. Por example, one or more amino acid residues within the sequenee ean be substituted by another arnino aeid 25 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) arnino acids include alanine, leucine, isoleueine, valine, proline, phenylalanine, L,y~tophan and methionine. The polar neutral amino acids include glycine, serine, ~hl~t,o~ e, cysteine, 30 tyrosine, asparagine, and gl~ l.i.,P The positively charged (basic) amino acids include arginine, Iysine and histidine. The negatively charged (acidic3 amino acids include aspartic aeid and ~ t~mic acid.
In a specific embodiment of the invention, proteins consi~Li.lg of or 35 Co.~ illg a fragment of a vertebrate Deltex protein consisting of at least lO (comim amino aeids of the vc~Lt:b.dL~ Deltex protein is provided. In other emborlim~nt~, the W O 971'L8822 PCT~US96/18675 fragment consists of at least 20 or 50 amino acids of the vel~b-dte Deltex protein. In specific emborlimPntc, such fr~gmPnt~ are not larger than 35, 100 or 200 amino acids.
Dl,.ivdLives or analogs of vertebrate Deltex include but are not limited to tbose peptides which are ~ )s~ Ty homologous to human Deltex or fr~m~nt~ thereof.
In a specific embodiment, deliv~Liv~s or analogs of v~l~bl~l~ Deltex include those peptides which are 5nhct~nti~l1y homologous to the amino terminal 180 arnino acids (1-1803 of human Deltex. In one embodiment, the amino terminal region of the vertebrate Deltex protein has at least 30% identity over the amino terminal a.nino acid sequçn~e of 0 iri~ntir~lly sized human Deltex. In another embodirnent this identity is greater than 35%.
In a preferred embodiment, the amino tPrrnin~l amino acid identity of the v~lL~:bldLe Deltex is greater than 45 %. In a more L~l~ir~ ,d embotiiment this identity is greater than 55%. In a most preferred embodiment, t'ne homology of the amino terminal amino acids of ~e vertebrate Deltex is greater than 65% over the corresponding human Deltex amino terrninal amino acid se~uence of i~l~ntit~l size.
In another specific embodiment, delivaLi~es or analogs of vertebrate Deltex include those peptides which are substantially homologous to the central region (arn~no acids 181 441) of human Deltex, or fr~gmPntc thereof. In one embodiment, the central region of 20 the vertebrate Deltex protein has at least 30% identity with the c.,.l~onding human Deltex sequence of it~ tit Z~l size. In another embodiment this identitv 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 embodirnent this identity is greater 25 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 65%.
Additionally, derivatives or analogs of vertebrate Deltex include but are not limited to those peptides which are sllbst~nti~lly homologous to the carboxy terminal amino 30 acids of human Deltex or fr~gmp-nt~ thereof. In one embodiment, the carboxy terminal region of the velL~bld~ Deltex protein (the carboxy terminal 180 amino acids) has at least 45% identity over the amino acid sequence of i~Pnt;~l size. In another embodirnent this identity is greater than 50%. In a pre~l.ed embotlimPnt, the amino terminal amino acid 3 5 identity of the vertebrate Deltex is greater than 5~ % . In a more preferred embodiment this identity is greater than 60~. In a most pref~l~ed embodiment, the homology of the amino terminal amino acids of the vertebrate Deltex is greater than 65%.

W O 97/18822 PCT~US96/18675 In another preferred embodiment, de~ivdli~,s or analogs of vclLcbldLc Deltex comprise regions collsc.v~d between Drosophila and human Deltex (see Section 8).The velLti iate Deltex protein d.,HvdLivcs and analogs of the invention can be produced by various mrthorlc known in the art. The m~nip~ tjo~C which result in their pro~ rtion can occur at the gene or protein level. For example, the cloned vertebrate deltex gene sequenre can be mo lifiPd by any of llulllel~us strategies known in the art (Sambrook et al., 1990, Molecular Cloning, A Laboratory l~T~m~l 2d ed., Cold Spring HarborLaboratory, Cold Spring Harbor, New York). The seqUPnrç can be cleaved at apploplialc 10 sites with restriction çnflonllclp~se(s)~ followed by further el~yllldlic mndific~t;nn if desired, isolated, and ligated in vitro. In the production of the gene enrofling a dcLivdLive or analog of a vertebrate Deltex protein, care should be taken to ensure that the modified gene remains within the same tr~ncl~tinr~l reading frarne as the vertebrate deltex gene, ~ c~upled by translational stop signals, in the gene region where the desired vertebrate Deltex activity is e~nro~lP~
Ad~itinn~lly, the vcll~b~dtc Deltex-encoding nucleic acid sequence can be mutated in vitro or in Yivo, to create and/or destroy tr~ncl~tinn initiation, and/or tPrmin~tion seqllrnres, or to create variations in coding regions and/or forrn new l~ ion 2 0 en~lonllr-le~ce sites or destroy preexisting ones, to f~rilit~t~ further in vitro modification.
Any technique for mllr~gt~nPcic known in the art can be used, inrlllr~ing but not limited to, in vitro site-directed ~ rllPsi~ (Hutchinson et al., 1~78, J. Biol. Chem 253:6551), use of TAB~ lin}cers (Phdlll,acia), etc.
2~; Manipulations of the vertebrate deltex sequence may also be made at the protein level. Tnrlllde(i within the scope of the il~vtl,Lion are vertebrate Deltex protein fr~gm~lltc or other derivatives or analogs which are dirrtlenlially modified during or after translation, e.g., by acetylation, phosphorylation, carboxylation, ~mitl~tion, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or 3 ~ other cellular ligand, etc. Any of numerous chf mir~l mn~ifir~tin11c may be carried out by known tprhniques~ inrln~inE but not limited to specific rhlomir~ cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, forrnylation, oxidation, reduction, etc.
3 5 In a preferred aspect, phosphorylation or, allc.~ ivcly, dephosphorylation is carried out, which can be to various extents, on the purified vertebrate Deltex protein or , W ~ 97~882Z P ~ ~US96/~8675 d~"iv~Live t'nereof. The phosphnrylation state of the mr.lPrlllP may be i~ ull~ll to its role in imTare~ r signal LLn~ of Notch filnrtir~n Phosphorylation can be carried out by reaction with an ap~liJplidle kinase (e.g., possibly cdc2 or CK II). Dephnsph~rylation can be carried out by reaction with an a~lol,.;aL~ ph~ .h~l~ce In addition, analogs and derivatives of ~ eb,dLe Deltex proteins can be rhPrn;r~lly 7yl"l-P~ rl For eY~imple, a peptide corresponding to a portion of a ~ bLdlt~
Deltex protein which co~ cs ~e desired domain, or which mP~ tP~ the desired activity in t~itro, can be synth~si7Pi~ by use of a peptide synthPci7Pr. Furthe~more, if desired, 10 nr,nt~l~ccjr.~l amino acids or c~ mi~l amino acid analogs can be introduced as a sllbstit.lti~ n or addition into the V~l Lebla.~ Deltex protein seq ler r~. Non~l~ccir~l amino acids include but are not limited to the D-isomers of ~e eol~ amino acids, ~-arnino iso~uLylic acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine"~-alanine, c7~sign~r arnino acids such as ,~-methyl amino acids, C~-methyl amino acids, and NcY-methyl amino acids.
In a specific embodirnent, the velLebldte Deltex delivdli\,., is a cl~i~clic, orfusion, protein comprising a vertebrate Deltex protein or fragment thereof (p.~ bly conC;cting of at least a domain or motif of the vertebrate Deltex protein, or at least 10 amino 2 0 acids of the vertebrate Deltex protein) joined at its amino or carboxy-terminus via a peptide bond to an amino acid sequence of a dirf~ nt protein. In one embo~limPnt such a chi ll.,lic protein is produced by recombinant expression of a nucleic acid enro ling the protein (co~llplising a vertebrate Deltex-coding sequence joined in-frame to a coding sequence for a 25 different protein). Such a chimeric product can be made by ligating the a~plo~ L~ nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and e~ ,s~h~g the chimeric product by mçth~s commonly known in the art. Alternatively, such a chill~e~ic product may be made by protein synthetic techniques, e.g., by use of a peptide sy"~ ,. A specific embodiment 3 ~ relates to a ChhllliC protein cu~l~li~s~ng a fragment of a vertebrate Deltex protein which coll.~,ises a domain or motif of the ~c.t~,b~e Deltex protein, e.g., 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 embo-limPn~ a chirneric nucleic acid can be c0l3~.L,ucLGd, 35 encoding a fusion protein co~ Lhlg of a vertebrate Deltex Notch-binding fragment joined to a non-Deltex protein. As another example, and not by way of limitation, a lecu-l-bil~lL

W O 97/18822 PCTnUS96/18675 PC~11P can be constructed according to the invention, co~ ..lg coding portions of both a ~ ;bldL~ deltex gene and another gene which is a lll~,~(" of the "Notch group."
Another specific embodirnent relates to a cl~ .ic protein colll~ lg a fragrnent of a ,b.dte Deltex protein of at least six amino acids. Particular examples of the construction and expression of fusion plOL~illS co~ )lisillg human Deltex or various Notch fragmenr~, are described in Section 7.
Other specific embodirnents of derivatives and analogs are described in the s~lhsectirn below and exarnples sections infra.

~;.6.1. DERIV~TIVES OF V~;~ll~;~KATE DELTEX CONTAINING
ONE OR MORE DOMAINS OF 'l'H~: PROTEIN
In a specific embodiment, the invention provides vertebrate Deltex de~ivdLives and analogs, in particular velt.,blal~ Deltex fragrnents and derivatives of such 15 fragments, that comprise or consist of one or more domains of the vertebrate Deltex protein, inrlnriing but not lirnited to a region which binds to a Notch protein (or a m~lPcuie comprising the ANK repeats thereofl, a region which binds to a second Deltex protein or portion thereof, an SH3-binding domain, or a ring-H2-zinc f~ger domain. In specific 2 o embodiments, the vertebrate Deltex derivative may lack all or a portion of one or more of the foregoing domains.
In specific embofiim~ntc directed to the domains of the human Deltex protein, ~e aforesaid domains consist of approximately the following amino-acid sequences (see Section 6.1.1 infra):
SH3 binding rlom~in~ SEQ ID NOS: 17-21 Ring-H2-zinc finger dom~in: SEQ ID NO:25 Other binding fr~gm~nt~, e.g., smaller than those set forth above, can be 30 iflentifi~d by routine methods, e.g., by co~ ,uclion of nucleic acids encoding such fragments and assays for binding (e.g., via the interaction kap methoa described in Section 7 infra.
In a specific embodiment, relating to a vertebrate Deltex protein of a species other than human, fr~gm~ont~ eo~ isillg 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.

WO 97/18822 PCT/US96/1867~;

We have delllo~at~d that Drosophila Deltex binds to human Notch-l and 2, sllg~sting evolutionary cons~ a~ion of bio( h~n~ir~l activity between human and Drosophila Deltex. We have also tlrl,.o~ .altd that human Deltex binds to human Notch-1 and 2, and that human Deltex binds to Drosophila Notch. Using the iut,~clion trap system (described infra) as our assay we ~y~ l;r~lly Çx;~ r~l by deletion analysis, the dom~in.
of Notch and Deltex which are responsible for protein-protein ~l~.acLious. Both Deltex-Deltex as well as Deltex-Notch intPr~etinnc were ~etrcte~ Deletion CO1~LLUCL~ encoding various fr~gm~ntc (described below) of Drosophila Deltex, Drosophila Notch and human 10 Notch were ~ ssed as fusion constructs (LexA or ACT fusions), and assayed.
The seq~ nres of fr~m~ntc A-D (SEQ ID NOS:13-16, ~csL~ecLiv~ly) of Drosophila Deltex which were expressed are shown in Fig. 4A-B.
Figure 5 :~UllUllali~t~S the Deltex-Deltex interactions we have rlPtPctrri 15 Fragment A interacts with Fragment A (homoypic inLc-~ iu~ls). Fragment B iu~la~,~ wi~
Fragment B (homotypic interactions). Fragment C illt~,ld~ with Fragment C (homotypic interactions). In ~d~itinn, we ~1~t~ct~od ulL~l~CLionS between rl;.~ C and B. However, we can only detect the fragment C-B interaction if r~a~lllent C is tested as the "bait" (i.e., as the LexA fusion). If Fragment B is the bait, this interaction is not d~tect~d All the other 20 aforesaid interactions occur u~ e-;~ive of which fragment is used as the bait. Fragment A
consists of arnino acids 1-303. Fl~gn~ll~ B consists of arnino acids 306-486. Fragment C
consists of amino acids 514-737.
--- The heterotypic interaction between Notch and De}tex is occurred between 25 the ANK repeat region of Notch and fragment D of Deltex (which is part of fragment A and includes arnino acids 1-204). Drosophila Notch ANK repeats as well as the ANK repeats of both human Notch proteins (enro~rd by TAN-1 and hN) were tested in this interaction assay and showed positive binding to Ld~,~uenL D. The following rl,.g.,-~-."~ cont~ining the ANK
repeat region were used: Drosophila Notch.amino acids: 1889-2076 (nurnbering per30 Wharton et al., 1985, Cell 43:567-581); Human Notch TAN-l arnino acids: 1826-2146;
Human Notch hN amino acids: 1772-2093. All displayed ullel~cLions with fragment D.
Figure 6 ~iUllUllali~.t~ s(h~m~tir~lly this interaction.
In specific embodiments, verte~rate Deltex regions are provided that are most 35 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.

W O97/18822 PCT~US96/18675 Binding i l~ld-,Lions between fr~m~ntc are in~ tf~ by arrows in Figures 5 and G. Such regions homologous to A-D are predicted also to display the binding interactions shown in Figures 5 and 6. Thus, arnino acids 1-237, 238-391, 392-620, and 1-17~ of SEQ ID NO:12 correspond to Drosophik rla~ A-D, l~ c~,Lively. Molecules CO~ g one or more of the foregoing regions are provided. Accordingly, by way of exarnple, a mnlPcnlr cul~ g amino acid nurnbers 1-237 of SLQ ID NO: 12 is predicted to bind the Notchankyrin repeats.
Also provided are inhibitors (e.g., peptide inhibitors) of the folcgoing protein10 interactions with Notch or with a second Deltex protein.
The ability to bind to a Notch protein or a Deltex protein (or deliv~Liv thereof) can be firmnnctrated by in vitro assays such as the interaction trap lr~ l,n;.l,..o (Section 7, infra).
1~; The nucleic acid se~uenr~o-s encoding Notch or vertebrate Deltex proteins or fragments thereof, for use in such assays, can be isolated from porcine, bovine, equine, feline, car~ine, as well as primate sources and any other m~mm~lc in which homologs of known genes can be i~lentifi~od. For example, the Notch protein or portion thereof CO~Ilpli~iulg the ANK repeats which can be expressed and assayed for binding to Deltex or a 2 0 Deltex derivative can be derived from any of the Notch homologs: human hN, human TAN-1, Xenopus, and Drosophik.
Due to the degenel~ey of nucleotide coding sequences, other DNA seq~lenres which encode substantially the sarne amino acid sequence as the aforesaid domains may be 25 used in the practice of the present invention. These include but are not lirnited to nucleotide seqnenres colll~lismg all or portions of the vertebrate deltex genes which are altered by the ~ub~LiLIlLion of different codons that encode a functionally equivalent arnino acid residue within the sequence, thus producing a silent change. Likewise, the vertebrate Deltex proteins, fr~mrntc or derivatives thereof, of the invention include, but are not limited to, 3 ~ those cnnt~inin~, as a primary arnino acid sequence, all or part of the amino acid sequence of the domains inr!n~iing altered sequ~nres in which functionally equivalent arnino acid residues are substituted for residues within the sequence ("conselvd~ivt~" changes).
The derivatives, analogs, and peptides of the invention can be produced by 3 5 various methods known in the art. The manipulations which result in their production can occur at the gene or protein level.

~ 34 -W 097/18822 PCTrUS96/18675 A(T~lititllT~11y, the nucleic acid se~lut~c can be mutated in vitro or in vivo;
and maniFul~tit)nc of the seqllenre rnay also be made at the protein level.
In ~lrl;tin~, analogs and peptides can be cht~mir~11y syntht~ci7 5.7. IN VI~RO ASSAYS OF V~ ;~ATE DELTEX
PROTEINS, I)ERIVAT~VES AND ANALOGS
The fi1nrtit)n~1 activity of vc~ .aL~ Deltex pluLGills, de-iv~ivts and analogs, can be assayed in vitro by various methods.
For example, in one embo Tim-~nf where one is assaying for the ability to bind or compete with wild-type VG1~b1aL~ Deltex for binding to anti-v.,llG~-~L~ Deltex antibody, various imm~-ntl~cs~ys known in the art can be used, inr1ntlin~ but not limited to competitive and non-c~ .GI;l;vG assay systems using techniques such as radioimml1nn~csays, ELISA (enzyme linked imm1~noSn~I,e~L assay), "sar.~lwicl~ Ino~c!;,.y~,~
15 imrnunor~ liomt~tric assays, gel diffusion ~lGcipiLi,~ reactions, imml1ntmiffusion assays, in situ immtlno~cc-~ys (using colloidal gold, enzyme or radioisotope labels, for example), western blots, p ~~ci~ ion r~,aL;liOnS, ~g~ ion assays (e.g., gel ~gg1~Ttin~tjon assays, hPTn~pg111tin~tion assays), complement fixation assays, immunofluolc~sc~llce assays, protein 20 A assays, and illllll l,,nc1~l.u~horesis assays, etc. In one embodiment, antibody binding is t1etectetl by lett~cting a label on the primary antibody. In another embodiment, the primary antibody is dt~tect~-d by t~iPtt~cting binding of a secondary antibody or reagent to the primary antibody. In a further embo-1im-~nt, the secondary antibody is labelled. Many means are known in the art for r1Pt~cting binding in an immnno~cc~y and are within the scope of the present mventron.
In another embodirnent, where one is assaying for the ability to mediate binding to Notch or portion thereof (e.g., Notch ankyrin repeats) to a second Deltex protein or portion thereof, one can carry out assays such as that described infra in Section 7.
3 o Other mt~thotlc will be known to the skilled artisan and are within the scope of the inv~ ioll.
In another embodirnent, 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 manner, agûnists and antagonists of Deltex can be itltntifit~tl 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 presenc~ of one or more - 3~ -mnlrcllies which are desired to be tested for the ability to inhibit or reduce binding between the Notch protein and the Deltex protein, and idellliryillg the mnl~clll~s 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 m~othn-l inrlll~ling but not limited to yeast interac~ion trap assays, cell culture in vitro aggregation assays, and soluble binding assays using purified Notch and Deltex proteins. A specific embodirnent is as follows: Cultured cells are colldl~recl~d with plasmid expression CO1~ that place Notch and deltex under distinct inducible promoters. Notch ex~ iOn in these cells is first induced to ensure 10 proper cell surface lor~li7~tinn; Deltex expression is then inrinrer~ These cells are then ag~ a~ed with cells e~lessillg Delta, to produce mutual capping of Notch and Delta at the point of mutual contact (see Singer J., 1992, Science, 255:1671-1677; Fehon et al., 1990, Cell, 61:523-534; Heitzler, et al., 1991, Cell, 64:1083-1092). Under these co~ itit)nc~
Deltex colocalizes with the capped Notch by virtue of its binding to Notch. The cells are then inr~lb~trd in the plesel~ce 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 De}tex to Notch will result in an ill~ .edsed loc~li7~tion of Deltex throughout the cell cytoplasm. This increased loç~li7~tinn can be 2 0 ~~etermin~d according to m~thorlc known in the art (e.g., immunofluol~sc~llt staining with antibody to Deltex). The method can also be carried out using d~livaliv~s 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 ~tlminictration of a therapeutic compound of the invention. Such therapeu~ic compounds (termed herein "Theldpt;uLi~s") include: vc.~bldL~ Deltex proteins and analogs and derivatives (inr.!nflin~ fr~m~-nt~) thereof (e.g., as described hereinabove);
30 antibodies thereto (as ~I~5~ C~1 hereinabove); nucleic acids encoding the VG1~IJ1d~t: Deltex proteins, analogs, or derivatives (e.g., as described hereinabove); and vt;lLt;l~ldt~ delte~c ~nti~ence nucleic acids. As stated supra, the Antagonist Thel.~ ics of Ihe invention are those Therapeutics which ~nt~gQni7l?, or inhibit, a vertebrate Deltex function and/or Notch 35 function. Such Antagonist Thel~p~ .l;rs are most preferably i(1r-ntifi~d by use of known convenient in vitro assays, e.g., based on their ability to inhibit binding of vertebrate Deltex W O 97/18~22 PCTAUS96/18675 to another protein (e.g., a Notch protein), or inhibit any known Notch or v~ dL~ Deltex r,."r!ir"~ as preferably assayed in vitro or in cell culture, ~lthol~gh genetic assays (e.g., in Drosophila or mouse) may also be employed. In a p..~r.,.l~d embodiment, t'ne Antagonist Therapeutic is a protein or dclivdLiv~ thereof CO~ ug a fi-nrtion~lly active fid~uell~ such as a fragment of vertebrate Deltex which l..~liz~rs binding to Notch, or an antibody thereto.
In other specific emborlimrnt~ such all AllLdgc,~ L Thf~ ;c is a nucleic acid capable of ...iUg a mr)lloclllr co~ ,i..Llg a fragment of v~lL~l>iaL~ Deltex which binds to Notch, or a ~ dL~ deltex ~nti.c~n.~e nucleic acid (see Section 5. I l herein). It should be noted that 10 preferably, suitable in vitro or in vivo assays, as described infra, should be utiiized to rlel~l",;"r the effect of a specific Therapeutic and whether its ~r~ dLion is in~ir~trA for l.~dl.U~ of the affected tissue, since the develu~ Ldl history of the tissue may clrlr..f.;.~f whether an Antagonist or Agonist Tht;ld~eulic is desired.
In another embodiment of the invention, a nucleic acid containing a portion of a vertebrate de~tex gene is used, as an Antagonist Therapeutic, to promote ve~bld~e del~ex inactivation by homologous recombination (Koller and SmithiPs, 1989, Proc. Natl.
Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
The Agonist Thel~t:uLics of the iulvellliOll, as described supra, promote 20 v~lL~l,ldte Deltex function. Such Agonist Therapeutics include but are not limited to proteins and de.ivaLives con~lisi~lg the portions of Notch that mediate binding to v,_~b Deltex, i.e., the ANK repeats, and nucleic acids encoding the l~o.~:gou.g (which can be d 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 ~ ively inhibit, a desired vertebrate Deltex piup~,Ly, e.g., binding to Notch, binding to an intr~celltTl~r ligand, can be used thc.,,l,eu~ic~lly as i1~.1UCeL;~ or inhibitors, l~s~ lively~ of such propery and its physiological 3 ~ correlates. In a specific ernbodirnent, a peptide (e.g., in the range of 6-50 or 15-25 amino acids; and particularly of about 10, 15, 20 or 25 arnino acids) containing the seqnPnre of a portion of vertebrate Deltex which binds to Notch is used to antagonize Notch function. In a specific embodiment, such an Antagonist Thel~pt;uLic is used to treat or prevent human or 35 other m~lign~nries ~soci~trd with iul~;-eased Notch expression (e.g., cervical cancer, colon cancer, breast cancer, squamous adenocarcinomas (see infra)). Derivatives or analogs of W O 97/18822 PCTnJS96/18675 vertebrate Deltex can be tested for the desired activity by procedures known in the art, inr.ln~iing but not limited to the assays described in the examples infra. For example, molPclllPs co~ .ulg Deltex fragments which bind to Notch ANK repeats ~see Section 7), can be obtained and selected by ~A~lC;,sulg deletion mutants of human Deltex (or of a nucleotide sequence of another species and assaying for bihding of the e~ ssed product to Notch by any of several methods, such as the interaction trap system described in the Examples Sectinn.~ infra. In one specific embo limPnt peptide libraries can be screened to select a peptide with the desired activ}ty; such scre~ g can be carried out by assaying, 10 e.g., for binding to Notch or a molecule c~ the Notch ANK repeats.
The Agonist and Antagonist Thela~,uLics of the invention have the,d~t;u~ic utility for disoldc,s of cell fate. The Agonist Th~la~uLics are ~ cd therapeutically (inr.l~tling prophylactically): (1) in diseases or disorders involving an absence or decreased (relative to normal, or desired) levels of Notch or vel~bldte~ Deltex function. for example, in patients where Notch or vertebrate Deltex protein is lacking, genetically defective, biologically inactive or underactive, or undc.ex~lessed; and (2) in diseases or disorders wherein in vitro (or in vivo) assays (see infra) indicate the utility of vertebrate Deltex agonist ~-lmini~t ation. The absence or decreased levels in Notch or vertebrate Deltex 2 0 function can be readily detectPA e.g., by obL~ g a patient tissue sample (e.g., from biopsy tissue) and assaying it in l~itro for protein levels, ~.Llu-;Lu~c and/or activity of the e,~ essed Notch or Vt~ ld~ Deltex protein. Many mPth~dc standard in the art can be thus - employed, inrln-ling but not limited to immnno ~Ccays to detect andlor visualize Notch or 25 vertebrate Deltex protein (e.g., Western blot, immnn~ ec;ipi~tion followed by sodium dodecyl sulfate pol~,dclylalllide gel electrophoresis, ;,.""",~ncyl~ch.~.";~ , etc.) and/or hybridization assays to detect Notch or v~lL~ ld~ Deltex e~ ession by detectin~ and/or vi~u~li7ing respectively Notch or velL~l.ldt~ deltex mRNA (e.g., Northern assays, dot blots, in sitrl hybridization, etc.) In vitro assays which can be used to flPtpnninp- whether adlllill~sLldLion of a specific Agonist Thc.a~t;ulic or Antagonist The-dptuLic is inrlir~tP-I, include in vitro cell culture assays in which a patient tissue sarnple is grown in culture, and exposed to or ot'nerwise ~ll~llil.;~l. rcd a Thc.d~ ic, and the effect of such Thcldpc;ulic upon the tissue 3 5 sarnple is observed. In one embodiment, where the patient has a m~lign~nry, a sample of cells from such m~lign~nr.y is plated out or grown in culture, and the cells are then exposed W O 97/18822 PCT~US96/18675 to a Therapeutic. A Thc~d~ ic which inhibits survival or growth of the m~lign~nt cells (e.g., by promoting t~rmin~l difr~ rl~ is selected for thP~ 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 ~ 3H-Lllyll~idille incorporation, by direct cell count, by clPtPcting changes in LldllscliyLional activity of known genes such as proto-oncogenes (e.g., fos, m~c) or cell cycle lll~h~; cell viability can be ~ssessed by trypan blue staining, di~re,~llLiation can be ~essed visually based on changes in morphology, etc.
In a specific aspect, the m~lign~nt cell cultures are separately exposed to (1) an Agonist 10 The,d~euLic, and (2) an Antagonist Therapeutic; t'ne result of the assay can indicate which type of Therapeutic has therapeutic efficacy.
In another emboriimPnt, a The,apeuLic is infiir~ted for l~ie which exhibits the desired effect, inhibition or promotion of cell growth, upon a patient cell sarnple from tissue having or suspected of having a hyper- or hy~roliferative disorder. l~s~ecLively. Such hyper-or 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 Thel~e~lLic is in-lir~trd for use in treating nerve injury or a nervous system degellel~Live disorder (see Section 5.8.2~ which exhibits in 20 vitro promotion of nerve legenel~Lion/neurite ext~n~jon from nerve cells of the affected patient type.
In addition, adlllilli~L~ILion of an Antagonist ThcLdy~u~ic of the invention is ~ also in~lir~t~d in diseases or disorders determined or known to involve a Notch or Deltex 25 dr~min~nt activated phenotype ("gain of function" mutations.) ~1mini~fration of an Agonist Therapeutic is inrlic~ted in ~ e~c~c or disorders d~L~ cd or known to involve a Notch or Deltex dominant negative pheno~pe ("loss of function" mnt~tiorl~) The fi-nrtion~ of ' various structural domains of the Notch protein have been illv~Ligated in vivo, by ectopically ~Ayl~Ssillg a series of Drosophila Notch deletion mutants under the hsp70 heat-3 ~ shock promoter, as well as eye-specific promoters (see Rebay et al., 1993, Cell 74:319-329). Two classes of d-lmin~nt phenotypes were observed, one suggestive of Notch loss-of function mutations and the other of Notch gain-of-function mnt~tic)n~. Dominant "activated" phenotypes resulted from overexpression of a protein lacking most extr~re~ r 3~; sequen~-es, while dominant "negative" phenotypes resulted from overeA~ ioll of a protein lacking most intr~celii-l~r sequences. The results in~lirate~l that Notch functions as a W O 97/18822 PCT~US96/18675 iece~,Lol whose extrpr~ r domain l..rli~ s ligand-binding, resulting in the I~A~ i;nn of devel~.llc.lL~l signals by the cytoplasmic domain. The phenotypes observed also sl~g~s that the ANK repeat region within the intraCç~ r domain plays an escentiAl role in Notch IllF~l;Al d signal LIA~ ion events (intrPr~ lAr Çu~ l). We have shown that Drosophila Deltex binds to the Notch ANK repeat region.
In various specific embodimPnfs~ in vitro assays can be carried out with l~,prcs.,llLaLiv~ cells of cell types involved in a patient's disorder, to ~ll t~ . ",;,~ if a Thc.dlJt;ulic has a desired effect upon such cell types.
In another embodiment, cells of a patient tissue sample ~ d of being pre-neoplastic are similarly plated out or grown in vitro, and exposed to a ThclalJ~uLic. The l'llc.d~ Lic which results irl a cell ~hencly~e that is more normal (i.e., less r~lcsellLaliv~
of a pre-neoplastic state, neoplastic state, mAIign~nt state, or llal~fulllled ph~uo~y~e) is selected for Illel~eulic use. Many assays ~L~ndard in the art can be used to assess whether a pre-neoplastic state, neoplastic state, or a Ll~Ç~,lllled or m~lignA~t phenotype, is present.
For ~xAmrlto. cllAId.;L~ ics ACSoci~t~d with a L-~rolllled phenotype (a set of in vitro chara~L~ L~ associated with a tumorigenic ability in vivo) include a more rounded cell morphology, looser s ~h~llA~ ll att~rl-",.~"l loss of contact inhibition, loss of anchorage 20 depen-len~e, release of proL!Gases such as plasminogen activator, .ncl~_dsed sugar lld~Oll, dccll,ased serum ic.lui~ , expression of fetal :mt;genc, disappearance of the 250,000 dalton surface protein, etc. (see Luria et al., 1978, General Virology, 3d Ed., ~ohn Wiley &
Sons, New York pp. 436~L6).
2 5 In other specific embotlim~ntc, 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 chaldcL~ Lic(s) associated with the mA~ignAnt neoplastic or pre-neoplastic disorder desired to be treated or p~e~e.lLc:d, 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 andlor Deltex function that can be used as an Antagonist The.d~eulic is a molecule co~ isi.lg a Deltex protein or portion thereof that m~ tes binding to Notch, covalently bound to a protease or 3 5 proteolytically active fragment thereof. Such protease preferably is able to cleave a Notch protein. The molecule is preferably a fusion protein (i.e., the covalent bond is a peptide - 4û -W ~ 97/~8822 PCT~US96/18G75 bond). The Deltex protein is preferably a vertebrate protein, most preferably human.
Accordingly, the invention provides a method of targeting or ~laclivdlillg proteins to which Deltex binds (e.g., Notch) in a cell. According to this m~thr~d, the molecule co~ risillg the Deltex protein or portion thereof and the pro~ase se~uenres is produced through rhPmi or via molecular biological techniques. This molecule (e.g., fusion protein) is introduced into the cell by techniques known in the art (e.g., Lld~,f~,,Lion of the cell with a nucleic acid encoding the mol~cnl~ such that its e~l~ssion occurs intr~r~ rly). Inside the cell, the molecule can bind to Notch and/or other Deltex binding pd~ ,L;7. Upon such binding, the 10 protease portion of the molecule cleaves the proteirl 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 ~i~n~ling pathway. Molecules which would illacLivdte protein function e.g., by binding thereto, can be used as an altellldLive to proteases.
The Antagonist Theld~,ru~ics are a~l".i"i~~ltd the,i~r~ ly (inr~ in~
prophylactically): (l) in diseases or disorders involving increased ~relative to norrnal, or desired) levels of Notch or v~lL~bl~ Deltex function, for example, where the Notch or vertebrate Deltex protein is uve~ ssed or ove,a~ e; and (2) in diseases or disorders 2 0 wherein in vitro (or in vivo) assays indicate the utility of vertebrate Deltex antagonist ~lminic~ration. The increased levels of Notch or vertebrate Deltex ~lnrtion can be readily dç~ctPd by methods such as those described above, by quallliryil~g protein and/or RNA. ~n - vitro assays with cells of patient tissue sample or the ap~,ul"idte cell line or cell type, to 25 determine therapeutic utility, can be carried out as described above.

5.8.1. MALIGNANCIES
~ vr~ n~nt and pre-neoplastic cnn-iition~ which can be tested as described supra for eff~cacy of intervention with Antagonist or Agonist Therapeutics, and which can 30 be treated upon thus observing an inriir:~tirn of therapeutic utility, include but are not limited to those described below in Sections 5.8. l and 5.9. l.
I~lign~nries and related disorders, cells of which type can be tested in vitro (and/or i~z vivo), and upon observing the al~lol,liate assay result, treated according to the 35 present invention, include but are not limited to those listed in Table l (for a review of such disorders, see Fishrnan et al., 1985, Medicine, 2d Ed., J.B. LilJ~hlcolL Co., Philadelphia):

WO 97/18822 PCT~US96/18675 MALIGNANCIES AND RELATED DISORDERS
T Pnk~m;~
aeute l~llkPmi~
aeute Iymphoeytie l~l-k~mi~
aeute myeloeytie l~nk~mi~ -myeloblastie promyeloeytie myelomonoeytie 1 o monoeytie erythrolP-lk~mi~
ehronie lellk~mi~
ehronie myeloeytie (granuloeytie~ lellkt~mi~
ehronie lymphoeytie l~uk~mi~
Polyeythemia vera Lymphoma Hodgkin's disease non-Hodgkin's disease Multiple myeloma Waldenstrom's maeroglobulinemia Heavy ehain disease Solid tumors 2 ~ sareomas and eareinomas rbrosal collla myxosal co.lla liposarcoma chondrosaicollla - osteogenie sareoma chordoma 2 5 ~ngioc~rcoma endotheliosarcoma lymphangiosal co.lla lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor leiomyosa~ 1onla rhabdomyosarcoma colon carcinoma pancreatic eaneer breast eaneer ovarian eaneer 3 5 prostate caneer squamous eell eareinoma basal eell eal.;hlo~lla -CA 02238404 l998-05-22 W~ g7/18822 PCT~US96/18675 a~n~
sweat gland c~.,i.~ollla seh~r~ouc gland cdleillul~la papillary (.dl'UinCl~ld papillary a~ nç~ ~ ~;illulllds eyst~1el-nc~ici,lo,~a mPd~ ry calcil~ la bronchogenic carcinoma renal eell careinoma h- ~A~
bile duct carcinoma chorioc~ wllla 1 0 s~
~,llI~lyollal carcinoma Wilms' tumor cervical cancer tostirnl~r tumor lung cal~ lul~la small cell lung cdl-;illolna bladder carcinoma epithelial c~u-;illoll.a glioma astrocytoma medulloblastoma craniopharyngioma 2 û ~ellllylllullla pinr~ m~
hrm~n~ioblastoma acoustic n~.l.ullla oligodendroglioma ,, I I IP~ iCII I I~
mrl~n~m~
2 5 neuroblastoma retinoblastoma In specific embor-;m-ont~, m~lign~nry or dysprolire-dlive changes (such as metaplasias and dysplasias) are treated or pl~vellL~d in epithelial tissues such as those in the cervix, esophagus, and lung.
M~ ies of the colon and cervix can exhibit increased e~ ssion of human Notch relative to such non-m~lign~nt tissue (see PCT Publication WO 94/07474 published April 14, 1994, incorporated by l~relellce herein in its entirety). Thus, in specific 35 embodiments, m~ "~ ;rs of the colon or cervix are treated or p~,v~llL~d by admh~ eli,lg an errt:clive arnount of an Antagonist Therapeutic of the hlv~llLioll. The presence of W O97/18822 PCT~US96/1867S

increased Notch eA~ ,S~ in colon, and cervical cancer s~l~gestc that many more cance~u~
and hy~cl~roli~dLive con~iitinn.~ exhibit upregulated Notch. 'rhus, in specific embodimP!ntc.
various cancers, e.g., breast cancer, s~ u~ ~nnr~. ~iuOl, a, seminom~ mPI~nr~m~, and lung cancer, as well as other hyperproli~ldlive disorders, can be treated or prevented by dLion of an Antagonist Thcld~uLic.

5.8.2. NERVOUS ~;Y~ ;~ DISORDERS
Nervous system disorders, involving cell types which can be tested as 10 described supra for efficacy of mL~l venLion with Antagonist or Agonist Therapeutics, and which can be treated upon thus observing an inr1ir~tion of thela~ Lic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a discolmecLion of axons, a ~ ;nn or dcge~ldLion of neurons, or demyelin~tion.
15 Nervous 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:
(i) ILdulllaLic lesions, inrl-lriin~ lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or conl~.. ,ssion injulies, ~ii) ic-.l., ic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, inr~ in~ cerebral ~ infarction or icch~mi~ or spinal cord infarction or ;.~ "~i~
2 5 (iii) m~ n~nt lesions, in which a portion of the nervous system is destroyed or injured by m~ n~nt tissue which is either a nervous system associated m~ n~nry or a m~ n~nry derived from non-nervous system tissue;
(iv) infectious lesions, in which a portion of the nervous system is 3 ~ destroyed or injured as a result of infectirJ~, for example, by an abscess or associated with infection by human i~ r,defiriency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;
3 5 (v) dc~ene.dLive lesions, in which a portion of the nervous system is destroyed or injured as a result of a dege.leldLiv~ process including W O g7~18822 PCT~US96/I8675 but not lirmited to degen~atirtn ~sociAI~d with p,..k;...cOll~s disease, 's disease, ~llntin~t~n s chorea, or ~uyollo;~llic lateral scl~losis;
(vi) lesions associated with lluL,ili~)na} 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, vitarnin B12 derleie.l~;y, folic acid derlcL,.~.,y, Wernicke disease, tobacco-alcohol amblyopia, Ma~ idr~v~-Bignami disease (primary de~t;ne.~ioll of the corpus r~llosl-m), and alcoholic cerebellar degell~,.dLion;
(vii) neurological lesions associated with ~y~,.llic diseases inr.~ ing but not limited to diabetes (diabetic n~u,op~ y, Bell's palsy), ~y~uuc lupus eryth~ , ca.. ,,llollla, or sarcoidosis;
(viii) lesions caused by toxic sT-bs~;.llr~s inrlllrling 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 2 0 limited to multiple sclerosis, human irnmunorlef;r,ienry virus-associated myelopathy, lld~v~e myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine ~ myelinolysis.
2 5 Therapeutics which are useful according to the inveMion for treatrnent of a nervous system disorder may be selected by testing for biological activity in promoting the survival or dirrerel,l,ation of neurons (see also Section 5.8). :For example, and not by way of limit~ti~n, Therapeutics which elicit any of the following effects may be useful according to ~e invention:
(i) increased survival time of neurons in culture;
(ii) increased ~ u~ing of neurons in culture or in vivo;
(iii) increased production of a neuron-associated molecule in culture or in YiVo, e.g., choline acetyll,~rel~se or acetylcholinrsterase with 3 5 respect to motor neurons; or (iv) decreased ~ylll~tollls of neuron dysfunction in vivo.

W O 97/18822 PCT~US96/18675 Such effects may be measured by any method known in the art. In pler~-lcd, non-limiting embodiments, increased survival of neurons may be lllea~uled by the method set forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sL~ ululg of neurons may be detected by m~fh~ rlc set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) or 13rown et al. (1981, Ann. Rev. Neurosci. 4:17~L2); i,lei~,a5ed production of neuron-associated mnll~cllles may be measured by bioassay, e~yllldlic assay, antibody binding, Northern blot assay, etc., depending on the mr~eclll~ to be ,lleasuled; and motor neuron dysfunction may be measured by ~sse~ g the physical ll-anirc;~L~Lion of motor neuron disorder, e.g., 10 w~ak~less, motor neuron ctmtlucti--n ve}ocity, or functional disability.
In specific embodirnents, motor neuron disorders that may be treated accol.ling to the illvellLioll include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degeneldLive disease or m~lign~nry that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyoL~uphic lateral sclerosis, and including but not lirnited to progressive spinal mllcclll:lr atrophy, progressive bulbar palsy. primary lateral sclerosis, infantile and juvenile mllcclll~r atrophy, progressive bulbar paralysis of childhood (Fazio-Londe ~ylldlollle), poliomyelitis and the post polio syndrome, and 20 Hereditary Motorsensory Neulo~dLlly (Charcot-Marie-Tooth Disease).

5.8.3. TISSUE REPAIR AND REGENERATION
- In another embodiment of the invention, a Therapeutic of the invention is 25 used for promotion of tissue regeneration and repair, including but not limited to treatment of benign dysproliferative disorders. Specific emborlim~ntc are directed to treatment of cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes), Llea~lllel1l of keloid (hypertrophic scar) formation (disfiguring of the skin in which the scarring process i~lLelrt;les with normal renewal), psoriasis (a common skin 3 ~ condition characterized by excessive proliferation of the skin and delay in proper cell fate determination), and baldness ~a condiLiol1 in which terminally dirr.,lel1liated hair follicles (a tissue rich in Notch) fail to function properly).
Deltex agonists and antagonists can also be used to manipulate the 3 5 differentiation state of non-terminally dirrel~llLidL~d (e.g., stem and progenitor) cells. ~or example, a stem cell can be exposed to such an agonist to inhibit its dirrelellLiation and W O 97J18822 PCT~US96/18675 achieve cxl~An~iQn of the stem cell population by in-~nhAfi~n in vitro under growth COll~ C. Such stem cells have use in l,~ .lA.,IA~inn for in vivo repopulation of their plogcl~y cells and tissue ~gc.lel~lion. (For m~tl o~lC that can be used in the foregoing, see United States patent application Serial No. 08/537,210 filed Sept~mher 29, 1995 by Artavanis-Tsakonas et al., entitled "Manipulation of Non-Terminally DirrclelLa~d Cells Using the Notch Pdlhway," which is iulccJ~l~ula~cd by lcrclcllce herein in its entirety.) For example, a method for the ~ A..~ n of a ~ Cul .or cell (e.g., a hurnan stem or plogc~ u cell) col~ ises colll~(;lillg the cell with an arnount of a vertebrate (e.g., hurnan) Deltex 10 protein or functionally active portion thereof crrc~,livc to inhibit dir~,~ A~i~n of the cell, and exposing the cell to cell growth c~ n-lition.~ such that the cell proliferates. In various embodiments, the ~ ,ul~or cell can be but is not limited to a hematopoietic p,~ ~,ul~or cell, epithelial precursor cell, Icidney ~ euis~l cell, neural p.e~;ul~or cell, skin precursor cell, 15 osetoblast precursor cel}, chondrocyte precursor cell, liver precursor cell, and muscle cell.

5.9. PROPHYLACTIC USES
5.9.1. MALIGNANCIES
The Thcld~cu~ics of the invention can be administered to prevent progression 2 0 to a neoplastic or mAlignAnt state, inrlllriing but not limited to those disorders listed in Table 1. Such ~"."i~;l.dtion is in~irAt.orl where the Theldy~.lLic is shown in assays, as described supr~, to have utility for Ll~;dllncllt or l~.cve.lLion of such disorder. Such prophylactic use is - infli~ted in conditions known or ~u~yeclcd of l.lccedillg progression to neoplasia or cancer, 25 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 Patho~og;y, 2d Ed., W.B. Saunders Co., PhiiA~elrhi~, pp. 68-79.) Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without si~nifirAnt alteration in structure or function. As 3 ~ but one example, ~n~ m~?trial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully di~lclllid~cd cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epirh~ m.
35 Dysplasia is frequently a ~clu~lner of cancer, and is found mainly in ~he epithelia; it is the most disorderly forrn of non-neoplastic cell growth, involving a loss in individual cell W O 97/18822 PCT~US96/18675 uniru.l"iLy and in the al~ r~ dl oripnt~tinn of cells. Dysplastic cells often have abnorrnally large, deeply stained nuelei, and exhibit pleomo.~his.l,. Dysplasia ehalacL~,iisLically occurs where there exists chronic illil~LiOll or ;I-n~ nll, and is often found in the cervix, lc~ Loly passages, oral cavity, and gall bladder.
AlLt.l,ativ~ly or in addition to the p.~,sellce of ~)nnrm~l cell growth characterized as hyperplasia, metaplasia, or dysplasia, the prcsellce of one or more chdldclG.i~l;cs of a ~an~Çu,-~ed phenotype, or of a m~lign~nt phenotype, displayed in vivo or displayed in vitro by a eell sarnple from a patient, ean indieate the desirability of lû prophylactic/Lllt;ld~!GuLic a~i.";,.;~l.dLion of a Th~ culic of the invention. As m~rltinnP~
srlpra, such clla~cL~Iisties of a ~a~r~,..-.ed phenotype inelude morphology ehanges, looser Sub~Ll~Lul~ , loss of eontact inhibition, loss of anchorage depen-l.onre, protease release, increased sugar L~ oll, decreased serum re4uuG~ t, expression of fetal antigens, disappearance of the 25Q,ûOQ dalton cell surface protein, etc. (see also id., at pp.
84-90 for characteristics associated with a Ll~L~----ed or m~iign~n~ phenotype).In a specific embodiment, leukoplakia, a benign-~l~aliulg hyperplastic or dysplastic lesion of the epithPlinm, or Bowen's disease, a eareinoma in situ, are pre-neoplastic lesions indicative of the desirability of prophylactic intervention.
In another emborlim~nt. r,bro~;y~lic disease (eystic hyperplasia""~"".,~,y dysplasia, particularly ~t?Pnncic (benign epithelial hyperplasia)) is indicative of the desirability of prophylactic intervention.
In other embodiments, a patient which exhibits one or more of the following 25 predisposing factors for m~lign~nfy is treated by a-l..,;";cl,d~ion of an effective amount of a Therapeutic: a chromosomal translocation ~CSoc;~t~d with a m~lign~nry (e.g., thePhiladelphia chromosome for chronic myelogenous l~lkPmi~, t(l4;18) for follicular Iymphoma, etc.), familial polyposis or Gardner's ~yll;hulllc (possible folG~ullnel~ of colon cancer), benign monoclonal gammopathy (a possible role.u,l"e, of multiple myeloma), and a 3 û first degree kinship with persons having a cancer or }"eca,lce,..us disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the eolon, Gardner's syndrome, hereditary exostosis, polyendocrine adennm~tncic, mP(~ ry thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers ~yll;llollle, 3 j neurofibromatosis of Von Rerklingh~llcen~ retinoblastoma, carotid body tumor, cutaneous mPl~noc~reinoma, intraocular melanocarcinoma, xeroderma pigmentûsum~ ataxia WO 97~18822 PC'r~US96/18675 trl~ ci~, Chf~ k-Higashi :,yll~Lollle, albinism, Faneoni's aplastie anemia, and Bloom's ~ylldlulllc; see Robbins and Angell, 1976, Basic Pa~hology, 2d Ed., W.B. Saunders Co., phil~ phi~ pp. 112-113) etc.) In another speeific embo limPnt an Antagonist Thr-ld~ lir of the invention is a~ ,;,,ic~ d to a human patient to prevent ~lu~ ,s~ioll to brea t, colon, or eervical caneer.

5.9.2. OTHER DISORDERS
In other embo~ ;, a The.d~ ic of the invention ean be ~ ed to 10 prevent a nervous system disorder described in Section 5.8.2, or other disorder (e.g., liver eirrhncic, psoriasis, keloids, b~ nf~cs) deseribed in Section 5.8.3.

.10. DEMONSl~TION OF T~RAPEUTIC
OR PROPHYLACTIC UTILITY
The Therapeutics of the invention can be tested in vivo for the desired t~.C~d~u~iC or prophylactie aetivity. For example, such colll~uullds ean be tested in suitable animal model systems prior to testing in humans, inrll1(iin~ but not limited to rats, mice, ~hi(~l~Pn, cows, monkeys, rabbits, ete. For in vivo testing, prior to ~ .dlion to 2 o hl~m~nc, any animal model system known in the art may be used.

5.11. ANTISENSE REGULATION OF
VERTEBRATE DELTEX EXPRESSION
The present invention provides the theLdp~ ic or prophylactic use of nucleic 25 acids of at least six nucleotides that are ~ntic~nce to a gene or cDNA encoding vertebrate Deltex or a portion thereof. ".Anti.c~n~e" 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 complem~n~rity. Such ~ e~lce nucleic acids have utility as Antagonist 30 The~al,eulics of ~e iulv~lllioll, and can be used in the LledllllGllL or prevention of disorders as described s~pra in Seetion 5.8 and its s--bsec~ic)nc.
The ~ ce nucleic acids of the invention can be oligonucleotides that are double-stranded or single-stranded, RNA or DNA or a modification or deriva~ive thereof, which can be directly ~ c:d to a cell, or which can be produced intracellularly by 35 I.~ns~ Lion of exogenous, introduced se4uences.

W O 97/18822 PCT~US96/18675 .

In a specific embodiment, the vertebrate deltex ~ ei~e nucleic acids provided by the instant i~lvelllioll can be used for the treatment of tumors or other disorders, the cells of which tumor type or disorder can be delllo~ dtcd (in vitro or in vivo) to express a v~blate deltex gene or a Notch gene. Such ~l~rnnn~tration can be by detection of RNA or of protein.
The invention further provides phd~ ;e~l compositions CU~ lg an err~cLivc amount of the vclLcb~aLe deltex ~..I;cc.~e nucleic acids of the invention in a rh~rm~reutir~11y acceptable carrier, as described infra in Section 5.12. Methods for lû treatment and ~,cven~ion of disorders (such as those described in Sections 5.8 and 5.9) co~ isillg a~l...;,.;~. Li~lg the ph;.. ~ r~ l compositions of the invention are also provided.
In another emboflim~nt, the invention is directed to m~tho~lc for inhibiting the15 expression of a V-,l Lcl,~ate deltex nuclelc acid seqllenre in a prokaryotic or eukaryotic cell colll~lisillg providing the cell with an effective amount of a composition Colll~lisillg an ~nti~n~e vertebrate deltex nucleic acid of the i~ cllLion.
Vertebrate deltex ~nti~?nce nucleic acids and their uses are described in detail below.

5.11.1. VERTEBRATE DELTEX ANTISENSE NUCLEIC ACIDS
The vertebrate deltex ~ e~ce nucleic acids are of at least six nucleotides and are preferably oligonucleotides (ranging from 6 to about 50 oligonucleotides). In 25 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 chilllc.ic mixtures or derivatives or modified versions thereof, single-stranded or double-str~n-l~d The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate baclcbone. The oligonucleotide may include other appending groups such as 30 peptides, or agents f~rilit~ting LI~ OlL across the cell membrane (see, e.g., 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, e.g., PCT Publication No. WO 89/10134, published April 25, 35 1988), hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, BioTechniques

6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549).

O 97/18822 PCT~US96/I~675 In a preferred aspeet of the inve,ltion, a ~,~.Lcbldtc deltex ~ ;Cf n.ce oliglJ~ r~Lide is provided, ~l~rc.a'vly of single-stranded DNA. In a most ~lcrcll~d aspeet, sueh an oligomlr!eoti-lP Co~ iScs a se-lu~nee ~ e to the sequPnreenroriing an SH3-binding domain or a Noteh-binding domai~l of v~,~Lcl)ldLc deltex or zine finger do nain, most plcfcldbly7 of human deltex. The oligonueleotide may be mnfiifiPfl at any position on its ,Llu~;Lule with sl-h"i~ generally known in the art.
The vellcb~atc deltex ~ rlc~ oligollllrl~ol;~f~ m-ay eomprise at least one mf~ifi-od base ~moiety whieh is seleeted from the group inr~ ing but not limited to S-nuuloL~ldeil, 5-brnmmlr~oil~ S-ehlorouraeil, S-iodouraeil, llylJux~ T~ P~ xantine, 4-aeetyl~;ylu~ule, 5-(earboi~yllydlo~yl u~;hyl) uraeil, 5-earbo~y~ nllyl~...;.~o~. ;hyl-2-lhiuulidiue, S-c~llo~yl~ thyl~minlllll~ylulacil7 dillydluul.acil, beta-D-galactosy~ Po~inP7 inosine, N6-isope~llt;llyl~rlenin~ ly1~ Ih~P, I-methylinosine, 2,2-dimethyl~l~ninP, 2-methyl~-le~linP, 2-methylguanine, 3-methyleytosine, 5-llltLllyl-;y~usine, N6-ade une,

7-methyi~-~ninP, S-methyl;..";"~"". Illyluracil, 5-methoxy~minnm~ofhyl-2-thiouraeil, beta-D-maunosylqueosin~, S'-metnoxyearboxymclllylulaeil, S-metho~yulacil, 2-1llt~hyllllio-N6-isopentenyl~lPnin~, uraeil-5-o~.yde~:lic aeid (v), wyb..l~lxocin~ psell-lollraeil, queosine, 2-thioeytosine, 5-methyl-2-thiouracil, 2-thiouraeil, 4-1lliUUlaCil, 5-n~lllyluiacil, uraeil-S-oxyaeetic aeid methylester, uracil-5-oxyacelic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-earbo~y~lo~yl) uraeil, (aep3)w, and 2,6-diaminopurine.
In another embo-limPnt, the oligolluclf,otide colll~lises at least one modifi~d sugar moiety se!~et.-d from the group inrlll-ling but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the oiigonll~l.ootiriecolll~lis~s at least one mo-lifiPd phc.s,ph~tf~ backbone selected from the group eo~ Lulg of a phosphorothioate, a ~hosFh~ro~lithioato, a phnsph~ramidothioate~ a phosphol~"~ 7 a phosphortli~m~ t~ a methyl~hosphorl~t.q, an allyl phosphuL~i.,s~el, and a rolll.ac~,L~l or analog thereof.
In yet another embo~1im~-nt the oligonucleotide is an cY-anomeric oligonucleotide. An ~x-a~lomeric oligonucleotide forms specific double-stranded hybrids with complem~ont~ry RNA i n which, CollLIdlr to the usual ,~-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641).

W O97/18822 PC~nJS96/18675 The oligonucleotide may be conj~ ~d to another molecllle, e.g., a peptide, hybri-1i7~tion Lliggel~d cross-linking agent, transport agent, h~ L;fT~ inn-triggered cleavage agent, etc.
Oligonucleotides of the invention may be synthPsi7Pd by standard m~th~l~
known in the art, e.g., by use of an ~uLc~lllaL~d DNA synth~i7~r (such as are co~ elcially available from Biosearch, Applied Biosystems, etc.). As examples, ~hosphorothioate oligonucleotides may be synth~s;~PA by the method of Stein et al. (1988, Nucl. Acids Res.
16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore 10 glass polymer supports (Sarin et al., 1988. Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
In a specific embo-1im~nt the vertebrate deltex ~nti~n~e oligonucleotide comprises catalytic ~NA, or a ribozyme (see, e.g., PCT International Publication15 WO 90/11364, published October 4, 1990: Sarver et al., 1990, Science 247:1222-1225). In another embodimene, the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chirneric RNA-DNA analogue (Inoue et al., 1987, FEBS ~ett. 215:327-330).
In an alternative embodiment, the vertebrate deltex ~nticçn~e nucleic acid of 2 0 the invention is produced intr~re~ rly 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 ~nti~en~e nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the vertebrare deltex 25 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 se~n~nre encoding the vertebrate deltex 3 ~ antisense RNA can be by any promoter known in the art to act in vertebrate, preferably human, cells. Such plo~ L~ls can be inducible or co~ iLuLi~/e. 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 35 (Yamarnoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner WO 97/18822 PCTnJS96~18675 et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory seqllenr~s of the metallo~ionein gene (Brinster et al., 1982, Nature 296:39~2), etc.
The ~nti~en~e nucleic acids of the inv~ co~ ise a sequence compk-..~ ,y to at least a portion of an RNA l~d~C~ t of a ve.L~bldte deltex gene, preferably a human deltex gene. However, ~hs~ te comple.~r,.l;.,iLy, ~lth~ h preferred, is not required. A seql-enre "C~ 3~ nt~ry to at least a portion of an RNA," as leÇ~ d to herein, means a sequenre having ~",rr~ compl~"~"l~,iLy to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded V~;lL~;bLdL~ deltex ~tiCr=.~ce 10 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 bo~ the degree of compi~.,.r"li..iLy and the length of the ~ntiC~nce nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base micm~trh~s with a ~ k bldte deltex RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of Il~ l h by use of standard procedures to deterrnine the melting point of the hybridized complex.

5.11.2. T~FRAPEUTIC UTILITY OF VERTE~BRAT~

The ~,el~bldL~ deltex ~ ;ce.~ce nucleic acids can be used to treat (or prevent) m~ n~n~ ies or other disorders, of a cell type which has been shown to express vertebrate deltex or Notch. In specific embodiments, the m~ y is cervical, breast, or coloncancer, or squamous adenocal~hlollla. ~ n~nt, neoplastic, and pre-neoplaslic 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 ~nrice,lce vertebrate deltex oligonucleotide is used.
~ n~nt (particularly, tumor) cell types which express vertebrate deltex or 3 o Notch RNA can be if l~ntifi~d by various m~thollc known in the art. Such methods include but are not limited to hybridization with a vG.l~lJldt~ dellex or Notch-specific nucleic acid (e.g., by Northern hybridization, dot blot hybri-li7~tinn, in situ hybridization), observing the abiliy of RNA from the cell ype to be translated in vitro into Notch or vertebrate Deltex, imml~nn~cs~y, etc. In a preferred aspect, primary tumor tissue from a patient can be assayed for Notch or vertebrate Deltex expression prior to L~ e.g., by immunocytochemistry or in situ hybri~li7~tion W O 97/18822 PCT~US96/18675 ph:lrrn~relltir~1 colllL\o~iLiolls of the invention (see Section 5.12), CO~ illgan effective amount of a Y~ bldL~ deltex ~ e nucleic acid in a ~ cu~i~ ,.liyacceptable carrier, can be ~ ,d to a patient having a m~lign~nry which is of a ~ype that expresses Notch or vel le~l at~ deltex RNA or protein.
The arnount of ve}tebrate deltex ~ ce nucleic acid which will be ~r~CLi in the Lleal~ of a particular disorder or collriitit~n will depend on the nature of the disorder or condition, and can be ~elr.~lll;ll~cl by ~l~udard clinical lecl~.lues. Where possible, it is desirable to delf~...inP- the ~ ;.cc-l~e ~;yLOluxiciLy of the tumor type to be 10 treated in vitro, and then in useful animal model systems prior to testing and use in h~lm~n.c In a speci~lc embodirnent, pharrn~- e~-tic~l culll~osiliolls Cull~ vertebrate deltex ~nti~çnce nucleic acids are a~ d via liposomes, Illiclu~d-Licles, ormicror~rs~ os. In various ell.bodilllents of the i.l~e.lLion, it may be useful to use such 15 compositions to achieve sllst~in~d release of the vertebrate deltex ~ P ..~e nucleic acids. In a specific embodiment, it may be desirable to utilize liposûmes targeted via antibodies to specific identifiable tumor antigens (T PonPtti et al., 1990, Proc. Natl. Acad. Sci. U.S.A.
87:2448-2451; Re.llleisen et al., 1990, J. Biol. Chem. 265:16337-16342).

2 0 5.12. T~3:RAPEUTIC/PROPHYLACTIC
ADMINISTRATION AND COMPOSITIONS
The invention provides methods of treatment (and prophylaxis) by ~rlmintctration to a subject of an effective amount of a Theldpculic of the il~vt;lllioll. In a preferred aspect, the Therapeutic is substantially purified. The subject is preferably an animal, including but not lirnited to animals such as cows, pigs, chickens, etc., and is preferably a m~rnm~l, and most preferably human.
Various delivery systems are known and can be used to ~ , . a Therapeutic of the invention, e.g., encapsulation in liposomes, lliic~ Licies, 30 microcapsules, expression by recombin~nt cells, ~ce~ m~di~-od endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:442~4432), consLIuL;Lion of a The.~cuti-: nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intr~mllscnl~r, illLlapeli~oneal~ intravenous, sub~;u~neous, intranasal, epidural, and oral routes. The compounds may be allll,,,,ict..,cd by any convenient route, for example by infusion or b~lus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral ml-c-~s~ rectal and intestinal mucosa, etc.) and may be W O97/18822 PCT~US96/18675 ahl,ini~,L.ed together with other bi~lo~ir~lly active agents. A.l...;,.;~.aLion can be sy~
or local. In ~rlrlition~ it may be desirable to introduce the r~ r~l;r~l compositions of the invention into the central nervous system by any suitable route, inri~-lin~ iu~ v~ ;ular and i~-l,dll-ecal illje.,~ioll; intraventricular i..je~lin~ may be f~oilit~tpd by an i~L,d~ ;ular catheter, for example, ~tt~rhPd to a ieS~VOil. such as an Ommaya lese,vou. Pul~on~y administration can also be employed, e.g., by use of an inhaler or nebulizer, and f~lrrn~ ti~,n with an aerosolizing agent.
In a specific ellll)or~ t it may be desirable to a~l.llilli~r the 10 rh,,~ e~ l colll~o~ ions of the invention locally to the area in need of tre~tTn~nt- this may be achieved by, for example, and not by way of limit~tion~ local infusion during surgery, topical application, e.g., in CO~ n with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said irnplant being of a porous, non-porous, or g~l~tin~u~ material, including Illtn~ e:s.
such as sialastic membranes, or fibers. In one embodiment, ~ dtion can be by direct injection at d~e site (or former site) of a m~ n~nt tumor or neoplastic or pre-neoplastic tissue.
In another embodiment, the Therapeutic can be delivered in a vesicle, in 20 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 (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.) In yet another embo-limPnt the Therapeutic can be delivered in a controlled 25 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 Applir~til ng of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida ~1974); Controlled Drug Bioavailability, Drug Product Design and 3 ~ Ptlrur,llance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.
Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see a~'so Levy et al., Science228:190 (1985); During et al., Arm. Neurol. 25:351 (1989); Howard et al., J. Nt:ulo~ g.
71:105 (1989)). In yet another embo~l-m~nt a controlled release system can be placed in 35 prox rnity of the the.~t;u~ic target, i.e., the brain, thus requiring only a fraction of the -W O 97/18822 PCT~US96/18675 ~y~lelllic dose (see, e.g., Goodson, in ~Air~l Applir~tio~.c of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Other controlled release systems are ~ c~.cs~nd in the review by Langer (Science 24~:1527-1533 (1990)).
In a specific embodiment, ~ dtion of a Thc~dy.,.lLic into a Notch-e,~yu~ssillg cell is accomplished by linkage of the Thclaptulic to a Delta (or other L Jyulylhlllic) protein or portion thereof capable of ...~ binding to Noteh. Contact of a Notch-e~ples~ g cell with the linked Thcldy~uLiu results in binding of the linked 10 Therapeutic via its Delta portion to Notch on the surface of the cell, followed by uptake of the linked The~d~culic into the Notch-~ ,si~ eell.
In a specific embodiment, the T~lc~dytuLic is delivered intr~r~ rly (e.g., by expression from a nucleic acid vector, or by linkage to a Delta protein capable of binding to Nouh followed by binding and internalization, or by l~;cc~,Lol-mto~ t~d or diffusion mech~nicmc) .
In a specific embodiment where the The.dpeuLic is a nucleic acid encoding a protein Thel~t:.lLic, the nucleic acid can be ~ ;L~.ed in vivo to promote e~l~ssion of its encoded protein, by cor~L,~;Lillg it as part of an ay~loplidle~ nucleic acid e~Lpl~sio 20 vector and ~ lillg it so that it becullles intr~relln~r, e.g., by use of a lellovi-~l vector (see U.S. Patent No. 4.980,286), or by direct inj~cti-n, or by use of ~ u~alLicle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating wi~ lipids or cell-surface - receptors or transfecting agents, or by a~ X~ lg it in linkage to a homeobox-like peptide 25 which is known to enter the nucleus ~see e.g., 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 e~l,lt;ssioll, by homologous recombination.
In specific embo~lim~n~ directed to L.eaLlll~lll or ~l~v~lllion of particular 3û disorders, preferably the following forrns of a(l..,ini~,dLion are used:

Disorder I ~ ~f~. . ed Forms of Ad~ LI ~lion Cervical cancer Topical Gastroi,.~sli.);~l cancer Oral; ~l~dvenous Lung cancer Inhaled; intravenous W O 97/18822 PCTnUS96/18675 TPnk~mi~ ~ILl~ luuS;e~aC~l~ulc MPt~ctsttir carcin-m~c Il,Lld~ us; oral Brain cancer Targeted; i~ ous; ;1lllall~r~
Liver ci~lhosis Oral; illLlay~noùs Psoriasis Topical - Keloids Topical R~l-lnrss Topical Spinal cord injury Targeted; illLld~,.wuS; intrathecal Pa~ soll7s disease Targeted; ~llLIavellOuS; intra~ecal Motor neuron disease Targeted; ~IlLld~ .uuS; intrathecal ~17hrimPr's disease Targeted; intravenous; intrathecal The present invention also provides pha~ r~ l compositior~. Such compositions con.~lise a thel~peul;r~lly er~cLi~le amount of a TheldpL.lLic, and a ph~rm~treutir~tlly acceptable carrier. ~n a specific emborlimPnt, the term "ph~ r~ ;rally acceptable" means approved by a regulatory agency of the Federal or a state g~ rnt or listed in the U.S. Pharmacopeia or other generally recognized ph~ acopeia for use in 20 animals, and more particularly in humans. The terrn "carrier" refers to a diluent, ad.juvant, excipient, or vehicle with which the thel~tuLic is <t~ J ~d. Such ph~ r~
carriers can be sterile liquids, such as water and oils, inrll-~ing those of petroleum, animal, vegetable or ~yl~ ic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a ~lefe~l~d carrier when the pharmt~eutir~l colll~osiLion is ~.l",;,~ led intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solntionc. Suitable pharrn,treutir tl excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monosLea.a~, talc, sodium chloride, dried skim milk, 3 ~ glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor ~,,...~1.,l~; of wetting or emulsifying agents, or pH buffering agents.
These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sl-~t~inrd-release formulations and the like. The composition can be 35 form--l,ttrd as â suppository, with traditional binders and carriers such as ~ ly~e~ides. Oral formulation can include standard carriers such as pll~""~r~"l;r~l grades of mannitol, lactose, starch, m,t~"~Sj,l", stearate, sodium saccha~ e, cellulose, magnesium carbonate, W O 97/18822 PCT~US96/18675 etc. Examples of suitable ph,...,.~r~ l carriers are described in rRPmin~tol7's Pharm~rentic~t Sciences" by E.W. Martin. Such co~ ositions will contain a thc~Al.c..lir~lly effective arnount of the The~ape~-Lic. plcr~lably in purified form, together with a suitable amount of carrier so as to provide the form for proper ~.I.,,;.~;.~I.~lion to the patient. The forrnulation should suit the mode of ~ ation In a preferred embodiment, the composition is form~ tPd in accordance with routine procedures as a ph~ ;r~l coll~osilion adapted for i~ d~ ouç a~l~";,.i~il.alion to hurnan beings. In another pltr~l.ed embodimP~lt the composition is fnrrmll~tPd in 10 accordance with routine pro-,e-lul-,s as a pharrn~reutir~l composition adapted for .IlL.a~.,..uus ~.l",i"i~L!dlion to m~mm~lc. Typically, compositions for illlld~tllous ~ l.dtion are solutions in sterile isotonic aqueous buffer. Where ~-~ ceC~, y, the composition may also include a solubilizing agent and a local anPsthPtiç such as lignltç~inP to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mLlced together in unit dosage form, for example, as a dry lyophilized powder or water free conce~ dL~ in a hermetically sealed container such as an arnpoule or sachette in~ fing the quantity of active agent. Where the composition is to be ~.l..li..;~iL~d by infusion, it can be ~ ensed with an infusion bottle conl ~;";,.g sterile pharm~celTtir:ll grade water or saline. Where the 2 0 composition is ~ rred by injection, an arnpoule of sterile water for injecti(m or saline can be provided so that the ingredients may be mixed prior to ~ tion.
The Therapeutics of the invention can be formulated as neutral or salt forms.
- Pharm~reutic~lly acceptable salts include those formed with free amino groups such as those 25 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, h;ctiriin~?, procaine, etc.
The amount of the Therapeuic of the invention which will be effective in the 3 ~ treatrnent of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical ter.hniql1~s. In addition, in vitro assays may optionally be employed to help identify optirnal dosage ranges. The precise dose to be employed in the formnl~tion will also depend on the route of a~lmini~tration, and 3 5 the seriousness of the disease or disorder, and should be decided according to the ,url~mrnt of the practitioner and each patient's circnmct~nre~. However, suitable dosage ranges for W O 97/18822 PCTrUS96/18675 intravenous A~ are generally about 20-5û0 lll~ lOgldlllS~ of active compound per kilogram body weight. Suitable dosage ranges for iuLl~dsal A~ ";~ Lion are generally about 0.01 pg/kg body weight to 1 mg/lcg body weight. Effective doses may be extrapolated from dose-.~,s~o~se curves derived from in vitro or animal model test systems.
Su~posiLulies generally contain active Jll~rediCll~ in the range of 0.5% to 10%
by weight; oral fnnmll~Ati-~n~ ~lerGldbly contain 10% to 95% active ingredient.
The invention also provides a l~tlA. ~ r~ ;rAl pack or kit CO~ ~g one or more containers filled with one or more of the ingredients of the phA~ i. Al lû compositios of the UlVGllLlOn. Optionally A~soc;~lrd with such co.,lA;..f . (s) can be a notice in the form ~ ,s~,libcd by a guYc~ llAl agency regnl-Atin~ the l,.~ rA. l~..G, use or sale of hal~l~Are~llirAlc or biological pio~lu.;L~, which notice reflects approval by the agency of m~An-lf~rtnre, use or sale for human ~,1",i"i~l,dli-~

5.13. DIAGNOSTIC UTILITY
Vertebrate Deltex proteins, analogues, dcL;vdL~es, and sllhseqtl~onres thereof,VGlLG7l/ldLG deltex nucleic acids (and se.luel-~fc compl .,.~ ..l,.,y thereto), anti-vertebra te Deltex antibodies, have uses in (li~gnostir.S. Such molecules can be used in assays, such as 2û immllnn~cs~ys, to detect, prognose, di~gnl~se~ or monitor various wn-litionc, diseases, and disorders affecting vt:lLel,ldLe Deltex e~ ,ioll, or monitor the LLedLI~e~L thereof. In particular, such an imml~no~c~y is carried out by a method Culllyli~ lg co..l~. Ii"g a sample derived from a patient with an anti-vG~LGbldLe Deltex antibody under conflitinn~ such that 25 immnnnspeciFlc binding can occur, and detecting or measuring the amount of any immnnospecific 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 abc.ld"L Notch andlor vertebrate Deltex loc~li7~ti-n or aberrant levels of Notch-vertebrate Deltex coloc~li7~tion in a disease state. In a specific embodiment, antibody to V~lLG13~d~G
30 Deltex can be used to assay in a patient tissue or serum sample for the p,.,sencc of velLebldle De}tex where an ab~ ~lL level of VG~LGbldte Deltex is an ;n~lir.~tion of a (lice~ed conniti~-n 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 3 5 an endogenous vertebrate Deltex protein may be indicative of a disorder of cell fate (e.g., cancer, etc.) By "aberrant lëvels," is meant illcleased or dec,cased levels relative to that WO97/18822 PCT~US96/18675 present, or a standard level le~l~ sc ~ g that present, in an analogous sarnple from a subject not having the disorder.
The immllno~cs~ys which can be used include but are not limited to competitive and non-c~ )t~;liv~ assay systerns using techniques such as western blots, r~riinimmlmn~c~ays, ELISA (enzyme linked i- ....~ os~ L assay), "sandwich"
immllnc~ ys, i lll~ no~l~,ciyil~Lion assays, LJl~ iLill reactions, gel diffusion precipitin re~cti- n~, imml-nnlliffusion assays, a~ n assays, complement-fixation assays, irmnunoradiometric assays, fluo~eent ;~ n~cs~y~, protein A immlmn~csays, to name:L O but a few.
Vertebrate deltex genes and related nucleic acid seqllenres and subsequences, inrlllfling c-~mrl-om~t~ry sequPn~ç~, and other ~polyLl~ , gene sequ~n~es~ can also be used in hybri~ ri~n assays. Vt~ lale deltex nucleic acid sequ~rlres, or subsequences thereof corll~ ih-g about at least 8 nucleotides, can be used as hybridization probes.
Elybridization assays can be used to detect, ~u~ ose, rli~gn~se, or monitor coIl~ition~, disorders, or disease states associated with abc.ldlll changes in vertebrate Deltex expression and/or activity as described supra. In particular, such a hybridization assay is carried out by a method colll~ g cullL~l;Lillg a sample COl~ nucleic acid with a nucleic acid 2 0 probe capable of hybridizing to vertebrate deltex DNA or RNA, under cc)~-litionc such that hybridization can occur, and de~cting or lllea~ulillg any resulting hybri~i7~tign 6. EXAl\~PLE: CLONING AND CHARACTE:RIZATION OF HUMA~' DELTEX
2 5 As described herein, we have accomplished the isolation and molecular characterization of human deltex. We report the clor~ing and sequencing of human deltex.
Human deltex encodes five putative SH3 domain binding sites and a ring-H2-zinc ~mger in similar locations to the coll~~ollding motifs found in Drosophila Deltex.

6.1. RESULTS
6.1.1. MOLECULAR CLONING OF 'lHE HUMAN DELTEX LOCUS
Human deltex was isolated through a combination of cO~ Lt;l and biorh~omir~l screens. Initially, a human expressed sequence tag ~l~t~ce was screened for 3 5 homology against the amino acid sequence of Drosophila Deltex. The critical part of this search involved the as~ul.~lion that stop codons in a particular reading frame of the ~l~t~h~ce are the result of se~ n~i"g mi~t~k~?s. Accordingly, stop codons were ignored and the open reading frame was PytPnrlPcl in a different frame. The predicted amino acid se.lu~ ce .onror~ by the hypothetical open reading frames were then u)~ aled with the protein product of the Drosophila deltex L~ lioll unit.
We previously i~l~ntifiPII the Drosophila deltex ~ scliplion unit by showing via gerrnline-~n~ tPd l,~lsrol~lion e~c.i nenls that a g~nl~"i~ fragment ~."l;.;,~;.-~ this l,ansc;li~tion unit is capable of complemPntin~ most deltex mutant defects. Moreover, this genomic r~a~lllelll rescues the normally lethal genetic intPrartion that results when flies are 10 doubly mutant for deltex and nd. Finally, Northern analysis in-lir~t~s a m~tern~l loading of deltex Llal~cJipl~ into the developing oocyte, a finding that is col-~ rl.~ 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 }5 transcription unit were isolated from an embryonic cDNA library, the complete nucleotide seq~-~nre (SEQ ID NO: 1) and predicted protein product were then determined (SEQ ID
NO:2).
Co~l~ydl i~on of ~e arnino acid sequence of Drosophila Deltex with that predicted for what we ~e(1l-ced to be hypothetical open reading frames in the rl~t~h~e 2 0 i-lPntifiPd a sequence:gnl I dbest I 24254 T05200 widl s~ r~ t homology to Drosophila Deltex. Within TQ5200, five collsel vGd stretches of amino acids were found in dirr~,r~,lL
reading frarnes, 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 545-25 555 (SEQ ID NO:3), 565-580 (SEQ ID NO:5), 581-616 (SEQ ID NO:7) and 6û2-638 (SEQ
ID NO:9) respectively. These sequences are shown in Table II, i~Pntie~l amino acids are shown in bold.

W O 97/18822 PCT~US96/18675 TABLEII

AMINO
ACID
NOS.
Drosophila 602-638 VYGEKVGVQPIGSM~W~ LPGHEGQNTIQrVYD
Deltex T05200 200-310 IY~;K l ~; l QPPGKMEF;HLIPHSLXFGPl~ l Q'l X~lVYD
Drosophi}a 565-580 LSRC~T.M~IT.QCLNGM
Deltex Drosophila 581-616 IIAQQNEMNKNLFIECPVCGIVYGEKVGNQPIGSMS
Deltex l~i T05200 138-245 LVAMYSNGNKDGSLQC:~PrCKPsMG~RRVRSRLGRWS
Dl osuphila 545-555 QPCPMCMEELV
Deltex 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 synthesi~d based on the DNA sequence of gnl I dbest I 24254 T05200.
PCR reactions were performed using the four dirr~.ent primer combinations and a 2 5 human fetal brain cDNA library (Invitrogene) as the templaee. The PCR product was sequenced and found to have the satne DNA seque~re as gnl I dbest I 24254 T05200.
The PCR product ge,lelal~d 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: 1 1? 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 35 probe to screen a northern blot cont~ining poly(A)t rnRNA isolated from various human tissue samples. This probe was observed to hy~ridize to a 5.4-kb RNA in -W O 97/18822 PCT~US96/18675 heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. Whenthis probe was used to screen a zoo blot (a blot c~ gennmir EcoRI rlig~st.od DNA of various species, obtained from Clontech) by Southern hybritli7~ti~
llyl~ ;t)n was obsened in genoll~ic hurnan, monkey, rat, mice, dog, cow, and yeast DNA. HylJ~ inn was not observed in tke rabbit and chicken g~nr"~
. DNA.

Sl~u~lul~l analysis of the human Deltex protein:
The predicted human Deltex product has 720 amino acids and an Psfim~tPd molecular mass of a~ xi...;~ly 80 kDa. The 180 amino terrninal residues of human Deltex have an d~iWLil~dl~ identity of 33 % with the corresponding amino acid residues of Drosophila Deltex and the nucleic acids e~ror1ing these arnino acids have an ap~ro~il-late 52% identity. The 180 carboxyterminal arnino acids of hurnan Deltex have an approximate 48% identity with thecu..~ ondillg arnino acid residues of Drosophila Deltex and the nucleic acids encoding these carboxy terrninal amino acids have an approximate 49% identity.
A structural analysis of human Deltex protein revealed a CO~ ,. ved 2 0 structure among Deltex proteins (see Figure 3). Like Drosophila Deltex, hurnan Deltex has both ring-H2-zinc finger (amino acids 411~71) (SEQ ID NO:25) and putative SH3-binding rlnm~in~. Noticeably absent from the human Deltex are the two opa repeats that subdivide the primary SL~ Lult: of the Drosophila Deltex into 25 three domains. Each of ehe Drosophila Deltex ~lom~in~ I, II, and III, has been found using the yeast "interaction trap assay" to be capable of m~ ting homotypic interactions ¢see infra).
i) Domain I:
Domain I corresponds to the N-tPrmin~1 303 amino acids of 3 ~ Drosophila and the first 237 amino acids of human Deltex. In Drosophila, we have demonstrated that the region of human Deleex corresponding to the firse 175amino acids of domain I is essenti~l and sufficient to bind the Notch ANK repeats and overexpression of this domain can rescue loss-of-function phenotypes of 35 Deltex. Since Drosophila Deltex can bind to human Notch-l and 2, conservation -W O 97/18822 PCT~US96/18675 of binding activity between human Deltex and Drosophila domain I is suggested.
Furtherrnore, this has been ~.omnn.ctrated. See infra.
ii) Putative SH3 binding domains:
Domain II of Drosophila Deltex contains a putative SH3-binding site (amino acids 476-484) (M~t.cun~ et al., 1995, Development 121(8):2633-2644).
Five ~live SH3-binding sites (SEQ ID NOS: 17-21) are found in human Deltex (within amino acids 226-377) in a position co~ ol1ding to the SH3-binding site in domain II of Drosophila Deltex (Table m).
SH2 and SH3 dom~inc are conserved protein mnrlnl~oc so named based on their homology to the oncogene Src (~rc Homology). These motifs have been implicated in mP~;~tin~ protein-protein interactions in a number of signal tr~ncdnr~iorl pathways (reviewed in Cell 71:359-362;Science 252:668-674; Trends Cell Biol. 3:8-13; FEBS 307:55-61). Recently, a comp~ n~.y motif that binds to the SH3 dornain has been iclrntifi~d and called simply an 'SH3-binding domain' (nSH3-BDn) (Science 259:1157-1161). The core binding region of SH3-BD is proline-rich and a~l,ro~ dL~ly ten residues in length. As shown in Table III, tbis motif, as defined from a mouse protein that exl,c.i,l~ell~lly bound an SH3 domain (SEQ ID NO:23), is shown aligned to the putative SH3-binding site in Drosophi~a Deltex (SEQ ID NO:22) and the five regions (SEQ ID NOS: 17-21) that may esellL human versions of this motif. These regions are located centrally in the Deltex protein. For lt;r~lcnce, regions of the protein encoded by the Drosophila Son of sevenless (SOS) protein (SEQ ID NO:24), which may also contain SH3-BD, is shown. The Son of sevenless encoded protein, a putative guanine mlrlPotirie ç~rh~nge factor (GNEF), has been shown to bind to an 'adaptor' protein (drk) c(~ g only SH2 and SH3 m~ lPs, although the actual residues that mediate binding have not been accurately defined (Simon M., et al., 1993, Cell 73:169-177 and Olivier J., et al., 1993, Cell 73:179-191).

W O 97/1882Z PCT~US96/18675 TABLF III

tive SH-3 Domain Rin-lin~ Sites in Deltex E~-~IL~.S SEQ ID NO:
Fly Deltex RAP-VPPPLPI~IPRQQ 22 Mouse 3BP-1RAPTMPPPLPPVPPQP 23 Fly Son of sevenless RA--VPPPLPPRRKER 24 ~llm~n Deltex 226-244 VXI~APPLSXPX~GGPPG.A 17 IV 353-36~ RAPKPILHPPPVS 20 There are ~U11~LILIY only six SH3-c.,~ ;,.;"g proteins ~ ntifi~d in Drosophila, any one of which may be a direct binding partner of Deltex, and thusan indirect partner of Notch.
The functional re~uirement of the Deltex domain II-III in Drosophila 25 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 ~i~";r,~ t re~ rtinn of the ability to activate the Notch .~ign~ling paLllw~. These results indicate that domains II and III are not reAI-nrl~nt iii) Ring-H2-zinc finger:
Human deltex (nucleotides 1734-1916 of SEQ ID N0: 11) encodes a ring-H2-zinc finger (SEQ ID NO: 25), ~pea~ g as amino acids 411~71 of SEQ
ID NO: 12 in that part of human Deltex which corresponds to domain III of Drosophila Deltex. This ~ype of zinc finger is believed to be involved in 3 ~ protein/protein interactions.

6.2. ~AT~3RIAL AND METHO~S
6.2.1. SEOUENCh~ DETERM~ATION AND ANALYSIS
The EcoRI-cDNA insert was subcloned directly in both o.i~ ion~
into Bluesc~i~t KS. Ovell~ g deletions were produced on the insert using the DNAse I method to gelleldLt bidirectional ~elPti(~nc (Eberle et al., 1993, BiotPrhnirllles 14:408). The resulting deletions were analyzed using an IBI
AI;C sequencer.
DNA sequence manipulations were p~lrolllled using Intelligenetic's l0 PC-GENE software. Open reading frame prediction and plotting were perforrned using the Uli-v~sily of Wi~con~in program CODONPREFERENCE (Gribshov et al., 1984, Nucl. Acids Res. 12:539-549). The GenPept and SWISS-PROT
.IAtAhA~eS were searched with all or part of the ~le(l~-red arnino acid sequence using 15 the FASTA program (Pearson and T irmAn, 1988, Proc. Natl. Acad. Sci. USA, 85:2444-2448) available by the ~er~ nk FASTA server through BITNET.

7. EXAMPLE: HUMAN DELTEX BIr'lDS
HUMAN AND DROSOPHILA NOTCH
2 0 We have ~i.omon~trated in Drosophila a specific and direct physical interaction between Deltex and Notch ANK repeats (Diederich et al., 1994, Development 120:473481). 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 a~;livdLillg l dllscri~tion. These constructs are referred as pEG.
30 A second foreign protein segm~nt is fused to an acidic l.dnsc.i~tional activation domain that does not bind DNA on its own. These CO1I~lL~1C~ are referred to as pJG. Coexpression of these two proteins in yeast cells results in the functionalreconstruction of an active LexA "hybrid" l.dnscli~lion factor if the foreign proteins physically interact with one another. Activity of the hybrid LLdnscli~lion factor is monitored by L~dns.;.i~lion of the ~-galactosidase reporter gene.
Expression of fusion proteins from the pJG construct is induce-i when yeast cells =

W O 97/18822 PCT~US96/18675 are cultured in the ga}actose media but not in the glucose media. Therefore, positive interaction should be observed on~y in ~ rt~)se media.
The constructs eY;~ od using the yeast interaction were as fol}ows:
the pEGhDeltex costruct co~ the entire coding region of human Deltex;
pJGhNotch-l encodes the ankyrin repeats region of hurnan Notch-1 from amino acids 1826-2147; pJGhNotch-2 encodes the ankyrin repeats region of human Notch-2 from arnino acids 1772-2084; pJGhNotch encodes the ankyrin repeats of Drosophila Notch from arnino acids 1827-2259; and JGfHairless contains the entire 10 coding region of Drosop~ila Hairless.
As ~,esellL~d in Table IV, ~i~"ilir~"l in~ C.tiC)Il of ~B-g~l~rt~ ce activity was observed when yeast cells co~l~reeltd with pEGhDeltex and pJGhNotch-1, pJG~INotch-2 or pJGHfNotch, were cultured in galactose media 15 (Table IV). These results indicate that human Deltex binds to the ankyrirl repeats human Notch-1, human Notch-2 as well as that of Drosophila Notch. Standard deviation is picse.ll~d in the pare~th~ses~

TABLE IV

Media Coexpressed Coll~L,uc~ Galactose Glucose pEGhDeltex/pJGhNotch-l732(35) 4(1) 2 5 pEGhDeltex/pJGhNotch-2195(25) 11(5) pEGhDeltex/pJGfNotch892(184) 14(4) pEGhDeltex/pJGHairless 61(13) 20(2) pEGhDeltex/pJG 38(1) 21(5) 3 o pEG/pJGhNotch-l 47(13) 13(5) pEG/pJGhNotch-2 48(7) 19(8) pEG/pJGfNotch 69(18) 20(5) pEG/pJGHairless 59(4) 39(14) 3 5 pEG/pJG 60(10) 32(4) W O 97/18822 PCTnUS96/18675

8. E~ DPLE: CLO~n~G OF V~K'l~E~ E DELllE~ GErES
The evolution of hurnans and Drosophila diverged about 600 million years ago. As rli~cllg~iod supra, Deltex protein d~ dl~s a conserved structure in these two evolutionary distant species. Knowledge of the conserved regions ofthe protein allows one to design synthetic de~nc~dl~ prirners for use in hybridization and PC~ reactions which enable the cloning of Deltex encoding nucleic acids in other Ol~ dl~isln5.
Five regions of high cons~l ~dtion between human and Drosophila are found in arnino acid ~ ;hes of human Deltex arnino acid numbers 414419 (SEQ ID NO:30), 475~80 (SEQ ID NO:31), 504-511 (SEQ ID NO:32), 531-539 (SEQ ID NO:33) and 5~7-564 (SEQ ID NO:34). These sequences are conserved in Drosophila Deltex arnino acid stretches 549-555 (SEQ ID NO:35), 603-608 (SE~Q 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 ellro-l;"g nucleic acids of other 01 gani~ll,s.
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' orient~tinn A second series of degenerate primers corresponding to the ~nti~en~e strand of the nucleic acids encoding amino acids 475-480 in Drosophila (S~Q ID NO:31) and 603-608 in human (SEQ ID
NO:36) is also synth~ci7~od The two series of primers are added to a mixmre co~t~ining 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 3 ~ sequences and the mouse nucleic acid homolog being iso}ated.
After surçeccful PCR amplifir~tinn, 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 3~ complete nucleotide sequence of the mouse deltex homolog is determined by sequence analysis.

W O 97/18822 PC~nUS96/18675

9. DEPOSIT OF MICROORGANISMS
Plasmid pBS hdx cont~ining 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 & FAmon-l.c, 1155 Avenue of the Americas, New York, New York 5 10036 on behalf of Yale Univ~l~ily on November 17, 1995, with the American Type Culture Collection, 1201 Parklawn Drive, Rockville, Maryland 20852, under the provisions of the Budapest Treaty on the TntPrn~tional Recognition of the Deposit of Microul~ lc for the Purposes of Patent Procedures, and ~csign~d arcesciQn number97341.

The present invention is not to be limited in scope by the microul~,dni~ln deposited or the specific embo~iimPnt~ described herein. Indeed, various mo~ifir~tions of the invention in ~ itinrt to those described herein will become a~ar~,lL to those 15 skilled in the art from the foregoing description and acco~ allyillg figures. Such modifications are int~on~l~d to fall within the scope of the appended claims.
Various publications are cited herein, the disclosures of which are incorporated in their t;~ iies.

~UU~N~: LISTING
( 1 ) Q~N~RAT. INFORMATION:
ti) APPLICANT: Ar~avanis-T~ k~n~c, Spyridon Mat~uno, Kenji (ii) TITLE OF lNV~w ~ lON: ~ ~XATE DELTEX PROTEINS, NUCLEIC
ACIDS, AND ANTIBODIES, AND ~RT.~T~n MET~ODS AN~
COMPOSITIONS
(iii) NUMBER OF S~UU~N~S: 3 9 (iv) CORR~POh~EN~E pnDR~S:
~A~ AnD~qs~ Pennie ~ r~ ~
B~ STREET: 1155 Avenue o~ the Americas C~ CITY: New York D~ STATE: New York ~E~ ~Y: U.S.A.
~FJ ZIP: 10036-2711 (v) COMPUTER READABLE FORM:
(A~ MEDIUM TYPE: Floppy disk (B COMPUTER: IBM PC compatible (C OPERATING 5YSTEM: PC-DOS/MS-DOS
(D~ SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: To Be Assigned (B) FILING DATE: 22-NOV-1995 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Mi~rock, S. Leslie (B) REGISTRATION NUMBER 18,872 (C) REFERENCE~DOCRET NUMBER: 7326-036 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 790-9090 (B) TELEFAX: (212) 869-9741/8864 (C) TELEX: 66141 PENNIE
(2) INFORMATION FOR SEQ ID NO:l:
( i ) ~EUU~N~ CHARACTERISTICS:
(A~ LENGT~: 3771 base pairs (B) TYPE: nucleic acid (C STRANn~T)N~C:s: double (D~ TOPOLOGy l-n'-~
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME~KEY: CDS
(B) LOCATION: 345..2555 (xi) SEQUENCE DESCRIPTION: SEQ I~ NO:1:

ACCGAGTGAC TAAAGAACCA GAArrAAAAC llCGG~AIA~A TGGAAGCCAG GGAAAATCAG 120 GGATAACTAA CGCTGGCAGC ~G~LC~ACCA TTTTTAATTT ~ ~lllAT lll~lGCC'~A 180 ull~GC'GAG Cr~QCQAr.~T AGCGCGACAG CAACAGCAAG AGAGAGCGAG AGAGA~AGTG 240 CA 02238404 1998-0~-22 W O 97/188Z2 P ~ ~US96/18675 AGTGAGTGAG AGCTAGTGAA GAGAGCGCAG GAGGAGTTGG ATATGGA~AT GGGcATGGAT 300 ATGGCAATGG GCTCACTCCA CGGATAACGG ATcAAcTGcA AGCA ATG GCC AGC AGC 356 Met Ala Ser Ser Ala Gly Ser Ala Ala Ser Gly Ser Val Val Pro Gly Gly Gly Gly Ser Ala Ala Ser Ser Cy6 Ala Thr Met Ala Leu Ser Thr Ala Gly Ser Gly Gly Pro Pro Val Agn His Ala His Ala Val Cys Val Trp Glu Phe Glu Ser Arg Gly Lys Trp Leu Pro Tyr Ser Pro Ala Val Ser Gln His Leu GAA CGC GCC CAC GCC AAG A~A CTG ACG CGC GTC ATG CTG AGC GAT GCG 596 Glu Arg Ala His Ala Lys Lys Leu Thr Arg Val Met Leu Ser ABP Ala Asp Pro Ser Leu Glu Gln Tyr Tyr Val Asn Val Arg Thr Met Thr Gln Glu Ser Glu Ala Glu Thr Arg Ser Gly Leu Leu Thr Ile Gly Val Arg Arg Met Leu Tyr Ala Pro Ser Ser Pro Ala Gly Lys Gly Thr Lys Trp 120 125 .130 Glu Trp Ser Gly Gly Ser Ala Asp Ser Asn Asn Asp Trp Arg Pro Tyr Asn Met His Val Gln Cys Ile Ile Glu Asp Ala Trp Ala Arg Gly Glu ~150 155 160 Gln Thr Leu Asp Leu Cys Asn Thr His Ile Gly Leu Pro Tyr Thr Ile Asn Phe Cys Asn Leu Thr His Val Arg Gln Pro Ser Gly Pro Met Arg Ser Ile Arg Arg Thr Gln Gln Ala Pro Tyr Pro Leu Val Lys Leu Thr Pro Gln Gln Ala A~n Gln Leu Lys Ser Asn Ser Ala Ser Val Ser Ser Gln Tyr Asn Thr Leu Pro Lys Leu Gly Asp Thr Lys Ser Leu ~is Arg Val Pro Met Thr Arg Gln Gln His Pro Leu Pro Thr Ser His Gln Val -71- ~

CA 02238404 1998-0~-22 WO 97/1882~ PCTAU$96/l8675 Gln Gln Gln Gln His Gln Leu Gln His Gln Gln Gln Gln Gln Gln Gln His ~is His Gln His Gln Gln Gln Gln His Gln Gln Gln Gln Gln His Gln Met Gln His His Gln Ile His His Gln Thr Ala Pro Arg Lys Pro Pro Lys Lys His Ser Glu Ile Ser Thr Thr Asn Leu Arg Gln Ile Leu Asn Asn Leu A6n Ile Phe Ser Ser Ser Thr Lys His Gln Ser Asn Met Ser Thr Ala Ala Ser Ala Ser Ser Ser Ser Ser Ser Ala Ser Leu His His Ala Asn His Leu Ser His Ala His Phe Ser His Ala Lys Asn Met Leu Thr Ala Ser Met Asn Ser His His Ser Arg Cys Ser Glu Gly Ser Leu Gln Ser Gln Arg Ser Ser Arg Met Gly Ser His Arg Ser Arg Ser Arg Thr Arg Thr Ser Asp Thr Asp Thr Asn Ser Val Lys Ser ~is Arg Arg Arg Pro Ser Val Asp Thr Val Ser Thr Tyr Leu Ser His Glu Ser Lys Glu Ser Leu Arg Ser Arg Asn Phe Ala Ile Ser Val Asn Asp Leu Leu Asp Cys Ser Leu Gly Ser Asp Glu Val Phe Val Pro Ser Val Pro Pro Ser Ser~Leu Gly Glu Arg Ala Pro Val Pro Pro Pro Leu Pro Leu CAT CCG CGA CAG CAA CAG CAG CAG CAA CAA CAG CAG CAA CAG CTG CAG lB44 His Pro Arg Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Leu Gln Met Gln Gln Gln Gln Gln Ala Gln Gln Gln Gln Gln Gln Ser Ile Ala ~05 510 515 Gly Ser Ile Val Gly Val Asp Pro Ala Ser Asp Met Ile Ser Arg Phe GTC AAG GTG GTG GAG CCA CCG CTG TGG CCC AAT GCC CAG CCC TGl CCC 1988 Val Lys Val Val Glu Pro Pro Leu Trp Pro Asn Ala Gln Pro Cys Pro ~ -72-CA 02238404 l998-0~-22 W O 97)1~822 PCT~US96/18675 Met Cys Met Glu Glu Leu val His Ser Ala Gln Asn Pro Ala Ile Ser Leu Ser Arg Cys Gln His Leu Met His Leu Gln Cys Leu Asn Gly Met Ile Ile Ala Gln Gln Asn Glu Met Asn Lys Asn Leu Phe Ile Glu Cys Pro Val Cys Gly Ile Val Tyr Gly Glu Lys Val Gly Asn Gln Pro Ile Gly Ser Met Ser Trp Ser Ile Ile Ser Lys Asn Leu Pro Gly His Glu Gly Gln A8n Thr Ile Gln Ile Val Tyr Asp Ile Ala Ser Gly Leu Gln Thr Glu Glu His Pro Hi8 Pro Gly Arg Ala Phe Phe Ala Val Gly Phe CCG CGG ATC TGC TAC TTG CCG GAC TGC CCG CTG GGG CGA AAG GTT TTG 2~72 Pro Arg Ile Cys Tyr heu Pro Asp Cys Pro Leu Gly Arg Lyfi Val Leu ~Arg Phe Leu Lys Ile Ala Phe Asp Arg Arg Leu heu Phe Ser Ile Gly Arg Ser Val Thr Thr Gly Arg Glu Asp Val Val Ile Trp Asn Ser Val Asp His Lys Thr Gln Phe Asn Met Phe Pro Asp Pro Thr Tyr Leu Gln Arg Thr Met Gln Gln Leu Val His Leu Gly Val Thr Asp CTCGATCATT ~l~LLc~ATT C~1C~L lAAG TTACTTTCTA CATAATCTCA ~L~L~~ ~C 2685 AAlC~LC~ll TACTATGATA TAlll L L 111 ATAGATATAT TGTAATAGCG TTCGAGCTGC 2745 TCGAACCCTA AAACAACAGC AAACCACAAT TGCAATTGTA G~llC~lllC CG~l~LLC~A 2805 CTAGTATTAG GCATTATCCT GA~ L~AT TCCTGATTCG ATTCAAGCCA AGCCAAGCCA 2985 TGTTGATATA GCTAGCTATA ACCATTGCCC AL~L~-lC~AT ~L~-l~lCG~-L TTCGAATTTG 3105 ~AT CAGATCCATG TGAATTTTCT TTATATCGGA TTTATATAGG ATTA~AATAG 3165 WO 97/l8822 PCT~US96/18675 TATTTTGAGA ~r~ ~GG AGATGGGTAA ATTC~A~A ~ ~AC ~ GGC 3225 A~rGr.A~T TTAGGAAGCA TAGTTGTAAC GCAGCCAGAT ATTCCATTAC Gc~T~T~r~T 3345 ATACATATAC ~TATA~ATAC ATACATAAAC ATATTTTAAC ATAGCCCCAT AGCCATACGA 340s CATAACAATA Alllll l l lA TCGAATCCCT TGr~T~TT TGATGAATTG llG~ ~AT 3465 ATTGATATCA TCGAGCATCG AACGAACTAT CGT~T~rATC GCCAATATAT AGCATATATA 3525 GCATATAGTA TGTAr~A~-ATc GTACGGACAG CTAGCGGCTA CTGACCGCGC CACCATATTT 3585 TTT~.~ AT ATTCCACAAC AAATTCCACA CCATTTATGT ATGCATATTA CGCATATATA 3705 (2~ INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 737 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (Xi ) S~Q~N~ DESCRIPTION: SEQ ID NO:2:
Met Ala Ser Ser Ala Gly Ser Ala Ala Ser Gly Ser Val Val Pro Gly Gly Gly Gly Ser Ala Ala Ser Ser Cys Ala Thr Met Ala Leu Ser Thr Ala Gly Ser Gly Gly Pro Pro Val Asn His Ala His Ala Val Cys Val Trp Glu Phe Glu Ser Arg Gly Lys Trp Leu Pro Tyr Ser Pro Ala Val Ser Gln His Leu Glu Arg Ala His Ala Lys Lys Leu Thr Arg Val Met Leu Ser Asp Ala Asp Pro Ser Leu Glu Gln Tyr Tyr Val Asn Val Arg Thr Met Thr Gln Glu Ser Glu Ala Glu Thr Arg Ser Gly Leu Leu Thr Ile Gly Val Arg Arg Me~ Leu Tyr Ala Pro Ser Ser Pro Ala Gly Lys Gly Thr Lys Trp Glu Trp Ser Gly Gly Ser Ala Asp Ser A6n Asn Asp Trp Arg Pro Tyr Asn Met His Val Gln Cys Ile Ile Glu Asp Ala Trp Ala Arg Gly Glu Gln Thr Leu Asp Leu Cys Asn Thr His Ile Gly Leu Pro Tyr Thr Ile Asn Phe Cys Asn Leu Thr His Val Arg Gln Pro Ser 180 185 1~0 CA 02238404 1998-0~-22 WO 97/18822 PCT~US96/18675 Gly Pro Met Arg Ser Ile Arg Arg Thr Gln Gln Ala Pro Tyr Pro Leu 195 20~ 205 Val Lys Leu Thr Pro Gln Gln Ala Asn Gln Leu Lys Ser Asn Ser Ala Ser Val Ser Ser Gln Tyr Asn Thr Leu Pro Lys Leu Gly Asp Thr Lys Ser Leu His Arg Val Pro Met Thr Arg Gln Gln His Pro Leu Pro Thr Ser Hifi Gln Val Gln Gln Gln Gln Hi6 Gln Leu Gln ~ His Gln Gln Gln Gln Gln Gln Gln His His His Gln His Gln Gln Gln Gln His Gln Gln Gln Gln Gln His Gln Met Gln His His Gln Ile His His Gin Thr Ala Pro Arg Lys Pro Pro Lys Lys His Ser Glu Ile Ser Thr Thr Afin Leu Arg Gln Ile Leu Asn Asn Leu Asn Ile Phe Ser Ser Ser Thr Lys His Gln Ser Asn Met Ser Thr Ala Ala Ser Ala Ser Ser Ser Ser Ser Ser Ala Ser Leu His His Ala Asn His Leu Ser His Ala His Phe Ser His Ala Lys Asn Met Leu Thr Ala Ser Met Asn Ser His His Ser Arg Cys Ser Glu Gly Ser Leu Gln Ser Gln Arg Ser Ser Arg Met Gly Ser Hi8 Arg Ser Arg Ser Arg Thr Arg Thr Ser Asp Thr Asp Thr Asn Ser Val 405 41~ 415 Lys Ser His Arg Arg Arg Pro Ser Val Asp Thr Val Ser Thr Tyr Leu Ser His Glu Ser Lys Glu Ser Leu Arg Ser Arg Asn Phe Ala Ile Ser Val Asn Asp Leu Leu Asp Cys Ser Leu Gly Ser Asp Glu Val Phe Val Pro Ser Val Pro Pro Ser Ser Leu Gly Glu Arg Ala Pro Val Pro Pro Pro Leu Pro Leu His Pro Arg Gln Gln Gln Gln Gln Gln Gln Gln Gln 485 490 4gS
Gln Gln Leu Gln Met Gln Gln Gln Gln Gln Ala Gln Gln Gln Gln Gln Gln Ser Ile Ala Gly Ser Ile Val Gly Val Asp Pro Ala Ser Asp Met Ile Ser Arg Phe Val Lys Val Val Glu Pro Pro Leu Trp Pro Asn Ala Gln Pro Cyfi Pro Met Cys Met- Glu Glu Leu Val His Ser Ala Gln Asn Pro Ala Ile Ser Leu Ser Arg CYF: Gln His Leu Met His Leu Gln Cys --75 ~

Leu Asn Gly Met Ile Ile Ala Gln Gln Asn Glu Met Asn Lys Asn Leu Phe Ile Glu cys Pro Val CYB Gly Ile Val Tyr Gly Glu Lys Val Gly Asn Gln Pro Ile Gly Ser Met Ser Trp Ser Ile Ile Ser Lys Asn Leu Pro Gly His Glu Gly Gln Asn Thr Ile Gln Ile Val Tyr Asp Ile Ala Ser Gly Leu Gln Thr Glu Glu His Pro His Pro Gly Arg Ala Phe Phe Ala Val Gly Phe Pro Arg Ile Cys Tyr Leu Pro Asp Cys Pro Leu Gly A~g Ly~ Val Leu Arg Phe Leu Lys Ile Ala Phe Asp Arg Ary Leu Leu 675 680 6~5 Phe Ser Ile Gly Arg Ser Val Thr Thr Gly Arg Glu Asp Val Val Ile Trp Asn Ser Val Asp His Lys Thr Gln Phe Asn Met Phe Pro Asp Pro ~hr Tyr Leu Gln Arg Thr Met Gln Gln Leu Val His Leu Gly Val Thr Asp t2) INFORMATION FOR SEQ ID NO:3:
UhN~h CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: tlnknl ..
(ii) MOLECULE TYPE: peptide (xi) ~hQu~N~h DESCRIPTION: SEQ ID NO:3:
Gln Pro Cys Pro Met Cys Met Glu Glu Leu Val (2) INFORMATION FOR SEQ ID NO:4:
(i) ~EQuhN~h CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY llnkn (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Glu ABP Cys Thr Ile Cys Met Glu Arg Leu Va (2) INFoRMATroN FOR SEQ ID NO:5:

W O g7/18822 PCT~US96/l8675 5uu~iN~-~; CHARAcTERIsTIcs:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY t ln kn~
(ii) MOLECULE TYPE: peptide (Xi) ~UU~N~ DESCRIPTION: SEQ ID NO:5:
Leu Ser Arg Cys Gln His Leu Met His Leu G}n Cys Leu Asn Gly Met (2) INFORMATION FOR SEQ ID NO:6:
( i ) ~UU~N~ CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B~ TYPE: amino acid (D) 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 (2) INFORMATION FOR SEQ ID NO:7:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ile Ile Ala Gln Gln Asn Glu Met Asn Lys Asn Leu Phe Ile Glu Cys Pro Val Cys Gly Ile Val Tyr Gly Glu Lys Val Gly Asn Gln Pro Ile Gly Ser Met Ser (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Leu Val Ala Met Tyr Ser Asn Gly Agn Lys Asp Gly Ser Leu Gln cy8 l 5 l0 15 Pro Thr Cys Lyg Pro Ser Met Gly Arg Arg Arg Val Arg Ser Arg Leu Gly Arg Trp Ser (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE ~AR~CTERISTICS:
(A) LENGTH: 37 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOT~.~ TYPE: peptide (Xi) ~UU~'N~ DESCRIPTION: SEQ ID NO:9:
Val Tyr Gly Glu Lys Val Gly Val Gln Pro Ile Gly Ser Met Ser Trp l 5 l0 15 Ser Ile Ile Ser Lys Asn Leu Pro Gly His Glu Gly Gln Asn Thr Ile Gln Ile Val Tyr Asp (2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQu~N~ CHARACTERISTICS:
(A) LENGTH: 37 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (Xi ) S~uU~N~ DESCRIPTION: SEQ ID NO:l0:
Ile Tyr Gly Glu Lys Thr Gly Thr Gln Pro Pro Gly Lys Met Glu Phe l 5 l0 15 His Leu Ile Pro His Ser Leu Xaa Phe Gly Pro Asp Thr Gln Thr Xaa Arg Ile Val Tyr Asp (2) INFORMATION FOR SEQ ID NO:ll:
( i ) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 2547 base pairs (B) TYPE: nucleic acid (C) STR~N~ N~ : double (D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/REY: CDS
(B) LOCATION: 504..2363 CA 02238404 1998-0~-22 W O 97/18822 PCT~US96/18675 (xi) SEQUENCE DESCRIPTION: SEQ ID NO~
Gr~.~.AA~CC CCACTGAAGC CGGGCGCAGG ~L~lGG~ACG CA~ll~G~AG TGCAAAGGGC 60 TGGCTGAGAG CCGCAGGAGC AGCAGGCTGT GGCCCAGGCC lC~lGG~l~A CAGGCC'~l 120 CTGGCGGGGA ~cA~G~A~ AGAGACAACA CAGAAGAGGC TGGACCTCGA ACAGGGGCGG 180 ~C~l~ACT CCCTACCTGA G~rAr~ccr~ GGGGCCAAGG ACTTTAGAGC 'L~ ' 1 lC~'l'CC 240 GAAGGG~l~l TTGCAGCCTG GATGGCCATC CCACATTCCT TTAACGGAGG TCTCTAGGCC 360 TCAGAGAGAA cc~Ar~Ar~TTA GAAAGGAGGC CAGACGGTCC TTG~ ~C ~LGGGGAGA 420 GAGGAAGTTG CCGCCTGCTG CCAGGCCCAG GAGGAGCTGG GCCTGCAATA ~GGGG~ACC 480 TGGCCC~lGA GGCAGTGGCG GCC ATG TCA CGG CCA GGC CAC GGT GGG CTG 530 Met Ser Arg Pro Gly His Gly Gly Leu Met Pro Val Asn Gly Leu Gly Phe Pro Pro Gln Asn Val Ala Arg Val

10 15 20 25 Val Val Trp G1U Cys Leu Asn Glu His Ser Arg Trp Arg Pro Tyr Thr Ala Thr Val Cys His His Ile Glu Asn Val Leu Lys Glu A8p Ala Arg Gly Ser Val Val Leu Gly Gln Val Asp Ala Gln Leu Val Pro Tyr Ile Ile Asp Leu Gln Ser Met His Gln Phe Arg Gln Asp Thr Gly Thr Met Arg Pro Val Arg Arg Asn Phe Tyr Asp Pro Ser Ser Ala Pro Gly Lys go g5 100 105 Gly Ile Val Trp Glu Trp Glu Asn Asp Gly Gly Ala Trp Thr Ala Tyr Asp Met Asp Ile Cys Ile Thr Ile Gln Asn Ala Tyr Glu Lys Gln His Pro Trp Leu Asp Leu Ser Ser Leu Gly Phe Cy8 Tyr Leu Ile Tyr Phe AAC AGC ATG.TCG CAG ATG ARC CGC CAG ACG CGC CGG CGC CGC CGT CTG 1010 Asn Ser Met Ser Gln Met Xaa Arg Gln Thr Arg Arg Arg Arg Arg Leu Arg Arg Arg Leu Asp ~eu Ala Tyr Pro Leu Thr Val Gly Ser Ile Pro Lys Ser Gln Ser Trp Pro Val Gly Xaa Ser Ser Gly Gln Pro Cys Ser CA 02238404 1998-0~-22 O 97/18822 PCTnJS96/l8675 l'~M ~A~ ~A~ TGC CTG YTG GTC AAC AGC ACG CGC GCC GTC TCC AAC GTC 1154 Xaa &ln Gln Cys Leu Leu Val Asn Ser Thr Arg Ala Val Ser Asn Val I}e Leu Xaa Ser Gln Arg Arg Lys Val Xaa Pro Ala Pro Pro Leu Ser Xaa Pro Xaa Xaa Pro Gly Gly Pro Pro Gly Ala Leu Gly Val Arg Pro Ser Val Thr Phe Thr Gly Xaa Xaa Leu Xaa Glu Val Xaa Phe Xaa Gly Pro Val Glu Pro Xaa Xaa Ser Pro Gly Xaa Pro Pro Arg Ser Pro Gly Ala Pro Gly Gly Ala Arg Thr Pro Gly Gln Asn Asn Leu Asn Arg Xaa Gly Pro Gln Arg Thr Thr Xaa Val Ser Ala Arg Ala Ser Ile Pro Pro Gly Val Pro Ala Leu Pro Val ~ys Asn Leu Asn Gly Thr Gly Pro Val His Pro Ala Leu Ala Gly Met Thr Gly Ile Leu Leu Cys Ala Ala Gly Leu Pro Val Cys Leu Thr Arg Ala Pro Lys Pro Ile Leu ~is Pro Pro Pro Val Ser Lys Ser Asp Val Lys Pro Val Pro Gly Val Pro Gly Val Cys Arg Lys Thr Lys Lys Lys His Leu Lys Lys Ser Lys Asn Pro Glu Asp Val Val Arg Arg Tyr Met Gln Lys Val Lys Asn Pro Pro Asp Glu Asp Cys Thr Ile Cys Met Glu Arg Leu Val Thr Ala Ser Gly Tyr Glu Gly Val Leu Arg His Lys Gly Val Arg Pro Glu Leu Val Gly Arg Leu Gly Arg Cys Gly His Met Tyr ~is Leu heu Cys Leu Val Ala Met Tyr Ser Asn Gly Asn Lys Asp Gly Ser Leu Gln Cys Pro Thr Cys Lys Ala Ile Tyr Gly Glu Lys Thr Gly Thr Gln Pro Pro Gly Lys Met Glu Phe ~ CA 02238404 l998-0~-22 W O 97/188Z2 PCT~US96/18675 ~/~ 480 485 His Leu Ile Pro His Ser Leu Pro Gly Phe Pro Asp Thr Gln Thr Ile Arg Ile Val Tyr Asp Ile Pro Thr Gly Ile Gln Gly Pro Glu His Pro Asn Pro Gly Lys Lys Phe Thr Ala Arg Gly Phe Pro Arg His Cys Tyr Leu Pro Asn Asn Glu Lys Gly Arg Lys Val Leu Arg Leu Leu Ile Thr Ala Trp Glu Arg Arg Leu Ile Phe Thr Ile Gly Thr Ser Asn Thr Thr Gly Glu Ser Asp Thr Val Val Trp A8n Glu Ile His His Lys Thr Glu Phe Gly Ser Asn Leu Thr Gly His Gly Tyr Pro Asp Ala Ser Tyr Leu Asp Asn Val Leu Ala Glu Leu Thr Xaa Gln Gly Val Ser Glu Ala Ala GGC AAG GCT TGAGGSCCAA GGCTGCCCAC CTTCCCTCCT G~lll~GCCC 2403 Gly Lys Ala TGGTCCGGCA AATGCCTCCT TCGCCAGGTG L~lC~~ A GCCCAGGTTC AGGGCTGGGG 2463 AGGAGCCTGC GGAAGGGGCC GCAGCCATTC AGGGGACTGN ~l~N~AAG TTGGATGAGG 2523 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 620 amino acids ~B) TYPE: amino acid (D) 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 } 5 10 15 Phe Pro Pro Gln Asn Val Ala Arg Val Val Val Trp Glu Cys Leu Asn Glu His Ser Arg Trp Arg Pro Tyr Thr Ala Thr Val Cys His His Ile Glu Asn Val Leu Lys Glu Asp Ala Arg Gly Ser Val Val Leu Gly Gln Val Asp Ala Gln Leu Val Pro Tyr Ile Ile Asp Leu Gln Ser MeL His CA 02238404 1998-0~-22 WO 97/18822 PCT~US96/18675 .
Gln Phe Arg Gln Asp Thr Gly Thr Met Arg Pro Val Arg Arg Asn Phe Tyr Asp Pro Ser Ser Ala Pro Gly Lys Gly Ile Val Trp Glu Trp Glu Asn Asp Gly Gly Ala Trp Thr Ala Tyr Asp Met Asp Ile Cy8 Ile Thr Ile Gln Asn Ala Tyr Glu Lys Gln ~is Pro Trp Leu Asp Leu Ser Ser Leu Gly Phe Cys Tyr Leu Ile Tyr Phe Asn Ser Met Ser Gln Met Xaa Arg Gln Thr Arg Arg Arg Arg Arg Leu Arg Arg Arg Leu Asp Leu Ala Tyr Pro Leu Thr Val Gly Ser Ile Pro Lys Ser Gln Ser Trp Pro Val Gly Xaa Ser Ser Gly Gln Pro Cys Ser Xaa Gln Gln Cys Leu Leu Val Asn Ser Thr Arg Ala Val Ser Asn val Ile Leu Xaa Ser Gln Arg Arg Lys Val Xaa Pro Ala Pro Pro Leu Ser Xaa Pro Xaa Xaa Pro Gly Gly Pro Pro Gly Ala Leu Gly Val Arg Pro Ser Val Thr Phe Thr Gly Xaa Xaa Leu Xaa Glu Val Xaa Phe Xaa Gly Pro Val Glu Pro Xaa Xaa Ser Pro Gly Xaa Pro Pro Arg Ser Pro Gly Ala Pro Gly Gly.Ala Arg Thr Pro Gly Gln Asn Asn Leu Asn Arg Xaa Gly Pro Gln Arg Thr Thr Xaa Val Ser Ala Arg Ala Ser Ile Pro Pro Gly Val Pro Ala Leu Pro Val Lys Asn Leu Asn Gly Thr Gly Pro Val E~is Pro Ala Leu Ala Gly Met Thr Gly I le Leu Leu Cys Ala Ala Gly Leu Pro Val Cys Leu Thr Arg Ala Pro Lys Pro Ile Leu His Pro Pro Pro Val Ser Lys Ser Asp Val Lys Pro Val Pro Gly Val Pro Gly Val Cys Arg Lys Thr Lys Lys Lys His Leu Lys Lys Ser Lys Asn Pro Glu Asp Val Val Arg Arg Tyr Met 385 3gO 395 400 Gln Lys Val Lys Asn Pro Pro Asp Glu Asp Cys Thr Ile cy5 Met Glu Arg Leu Val Thr Ala Ser Gly Tyr Glu Gly Val Leu Arg His Lys Gly 420 425 .430 Val Arg Pro Glu Leu Val Gly- Arg Leu Gly Arg Cys Gly His Met Tyr His Leu Leu Cys Leu Val Ala Met Tyr Ser Asn Gly Asn Lys Asp Gly CA 02238404 1998-0~-22 4~0 455 460 Ser Leu Gln Cys Pro Thr Cys Lys Ala Ile Tyr Gly Glu Lys Thr Gly Thr Gln Pro Pro Gly Lys Met Glu Phe Hifi Leu Ile Pro His Ser Leu Pro Gly Phe Pro Asp Thr Gln Thr Ile Arg Ile Val Tyr Asp Ile Pro Thr Gly Ile Gln Gly Pro Glu His Pro Asn Pro Gly Lys Lys Phe Thr Ala Ary Gly Phe Pro Arg His Cys Tyr Leu Pro Asn Asn Glu Lys Gly Arg Lys Val Leu Arg Leu Leu Ile Thr Ala Trp Glu Arg Arg Leu Ile Phe Thr Ile Gly Thr Ser Asn Thr Thr Gly Glu Ser Asp Thr Val Val Trp Asn Glu Ile His His Lys Thr Glu Phe Gly Ser Asn Leu Thr Gly His Gly Tyr Pro Asp Ala Ser Tyr Leu Asp Asn Val Leu Ala Glu Leu 5g5 600 605 Thr Xaa Gln Gly Val Ser Glu Ala Ala Gly Lys Ala (2) INFORMATION FOR SEQ ID NO:13:
t i ) S~UU~N~ CHARACTERISTICS:
(A) LENGTH: 303 amino acids tB) TYPE: amino acid tD) TOPOLOGy: llnkn~
tii) MOLECULE TYPE: protein -- txi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Ala Ser Ser Ala Gly Ser Ala Ala Ser Gly Ser Val Val Pro Gly Gly Gly Gly Ser Ala Ala Ser Ser Cys Ala Thr Met Ala Leu Ser Thr Ala Gly Ser Gly Gly Pro Pro Val Asn His Ala His Ala Val Cys Val Trp Glu Phe Glu Ser Arg Gly Lys Trp Leu Pro Tyr Ser Pro Ala Val Ser Gln His Leu Glu Arg Ala His Ala Lys Lys Leu Thr Arg Val Met Leu Ser Asp Ala Asp Pro Ser Leu Glu Gln Tyr Tyr Val Asn Val Arg Thr Met Thr Gln Glu Ser Glu Ala Glu Thr Arg Ser Gly Leu Leu Thr 100 lQ5 110 Ile Gly Val Arg Arg Met Leu Tyr Ala Pro Ser Ser Pro Ala Gly Lys CA 02238404 l998-0~-22 WO 97/18822 PCTnUS96/l8675 Gly Thr Lys Trp Glu Trp Ser Gly Gly Ser Ala Asp Ser Asn AGn Asp Trp Arg Pro Tyr Asn Met His Val Gln Cys Ile Ile Glu Asp Ala Trp Ala Arg Gly Glu Gln Thr Leu Asp Leu Cys Asn Thr His Ile Gly Leu Pro Tyr Thr Ile Asn Phe Cys Asn Leu Thr His Val Arg Gln Pro Ser Gly Pro Met Arg Ser Ile Arg Arg Thr Gln Gln Ala Pro Tyr Pro Leu Val Lys Leu Thr Pro Gln Gln Ala Asn Gln Leu Lys Ser Asn Ser Ala Ser Val Ser Ser Gln Tyr Asn Thr Leu Pro Lys Leu Gly Asp Thr Lys Ser Leu His Arg Val Pro Met Thr Arg Gln Gln His Pro Leu Pro Thr Ser His Gln Val Gln Gln G}n Gln His Gln Leu Gln His Gln Gln Gln Gln Gln Gln Gln His ~is His Gln His Gln Gln Gln Gln ~is Gln Gln Gln Gln Gln His Gln Met Gln His His Gln Ile His His Gln Thr (2) INFORMATION FOR SEQ ID NO:14:
( i ) S ~:~U~N~'~ CHARACTERISTICS:
(A) LENGTH: 181 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Arg Lys Pro Pro Lys Lys His Ser Glu Ile Ser Thr Thr Asn Leu Arg ; 5 10 15 Gln Ile Leu Asn Asn Leu Asn Ile Phe Ser Ser Ser Thr Lys His Gln Ser Asn Met Ser Thr Ala Ala Ser Ala Ser Ser Ser Ser Ser Ser Ala Ser Leu His His Ala Asn His Leu Ser His Ala His Phe Ser His Ala Lys Asn Met Leu Thr Ala Ser Met Asn Ser His His Ser Arg Cys Ser Glu Gly Ser Leu Gln Ser Gln Arg Ser Ser Arg Met Gly Ser His Arg Ser Arg Ser Arg Thr Arg Thr Ser Asp Thr Asp Thr Asn Ser Val Lys Ser His Arg Arg Ary Pro Ser Val Asp Thr Val Ser Thr Ty_ Leu Ser CA 02238404 l998-05-22 W O 97/18822 PCTnUS96/1867 His Glu Ser Lys Glu Ser Leu Arg Ser Arg Asn Phe Ala Ile Ser Val Asn Asp Leu Leu A~p Cy8 Ser Leu Gly Ser Asp Glu Val Phe Val Pro Ser Val Pro Pro Ser Ser Leu Gly Glu Arg Ala Pro Val Pro Pro Pro Leu Pro Leu His Pro (2) I~FORMATION FOR SEQ ID NO:15:
( i ) :i~iUU l':N~ ARAt~TF~R T-':TICS:
~A) LENGTH: 224 amino acids (3) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) ~h:UU~N~ DESCRIPTION: SEQ ID NO:15:
Ser Ile Ala Gly Ser Ile Val Gly Val Asp Pro Ala Ser Asp Met Ile Ser Arg Phe Val Lys Val Val Glu Pro Pro Leu Trp Pro Asn Ala Gln Pro Cys Pro Met Cys Met Glu Glu Leu Val His Ser Ala G}n Asn Pro Ala Ile Ser Leu Ser Arg Cys Gln His Leu Met His Leu Gln Cys Leu Asn Gly Met Ile Ile Ala Gln Gln Asn Glu Met Asn Lys Asn Leu Phe Ile Glu Cys Pro Val Cys Gly Ile Val Tyr Gly Glu Lys Val Gly Asn - Gln Pro Ile Gly Ser Met Ser Trp Ser Ile Ile Ser Lys Asn Leu Pro Gly His Glu Gly Gln Asn Thr Ile Gln Ile Val Tyr Asp Ile Ala Ser Gly Leu Gln Thr Glu Glu His Pro Hi8 Pro Gly Arg Ala Phe Phe Ala Val Gly Phe Pro Arg Ile Cyg Tyr Leu Pro Asp Cys Pro Leu Gly Arg 145 15~ 155 160 Lys Val Leu Arg Phe Leu Lys Ile Ala Phe Asp Arg Arg Leu Leu Phe Ser Ile Gly Arg Ser Val Thr Thr Gly Arg Glu Asp Val Val Ile Trp Asn Ser Val Asp His Lys Thr Gln Phe Asn Met Phe Pro Asp Pro Thr Tyr Leu Gln Arg Thr Met Gln Gln Leu Val His Leu Gly Val Thr Asp (2) INFORMATION FOR SEQ ID NO:16:

W O97/18822 PCT~US96/18675 (i) Shyu~ ~A~T~RISTICS:
(A) LENGTH: 20~ amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MO~ TYPE: protein (xi) SEgu~N~ DESCRIPTION: SEQ ID NO:16:
Met Ala Ser Ser Ala Gly Ser Ala Ala Ser Gly Ser Val Val Pro Gly Gly Gly G}y Ser Ala Ala Ser Ser Cys Ala Thr Met Ala Leu Ser Thr Ala Gly Ser Gly Gly Pro Pro Val Asn His Ala His Ala Val Cy8 Val Trp Glu Phe Glu Ser Arg Gly Lys Trp Leu Pro Tyr Ser Pro Ala Val Ser Gln His Leu Glu Arg Ala His Ala Lys Lys Leu Thr Arg Val Met ~o ~eu Ser Asp Ala Asp Pro Ser Leu Glu Gln Tyr Tyr Val Asn Val Arg g0 95 Thr Met Thr Gln Glu Ser Glu Ala Glu Thr Arg Ser Gly Leu Leu Thr Ile Gly Val Arg Arg Met Leu Tyr Ala Pro Ser Ser Pro Ala Gly Lys Gly Thr Lys Trp Glu Trp Ser Gly Gly Ser Ala Asp Ser Asn Asn Asp Trp Arg Pro Tyr Asn Met His Val Gln Cy8 Ile Ile Glu Asp Ala Trp Ala Arg Gly Glu Gln Thr Leu Asp Leu Cys Asn Thr His Ile Gly Leu Pro Tyr Thr Ile Asn Phe Cys Asn Leu Thr His Val Arg Gln Pro Ser Gly Pro Met Arg Ser Ile Arg Arg Thr Gln Gln Ala (2) INFORMATION FOR SEQ ID NO:17:
( i ) S ~:UU ~N~ CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (D) 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 Pro Gly Ala -ô6-W O 97/18822 PCT~US96/18675 (2) INFORMATION FOR SEQ ID NO:18:
( i ) ~UU~N~'~ CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) M~T~cu~ TYPE: peptide (Xi) ~UU~N~'~' DESCRIPTION: SEQ ID NO:18:
Xaa Ser Pro Gly Xaa Pro Pro Arg Ser Pro Gly Ala Pro Gly Gly (2) INFORMATION FOR SEQ ID NO:l9:
Q J~'N~'~ CHARACTERISTICS:
~AI LENGTH: 12 amino acidG
~B~ TYPE: amino acid ~D, TOPOLOGy l-n'- - - "
(ii) MOLECULE TYPE: peptide (xi) SEUU~N~ DESCRIPTION: SEQ ID NO:19:
Ser Ile Pro Pro Gly Val Pro Ala Leu Pro Val Lys (2) INFORMATION FOR SEQ ID NO:20:
( i ) ~'~UU~N~'~' CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (iit MOLECULE TYPE: peptide (xi) SEQu~N~ DESCRIPTION: SEQ ID NO:20:
Arg Ala Pro Lys Pro Ile Leu HiG Pro Pro Pro Val Ser (2) INFORMATION FOR SEQ ID NO:21:
U~N~ CHARACTERISTICS:
(A) LENGTH: 10 amino acid (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (Xi ) ~EQU~:N~ DESCRIPTION: SEQ ID NO:21:

Val Lys Pro Val Pro Gly Val Pro Gly Val 1 5 lO
(2) INFORMATION FOR SEQ ID NO:22:

WO 97/18822 PCTrUS96/1867 (i) ~EUU~N~ CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) M~r~.~ TYPE: peptide (xi) ~uu~N~ DESCRIPTION: SEQ ID NO:22:
Arg Ala Pro Val Pro Pro Pro Leu Pro Leu His Pro Arg Gln Gln l 5 l0 15 (2) INFORMATION FOR SEQ ID NO:23:
(i) ~Q~N~ CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) M~r~cur~ TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Arg Ala Pro Thr Met Pro Pro Pro Leu Pro Pro Val Pro Pro Gln Pro l 5 l0 15 (2) INFORMATION FOR SEQ ID NO:24:
(i) ~Uu~NL~: CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQu~N~ DESCRIPTION: SEQ ID NO:24:
Arg Ala Val Pro Pro Pro Leu Pro Pro Arg Arg Lys Glu Arg i 5 lO
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE r~A~rTERIsTIcs:
(A) LENGTH: 61 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQu~N-~ DESCRIPTION: SEQ ID NO:25:
Cys Thr Ile Cys Met Glu Arg Leu Val Thr Ala Ser Gly Tyr Glu Gly l 5 l0 15 Val Leu Arg HiS Lys Gly Val Arg Pro Glu Leu Val Gly Arg Leu Gly W O 97tl8822 PCT~US96/18675 Arg Cys Gly His Met Tyr His Leu Leu Cys Leu Val Ala Met Tyr Ser Asn Gly Asn Lys Asp Gly Ser Leu Gln Cys Pro Thr Cys (2) INFORMATION FOR SEQ ID NO:26:
(i) ~;UU~N~ r~R~TERIsTIcs:
- ~A) LENGTH: 24 ba~e pairs ~,B) TYPE: nucleic acid .C) ST~N~ N~ S: single ,D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(xi) ~QU~N-~ DESCRIPTION: SEQ ID NO-26:
CACCATCTGC ATGr~Arcr~r TGGT 24 (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CXARACTERISTICS:
(A) LENGTH: 24 base pairs (8) TYPE: nucleic acid tC) STR~NI)~ N~:~S: single (D) TOPOLOGY: linear tii) MOLECULE TYPE: DNA

(Xi ) ~UU~N~ DESCRIPTION: SEQ ID NO:27:
GGC~CA TGTACCACCT GCTG 24 (2) INFORMATION FOR SEQ ID NO:28:
(i) S:QUENCE CHARACTERISTICS:
A) LENGTH: 24 base pairs - IB) TYPE: nucleic acid ~C) STRANDEDNESS: single ;D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(xi) ~'UU~NL'~ DESCRIPTION: SEQ ID NO:28:

(2) INFORMATION FOR SEQ ID NO:29:
(i) ~UU~N~'~ CHARACTERISTICS:
'A) LENGTH: 23 base pairs B) TYPE: nucleic acid C) STRA~ )N~ : single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

W O 97/1882~ PCT~US96/18675 ~Xl) 5EQUENCE DEscRIPTION; SEQ ID NO:29:

(2) INFORMATION FOR SEQ ID NO:30:
(i) ~. iUU~N~'~ CHARACTERISTICS:
~A) LENGTH~ 6 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (Xi ) ~EUU~N~ DESCRIPTION: SEQ ID NO:30:
Cys Met Glu Ar~ Leu Val (2) INFORMATION FOR SEQ ID NO:31:
(i) SEUU~:N~' CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY:.unknown ~ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3}:
Tyr Gly Glu Lys Thr Gly (2) INFORMATION FOR SEQ ID NO:32:
( i ) ~UU~N - '~ CHARACTERIsTICS:
(A) LENGTH- 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (Xi ) ~ U~'N~'~ DESCRIPTION: SEQ ID NO:32:
Thr Ile Arg Ile Val Tyr Asp Ile (2) INFORMATION FOR SEQ ID NO:33:
( i ) ~UU~N~' CHARACTERISTICS
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Gly Phe Pro Arg His Cys Tyr Leu Pro W O 97~18822 PCT~US96/18675 (2) INFORMATION FOR SEQ ID NO:34:
(i) SEQu~N~ CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Arg Arg Leu Ile Phe Thr Ile Gly (2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERIS$ICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Met Cys Met Glu Glu Leu Val (2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Tyr Gly Glu Lys Val Gly (2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Thr Ile Gln Ile Val Tyr Asp Ile WO 97/18822 PCT~US96/18675 (2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUBNCE CHARACTBRISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Gly Phe Pro Arg Ile Cys Tyr Leu Pro l 5 (2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Arg Arg Leu Leu Phe Ser Ile Gly l 5 CA 02238404 l998-05-22 W O 97/18822 PCTt~S96/18675 T ~ Arr" ~' No:PCT/
MICROORGANISMS
Option~l Shoot in connoction with tho ' ~ rPforrod to on po~o 69 lines 1~20 o~ the description A IDENTIFICATION OF DEPOSIT ' Furthor doposits ~n~ idonti6Od on i~n ~dditiomll shoùt ' Name of deposoary institurion Atneric~ n Type Culture Collct~ffon Address of depositary institution (includin~ postal code and country~ ' 12301 Parklawn Drive Rockvillo, MD 20852 US
Dato of deposit ' N~o_, B~ 17,1995 AccrlssionNumber',97341 B ADDI 11 Ni/' l INDICATIONS (Ictv~ bl~t if nr,t ~pplic ble) ~i~ infom~ tion u r~omiDued on~~cr~nte ~tl~ChCd Jhet C DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE ' IdU

D SEPARATE FL ' ' '' OF INDICATIONS ~ (lctve blt~nk u p0t poiic ble) Th~ indic-tion- Iist-d b.lDw will b. ,ubmittr d to th~ Int~rn~tion-l ~ur~-u l~t,,, ~Sp-ci~y th- c-n-r-l n-tur- Ot th- ~ndic.tion- . 0 'Acc-v~lon Numbr~r ol D~po~it~) E~This sheet was received with the I ' ' application when filed (to be checked by thc receivilig Office) (Authorizèd Officer) The date of receipt (from dte applicant) by the I ' ' Bureau was (Authorized Offlcer) Form P~; NROtl34 (January 1981 ) -92.1-

Claims (102)

1. WHAT IS CLAIMED IS:
   
   l. A purified vertebrate Deltex protein.
2. 2. The protein of claim 1 which is a mammalian protein.
3. 3. The protein of claim 1 which is a human protein.
4. 4. The protein of claim 1 having the amino acid sequence depicted in Figure 2A-C (SEQ ID NO:12).
5. 5. 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.
6. 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.
7. 7. A purified fragment of a vertebrate Deltex protein consisting of at least 10 continuous amino acids of a vertebrate Deltex protein, which displays one or more functional activities associated with a full-length vertebrate Deltex protein.
8. 8. The fragment of claim 7 which consists of at least 20 continuous amino acids of the Deltex protein.
9. 9. The protein of claim 5 in which the protein is able to be bound by an antibody to a Deltex protein.
10. 10. A purified protein comprising a fragment of a vertebrate Deltex protein, which fragment binds to a Notch protein or to a molecule comprising the cdc10/SW16/ankyrin repeats of a Notch protein.
11. 11. A purified protein comprising a fragment of a first vertebrate Deltex protein, which fragment binds to a second Deltex protein or to a moleculecomprising a fragment of a second Deltex protein.
12. 12. A purified protein comprising a fragment of a vertebrate Deltex protein, which fragment comprises a SH3-binding domain of the vertebrate Deltex protein.
13. 13. A chimeric protein comprising a functionally active fragment of a vertebrate Deltex protein joined via a peptide bond to an amino acid sequence of a protein other than a vertebrate Deltex protein.
14. 14. The protein of claim 13 in which the fragment binds to a Notch protein or to a molecule comprising the cdc10/SW16/ankyrin repeats of a Notch protein.
15. 15. The protein of claim 13 in which the fragment comprises an SH3-binding domain.
16. 16. The protein of claim 13 in which the fragment comprises a zinc finger domain.
17. 17. A purified derivative of the protein of claim 4, which derivative (a) is characterized by the ability to be bound by an antibody to the protein of claim 4, which antibody does not bind to Drosophila Deltex protein, and (b) has one or more insertions, deletions, or substitutions relative to the protein.
18. 18. A purified peptide having an amino acid sequence in the range of 10-35 amino acids, said sequence being a portion of a vertebrate Deltex protein sequence.
19. 19. A purified derivative of the protein of claim 4, which derivative (a) is characterized by the ability to be bound by an antibody to the protein of claim 4, which antibody is unable to bind to Drosophila Deltex protein, and (b) is able to display one or more functional activities of the protein of claim 4.
20. 20. A molecule comprising the sequence of a human Deltex protein.
21. 21. An antibody which binds to a vertebrate Deltex protein, and which does not bind to a Drosophila Deltex protein.
22. 22. The antibody of claim 21 which binds to a human Deltex protein.
23. 23. The antibody of claim 21 which is monoclonal.
24. 24. A fragment or derivative of the antibody of claim 23 containing the binding domain of the antibody.
25. 25. A purified nucleic acid encoding a vertebrate Deltex protein.
26. 26. The nucleic acid of claim 25 which lacks introns.
27. 27. The nucleic acid of claim 25 which encodes a protein having the amino acid sequence depicted in Figure 2A-C(SEQ ID NO:12).
28. 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. 29. A purified nucleic acid complementary to the nucleic acid of claim 25.
30. 30. The nucleic acid of claim 25 which encodes a human Deltex protein.
31. 31. A purified first nucleic acid which is hybridizable to a second nucleic acid under conditions of low stringency, said second nucleic acidcomprising the nucleotide sequence depicted in Figure 2A-C(SEQ ID NO:11), said first nucleic acid comprising at least 110 continuous nucleotides of SEQ ID NO:11.
32. 32. A purified first nucleic acid which is hybridizable 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), said first nucleic acid comprising at least 110 continuous nucleotides of SEQ ID NO:11.
33. 33. A purified first nucleic acid which is hybridizable to a second nucleic acid under conditions of low stringency, said second nucleic acidencoding 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.
34. 34. A purified first nucleic acid which is hybridizable to a second nucleic acid 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.
35. 35. A purified first nucleic acid which is hybridizable to a second nucleic acid under conditions of high stringency, said second nucleic acid comprising nucleotide numbers 500-1044, 1045-1821, or 1822-2380 of SEQ
    ID NO:11.
36. 36. A purified first nucleic acid which is hybridizable to a second nucleic acid under conditions of low stringency, said second nucleic acidcomprising nucleic acids 500-1044, 1045-1821, or 1822-2370 of SEQ ID N0:11.
37. 37. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 25 amino acids of SEQ ID NO:12.
38. 38. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 50 amino acids of SEQ ID NO:12.
39. 39. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 100 amino acids of SEQ ID NO:12.
40. 40. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 150 amino acids of SEQ ID NO:12.
41. 41. A purified nucleic acid comprising a nucleotide sequence encoding a protein comprising the first 230 amino acids of SEQ ID NO:12.
42. 42. A purified nucleic acid comprising 110 continuous nucleotides of SEQ ID NO:11
43. 43. A purified nucleic acid encoding the protein of claim 5.
44. 44. A purified nucleic acid encoding the protein of claim 10.
45. 45. A purified nucleic acid encoding the protein of claim 11.
46. 46. A purified nucleic acid encoding the protein of claim 12.
47. 47. A purified nucleic acid encoding the protein of claim 13.
48. 48. The nucleic acid of claim 25 as contained in plasmid pBS
    hdx as deposited with the ATCC and assigned accession number 97341.
49. 49. A nucleic acid vector comprising the nucleic acid of claim 25.
50. 50. A nucleic acid vector comprising the nucleic acid of claim 26.
51. 51. A recombinant cell containing the nucleic acid vector of claim 49.
52. 52. A recombinant cell containing the nucleic acid vector of claim 50.
53. 53. A method for producing a vertebrate Deltex protein comprising growing the recombinant cell of claim 51, such that the vertebrate Deltex protein is expressed by the cell; and recovering the expressed vertebrateDeltex protein.
54. 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.
55. 55. A pharmaceutical composition comprising a therapeutically effective amount of a vertebrate Deltex protein; and a pharmaceutically acceptable carrier.
56. 56. The composition of claim 55 in which the vertebrate Deltex protein is a human Deltex protein.
57. 57. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 7; and a pharmaceutically acceptable carrier.
58. 58. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 10; and a pharmaceutically acceptable carrier.
59. 59. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 11; and a pharmaceutically acceptable carrier.
60. 60. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 12; and a pharmaceutically acceptable carrier.
61. 61. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 13; and a pharmaceutically acceptable carrier.
62. 62. A pharmaceutical composition comprising a therapeutically effective amount of a derivative of a vertebrate Deltex protein, which derivative is characterized by the ability to bind to a Notch protein or to a molecule comprising the cdc10/SW16/ankyrin repeats of a Notch protein.
63. 63. A pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid encoding a vertebrate Deltex protein; and a pharmaceutically acceptable carrier.
64. 64. A pharmaceutical composition of claim 63 in which the Deltex protein is a human Deltex protein.
65. 65. A pharmaceutical composition comprising a therapeutically effective amount of an antibody which binds to a vertebrate Deltex protein but not to a Drosophila Deltex protein, or a fragment or derivative of the antibody containing the binding domain thereof; and a pharmaceutically acceptable carrier.
66. 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 molecule which antagonizes the function of a vertebrate Deltex protein.
67. 67. A method according to claim 66 in which the disease or disorder is a malignancy 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. 68. The method according to claim 66 in which the disease or disorder is cervical cancer.
69. 69. The method according to claim 66 in which the disease or disorder is breast cancer.
70. 70. The method according to claim 66 in which the disease or disorder is colon cancer.
71. 71. The method according to claim 66 in which the malignancy is selected from the group consisting of melanoma, seminoma, and lung cancer.
72. 72. The method according to claim 67 in which the subject is a human.
73. 73. The method according to claim 66 in which the molecule is a]
    antibody to vertebrate Deltex or a derivative of said antibody containing the binding domain thereof, which antibody does not bind to Drosophila Deltex.
74. 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.
75. 75. The method according to claim 66 in which the molecule is a protein comprising the SH3 binding domain of a vertebrate Deltex protein.
76. 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. 77. The method according to claim 66 in which the molecule is an oligonucleotide which (a) consists of at least six nucleotides; (b) comprises a sequence complementary to at least a portion of an RNA transcript of a vertebrate deltex gene;
    and (c) is hybridizable to the RNA transcript.
78. 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 pharmaceutically effective amount of a molecule which promotes the function of a vertebrate Deltex protein.
79. 79. A method of treating or preventing a malignancy in a subject comprising administering to a subject in need of such treatment or prevention aneffective amount of a vertebrate Deltex protein.
80. 80. The method according to claim 79 in which the Deltex protein is a human Deltex protein.
81. 81. A method of treating or preventing a malignancy in a subject comprising administering to a subject in need of such treatment or prevention aneffective amount of the nucleic acid of claim 25.
82. 82. A method of treating or preventing or malignancy in a subject comprising administering to a subject in need of such treatment or prevention aneffective amount of the antibody of claim 21.
83. 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 oligonucleotide, which oligonucleotide (a) consists of at least six nucleotides; (b) comprises a sequence complementary to at least a portion of an RNA transcript of the vertebrate deltex gene; and (c) is hybridizable to the RNA transcript.
84. 84. An isolated oligonucleotide consisting of at least six nucleotides, and comprising a sequence complementary to at least a portion of anRNA transcript of a vertebrate deltex gene, which oligonucleotide is hybridizable to the RNA transcript.
85. 85. A pharmaceutical composition comprising the oligonucleotide of claim 84; and a pharmaceutically acceptable carrier.
86. 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. 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 thepatient to bind to a vertebrate Deltex protein, in which an increase or decrease in the ability of the Notch protein 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. 88. A method of identifying a molecule that inhibits or reduces the binding of a vertebrate Deltex protein to a Notch protein, comprising:
    (a) contacting (i) a Notch protein or fragment thereof that mediates binding to a Deltex protein, and (ii) a vertebrate Deltex protein 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 (b) 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. 89. The method of claim 88 in which the Deltex protein is a human Deltex protein.
90. 90. A method of inactivating Notch function in a cell comprising introducing into the cell a molecule, said molecule comprising (a) a vertebrate Deltex protein or portion thereof that mediates binding to a Notch protein; and (b) a protease or proteolytically active portion thereof.
91. 91. A method for the expansion of a precursor cell comprising contacting the cell with an amount of a vertebrate Deltex portion 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. 92. The protein of claim 1, which protein has the amino acid sequence depicted in Figure 2A-C (SEQ ID NO:12); or (a) is able to be bound by an antibody to a human Deltex protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the double stranded human Deltex nucleotide sequence contained in plasmid pBS hdx as deposited with the ATCC and assigned accession number 97341.
93. 93. A purified fragment of the protein of claim 3, said fragment (a) 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 (b) able to be bound by an antibody to a human Deltex protein having said sequence.
94. 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), (b) 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 (c) is able to be bound by an antibody to said human Deltex protein.
95. 95. A purified fragment of the protein of claim 3, said protein having 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, and (b) is able to be bound by an antibody to said human Deltex protein.
96. 96. A chimeric protein comprising a functionally active 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), and (b) is joined via a peptide bond to an amino acid sequence of a protein other than said human Deltex protein.
97. 97. A purified fragment of the protein of claim 3, said protein having 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 cdc10/SW16/ankyrin repeat binding domain of the protein, or the Deltex binding domain of the protein, and (b) is able to bind to an antibody to said human Deltex protein.
98. 98. A molecule comprising a fragment of the protein of claim 3, in which said fragment 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); or said fragment (a) is able to be bound by an antibody to a human Deltex protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the double stranded human Deltex nucleotide sequence contained in plasmid pBS hdx as deposited with the ATCC and assigned accession number 97341.
99. 99. The protein or molecule of claim 92 or 98, in which the first nucleic acid is hybridizable to the second nucleic acid under highly stringent conditions.
100. 100. The protein or molecule of claim 92 or 98, in which the first nucleic acid is hybridizable to the second nucleic acid under low stringency conditions.
101. 101. The protein, fragment, derivative, or molecule of any one of claim 9, 92-95, or 97-100, in which the antibody does not bind to Drosophila Deltex protein.
102. 102. A pharmaceutical composition comprising a the therapeutically effective amount of the protein, fragment, or molecule of any one of claim 92-101;
     and a pharmaceutically acceptable carrier.