ZA200203366B - Nucleic acids encoding endotheliases, endotheliases and uses thereof. - Google Patents

Description

NUCLEIC ACIDS ENCODING ENDOTHELIASES, ENDOTHELIASES AND USES

THEREOF

RELATED APPLICATIONS

Benefit of priority to the following applications is claimed herein:

U.S. provisional application Serial No. 60/166,391 to Edwin L. Madison and

Edgar O. Ong, filed November 18, 1999 entitled "NUCLEOTIDE AND PROTEIN

SEQUENCES OF PROTEASE DOMAINS OF ENDOTHELIASE AND METHODS

BASED THEREON"; and U.S. provisional application Serial No. 06/234,840 to

Edwin L. Madison, Edgar O. Ong and Jiunn-Chern Yeh, filed September 22, 2000, entitled "NUCLEIC ACID MOLECULES ENCODING TRANSMEMBRANE

SERINE PROTEASES, THE ENCODED PROTEINS AND METHODS BASED

THEREON."

This application is also related to U.S. provisional application Serial No. - 60/179,982, to Edwin L. Madison and Edgar O. Ong, filed February 3, 2000, entitled "NUCLEOTIDE AND PROTEIN SEQUENCES OF A TRANSMEMBRANE

SERINE PROTEASE AND METHODS BASED THEREOF"; U.S. provisional application Serial No. 60/183,542, to Edwin L. Madison and Edgar O. Ong, filed

February 18, 2000, entitled "NUCLEOTIDE AND PROTEIN SEQUENCES OF A

TRANSMEMBRANE SERINE PROTEASE AND METHODS BASED THEREOF"; U.S. provisional application Serial No. 60/213,124, to Edwin L. Madison and Edgar O.

Ong, filed June 22, 2000, entitled "NUCLEOTIDE AND PROTEIN SEQUENCES

OF A TRANSMEMBRANE SERINE PROTEASE AND METHODS BASED

THEREOF"; U.S. provisional application Serial No. 60/220,970, to Edwin L.

Madison and Edgar O. Ong, filed July 26, 2000, entitled "NUCLEOTIDE AND

PROTEIN SEQUENCES OF A TRANSMEMBRANE SERINE PROTEASE AND

METHODS BASED THEREOF."

Where permitted the above-noted provisional applications are incorporated by reference in their entirety.

05-12-2001 BE ki US003180¢ a h J

Co FIELD OF INVENTION TE

Nucleic acid molecules that encode proteases and portions thereof, particularly protease domains, that are expressed endothelial cells and the use of the proteases and nucleic acids in methods of screening} Also provided are § prognostic, diagnostic and therapeutic methods using the proteases and domains thereof and the encoding nucleic acid molecules. -

BACKGROUND OF THE INVENTION EE . :

Angiogenesis is the generation of new blood vessels from parent - - © microvessels. Controlled and uncontrolled angiogenesis proceed in 2 similar. 70 manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukoeytes.

The endothelial cells, which line the lumen of biood vessels, then protrude. .~ - through the basement membrane. Angiogenic stimulants induce the :endothelial : ; a ’ 18 cells to migrate through the eroded. basement mernbrane. The rhigrating célls Ty Ee N rT meet "form a “sprout” off the parent blood vessel, wheré'thie endothelial cells undergo = 1% == . 130s

RE mitosis and proliferate. The endothelial sprouts mérgeswith. each other:to form... ~~ capillary loops, creating the new blood vessel. | ER A A :

Angiogenesis, modulators and associated diseases - ~~. - :

Angiogenesis is highly regulsted by a system Of GHGICHHs Sumatra x mama ws and inhibitors. Known examples of angiogenesis stimulators include’ certain - : - growth factors, cytokines, proteins, peptides, carbohydrates and lipids: (Norcby, :

APMIS, 105:417-437 (1997); Polverini, Crit. Rev. Oral. Biol. Med., 6:230-247 - {1 995). A variety of endogenous and exogenous arigiogenesis. inhibitors are . known in the art (Jackson et al., FASEB, 11:457-465 (1997); Notrby, APMIS, 105:417-437 (1997); and O'Reilly, Investigational New Drugs, 15 5-13 (1 997)).

In adult organisms, capillary endothelial cells divide relatively infrequently.

When triggered by appropriate signals, e.g., in response to hormonal signals during menses or following the release of [pro-angiogenic mediators sequestered in the extracellular matrix, endothelial cells lining venules will systematically |. degrade their basement membrane and proximal extracellular matrix, migrate directionally, divide, and organize into new functioning capillaries, within a

Empf .zeit:05/12/2001 12:34 Empf.nr 2972 P.018

AMENDED SHEET

05-12-2001 co Ee US0031803 . . 3 | « _ 2. matter of days (Polvarini, Crit. Rev. Oral. Biol. Med., §:230-247 {1995}. This dramatic amplification of the microvasculature is nevertheless temporary, for as rapidly as the new capillaries are formed, they virtually disappear within a matter ‘of days or weeks, returning the tissue microvasculature to its stafus guo. itis this feature of transient growth and regression of capillaries that primarily distinguishes physiological angiogenesis from pathological one (Polverini, Cr.

Rev. Oral, Biol. Med., 5:230-247 (1995)). In contrast, pathelogical angiogenesis is caused by a shift in the net balance between stimulators and inhibitors of : Co angiogenesis, e.g., dug to the overproduction of normal or aberrant forms of angiogenic mediators, or due to a relative deficiency in inhibitors of this process (Polverini, Crit. Rev. Oral. Biol. Med., 6:230-247 (1995). . i . . : Angiogenesis is essential for normal placental, smbryonic, fetal and post- natal development and growth, but almost never occurs physiologically in = - aduithood except In very specific restricted situations. For example, :

Lo. . 15 angiogenesis _is normally observed in wound healing, :fetal and émbryonal:. - iwi. ERE Ee a Co. ‘development and formation of the corpus luteum, -endometriam-and placenta. ce

RTT Angiogenesis in the adult Is often associated with disease. States. © .. ivi te gino linen

Persistent, unregulated angiogenesis oes in a multiplicity. of disease Te states, tumor metastasis and abnormal growth by, endothelial cells and supports mn veri ww B80 th pathcloyisal Yamsge sei hese coins. The GIVE SE PEIOTOGICG) = TEE I TTR an, disease states in which unregulated angiogenesis is present have been grouped = together as angiogenic dependent or angiogenic associated diseases,” - - : - .

The contral of angiogenesis is altered in certain disease states and, in many cases, the pathological damage associated with the disease Is related to uncontrolled angiogenesis (see generally Norrby, APMIS, 105:417-437 (1 997); and O'Reilly, /nvestigational New Drugs, 15:5-13 (1997)). Thus, angiogenesis - is involved in the manifestation or progress of various diseases, for example, various inflammatory diseases, such as rheumatoid arthritis, psoriasis, diaborid - retinopathies, certain ocular disorders, including recurrence of pterygii, scarring excimer laser surgery and glaucoma filtering surgery, various disorders of the. anterior eye, cardiovascular disorders, chronic inflammatory diseases, wound repair, circulatory disorders, crest syndromes, dermatological disorders (see,

Empf .zei t:05/12/2001 12:34 Enpf nr .2972 P.019

AMENDED SHEET

05-12-2001 : US003180. bs-peC—2em1 11:37 FROM BOUT WADE TENNANT TO passes Pe? : a ~ e.g.. U.S. Patent Nos. 5,593,990, 5,629,327 and 5,712,239 Tlgand notably oo cancer, including solid neoplasms and vascular tumors. - Several lines of direct evidence indicate that angiogenesis is essential for the growth and persistence of : solid tumors and their metastases. EEE ’

Thus, it is clear that angiogenesis plays a major role: in the metastasis of cancer and in the pathology of a variety of other disorders.” ‘Repressing. : eliminating or modulating this activity, should impact the etiology ‘of these ~~ . diseases and serve as a point of therapeutic intervention. In the disease state, : prevention of angiogenesis could avert the damage caused by the invasion of the new microvascular system. Therapies directed at control of the angiogeitic: : processes could lead to the abrogation or mitigation of these diseases.

Soa . . . Hence there is a need to develop therapeutics that target angiogenesis ancl: he, Li oo wo i oo B modulate, particularly, inhibit. aberrant or uncontrolled angiogenesis: = Therefore: *"* Sa

Ce is-an.object herein to provide assays for identification of suchagents. It is Tela Te i5 ‘also an object herein to provide nucleic acids encoding the proteins and” . 0 © .. .. polypeptides and aiso to provide the proteins and polypeptides that are involved -

SUMMARY OF THE INVENTION Vo ee ~

Provided herein are a cias§mermbrans proteases that are expressed on cells, particularly endothelial ceils, and that participate .in angiogenesis. Also provided herein are methods of modulating the activity of the proteases and’. . screening for compounds that modulate the activity thereof. Such modulation includes inhibiting, antagonizing agonizing and othérwise altering the activity of the protease. Of particular interest is the extracellular doniain of these proteases that includes the proteolytic portion of the protein. in particular, proteases, especially protease domains thereof, of proteins designated herein as endotheliase proteins are provided. Also provided bref herein are nucleic acids encoding the protease domains of the endotheliases and also the full-length endothefiases. in exemplary embodiments endotheliases, designated 1 and 2, particularly the protease domains thereof are provided. fA

Full-length endotheliase 2 variants are also provided. Nucleic acid molecules encoding the endotheliases are provided as are vectors containing the nucleic

AMENDED SHEET

05-12-2001 a | US0031803 'GSDEC-ZAP1 11:37 FROM BOLLT URDE TENNANT TO @P498323934465 P.21 i 8- : . acids and cells containing the vectors. Preferred endotheliases are those that. include the sequence of amino acids set forth in SEQ ID No. 2, 4, 6 or 22, and particularly the protease domain encoding portions (SEQ ID No. 2, amino acids 321.5851 SEQ ID No. 4, and amino acids 321-556 of SEQ ID No. 6; aming 85 acid 320 of each SEQ ID is optionally included as part of the protease domain).

Also provided are nucleic acid molecules. that hybridize, preferably along their full length, to a nucleic acid molecule having the nucleotide sequence of nucleotides set forth in SEQ. ID NO. 1, 3.5 or 22 or portions thereof, preferably, the protease domain encoding portions or a sufficient portion thereof such that the © 10 encoded protein exhibits protease activity. Co a. in preferred embodiments, the isolated nucleic acid molecule hybridizes to «7 . thenucleic acid having the nuclectide sequence set forth in the SEQ.ID.Ne. 1,3... ::.a

EAR or§ Under high stringency conditions.” in another prefsied. embodiment, the wire i . ©. 0. isolated nucleic-acid molecule contains sequence of-nucieotides.setforth:in: Lo Lo

SEQ. ID No.1,3or5. Nooo - In ane embodiment; the isolated nucleic acid molecule contains only the ev apres co ie ee BEAYENCE. OL PR aay in SEQ. 1D.MNe 1.3 or.8 or.portinns Thereof, , cum amis including molecules [eortairy 14. 16, 30, 100 or up to the full length sequence - thereof. in another embodiment, an isolated nucleic acid molecule has a nucleotide sequence complementary to the nucleotide sequence encoding the protease domain of the endotheliase is provided.

Vectors and plasmids containing the above-noted nucleic acid molecules

Qe provided. Celis containing the plasmids or vectors are provided herein. More preferably, the cell is a bacterial cell, a yeast cell, a fungal celi, a plant cell, . insect cell or an ahimal cell.

Cells that contain the nucleic acids, and preferably express the encoded endotheliases or portion thereof, preferably the protease domain thereof, are also ) provided. The cells and vector can be engineered to express the endotheliase oh the surface; such cells preferably contain the full-length coding portion of an endotheliase. In other embodiments, the cells and vectors are designed to secrete the encoding endotheliase or protease domain thereof, such as by including a sequence of nucleotides that directs secretion.

AMENDED SHEET

Front a1 4° NEI, apr 1rie2n Conf —. e070 PN NAA

© 05-12-2001 } | US003180¢ bs-DEC-2721 11:37 FROM BOULT WADE TENNANT = . 10 B245BI23334465 P.22 . 6. ~

Methods for producing a protease domain of an endotheliase. or an oT endotheliase by growing the above-described cells under conditions ‘whereby the ) _ encoded protease domain of the endotheliase is expressed by the cell, and ST recovering the expressed protease domain protein, are-provided herein... =~ :

Also provided herein are antibodies that specifically bind, particularly ~~ immunospecifically, to the endotheliases, preferably to the protease domains of.- - the endotheliases. Cees

Compositions, including pharmaceutical containing the endotheliase or toe ' protease domains of the endotheliase are provided. -Combinations; kits ~ Co containing the combinations and articles of manufacture containing the-.. ... Co compositions or proteins are also provided. In one'embodiment. combinations w..... .. -. areprovided herein that include an inhibitor of an endotheliase -or-an: inhibitor.-of.: STRENET

ER: on LL the protease activity. thereof and another anti angiogenic treatment or agents: win Sortie: coe - oe | “The. endotheliase inhibitor and the anti-angiogenic “gentican be formulatedin'a “dl st a 45 single pharmaceutical composition or each cari be formulated ina separate SE pharmaceutical composition. Kits containing the combinations are:provided: So : endotheliases and bearing the genes encoding an endotheliase; preferably under. - TL. control of a non-native promotor control are provided. = = 7 : ce

Conjugates of the endotheliases or protease domain portion thereof, such as conjugates of an endotheliase with a targeting agent to direct the Co - endotheliase to a particular ceilgior tissuagfand conjugates witha linker or : a detection moiety for linkage to a solid support and. detection; respectively, sre provided. Methods using the conjugates are also provided. For exampie, the endatheliases or protease domain thereof may be linked toa cell specific =. targeting agent, particularly an agent, such as a growth factor or monoclonal antibody that binds to a ceil surface protein that results in internalization of the conjugate or endotheliase portion thereof. These conjugates are administered with, before or following administration of a prodrug, such as a daunomycin or other cytotoxic agent. The prodrug is designed to be activated by the } endotheliase. Upon binding to the targeted celis and internalization, the endotheliase Anteracis-witly activates the prodrug, thereby providing a means for

AMENDED SHEET

EE A Ta Rea aL EE Lat Ta R Feed oo. 20ND 0D NOD

05-12-2001 oo | US0031805 . oS-DEC-2001 11:37 FROM BOLT WADE TENNANT TO BB49B83 23934465 P.23 < oR . target specific activation of a drug and selective killing or inhibition of targeted cells. Aliso provided herein are modulators of the activity of the endotheliase or protease domain. He :

Further provided herein are prognostic, diagnostic and therapeutic screening methods using the protease domains of the endotheliase and the nucleic acids encoding such domains. In particular, the prognostic, diagnostic and therapeutic screening methods are used for preventing or treating, or for finding agents useful in preventing or treating, diseases or disorders associated } with aberrant level of angiogenesis.

Methods for screening for compounds that modulate the activity of the endotheliase fipprovided. In vitro assays in which the compounds are identified one by contacting them .with the endotheliase or protease domain. thereof and:a-- REE

Cea - substrate for the endotheliase are provided: A-changetin'the amount-of:" = - seu rina . 3 B : B Coe substrate cleaved in the. presence of the compounds’ cérfipared tothe absence-of 0. 0 the compound indicates that the compound modulates the sctivity of the

EER endotheliase. ‘Such. compounds are selected for further analyses or for use 10 -. om... inhibit the activity of the endotheliase, such as incr or dgonists, Thein vitro assays can be performed in liquid phase or of solid phase substrates by I - linking the endotheliase. or protease domain thereof directly or via a linker to 2 - sold support. Cell-based screening assays are also provided. The compounds can also be identified by contacting the substrates with a cell that expresses the endotheliase or the extracellular domain or proteolytically active portion thereof.

In one embodiment, the method for identifying a modulator of an endotheliase includes: a) contacting an endotheliase with a substrate of the endotheliase, and detecting the proteolysis of the substrate, whereby the activity of the endotheliase is assessed; b) contacting the endotheliase with a substrate of the endotheliase in the presence of a test substance, and detecting the : proteolysis of the substrate, whereby the activity of the endotheliase is assessed; and c¢) comparing the activity of the endotheliase assessed in steps a) and b), whereby a difference in activity measured in step a) from the activity . measured in step b) indicates that the test substance modulates the activity of

AMENDED SHEET

— ’ CL eA PAA ANY MeN Cem 0 0070 ND NND

° WO 01/36604 PCT/US00/31803 the endotheliase. In a preferred embodiment, a plurality of the test substances are screened for simultaneously in the above screening method.

In another embodiment, the endotheliase to be screened against is isolated from a target cell and the test substance is a therapeutic compound, A difference of the endotheliase activity measured in the presence and in the absence of the test substance indicates that the target cell responds to the : therapeutic compound. For example, detection of protease domains in the blood or other body fluid can be indicative of cancer, particularly metastatic cancer.

Method for diagnosing diseases or disorders by detecting levels of endotheliases or protease domains thereof or encoding nucleic acids in body tissues and body fluids are provided. The methods require testing a body fluid or tissue for the presence or level of an endotheliase or protease domain thereof and assessing whether the level is higher or lower than in non-disease state.

For example, methods of diagnosing a disease or disorder by detecting an aberrant level of an endotheliase in a subject. The methods include the steps of measuring the level of the DNA, RNA, protein or functional activity of an endothelial endotheliase, in a sample derived from a subject, where an increase or decrease in the level of the DNA, RNA, protein or functional activity of the endotheliase, relative to the level of the DNA, RNA, protein or functional activity found in an analogous sample not having the disease or disorder indicates the presence of the disease or disorder in the subject.

In another embodiment, a method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder associated with undesired and/or uncontrolled angiogenesis in a subject is provided. In this method, the level of DNA, RNA, protein, or functional activity of an endotheliase in a sample derived from the subject, where an increase in the level of the DNA,

RNA, protein, or functional activity in the sample, relative to the level of the

DNA, RNA, protein, or functional activity found in an analogous sample not having the undesired and/or uncontrolled angiogenesis, indicates the presence of the undesired and/or uncontrolled angiogenesis.

In another embodiment, a method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder associated with deficient angiogenesis in a subject is provided herein. This method . includes measuring the level of DNA, RNA, protein, or functional activity of an . endotheliase in a sample derived from the subject, where a decrease in the level .. of the DNA, RNA, protein, or functional activity in the sample, relative to the 5: level of the DNA, RNA, protein, or functional activity found in an analogous = sample not having the deficient angiogenesis, indicates the presence of the deficient angiogenesis. : Also provided are methods for treatment or prophylaxis of a disease or disorder associated with undesired and/or uncontrolled angiogenesis in a 10: mammal by administering to a mammal an effective amount of an inhibitor of an . endotheliase, whereby the disease or disorder is treated or prevented are + provided. The endotheliase inhibitor used in the treatment or prevention is = preferably administered with a pharmaceutically acceptable carrier or excipient = and the mammal treated is a human. Preferred inhibitors herein are antibodies - that specifically bind to the endotheliase or to the protease domain thereof, and inhibitors of translation of the encoding mRNA and inhibitors of transcription of the mRNA, including antisense nucleic acid molecules.

In another embodiment, the treatment or prevention method also includes administering an additional anti-angiogenic treatment(s) or agent(s} in combination therewith. The further anti-angiogenic agent or treatment may be administered simultaneously, subsequently or prior to administration of the endotheliase inhibitor, which can be, for example, an antibody or a fragment or derivative thereof containing the binding region thereof against the endotheliase, an antisense nucleic acid encoding the endotheliase, or a nucleic acid containing at least a portion of a gene encoding the endotheliase into which a heterologous nucleotide sequence has been inserted such that the heterologous sequence inactivates the biological activity of at least a portion of the gene encoding the endotheliase, in which the portion of the gene encoding the endotheliase flanks the heterologous sequence so as to promote homologous recombination with a genomic gene encoding the endotheliase. - :

The undesired angiogenesis to be treated or prevented is associated with disorders and diseases that include, but are not limited to, solid neoplasms,

05-12-2001 US003180¢ . 25-DEC-2001 11:37 FROM BOULT WADE TENNANT TO BB4SEIZTIAAES p.24 : vr : -10- vascular malformations find cardiovascular disorders, chronic inflammatory diseases, aberrant wound repairs, such as observed following glaucoma filtering surgeries and excimer laser eye surgery, circulatory disorders, crest syndromes, oC dermatological disorders and ocular disorders. The vascular malformations and § cardiovascular disorders to be treated or prevented include angiofibroma, angiolipoma, atherosclerosis, restenasis/reperfusion injury, arteriovenous malformations, hemangiomatosis and vascular adhesions, dyschondroplasia “with vascular hamartomas (Fafuccl’s syndrome}, hereditary hemorrhagic telan- giectasia {(Rendu-Oslet-Weber syndrome), or Von Hipple Lindau syndrome; the ] 10 chronic inflammatory diseases to be treated or prevented are diabetes mellitus, . hemophiliac joints, Inflammatory bowel disease, nonhealing fractures,

Ce _. .... periodontitis {rapidly progressing and juvenile), psoriasis; rheumatoid arthritis; =~ <5... & <- — an Chet --Venous stasis.ulcers, granulations-bumns, hypertrophic: scars=liver circhosis;=" IT ne veo. 1. .osteoradionecrosis, postoperative. adhesions, 'pyogeriic-graniuloma, -or. systemic: -. EE ) 15 sclerosis; the circulatory disorder to be wreatéd,or prevented is Raynaud's ’

X phenomenon; the crest syndromes to be treated or prevented are calcinosis, -- oo . ...._..... esophageal, dyomotioty, sclerodactyly and refute the dermatclogleal disorders to be treated or prevented are systemic vasculitis, scleroderma; CLT pyoderma gangrenosum, vasculopathy, venous, arterial ulcers, Sturge-Weber = syndrome. Port-wine stains, blue rubber bleb nevus syndrome, Klippel .

Trenaunay-Weber syndrome or Osler-Weber-Rendu syndrome; and the ocular disorders to be treated or prevented are blindness caused by ocular neovascular disease, corneal graft neovascularization, macular degeneration in the eye, neovascular glaucoma, trachoma, diabetic retinopathy. ‘myopic degeneration, retinopathy of prématurity, retrolental fibroplasia, or corneal neovascularization.

In another embodiment, a method for treating or preventing a disesss or disorder associated with deficient angiogenesis in 2 mammal is provided herein.

The method includes administering to a mammal an effective amount of an endotheliase protein, a nucleic acid encoding the protein, and a nucleic acid encoding a derivative or analog of the protein that is active in promoting angiogenesis, whereby the disease or disorder is treated or prevented. In a preferred embodiment, the endotheliase protein, a derivative or analog of the

AMENDED SHEET

~ . BP EY Wins a ERs Lan Com me 2070 1) MOA

05-12-2001 a | | US0031805 . bS-pEC-2801 11:38 FROM BOLT WADE TENNANT TO AB4SBIZ3IF4465 P.25 . protein, a nucleic acid encoding the protein, and a nucleic acid encoding a derivative or analog of the protein is administered with & pharmaceutically ’ acceptable carrier or excipient. The mammal to be treated, preferably, is a human. in another embodiment, the treatment or prevention method further : 8 includes administering an pro-angiogenic treatment or agent.

Also provided are methods for treating or preventing a disease or disorder associated with deficient or defective angiogenesis in a mammal, such as a human, by administering to a mammal an effective amount of an endotheliase or a catalytically or functionally. active portion, such as a protease domain, thereof, ~ 10 whereby the disease or disorder is treated or prevented or the symptoms are ameliorated. The method can further include, administeringfary pro-angiogenic

SE EEE treatment or. agent that promotes angiogenesis simultaneously; prior to or-after. .

DERE To administration of the endotheliase or portion thereof «= caus etn pS ere mgs ie

EI cee. =... Among the endotheliases provided herein are those whose activity is: Ee upregulated during angiogenesis. Such endothéiiases can serve targets for -. activation of prodrugs that are designed to be selectively activated by these Cee ee eos ww... . . endetheliases. Hence therapeutic methods for tating disorders that involve endothelial cells that express such upregulated endotheliases are provided. . oo

Also provided herein are transgenic non-human animals. in these - animals, an endogenous gene of an endotheliase is deleted or inactivated by homologous recombination or insertional mutagenesis of the animal or an - ancestor thereof. oo

Also provided herein are articles of manufacture that contain: a) : packaging material; b) an endotheliase or protease domain thereof. ora composition containing the endotheliase or protease domain thereof; and c) : label indicating that the article is for use in identifying a modulator of the activity of an endotheliase in a sample or for diagnostic, therapeutic or drug screening use. "BRIEF DESCRIPTION OF DRAWING

Figure 1 depicts the domain organization of the endotheliase 2 splice variants. Each variant includes three repetitive sequences composed of

AMENDED SHEET

ASPAGTPPGRASP (SEQ ID NO. 14) a sequence motif for N-myristoylation modification just before the transmembrane domain.

DETAILED DESCRIPTION OF THE INVENTION

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications and sequences from GenBank and other data bases referred to herein are incorporated by reference in their entirety.

As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the [IUPAC-IUB Commission on

Biochemical Nomenclature (see, (1972) Biochem. 11:942-944).

As used herein, angiogenesis is intended to broadly encompass the totality of processes directly or indirectly involved in the establishment and maintenance of new vasculature (neovascularization), including, but not limited to, neovascularization associated with tumors.

As used herein, anti-angiogenic treatment or agent refers to any therapeutic regimen and compound, when used alone or in combination with other treatment or compounds, that can alleviate, reduce, ameliorate, prevent, or place or maintain in a state of remission of clinical symptoms or diagnostic markers associated with undesired and/or uncontrolled angiogenesis. Thus, for purposes herein an anti-angiogenic agent refers to an agent that inhibits the establishment or maintenance of vasculature. Such agents include, but are not limited to, anti-tumor agents, and agents for treatments of other disorders associated with undesirable angiogenesis, such as diabetic retinopathies, restenosis, hyperproliferative disorders and others.

As used herein, pro-angiogenic agents are agents that promote the establishment or maintenance of the vasculature. Such agents include agents for treating cardiovascular disorders, including heart attacks and strokes.

Q

. \ .

As used herein, undesired and/or uncontrolled angiogenesis refers to . pathological angiogenesis wherein the influence of angiogenesis stimulators = outweighs the influence of angiogenesis inhibitors.

As used herein, deficient angiogenesis refers to pathological angiogenesis b.r associated with disorders where there is a defect in normal angiogenesis =z resulting in aberrant angiogenesis or an absence or substantial reduction in angiogenesis.

As used herein, endotheliase refers to a mammalian protein, including humans, that has a transmembrane domain and is expressed on the surface of endothelial cells and includes a protease domain, particularly an extracellular -. protease domain, and is preferably a serine protease. Thus, reference, for . = example, to endotheliase encompasses all proteins encoded by the endotheliase : - gene family, or an equivalent molecule obtained from any other source or that : =» has been prepared synthetically or that exhibits the same activity. The - 15 endotheliase gene family are transmembrane proteases expressed in endothelial cells. These proteases include serine proteases. When more particularity is need, it refers to proteins that have these features and also include a protease domain that exhibits sequence homology to the endotheliases 1 and 2 exemplified herein. Endotheliase 1 and 2, for example exhibit about 40% or 45% identity. Sequence homology means sequence identity along its length when aligned to maximize identity of at least about 25%, 40%, 60%, 80%, 90% or greater number of residues. Sequence homology also is assessed by determining whether the encoding sequences of nucleic acids hybridize under conditions of at least moderate, or for more closely related proteins, high stringency to the nucleic acid molecules provided herein or to those that encode the same proteins but differ in sequence by virtue of the degeneracy of the genetic code. In addition, endotheliases encompass endotheliases with conservative amino acid substitutions, such as those set forth in Table 1, that do not substantially alter its proteolytic activity. Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential

© 05-12-2001 - US003180: : A — 11:38 FROM BOLT WADE TENNANT TO BB49BIZ3IIA4ES P.26 ’ -i4- . regions of a polypeptide do not substantially alter biological activity (see, e.q.,

Watson et al. Molecular Biology of the Gene. 4th Edition, 1987, The (EAR Cummings Pub. co., p.224). Also included within the definition, is the catalytically active fragment of an endotheliase. : :

As used herein, the protease domain of an endotheliase refers to te polypeptide portion of the endothellase. that is the extracellular portion that exhibits protease activity. The protease domain is a polypeptide that includes at least the minimum number of amino acids, generally more than 50 or 100, required for protease activity. Protease activity may be assessed empirically. : 10 . such-as by testing the polypeptide for its ability to act asa protease. - Assays, - CT such as in the assays described in the EXAMPLES, except employing a known

Lan substrate in place ofthe test compounds as described ‘iff the Examples vay be TVET F sees i used. Furthérmions, since proteases, particlis iy Séfine proteases, Rave iT rial Fe co ie .... . . characteristic structures and sequences or motifs; the protease domain may be TT - . 15 readily identified by such structure and sequence or motif. - ~~ + =~ - : Sot Te ; As used herein, the portion of protease Yomsin of endotheliase refers'to oC eet ae ee dE -PrOtEGSE: demain of endotheliase that ie lecsted within or is the.exiracalllar, «crm rgmicen : domain of an endotheliase and exhibits serine proteolytic activity. ‘Hence it is at - least the minimal portion of the extracellular domain that exhibits protealytic : activity as assessed by standard assays. An exemplary protease domain of an endotheliase is set forth in SEQ ID No. 2 and as amino acids 321-688 and 321- 344 of SEQ ID Nos. 4 and 68. Smaller portions thereof that retain protease activity are contemplated. The protease domains vary in size and constitution, including insertions and deletions jn surface loops. Such dornains exhibit conserved Structure, including at least one structural feature, such as the active site triad, primary specificity pocket, oxyanion hole and/or other features of serine protease domains of proteases. Thus, for purposes herein, the protease domain is a portion of an endotheliase, as defined herein, but is homologous in terms of structural features and retention of sequence of similarity or homology the protease domain of chymotrypsin or trypsin. : :

As used herein, homologous means about greater than about 25% sequence identity. By sequence identity, the number of conserved amino acids -

AMENDED SHEET : «aa LRA TY ~ Cond me SUD (IR

. as determined by standard alignment algorithms programs, and used with default gap penalties established by each supplier. Also homology may be assessed by - conserved nucleic acid sequence, which includes anything that hybridizes under at least low stringency conditions and encodes the domain. Similarly nucleic 5. acid sequence alignment programs are commercially available (DNAStar -. "MegAlign" program (Madison, WI) and the University of Wisconsin Genetics

Computer Group (UWG) "Gap" program (Madison WI}}. Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency ali along the length of the nucleic acid of 10... interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule. ) As used herein, recitation that a polypeptide consists essentially of the ) - protease domain means that the only endotheliase portion of the polypeptide is a . protease domain or a catalytically active portion thereof. The polypeptide may . 15 optionally include additional non-endotheliase-derived sequences of amino acids.

As used herein, pro-angiogenic treatment or agent refers to any therapeutic regimen and compound, when used alone or in combination with other treatment or compounds, that can alleviate, reduce, ameliorate, prevent, or place or maintain in a state of remission of clinical symptoms or diagnostic markers associated with deficient angiogenesis.

As used herein, domain refers to a portion of a molecule, e.g., proteins or nucleic acids, that is structurally and/or functionally distinct from other portions of the molecule.

As used herein, protease refers to an enzyme catalyzing hydrolysis of proteins or peptides.

As used herein, serine protease refers to a diverse family of proteases wherein a serine residue is involved in the hydrolysis of proteins or peptides.

The serine residue can be part of the catalytic triad mechanism, which includes a serine, a histidine and an aspartic acid in the catalysis, or be part of the hydroxyl/e-amine or hydroxyl/a-amine catalytic dyad mechanism, which involves a serine and a lysine in the catalysis. Examples of serine proteases include, but are not limited to, chymotrypsin, trypsin, plasmin, thrombin and elastase.

05-12-2001 oo | US0031803 : B5-DEC-2001 11:38 FROM BOT WADE TENNANT : T0 eP498323954465 pP.27

As used herein, catalytic activity refers to the activity of the endotheliase as a proteases. Function of the endothaliase refers to its function in endothelial : cell biology. including promotion of angiogenesis of involvement in angiogenesis.

As used herein, nucleic acids include DNA, RNA and riage tharsof, including protein nucleic acids (PNA) and mixture thereof. - Nucleic acids can be Cees single or double stranded. When referring to probes or primers, single-stranded molecules are contemplated. . N : cen

As used herein, nucleic acid encoding 8 fragment or portion of an - endotheliase refers to a nucleic acid encoding only the recited fragment or . : . 10 portion of endotheliase protein, and not the other contiguoiss portions of the SLT endotheliase as a continuous sequence. : ~ E met Agiused herein, heterologous of foreigfi DNA and RNA‘E(E used” =r =o 0. TT “swe oof 7 Tintérchangéably ‘and refer to DNA or RNA that does’fide Seelr Ratorally as pan of » TF c= wo cs -- the geriome in which'it is present or which is found ir aiocation oF locations in: HEE the genome that differ from that in which it octurs in-naturé., Heterologous © CoE : - - nucleic acid is generally not endogenous to the tell into whiéh it is introduced, ’ a rma ees senses DUE WAS been. obtained. from.another cell.or prep: : ed_syntherically...Genet@ly,.... ..scwimmmsms although not necessarily, such nucleic acid RNA arid proteins’ that are Co not narmally produced by the cell in which itis expressed.’ * Any DNA or RNA - - - that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed is herein encompassed by heterologous DNA.

Heterologous DNA and RNA may aiso encode RNA Or protéins that mediate or alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes.

As used herein, operative linkage of heterologous DNA to regulatory and effector sequences of nucleatides, such as promoters, enhancers, wanscriptional and translational stop sites, and other signal sequences refers to the relationship between such DNA and such sequences of nucleotides. For example, operative linkage of heterologous DNA to a promoter refers to the physical relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA in reading frame. :

AMENDED SHEET

Comf — tae ION] Trae FmDt Ar SMES Ed

05-12-2001 oo : | US003180% -17- CL

As used herein, a sequence complementary to at least a portion of an

RNA, with reference to antisense oligonucieotides, means a sequence having sufficient complementarily to be able to hybridize with the RNA, preferably under - : moderate or high stringency conditions, forming a stable duplex; in the case of : 5 double-stranded endotheliase antisense nucleic acids, a single strand of the . duplex DNA may thus be tested, or triplex formation may be assayed. - The ) ability to hybridize depends on the degree of complementarily and the length of the antisense nucleic acid. Generally, the longer the hybridizing ‘nucleic acid, the - ~~ more base mismatches with®’an endotheliase encoding RNA it can contain and still form a stable duplex (or triplex, as the case may be.” One skilled. in the ant can ascertain a tolerable degree of mismatch by use of standard: procedures to : o. . determine the meiting point of the hybridized complex. : }

To ~ For purposes herein, conservative aming acid substitutions. may be.made- . : : in any of endotheliases or protease domains thereof provided that the. resulting © 1" }5 protein exhibits protease activity. Amino acidisubstititions.@re preferably made. - . STR

TEE es cordance with those set forth in TABLE as follows: suicides em NL a . nt we . ate re ar aan ) ae no . : - : : k - "TABLE 1 I. A . “ . . . . . . . - nig .. : Original residue Conservative substitution =. = © .

Ala (A) | Gly; Ser Co : 20 Arg (R) : Sh Lys :

SUPE Von. AUN , Gin; His :

REN ET al LL tr NEE CASAC ad 3 ts (6) TTD SRN I TY a Te A Bar a rv TLS Hoc IRE AS ASE YET

Gin (Q) © Asn -

Glu (E) , Asp

Gly (G) i : Ala; Pro .

His (H) Asn; Gin "- . We (I) ] Leu; Val

Leu (L) lle; Val . :

Lys (K) Arg: Gin: Glu

Net (M) Leu; Tyr; lle

Phe (F) Met; Leu; Tyr | :

Ser (S) Thr ,

The (T) Ser

Trp (W) Tyr !

Tyr (V) Trp; Phe : Val {V) or lle; Lov } Other substitutions are also permissible and may be determined empirically or in accord with known conservative substitutions. :

Empf .zei t:05/12/2001 12:35 Enpf.nr.:972 P.028 i ;

AMENDED SHEET

\

As used herein, the amino acids, which occur in the various amino acid sequences appearing herein, are identified according to their well-known, three- letter or one-letter abbreviations. The nucleotides, which occur in the various

DNA fragments, are designated with the standard single-letter designations used routinely in the art.

As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.

As used herein, a probe or primer based on a nucleotide sequence disclosed herein, includes at least 10, 14, preferably at least 16 or 30 or 100 contiguous sequence of nucleotides of SEQ ID Nos. 1, 3 or 5.

As used herein, amelioration of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.

As used herein, antisense polynucleotides refer to synthetic sequences of nucleotide bases complementary to mRNA or the sense strand of double stranded DNA. Admixture of sense and antisense polynucleotides under appropriate conditions leads to the binding of the two molecules, or hybridization. When these polynucleotides bind to (hybridize with) mRNA, inhibition of protein synthesis (translation) occurs. When these polynucleotides bind to double stranded DNA, inhibition of RNA synthesis (transcription) occurs.

The resulting inhibition of translation and/or transcription leads to an inhibition of the synthesis of the protein encoded by the sense strand.

As used herein, an array refers to a collection of elements, such as antibodies, containing three or more members. An addressable array is one in which the members of the array are identifiable, typically by position on a solid phase support. Hence, in general the members of the array will be immobilized to discrete identifiable loci on the surface of a solid phase.

As used herein, antibody refers to an immunoglobulin, whether natural or partially or wholly synthetically produced, including any derivative thereof that retains the specific binding ability the antibody. Hence antibody includes any

05-12-2001 SE a. US003180¢ protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. Antibodies include members of any immunoglobulin claims, including IgG, IgM, IgA, IgD and IgE.

As used herein, antibody fragment refers to any derivative of an antibody § that is less then full length, retaining at least a portion of the full-length antibody’s specific binding ability. Examples of antibody fragments include Jout are not limited to, Fab, Fab’, Flab),, single-chain Fvs (scFV), FV, dsFV diabody and Fd fragments, The fragment can include muitiple chains linked together, such as by disulfide bridges. An antibody fragment generally contains at least 40 about 50 amino acids and typically at least 200 amino acids.” ne

As used herein, an Fv antibody fragment is composed of one variable - . heavy domain {V,) and one variable light domain linked by noncovalent interactions. A

As used herein, a dsFV refers to an Fv with an engineered intermolecular } . ... ..35.-. disulfide bond, which stabilizes the Vy-Vi- pairs” iu ioiw 1 1 70 in mabe imi woo

TET ATT Uae used herein, an F(ab), fragment is an antibody fragment ‘that results ET enan

CLT TTT from digestion of an immunoglobulin with pepsin avpH 4.0:4.5; it may be: Car wl recombinantly produced. \ . ) Co As used herein, Fab fragments is an antibhdy fragment that results from

SE "i I digestion” of ‘ih irimunogiobulin with papain; it dy be recormbinstitly™ produces «ws: may me

As used herein, scFVs refer to antibody fragments that contain a variable - light chain (V,} and variable heavy chain (V,) covalently connected by a- Coe polypeptide linker in any order. The linker is of a length such that the two variable domains are bridged without substantial interference. Preferred linkers are (Gly-Ser), residues with some Glu or Lys residues dispersed throughout to increase solubility. i \

As used herein, humanized antibodies refer 10 antibodies that are modified to include human sequences of amino acids so that administration a human will not provoke an immune response. Methods for preparation of such antibodies are known. For example, the hybridoma that expresses the i © monoclonal antibody is altered by recombinant DNA techniques to' express an antibody in which the amina- acid composition of the non-variable regions is .

Enpf .zei 05/12/2001 12:36 Enpf nr .2972 P.029

AMENDED SHEET based on human antibodies. Computer programs have been designed to identify such regions.

As used herein, diabodies are dimeric scFV; diabodies typically have shorter peptide linkers than scFvs, and they preferentially dimerize.

As used herein, humanized antibodies refer to antibodies that are modified to include human sequences of amino acids so that administration to a human will not provoke an immune response. Methods for preparation of such antibodies are known. For example, the hybridoma that expresses the monoclonal antibody is altered by recombinant DNA techniques to express an antibody in which the amino acid composition of the non-variable regions is based on human antibodies. Computer programs have been designed to identify such regions.

As used herein, production by recombinant means by using recombinant

DNA methods means the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA.

As used herein the term assessing is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the activity of an endotheliase, or a domain thereof, present in the sample, and also of obtaining an index, ratio, percentage, visual or other value indicative of the level of the activity. Assessment may be direct or indirect and the chemical species actually detected need not of course be the proteolysis product itself but may for example be a derivative thereof or some further substance.

As used herein, biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Biological activities may be observed in in vitro systems designed to test or use such activities. Thus, for purposes herein the biological activity of a luciferase is its oxygenase activity whereby, upon oxidation of a substrate, light is produced.

05-12-2001 oo | US0031803 $5-DEC-20@1 11:38 FROM BOULT WADE TENNANT TO @P49B9Z3INA4ES P.38

As used herein, a combination refers to any association between two or fameng’ more items. woe - As used herein, a composition refers to ffany mixture. it-may be a : solution, a suspension, liquid, powder, a paste, aqueous, NonN-8queous or any’ combination thereof. : TL

As used herein, a conjugate refeis to the compounds provided: herein that : include one or more endotheliases, or domains thereof, and one ormore =. . targeting agents. These conjugates include those produced by reconibinant : means as fusion proteins, those produced by chemical means, such as by chemical coupling, through, for example, coupling ‘to sulfhydryl. groups, and : those produced by any other method whereby 3t least one endothellase, or a : . + domain thereof, is finked, directly.or indirectly via linkerls) to a targeting agent. :'- ©" 8

Lhd w + As used herein, a targeting agent; is any moisty, such-asa protein.or. 4 =i. 1 in cee ees effective portion thereof, that provides specific binding..of the-conjugate to EC BREA Ad surface receptor, which preferably internalizes the conjugate or endotheliase... .. . . .portion thereof. A targeting agent may also be I that promotes or facilitates, . . ane rs areeran for @xaMPlo. affinity isolation or purification of the conjugate; attachment of the conjugate to a A i SANE ro m—— or detection of the conjugate or complexes containing the conjugate.” oT ’ =

As used herein, an antibody conjugate refers to a conjugate in‘which the targeting agent is an antibody. oo

As used herein, humanized antibodies refer to antibodies that are . : modified to include human sequences of amino acids so that administration to a human will not provoke an immune response. Methods for preparation ‘of such antibodies are known. For example, the hybridoma that expresses the ! : monoclonal antibody is altered by recombinant DNA techniques to express sh antibody in which the amino acid composition of the non-variable regions Is based on human antibodies. Computer programs have been designed to identify such regions.

As used herein, derivative or analog of a molecule refers to a portion derived from or a modified version of the molecule. } : | . :

AMENDED SHEET

— rr emrAAANAY TROD Com mw olf) U3 (Tad

As used herein, fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.

As used herein, an effective amount of a compound for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease.

Repeated administration may be required to achieve the desired amelioration of symptoms.

As used herein equivalent, when referring to two sequences of nucieic acids means that the two sequences in question encode the same sequence of amino acids or equivalent proteins. When equivalent is used in referring to two proteins or peptides, it means that the two proteins or peptides have substantially the same amino acid sequence with only conservative amino acid substitutions (see, e.g., Table 1, above) that do not substantially alter the activity or function of the protein or peptide. When equivalent refers to a property, the property does not need to be present to the same extent [e.g., two peptides can exhibit different rates of the same type of enzymatic activity], but the activities are preferably substantially the same. Complementary, when referring to two nucleotide sequences, means that the two sequences of nucleotides are capable of hybridizing, preferably with less than 25%, more preferably with less than 15%, even more preferably with less than 56%, most preferably with no mismatches between opposed nucleotides. Preferably the two molecules will hybridize under conditions of high stringency.

As used herein, inhibitor of an endotheliase encompasses any substances that prohibit or decrease production, post-translational maodification(s), maturation, or membrane localization of the endotheliase, or any substances that interfere with or decrease the proteolytic efficacy of the endotheliase.

As used herein, a method for treating or preventing disease or disorder associated with undesired and/or uncontrolled angiogenesis means that the

. . .; diseases and the symptoms associated with the undesired and/or uncontrolled =, angiogenesis are alleviated, reduced, ameliorated, prevented, placed in a state of + remission, or maintained in a state of remission. It also means that the hallmarks of pathological angiogenesis are eliminated, reduced or prevented by the treatment. Non-limiting examples of the hallmarks of the pathological = angiogenesis include uncontrolled degradation of the basement membrane and proximal extracellular matrix of the endothelial cells, migration, division, and organization of the endothelial cells into new functioning capillaries, and the . persistence of such functioning capillaries. 10-3: As used herein, a method for treating or preventing disease or disorder .. associated with deficient angiogenesis means that the diseases and the .. Symptoms associated with the deficient angiogenesis are alleviated, reduced, : ~~: ameliorated, prevented, placed in a state of remission, or maintained in a state of . «remission.

As used herein, operatively linked or operationally associated refers to the functional relationship of DNA with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. For example, operative linkage of DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA. In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5’ untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation (i.e., start) codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation.

Alternatively, consensus ribosome binding sites (see, e.g., Kozak, J. Biol.

Chem., 266:19867-19870 (1991)) can be inserted immediately 5’ of the start codon and may enhance expression. The desirability of (or need for) such modification may be empirically determined. :

As used herein, pharmaceutically acceptable salts, esters or other derivatives of the conjugates include any salts, esters or derivatives that may be

. ~WO 01/36604 PCT/US00/31803 readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects and that either are pharmaceutically active or are prodrugs.

As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. To produce a prodrug, the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes. The prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs of the compound (see, e.q., Nogrady (1985) Medicinal Chemistry A Biochemical

Approach, Oxford University Press, New York, pages 388-392).

As used herein, a drug identified by the screening methods provided herein refers to any compound that is a candidate for use as a therapeutic or as lead compound for designed a therapeutic. Such compounds can be small molecules, including small organic molecules, peptides, peptide mimetics, antisense molecules, antibodies, fragments of antibodies, recombinant antibodies and other such compound which can serve as drug candidate or lead compound.

As used herein, production by recombinant means by using recombinant

DNA methods means the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA.

As used herein, a promoter region or promoter element refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operatively linked. The promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation.

This portion of the promoter region is referred to as the promoter. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be

»- cfs acting or may be responsive to trans acting factors. Promoters, depending i= upon the nature of the regulation, may be constitutive or regulated. Exemplary promoters contemplated for use in prokaryotes include the bacteriophage T7 and

T3 promoters. . 5: As used herein, a receptor refers to a molecule that has an affinity for a + given ligand. Receptors may be naturally-occurring or synthetic molecules.

Receptors may also be referred to in the art as anti-ligands. As used herein, the receptor and anti-ligand are interchangeable. Receptors can be used in their : . unaltered state or as aggregates with other species. Receptors may be attached, covalently or noncovalently, or in physical contact with, to a binding member, ) -- either directly or indirectly via a specific binding substance or linker. Examples : ;; of receptors, include, but are not limited to: antibodies, cell membrane receptors . .=. surface receptors and internalizing receptors, monoclonal antibodies and antisera a + reactive with specific antigenic determinants [such as on viruses, cells, or other - : 15:- materials], drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles. } - Examples of receptors and applications using such receptors, include but are not restricted to: a) enzymes: specific transport proteins or enzymes essential to survival of microorganisms, which could serve as targets for antibiotic [ligand] selection; : b} antibodies: identification of a ligand-binding site on the antibody molecule that combines with the epitope of an antigen of interest may be investigated; determination of a sequence that mimics an antigenic epitope may lead to the development of vaccines of which the immunogen is based on one or more of such sequences or lead to the development of related diagnostic agents or compounds useful in therapeutic treatments such as for auto-immune diseases ¢) nucleic acids: identification of ligand, such as protein or RNA, binding sites; d) catalytic polypeptides: polymers, preferably polypeptides, that are capable of promoting a chemical reaction involving the conversion of one or more reactants to one or more products; such polypeptides generally include a binding site specific for at least one reactant or reaction intermediate and an active functionality proximate to the binding site, in which the functionality is capable of chemically modifying the bound reactant [see, e.q., U.S. Patent No. 5,215,899]; e) hormone receptors: determination of the ligands that bind with high affinity to a receptor is useful in the development of hormone replacement therapies; for example, identification of ligands that bind to such receptors may lead to the development of drugs to control blood pressure; and f) opiate receptors: determination of ligands that bind to the opiate receptors in the brain is useful in the development of less-addictive replacements for morphine and related drugs.

As used herein, sample refers to anything which may contain an analyte for which an analyte assay is desired. The sample may be a biological sample, such as a biological fluid or a biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like. Biological tissues are aggregate of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues.

Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual celi(s).

As used herein: stringency of hybridization in determining percentage mismatch is as follows: 1) high stringency: 0.1 x SSPE, 0.1% SDS, 65°C 2) medium stringency: 0.2 x SSPE, 0.1% SDS, 50°C 3) low stringency: 1.0 x SSPE, 0.1% SDS, 50°C

It is understood that equivalent stringencies may be achieved using alternative buffers, salts and temperatures.

The term substantiaily identical or homologous or similar varies with the context as understood by those skilled in the relevant art and generally means at least 70%, preferably means at least 80%, more preferably at least 90%, and most preferably at least 95% identity.

As used herein, substantially. identical to a product means sufficiently =. similar so that the property of interest is sufficiently unchanged so that the - substantially identical product can be used in place of the product. = As used herein, substantially pure means sufficiently homogeneous to : b.-. appear free of readily detectable impurities as determined by standard methods + of analysis, such as thin layer chromatography (TLC), gel electrophoresis and : high performance liquid chromatography. (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would _. not detectably alter the physical and chemical properties, such as enzymatic and : 10.+ biological activities, of the substance. Methods for purification of the _-- compounds to produce substantially chemically pure compounds are known to -; those of skill in the art. A substantially chemically pure compound may, : + however, be a mixture of stereoisomers or isomers. In such instances, further « purification might increase the specific activity of the compound. p 16. As used herein, target cell refers to a cell that expresses an endotheliase naturally.

As used herein, test substance refers to a chemically defined compound (e.g., organic molecules, inorganic molecules, organic/inorganic molecules, proteins, peptides, nucleic acids, oligonucleotides, lipids, polysaccharides, saccharides, or hybrids among these molecules such as glycoproteins, etc.) or mixtures of compounds (e.g., a library of test compounds, natural extracts or culture supernatants, etc.) whose effect on an endotheliase, or a domain thereof, is determined by the disclosed and/or claimed methods herein.

As used herein, the terms a therapeutic agent, therapeutic regimen, radioprotectant, chemotherapeutic mean conventional drugs and drug therapies, including vaccines, which are known to those skilled in the art. Radiotherapeutic agents are well known in the art, :

As used herein, treatment means any manner in which the symptoms of a conditions, disorder or disease are ameliorated or otherwise beneficially altered.

Treatment also encompasses any pharmaceutical use of the compositions herein.

As used herein, vector {or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. The vectors typically remain episomal, but may be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art. An expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

As used herein, protein binding sequence refers to a protein or peptide sequence that is capable of specific binding to other protein or peptide sequences generally, to a set of protein or peptide sequences or to a particular protein or peptide sequence.

As used herein, epitope tag refers to a short stretch of amino acid residues corresponding to an epitope to facilitate subsequent biochemical and immunological analysis of the epitope tagged protein or peptide. Epitope tagging is achieved by appending the sequence of the epitope tag to the protein- encoding sequence in an appropriate expression vector. Epitope tagged proteins can be affinity purified using highly specific antibodies raised against the tags.

As used herein, metal binding sequence refers to a protein or peptide sequence that is capable of specific binding to metal ions generally, to a set of metal ions or to a particular metal ion.

As used herein, a composition refers to a any mixture. It may be a solution, a suspension, liquid, powder, a paste, agueous, non-aqueous or any combination thereof.

“ - As used herein, a combination refers to any association between two or .... among more items. - As used herein, fluid refers to any composition that can flow. Fluids thus _ encompass compositions that are in the form of semi-solids, pastes, solutions, 5. aqueous mixtures, gels, lotions, creams and other such compositions.

For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.

B. ENDOTHELIASE PROTEINS AND DERIVATIVES AND ANALOGS . 3 Protease involvement in angiogenesis . 10... In the initial stages of angiogenesis, microvascular endothelial cells of - preexisting blood vessels locally degrade the underlying basal lamina and invade into the stroma of the tissue to be vascularized. It has been shown that this . .. process requires a wide array of degradative enzymes (Mignatti and Rifkin, .. Enzyme Protein, 49(1-3):117-37 (1996)). Components of the plasminogen 15 ...activator (PA)-plasmin system and the matrix metalloproteinase (MMP) family play important roles. PAs trigger a proteinase cascade that results in the generation of high local concentrations of plasmin and active MMPs. This increase in proteolytic activity has three major consequences: it permits extracellular matrix degradation and invasion of the vessel basal lamina, generates extracellular matrix (ECM) degradation products that are chemotactic for endothelial cells, and activates and mobilizes growth factors localized in the

ECM. Five proteases, urokinase-type plasminogen activator, factor Xll, protein

C, trypsinogen IV, and a membrane-type serine protease 1 (MTSP1) that may be involved in these processes have been identified.

At least nine different MMPs have been identified. Among these are gelatinase A, which degrades collagen types IV and V, elastin and laminin and which is frequently overexpressed in stromal cells of malignant tumors (Vassalli and Pepper, Nature, 370:14-5 {(1994)). Nucleic acid encoding a matrix metalloproteinase with a potential transmembrane domain has been cloned (Sato etal, Nature, 370:61-5 {1994}). Expression of the cioned gene product on the cell surface induces specific activation of pro-gelatinase A in vitro and enhances cellular invasion of the reconstituted basement membrane. Tumor cells of a WO 01/36604 PCT/US00/31803 invasive lung carcinomas, which contain activated forms of gelatinase A, were found to express the transcript and the gene product. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 b prostatic cancer tumors (see, Takeuchi et al. Proc. Natl. Acad. Sci. (USA), 96(20):11054-61 (1999)).

Thus, endothelial cells and proteases play key roles in angiogenesis and related processes. Furthermore, proteases can serve as therapeutic targets and points of intervention in processes relying on the protease activities.

As noted above, aberrant angiogenesis and processes related thereto have a role in a variety of disorders, including cancers, diabetic retinopathies, . hyperproliferative disorders, restenosis, and others. As provided herein, because proteases are involved either directly or indirectly in angiogenesis, which is aberrant in a variety of disorders, altering the activity of proteases involved in angiogenesis could treat the resulting disorders. Thus, proteases involved in angiogenesis can serve as therapeutic targets and also in drug screening methodologies to identify compounds that modulate, particularly inhibit, angiogenesis.

Provided herein is a class of proteases, designated herein as endotheliases. The proteases are endothelial cell transmembrane proteins proteins, which view of the role(s) of endothelial cells and proteases in angiogenesis and related processes, are directly or indirectly involved in the process of angiogenesis or processes related thereto. Thus, these proteases are therapeutic, diagnostic, prognostic targets for intervention in the process of angiogenesis, either for inhibition or activation thereof. In addition, these proteases, particularly the protease domains {extracellular domains) provide a means to screen for compounds that modulate angiogenesis.

The endotheliases provided herein and the screening methods provided herein permit discovery of candidate compounds that modulate processes involved in the establishment and maintenance of the vasculature. These methods provide a means to select compounds that selectively bind to the endotheliases or interact therewith resulting an increase or decrease in the

“ 4 protease activity of the endotheliase. Since the endotheliases occur on «s endothelial cells, which are intimately involved in processes related to + establishment and maintenance of the vasculature, the compounds identified by _ the methods herein can have activity as anti-angiogenic or pro-angiogenic 5.. agents. . ps Endotheliases

Thus, provided herein are endotheliases, including protease domains thereof. As noted, endotheliases are endothelial cell transmembrane proteins . that include an extracellular domain that exhibits protease activity. By virtue of : 10-... its expression on endothelial cells, these proteases participate directly or + indirectly in processes involved in angiogenesis. :

Ee Substantially purified protease domains of endotheliase proteins and . -- derivatives or analog thereof that can be bound by an antibody directed against : such protease domain are provided. Also included are substantially purified : 16 . protein that contain amino acid sequences that have at least 40%, more preferably 60% or greater, identity to the protease domain of the endotheliase proteins exemplified herein, where the percentage identity is determined over an amino acid sequence of identical size to the protease domain. More preferably, the sequence identity is at least 90% identical.

In exemplary embodiments, two different endotheliases, including variant forms thereof, are provided. These include an endotheliase 1 and two splice variants of an endotheliase 2. Other members of the family may be identified by probing for genes or searching libraries for genes that have sequence identity, particularly at least 40%, 60%, 80%, 90% or greater sequence identity to the protease domain of an endotheliase identified herein, or that hybridize under conditions of high stringency to the full-length of the nucleic acid encoding a protease domain of an endotheliase provided herein.

Alternatively, and as a way of identifying endotheliases that may have lower sequence identity, an endotheliase may be identified by the methods exemplified herein, such by identifying ESTs or other nucleic acid fragments that have sequences similar to a protease and then using such fragments as probes to identify and select cDNA clones encoding full-length proteases or protease domains thereof, identifying those that have the characteristics of transmembrane proteins, and then determining the gene expression profile to identify those that are expressed on the surface of endothelial cells. Encoded proteins that have protease activity, that include a transmembrane domain and an extracellular domain, and that are expressed in endothelial cells are endotheliases. Any method for identification of genes encoding proteins (or proteins) that encode a transmembrane protease expressed on an endothelial cell is contemplated herein.

As described below, exemplary nucleic acid molecules encoding an endotheliase and/or protease domain therein are provided. Full-length clones encoding endotheliase or a protease domain thereof can be obtained by any methods known in the art, including, but are not limited to, PCR amplification using synthetic primers that hybridize to the 3° and 5’ ends of an endotheliase and/or by cloning from a cDNA or genomic library using a PCR amplification product or an oligonucleotide specific for the gene sequence Homologs (e.g., nucleic acids of the above-listed genes of species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular sequence provided as a probe using methods well known in the art for nucleic acid hybridization and cloning.

For recombinant expression of one or more of the proteins, the nucleic acid containing all or a portion of the nucleotide sequence encoding the protein can be inserted into an appropriate expression vectar, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein coding sequence. The necessary transcriptional and translational signals can also be supplied by the native promoter for endotheliase genes, and/or their flanking regions.

Full-length endotheliases and domains thereof, particularly the protease domain of an endotheliase are provided herein. The domain or portion there of the endotheliase contains at least 10, 20, 30, 40, or 50 contiguous amino acids of an endotheliase. In specific embodiments, such domains or fragments are not larger than 35, 100, 200 or 500 amino acids. Derivatives or analogs of

“ ~. endotheliases include but are not limited to molecules containing regions that are substantially homologous to endotheliase in various embodiments, at least 30%, ; . +: 40%, 50%, 60%, 70%, 80%, 90% or 95% identity over an amino acid } . sequence of identical size or when compared to an aligned sequence in which ; b.. the alignment is done by a computer homology program known in the art or } -»- Whose encoding nucleic acid is capable of hybridizing to a sequence encoding endotheliase under stringent, moderately stringent, or nonstringent conditions.

Endotheliase derivatives can be made by altering their sequences by _ substitutions, additions or deletions that provide for functionally equivalent 10... molecules. Due to the degeneracy of nucleotide coding sequences, other DNA -»: sequences which encode substantially the same amino acid sequence as an : +; endotheliase gene can be used herein. These include but are not limited to , :_ nucleotide sequences containing all or portions of endotheliase genes that are : - -altered by the substitution of different codons that encode the amino acid . 15 . residue within the sequence, thus producing a silent change. Endotheliase derivatives include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of endotheliase, including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar {hydrophobic} amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Exemplary of the endotheliase provided herein are endotheliase 1 and endotheliase 2.

05-12-2001 a | | oo US0031803

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Endotheliase 1 and nucleic acids encoding endotheliase 1 )

Nucleic acids . on

The identification and cloning of nucleic Ad encoding the protease. . domain of endotheliase 1 is described in Example 1. Provided herein Isfhucieic § acid that encodes the protease domain of an endotheliase 1. Such nucleic acid enocins Coe molecule ffrose-that-eneeds the sequence of amino’ acids set forth'in + = - :

SEQ. ID NO:2; it also includes the amino acid 120- or 191-424 of sequence of

SEQ ID NO. 22;fhe Arg is optionally included). Also provided herein:are nucleic acids that hybridize tc a nucleic acid containing the sequence of nucleotides set : forth in the SEQ. ID No. 1. Hybridization is préferably under moderate conditions and more preferably high stringency conditions and is such that the nucleic acid

Cs ... .of interest hybridizes along its full length the nucleic acid-that encodes the wove. wu we

CE case Gomain endothelisse.1 and is considered tobe anerothellased By © © 5 5 7

CL } N virtue of its expression. in endothelial cells, ‘activity. as. a. protease and Similarity = x i: Tie a oo ‘15 as assessed by hybridization or by having sequence identify of at least about } ] 60%, more preferably more than about 75% sequence identity, mare ‘preferably ST- ... more than about 30% to the protease domain of endothélisse 1 exemplified : herein, Such proteins will also exhibit protease adtivity. Also contemplated Comm herein are proteins that include conservative amino acid sequence changes, such - oT as those set forth in Table 1 above, and retain protease activity.

The endotheliase 1 protease domain and encoding nucleic acid is contemplated for use in the methods and cells and vectors described herein.

The protease domain may be used as purified protein or may be part of a larger protein, such as the full length endotheliase 1. ;

Also contemplated for use in the methods, vectors and cells is a full- length endotheliase 1. Exemplary of full-length endotheliase are endotheliasts that include the sequence of amine acids set forth in SEQ ID No. 22, which provides, Juli length endotheliases (see, international PCT application No. WQ ozeoR. International PCT application No. wo 242000 provides a gene designated DESC1 used for diagnosis of squamous cell carcinoma or prostate. cancer. DESC1 is shown herein to encode an endotheliase 1. DESC1 and the encoded protein (see SEQ ID No. 22) are contemplated for use in the methods,

AMENDED SHEET

TL La Ta Feel .. enmA MAD

- cells and vectors provided herein. The PCT application does not suggest using the protease domain thereof for any purpose. : E Proteins

Provided herein are protease domains of an endotheliase, including endotheliase 1. A substantially purified protease domain of an endotheliase .- protein that is encoded by the nucleic acid molecules that encode the nucleic acids are provided. In particular, the protease domain contains the sequence of amino acids set forth in SEQ ID No. 2 or a fragment thereof that exhibits . ~ protease activity or contains a sequence of amino acids that hybridizes to a 10. nucleic acid molecule that encodes the protein of SEQ ID NO. 2.

In one exemplary embodiment, the substantially purified protease domain : ~ is encoded by the sequence of nucleic acids set forth in SEQ. ID No. 1 or y ~ hybridizes thereto along its full length under high stringency conditions and has activity as a protease. In other embodiments, a substantially purified derivative . 15 .. or analog of the protease domain of the endotheliase protein is provided, which derivative or analog is able to be bound by an antibody directed against such protease domain.

In another embodiment, the substantially purified protein contains a sequence of amino acids that has at least 60% identity to the protease domain of the endotheliase 1 protein is provided, wherein the percentage identity is determined over an amino acid sequence of identical size to the protease domain. More preferably, the sequence identity is at least 90% identical.

In specific aspects, the protease domain peptide, derivatives or analogs of the protease domain of the endotheliase protein are from animals, including mammals, such as, but are not limited to, mouse, rat, chicken and human origin and is functionally active, /.e., capable of exhibiting one or more functional activities associated with the domains, e.g., serine protease activity, immunogenicity or antigenicity.

Endotheliase 2 and nucleic acids encoding endotheliase 2

The cloning of endotheliase 2 and expression profile thereof is described in Example 2. Two splice variant forms of endotheliase 2 designated endotheliase 2-S and endotheliase 2-L are also provided (see FIG. 1 for the domain organization thereof; see also EXAMPLE 2}. The open reading frame of the nucleic acid encoding endotheliase 2-S (SEQ ID No. 3) is composed of 1,689 bp, which translates to a 562-amino acid protein (SEQ ID No. 4), while the ORF of endotheliase 2-L is composed of 2,067 bp (SEQ ID No. 5), which translates to a 688-amino acid protein (SEQ ID No. 6).

The nucleic acid encoding the protease domain of endotheliase 2-S is composed of 729 bp which translates to a 242-amino acid protein (amino acids 321-562 of SEQ ID Nos. 3 and 4), while that of endotheliase 2-L is composed of 1,107 bp, which translates to a 368-amino acid protein (amino acids 321-688 of

SEQ ID Nos. 5 and 6). The domain organization of each form is depicted in

Figure 1.

The full-length and protease domains of endotheliase 2 as well as other domains (see Fig. 1) are provided herein as are cells, vectors and methods that use the proteins and/or encoding nucleic acid.

Also provided herein are nucleic acids that hybridize to a nucleic acid containing the sequence of nucleotides set forth in SEQ. ID No. 3 or 5.

Hybridization is preferably effected under moderate conditions and more preferably high stringency conditions and is such that the nucleic acid of interest hybridizes along its full length the nucleic acid that encodes the protease domain of endotheliase 2 and is considered to encode an endotheliase 2 by virtue of endogenous expression of the protein in endothelial cells, activity as a protease and similarity as assessed by hybridization or by having sequence identify of at least about 60%, more preferably more than about 85% sequence identity, more preferably more than about 90% to the protease domain of endotheliase 2 exemplified herein. Such encoded proteins will also exhibit protease activity.

The nucleic acids are contemplated for use in vectors, cells and methods provided herein.

Endotheliase-2 proteins

Any and all of the above-noted endotheliases and/or protease domains thereof, such as those that include the sequences of amino acids in SEQ ID Nos. 2,4, 6 and 22 or are encoded by nucleic acid that hybridize thereto under the conditions as described above are contemplated for use in the methods herein.

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Also contemplated herein are proteins that include.conservative amino acid sequence changes, such as those set forth in Table 1 above, and retain protease activity. So : Vectors, plasmids and ceils that contain nucleic acids encoding an ’ endotheliase or protease domain thereof. : . :

Also provided are vectors that contain nucleic acid encoding the. : endotheliases. Cells containing the vectors are siso provided. The cells-include’ eukaryotic and prokaryotic cells, antl the vectors are Any suitable for use therein.

Prokaryotic and eukaryotic cells, including endothelial cells, containing the vectors are provided. Such cells include bacterial cells, yeast cells, fungal cells. : Co. .plant cells, insect cells and animal cells. The cells are.used to produce an... -.. . . . . . i Ce endotheliase.or. protease ‘domain thereof, by.growing-the, above-described:cells: EY a NET eee ae. under conditions whereby the encoded endotheliase or protease domain. of:the: :- ~~. -:.. endatheliase is expressed by the cell, and recovering the expressed protease . domain protein. For purposes herein, the protease- domain is-preferably secreted imothemedum. 4 one embodiment, the vectors include a sequence of nucleotides that encodes a polypeptide that has protease activity and contains all ora portion of - only the protease domain, or multiple copies thereof, of an endotheliase are provided. Also provided are vectors that comprise a sequence of nucleotides that encodes the protease domain and additional portions of an endotheliase up to and including a full length endotheliase, as well as multiple copies thereof, are also provided. The vectors mayfSeiected for expression of the endatheliase or protease domain thereof in the cell or such that the endotheliase is expressed as a transmembrane protein. Alternatively, the vectors may include signals necessary for secretion of encoded proteins. When the protease domain is : expressed the nucleic acid is preferably linked to a secretion signal, such as the

Saccharomyces cerevisiae a mating factor signal sequence or a portion thereof.

A variety of host-vector systems may be used to express the protein coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g. vaccinia virus, adenovirus, etc.); insect cell systems

AMENDED SHEET

R WO 01/36604 PCT/US00/31803 infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system used, any one of a number of suitable transcription and translation elements may be used.

Any methods known to those of skill in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene containing of appropriate transcriptional/translational control signals and protein coding sequences. These methods may include jn vitro recombinant DNA and synthetic techniques and in vivo recombinants {genetic recombination). Expression of nucleic acid sequences encoding endotheliase, or domains, derivatives, fragments or homologs thereof, may be regulated by a second nucleic acid sequence so that the genes or fragments thereof are expressed in a host transformed with the recombinant DNA molecule(s). For example, expression of the proteins may be controlled by any promoter/enhancer known in the art. In a specific embodiment, the promoter is not native to the genes for endotheliase. Promoters which may be used include but are not limited to the SV40 early promoter (Bernoist and Chambon, Nature 290:304-310 (1981)), the promoter contained in the 3’ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 {1980}), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA. 78:1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Mature 296:39- 42 (1982)); prokaryotic expression vectors such as the f-lactamase promoter {Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 1978)) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)); see also “Useful Proteins from Recombinant Bacteria”: in Scientific American 242:79-94 (1980)); plant expression vectors containing the nopaline synthetase promoter (Herrar-Estrella et al., Mature 303:209-213 (1984)) or the cauliflower mosaic virus 358 RNA promoter (Garder et al., Nucleic Acids Res. 9:2871 (1981}), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., Mature 310:115-120 (1984)); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol

. - :» dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline g =- phosphatase promoter, and the following animal transcriptional control regions - = that exhibit tissue specificity and have been used in transgenic animals: elastase .. 1 gene control region which is active in pancreatic acinar cells (Swift et al., Cell . : 5;r 38:639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. ; -z» 50:399-409 (1986); MacDonald, Hepatology 7:425-515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan et al., Nature 315:115-122 (19856), immunoglobulin gene control region which is active in : _ lymphoid cells (Grosschedl et al., Cell 38:647-658 (1984); Adams et al., Nature on : 102 318:533-538 (1985); Alexander et al., Mol. Cell Biol. 7:1436-1444 (1987)), mouse mammary tumor virus control region which is active in testicular, breast, - . x lymphoid and mast cells (Leder et al., Cell 45:485-495 (1986)), albumin gene = : = control region which is active in liver (Pinckert et al., Genes and Devel. 1:268-

E : + 276 (1987)), alpha-fetoprotein gene control region which is active in liver ~ . 15» (Krumlauf et al., Mol. Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science 235:53-58 1987)), alpha-1 antitrypsin gene control region which is active in liver {Kelsey et al., Genes and Devel. 1:161-171 {1987)), beta globin gene control region which is active in myeloid cells (Mogram et al., Nature 315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al., Ce// 48:703-712 (1987)}, myosin light chain-2 gene control region which is active in skeletal muscle {Sani, Nature 314:283-286 {1985)), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al., Science 234:1372-1378 (1986).

In a specific embodiment, a vector is used that contains a promoter operably linked to nucleic acids encoding an endotheliase, or a domain, fragment, derivative or homolog, thereof, one or more origins of replication, and optionally, one or more selectable markers {e.g., an antibiotic resistance gene}.

Expression vectors containing the coding sequences, or portions thereof, of an endotheliase, is made, for example, by subcloning the coding portions into the

EcoRI restriction site of each of the three pGEX vectors (glutathione S- transferase expression vectors {Smith and Johnson, Gene 7:31-40 {1988)). This allows for the expression of products in the correct reading frame. Preferred vectors and systems for expression of the protease domains of the endotheliases are the Pichia vectors, particularly those designed for secretion of the encoded proteins. One exemplary vector is described in the EXAMPLES.

The vectors are introduced into host cells and the proteins expressed therein. Once a recombinant cell expressing an endotheliase protein, or a domain, fragment or derivative thereof, is identified, the individual gene product can be isolated and analyzed. This is achieved by assays based on the physical and/or functional properties of the protein, including, but not limited to, radioactive labeling of the product followed by analysis by gel electrophoresis, immunoassay, cross-linking to marker-iabeled product

The endotheliase proteins may be isolated and purified by standard methods known in the art (either from natural sources or recombinant host cells expressing the complexes or proteins), including but not restricted to column chromatography (e.g., ion exchange, affinity, gel exclusion, reversed-phase high pressure, fast protein liquid, etc.), differential centrifugation, differential solubility, or by any other standard technique used for the purification of proteins. Functional properties may be evaluated using any suitable assay known in the art.

Alternatively, once an endotheliase or its domain or derivative is identified, the amino acid sequence of the protein can be deduced from the nucleotide sequence of the gene which encodes it. As a result, the protein or its domain or derivative can be synthesized by standard chemical methods known in the art (e.g. see Hunkapiiler et al, Nature 310:105-111 (1984).

Manipulations of endotheliase sequences may be made at the protein level. Also contemplated herein are endotheliase proteins, domains thereof, derivatives or analogs or fragments thereof, which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, . including but not limited to specific chemical cleavage by cyanogen bromide,

trypsin, chymotrypsin, papain, V8 protease, NaBH,, acetylation, formylation, : . = oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc. . z In specific embodiments, the endotheliases are modified to include a . fluorescent label. In other specific embodiments, the endotheliase is modified to . 5, have a heterofunctional reagent, such heterofunctional reagents can be used to : . -- crosslink the members of the complex. : In addition, domains, analogs and derivatives of an endotheliase can be chemically synthesized. For example, a peptide corresponding to a portion of an endotheliase, which includes the desired domain or which mediates the desired 10:x. activity in vitro can be synthesized by use of a peptide synthesizer. . Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs . : -: can be introduced as a substitution or addition into the endotheliase sequence. oe : . Non-classical amino acids include but are not limited to the D-isomers of the - -~ common amino acids, a-amino isobutyric acid, 4-aminobutyric acid, Abu, + 15 . 2-aminobutyric acid, e-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionoic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, R-alanine, fluoro-amino acids, designer amino acids such as R-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

In cases where natural products are suspected of being mutant or are isolated from new species, the amino acid sequence of the endotheliase isolated from the natural source, as well as those expressed in vitro, or from synthesized expression vectors in vivo or in vitro, can be determined from analysis of the

DNA sequence, or alternatively, by direct sequencing of the isolated protein.

Such analysis may be performed by manual sequencing or through use of an automated amino acid sequenator.

C. Identification and isolation of endotheliase genes

Any method known to those of skill in the art for identification of nucleic acids that encode desired genes may be used. Any method available in the art can be used to obtain a full length (i.e., encompassing the entire coding region)

cDNA or genomic DNA clone encoding an endotheliase. In particular, the polymerase chain reaction (PCR) can be used to amplify a sequence identified as being differentially expressed in tissues with aberrant level of angiogenesis, e.g., nucleic acids containing the nucleotide sequences of endotheliase (SEQ. NO: 1, : 5 3, 5 or 22), in a genomic or cDNA library. Oligonucleotide primers that hybridize to sequences at the 3’ and 5’ termini of the identified sequences can be used as primers to amplify by PCR sequences from a nucleic acid sample (RNA or DNA), preferably a cDNA library, from an appropriate source (e.g., tumor or cancer tissue}.

PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp’). The DNA being amplified can include mRNA or cDNA or genomic DNA from any eukaryotic species. One can choose to synthesize several different degenerate primers, for use in the PCR reactions.

It is also possible to vary the stringency of hybridization conditions used in priming the PCR reactions, to amplify nucleic acid homologs (e.g., to obtain endotheliase sequences from species other than humans or to obtain human sequences with homology to endotheliase) by allowing for greater or lesser degrees of nucleotide sequence similarity between the known nucleotide sequence and the nucleic acid homolog being isolated. For cross species hybridization, low stringency conditions are preferred. For same species hybridization, moderately stringent conditions are preferred. After successful amplification of the nucleic acid containing all or a portion of the identified endotheliase sequence or of a nucleic acid encoding all or a portion of an endotheliase homolog, that segment may be molecularly cloned and sequenced, and used as a probe to isolate a complete cDNA or genomic clone. This, in turn, will permit the determination of the gene's complete nucleotide sequence, the analysis of its expression, and the production of its protein product for functional analysis. Once the nucleotide sequence is determined, an open reading frame encoding the endotheliase gene protein product can be determined by any method well known in the art for determining open reading frames, for example, using publicly available computer programs for nucleotide sequence analysis.

Once an open reading frame is defined, it is routine to determine the amino acid

: -- sequence of the protein encoded by the open reading frame. In this way, the . =- nucleotide sequences of the entire endotheliase genes as well as the amino acid - - - sequences of endotheliase proteins and analogs may be identified. : - Any eukaryotic cell potentially can serve as the nucleic acid source for - - 5. the molecular cloning of the endotheliase gene. The nucleic acids can be - : «= isolated from vertebrate, mammalian, human, porcine, bovine, feline, avian, . equine, canine, as well as additional primate sources, insects, plants, etc. The

DNA may be obtained by standard procedures known in the art from cloned DNA . . le.g., a DNA "library"), by chemical synthesis, by cDNA cloning, or by the - . 10+: cloning of genomic DNA, or fragments thereof, purified from the desired cell . -; (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory 3 : - Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New o = York; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, vs - = Ltd., Oxford, U.K. Vol. |, Il). Clones derived from genomic DNA may contain o. . 15 :; regulatory and intron DNA regions in addition to coding regions; clones derived fromm cDNA will contain only exon sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.

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

Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired gene may be accomplished in a number of ways. For example, a portion of the endotheliase (of any species) gene (e.g., a

PCR amplification product obtained as described above or an oligonucleotide having a sequence of a portion of the known nucleotide sequence) or its specific

RNA, or a fragment thereof be purified and labeled, and the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe

© 05-12-2001 - : | US003180:

PS-DEC-2881 11:33 FROM BOLT WADE TENNANT TO epasEsRIINAES P.33 ’ <44- Ca (Benton and Davis, Science 196:180 (1977); Grunstein and Hogness, Proc. Nat,

Acad. Sci. U.S.A. 72:3961 [1975)). Those DNA fragments with substaritial homology to the probe will hybridize. It is also possible to identify the appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected according to 3 known restriction map if such : is available or by DNA sequence analysis and comparison to the known nucleotide sequence of endotheliase. Further selection can be carried out on the basis of the properties of the gene. ‘ Alternatively, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones, or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein

Lo that, or has similar rdontion electrophoretic raion a ) .. .

TL. A Cima behavior. proteolytic digestion maps, antigenic. piopértiés; serine proteases - ri LIT nat a activity or ability to promote angiogenesis, as known for the:eridotheliaser Ifan.. © wv ;

N 15 anti-endotheliase antibody is available, the protein ‘may be identified" by binding of labeled antibody to the putatively endotheliase synthesizing ‘clones, inan ~~... "7

ELISA (enzyme-linked immunosorbent assay) type procedure. : . oo }

Co © Alternatives to isolating the endotheliase genomic DNA include, butare ~~ not limited to, chemically synthesizing the gene sequence from a krown - sequence or making cDNA to the mRNA that encodes the endotheliase protein. ’

For example, RNA for cDNA cloning of the endotheliase gene can be isolated from cells expressing the protein. The identified and isolated nucleic acids can then be inserted into an appropriate cloning vector. A large number of vector- host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be | - compatible with the host cell used. Such vectors include, but are not fimited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene, La Jolia, CA). The insertion into a cloning vector can, for example, be accomplished by ligating the

DNA fragment into a cloning vector which has complementary cohesive termini.

However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be : :

AMENDED SHEET

- Ne La as ara tals Feel _. MTA NM ANN

: enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers . .. may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the : . 5... cleaved vector and endotheliase gene may be modified by homopolymeric tailing. . .. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated. » In an alternative method, the desired gene may be identified and isolated 10 .. after insertion into a suitable cloning vector in a "shot gun" approach. . Enrichment for the desired gene, for example, by size fractionization, can be » ...done before insertion into the cloning vector. } . . In specific embodiments, transformation of host cells with recombinant i } .. DNA molecules that incorporate the isolated endotheliase gene, cDNA, or i 15 . synthesized DNA sequence enables generation of multiple copies of the gene. . Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.

D. SCREENING METHODS 1. Methods for screening for compounds that modulate the activity of an endotheliase

Methods for identifying compounds that bind to or interact with an endotheliase, particularly, the protease domain thereof, are provided. The identified compounds are candidates or leads for identification of compounds for treatments of tumors and other disorders and diseases involving aberrant angiogenesis. The endotheliases used in the methods include any endotheliase as defined herein, and preferably use the endotheliases 1 and 2 provided herein, and preferably the protease domains thereof. A variety of methods are provided herein. These methods may be performed in solution or in solid phase reactions in which the endotheliase(s) or protease domain(s) thereof are linked, either directly or indirectly via a linker, to a solid support. Screening assays are described in the Examples, and these assays have been used to identify candidate compounds.

One method include the steps of (a) contacting the endotheliase or protease domain thereof with one or a plurality of test compounds under conditions conducive to interaction between the ligand and the compounds; and (b) identifying one or more compounds in the plurality that specifically binds to the ligand.

Another method provided herein includes the steps of a) contacting an endotheliase or protease domain thereof with a substrate of the endotheliase, and detecting the proteolysis of the substrate, whereby the activity of the endotheliase is assessed; b) contacting the endotheliase with a substrate of the endotheliase in the presence of a test substance, and detecting the proteolysis of the substrate, whereby the activity of the endotheliase is assessed; and c) comparing the activity of the endotheliase assessed in steps a) and b), whereby the activity measured in step a) differs from the activity measured in step b) indicates that the test substance modulates the activity of the endotheliase. in another embodiment, a plurality of the test substances are screened for simultaneously in the above screening method. In another embodiment, the endotheliase to be screened against is isolated from a target cell. In another embodiment, the test substance is a therapeutic compound, such that a difference of the endotheliase activity measured in the presence and in the absence of the test substance indicates that the target cell responds to the therapeutic compound.

In comparing the activity of an endotheliase in the presence and absence of a test substance to assess whether the test substance is a modulator of the endotheliase, it is unnecessary to assay the activity in parallel, although such parallel measurement is preferred. It is possible to measure the activity of the endotheliase at one time point and compare the measured activity to a historical value of the activity of the endotheliase. For instance, one can measure the activity of the endotheliase in the presence of a test substance and compare with historical value of the activity of the endotheliase measured previously in the absence of the test substance, and vice versa. This can be accomplished,

: for example, by providing the activity of the endotheliase on an insert or - } . pamphlet provided with a kit for conducting the assay.

Preferably, the endotheliase to be screened against is isolated from a ] ., target cell. More preferably, the test substance to be screened for is a - - 5: therapeutic compound, and whereby a difference of the endotheliase measured ] = in the presence and in the absence of the test substance indicates whether the target cell responds to the test substance.

Combinations and kits containing the combinations optionally including instructions for performing the assays are provided. The combinations include . . 10 .-an endotheliase and a substrate of the endotheliase to be assayed; and, optionally reagents for detecting proteolysis of the substrate. The substrates, ~ - ~which are typically proteins subject to proteolysis by a particular endotheliase, _ .: ¢an be identified empirically by testing the ability of the endotheliase to cleave ww : =the test substrate. Substrates that are cleaved most effectively li.e., at the 3 16 . lowest concentrations and/or fastest rate or under desirable conditions), are : identified.

Additionally provided herein is a kit containing the above-described combination. Preferably, the kit further includes instructions for identifying a modulator of the activity of an endotheliase. Any endotheliase is contemplated as target for identifying modulators of the activity thereof.

A variety of formats and detection protocols are known for performing screening assays. Any such formats and protocols may be adapted for identifying modulators of endotheliase activities. The following includes a discussion of exemplary protocols. 1. High throughput screening assays

Although the above-described assay can be conducted where a single endotheliase is screened against, and/or a single test substance is screened for in one assay, the assay is preferably conducted in a high throughput screening mode, /.e., a plurality of the endotheliases are screened against and/or a plurality of the test substances are screened for simultaneously (See generally, High

Throughput Screening: The Discovery of Bioactive Substances (Devlin, Ed.)

Marcel Dekker, 1997; Sittampalam et al., Curr. Opin. Chem. Biol., 1{3):384-91 :

05-12-2001 | | Te US0031802 3 : 4s. } (1997): and Silverman et al., Curr. Opin. Chem. Biol., 2{3):397-403 (1998)). For example, the assay can be conducted in a multi-well (e.g., 24-, 48-, 96-, or 384- welll, chip or array format.

High-throughput screening (HTS) is the process of testing a large number of diverse chemical structures against disease targets to Identify. “hits” . (Sittampalam et al., Curr. Opin. Chem. Biol., 113):384-91 {1997)). Current state-of-the-art HTS operations are highly automated and computerized to handle sample preparation, assay procedures and the subsequent processing of large . . volumes of data. ” : : iC Detection technologies employed in high-throughput screens depend on the type of biochemical pathway being investigated (Sittampalam. et ai., Cur. . Opin. Chem. Biol., 1{3):384-91 (1997}). These methods include, radiochemical methods, such as the scintillation proximity assays (SPA), which canbe-adapted = =~ °° to a variety of enzyme assays (Lemer et al., J. Biomol. Screening, 1:1 35-143 45 (1936); Baker et al.. Anal. Biochem., 239:20:24.{1936); Baum etal: Anak: tT i

SE Biochem. 237:129-134 0 996); and Sullivan et al., J. Biomol. .Scréening; 2:19- re

I 23 (1997) and protein-protein interaction assays {Braunwalder et al., J. Biomol. © i..."

Screening, 1:23-26 (1996); Sonatore et 3l., Ans]. Biochem., 240:289-287 °° " (1996); and Chen et al., J. Biol. Chem.. 271 :25308-26315 (1996)). and non- a ‘2G isotopic detection methods, including but are not Vmod to calorimetric aiid--- mee luminescence detection methods, resonance energy transfer (RET) methods, - time-resolved fluorescence (HTRF) methods, cell-based fluorescence assays, . such as fluorescence resonance energy transfer (FRET) procedures ‘{see, e.g.,Gonzalez et al, Biophys. J., 69:1272-1280 (1995)), fluorescence polarization or aniotropy methods (see, e.g., Jameson et al., Methods Enzymol., 246:283-300 {1995); Jolley, J. Biomol. Screening, 1:33-38 (1996); Lynch et al., - Anal. Biochem., 247:77-82 (1997)), fluorescence correlation spectroscopy FCS) and other such methods. 2. Test Substances

Test compounds, including small molecules and libraries and collections thereof can be screened in the above-described assays and assays described below to identify compounds that modulate the activity an endotheliase.

Enpf .zeit:06/12/2001 12:36 Enef nr .:972 P.034

AMENDED SHEET

/ \ -49- _- Rational drug design methodologies that rely on computational chemistry may be -. : -~used to screen and identify candidate compounds. . The compounds identified by the screening methods include inhibitors, including antagonists, and may be agonists Compounds for screening are any . 5 .. compounds and collections of compounds available, know or that can be «prepared. . a. Selection of Compounds

Compounds can be selected for their potency and selectivity of inhibition ; of serine proteases, especially endotheliase. As described herein, and as 10 ...generally known, a target serine protease and its substrate are combined under . assay conditions permitting reaction of the protease with its substrate. The _ : ,assay is performed in the absence of test compound, and in the presence of " increasing concentrations of the test compound. The concentration of test — compound at which 50% of the serine protease activity is inhibited by the test — 15 .compound is the IC, value (Inhibitory Concentration) or EC, (Effective . Concentration) value for that compound. Within a series or group of test compounds, those having lower ICy, or EC, values are considered more potent inhibitors of the serine protease than those compounds having higher IC, or

ECs, values. The IC5, measurement is often used for more simplistic assays, whereas the EC, is often used for more complicated assays, such as those employing cells.

Preferred compounds according to this aspect have an IC, value of 100 nM or less as measured in an in vitro assay for inhibition of endotheliase activity.

Especially preferred compounds have an IC, value of less than 100 nM.

The test compounds also are evaluated for selectivity toward a serine protease. As described herein, and as generally known, a test compound is assayed for its potency toward a panel of serine proteases and other enzymes and an ICs, value or EC, value is determined for each test compound in each assay system. A compound that demonstrates a low IC, value or EC, value for the target enzyme, e.g., endotheliase, and a higher IC, value or EC, value for other enzymes within the test panel (e.g., urokinase tissue plasminogen activator, thrombin, Factor Xa), is considered to be selective toward the target enzyme. Generally, a compound is deemed selective if its IC, value or EC, value in the target enzyme assay is at least one order of magnitude less than the next smallest IC, value or EC, value measured in the selectivity panel of enzymes.

Presently preferred compounds have an IC, value of 100 nM or less as measured in an /n vitro assay for inhibition of urokinase activity. Especially preferred compounds have an IC, value in the in vitro urokinase inhibition assay that is at least one order of magnitude smaller than the IC, value measured in the in vitro tPA inhibition assay. Compounds having a selectivity ratio of IC, u-

PA assay: IC, endotheliase assay of greater than 100 are especially preferred.

Compounds are also evaluated for their activity in vivo. The type of assay chosen for evaluation of test compounds will depend on the pathological condition to be treated or prevented by use of the compound, as well as the route of administration to be evaluated for the test compound.

For instance, to evaluate the activity of a compound to reduce tumor growth through inhibition of endotheliase, the procedures described by Jankun et al., Canc. Res., 57:559-563 (1997) to evaluate PAI-1 can be employed. Briefly, the ATCC cell lines DU145 and LnCaP are injected into SCID mice. After tumors are established, the mice are given test compound according to a dosing regime determined from the compound's in vitro characteristics. The Jankun et al. compound was administered in water. Tumor volume measurements are taken twice a week for about five weeks. A compound is deemed active if an animal to which the compound was administered exhibited decreased tumor volume, as compared to animals receiving appropriate control compounds.

Another in vivo experimental model designed to evaluate the effect of p- aminobenzamidine, a swine protease inhibitor, on reducing tumor volume is described by Billstrom et al., Int. J. Cancer, 61:542-547 (1995).

To evaluate the ability of a compound to reduce the occurrence of, or inhibit, metastasis, the procedures described by Kobayashi et al., /nt. J. Canc., 57:727-733d (1994) can be employed. Briefly, a murein xenograft selected for high lung colonization potential in injected into C57B1/6 mice i.v. {experimental metastasis) or s.c. into the abdominal wall {spontaneous metastasis). Various

05-12-2001 - | | : US003180: bS-DEC-2@P1 11:33 FROM BOLT WADE TENNANT TO maage9zI9eadss P35 cancentrations of the compound to be tested can be admixed with the tumor cells in Matrigel prior to injection. Daily i.p. injections of the test compound are made either on days 1-6 or days 7-13 after tumor inoculation. . The animals are sacrificed about three or four weeks after tumor inoculation, and the lung tumor colonies are counted. Evaluation of the resulting data permits a determination as to efficacy of the test compound, optimal dosing and route of administration.

The activity of the tested compounds toward decreasing mor volume and metastasis car be evaluated infmodel described in Rabbani et al., /nt. J. :

Cancer 63:840-845 (1995) to evaluate their inhibitor. There, Mat LyLu tumor ] 10 cells were injected into the flank of Copenhagen rats. The animals were implanted with osmotic minipumps to continuously administer various doses of i test compound for up to three weeks. The tumor mass and volume of . | .: " 2

I experimental and control animals were evaluated during’ the-experiment; as were Seen eis ce metastatic growths. Evaluation of the resulting data permitsa:detérmination as: Le Re 10 efficacy of the test compound, optimal sora and route of administration. .Some of these authors described a related pr yeol in Xing et al., Canc. Res., “= ) "To evaluate the inhibitory activity of a compound toward neovascularization, s rabbit cornea neovascularization model can be employed. ”

Avery et al., Arch. Ophthaimol., 108:1474-1475 (1990) describe anestherizing

New Zealand albino rabbits and then making a central comeal incision and forming a radiai corneal pocket. A slow release prostaglandin pellet was placed

Co in the pocket to induce neovascularization. Test compound was administered ip. for five days, at which time the animals were sacrificed. The effect of the test compound is’evaluated by review of periodic photographs taken of the! limbus, which can be used to calculate the area of neovascular response ant, therefore, limbal neovasecuiarization. A decreased area of neovascularization as compared with appropriate controls indicates the test compound was effective at decreasing or inhibiting neovascularization.

As angiogenesis model used to evaluate the effect of 3a test compound in preventing angiogenesis is described by Min et al., Canc. Res., 56:2428-2433 {1996). C57BLS mice receive subcutaneous injections of a Matrigel mixture.

AMENDED SHEET

— em amAAnt mend - _r aAme on Aer ~

05-12-2001 oo | | | US0031808 g5-pEC-28P1 11:33 FROM BOLT WADE TENNANT | TO mpagEIZIIIMES P36 containing bFGF, as the angiogenesis-inducing agent, with and without the test compound. After five days, the animals are sacrificed and the Matrigel plugs, in } which neovascularization can be visualized, are photographed. An experimental animal receiving Matrigel and an effective dose of test compound will exhibit less vascularization than 2 control animal or an experimental animal receiving a less- or non-effective Foeg of compound.

An in vivo system designed to test compound for their ability to limit the spread of primary Mors is described by Crowley et al., Proc. Natl, Acad. Sci, ) 90:5021-5025 {1993}. Nude mice are injected with tumor cells (PC3) engineered to express CAT (chloramphenicol acetyltransferase). Compounds to a be tested for their ability to decrease tumor size and/or metastases are oe administered to the animals, and subsequent measurements’ of turivor size'andfort T+ Te

Ls ands metastatic growths are ‘made; Tn addition; the level: GAT: detected intufios i © 7s . CD organs-provides an indication of the ability of the tést'compound to inhibit “© <7 TRE 748 metastasis; detection of less CAT in tissues of treated animal versus a control . .. animal indicates less CAT-expressing cells migrated to that-tissue. CT ’ oo. ... ._fnvivo experimental modes designed to eYaluste the inhibitory patential of a test serine protease inhibitors, using a tumor cell line F3lI, the to be highly invasive, are described by Alonso et al., Breast Canc. Res. Treat., 40:209-223 ” (1996). This group describes in vivo studies for toxicity determination, tumor growth, invasiveness, spontanecus metastasis, experimental lung metastasis, and an angiogenesis assay. Co

The CAM model (chick embryo choricallantoic membrane model), flrst described by L. Ossowski in 1998 (J. Call Biol, 107:2437-2445 (1988), provides another method for evaluating the urokinase inhibitory activity of a ‘test compound. In the CAM model, tumor cells invade through the chorioallantolb © membrane containing CAM with tumor cells in the presence of several serine, protease inhibitors results in less or no invasion of the tumor cells through the membrane. Thus, the CAM assay is performed with CAM and tumor cells in the - presence and absence of various concentrations of test compound. The invasiveness of tumor cells is measured under such conditions to provide an

AMENDED SHEET mL eAr iA PART AREA Comf sO) Lf

05-12-2001 © | | | US0031803

BS-DEC-2081 11:48 FROM BOLT WADE TENNANT TO BB49SI23IF44ES P.37 toed ol -63- Ll indication of the compound's inhibitory activity. A compound having inhibitory activity correlates with less tumor invasion. . ;

The CAM model is also used in a standard assay of angiogenesis {i.e., . effect on formation of new blood vessels (Brooks et al., Methods in Molecular

Biology, 129:257-269 (1999)). According to this model, a fiter Sisg cQntaining an angiogenesis inducer, such as basic fibroblast growth factor #FBEl is placed onto the CAM. Diffusion of the cytokine into the CAM induces local’ ~ . angiogenesis, which may be measured in several ways such as by counting. the . number of blood vessel branch points within the CAM directly below the filter disc. The ability of identified compounds to inhibit. cytokine-induced ’ angiogenesis can be tested using this model. A test compound .can either be +... - added tothe filter disc that contains the angiogenesis inducer, be placed directly ~~ .:-- .-

TE Ce eae .+On the membrane or be administered systemically:<The: extent:of new ‘blood ii RIEL EE Ty ~ Ce vessel formation. ir the presence and/or absence of test compound: cai TRA oN Ca anes

Co 15 compared using this model. The formation of fewer new blood vessels in the presence of a test compound would be indicative of anti-angiogenesis activity. ~~ = ©... Ambanclogenegis activy for inhibitors of endothelvse would suggest thet endotheliase plays a significant role in angiogenesis. . b. Known serine protease inhibitors : =

Compounds for screening can be serine protease inhibitors, which can be oo tested for their ability to inhibit the activity of the endotheliase protein at least Co partially encoded by a nucleic acid that hybridizes to a DNA having a nuclestide sequence set forth in the SEQ. ID NO:1. To | :

Exemplary, but not limiting serine proteases, are the following known serine protease inhibitors are used in the Screening assays: Serine Protease inhibitor 3 (SPI-3) (Chen, M.C.,, et al, Fhickng, 11(11):856-862 (1999);

Aprotinin (lijima, &., et al., J. Biochem. (Tokyo), 126(5):912-916 (1999)); Kazal- : type serine protease inhibitor-like proteins (Niimi, T., et al., Eur. J. Biochem., 266(1):282-292 (1998)); Kunitz-type serine protease inhibitor (Ravichandran, S., etal, Acta Crystallogr. D. Biol. Crystallogr., 55(11):1814-1821 (1999)); Tissue factor pathway inhibitor-2/Matrix-associated serine protease inhibitor (TFPI- 2MSPI), (Liv, Y., et al., Arch. Biochem. Biophys., 370(1):112-8 (1998); .

AMENDED SHEET

EET FL lal a'a a RINE Ia¥T ow) Cede 2070 13 HY

05-12-2001 oo | | | US0031803 d5-DEC-2@A1 11:42 FROM BOUT WADE TENNANT TO ©A498323994465 Pp. : -54- LE

Bukunin, (Cui, C.Y., et al., J. /nvest. Dermatol., 113(2):182-8 (1999));

Nafmostat mesilate {Ryo. R., et al., Vox Sang., 76(4):241-6 (1999); TPCK (Huang, Y., et al., Oncogene, 18{23):3431-9 (1999)); A synthetic cotton-bound serine protease inhibitor (Edwards, J.V., et al.. Wound Repair Regen.. 7(2):106- 18 (1999): FUT-175 (Sawada, M., et al., Stroke, 30(3):644-50 (1999));

Combination of serine protease inhibitor FUT-01 75 and thromboxane synthetase inhibitor OKY-046 (Kaminogo, M., et al., Neurol. Med. Chir. (Tokyo), 38(11):704-8; discussion 708-9 (1 938); The rat serine protease inhibitor 2.3 } gene {LeCam, A., et al., Biochem. Biophys. Res. Commun.. 253(2):31 1-4 (1998); A new intracellular serine protease inhibitor expressed in the rat fotsitory Liana complexes with granzyme B (Hill, R.M,, et al., FEBS Lett, a ~ 440(3):361-4.(1998)); 3,4-Dichloroisocoumarin ‘(Hammed, A.;-et al: Proc: Soc, ot une we sane Exp Biol MEM: 2Y8(2):132571988)); LEX032 (Bains AS. Tet al., Baie J CREE PI . i. si Pharmacol; 356(1):67-72 (1998); N-tosyl-L-phenylalanine cchigromethyl ketone: =~ <i (Dryjanski, M., et al., Biochemistry, 37(40):1 4151-8 (1998}); Mouse gene for : the serine protease inhibitor neuroserpin (P112) (Berger, P., et al., Gene, 214(i--

Ca .. 2125-33 (1998)); Rat serine protease inhibitor 23 gene (Paul, C,, et al., Eur. J. . vor mi

Biochem., 254(3):538-46 (1998)); Ecotin (Yang, S.Q., etal., J. Mol. Biol., 279(4):945-57 (1998); A 14 kDa plant-related serine protease inhibitor (Roch, -

P., et al, Dev. Comp. Immunol., 22(1}):1-12 (1998); Matrix-associated serine protease inhibitor TFPI-2/33 kDa MSP! (Rao, C.N., etal., for. J. Concer, 76(5):749-56 (1998); ONO-3403 (Hiwasa, T., et al., Cancer Lett, 126(2):221- 5 (1998)); Bdellastasin (Moser, M., et al., Eur. J. Biochem., 253(1):212-20 (1998)); Bikunin (Xu, Y., et al., J. Mol. Biol., 276(5):955-66 (1 998);

Nafamostat mesildte (Meligren, K., et al., Thromb. Haemost., 19(2):342-7 (1998)); The growth hormone dependent serine protease inhibitor, Spi 2.1 {Maake, C., et al., Endocrinology. 138(12):5630-6 {1997}); Growth factor activator inhibitor type 2, a Kunitz-type serine protease inhibitor (Kawaguchi, T., et al., J. Biol. Chem., 272(44):27558-64 (1997); Heat-stable serine protease inhibitor protein from ovaries of the desert locust, Schistocerga gregaria (Hamdaoui, A., et al., Biochem. Biophys. Res. Commun. 23812):357-60 (1997); Bikunin, (Delaria, K.A., et al., J. Biol. Chem., 272(18):12208-14 =

AMENDED SHEET roof __t1eNC AN ONNT A002 I TN FTI

(1997)); Human placental bikunin {Marlor, C.W., et al., J. Biol. Chem., ~ 272(10):12202-8 (1997)); Hepatocyte growth factor activator inhibitor, a novel = -. Kunitz-type serine protease inhibitor (Shimomura, T., et al., J. Biol. Chem., ~. 272(10):6370-6 (1997}); FUT-187, oral serine protease inhibitor, (Shiozaki, H., . g 5+. et al., Gan To Kaguku Ryoho, 23(14): 1971-9 (1996)); Extracellular matrix- 3 ‘~ associated serine protease inhibitors (Mr 33,000, 31,000, and 27,000 (Rao, - C.N., et al., Arch. Biochem. Biophys., 335(1):82-92 (1998)); An irreversible isocoumarin serine protease inhibitor (Palencia, D.D., et al., Biol. Reprod., 55(3}:536-42 {1996)); 4-(2-aminoethyl}-benzenesulfony! fluoride (AEBSF) 10:: (Nakabo, Y., et al., J. Leukoc. Biol., 60(3):328-36 {1996)); Neuroserpin ~ (Osterwalder, T., et al., EMBO J., 15(12):2944-53 {1996)); Human serine + protease inhibitor alpha-1-antitrypsin (Forney, J.R., et al., J. Parasitol.. = - 82(3):496-502 (1996)); Rat serine protease inhibitor 2.3 (Simar-Blanchet, A.E., . ) ~ et al., Eur. J. Biochem., 236(2):638-48 (1996)); Gebaxate mesilate (parodi, F., 1 . 165. -et al., J. Cardiothorac. Vasc. Anesth., 10(2):235-7 (1996)}; Recombinant serine : protease inhibitor, CPTI Il (Stankiewicz, M., et al., (Acta Biochim. Pol., 43(3):525-9 (1996)); A cysteine-rich serine protease inhibitor (Guamerin II) (Kim,

D.R., et al., J. Enzym. Inhib., 10(2):81-91 (1996)); Diisopropylfluorophosphate (Lundqvist, H., et al., Inflamm. Res., 44(12):510-7 (1995)); Nexin 1 (Yu, D.W,, et al., J. Cell Sci., 108(Pt 12):3867-74 (1995)); LEXO32 (Scalia, R., et al.,

Shock, 4(4):251-6 {1995)); Protease nexin | (Houenou, L.J., et al., Proc. Natl.

Acad. Sci. U.S.A., 92(3):895-9 (1995}); Chymase-directed serine protease inhibitor (Woodard S.L., et al., J. /Immuno/., 153(11):5016-25 (1994}}; N-alpha- tosyl-L-lysyl-chloromethy! ketone (TLCK) (Bourinbaiar, A.S., et al., Cell Immunol., 155(1):230-6 (1994); Smpi56 (Ghendler, Y., et al., Exp. Parasitol., 78(2):121- 31 (1994)); Schistosoma haematobium serine protease (Blanton, R.E., et al.,

Mol. Biochem. Parasitol. , 63(1):1-1 1 (1994)); Spi-1 (Warren, W.C., et al., Mol.

Cell Endocrinol., 98(1):27-32 (1993)); TAME (Jessop, J.J., et al., Inflammation, ~ 17(5):613-31 (1993); Antithrombin Ill (Kalaria, R.N., et al., Am. J. Pathol., 143(3):886-93 (1993)); FOY-305 (Ohkoshi, M., et al., Anticancer Res., 13(4):963-6 (1993)); Camostat mesilate (Senda, S., et al., Intern. Med., 32(4):350-4 (1 993): Pigment epithelium-derived factor (Steele, F.R., et al.,

05-12-2001 A gh US003180¢

B ; 56- .

Proc. Natl. Acad. Sci. U.S.A., 90(4):1526-30 (1993)); Antistasin (Holstein,

T.W., et al., FEBS Lett., 309(3):288-92 (1982)); The vaccinia virus K2L gene encodes a serine protease Inhibitor (Zhou, J., et al., Virology, 189(2):678-86 (1992); Bowman-Birk serine-protease inhibitor (Werner, M.H., et al., J.- Mol.

Biol, 225(3):873-89 (1992); FUT-175 (Yahamoto, H., et al., Neurosurgery, 30(3):358-63 (1992); FUT-175; (Yanamoto, H., et al., Neurosurgery, 30(3):351-6, discussion 356-7 (1992); PAI (Yreadwell, B.V., et al.,-J. Orthop.

Res., 9(3):309-16 (1981}); 3,4-Dichloroisocoumarin (Rusbridge, N.M., et al., i

FEBS Lett, 268(1):133-6 (1990); Alpha 1-antichymotrypsin (Lindmark, B.E., et al., Am. Rev. Respir. Des., 141(4 Pt 1):884-8 (1990); P-toluenesulfonyl-L- arginine methyl ester (TAME) {Scuderi, P., J. /mmunol., 143(1):168-73 (1989)); ) Aprotinin (Seto, S., et al., Adv. Exp. Med. Biol., 2478:48-54 (1989): Alpha 1- antichymotrypsin (Abraham, C.R., et al., Ce/l, 52(4):487-501 (1988); Contrapsin [Modha, J., et al., Parasitology; 96 (Pt 1):99-109 (1988); (FOY-305) 18 (Yamaichi, ¥., et al., Hiroshima J. Med. Sci.; 36Li1:81-7 No abstract aviiigble = + i 1 win (398) Alpha 2-antiplasmin (Holmes, WEL, et al, U- Biol. Chiern.; 26214):1 659 CE

Co oe 64°(1987)); 3,4 dichloroisocournarin (Harper, J.W.. et ai, Biochemistry, ~~ Tl ] Hf Ee ad Le Hi a vd N oo Co 24(8):1831-41 (1985}); 2 (Tsutsui, K., etal. } Biochem. Biophys. Res, Commun. 123011:271-7 (1984); Gabexate mesilate

Does —. 20 (Hesse, B., et al., Pharmacol. Res. Commun. , 1647):637-45.01 9841); Eherrybin.. TITS methyl sulfonyl! fluoride (Dufer, J., et al., Scand. I Haematol, 32(1):25-32 - (1984); Aprotinin (Seto, S., et al., Hypertension, '5(6):893-9 (1983)); Protease - inhibitor CI-2 (McPhalen, C.A., et al., J. Mol. Biol., 168(2):445-7 (1983);

Phenylmethyisulfony! fiuoride {Sekar V., et al., Biochem. Biophys. Res.

Commun., 89(2):474-8 (1979); PGE (Feinstein, M.D., et al., Prostaglandine, 14(6):1075-93 (1977)a : ; \ c. Combinatorial libraries and other libraries :

The source of compounds for the screening assays, can be libraries, : including, but are not limited to, combinatorial libraries. Methods for synthesizing combinatorial libraries and characteristics of such combinatorial \ libraries are known in the art (See generally, Combinatorial Libraries: Synthesis,

Screening and Application Potentis/ (Cortese Ed.) Walter de Gruyter, Inc., 1995;

Emp{ .zeit:05/12/2001 12:37 Empf.nr.2972 P.OS3

AMENDED SHEET

Tietze and Lieb, Curr. Opin. Chem. Biol., 2{3):363-71 (1998); Lam, Anticancer

E - Drug Des., 12(3):145-67 (1997); Blaney and Martin, Curr. Opin. Chem. Biol., a ~ 1{1):54-9 (1997); and Schultz and Schultz, Biotechnol. Prog., 12{6):729-43 . (1996). i - 5. Methods and strategies for generating diverse libraries, primarily peptide-

L _ and nucleotide-based oligomer libraries, have been developed using molecular biology methods and/or simultaneous chemical synthesis methodologies (see, e.g., Dower et al., Annu. Rep. Med. Chem., 26:271-280 (1991); Fodor et al., ) Science, 251:767-773 (1991); Jung et al., Angew. Chem. Ind. Ed. Engl., 10: 31:367-383 (1992); Zuckerman et al., Proc. Natl. Acad. Sci. USA, 89:4505- = 4509 (1992); Scott et al., Science, 249:386-390 (1990); Devlin et al., Science, = --249:404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA, 87:6378-6382 - - (1990); and Gallop et al., J. Medicinal Chemistry, 37:1233-1251 (1994)). The a . -- resulting combinatorial libraries potentially contain millions of compounds and = . 15 ... that can be screened to identify compounds that exhibit a selected activity. . The libraries fall into roughly three categories: fusion-protein-displayed peptide libraries in which random peptides or proteins are presented on the surface of phage particles or proteins expressed from plasmids; support-bound synthetic chemical libraries in which individual compounds or mixtures of compounds are presented on insoluble matrices, such as resin beads (see, e.g.,

Lam et al., Nature, 354:82-84 (1991)) and cotton supports (see, e.g., Eichler et al., Biochemistry 32:11035-11041 (1993)); and methods in which the compounds are used in solution (see, e.g., Houghten et al., Nature, 354:84-86 (1991); Houghten et al., BioTechnigques, 313:412-421 (1992); and Scott et al,,

Curr. Opin. Biotechnol., 5:40-48 (1994)). There are numerous examples of synthetic peptide and oligonucleotide combinatorial libraries and there are many methods for producing libraries that contain non-peptidic small organic mole- cules. Such libraries can be based on basis set of monomers that are combined to form mixtures of diverse organic molecules or that can be combined to form a library based upon a selected pharmacophore monomer.

Either a random or a deterministic combinatorial library can be screened by the presently disclosed and/or claimed screening methods. In either of these

05-12-2001 To | US0031803

PS-DEC-29P1 11:48 FROM BOLT WADE TENNANT TO BP4SB923994465 P. 48 . two libraries, each unit of the library is isolated and/or immobilized on a solid support. In the deterministic library, one knows 2 prion a particular unit's location on each solid support. In a random library. the location of a particular unit is not known a priori although each site still contains a single unique unit.

Many methods for preparing libraries are known to those of skill in this art (see, e.g., Geysen et al., Proc. Natl, Acad. Sci. USA, 81:3998-4002 (1984),

Houghten et al., Proc. Nstl. Acad. Sci. USA, 81:5131-5135 (1985).

Combinatorial library generated by the any techniques known to those of . skill in the art are contemplated (see, e.g., Table 1 of Schultz and Schultz,

Biotechnol. Prog., 12(6):729-43 (1996}) for screening; Bartel et al., Science, 261:1411-1418 (1993); Baumbach ef al BioPharm, (May):24-35 (1992); Bock

Ce et ol. Nature. 355:564-566 (1992); Borman, S., Combinatorial chemists. focus = -

Ce oo on fami molecules, molecular. recognition, and sutemgtion, Chem. Eng. News, eT oo ELi21:29 (1996); Boubiik, et 8l.,-Eukarystic Virus Display: Engineering the Major." . co - "45 Surface Glycoproteins of the Autographa California Nuclesr Polyhedrosis ‘Virus ~~ ° (ACNPV) for the Presentation of Foreign proteins on-the Virus Surface, - : :

Bio/T echnology, 13:1079-1 084 (1995); Brenner, \et al., Encoded Combinatorial

ETS “Chaffiistry, Proc ‘Wail. Acsd Sci. $30, Be 15983 (1992); Cafiisch, et &i,, =e anes

Computational Combinatorial Chemistry for De Novo Ligand Design: Review and —-

Assessment, Perspect. Drug Discovery Des., 3:51-84 1 935); Cheng, et al.,

Sequence-Selective Peptide Binding with a Peptido-A,B-trans-steroidal Receptor

Selected from an Encoded Combinatorial Library, J. Am. Chem. Soc., 118:1813- 1814 (1996); Chu, et al., Affinity Capillary Electrophoresis to Identify the

Peptide in A Peptide Library that Binds Most Tightly to Vancomycin, J. Org. t

Chem., 58:648-652 (1993); Clackson, et al., Making Antibody Fragments Using

Phage Display Libraries, Nature, 352:624-628 (1991); Combs, et al., Protein

Structure-Based Combinatorial Chemistry: Discovery of Non-Peptide Binding

Elements to Src SH3 Domain, J. Am, Chem, Soc., 118:287-288 (1896); Cwira, et al., Peptides On Phage: A Vast Library of Peptides for Identifying Ligands,

Proc. Nat. Acad. Sci. U.S.A., 87:6378-6382 (1930); Ecker, et al., Combinatorial

Drug Discovery: Which Method will Produce the Greatest Value?,

Bio/Technology, 13:351-36Q (1995); Ellington, et al., /n Vitro Selection of RNA

AMENDED SHEET mor aPC NOON 10807 Lom me on DA

~ Molecules That Bind Specific Ligands, Mature, 346:818-822 (1990); Ellman, . . J.A., Variants of Benzodiazephines, J. Am. Chem. Soc., 114:10997 (1992); .. Erickson, et al., The Proteins; Neurath, H., Hill, R.L., Eds.: Academic: New York, 1976; pp. 255-257; Felici, et al., J. Mol. Biol., 222:301-310 (1991); Fodor, et 5. al., Light-Directed, Spatially Addressable Parallel Chemical Synthesis, Science,

E .. 251:767-773 (1991); Francisco, et al., Transport and Anchoring of Beta-

Lactamase to the External Surface of £. Coli., Proc. Natl. Acad. Sci. U.S.A., 89:2713-2717 (1992); Georgiou, et al., Practical Applications of Engineering

Gram-Negative Bacterial Cell Surfaces, 7/BTECH, 11:6-10 (1993); Geysen, et al., 10; Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a _ single amino acid, Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Glaser, . et al., Antibody Engineering by Condon-Based Mutagenesis in a Filamentous _. Phage Vector System, J. Immunol., 149:3903-3913 (1992); Gram, et al., In . vitro selection and affinity maturation of antibodies from a naive combinatorial - . 15 . immunoglobulin library, Proc. Natl. Acad. Sci., 89:3576-3580 (1992); Han, et - al., Liquid-Phase Combinatorial Synthesis, Proc. Natl. Acad. Sci. U.S. A., 92:6419-6423 (1995); Hoogenboom, et al., Multi-Subunit Proteins on the

Surface of Filamentous Phage: Methodologies for Displaying Antibody (Fab)

Heavy and Light Chains, Nucleic Acids Res., 19:4133-4137 (1991); Houghten, et al., General Method for the Rapid Solid-Phase Synthesis of Large Numbers of

Peptides: Specificity of Antigen-Antibody Interaction at the Level of Individual

Amino Acids, Proc. Natl. Acad. Sci. U.S.A., 82:5131-56135 (1985); Houghten, et al., The Use of Synthetic Peptide Combinatorial Libraries for the Determination of Peptide Ligands in Radio-Receptor Assays-Opiod-Peptides, Bioorg. Med.

Chem. Lett., 3:405-412 (1993); Houghten, et al., Generation and Use of

Synthetic Peptide Combinatorial Libraries for Basic Research and Drug Discovery,

Nature, 354:84-86 (1991); Huang, et al., Discovery of New Ligand Binding

Pathways in Myoglobin by Random Mutagenesis, Nature Struct. Biol., 1:226-229 (1994); Huse, et al., Generation of a Large Combinatorial Library of the

Immunoglobulin Repertoire In Phage Lambda, Science, 246:1275-1281 (1989);

Janda, K.D., New Strategies for the Design of Catalytic Antibodies, Biotechnol.

Prog., 6:178-181 (1990); Jung, et al., Multiple Peptide Synthesis Methods and

© 05-12-2001 | | US0031802 } os DEC-2001 11:48 FROM BOLT WADE TENNANT 10 EB4SEIZITIAAES P.41 ’ Their Applications, Angew. Chern. Int. £d. Engl., 31:367-486 {1992); Kang, et al., Linkage of Recognition and Replication Functions By Assembling

Combinatorial Antibody Fab Libraries Along Phage Surfaces. Proc. Natl. Acad.

Sci. U.S.A., 88:4363-4366 (19913); Kang, et al., Antibody Redesign by Chain

Shuffling from Random Combinatorial Immunoglobulin Libraries, Proc. Nat.

Acad. Sci. U.S.A., 88:11120-11123 (1991b}; Kay, et al., An-M13 Phage Library ’

Displaying Random 38-Amino-Aéid-Peptides as a Source of Novel Sequences with Affinity to Selected Targets Genes, Gere, 128:59-65 (1993); Lam, et al.. A : new type of synthetic peptide library for identifying ligand-binding activity,

Nature, 354:82-84 (1991) (published errata Sean Nature, 358:434 (1992) and Nature, 360:768 (19582); Lebl, et al., One Bead One Structure Combinatorial - -

Libraries, Biopolymers Pept. Sci.), 37:177-198 (1995); Lerner, et al.. Antibodies without Immunization, Science, 258:13131314 (199Z)ELL etal Minimizatién” TT 0

Soe orief 5 Polypeptide Hormone, Science, 270:1657-16601995}; Ligh, at al: IE

Lo ~~ 18. - Display of Dimeric Bacterial Alkaline: Phosphatase onthe Major Cat Protein of ~~ % 77 77

Filamentous Bactériophage, Bioorg. Med. Chem) Lett., 3:1073-1078 (1992);

Little, et al., Bacterial Surface Presentation of Proteins and Peptides: An iver Sp cae on inns c6IEENtIVE to Phage Technology?, Trends Biotechnol +. 11:3:5.11993)k Marks. ek... comm aor al... By-Passing Immunization. Human Antibodies trom V-Gene Libraries -

Displayed on Phage, J: Mol. Biol., 222:681-597 (1991); Matthews, et al.,

Substrate Phage: Selection of Protease Substrates by Monovalent Phage Display,

Science, 260:1113-1117 (1993); McCafferty, et al., Phage Enzymes: Expression and Affinity Chromatography of Functionat Alkaline Phosphatase on the Surface ‘ of Bacteriophage, Protein Eng., 4:955-961 (1991); Menger, etal, Phosphatase

Catalysis Developed Via Combinatorial Organic Chemistry, J. Org. Chem., 60:6666-6667 (1995); Nicolaou, et sl., Angew. Chem. Int. Ed. Engl., 34:2289- 2291 (1995); Oidenburg, et al., Peptide Ligands for A Sugar-Binding Protein

Isolated from a Random Peptide Library, Proc. Natl. Acad. Sci. U.S.A., 89:5393- 5397 (1992); Parmley, et al., Antibody-Selectable Filamentous td Phage Vectors:

Affinity Purification of Target Genes, Genes, 73:305-318 (1988); Pinilla, et al, :

Synthetic Peptide Combinatorial Libraries (SPCLS)--Identification of the Antigenic .

Determinant of Beta-Endorphin Recognized by Monoclonal Antibody-3E7, Gene, . :

AMENDED SHEET

-_ eA 21M ANNY TNE mh aU DAP cl RVI

05-12-2001 oo : | US0031803 “ ’ © pS-DEC-28@1 11:4@ FROM BOULT WADE TENNANT TO BB4S8323934465 p.a2 _____ -51- I. 128:71-76 (1993); Pinilla, et al., Review of the Utility of Soluble Combinatorial

Libraries, Biopolymers, 37:221-240 (1995); Pistor, et al., Expression of Viral -

Hemegglutinan On the Surface of £. Cofi., Klin. Wochenschr., §6:110-116 (1989); Pollack, et al., Selective Chemical Catalysis by an Antibody, Science, 234:1570-1572 (1986); Rigler, et al., Fluorescence: Correlations, Single Molecule

Detection and Large Number Screening: Applications in Biotechnology, J. ~

Biotechnal., 41:177-186 (1935); Sarvetnick, et al., Increasing the Chemical

Potential of the Germ-Line Antibody Repertoire, Proc. Nati. Acad. Se. USA. . 90:4008-4011 (1993); Sastiy, et al., Cloning of the-immunological Photon In To

Escherichia Coll for Generation of Monoclonal Catalytic Antibodies: “Construction "of a Heavy Chain Variable Region-Specific cDNA Library, Proc. Natl. Acad. Sci. . EE U.S.A. 86:5728-5732 “1 983); Scott, et al.. Searching for-Peptids Ligahds With: A AR E <TC ET an Epitope Library, Science, 249:386-390 (1990); Sears, of af. Enginaddng: = TT

Uw Tmeste proves for Bioorganic Synthesis: Peptide Bond Eorwiation, Biotechnol Prog., 12:423-433 (1996); Simon, et. al., Peptides: ‘A\Meditar Approach to Drug’ Ce

Discovery, Proc. Natl. Acad. Sci. U.S.A., 89:9367V-9371 (1892); Still, et al.,

Ha ary of Sedudnce- Selective Peptide Binding py Synthetic Receptors Using ——

Encoded Combinatorial Libraries, Ace. Chem. Res., 29:155-163 (1996); =

Thompson, et al., Synthesis and Applications of Small Molecule Libraries, Chem. )

Rev., 96:555-600 (1996); Tramontano, et al., Catalytic Antibodies, Science. 234:1566-1570 (1986); Wrighton, et al., Small Peptides as Potent Michetics of the Protein Hormone Erythropoietin, Science, 273:458-464 (1996); York, etal,

Combinatorial mutagenesis of the reactive site region in plasminogen activator inhibitor], J. Biol. Chem., 266:8595-8600 (1991); Zebedee, et al., Human

Combinatorial Anfibody Libraries to Hepatitis B Surface Antigen, Proc. Natl. 2uckermann

Acad. Sci. U.S.A., 89:3175-3178 (1 992); Zuekermer, et al., identification of

Highest-Affinity Ligands by Affinity Selection from Equimolsr Peptide fMmarey

Generated by Robotic Synthesis, Proc. Natl. Acad. Sci. U.S.A., 89:4505-4509 (1992). )

For example, peptides that bind to an endotheliase or a protease domain of an endotheliase can be Fred ting phage display libraries. In an exemplary embodiment, this method can include a) contacting phage from a :

AMENDED SHEET

Conf mm 2e Beran VOI LUM 1 all somitc | oo Te

05-12-2001 SR | So US003180¢

A — 11:41 FROM BOUT WADE TENNANT TO BP498323994465 P43 ____. -62- i. * phage library with the endotheliase protein or a protease domain thereof; {b) isolating phage that bind to the protein; and lc) determining the identity of at least one peptide coded by the isolated phage to identify a peptide that binds to an endotheliase. . § E. Moduistors of the activity of endothefiases

Provided herein are compounds, identified by screening or produced using “the endotheliases or protease domain in other screening methods, ‘that modulate’ the activity of an Andtnoieos These compounds act by directly interacting with the endotheliase or by altering transcription or translation thereof. Such molecules include, but are not limited to, antibodies that specifically react with an endotheliase, particularly with the protease domaip thereof, antisense nucleic oo

Co acids. that alter expression of the endotheliase, gndibodied,. peptide mimetics”. * Te

Jv... andothériich compounds. © © © RT FETE ene - | 3 | jp C1 : Antibodies. oo | \ Co Cs LL . . }

I 15 Provided herein are antibodies that spec] ically bind to an endotheliase, : preferably to the protease domain of the A protein. Preferably, the B ._..__ s;tibody isa monoclonal antibody, and preferably. the antibody immuno specifically binds to the protease domain of the endotheliase protein. In particular embodiments, antibodies to the protease domain of endotheliase 1 are - provided. Also provided are to endotheliase 2 and to the protease domain thereof are aiso provided. . The endotheliase protein and domains, fragments, homologs and - derivatives thereof may be used as immunogens to generate antibodies that specifically bind such immunogens. Such antibodies include but are not limited to polyclonal, morioclonal, chimeric, single chain, Fab fragments, and an Fab : expression library. In a specific embodiment, antibodies to human endotheliase are produced. In another embodiment, complexes formed from fragments of endotheliase, which fragments contain the serine protease domain, are used as immunogens for antibody production.

Various procedures known in the art may be used for the production of : polycional antibodies to endotheliase protein, its domains, derivatives, fragments or analogs. For production of the antibody, various host animals can be oo

AMENDED SHEET

Font nto R param) pre LIE) ofl sosize 1 olor immunized by injection with the native endotheliase protein or a synthetic ; i . version, or a derivative of the foregoing, such as a cross-linked endotheliase. : = Such host animals include but are not limited to rabbits, mice, rats, etc. Various

N .. adjuvants can be used to increase the immunological response, depending on the host species, and include but are not limited to Freund's (complete and - i} «: incomplete), mineral gels such as aluminum hydroxide, surface active substances : . such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, and potentially useful human adjuvants such as bacille Calmette- - Guerin (BCG) and corynebacterium parvum. - 10. For preparation of monoclonal antibodies directed towards an : =. endotheliase or domains, derivatives, fragments or analogs thereof, any technique that provides for the production of antibody molecules by continuous ~ . -- cell lines in culture may be used. Such techniques include but are not restricted - . to the hybridoma technique originally developed by Kohler and Milstein {Nature - : 15... 256:495-497 (1975)), the trioma technique, the human B-cell hybridoma : technique {Kozbor et al., Immunology Today 4:72 {1983)), and the EBV hybridoma technique to produce human monoclonal antibodies (Cole et al., in

Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 {(1985)). In an additional embodiment, monocional antibodies can be produced in germ-free animals utilizing recent technology (PCT/US90/02545). Human antibodies may be used and can be obtained by using human hybridomas (Cote et al., Proc. Natl. Acad. Sci. USA 80:2026-2030 (1983)). Or by transforming human B cells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies and

Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). Techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad.

Sci. USA 81:6851-6855 (1984); Neuberger et al., Nature 312:604-608 (1984);

Takeda et al., Nature 314:452-454 (1985)) by splicing the genes from a mouse antibody molecule specific for the endotheliase protein together with genes from a human antibody molecule of appropriate biological activity can be used.

Techniques described for the production of single chain antibodies (U.S. patent 4,946,778) can be adapted to produce endotheliase-specific single chain antibodies. An additional embodiment uses the techniques described for the

05-12-2001 I US0031803 : P5-DEC-2081 11:41 FROM BOULT WADE TENNANT TD BB498323934465 P.ad ___ __ construction of Fab expression libraries (Huse et al., Science 246:1275-1281 (1989) to allow rapid and easy identification of monocional Fab fragments with the desired specificity for endotheliase or endotheliasefef domains, derivatives, or analogs thereof. Nen-human antibodies can be “humanized” by known 5 methods (see, e.g., U.S. Patent No. 5,225,538).

Antibody fragments that contain the idiotypes of endotheliase can be

Co generated by techniques known in the art. For example, such fragments include but are not limited to: the F(ab’)2 fiagment which can be produced by pepsin digestion of the antibody molecule; the Fab’ fragments that can be generated by ; reducing the disulfide bridges of the Flab2 fragment. the Fab fragments that ) can be generated by treating the antibody greleeulag with papain and a reducing : agent, and Fv fragments. cL

CU Inthelproduction of antibodies, screening for the desitad antibody canbe’ Tt 2TH

Ts aceomplished by techniques known in the art) e.g., ELSA (enzymiedinked Cone

Co 15 immunosorbent assay). To select antibodies ‘specific to a particular domain of the endotheliase one may assay generated hybridomas for a product that binds to the fragment of the endothaliase that contains, such a domain - N oo

Cr “The foregoing antibodies can be used in mbthods known in the ant relating to the localization and/or quantitation of endotheliase proteins, e.g., for - imaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc. in another embodiment, (see /n/7a), anti-endotheliase antibodies, or fragments thereof, containing the binding ‘domain are used as therapeutic agents. 2. Peptides and Peptide Mimetics

Provided hérein are methods for identifying molecules that bind to and modulate the activity of endotheliases. Included among molecules that bind to wncorhelioses £ndeethelrased are peptides and peptide mimetics. Peptide mimetics are molecules or compounds that mimic the necessary molecular conformation of a ligand or polypeptide for specific binding to a target molecule such as, e.g., an endotheliase. In an exemplary embodiment, the peptides or peptide mimetics bind to the protease domain of the endotheliase. Such peptides and peptide mimetics include those of antibodies that specifically bind an endotheliase and,

AMENDED SHEET

— __trefC FIO NNT Ve FMUT FE ST Lee

05-12-2001 no | | | US0031803

PS-DEC-20@1 11:41 FROM BOLT WADE TENNANT TO @BASESZIIIALES P.a5 : -65- a fonitt, Hino to the protease domain of an endotheliase. The peptides and peptide mimetics identified by methods provided herein can be agonists or antagonists of endotheliases.

Such peptides and peptide mimetics are useful for diagnosing, treating, preventing, and screening for a disease or disorder associated with endotheliase activity in a mammal. In addition, the peptides and peptide mimetics are useful for identifying, isolating, and purifying molecules or compounds that modulate . the activity of an endotheliase, or specifically bind to an endotheliase,

Dery he protease domain of an endotheliase. Low molecular weight peptides and peptide mimetics can have strong binding properties to a target molecule, e.g.. an endotheliase or, Lins sme 8 the protease domain of an a endotheliase. RE rem eee UE iw... . i Peptides nd peptide miinetics that bind to eridotheliasas ‘as described ~~

AC _. .. hergin can be administered to mammals, including: humans, fo'rodulate ~ NE - 15 endotheliase activity. Thus, methods for therapeutic treatment and prevention of diseases or disorders associated with angiogenesis that comprise’ =

Lo _. ..administering a.peptide or peptide mimetic compound in an amount sufficiantte |, modulate such activity are provided. Thus, also provided herein are methods for - treating a subject having such a disease or disorder in which a peptide or peptide mimetic compound is administered to the subject in a therapeutically effective : dose or amount. :

Compositions containing the peptides or peptide mimetics can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions can be administered to a patient already suffering from a disease, a described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient. \ :

In prophylactic applications, compositions contsining the peptides and peptide mimetics are administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be a "prophylactically

AMENDED SHEET : me ieAm A OAR TEL TE I effective dose”. In this use, the precise amounts again depend on the patient's state of health and weight.

Accordingly, the peptides and peptide mimetics that bind to an endotheliase can be used generating pharmaceutical compositions containing, as an active ingredient, at least one of the peptides or peptide mimetics in association with a pharmaceutical carrier or diluent. The compounds can be administered, for example, by oral, pulmonary, parental (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation (via a fine powder formulation), transdermal, nasal, vaginal, rectal, or sublingual routes of administration and can be formulated in dosage forms appropriate for each route of administration (see, e.g., International PCT application Nos. WO 93/25221 and WO 94/17784,; and European Patent Application 613,683).

Peptides and peptide mimetics that bind to endotheliases are useful in vitro as unique tools for understanding the biological role of endotheliases, including the evaluation of the many factors thought to influence, and be influenced by, the production of endotheliase. Such peptides and peptide mimetics are also useful in the development of other compounds that bind to and modulate the activity of an endotheliase, because such compounds provide important information on the relationship between structure and activity that should facilitate such development.

The peptides and peptide mimetics are also useful as competitive binders in assays to screen for new endotheliases or endotheliase agonists. In such assay embodiments, the compounds can be used without modification or can be modified in a variety of ways; for example, by labeling, such as covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal. In any of these assays, the materials thereto can be labeled either directly or indirectly. Possibilities for direct labeling include label groups such as: radiolabels such as '?°l enzymes (U.S. Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring the change in fluorescence intensity, waveiength shift, or fluorescence polarization. Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label

: .. groups. The compounds may also include spacers or linkers in cases where the compounds are to be attached to a solid support. — Moreover, based on their ability to bind to an endotheliase, the peptides and peptide mimetics can be used as reagents for detecting endotheliases in

N 5.. living cells, fixed cells, in biological fluids, in tissue homogenates, in purified, .. Natural biological materials, etc. For example, by labelling such peptides and

B . peptide mimetics, one can identify cells having endotheliases. In addition, based on their ability to bind an endotheliase, the peptides and peptide mimetics can be _ used in situ staining, FACS (fluorescence-activated cell sorting), Western ] 10... blotting, ELISA, etc. In addition, based on their ability to bind to an endotheliase, the peptides and peptide mimetics can be used in purification of endotheliase polypeptides or in purifying cells expressing the endotheliase _ - polypeptides, e.g., a polypeptide encoding the protease domain of an ) _. endotheliase. ~ 15 The peptides and peptide mimetics can also be utilized as commercial ) reagents for various medical research and diagnostic uses.

The activity of the peptides and peptide mimetics can be evaluated either in vitro or in vivo in one of the numerous models described in McDonald (1992)

Am. J. of Pediatric Hematology/Oncology, 14:8-21, which is incorporated herein by reference.

Peptide and peptide mimetic therapy

Peptides and peptide mimetics that can bind to endotheliases or the protease domain of endotheliases and modulate the activity thereof, or have endotheliase activity, can be used for treatment of diseases and disorders associated with angiogenesis. The peptides and peptide mimetics may be delivered, in vivo or ex vivo, to the cells of a subject in need of treatment.

Further, peptides which have endotheliase activity can be delivered, in vivo or ex vivo, to cells which carry mutant or missing alleles encoding the endotheliase gene. Any of the techniques described herein or known to the skilled artisan can be used for preparation and in vivo or ex vivo delivery of such peptides and peptide mimetics that are substantially free of other human proteins. For example, the peptides can be readily prepared by expression in a microorganism or synthesis /n vitro.

The peptides or peptide mimetics can be introduced into cells, in vivo or ex vivo, by microinjection or by use of liposomes, for example. Alternatively, the peptides or peptide mimetics may be taken up by cells, in vivo or ex vivo, actively or by diffusion. In addition, extracellular application of the peptide or peptide mimetic may be sufficient to effect treatment of a disease or disorder associated with angiogenesis. Other molecules, such as drugs or organic compounds, that: 1) bind to an endotheliase or protease domain thereof; or 2) have a similar function or activity to an endotheliase or protease domain thereof, may be used in methods for treatment.

Rational drug design

The goal of rational drug design is to produce structural analogs of biologically active polypeptides or peptides of interest or of small molecules or peptide mimetics with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, e.g., more active or stable forms thereof; or which, e.g., enhance or interfere with the function of a polypeptide in vivo (e.g., ’ an endotheliase). In one approach, one first determines the three-dimensional structure of a protein of interest (e.g., an endotheliase or polypeptide having a protease domain) or, for example, of a endotheliase-ligand complex, by X-ray crystallography, by computer modeling or most typically, by a combination of approaches (see, e.g., Erickson et al. 1990). Also, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins. In addition, peptides can be analyzed by an alanine scan. In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide’s activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.

Also, a polypeptide or peptide that binds to an endotheliase or, preferably, the protease domain of an endotheliase, can be selected by a functional assay. and then the crystal structure of this polypeptide or peptide can be determined. The polypeptide can be, for example, an antibody specific

. -« for an endotheliase or the protein domain of an endotheliase. This approach can 5 . ~ yield a pharmacore upon which subsequent drug design can be based. Further, - itis possible to bypass the crystallography altogether by generating anti-idiotypic ] .. polypeptides or peptides, (anti-ids} to a functional, pharmacologically active . . 5.. polypeptide or peptide that binds to an endotheliase or protease domain of an - -=- endotheliase. As a mirror image of a mirror image, the binding site of the anti-ids is expected to be an analog of the original target molecule, e.g., an endotheliase or polypeptide having an endotheliase. The anti-id could then be _ used to identify and isolate peptides from banks of chemically or biologically : 10. produced banks of peptides. Selected peptides would then act as the . pharmacore. - - Thus, one may design drugs which have, e.g., improved activity or - stability or which act as modulators (e.g., inhibitors, agonists, antagonists, etc.)

E of endotheliase activity, and are useful in the methods of the present invention, - © 15.. particularly the methods for diagnosis, treatment, prevention, and screening of a disease or disorder associated with angiogenesis. By virtue of the availability of cloned endotheliase sequences, sufficient amounts of the endotheliase polypeptide may be made available to perform such analytical studies as X-ray crystallography. In addition, the knowledge of the amino acid sequence of an endotheliase or the protease domain thereof, e.g., the protease domain encoded by the amino acid sequence of SEQ ID NO: 2, can provide guidance on computer modeling techniques in place of, or in addition to, X-ray crystallography.

Methods of identifying peptides and peptide mimetics that bind to _ : -endotheliases

Peptides having a binding affinity to the endotheliase polypeptides provided herein (e.g., an endotheliase or a polypeptide having a protease domain of an endotheliase) can be readily identified, for example, by random peptide diversity generating systems coupled with an affinity enrichment process.

Specifically, random peptide diversity generating systems include the "peptides on plasmids" system (see, e.g., U.S. Patent Nos. 5,270,170 and 5,338,685); the "peptides on phage” system (see, e.g., U.S. Patent No. 6,121,238 and

Cwirla,et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:6378-6382); the

05-12-2001 oo US0031803 © PS-DEC-28@1 11:41 FROM BOULT WADE TENNANT TO BP4SESZ3934465 P.46 -70- ~ “polysome system;” the "encoded synthetic library (ESL)” system: and the “very large scale immobilized polymer synthesis” system (see, e.g., U.S. Patent No. 6,121,238; and Dower et &l. (1991) Ann. Rep. Med. Chem. 26:271-280)-

For example, using the procedures described above, random peptides can generally be designed to have a defined number of amino acid residues in length (e.g., 12). To generate the collection of oligonuciectides encoding the random - peptides, the codon motif (NNK}x, where N is nucleotide A, c, G, or T {equimolar; depending on the methodology employed, other nuclectides can be oo employed), Kis G or T (equimolar), and x is an integer corresponding to the number of amine acids in the peptide (e.g., 12) can be used to specify any one of the 32 possible codons resulting from the NNK motif: 1 for each of 12 amino :

Co acids, 2 for each of 5 amino acids; 3 for each of 3 ‘aiing_4GidE: dnd oRly Gre. of! - BRC

Ee “the three stop codons. Thus, the NNK motif encodes sii of the'amino acids, = ©

Cn ©. "encodes only one stop codon, and reduces coon ‘bias TER Ee no E

The random peptides can be presented, \for example, either onthe surface : of a phage particle, as part of a fusion protein containing either the pill or the rr PIII caat protein of a phage 1d derivative (peptides on phage) orasatfusion =~ 7 °° protein with the Lacl peptide fusion protein bound to a plasmid (peptides on - plasmids). The phage or plasmids, including the DNA encoding the peptides, can be identified and isolated by an affinity enrichment process using immobilized endotheliase polypeptide having a protease domain. The affinity enrichment y process, sometimes called “panning,” typically involves multiple rounds of incubating the phage. plasmids, or polysomes with the immobilized endotheliase polypeptide, collecting the phage. plasmids, or polysomes that bind to the : endotheliase polypeptide (along with the accompanying DNA or mRNA), and producing more of the phage or plasmids (along with the accompanying

Laci-peptide fusion protein) collected. :

Characteristics of peptides and peptide mimetics

Typically, the molecular weight of preferred peptides or peptide mimetics is from about 250 to about 8,000 daltons. If the peptides are oligomerized, dimerized and/or derivatized with a hydrophilic polymer (e.g., to increase the affinity and/or activity of the compounds), the molecular weights of such :

AMENDED SHEET : mor rae ND an mae CYTES ITY IE SREY

- - peptides can be substantially greater and can range anywhere from about 500 to . - about 120,000 daltons, more preferably from about 8,000 to about 80,000 . daltons. Such peptides can comprise 9 or more amino acids wherein the amino _ acids are naturally occurring or synthetic (non-naturally occurring) amino acids.

One skilled in the art would know how to determine the affinity and molecular : -- weight of the peptides and peptide mimetics suitable for therapeutic and/or . diagnostic purposes (e.g., see Dower et al., U.S. Patent No. 6,121,238).

The peptides may be covalently attached to one or more of a variety of ] hydrophilic polymers. Suitable hydrophilic polymers include, but are not limited

BN 10... to, polyalkylethers as exemplified by polyethylene glycol and polypropylene -- glycol, polylactic acid, polyglycolic acid, polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran and dextran . oo derivatives, etc. When the peptide compounds are derivatized with such ) . .. polymers, their solubility and circulation half-lives can be increased with little, if . . 16... any, diminishment in their binding activity. The peptide compounds may be . dimerized and each of the dimeric subunits can be covalently attached to a hydrophilic polymer. The peptide compounds can be PEGylated, i.e., covalently attached to polyethylene glycol (PEG).

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide.

These types of non-peptide compounds are termed "peptide mimetics” or "peptidomimetics" (Luthman et a/., A Textbook of Drug Design and

Development, 74:386-406, 2nd Ed., Harwood Academic Publishers (1996);

Joachim Grante (1994) Angew. Chem. Int. Ed. Engl., 33:1699-1720; Fauchere (1986) J. Adv. Drug Res., 15:29; Veber and Freidinger (1985) TINS, p. 392; and

Evans et al. (1987) J. Med. Chem. 30:1229). Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Preparation of peptidomimetics and structures thereof are known to those of skill in this art.

Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of

L-lysine) may be used to generate more stable peptides. In addition, constrained

05-12-2001 ) | | US0031803 : as—pEC-2001 11:41 FROM BOULT WADE TENNANT TO .DP498323F34465 P.47 peptides containing a consensus sequence or a substantisily identical consensus sequence variation may be generated by methods known in the art [Rize ef 3/. (1992) Ann. Rev. Biochem., 67:387, incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular : 5 disulfide bridges which cyclize the peptide. - co

Those skilled in the art would appreciate that modifications may be made to the peptides and mimetics without deleteriously effecting the biological or functional activity of the peptide. Further, the skilled artisan would know how to design non-peptide structures in three dimensional terms, that mimic the peptides that bind to a target molecule, e.g., an endothefiase or, FA oral the ) protease domain of endotheliases (see, e.g.. Eck and Sprang (1989) J. Biol.

EE Chem., 26: 17605-18795). B J oo FEET. pd IETERL ~ Ce

CE co When ased for diagnostic purposes, the peptides an peptide ‘mimetics SRE © =". may be labeled .with a detectable label and, accordingly, tHe peptides dnd TI Te CC peptide mimetics without such a label can serve as taimediates in the preparation of labeled peptides and peptide mimetics. Detectable labels can be

Cede moleeiies oF compounds, which when covalently ltcaciied 10 thie pepéides ang~~~~ re peptide mimetics, permit detection of the peptide and peptide mimetics in vivo, - for example, in a patient to whom the peptide or peptide mimetic has been ~~ administered, or in vitro, €.g., in a sample or cells. Suitable detectable labels are well known in the art and include, by way of example, radioisotopes, fluorescent labels (e.g., fluorescein), and the like. The particular detectable label employed is not critical and is selected relative to the amount of label to be employed as well as the toxicity of the label at the amount of label employed. Selection of the label relative to such factors is well within the skill of the art. :

Covalent attachment of a detectable label to the peptide or peptide ; mimetic is accomplished by conventional methods well known in the art. For' example, when the '“| radioisotope is employed as the detectable label, covalent attachment of '¥% to the peptide or the peptide mimetic can be achieved by incorporating the amino acid tyrosine into the peptide or peptide mimetic and thenAstimenng the peptide (see, e.g., Weaner et al. (1994) Synthesis and

Applications of Isotopicslly Labelled Compounds, pp. 137-140). If tyrosine is’ :

IE

AMENDED SHEET '

Fond 1 aSOE fernnny rm I I TARY not present in the peptide or peptide mimetic, incorporation of tyrosine to the N or C terminus of the peptide or peptide mimetic can be achieved by well known chemistry. Likewise, 3?P can be incorporated onto the peptide or peptide mimetic as a phosphate moiety through, for example, a hydroxyl group on the peptide or peptide mimetic using conventional chemistry.

Labeling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer {e.g., an amide group), to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling. Such non-interfering positions generally are positions that do not form direct contacts with the macromolecules(s) to which the peptidomimetic binds to produce the therapeutic effect. Derivatization (e.g., labeling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic.

Methods of preparing peptides and peptide mimetics

Peptides that bind to endotheliases can be prepared by classical methods known in the art, for example, by using standard solid phase techniques. The standard methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis, and even by recombinant DNA technology (see, e.g., Merrifield (1963) J. Am. Chem.

Soc., 85:2149, incorporated herein by reference.)

Using the "encoded synthetic library” or "very large scale immobilized polymer synthesis" systems (see, e.g., U.S. Patent No. 5,925,525, and 5,802,723); one can not only determine the minimum size of a peptide with the activity of interest, one can also make all of the peptides that form the group of peptides that differ from the preferred motif {or the minimum size of that motif) in one, two, or more residues. This collection of peptides can then be screened for ability to bind to the target molecule, e.g., and endotheliase or, preferably, the protease domain of an endotheliase. This immobilized polymer synthesis system or other peptide synthesis methods can also be used to synthesize truncation analogs and deletion analogs and combinations of truncation and deletion analogs of the peptide compounds.

BE an CIEE ALS ee Lo . 05-12-2001 I 7 US0031803

These procedures can also be used to synthesize peptides in which amino acids other than the 20 naturally occurring, genetically encoded amino acids are substituted at one, two, or more positions of the peptide. For instance, naphthylalanine can be substituted for tryptophan, facilitating synthesis. Other synthetic amino acids that can be substituted into the ‘peptides include

L-hydroxypropyl, L-3, 4-dihydroxy-phenylalanyl, d amino acids such-as

L-g-hydroxylysyl and D-d-methylzlanyl, L-o-methylalanyl, # amino acids, and isoguinolyl. D amino acids and non-naturally occurring synthetic amino acids can also be incorporated into the peptides (see, e.g., Roberts et o/, (1983) ) )

Unusual Amino/Acids in Peptide Synthesis, 56):341-449)." -

The peptides may also be modified by phosphorylation (see. e.g., W.

[21] a) UAC To oo . Bannwarth et al. (1996) Blosvanty and Medicinal Chemistry Letters, . 6(17):2141-2146), and other methods for making peptide derivatives (see, &.g.. oo Hruby et sl. (1990) Biochem. J., 268(2):249-262). Thus, peptide compounds *

SR 15. “also serve-as a basis to prépare peptide mimétics with SimitaF Biological activity. * » TI i oo : CT ‘Those of skill in the art recognize that a variety of techniques arg vo TITRE NE

AE © available for constructing ‘peptide mimetics with the ‘same’or similar desired ] . biological activity as the corresponding peptide ‘compound ‘but with riore oT © favorable sctivity than the peptide with respect to solubility, stability, and -

Th susceptibility to hydrolysis and proteolysis (see, e.g., Morgan ef of. (1989) Ann,

Rep. Med. Chem., 24:243-252). Methods for preparing peptide mimetics’ ha modified at the N-terminal amino group, the C-terminal carboxy! group, and/or changing one or more of the amido linkages in the peptide to a non-amido : : linkage are known to those of skill in the art. oo © 25 Amino terminus modifications include alkylating, acetylating, adding a carbobenzoy! group, forming a succinimide group, etc, see. e.g., Murrey etal (1995) Burger's Medicinal Chemistry and Drug Discovery, 5th ed., Vol. 1,

Manfred E. Wolf, ed., John Wiley and Sons, Inc.). C-terminal modifications include mimetics wherein the C-terminal carboxyl group Is replaced by an ester, an amide or modifications to form a cyclic peptide.

In addition to N-terminal and C-terminal modifications, the peptide oo compounds, including peptide mimetics, can advantageously be modified with or

Empf .zeit:05/12/2001 12:33 Empf .nr.2972 P.048

AMENDED SHEET covalently coupled to one or more of a variety of hydrophilic polymers. It has been found that when peptide compounds are derivatized with a hydrophilic polymer, their solubility and circulation half-lives may be increased and their immunogenicity is masked, with little, if any, diminishment in their binding

Bb activity. Suitable nonproteinaceous polymers include, but are not limited to, polyalkylethers as exemplified by polyethylene glycol and polypropylene glycol, polylactic acid, polyglycolic acid, polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran and dextran derivatives, etc. Generally, such hydrophilic polymers have an average molecular weight ranging from about 500 to about 100,000 daltons, more - preferably from about 2,000 to about 40,000 daltons and, even more preferably, from about 5,000 to about 20,000 daltons. The hydrophilic polymers also can have an average molecular weights of about 5,000 daltons, 10,000 daltons and -, 20,000 daltons. 16 .. . Methods for derivatizing peptide compounds or for coupling peptides to such polymers have been described (see, e.g., Zallipsky (1995) Bioconjugate

Chem., 6:150-165; Monfardini et al. (1995) Bioconjugate Chem., 6:62-69; U.S.

Pat. No. 4,640,835; U.S. Pat. No. 4,496,689; U.S. Pat. No. 4,301,144; U.S.

Pat. No. 4,670,417; U.S. Pat. No. 4,791,192; U.S. Pat. No. 4,179,337 and WO 95/34326, all of which are incorporated by reference in their entirety herein).

Other methods for making peptide derivatives are described, for example, in Hruby et al. (1990), Biochem J., 268(2):249-262, which is incorporated herein by reference. Thus, the peptide compounds also serve as structural models for non-peptidic compounds with similar biological activity. Those of skill in the art recognize that a variety of techniques are available for constructing compounds with the same or similar desired biological activity as a particular peptide compound but with more favorable activity with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis (see, e.g.,

Morgan et al. (1989) Ann. Rep. Med. Chem., 24:243-252, incorporated herein by reference). These techniques include replacing the peptide backbone with a backbone composed of phosphonates, amidates, carbamates, sulfonamides, secondary amines, and N-methylamino acids.

. osMEC-ZAR] 11:42 FRUM CCERLI WALT 1S A Saad 05-12-2001 : p LO US0031803

Peptide compounds may exist in a cyclized form with an intramolecular disulfide bond between the thiol groups of the cysteines. Alternatively, an _ intermolecular disulfide bond between the thiol groups of the cysteines can be produced to yield a dimeric {or higher oligomeric) compound. One or more of the 6 cysteine residues may also be substituted with a homocysteine. :

F. CONJUGATES :

A conjugate, containing: a) a protease domain of an endotheliase protein encoded by a nucleic acid hybridizes to a nucleic acid having the nucleotide sequence set forth in the SEQ. JD NO:1; and b) a targeting agent linked to the 16 endotheliase directly or via a linker, wherein the agent facilitates: i) affinity isolation or purification of the conjugate; ii) attachment of the conjugate toa - surface; iii) detection of the conjugate; or iv) targeted delivery to a selected h tissue or cell, is provided herein. f——————— eee eee ey - Theg conjugate can be a chemical conjugate or a fusion protein mixture thereof. . .

SPR 15 - The targeting agent is preferably a protein or peptide fragment; “Stich 381 a" SE ol or - ‘tissue specific or tumor-specific monoclonal antibody br growth factor or “RET

SA fragment therecf linked either directly or via a linkér to"é ‘endothéliase ora” =~ : protease domain thereof. The targeting agent may also be a protein or peptide’ :

A | fragment that contains a protein binding sequence, a nucleic acid binding ©7777 720 sequence, a lipid binding” sequence, a polysaccharide binding seguence, ora =" TC TTT metal binding sequence, or a linker for attachment 10 a solid support. = in a particular embodiment, the conjugate contains a) an endotheliase or ~ protease domain of an endotheliase protein encoded by a nucleic acid hybridizes a nucleic acid molecule having the nucleotide sequence set forth in the

SEQ. ID NO:1, 3, Bor 22; and b) a targeting agent linked to the endotheliase directly or via a linkera

Conjugates, such as fusion proteins and chemical conjugates, of the : endotheliase with a protein or peptide fragment (or plurality thereof) that functions, for example, to facilitate affinity isolation or purification of the endotheliase domain, attachment of the endotheliase domain to a surface, or detection of the endotheliase domain are provided. The conjugates can be - produced by chemical conjugation, such as via thiol linkages, but are preferably .

Emef .zei t205/12/2001 12:33 Empf .nr.:972 P.O4AS ;

AMENDED SHEET produced by recombinant means as fusion proteins. In the fusion protein, the peptide or fragment thereof is linked to either the N-terminus or C-terminus of the endotheliase domain. In chemical conjugates the peptide or fragment thereof may be linked anywhere that conjugation can be effected, and there may be a plurality of such peptides or fragments linked to a single endotheliase domain or to a plurality thereof.

The targeting agent is preferably for /n vitro delivery to a cell or tissue, and includes agents such as cell or tissue-specific antibodies, growth factors and other factors expressed on specific cells; and other cell or tissue specific agents the promote directed delivery of a linked protein. ’ Most preferably the targeting agent specifically delivers the endotheliase - to selected cells by interaction with a cell surface protein and internalization of conjugate or endotheliase portion thereof. These conjugate are used in a variety of methods and are particularly suited for use in methods of activation of prodrugs, such as prodrugs that upon cleavage by the particular endotheliase are cytotoxic. The prodrugs are administered prior to simultaneously with or subsequently to the conjugate. Upon delivery to the targeted cells, the protease activates the prodrug, which then exhibits is therapeutic effect, such as a cytotoxic effect. 1. Conjugation

Conjugates with linked endotheliase domains can be prepared either by chemical conjugation, recombinant DNA technology, or combinations of recombinant expression and chemical conjugation. The endotheliase domains and the targeting agent may be linked in any orientation and more than one targeting agents and/or endotheliase domains may be present in a conjugate. a. Fusion proteins

Fusion proteins are proved herein. A fusion protein contains: a) one or a plurality of domains of an endotheliases and b) a targeting agent. The fusion proteins are preferably produced by recombinant expression of nucleic acids that encode the fusion protein.

b. Chemical conjugation

To effect chemical conjugation herein, the endotheliase domain is linked via one or more selected linkers or directly to the targeting agent. Chemical conjugation must be used if the targeted agent is other than a peptide or protein, such a nucleic acid or a non-peptide drug. Any means known to those of skill in the art for chemically conjugating selected moieties may be used. 2. Linkers

Linkers for two purposes are contemplated herein. The conjugates may include one or more linkers between the endotheliase portion and the targeting agent. Additionally, linkers are used for facilitating or enhancing immobilization : of an endotheliase or portion thereof on a solid support, such as a microtiter . plate, silicon or silicon-coated chip, glass or plastic support, such as for high throughput solid phase screening protocols. a. Exemplary Linkers

Any linker known to those of skill in the art for preparation of conjugates may be used herein. These linkers are typically used in the preparation of chemical conjugates; peptide linkers may be incorporated into fusion proteins. - Linkers can be any moiety suitable to associate a domain of endotheliase and a targeting agent. Such linkers and linkages include, but are not limited to, peptidic linkages, amino acid and peptide linkages, typically containing between one and about 60 amino acids, more generally between about 10 and 30 amino acids, chemical linkers, such as heterobifunctional cleavable cross-linkers, including but are not limited to, N-succinimidyl (4-iodoacetyl)-aminobenzoate, sulfosuccinimydil (4-iodoacetyl}-aminobenzoate, 4-succinimidyl-oxycarbonyl-a- (2-pyridyldithio)toluene, sulfosuccinimidyl-6- [a-methyi-a-(pyridyldithiol)- toluamido] hexanoate, N-succinimidy!-3-(-2-pyridyldithio) - proprionate, succinimidyl 6[3(-(-2-pyridyldithio)-proprionamido] hexanoate, sulfosuccinimidyi 6[3(-(-2-pyridyldithio)-propionamido] hexanoate, 3-(2-pyridyldithio}-propionyl hydrazide, Ellman’s reagent, dichlorotriazinic acid, and S-(2-thiopyridyl)-L- cysteine. Other linkers include, but are not limited to peptides and other moieties that reduce stearic hindrance between the domain of endotheliase and the targeting agent, intracellular enzyme substrates, linkers that increase the

© 05-12-2001 LL | | | US0031808 p5-DEC-20A1 11:42 FROM BOULT WADE TENNANT TO Bp4SB923334465 P.58 . flexibility of the conjugate, linkers that increase the solubility of the conjugate, linkers that increase the serum stability of the conjugate, photocleavable linkers and acid cleavable linkers.

Other exemplary linkers and linkages that are suitable for chemically

Jinked conjugates include, but are not limited to, disulfide bonds, thioether bonds, hindered disulfide bonds, and covalent bonds between free reactive groups, such as amine and thiol groups. These bonds are produced using heterobifunctional reagents to produce reactive thiol groups on one or both of : the polypeptides and then reacting the thiol groups on ane polypeptide with reactive thiol groups or amine groups to which reactive maleimide groups or thiol groups can be sttachied on the other, Other linkers include, acid cleavable : : linkers, such as bismaleimideathoxy propane, acid:labile-transferrin.conjugates * ©. =

CET . and Sdipic 3cid Ay raaag hat would be cleaved in bre Gili inraceliais ar Ea ~ . + ‘compamments; cross linkers tliat are cleaved upon expesird t6"UV or-visible I IS

Co 15 . light and linkers, such as the various domains, ‘such as C,1, C2, and C,3, from the constant region of human IgG, (see, Batra et al. Molecular Immunol. oo 30:379-386 (1993). In some embodiments, several linkers may be included In © oderto take advantage of desired properties of each linker.

Chemical linkers and peptide linkers may be inserted by covalently © coupling the linker to the domain of endotheliase and the targeting agent. The heterobifunctional agents, described below, may be used to effect such covalent coupling. Peptide linkers may also be linked by expressing DNA encoding the linker and TA, linker and targeted agent, or linker, targeted agent and TA as a . fusion protein. Flexible finkers and linkers that increase solubility of the conjugstes are contemplated for use, either alone or with other linkers are also contemplated herein. 7 Acid cleavable, photocleavable and heat sensitive linkers

Acid cleavable linkers, photocleavable and heat sensitive linkers may also be used, particularly where it may be necessary to cleave the domain of . endotheliase to permit it to be more readily accessible to reaction. Acid cleavable linkers include, but are not limited to, bismaleimideothoxy propane; .

AMENDED SHEET :

Lal ss arias Lomi me BUS Lr and adipic acid dihydrazide linkers (see, e.g., Fattom et al. (1992) Infection &

Immun. 60:584-589) and acid labile transferrin conjugates that contain a sufficient portion of transferrin to permit entry into the intracellular transferrin cycling pathway (see, e.g., Welhoner et al. (1991) J. Biol. Chem. 266:4309- 4314).

Photocleavable linkers are linkers that are cleaved upon exposure to light (see, e.g., Goldmacher et al. (1992) Bioconj. Chem. 3:104-107, which linkers are herein incorporated by reference), thereby releasing the targeted agent upon exposure to light. Photocleavable linkers that are cleaved upon exposure to light are known (see, e.g., Hazum et al. (1981) in Pept., Proc. Eur. Pept. Symp., 16th, Brunfeldt, K (Ed), pp. 105-110, which describes the use of a nitrobenzyl group as a photocleavable protective group for cysteine; Yen et al. (1989)

Makromol. Chem 790:69-82, which describes water soluble photocleavable copolymers, including hydroxypropylmethacrylamide copolymer, glycine copolymer, fluorescein copolymer and methylrhodamine copolymer; Gold- a macher et al. (1992) Bioconj. Chem. 3:104-107, which describes a cross-linker and reagent that undergoes photolytic degradation upon exposure to near UV light (350 nm); and Senter et al. (1985) Photochem. Photobiol 42:231-237, which describes nitrobenzyloxycarbonyl chloride cross linking reagents that produce photocleavable linkages), thereby releasing the targeted agent upon exposure to light. Such linkers would have particular use in treating dermatological or ophthalmic conditions that can be exposed to light using fiber optics. After administration of the conjugate, the eye or skin or other body part can be exposed to light, resulting in release of the targeted moiety from the 26 conjugate. Such photocleavable linkers are useful in connection with diagnostic protocols in which it is desirable to remove the targeting agent to permit rapid clearance from the body of the animal. 2) Other linkers for chemical conjugation

Other linkers, include trityl linkers, particularly, derivatized trityl groups to generate a genus of conjugates that provide for release of therapeutic agents at various degrees of acidity or alkalinity.

The flexibility thus afforded by the ability to preselect the pH range at

05-12-2001 | | | | US0031803

BS-DEC-20P1 11:42 FROM BOLLT WADE TENNANT TO @P49ES2II4ES P.51 which the therapeutic agent will be released allows selection of a linker based on the known physiological differences between tissues in need of delivery of a therapeutic agent (see, e.g., U.S. Patent No. 5,612,474). For example, the acidity of tumor tissues appears to be lower than that of normal tissues. 3) Peptide linkers

The linker moieties can be peptides. Peptide linkers can be employed in fusion proteins and also in chemically linked conjugates. The peptide typically : has from about 2 to about 60 ating acid residues, for example from about 5 to : about 40, or from about 10 to about 30 amino acid residues. The length selected will depend upon factors, such as the use for which the linker is included. : a Peptide linkers are advantageous when the Targeting 3gent:is:. » 1: wo noaleu em en

LR ces proteinaceous. ‘For example; the linker moiety canbe a-fléxible spacer amific: FEEL Teme ©. ©: acid sequence, such as those known in single-chain. antibody: research. © nieic ii

Co 15 Examples of such known linker moieties include, but are not limited to, ) peptides, such as (Gly, Ser), and (Ser, Gly),, in which nis 1 to 6, preferably- 1 1 {- JE 4. more preferably 2to 4, and mis 110 6, preferably 1 to 4, more preferably 2 Co 10 4, enzyme cleavable linkers and others. Vo Lo TT ee

Additional tinking moieties are described, for example, in Huston et a/., -

Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, 1988; Whitlow, M., et a/., Protein

Engineering 6:989-895, 1993; Newton et al., Biochemistry 35:545-563, 1996;

A. J. Cumber et al., Bioconj. Chem. 3:397-401, 1992; Ladumer et al.. J. Mol.

Biol, 273:330-337, 1997; and U.S. Patent. No. 4,894,443. Insome embediments, several linkers may be included in order to take advantage of : i - 25 desired properties of each linker. - : 4 } 3. Targeting agents

Any agent that facilitates detection, immobilization, or purification of the conjugate is contemplated for use herein. For chemical conjugates any moiety that has such properties is contemplated; for. fusion proteins, the targeting agent is a protein, peptide or fragment thereof thatfSufficient to effects the targeting activity. Preferred targeting agents are those that deliver the endotheliase or portion thereof to selected cells and tissues. Such agents include tumor specific

AMENDED SHEET

© 05-12-2001 US0031803 : p5_DEC-2071 11:42 FROM BOLT WADE TENNANT .T0 BB4SEIZIINAUES i pP.52 monoclonal antibodies and portions thereof, growth factors, such as FGF, EGF,

PDGF, VEGF, cytokines, including chemokines, and other such agents. 4, Nucleic acids, plasmids and celts : 0

Isolated nucleic acid fragments encoding fusion proteins are provided. § The nucleic acid fragment that encodes the fusion protein includes: aJ nucleic acid encoding a protease domain of an endotheliase protein encoded by a nucleic acid that hybridizes to a nucleic acid having the nucleotide sequence set forth in the SEQ. ID NO:1; and b} nucleic atid encoding a protein, peptide or effective fragment thereof that facilitates: I) affinity isolation oF purification of the fusion protein; ii) attachment of the fusion protein to a surface; or iii} detection of the fusion protein. Preferably, the nucleic acid is DNAgz ’ : “ Loree . Plasmids for replication snd vectors for expigssion that cantaid ‘the*sbove’ TE fe

Coie ee picleic Ed fraginiants ar Siso provided. Cells containing The plasmids and ro TT Le - Sn .. - ..--vectors-aré also-provided. The cells can bé any siitableshost including; bot are: | Pe

CT 15 not limited to, bacterial celis, yeast cells, fungal: cells, plant-cells-insect-cell and : animal cells. The nucleic acids, plasmids, and cells containing the ‘plasmids can i : LL _. be prapared according to methods known in tha an including any -dasoribad. . ocr mrss. herein. | © -

Also provided are methods for producing the above fusion proteins. An --

Sepia method includes the steps of growing, i.e. culturing the cells so that she proliferate, cells containing a plasmid encoding the fusion protein under conditions whereby the fusion protein is expressed by the cell, and recovering the expressed fusion protein. Methods for expressing and recovering : recombinant proteins are well known in the ast (See generally, Current Protocols in Molecular Biology (1998) § 16, John Wiley & Sans, Inc.) and such methods can be used for expressing and recovering the expressed fusion proteins.

Preferably, the recombinant expression and recovery methods disclosed in

Section B can be used. \

The recovered fusion proteins can be isolated or purified by ‘methods known in the art such as centrifugation, filtration, chromatograph, electrophoresis, immunoprecipitation, etc.. or by 3 combination thereof {See generally, Current Protocols in Molecular Biology (1998) § 10, John Wiley & ©

AMENDED SHEET ee i eAm rac adr Terai EE EY

Sons, Inc.). Preferably, the recovered fusion protein is isolated or purified through affinity binding between the protein or peptide fragment of the fusion protein and an affinity binding moiety. As discussed in the above sections regarding the construction of the fusion proteins, any affinity binding pairs can be constructed and used in the isolation or purification of the fusion proteins.

For example, the affinity binding pairs can be protein binding sequences/protein,

DNA binding sequences/DNA sequences, RNA binding sequences/RNA sequences, lipid binding sequences/lipid, polysaccharide binding sequences/polysaccharide, or metal binding sequences/metal. 10. 6. Immobilization and supports or substrates therefor

In certain embodiments, where the targeting agents are designed for -. linkage to surfaces, the endotheliase can be attached by linkage such as ionic or . covalent, non-covalent or other chemical interaction, to a surface of a support or .. matrix material. Immobilization may be effected directly or via a linker. The endotheliase may be immobilized on any suitable support, including, but are not limited to, silicon chips, and other supports described herein and known to those of skill in the art. A plurality of endotheliase or protease domains thereof may be attached to a support, such as an array (i.e., a pattern of two or more) of conjugates on the surface of a silicon chip or other chip for use in high throughput protocols and formats.

It is also noted that the domains of the endotheliase can be linked directly to the surface or via a linker without a targeting agent linked thereto. Hence chips containing arrays of the domains of the endotheliase.

The matrix material or solid supports contemplated herein are generally any of the insoluble materials known to those of skill in the art to immobilize ligands and other molecules, and are those that used in many chemical syntheses and separations. Such supports are used, for example, in affinity chromatography, in the immobilization of biologically active materials, and during chemical syntheses of biomolecules, including proteins, amino acids and other organic molecules and polymers. The preparation of and use of supports is well known to those of skill in this art; there are many such materials and preparations thereof known. For example, naturally-occurring support materials,

such as agarose and cellulose, may be isolated from their respective sources, and processed according to known protocols, and synthetic materials may be prepared in accord with known protocols.

The supports are typically insoluble materials that are solid, porous, deformable, or hard, and have any required structure and geometry, including, but not limited to: beads, pellets, disks, capillaries, hollow fibers, needles, solid fibers, random shapes, thin films and membranes. Thus, the item may be fabricated from the matrix material or combined with it, such as by coating all or part of the surface or impregnating particles.

Typically, when the matrix is particulate, the particles are at least about 10-2000 uM, but may be smaller or larger, depending upon the selected application. Selection of the matrices will be governed, at least in part, by their physical and chemical properties, such as solubility, functional groups, mechanical stability, surface area swelling propensity, hydrophobic or hydrophilic properties and intended use.

If necessary, the support matrix material can be treated to contain an appropriate reactive moiety. In some cases, the support matrix material already containing the reactive moiety may be obtained commercially. The support matrix material containing the reactive moiety may thereby serve as the matrix support upon which molecules are linked. Materials containing reactive surface moieties such as amino silane linkages, hydroxy! linkages or carboxysilane linkages may be produced by well established surface chemistry techniques involving silanization reactions, or the like. Examples of these materials are those having surface silicon oxide moieties, covalently linked to gamma-amino- propylsilane, and other organic moieties; N-[3-{triethyoxysilyl)propyllphthelamic acid; and bis-(2-hydroxyethyllaminopropyitriethoxysilane. Exemplary of readily available materials containing amino group reactive functionalities, include, but are not limited to, para-aminophenyltriethyoxysilane. Also derivatized polystyrenes and other such polymers are well known and readily available to those of skill in this art (e.g., the Tentagel® Resins are available with a multitude of functional groups, and are sold by Rapp Polymere, Tubingen, Germany; see,

U.S. Patent No. 4,908,405 and U.S. Patent No. 5,292,814; see, also Butz et al.,

Peptide Res., 7:20-23 (1994); and Kleine et al., /mmunobiol., 190:53-66

These matrix materials include any material that can act as a support matrix for attachment of the molecules of interest. Such materials are known to those of skill in this art, and include those that are used as a support matrix. - These materials include, but are not limited to, inorganics, natural polymers, and synthetic polymers, including, but are not limited to: cellulose, cellulose derivatives, acrylic resins, glass, silica gels, polystyrene, gelatin, polyvinyl _ pyrrolidone, co-polymers of vinyl and acrylamide, polystyrene cross-linked with 10... divinylbenzene and others (see, Merrifield, Biochemistry, 3:1385-1390 (1964), ~ polyacrylamides, latex gels, polystyrene, dextran, polyacrylamides, rubber, y .. silicon, plastics, nitrocellulose, celluloses, natural sponges. Of particular interest herein, are highly porous glasses (see, e.g., U.S. Patent No. 4,244,721) and .- others prepared by mixing a borosilicate, alcohol and water. . 15. Synthetic supports inciude, but are not limited to: acrylamides, dextran- derivatives and dextran co-polymers, agarose-polyacrylamide blends, other polymers and co-polymers with various functional groups, methacrylate derivatives and co-polymers, polystyrene and polystyrene copolymers (see, e.g.,

Merrifield, Biochemistry, 3:1385-1390 (1964); Berg et al., in /nnovation

Perspect. Solid Phase Synth. Collect. Pap., Int. Symp., 1st, Epton, Roger (Ed), pp. 453-459 (1990); Berg et al., Pept., Proc. Eur. Pept. Symp., 20th, Jung, G. et al. (Eds), pp. 196-198 (1989); Berg et al., J. Am. Chem. Soc., 111:8024-8026 (1989); Kent et al., /sr. J. Chem., 17:243-247 (1979); Kentet ~~ . ~ al., J. Org. Chem., 43:2845-2852 (1978); Mitchell ot al., Tetrahedron Lett., 42:3795-3798 (1976): U.S. Patent No, 4 507 230; U.S. Paieni No. 4,008,117; and U.S. Patent No. 5,389,448). Such materials include those made from polymers and co-polymers such as polyvinylalcohols, acrylates and acrylic acids such as polyethylene-co-acrylic acid, polyethylene-co-methacrylic acid, polyethy- lene-co-ethylacrylate, polyethylene-co-methyl acrylate, polypropylene-co-acrylic acid, polypropylene-co-methyi-acrylic acid, polypropylene-co-ethylacrylate, polypropylene-co-methyl acrylate, polyethylene-co-viny! acetate, poly- propylene-co-vinyl acetate, and those containing acid anhydride groups such as polyethylene-co-maleic anhydride and polypropylens-co-maleic anhydride.

Liposomes have also been used as solid supports for affinity purifications (Powell et al. Biotechnol. Bioeng., 33:173 (1989)).

Numerous methods have been developed for the immobilization of proteins and other biomolecules onto solid or liquid supports (see, e.g.,

Mosbach, Methods in Enzymology, 44 (1976); Weetall, Immobilized Enzymes,

Antigens, Antibodies, and Peptides, (1975); Kennedy et al., Solid Phase

Biochemistry, Analytical and Synthetic Aspects, Scouten, ed., pp. 253-391 (1983); see, generally, Affinity Techniques. Enzyme Purification: Part B.

Methods in Enzymology, Vol. 34, ed. W. B. Jakoby, M. Wilchek, Acad. Press,

N.Y. (1974); and Immobilized Biochemicals and Affinity Chromatography,

Advances in Experimental Medicine and Biology, vol. 42, ed. R. Dunlap, Plenum

Press, N.Y. (1974).

Among the most commonly used methods are absorption and adsorption 18 or covalent binding to the support, either directly or via a linker, such as the numerous disulfide linkages, thioether bonds, hindered disulfide bonds, and covalent bonds between free reactive groups, such as amine and thiol groups, known to those of skill in art (see, e.g., the PIERCE CATALOG,

ImmunoTechnology Catalog & Handbook, 1992-1993, which describes the preparation of and use of such reagents and provides a commercial source for such reagents; Wong, Chemistry of Protein Conjugation and Cross Linking, CRC

Press (1993); see also DeWitt et al., Proc. Natl. Acad. Sci. U.S.A., 90:6909 (1993): Zuckermann et al., J. Am. Chem. Soc., 114:10646 {1992); Kurth et al.,

J. Am. Chem. Soc., 116:2661 (1994); Ellman et al., Proc. Natl. Acad. Sci.

U.S.A., 91:4708 (1994); Sucholeiki, Tetrahedron Lttrs., 35:7307 (1994); Su-

Sun Wang, J. Org. Chem., 41:3258 (1976); Padwa et al., J. Org. Chem., 41:3550 (1971); and Vedejs et al., J. Org. Chem., 49:575 (1984), which describe photosensitive linkers).

To effect immobilization, a composition containing the protein or other biomolecule is contacted with a support material such as alumina, carbon, an ion-exchange resin, cellulose, glass or a ceramic. Fluorocarbon polymers have been used as supports to which biomolecules have been attached by adsorption

05-12-2001 | Co | | US0031803 @S-DEC-2881 11:42 FROM BOULT WADE TENNANT TO ©P84398323394465 P.53 {see, U.S. Patent No. 3,843,443; Published International PCT Application

WO0/86 03840).

G. Prognosis and diagnosis :

Endotheliase proteins, domains, analogs, and derivatives thereof,

B endotheliase nucleic acids (and sequences complementary thereto), and anti- endotheliase antibodies, have uses in diagnostics. Such molecules can be used in assays, such as immunoassays, 10 detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting endotheliase expressian, or monitor the treatment thereof. In particular, such an immunoassay is carried out by a method including contacting a sample derived from a patient with an anti- endotheliase antibody under conditions such that immunospecific binding can

Ce occur, and detecting. or measuring the amount of any immunospecific:-binding-by : 7 --- = ~ i wl -.. . .. the antibody.. In.a specific aspect; such: binding of ntibody in tissue sections r= om] zo Co ... .. .can be used to detect aberrant endotheliase localization. or aberrarit (&:g., 16w-or THT

Co 15 absent) levels of endotheliase protein. In a specific embodiment, antibody to - endotheligse protein can be used to assay in a patient tissue or serum sample for : the presence of endotheliase protein where an aberrant level of endotheliase ) rotein is an indication of a diseased condition. | TTT ~The immunoassays which can be used include but are not limited to = competitive and non-competitive assay systems using techniques such as western blots, radicimmunoassays, ELISA (enzyme linked immunosorbent ’ assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few,

Endothelisse genes and related nucleic acid sequences and subsequences, including complementary sequences, can also be used in hybridization assays.

Endotheliase nucleic acid sequences, or subsequences thereof containing about at Jeast 8 nucleotides, preferably 14 or 16 or more continufous nucleotides, can be used as hybridization probes. Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with aberrant changes in endotheliase expression and/or activity as

AMENDED SHEET :

© 05-12-2001 | | | US0031808

PS-DEC-20@1 11:42 FROM BOULT WADE TENNANT TO 88438523934465 P.54 -88- i. described suprs. In particular, such a hybridization assay is carried out by a method by contacting a sample containing nucleic acid with a nucleic acid probe capable of hybridizing to endutirefiasse DNA or RNA, under conditions such that hybridization can occur, and detecting or measuring any resulting: hybridization.

In a specific embodiment, a method of diagnosing a disease or disorder characterized by detecting an aberrant level of an endotheliase in a subject is provided herein by measuring the level of the DNA, RNA, protein or functional activity of the epithelial endotheliase at east partially encoded by a nucleic acid : that hybridizes to-a nucieic acid having the nucleotide sequence set forth in the

SEQ. ID NO:1 in a sample derived from the subject, wherein an increase or or decrease in the level of the DNA, RNA, protein or functional activity of the © =~ endotheiiase, relstive to the level of the DNA, RNA; protein: or furictional-activity’: Fe Go

EE oiindin an analogous sample nat having the diseéseror disorder indicates he. + 1 7 ee presence of the disease or disorder in the subject. megs ie nd re RETIRE

In another specific embodiment, a method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder : ie wn ene mgsgeiatad with undesined andlor viesnvolied angiogeiicsis in ETSULJECLAG ome sams min. kon provided by measuring the level of DNA, RNA. protein, or functional activity of - an endotheliase at least partially encoded by a nucleic acid that hybridizes to a : nucleic acid having the nucleotide sequence set forth in the SEQ. ID NO:1 in a sample derived from the subject, wherein an increase in the level of the DNA,

RNA, protein, or functional activity in the sample, relative to the level of the

DNA, RNA, protein, or functional activity found in an analogous sample not : having the undesired and/or uncontrolled angiogenesis, indicates the presence of the undesired and/or uncontrolled angiogenesis. \

In still another specific embodiment, a method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder ' associated with deficient angiogenesis in a subject is provided herein, which method by measuring the level of DNA, RNA, protein, or functional activity of an endotheliase at least partially encoded by a nucleic acid that hybridizes toa nucleic acid having the nucleotide sequence set forth in the SEQ. ID NO:1 ina sample derived from the subject. wherein agydecrease in the level of the DNA, .

AMENDED SHEET

EL Tal = a ae La TR Ld me SIE Len

05-12-2001 : a US0031803 - #5 DEC-2P1 11:43 FROM BOULT WADE TENNANT TO BASEIZIIRAAES P.55 - RNA, protein, or functional activity in the sample, relative to the level of the

DNA, RNA, protein, or functional activity found in an analogous sample not having the deficient angiogenesis, indicates the presence of the deficient angiogenesis. Co

S Kits for diagnostic use are also provided, that comprise in one or more containers an anti-endotheliase antibody, and, optionally, a labeled binding partner to the antibody. Alternatively, the anti-endotheliase antibody can be labeled {with a detectable marker, eg. a chemiluminescent, enzymatic, fluorescent, or radioactive moiety). A kitis also provided that includes in one or ) . 10 more containers a nucleic acid probe capable of hybridizing: to endotheliase- : encoding RNA. in a specific embodiment, a kit can comprise in" one or more “eo containers a pair of primers (e.g... each in the sizerange of 6-30 nucleotides) that-- = esi mri are capable of priming amplficatioijfe.g:, by polymerase chain feaction (388 YT 4 ew... .%. ..eg. Innis etal, 1990, PCR Protocols, Academic Press; Inc., San Diego; CA," 7-7 Sent ’ | 15 | ligase chain reaction (see EP 320,308) use of ag replicase, cyclic probe reaction, - - * - ar other methods known in the art under appropriate reaction conditions of at or rE a nn Jeast 3 portion of an. gndotheliase-encoding_nueleic. acid. A kit gan optionally... cremains further comprise in a container a predetermined amount of a purified ~~ - endotheliase protein or nucleic acid, e.g., for use és a standard or control. :

H. PHARMACEUTICAL COMPOSITIONS AND MODES OF ADMINISTRATION . 1. Components of the compositions :

Pharmaceutical compositions containing the identified compounds that modulate the activity of an endotheliase are provided herein. Also provided are = combinations of a compound that modulates the activity of an endotheliase and another treatment or compound for treatment of a disorder involving aberrant angiogenesis, such as a pro-angiogenic treatment or agent, or an anti-angiogenic treatment or agent. In certain embodiments, the compounds that modulate the activity of an endotheliase inhibit its activity and other compound is an anti- angiogenic treatment or agent. bo. .

The endotheliase modulator and the anti-angiogenic or oro-magetie = agent can be packaged as separate compositions for administration together or sequentially or intermittently. Ahernatively, they can be contained in a single

AMENDED SHEET

© 05-12-2001 : | US003180¢

OS-DEC-2881 11:43 FROM BOULT WADE TENNANT TO ER4SBIZIIRALES P.56 : -90- Lo composition for administration as a single composition. The combinations can be packaged as kits. : a. Endothebass inhibitors

Any endotheliase inhibitors, including those described herein when used alone or in combination with other compounds, that can alleviate, reduce, : ameliorate, prevent, or place or maintain in a state of remission of clinical symptoms or diagnostic markers associated with undesired and/or uncontrolled : angiogenesis, particularly vascular malformations and cardiovascular disorders, . chronic inflammatory diseases and aberrant wound repairs, circulatory disorders, - crest syndromes, dermatological disorders, or ocular disorders, can be used-in - -- + -- the present combinations. | Co “nae Dum = In‘one embodiment, the endotheliase inhibitor i$.af antibody. oF fragment. © + raz

Seer histeot tht specifically fasets with ah ndotheliass or the protedsd domain 1 i iL

REET Co thereof, an inhibitor of the endotheliase productish, ah iRhibItar of the epithelial +=" ¢ © - endotheliase membrane-localization, or any infiibitor of the expression of or =~ ~~ - ne activity of an endotheliase. | : rg a rr Sm emit si set rierenornlpen n ATANGinganic agent Se et ag PA RE Pe GE A CD MR

Any anti-angiogenic agents, including those described herein, when used - alone or in combination with other compounds, that can alleviate, reduce, ameliorate, prevent, or place or maintain in a state of remission of clinical symptoms or diagnostic markers associated with undesired and/or uncontrolled angiogenesis, particularly solid neoplasms, vascular malformations and cardiovascular disorders, chronic inflammatory diseases and aberrant wound repairs, circulatory disorders, crest syndromes, dermatological disorders, or ocular disorders, can be used in the combinations.

Ina specific embodirnent, the anti-angiogenic agent used in the oo combination Is an inhibitor of basement membrane degradation, sn inhibitor of cell migration, an inhibitor of endothelial cell proliferation, an inhibitor of three- dimensional organization and establishment of patency, or a physiological or physical anti-angiogenic treatment. :

Examples of anti-angiogenic agents include, but are not limited to, protease inhibitors, endostatin, taxol, fhsallyTGF-£, FGF inhibitors (see, Auerbach

AMENDED SHEET

EE a a LR Col sams 0 onL

05-12-2001 oo | | . US0031803 @5-DEC-20A1 11:43 FROM BOLT WADE TENNANT TO DB4S8923994465 P.57 and Auerbach, Pharmacol. Ther., 6313):265-311 (1994) for a.comprehensive listing of well known anti-angiogenic agents). Particular anti-angiogenic agents 'used in the combination include AGM-1470 (TNP-470), angiostatic steroids, - angiostatin, antibodies against avg3, antibodies against bFGF, ‘antibodies against

IL-1, antibodies against TNF-g, antibodies agsinst VEGF, auranofin, azathioprine,

BB-94, BB-2516, basic FGF-soluble receptor, carboxyamido-trizole (CAI), : cartilage-derived inhibitor (CDI, chitin, chloroquine, cisplatin, CM 101, cortisone/heparin, cortisone/hyaturofian, cortexoione/heparin, CT-2584, cyclophosphamide, cyclosporin A, dexamethasone, diciofenac/hyaluronan, eosinophilic major basic protein, fibronectin peptides, glioma-derived - --- -- : angiogenesis inhibitory factor (GD-AIF}, GM 1474, gold chloride, gold

Lee zs thiomalate,-heparinases, hyaluronan (high and low: molecular-weight speeiesy; +77 7 5

ERA 0 0 hydrdcortisonef/beta-cyclodextran, ibuprofen, Indofnethacin interferon-alpha; Fed

Cw Tee 0 interferon gamma-inducible protein 10, interferon-gamma; ‘IL-1’, eZ Ea aL 125 Te laminin, levamisole, linomide, LME09, marimastat (BB-2518)," = a coo medroxyprogesterone, Metastat {Col-3), methotrexate, minocycline, nitric oxide, iy ame ma ee PCITEOYIdE. {somatostatin analogue). Pachitaxel, D-penicillamine, DEFURETT oom Pv motets Sito RG: polysulfate, placental proliferin-related protein, placental Rnase inhibitor, - plasminogen activator inhibitor (PAls), platelet factor-4 (PF4); prednisolone, - ~~ : prolactin {16-Kda fragment), proliferin-related protein, prostaglandin synthase inhibitor, protamine, retinoids, Roquinimex {LS-2616. linomide), somatostatin, substance P, suramin, SU101, tecogalan sodium (DS-4152), tetrahydrocortisol- #rthrombospondins (TSPs), tissue inhibitor -of metalloproteinases (TIMP 1, 2, 3), vascular endothelial growth factor.inhibitors, vitamin A, Vitaxin, vitreous fiuids, thalidomide, 3-aminothalidomide, 3-hydroxythalidomide and metabolites or hydrolysis products of thalidomide, 3-aminothalidomide, or 3-hydroxythalidomide ((O° Reilly, Investigational New Drugs, 15:5-1 3 (1997); J. Natl Cancer Instit. y 88:786-788 (1996); U.S. Patent Nos, 5,583,990, 5,629,327 and 5,712,291). c. Pro-angiogenic agent

Any pro-angiogenic agents, including those described herein, when used alone or in combination with other compounds, that can promote physiological angiogenesis, particularly angiogenesis involved in normal placental, embryonic, :

AMENDED SHEET : — . EP aa tn IE Eat ia] ’ Limrmd mee SLI) INS fetal and post-natal development and growth, physiologically cyclical development in the ovarian follicle, corpus luteum and post-menstrual endometrium or wound healing, can be used in the present combinations.

The pro-angiogenic agent used in the combination can be a pro- angiogenic cytokine (Desai and Libutti, J. /mmunother., 22(3):186-211 (1999).

More preferably, the pro-angiogenic cytokine used is a basic fibroblast growth factor such as bFGF and FGF-2, a vascular endothelial growth factor/vascular permeability factor such as VEGF/VPF and vasculotropin, a platelet-derived endothelial cell growth factor such as PD-EDGF and thymidine phosphorylase, a transforming growth factor-beta (TGF-£), or angiopoietin-1 {Ang-1). 2. Formulations and route of administration

The compounds herein and agents are preferably formulated as pharmaceutical compositions, preferably for single dosage administration. The concentrations of the compounds in the formulations are effective for delivery of an amount, upon administration, that is effective for the intended treatment.

Typically, the compositions are formulated for single dosage administration. To formulate a composition, the weight fraction of a compound or mixture thereof is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is relieved or ameliorated.

Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.

In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with 2b other active ingredients. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared as described in U.S. Patent No. 4,522,811. 30 . The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo systems, such as the assays provided herein. : : The concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the active compound, . 5..- the physicochemical characteristics of the compound, the dosage schedule, and +, amount administered as well as other factors known to those of skill in the art.

Typically a therapeutically effective dosage is contemplated. The amounts administered may be on the order of 0.001 to 1 mg/ml, preferably about 0.005-0.056 mg/ml, more preferably about 0.01 mg/ml, of blood volume. 10.;- Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to ;. about 1000 mg and preferably from about 10 to about 500 mg, more preferably .~ about 25-75 mg of the essential active ingredient or a combination of essential : . ingredients per dosage unit form. The precise dosage can be empirically : . determined. . 15 The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. it is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or ’ "7" ‘supervising the administration of the compositions, and that the concentration _ 25 ranges set forth herein_are exemplary only and are not intended tc limit the scope or use of the claimed compositions and combinations containing them.

Preferred pharmaceutically acceptable derivatives include acids, salts, - = ~~ esters, hydrates, solvates and prodrug forms. The derivative is typically selected such that its pharmacokinetic properties are superior to the corresponding 308 neutral compound.

Thus, effective concentrations or amounts of one or more of the compounds provided herein or pharmaceutically acceptable derivatives thereof are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical or local administration to form pharmaceutical compositions. Compounds are included in an amount effective for ameliorating or treating the disorder for which treatment is contemplated. The concentration of active compound in the composition will depend on absorption, inactivation, excretion rates of the active compound, the dosage schedule, amount administered, particular formulation as well as other factors known to those of skill in the art.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.

In instances in which the compounds exhibit insufficient solubility, : methods for solubilizing compounds may be used. Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as Tween®, or dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.

For ophthalmic indications, the compositions are formulated in an opthalmically acceptable carrier. For the ophthalmic uses herein, local administration, either by topical administration or by injection is preferred. Time release formulations are also desirable. Typically, the compositions are formulated for single dosage administration, so that a single dose administers an effective amount.

Upon mixing or addition of the compound with the vehicle, the resulting = Mixture may be a solution, suspension, emulsion or other composition. The form .. of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the 5.. selected carrier or vehicle. If necessary, pharmaceutically acceptable salts or -.- other derivatives of the compounds may be prepared.

The compound is included in the pharmaceutically acceptable carrier in an } amount sufficient to exert a therapeutically useful effect in the absence of . undesirable side effects on the patient treated. It is understood that number and 10 ....degree of side effects depends upon the condition for which the compounds are administered. For example, certain toxic and undesirable side effects are } ... tolerated when treating life-threatening illnesses that would not be tolerated - ~ when treating disorders of lesser consequence. : - The compounds can also be mixed with other active materials, that do 15 . not impair the desired action, or with materials that supplement the desired action known to those of skill in the art for treating diseases or disorders associated with aberrant angiogenesis and other disorders for which the treatments provided herein are contemplated.

The formulations of the compounds and agents for use herein include those suitable for oral, rectal, topical, inhalational, buccal (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), transdermal administration or any route. The most suitable route in any given case will depend on the nature and severity of the condition being treated and on = "77 the nature of the particular active compound which is being used. - 25 The formulations are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oT “oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof. The pharmaceutically therapeutically active compounds and derivatives thereof are typically formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules.

Unit-dose forms may be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.

The composition can contain along with the active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acaciagelatin, glucose, molasses, polvinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company,

Easton, Pa., 15th Edition, 1975. The composition or formulation to be administered will, in any event, contain a quantity of the active compound in an amount sufficient to alleviate the symptoms of the treated subject.

05-12-2001 | | | US0031803 . PS-DEC-20P1 11:43 FROM BOULT WADE TENNANT TO 8438523334465 P.58

Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared. :

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by: conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl } : methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants le.g.,- i Ad or sodium starch glycolate): or wetting agents {e.g., sodium lauryl Pritaeshy The tablets may be coated by methods well- fae reeme co ---known.inthe.art. nL... Lo COE EEE Snwennim LL Se -

SRA CL The pharmaceutical preparation may also be in liquid form: forrexample; wel a

ETRE solutions, ‘syrups ‘of suspensions, of may be presenitedasa driig product for: Poem reconstitution with water or other suitable vehicle’ before use. “Such liquid . ' preparations may be prepared by conventional means with pharmaceutically en ere mie or ia BCC 2 Plable. additives such -o5-suspending agents deus. sorbitol syrugrveeliulessrrre sme rrr ve derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or - acacia); non-aqueous vehicles {e.g., almond oil, oily esters, or fractionated : vegetable oils}; and preservatives le.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).

Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active \ compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture, ;

Formulations suitable for topical application to the skin or to the eye © preferably take the form of an ointment, cream, lotion, paste, gel, spray. aerosol, or oil. Carriers which may be used include vaseline, lanoline, polyethylene glycols, alcohols, and combinations of two or more thereof. The 2opical formulations may further advantageously contain 0.05 to 15 percent by weight of thickeners selected from amang hydroxypropyl methyl cellulose, methyj cellulose, polyvinylpyrrolidone; polyvinyl! alcohol, poly (alkylene glycols), Co

AMENDED SHEET

~~ eeAr An EY Ane an bm me mdr Leo

- .WO0 01/36604 PCT/US00/31803 ( poly/hydroxyalkyl, (meth)acrylates or poly(meth}acrylamides. The topical formulations is most often applied by instillation or as an ointment into the conjunctival sac. It, however, can also be used for irrigation or lubrication of the eye, facial sinuses, and external auditory meatus. It may also be injected into the anterior eye chamber and other places. The topical formulations in the liquid state may be also present in a hydrophilic three-dimensional polymer matrix in the form of a strip, contact lens, and the like from which the active components are released.

For administration by inhalation, the compounds for use herein can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Formulations suitable for buccal (sublingual) administration include lozenges containing the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles containing the compound in an inert base such as gelatin and glycerin or sucrose and acacia.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water or other solvents, before use.

Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the

© 05-12-2001 TL A US0031803 -99- | . recipient for a prolonged period of time. Such patches suitably contain the active compound as an optionally buffered agueous solution of, for example, 0.1 to 0.2M concentration with respect to fe the active compound. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, e.g., Pharmaceutical Research 3 (6), 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound. : In addition to the common dosage forms set out above, the pharmaceutical compositions may also be administered by controlled release : means and/or delivery devices (see, e.g., in U.S. Patent Nos. 3,536,809; 3,698,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899; 4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,354,566; 5,591,787; 5,639,476; 5,674,533 and 5,733,566).

Desirable blood levels may be maintained by a continuous infusion of the active agent as ascertained by plasma levels. It should be noted that the

AEE 1 I attending physician would know how to and when Te términste; interrupt ‘or’ meen se EN

LT adjust therapy to lower dosage due to toxicity, or bore marrow, liver-of Kidney ~~ Le cu * dysfunctions. Conversely, the attending physician would ‘dlso kriow low to dnd © © when to adjust treatment to higher levels if the clinical. response is not adequate {precluding toxic side effects). ;

Ei andlor TOXIEHY oF the endethdilase inbibitorls) alone or in TTT combination with the anti-angiogenic agent or pro-angiogenic agent can aiso be - assessed by the methods known in the art (et generally, O'Reilly, -

Investigational! New Drugs, 15:5-13 {1997)). For example, the in vitro angiogenesis assays based on target compound's ability to inhibit endothelial cell proliferation, migration, and tube formation jn vitro can be used. Alternatively, the jn vivo angiogenesis assays such as the chicken chorioallantoic membrane (CAM) assay and the disc angiogenesis assays can be used. Preferably, the established pre-clinical models for the evaluation of angiogenesis inhibitors in vivo such as corneal angiogenesis assays, primate model of ocular angiogenesis, metastasis models, primary tumor growth model and transgenic mouse mode! of tumor growth can be used. :

Empf.zei t:05/12/2001 12:4] Empf.nr.:972 POSS

AMENDED SHEET

05-12-2001 : LC oo US0031803 oS-DEC-28D1 11:44 FROM BOULT WADE TENNANT TO BR4SEI23I34465 P.64 " -100- i.

The ‘active compounds or pharmaceutically acceptable derivatives may be prepared with carriers that protect the compound against rapid elimination from . the body, such as time release formulations or coatings.

Kits containing the compositions and/or the combinations with . instructions for mawation thereof are provided: The kit may further include a needle or syringe, preferably packaged in sterile form, for injecting the-complex, and/or a packaged alcohol pad. Instructions are optionally included for administration of the active agent by a clinician or by the patient. ) .

Finally, the compounds or endotheliases or protease domains thereof or

So ing aosiazng any of the Soacoading gents may be packaged as articles : of manufacture containing packaging material, a compound or suitable derivative a . ... thereof provided herein, which is effective for treatment of a diseases of - EI RA 7077 disorders. contemplated herein, within the packaging Wiatenal, and'a label tat © vc a indicates that the compound or a suitable derivativé-theréof is-for treating the ~ + =" 4

Bh 15 diseases or disorders contemplated herein. The! label can optionally include the So disorders for which the therapy.is warranted. N : ’ il... METHODSOFTREATMENT _ © er opecsrmss cm nes 1. Treatment of undesired angiogenesis : -

The compounds and combinations are used for treating or preventing a disease or disorder associated with undesired and/or uncontrolled angiogenesis i in a mammal is provided herein. In one embodiment, the method includes administering to a mammal an effective amount of an inhibitor of an endotheliase, whereby the disease or disorder is treated or prevented. Ina preferred embodiment, the endotheliase inhibitor used in the treatment or prevention is administered with a pharmaceutically acceptable carrier or . excipient. The mammal treated can be a human.

In other embodiments, the treatment or prevention method further includes administering an anti-angiogenic treatment or agent simultaneously with, prior to or subsequent to the endotheliase inhibitor, which can be any compound identified that inhibits the activity of an endotheliase, and includes an } antibody or a fragment or derivative thereof containing the binding region thereof against the endotheliase, an antisense nucleic acid encoding the endotheliase,

AMENDED SHEET

05-12-2001 11148 FROMTTOLLT WDE TERANT TO OF TIWAES | 1S0031803 : | i

X : . -101- . ] and a nucleic acid containing at least a portion of a gene encoding the endotheliase into which a heterologous nucleotide sequence has been inserted ‘such that the heterologous sequence inactivates the biological activity of at least a portion of the gene encoding the endotheliase, in which the portion of the gene 8 encoding the endotheliase flanks the heterologous sequence so as to promote homologous recombination with a genomic gene encoding the endotheliase.

The undesired or aberrant angiogenesis to be treated or prevented is associated with solid neoplasms, vascular malformations and cardiovascular } .disorders, chronic inflammatory diseases and aberrant wound repairs, circulatory ' disorders, crest syndromes, dermatological disorders, or ocular disorders.

Vascular malformations and cardiovascular disorders to be treated or prevented ! “ include :angiofibroma, angiolipoma, atherosclerosis, restenosis/reperfusion injury, ¢ artericvenous malformations, hemangiomatosis and vascular adhesions, ) dyschondroplasia with vascular hamartomas (Fafucci’s syndrome), hereditary hemorrhagic telangiectasia {Rendu-Osler-Websr syndrome}, or Von Hipple Lindau syndrome; the chronic inflammatory diseases t0 be treated or prevented are diabetes mellitus, hemophiliac joints, inflammatory bowel disease, nonhealing

Coe eases fractures, periodontitis {rapidly progressing and juvenilel, pseriesispsheumatoiharmerens arthritis, venous stasis ulcers, granulations-burns, hypertrophic scars, liver - cirrhosis, osteoradionecrosis, postoperative adhesions, pyogenic granuloma, or systemic sclerosis; the circulatory disorder to be treated or prevented is

Raynaud’s phenomenon; the crest syndromes to be treated or prevented are calcinosis, esophageal, dyomotiloty, sclerodactyly and teangiectasis; the dermatological disorders to be treated or prevented are systemic vasculitis, , scleroderma, pyoderma gangrenosum, vasculopathy, venous, arterial ulcers, :

Sturge-Weber syndrome, Port-wine stains, blue rubber bleb nevus syndrome, 3 ¢ Klippel-Trenaunay-Weber syndrome or Osler-Weber-Rendu syndrome; and the ocular disorders to be treated or prevented are blindness caused by ocular : neovascular disease, corneal graft neovascularization, macular degeneration in the eye,:neovascular glaucoma, trachoma, diabetic retinopathy, myopic

Soden retnopsthy of prematurity, retrolental fibroplasia, or corneal neovascularization. :

AMENDED SHEET

Emofangoce. . wveivver coo a. Antisense treatment

In a specific embodiment, as described hereinabove, endotheliase function is reduced or inhibited by endotheliase antisense nucleic acids, to treat or prevent disease or disorder associated with undesired and/or uncontrolled angiogenesis. The therapeutic or prophylactic use of nucleic acids of at least six nucleotides that are antisense to a gene or cDNA encoding endotheliase or a portion thereof. An endotheliase "antisense" nucleic acid as used herein refers to a nucleic acid capable of hybridizing to a portion of an endotheliase RNA (preferably mRNA) by virtue of some sequence complementarily. The antisense nucleic acid may be complementary to a coding and/or noncoding region of an endotheliase mRNA. Such antisense nucleic acids have utility as therapeutics that reduce or inhibit endotheliase function, and can be used in the treatment or prevention of disorders as described supra.

The endotheliase antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides {ranging from 6 to about 150 nucleotides, or more preferably 6 to 50 nucleotides). In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 125 nucleotides. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553- 6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. U.S.A. 84:648-652 (1987);

PCT Publication No. WO 88/09810, published December 15, 1988) or blood- brain barrier (see, e.g., PCT Publication No. WO 89/101 34, published April 25, 1988), hybridization-triggered cleavage agents (see, e.g., Krol et al.,

BioTechniques 6:958-976 (1988)) or intercalating agents (see, e.g., Zon, Pharm.

Res. 5:539-549 (1988). .

The endotheliase antisense nucleic acid is preferably an oligonucleotide, » more preferably of single-stranded DNA. In a preferred aspect, the oligonucleotide includes a sequence antisense to a portion of human

> endotheliase. The oligonucleotide may be modified at any position on its © structure with substituents generally known in the art.

The endotheliase antisense oligonucleotide may comprise at least one ... modified base moiety which is selected from the group including, but not limited 5: to b-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,

Ee xantine, 4-acetylcytosine, 5-({carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, : .. 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, : 10. 2-methylguanine, 3-methylcytosine, 5-methyicytosine, N6-adenine, 7-methyiguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl- i 2-thiouracil, beta-D-mannosylqueosine, 5’-methoxycarboxymethyluracil, 3 . b-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), . wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, . 15: 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid {v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

In another embodiment, the oligonucleotide includes at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose. The oligonucleotide can include at least one modified phosphate backbone selected from a phosphorothioate, a phosphorodithioate, a phospharamidothioate, a phosphoramidate, a _phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof. J

Z5 The oilgunuciedtide can be an g-anomeric oligonuciestide. An g-ancmeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which the strands run parallel to each other (Gautier et al., Nucl. Acids Res. 15:6625-6641 (1987). .

The oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent and hybridization-triggered cleavage agent.

05.13.2001 © 11148 FROM OUT URDE TENNANT TO & WIIUS 150031803 : | -104- .

The oligonucleotides may be synthesized by standard methods known in ; the art, e.g. by use of an automated DNA synthesizer (such as are commercially , available from Biosearch, Applied Biosystems, etc.}. As examples. 3 phosphorothioate oligonucleotides may be synthesized by the method of Stein et & al. (Nucl. Acids Res. 16:3209 (1988)), methyiphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc.

Natl. Acad. Sci. U.S.A. 85:7448-7451 (1988), etc.

Ii a specific embodiment, the endotheliase antisense oligonucieotide includes catalytic RNA, or a ribozyme (see, e.g.. PCT International Publication

WO 90/1 1364, published October 4, 1990; Sarver et al., Science 247:1222- 1225 (1990). In another embodiment, the oligonucleotide is a 2°-0- . methylribonucleotide (Inoue et al., Nucl. Acids Res. 18:6131-6148 (1987), ora _ chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327-330 (1987).

In an alternative embodiment, the endotheliase antisense nucleic acid Is produced intracellularly by transcription from an exogenous sequence. For : example, a vector can be introduced /n vivo such that it is taken up by a cell, ' within which cell the vector or a portion thereof. is transcribed, producing an .

SR ER dense nucleic acid TRINA]. “SUCH a vector Wolild Eontain a’sequence entoding™ the endotheliase 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 mammalian cells,

Expression of the sequence encoding the endatheliase antisense RNA can be by ~~ 25 any promoter known in the art to act in mammalian, preferably human, cells. :

Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, Nature 290:304-310 [1 981) the promoter contained in the 3‘ long terminal repeat of

Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980), the herpes i thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. ¢ © 78:1441-1445 {1 981), the regulatory sequences of the metallothionein gene so (Brinster et al., Nature 296:39-42 (1 982) etc. :

AMENDED SHEET

Fmofan2oioie wivoer vev=:

: The antisense nucleic acids include sequence complementary to at least a - portion of an RNA transcript of an endotheliase gene, preferably a human : - endotheliase gene. Absolute complementarily, although preferred, is not . . required. - 5. The amount of endotheliase antisense nucleic acid that will be effective in the treatment or prevention of disease or disorder associated with undesired and/or uncontrolled angiogenesis will depend on the nature of the disease, and can be determined empirically by standard clinical techniques. Where possible, it is desirable to determine the antisense cytotoxicity in cells in vitro, and then in : 10-- useful animal model systems prior to testing and use in humans. . 2. Treatment of deficient angiogenesis ; ws In another specific embodiment, a method for treating or preventing a : -- disease or disorder associated with deficient angiogenesis in a mammal is p - provided herein by administering to a mammal an effective amount of an ; 15. endotheliase protein at least partially encoded by a nucleic acid that hybridizes to a nucleic acid having the nucleotide sequence set forth in the SEQ. ID NO:1, a domain, a derivative or analog of the protein that is active in promoting angiogenesis, a nucleic acid encoding the protein, and a nucleic acid encoding a domain, a derivative or analog of the protein that is active in promoting angiogenesis, whereby the disease or disorder is treated or prevented.

In a preferred embodiment, the endotheliase protein, a domain, a derivative or analog of the protein, a nucleic acid encoding the protein, and a nucleic acid encoding a domain, a derivative or analog of the protein is administered with a pharmaceutically acceptable carrier or excipient.

In yet another preferred embodiment, the mammal to be treated is a : human. The treatment or prevention methods can further include administering an pro-angiogenic treatment or agent. In yet another preferred embodiment, the treatment is used to promote physiological angiogenesis, particularly angiogenesis involved in normal placental, embryonic, fetal and post-natal development and growth, physiologically cyclical development in the ovarian follicle, corpus luteum and post-menstrual endometrium or wound healing.

. .WO0 01/36604 PCT/US00/31803

The pro-angiogenic agent used in the treatment can be, for example, a basic fibroblast growth factor such as bFGF and FGF-2, a vascular endothelial growth factor/vascular permeability factor such as VEGF/VPF and vasculotropin, a platelet-derived endothelial cell growth factor such as PD-EDGF and thymidine

B phosphorylase, a transforming growth factor-beta (TGF-8), or angiopoietin-1 {Ang-1).

Gene Therapy

In an exemplary embodiment, nucleic acids that include a sequence of nucleotides encoding an endotheliase protein or functional domains or derivative thereof, are administered to promote endotheliase protein function, by way of gene therapy. Gene therapy refers to therapy performed by the administration of a nucleic acid to a subject. In this embodiment, the nucleic acid produces its encoded protein that mediates a therapeutic effect by promoting endotheliase protein function. Any of the methods for gene therapy available in the art can : 16 be used (see, Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and

Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and

Anderson, Ann. Rev. Biochem. 62:191-217 (1993); TIBTECH 11(5):1565-215 {1993). For example, one therapeutic composition for gene therapy includes an endotheliase-encoding nucleic acid that is part of an expression vector that expresses an endotheliase protein or domain, fragment or chimeric protein thereof in a suitable host. In particular, such a nucleic acid has a promoter operably linked to the endotheliase coding region, the promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, a nucleic acid molecule is used in which the endotheliase coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the endotheliase nucleic acid (Koller and

Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al.,

Nature 342:435-438 (1989).

Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying

-. vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, .~ respectively, as in vivo or ex vivo gene therapy. - In a specific embodiment, the nucleic acid is directly administered in vivo, 5.. where it is expressed to produce the encoded product. This can be -- accomplished by any of numerous methods known in the art, e.g., by } constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a __ defective or attenuated retroviral or other viral vector (see U.S. Patent No. 10-- 4,980,286), or by direct injection of naked DNA, or by use of microparticle - bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-

N ~ surface receptors or transfecting agents, encapsulation in liposomes, . microparticles, or microcapsules, or by administering it in linkage to a peptide . -. which is known to enter the nucleus, by administering it in linkage to a ligand . 15. . subject to receptor-mediated endocytosis {see e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand is a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT

Publications WO 92/06180 dated April 16, 1992 (Wu et al.); WO 92/22635 dated December 23, 1992 (Wilson et al.}; W092/20316 dated November 26,

T1992 (Findeis et al.); WO93/14188 dated July 22, 1993 (Clarke et al.), WO 93/20221 dated October 14, 1993 (Young)). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad.

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

In a specific embodiment, a viral vector that contains the endotheliase nucieic acid is used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-5699 (1993})). These retroviral vectors have been : modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. The endotheliase nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J.

Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994);

Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.

Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson,

Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431- 434 (1991); Rosenfeld et al., Cell §8:143-155 (1982); and Mastrangeli et al., J.

Clin. Invest, 31:225-234 (1993).

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

i i. In this embodiment, the nucleic acid is introduced into a cell prior to : . administration in vivo of the resulting recombinant cell. Such introduction can - be carried out by any method known in the art, including but not limited to ... transfection, electroporation, microinjection, infection with a viral or 5: bacteriophage vector containing the nucleic acid sequences, cell fusion, . += chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, Meth.

Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol, 217:618-644 - : 10: (1993); Cline, Pharmac. Ther. 29:69-92 (1985)) and may be used, provided that +... the necessary developmental and physiological functions of the recipient cells # are not disrupted. The technique should provide for the stable transfer of the - } _.. nucleic acid to the cell, so that the nucleic acid is expressible by the cell and . - preferably heritable and expressible by its cell progeny. - 15:: The resulting recombinant cells can be delivered to a patient by various methods known in the art. In a preferred embodiment, epithelial cells are injected, e.g., subcutaneously. In another embodiment, recombinant skin cells may be applied as a skin graft onto the patient. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not © limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc. in a preferred embodiment, the cell used for gene therapy is autoiogous to the patient. In an embodiment in which recombinant cells are used in gene therapy, an endotheliase nucleic acid Is introduced into the cells such that it is

R WO 01/36604 PCT/US00/31803 expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. in a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment. Such stem cells include but are not limited to hematopoietic stem cells (HSC), stem cells of epithelial tissues such as the skin and the lining of the gut, embryonic heart muscle ceils, liver stem cells (PCT Publication WO 94/08598, dated April 28, 1994), and neural stem cells (Stemple and Anderson,

Cell 71:973-985 (1992).

Epithelial stem cells (ESCs) or keratinocytes can be obtained from tissues such as the skin and the lining of the gut by known procedures (Rheinwald,

Meth. Cell Bio. 21A:229 {1980)). In stratified epithelial tissue such as the skin, renewal occurs by mitosis of stem cells within the germinal layer, the layer closest to the basal lamina. Stem cells within the lining of the gut provide for a rapid renewal rate of this tissue. ESCs or keratinocytes obtained from the skin or lining of the gut of a patient or donor can be grown in tissue culture (Rheinwald, Meth. Cell Bio. 21A:229 (1980); Pittelkow and Scott, Mayo Clinic

Proc. 61:771 (1986)). If the ESCs are provided by a donor, a method for suppression of host versus graft reactivity (e.g., irradiation, drug or antibody administration to promote moderate immunosuppression) can also be used.

With respect to hematopoietic stem cells (HSC), any technique which provides for the isolation, propagation, and maintenance /n vitro of HSC can be used in this embodiment. Techniques by which this may be accomplished include (a) the isolation and establishment of HSC cultures from bone marrow cells isolated from the future host, or a donor, or (b) the use of previously established long-term HSC cultures, which may be allogeneic or xenogeneic.

Non-autologous HSC are used preferably in conjunction with a method of suppressing transplantation immune reactions of the future host/patient. In a particular embodiment, human bone marrow cells can be obtained from the posterior iliac crest by needle aspiration (see, e.g., Kodo et al., J. Clin. Invest. 73:1377-1384 (1984)). In a preferred embodiment, the HSCs can be made highly enriched or in substantially pure form. This enrichment can be

. = accomplished before, during, or after long-term culturing, and can be done by any techniques known in the art. Long-term cultures of bone marrow cells can - be established and maintained by using, for example, modified Dexter cell culture . techniques (Dexter et al., J. Cell Physiol. 91:335 (1977) or Witlock-Witte culture 6.. techniques (Witlock and Witte, Proc. Natl. Acad. Sci. USA 79:3608-3612 . ~ (1982)).

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy includes an inducible promoter operably linked to the coding __ region, such that expression of the nucleic acid is controllable by controlling the 10...- presence or absence of the appropriate inducer of transcription.

NS ANIMAL MODELS

- Transgenic animal models are provided herein. In one embodiment, animal ; . models for diseases and disorders involving deficient angiogenesis are provided. -. Such an animal can be initially produced by promoting homologous 15 .. recombination between an endotheliase gene in its chromosome and an exogenous endotheliase gene that has been rendered biologically inactive {preferably by insertion of a heterologous sequence, e.g., an antibiotic resistance gene). In a preferred aspect, this homologous recombination is carried out by transforming embryo-derived stem (ES) cells with a vector containing the insertionally inactivated endotheliase gene, such that homologous recombination occurs, followed by injecting the ES cells into a blastocyst, and implanting the blastocyst into a foster mother, followed by the birth of the chimeric animal ("knockout animal”} in which an endotheliase gene has been inactivated (see

Ca pecchi , Science 244:1288- 1292 (1989). The chimeric animal can be bred to produce additional knockout animals. Such animals can be mice, hamsters, sheep, pigs, cattle, etc., and are preferably non-human mammals. In a specific embodiment, a knockout mouse is produced.

Such knockout animals are expected to develop or be predisposed to developing diseases or disorders involving deficient angiogenesis and thus can have use as animal models of such diseases and disorders, e.g., to screen for or test molecules for the ability to treat or prevent such diseases or disorders.

. me nme anand 44:48 FROM: ™OULT WADE TENNANT TO ©8@¢ 1923994465 P.R3/ 7H 05-12-2001 0! . US0031803 . } © : to © M2 .

In a separate embodiment, animal models for diseases and disorders : involving uncontrolled and/or undesired angiogenesis are provided. Such an animal ‘can be initially produced by promoting homologous recombination : between an endotheliase gene in its chromosome and an exogenous : endotheliase gene that would be over-expressed or mis-expressed (preferably by expression under a strong promoter). In a preferred aspect, this homologous recombination is carried out by transforming embryo-derived stem (ES) cells with + a vector containing the over-expressed or mis-expressed endotheliase gene, such - 2 that homologous recombination occurs, followed by injecting the ES cells into a : blastocyst, and implanting the blastocyst into a foster mother, followed by the birth of the chimeric animal in which an endotheliase gene has been over- . expressed or mis-expressed (see Capecchi, Science 244:1288-1292 (1989)).

The chimeric animal can be bred to produce additional animals with over- expressed or mis-expressed endotheliase. Such animals can be mice, hamsters, sheep, pigs, cattle, etc., and are preferably non-human mammals. in a specific embodiment, a:mouse with over-expressed of mis-expressed endotheliase is produced. . a a rn eae Such animals are expected to develop or be pradicoosed to developing... cweracs diseases or disorders involving diseases ar disorders involving uncontrolled - and/or undesired angiogenesis and thus can have use as animal madels of such diseases and disorders, e.g.. to screen for or test molecules for the ability to 7 inhibit function of endotheliase genes and proteins and thus treat or prevent } 2 such diseases or disorders. Lo :

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLE 1 . ~~ CLONING AND EXPRESSION 3 ENDOTHELIASE-1

Cell type and growth of cells !

Human umbilical vein endothelial cells (HUVEC P145, hereafter called

HUVEC) were purchased from Clonetics (catalog number CC-2519: BioWhittaker

Inc., Walkersville, MD). The cells were cultured at 37°C, 5% ez in endothelial growth medium (EGM; catalog number CC-3124; Clonetics) supplemented with

Ep fang AMENDED SHEET : p

. : u0.4% bovine brain extract with heparin, 2% fetal bovine serum, 1 yg/mL hydrocortisone, 10 ng/mL epidermal growth factor, 50 ng/mL amphotericin-B - - and 50 pg/mL gentamicin sulfate. All subsequent cell manipulations were carried . out according to the manufacturer's instructions. HUVEC were allowed to grow - Bb to about 90% confluent, then briefly washed with 1 x phosphate buffered saline. = = isolation of total RNA, and purification and enrichment of polyA + RNA

HUVEC were lysed in Trizol reagent (catalog number 155986; Life

Technologies, Rockville, MD) and total RNA was isolated according to the ~ manufacturer's protocol. The concentration of total RNA was estimated from » : 10. absorbance reading at 260 nm. PolyA + RNAs were purified and enriched using oligo-dT beads (catalog number 70061; Oligotex, Qiagen, Chatsworth, CA). ~ i. Reverse-transcription and polymerase chain reaction (PCR) . = HUVEC polyA + RNAs were converted to single-stranded cDNA (sscDNA) -. .- by reverse transcription using ProSTAR first-strand RT-PCR kit (catalog number " 15. 200420; Stratagene, La Jolla, CA) and SuperScript Il RNase H- reverse transcriptase (catalog number 18064-022; Life Technologies). An aliquot of

HUVEC sscDNA (4 ub) was subjected to PCR using 2 uM each of the sense and anti-sense degenerate oligonucleotide primers and Taq polymerase. The sequence of the sense primer was 5’-TGGRT{)VT{HWS{IHGC{)RC{)CAYTG-3’

SEQ ID NO. 9 and that of the anti-sense was 5'-(NGG(HCCNCCU)SWRTC(NCCYT{N)RCA(I)GHRTC-3’' SEQ ID No. 10, where

R=A.,G; V=G,AC, W=A,T;S=G,C; Y=C,T; H=A,T,C. The primer sequences correspond to two highly conserved regions in all serine proteases and amplify

PCR products ranging from 400 to 500 base pairs.

Clone screening and sequencing

The PCR products were separated on a 2% agarose gel and purified using a gel extraction kit (catalog number 28706; QlAquick gel extraction kit; Qiagen).

The purified DNA fragments were ligated into TA vectors (catalog number

K4500-01; TOPO-TA cloning kit, Invitrogen, Carlsbad, CA). After transformation into E. coli cells, plasmid DNAs were isolated and analyzed by digestion with

EcoRI restriction enzyme. Clones that had insert DNAs were further characterized by sequencing using a fluorescent dye-based DNA sequencing method (catalog number 4303149; BigDye terminator cycle sequencing kit with

AmpliTaqg DNA polymerase; Perkin Elmer, Lincoln, CA). A total of 170 clones were sequenced and analyzed. All sequences were analyzed by a multiple nucleotide sequence alignment algorithm (blastn) to identify identical or closely related cDNA clones deposited in GenBank (NCBI, Bethesda, MD). Those that did not show significant homology were further analyzed using blastx, which compares the six-frame conceptual translation products of a nucleotide sequence (both strands) against a protein sequence database (SwissProt). Two clones yielded a cDNA fragment that encodes a serine protease. One of these clones (H117) encodes the protease domain of an endothelial serine protease, the protein herein designated endotheliase 1.

Gene expression profile of the endothelial serine protease endotheliase 1

To obtain information regarding the tissue distribution of endotheliase 1, the DNA insert of clone H117 was used to probe an RNA blot composed of 76 different human tissues (catalog number 7775-1; human multiple tissue expression (MTE)} array; CLONTECH, Palo Alto, CA}. Significant expression was observed in the esophagus, with minor expression levels in the stomach, salivary gland, pancreas, prostate, bladder, trachea and uterus. Northern analysis using

RNA blots (catalog numbers 7765-1 & 7782-1; human muscle and digestive system multiple tissue northern (MTN) blots; CLONTECH) confirmed that the expression was restricted to the esophagus. Two transcripts (approximately 1.7 and 2 kb) were detected in the esophagus.

Presence of endotheliase 1 in human umbilical vein endothelial ceils

Single-stranded cDNA clones were reverse-transcribed from polyA + RNAs isolated and purified from human umbilical vein endothelial cells (HUVEC) using

Superscript Il (Life Technologies, Rockville, MD) and oligo-dT primer.

Endotheliase 1-specific primers (sense primer: 5’-CCTGCCAGATGGACTGCTTCCTTTG-3’ (SEQ ID No. 7) anti-sense primer: 5'-GGCATGCATCTGTTTTTCCTTCTAAGG-3’ (SEQ ID NO. 8) were used to amplify a 390-bp fragment from the sscDNAs of HUVEC. To prevent non-specific amplification of other serine proteases, the primers were designed so that they hybridize outside of the highly conserved sequences common to all

. trypsin-like serine proteases. RT-PCR of endotheliase 1 transcripts from HUVEC . cells showed a ~400-bp DNA fragment plus another smaller, non-specific band , after separation on a 2% agarose gel. The gel was blotted onto a positively charged nylon membrane and hybridized at 60°C with an endotheliase 1 cDNA ] 5... fragment isolated from the H117 clone using ExpressHyb{tm) hybridization ~ solution (CLONTECH). After washing at high stringency (68°C in Q.1x SSC; 0.1% SDS), a strong positive signal was observed on the ~400-bp fragment ) only, indicating that this band corresponds to the endotheliase 1 cDNA, and that .. HUVEC cells express endotheliase 1. . 10. 5’- and 3’- rapid amplification of cDNA ends (RACE)

To obtain cDNA that encodes the entire protease domain of . = endotheliase 1, 5'- and 3’-RACE reactions were performed. Since the presence . ot the transcript was detected in the prostate, a human prostate Marathon-Ready y . cDNA (catalog # 7418-1; Clontech) was used to isolate the 5’ and 3’ ends of . 15 , the cDNA encoding endotheliase 1. Marathon-Ready cDNAs are specifically made for RACE reactions. Two gene specific primers were used: 5'-GGCATGCATCTGTTTTTCCTTCTAAGG-3’ (SEQ ID No. 8) for 5’-RACE reaction and 5’-CCTGCCAGATGGACTGCTTCCTTTG-3’ (SEQ ID No. 7) for 3’-RACE reaction.

Two fragments, approximately 1.2 kbp and 1.4 kbp, were isolated that corresponded to the missing 5’ and 3’ end sequences. These fragments were confirmed by Southern analysis using the internal cDNA fragment as probe and by DNA sequence analysis. ee

TT TTT sequence analysis

All derived DNA and protein sequences were analyzed using MacVector (version 6.5; Oxford Molecular Ltd., Madison, WI). The cDNA encoding the protease domain of endotheliase1 is composed of 696 base pairs (SEQ ID No. 1) which translate to a 232-amino acid protein sequence (SEQ ID No. 2).

: 05.1 52001 + 11:48 FROM oT WADE TENNANT 0 ee 1923994465 u S 00318 03 ) g -116- ! ¢ : The cDNA encoding this pratein was cloned into a derivative of the Pichia ) pastoris vector pPICI9K (available from Invitrogen; see SEQ ID NO. 21), Plasmid pPIC9k features include the 5° AOX1 promoter fragment at 1-948; 6° AOX1 primer site at 855-8785; alpha-factor secretion signal(s) at 949-1218; . 6 alpha-factor primer site at 1152-1172; multiple cloning site at 1192-1241; 3 : AOX1 primer site at 1327-1347; 3’ AOX1 transcription termination region at 1253-1586; HIS4 ORF at 4514-1 980; kanamycin resistance gene at : 5743-4928; 3’ AOX1 fragment at 61 22-6879; ColE1 origin at 7961-7288; and the ampicillin resistance gene at 8966-8106. The plasmid used herein is derwived from pPIC9K by eliminating the Xho! site in the kanamycin resistance gene and the resulting vector is herein designated pPICIKX. \ For expression in Pichia pastoris, the ¢DNA encoding the protease domain . was amplified using the same pair of gene-specific primers, except that restriction sites were introduced at the 5’ ends of each primer to facilitate . * 4s cloning into the Pichia vector, pPIC9KX. The primers were as follows: forward (SEQ ID No. 23) y : 5"-TCTCTCGAGAAAAGAATCGTTGGTGGGACAGAAGTAGAAGAG-3'; and ee remot rn, ROVErSE {SEQ ID MO..28Y . Le 4 en ee mee Arr A © 5'-ATTCGCGGCCGCTTAGATACCAGTTTTTGAAGTAATCCA 3". - | EXAMPLE2Z

CLONING AND EXPRESSION OF ENDOTHELIASE 2 ’ {identification of endatheliase 2

The UniGene (http://www.ncbi.nim.nih.gov/UniGene) tool for data mining - at the National Center for Biotechnology Information was searched for a serine protease sequence cluster. A UniGene cluster is 8 non-redundant set of sequences that represents a unique human, mouse, or rat gene. The database R includes well-characterized genes and expressed sequence tag (EST) sequences, , including information regarding tissue distribution and chromosomal map . location. ; 3¢C A UniGene cluster (Hs.245327/Hs.266308) was identified that had ESTs ’ which were weakly similar to the human serine protease, hepsin {accession number PO5981).” This cluster contairis 12 EST sequences that were expressed

AMENDED SHEET

Emofanbe.v. . vowve- ooo

. . in the ovary, placenta, uterus, breast and colon. One EST sequence (Al909842) . ._ derived from human breast carcinoma was used to isolate the full-length cDNA - encoding this novel serine protease (hereafter referred to as endotheliase 2). ~The sequence contained in AI909842 did not show 100% identity to any known ~ 5... serine protease sequence deposited in GenBank. Using blastx to identify . ~. homologous protein sequences deposited in the protein database, the closest matches were found to be hepsin (53 to 55%), human TMPRSS2 (47%), human

MTSP2 (47%), and human plasma kallikrein B1 precursor (47%). A search of the unfinished human genome database {High Throughput Genomic Sequences (HTGS)) using blastn indicated that the gene encoding endotheliase 2 is located : “in chromosome 11 (11q23). “ he 5’- and 3’- rapid amplification of cDNA ends (RACE) = : To obtain a full-length cDNA clone encoding endotheliase 2, 5'- and

IS ) ..3'-RACE reactions were performed. The Marathon-Ready cDNA library from human mammary carcinoma {GI-101; CLONTECH; catalog number 7493-1) was ” used to isolate the 5’ and 3’ ends of the cDNA encoding endotheliase 2.

Marathon-Ready cDNAs are specifically made for RACE reactions. Two gene specific primers were used: 5'-GGAGGCAAGCAGGGTGGATGTGAGCGGAC-3’ (SEQ ID NO. 11) for 5’-RACE reaction and 5'-CGGATCGTGGGAGGGGCGCTGGCCTC-3’ (SEQ ID NO. 12) for 3'-RACE reaction. A ~1.5 kbp cDNA fragment was obtained from the 5’-RACE reaction.

The initial 3’-RACE reaction, however, did not produce any fragment. A nested

PCR was used on the initial 3'-RACE reaction products to obtain the rest of the oo 3’ end of endotheliase 2. The nested 3’ gene-specific primer used was 5'-CAAGTGAGTCTGCACTTCGGCACCACC-3’ SEQ ID NO. 13 and produced a ~1.2 kbp cDNA fragment. The fragments were subcloned into pCR2.1-TOPO

TA cloning vector (Invitrogen, Carlsbad, CA). The resulting clones were analyzed by Southern analysis using the cDNA insert of EST clone AI909842 as a probe and by DNA sequence analysis.

Domain organization of endotheliase 2

Sequence analysis of the translated endotheliase 2 coding sequence indicated that endotheliase 2 is a type-ll membrane-type serine protease. It has

~~ . WO 01/36604 PCT/US00/31803 ( a transmembrane domain at the N-terminus, followed by a single low density lipoprotein-A receptor domain and a single scavenger-receptor cysteine-rich domain. The C-terminus contains the trypsin-like serine protease domain characterized by the presence of the catalytic triad residues (histidine, aspartate b and serine) in 3 highly-conserved regions of the catalytic domain. In addition, three repetitive sequences composed of ASPAGTPPGRASP (SEQ ID NO. 14) are found just before the transmembrane domain, and represent a sequence motif for N-myristoylation modification.

PCR amplification of cDNA encoding full-length protease domain of endotheliase 2

To obtain the cDNA fragment encoding the protease domain of endotheliase 2, an end-to-end PCR amplification using gene-specific primers and the Marathon-Ready cDNA library from human mammary carcinoma was used.

The two primers used were: 5 -CGGATCGTGGGAGGGGCGCTGGCCTCG -3' (SEQ ID NO. 15) for the 5’ end, and 5'-CAGCAGGCCAGCTGGTTAGGATTTTATGAATCGCAC-3 (SEQ ID NO. 16) for the 3’ end. The 5‘ primer contained the sequence that encodes the start of the endotheliase 2 protease domain (RIVGGALAS SEQ ID NO. 17). The 3’ primer corresponds to the sequence flanking the stop codon (underlined). A ~730-bp fragment was amplified, subcloned into pCR2.1-TOPO TA cloning vector and sequenced.

For expression in Pichia pastoris, the cDNA encoding the protease domain was amplified using the same pair of gene-specific primers, except that restriction sites (Xho! site and Notl site for 5” and 3’ primers, respectively) were introduced at the 5’ ends of each primer to facilitate cloning into the Pichia vector, pPICOKX. The primers were as follows: forward (SEQ ID NO. 25) 5'-TCTCTCGAGAAAAGAATCGTGGGAGGGGCGCTGGCCTCG-3’ reverse (SEQ ID No. 26) 5 -ATAGCGGCCGCTGGTTAGGATTTTATGAATCGCACCTCGC-3'.

05-12.2001" 11748 FRO TOULT UADE TENNANT TO oe 7sRsIsdles US003180¢

Gene expression profile and transcript size of endotheliase 2 in normal and tumor tissues

To obtain information regarding the gene expression profile of the : endotheliase 2 transcript, the cDNA insert of EST clone AI909842 was used to 2 6 probe ani RNA dot blot composed of 76 different human tissues (human multiple . tissue expression (MTE)} array; catalog number 7775-1; CLONTECH, Palo Alta, :

CA) and a human tissue fornoy blot (human 12-lane multiple tissue northern oo (MTN) blot; catalog number 7780-1; CLONTECH). The RNA dot blot showed strong signals in placenta, pancreas, thyroid gland, liver and lung. Moderate signals vere observed in mammary gland, salivary gland, kidney, traches, esophagus, appendix, heart and fetal lung. Weak signals were seen in several other tissues. Endotheliase 2 is also expressed in several tumor cell lines including leukemia K-562 > Hela S3 = Burkitt's lymphomas (Raji and Daudi) = colorectal adenocarcinoma (SW480) = lung carcinoma {A548) = leukemia :

MOLT-4 = leukemia HL-60. . : Northern analysis detected several transcripts in the tissues tested with strong signals observed in the heart, skeletal muscle, kidney, liver and placenta. ¥ li... . Tne predominant transcript had a size of ~3 kb, while other transcripts were

I approximately 2.8 kb, 1.5 kband 1kb. © a

PCR amplification of the endotheliase 2 transcript from cDNA libraries oo made from several human primary tumors xenografted in nude mice (human © tumor multiple tissue cDNA panel, catalog number K15622-1, CLONTECH) was performed ‘using endotheliase 2-specific primers. The endotheliase 2 transcript was detected in breast carcinoma (GI-101), lung carcinomas (LX-1 > GI-117), colon adenocarcinomas (Gt-112 > CX-1), pancreatic adenocarcinoma (GI-103), and ovarian carcinoma (Gl-102). The endotheliase 2 transcript was also : detected in LNCaP and PC-3 prostate cancer cell lines as well as in HT-1080 ; human fibrosarcoma cell line.

Presence of endotheliase 2 in endothelial cells 30.» Single-stranded cDNAs were reverse-transcribed from polyA + RNAs isolated and purified from human umbilical vein endothelial cells (HUVEC) and human lung ! microvascular endothelial celis (HMVEC-L) using Superscript ii (Life 2 : : : ao fans AMENDED SHEET

Technologies, Rockville, MD) and oligo-dT primer. endotheliase 2-specific primers (sense primer: 5'-TCCAGGAAAGCCTCCACAGGTC-3’ SEQ ID NO. 18; anti-sense primer: 5'-GGAGGCAAGCAGGGTGGATGTGAGCGGAC-3’ SEQ ID

NO. 19} were used to amplify a 422-bp fragment from the sscDNAs of endothelial cells. This 422-bp fragment spans the scavenger receptor cysteine-rich domain and the serine protease domain. RT-PCR of endotheliase 2 transcripts from HUVEC and HMVEC-L cells showed a ~420-bp DNA fragment plus other non-specific bands after separation on a 2% agarose gel. The gel was blotted onto a positively charged nylon membrane and hybridized at 60°C with an endotheliase 2 cDNA originally isolated from human mammary gland carcinoma using ExpressHyb(tm) hybridization solution (CLONTECH). After washing at high stringency (68°C in 0.1x SSC; 0.1% SDS), a strong positive signal was observed on the ~420-bp fragment only, indicating that this DNA fragment corresponds to the endotheliase 2 cDNA, and that HUVEC and

HMVEC-L cells express endotheliase 2.

Presence of another form of endotheliase 2 in placenta and LNCaP cells.

Since multiple. endotheliase 2 mRNAs were present in placenta, another 3'-RACE reaction was performed using a endotheliase 2-specific primer (5'-TTCCTCCGGGAGGTGCAGGTCAATC-3’ SEQ ID NO. 20). Several products were observed, but only two fragments {~2.2 kbp and ~ 1 kbp) showed strong hybridization with the endotheliase 2 probe. Upon subcloning and sequence analysis, the longer fragment showed sequence identity to the endotheliase 2 cDNA initially isolated from human mammary carcinoma. The sequence of the shorter fragment showed sequence identity to that of the human mammary carcinoma-derived endotheliase 2 in all but the last 24 bp of the coding region.

Replacing these 24 bp was a 402-bp fragment which extended the coding region by an additional 134 amino acids. Interestingly, another transmembrane domain is found within this additional protein sequence. Using a set of endotheliase 2 primers (one primer common to both forms and the other one was specific to each form), the human mammary carcinoma and LNCaP cDNA libraries were screened for the presence of either or both forms. After subcloning and sequence analysis, the results showed that mammary breast carcinoma only

05.12.2001 1149 FROM LT WADE TENNANT To ees TERS US003180: : expressed the mRNA encoding the shorter protein (endotheliase 2-S), while

LNCaP only expressed the longer form {endotheliase 2-1).

Sequence analysis

Endotheliase 2-encoding DNA and the encoded protein were analyzed § using MacVector (version 6.5; Oxford Molecular Ltd., Madison, WI). The ORF of endotheliase 2-S is composed of 1,689 bp which translate to a 662-amino acid protein, while the ORF of endotheliase 2-L is composed of 2,067 bp which ; translate to a 688-amino acid protéin. The cDNA encoding the protease domain he in endotheliase 2-S is composed of 729 bp which translate to a 242-amino acid

J 10 protein sequence, while that of endotheliase 2-L is composed of 1,107 bp which translate to a 368-amino acid protein sequence. The nucleic acid sequences and the protein sequences of endotheliase 2-5 and endotheliase 2-L are set forth in -S; . - protein; SEQ ID NO. 5 : 15 sets forth the nucleic acid sequence of Endotheliase 2-L; and SEQ ID NO. 6 sets forth the Endotheliase 2-L encoded protein. : ; EXAMPLE 3 ee cee High throughput assay for identification of candidat angiogenesis TRHaGTa cs Tre case : Assay for identifying inhibitors of Endatheliase 1 -

The ability of test compounds to act as inhibitors of Endotheliase-1 (ET1) catalytic activity was assessed in an amidolytic assay. The inhibitor-induced : inhibition of amidolytic activity by the recombinant (rET 1) expressed in Pichia, as i measured by IC50 values was assessed. : : SE : s “The assay buffer was HBSA (10mM Hepes, 150mM sodium chloride, pH 7.4, 0.1% bovine serum albumin). All reagents were from Sigma Chemical . :

Co. (St. Louis, MO), unless otherwise indicated. Two IC50 assays at 30-minute {a 30-minute preincubation of test compound and enzyme) and at O-minutes {no : preincubation of test compound and enzyme) were conducted. For the IC50 : “assay at 30-minute, the following reagents were combined in appropriate wells of a Coming microtiter plate: 50 microliters of HBSA, 50 microliters of the test : compound, diluted (covering a broad concentration range) in HBSA (or HBSA alone for uninhibited velocity measurement), and 50 microliters of the rET-1 . toot yo AMENDED SHEET | oo

+ er nee Anns 11:49 FROM - MILT WADE TENNANT IU 60e "D23534400 r.or io 05-12-2001 Co Ll 2 US0031803 : i (Corvas International) diluted, in buffer, yielding a final enzyme concentration of 250 pM. Following a 30-minute incubation at ambient temperature, the assay . ¢ was initiated by the addition of 50 microliters of the substrate Spectrozyme tPA (Methyisulfonyi-D-cyclohexyityrosyl-L-glycyl-L-arginine-p-nitroaniline acetate, obtained from American Diagnostica, Inc. (Greenwich, CT) and reconstituted .in . deionized ‘water, followed by dilution In HBSA prior to the assay) were added to the wells, yielding a final volume of 200 microliters and a final substrate . concentration of 300 4M (about 1.5-times Km). .

For the IC50 assay at O-minute, the same reagents were combined: 50 microliters of HBSA, 60 microliters of the test compound, diluted {covering the identical concentration range) in HBSA (or HBSA alone for uninhibited velocity “ measurement), and 60 microliters of the substrate Spectrozyme tPAg The assay was initiated by the addition of 50 microliters of rET-1. The final concentrations . of all components were identical in both IC50 assays (at 30- and O-minute 7 incubations). oo . The initial velocity of chromogenic substrate hydrolysis was measured in . x both assays by the change of absorbance at 405 nM using a Thermo Max; . + acrsens mm smo. KiDETIC Microplate Reader (Molecular Devices) over a § minute period, In which less than:5% of the added substrate was used. The concentration of added | oo - inhibitor, ‘which caused a 50% decrease in the initial rate of hydrolysis was defined as the respective IC50 value in each of the two assays (30-and

O-minute).

Assay for identifying inhibitors of Endotheliase 2 Co

Test compounds for inhibition of the protease activity of the protease domain of endothéliase-2 was assayed in Costar 96 well tissue culture plates {Corning NY). Approximately 2-3 nM endotheliase 2 was mixed with varying concentrations of inhibitor in 29.2 mM Tris, pH 8.4, 29.2 mM imidazole, 217 mM NaCl (100 mL final volume), and allowed to incubate at room temperature ood for 30 miriutes. 400 mM substrate S 2765 (DiaPharma, Westchester, OH) was ; 30 added, and the reaction was monitored in a SpectraMAX Plus microplate reader : {Molecular Devices, Sunnyvale CA) by following the change in absorbance at . 405 nm for 1 hour at 37°C. .

Engr AMENDED SHEET.

Since modifications will be apparent to those of skill in this art, it is - .. intended that this invention be limited only by the scope of the appended claims.

EN FE :

Claims (1)

1. . OR-NFC-701 0 11:49 FROM “HUULY WHE TENNENT IU YY I925934400 F.o3/ ro 05-12-2001 US0031803 oo 2 -124- E CLAIMS / ,

   1. A substantially purified protein, comprising the protease domain of an endotheliase 2 protein or a ] 4 protein that is a catalytically active portion thereof, wherein: the endotheliase protease domain portion of the protein consists essentially of a sequence of amino acids that comprises the protease domain or a catalytically active portion thereof; and an endotheliase is an endothelial cell transmembrane protease.

   2. A substantially purified single chain protein, comprising the protease domain of an endotheliase 1 protein or a protein that is a catalytically active portion thereof, wherein: ; the endotheliase protease domain portion of the protein consists essentially of a sequence of . ¢ amino acids that comprises the protease domain or a catalytically active portion thereof; and an endotheliase is an endothelial cell ae mt hee LEAN SMEMbrane protease. Ce rer ya CL | ;

   i3. The substantially purified protein of claim 1 that consists essentially of the protease domain of an endotheliase 2 protein or a catalytically active portion thereof.

   4. The substantially purified protein of claim : 2 or 3, wherein the protease domain comprises the = sequence of amino acids set forth in SEQ ID No. 2 or . amino acids 320-562 of SEQ ID No. 4, or amino acids 320-688 of SEQ ID No. 6. or amino acids 190-424 of SEQ 4 ID No. 22 or a contiguous portion thereof that exhibits protease activity.

   5. A substantially purified endotheliase 2 £np fant AMENDED SHEET

   ¥ i

   ©. -%- 11:49 FROM JULT WADE TENNANT TO @8¢ 323994465 © 3 70 05-12-2001 ' usS0031803 ‘ i - ; -125- ; protein or a protein that is a catalytically active ' portion thereof. .

   6. The endotheliase protein or catalytically active portion thereof of claim 5 that is an : : endotheliase 2-L or is an endotheliase 2-S. ) " : "7. A protein of any of claims 1-6 that . $ specifically binds to an antibody that specifically binds to a protease domain of a protein comprising the sequence of amino acids set forth in SEQ ID No. 2, 4 or 6. )

   8. A protein of claim 1 or 2 that has at least about 40%, 60%, 80% or 90% sequence identity with a protease domain protein that comprises the sequence of B amino acids set forth in SEQ ID No. 2 or amino acids 320-562 of SEQ ID No. 4, or amino acids 320-688 of SEQ ID No. 6. or amino acids 190-424 of SEQ ID No. 22 or a contiguous portion thereof that exhibits protease activity. \ . ? PT TE ES TRE PSS CHE . : : so : | se RA ET FE Bh RPE RP

   9. A nucleic acid molecule, comprising a .- 2 : : sequence of nucleotides that encodes the protein of any 6f claim 1-8.

   10. A nucleic acid molecule that encodes an endotheliase protein or the protease domain. of an endotheliase or catalytically active portion of the ' protease domain and that hybridizes under conditions i of medium stringency along its full length to the nucleic acid molecule of claim 9.

   11. A nucleic acid molecule that encodes an endotheliase protein or catalytically active portion thereof and that hybridizes under conditions of high i

   Ld . : stringency along its full length .to the nucleic acid ¢ : . : AMENDED SHEET Cmnfanee ou Com. rw a. : © JLT WADE TENNANT TO ©8¢ 323994465 n 74 mo 05-12-2001 ' 11749 FRO ADE US0031803 molecule of claim 8.

   12. Aa vector comprising the nucleic acid : molecule of any of claims 9-11.

   13. The vector of claim 12 that is an expression vector. ' ! ¢ : 14, The vector of claim 12 or 13 that includes a ) sequence of nucleotides that direct secretion of any protein encoded by a sequence of nucleotides ~ operatively linked thereto.

   15. The vector of any of claims 12-14 that is a Pichia vector.

   16. A cell, comprising the vector of any of claims 12-14. . . 5

   17. The cell of claim 16 that is a prokaryotic cell. ‘ Ce Ch : EE SR SVE

   18. The cells of claim 16 that is a eukaryotic = ¢ cell. : }

   ‘19. The cell of claim 16 that is selected from among a bacterial cell, a yeast cell, a plant cell, an insect cell and an animal cell. 3 \

   30 . 20. The cell of claim 16 that is a mammalian cell.

   21. A cell of any of claim 16-20, wherein the endotheliase includes a transmembrane domain and a protease domain and is expressed in the cell as a transmembrane protein, wherein, if the cell is an . endothelial cell, the endotheliase is heterologous 2 Co : AMENDED SHEET : Confranveru: horror AE oy Pp m2, ag D007 11:49 FROM oT WADE TENNANT TO 88 "923994465 USOCS1 603 . . . ¥ : P - -127- : thereto.

   22. The cell of claim 21, wherein the endotheliase comprises the sequence of amino acids set forth in any of SEQ ID Nos. 2, 4, 6 and 22. + 23. The cell of claim 21, wherein the enddtheliase has at least about 90% sequence identity to the sequence of amino acids set forth in any of SEQ ID Nos. 2, 4, 6 and 22.

   24. A method for producing an endotheliase , protein or endotheliase protease domain protein, comprising: ) . ¢ 15 : culturing the cell of any of claims 16~19 under conditions whereby the encoded endotheliase protein or endotheliase protease domain protein is expressed by the cell; and recovering the expressed protein, | H : 25. An antisense nucleic acid molecule that Se comprises at least 14 contiguous naeleot ides om ama eres rn modified nucleotides complementary to the coding - portion of a nucleic acid molecule of claim 9.

   26. The antisense nucleic acid molecule of claim 1, wherein the contiguous nucleotides or modified ‘ nucleotides are complementary to a contiguous sequence of nucleotides in the protease domain of an | } ¢ 30 endétheliase. :

   ‘27. The antisense nucleic acid molecule of claim 26, wherein the endotheliase is an endotheliase 1 or endotheliase 2. ,

   28. An antibody that specifically binds to a protease domain of an endotheliase of any of claims 1- Co : tap fan AMENDED SHEET

   US=IHC=AML 1134Y FRUM-T SOUL WHUE TENNHNI IU UO 392599490D F308 05-12-2001 | y | | US0031803 E 8, or a fragment ox derivative of the antibody N containing a binding domain thereof, wherein the antibody is a polyclonal antibody or a monoclonal . ¢ antibody. : ;

   29. A kit, comprising a protein of any of claims 1-8 or a nucleic acid molecule encoding the protein, or vector comprising the nucleic acid, or a cell comprising the vector, and reagents for diagnostic, therapeutic or drug screening. 30 A conjugate, comprising: a) a protein of any of claims 1-8, and 1b) a targeting agent linked to the protein directly or via a linker.

   31. The conjugate of claim 30, wherein the : targeting agent permits | oo. ¢ i) affinity isolation or purification of ; 20 the conjugate; . . ii) attachment of the conjugate to a oo iii) detection of the conjugate; or - iv) targeted delivery to a selected tissue or cell. ’

   32. A combination, comprising: a) an inhibitor of the activity of an endotheliase or protease domain thereof; and b) an another treatment or agent selected from Co anti-tumor and anti-angiogenic treatments or agents. = i . 7

   33. The combination of claim 32, wherein the ; 35 endotheliase or protease domain thereof comprises a ot protein of any of claims 1-8. not an: AMENDED SHEET

   05-12-2001 °° 11:50. FROM La WADE TENNANT TO @@¢ 323994465 US00318 03 34, The combination of claim 32 or claim 33, wherein the.endotheliase inhibitor and the anti- ) angiogenic agent are formulated in a single pharmaceutical composition or each is formulated in - separate pharmaceutical compositions. ! 35. The combination of any of claims 32-34, wherein the endotheliase inhibitor is selected from ! antibodies and antisense oligonucleotides. ¢ 10 : ] :

   36. A solid support comprising two or more proteins of claim 1 linked thereto either directly or via a linker. ;

   "37. The support of claim 32, wherein the proteins comprise an array.

   38. A method for identifying compounds that modulate the activity of an endotheliase, comprising: contacting an endotheliase or protease domain of an endotheliase with a substrate proteolytically CT Se wee cleaved bythe endotheliase, and, either «= «mre Te ee simultaneously, before or after, adding a test f vo compound or plurality thereof; ¢ 25 , ‘measuring the amount of substrate cleaved in the ) presence of the test compound; and ‘selecting compounds that change the amount cleaved compared to a control, whereby compounds that modulate the activity of the endotheliase are identified. \ 39, A method of identifying a compound that specifically binds to an endotheliase or a protease domain thereof, comprising: contacting the endotheliase or protease domain with a test compound or plurality thereof under conditions conducive to binding thereof; .and . : : ¥ £ ov 1.., AMENDED SHEET . | :

   CU 41:5 s FROM LT TENNANT TO ©3 923994465 ° T= 05-12-2001" 11759 : FROM LT WADE US0031803 : } ~130- identifying compounds that specifically binds to . the endotheliase or to the protease domain thereof.

   40. The method of claim 38 or claim 39, wherein the endotheliase or protease domain thereof is a protein of any of claims 1-8. 41, The method of any of claims 38-40, wherein : the endotheliase or protease domain of an endotheliase i is expressed on the surface of a cell. 2 : ’

   42. The method of any of claims 38-40, wherein the endotheliase or protease domain thereof is linked either directly or indirectly via a linker to a solid support. h )

   43. The method of any of claims 38-40, wherein the compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof. Y FE VU CE SCAU SUL SHE NI NE Rr 44 Bol | The - method i. of Lan Y of cla im =a ims Ie - az, Ede. ae TE Tel TE a dA, wherein a plurality of the test substances are . screened for simultaneously. !

   2 . 45. The method of any of claims 38-44, wherein ) the change in the amount cleaved is assessed by comparing the amount cleaved in the presence of the test compound with the amount in the absence of the test compound. )

   46. The method of any of claims 38-45, wherein a plurality of endotheliases or protease domains thereof are linked to a solid support.

   47. Use of an endotheliase protein or protease domain thereof for the treatment or diagnosis of AMENDED SHEET Empfan C=. -. }

   -131- PCT/US00/31803 disorders associated with aberrant angiogenesis or undesired neovascularization.

   48. The use of claim 47, wherein the endotheliase or protease domain thereof is a protein of any of claims 1-8.

   49. A method for preventing a disease or disorder associated with undesired and/or uncontrolled angiogenesis or neovascularization, in a mammal, comprising administering to a mammal an effective amount of an inhibitor of an endotheliase.

   50. The method of claim 49, wherein the endotheliase comprises a protein of any of claims 1-8.

   51. The method of claim 49 or 50, wherein the mammal is a human.

   52. The method of any of claims 49-51, further comprising administering an anti-angiogenic treatment or agent, wherein the anti-angiogenic treatment or agent is administered prior to administration of the endotheliase, subsequent to, or simultaneously therewith.

   53. The method of any of claims 49-52, wherein the endotheliase inhibitor is selected from the group consisting of an antibody or a fragment or derivative thereof containing the binding region thereof against the endotheliase, an antisense nucleic acid encoding the endotheliase, and a nucleic acid comprising at least a portion of a gene encoding the endotheliase into which a heterologous nucleotide sequence has been inserted such that the heterologous sequence inactivates the biological activity of at least a portion of the gene encoding the endotheliase, in AMENDED SHEET

   PR-NEC-20R] 11:58 FROM.-ROULT WADE TENNANT TO 98 9923994465 P 77/7R 05-12-2001 | | US0031803 ~ which the portion of the gene encoding the . endotheliase flanks the heterologous sequence and promotes homologous recombination with a gene encoding the endotheliase. : ? ? : 54. The method of any of claims 43-53, wherein ) “the undesired angiogenesis is associated with disorders selected from the group consisting of solid neoplasms, vascular malformations and cardiovascular disorders, chronic inflammatory diseases and aberrant wound repairs, circulatory disorders, crest syndromes, dermatological disorders and ocular disorders.

   55. The method of claim 54, wherein: the vascular malformations and cardiovascular disorders are selected from the group consisting of angiofibroma, angiolipoma, atherosclerosis, restenosis/reperfusion injury, arteriovenous ’ malformations, hemangiomatosis and vascular adhesions, ’ dyschondroplasia with vascular hamartomas (Fafucci's ¢ syndrome), hereditary hemorrhagic telangiectasia ) eve ...... .|Rendu-Osler-Weber syndrome) and Von Hipple Lindau __ _. _. ._ ... syndrome; ! _ the chronic inflammatory diseases are selected from the group consisting of diabetes mellitus, hemophiliac joints, inflammatory bowel disease, nonhealing fractures, periodontitis (rapidly progressing and juvenile), psoriasis, rheumatoid arthritis, venous stasis ulcers, granulations-burns,

   -. 30 hypertrophic scars, liver cirrhosis, osteoradionecrosis, postoperative adhesions, pyogenic granuloma and systemic sclerosis; the circulatory disorder is Raynaud's phenomenon; . the crest syndromes are selected from the group f consisting of calcinosis, esophageal, dyomotiloty, ¢ sclerodactyly and teangiectasis; ) the dermatological disorders are selected from Eno fang AMENDED SHEET

   , Ae.nce-mni 11:50 FROM B0ULT WADE TENNANT oo TO B88 "7923994465 BD Pasa 05-12-2001 = US0031803 rR g . =133- - the group consisting of systemic vasculitis, . sclerbderma, pyoderma gangrenosum, vasculopathy, venous, arterial ulcers, Sturge-Weber syndrome, Port- wine stains, blue rubber bleb nevus syndrome, Klippel- Trenaunay-Weber syndrome and Osler-Weber-Rendu syndrome; ‘the ocular disorders are selected from the group consisting of blindness caused by ocular neovascular disease, corneal graft neovascularization, macular degeneration in the eye, neovascular glaucoma, trachoma, diabetic retinopathy, myopic degeneration, ' retinopathy of prematurity, retrolental fibroplasia 2 and: corneal neovascularization. :

   56. A recombinant non-human animal, wherein an endogenous gene of an endotheliase has been deleted or inactivated by homologous’ recombination or insertional mutagenesis of the animal or an ancestor thereof.

   "57. A recombinant non-human animal of claim 56, } ~ wherein the endotheliase comprises a protein of any of claims 1-8. :

   58. A recombinant non-human animal of claim 56 N or 57, wherein inactivation is effected by recombination with a nucleic acid molecule of any of d claims 9-11 that. has been modified by a deletion, 3 insertion or other mutation whereupon recombination - the endogenous endotheliase is inactivated.

   59. The recombinant non-human animal of any of claims 56-58, wherein the gene encoding an : endotheliase or protease domain thereof is under control of its native promoter. :

   ‘60. The cell of claim 21, wherein the endotheliase is an endotheliase 1 or endotheliase 2, ; Emp fant AMENDED SHEET . / TOTAL PIE C—— oo .

   -134- PCT/US00/31803

   61. Use of an inhibitor of an endotheliase in the manufacture of a preparation for treating or preventing a disease or disorder associated with undesired and/or uncontrolled angiogenesis or neovascularization, in a mammal .

   62. Use according to claim 61, wherein the endotheliase comprises a protein of any of claims 1-8.

   63. Use according to claim 61 or 62, wherein the mammal is a human.

   64. Use according to any of claims 61 to 63, further comprising administering an anti-angiogenic treatment or agent, wherein the anti-angiogenic treatment or agent is administered prior to administration of the endotheliase, subsequent to, or simultaneously therewith.

   65. Use according to any of claims 61-64, wherein the endotheliase inhibitor is selected from the group consisting of an antibody or a fragment or derivative thereof containing the binding region thereof against the endotheliase, an antisense nucleic acid encoding the endotheliase, and a nucleic acid comprising at least a portion of a gene encoding the endotheliase into which a heterologous nucleotide sequence has been inserted such that the heterologous sequence inactivates the biological activity of at least a portion of the gene encoding the endotheliase, in which the portion of the gene encoding the endotheliase flanks the heterologous sequence and promotes homologous recombination with a gene encoding the endotheliase.

   66. Use according to any of claims 61-65, wherein AMENDED SHEET

   -135- PCT/US00/31803 the undesired angiogenesis is associated with disorders selected from the group consisting of solid neoplasms, vascular malformations and cardiovascular disorders, chronic inflammatory diseases and aberrant wound repairs, circulatory disorders, crest syndromes, dermatological disorders and ocular disorders.

   67. Use according to claim 66, wherein: the vascular malformations and cardiovascular disorders are selected from the group consisting of angiofibroma, angiolipoma, atherosclerosis, restenosis/ reperfusion injury, arteriovenous malformations, hemangiomatosis and vascular adhesions, dyschondroplasia with vascular hamartomas (Fafucci’s syndrome), hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome) and Von Hipple Lindau syndrome; the chronic inflammatory diseases are selected from the group consisting of diabetes mellitus, hemophiliac joints, inflammatory bowel disease, nonhealing fractures, periodontitis (rapidly progressing and juvenile), psoriasis, rheumatoid arthritis, venous stasis ulcers, granulations-burns, hypertrophic scars, liver cirrhosis, osteoradionecrosis, postoperative adhesions, pyogenic granuloma and systemic sclerosis; the circulatory disorder is Raynaud's phenomenon; the crest syndromes are selected from the group consisting of calcinosis, esophageal, dyomotiloty, sclerodactyly and teangiectasis; the dermatological disorders are selected from the group consisting of systemic vasculitis, scleroderma, pyoderma gangrenosum, vasculopathy, venous, arterial ulcers, Sturge-Weber syndrome, Port-wine stains, blue rubber bleb nevus syndrome, Klippel-Trenaunay-Weber syndrome and Osler-Weber-Rendu syndrome; AMENDED SHEET

   -136- PCT/US00/31803 the ocular disorders are selected from the group consisting of blindness caused by ocular neovascular disease, corneal graft neovascularization, macular degeneration in the eye, neovascular glaucoma, trachoma, diabetic retinopathy, myopic degeneration, retinopathy of prematurity, retrolental fibroplasia and corneal neovascularization. :

   68. A substance or composition for use in a method for treating or preventing a disease or disorder associated with undesired and/or uncontrolled angiogenesis or neovascularization, in a mammal, said substance or composition comprising an inhibitor of an endotheliase, and said method comprising administering to a mammal an effective amount of said substance or composition.

   69. A substance or composition for use in a method of treatment or prevention according to claim 68, wherein the endotheliase comprises a protein of any of claims 1-8.

   70. A substance or composition for use in a method of treatment or prevention according to claim 68 or 69, wherein the mammal is a human.

   71. A substance or composition for use in a method of treatment or prevention according to any of claims 68-70, further comprising administering an anti- angiogenic treatment or agent, wherein the anti- angiogenic treatment or agent is administered prior to administration of the endotheliase, subsequent to, or simultaneously therewith.

   72. A substance or composition for use in a method of treatment or prevention according to any of AMENDED SHEET

   -137- PCT/US00/31803 claims 68-71, wherein the endotheliase inhibitor is selected from the group consisting of an antibody or a fragment or derivative thereof containing the binding region thereof against the endotheliase, an antisense nucleic acid encoding the endotheliase, and a nucleic acid comprising at least a portion of a gene encoding the endotheliase into which a heterologous nucleotide sequence has been inserted such that the heterologous sequence inactivates the biological activity of at least a portion of the gene encoding the endotheliase, in which the portion of the gene encoding the endotheliase flanks the heterologous sequence and promotes homologous recombination with a gene encoding the endotheliase.

   73. A substance or composition for use in a method of treatment or prevention according to any of claims 68-72, wherein the undesired angiogenesis is associated with disorders selected from the group consisting of solid neoplasms, vascular malformations and cardiovascular disorders, chronic inflammatory diseases and aberrant wound repairs, circulatory disorders, crest syndromes, dermatological disorders and ocular disorders.

   74. A substance or composition for use in a method of treatment or prevention according to claim 73, wherein: the vascular malformations and cardiovascular disorders are selected from the group consisting of angiofibroma, angiolipoma, atherosclerosis, restenosis/ reperfusion injury, arteriovenous malformations, hemangiomatosis and vascular adhesions, dyschondroplasia with vascular hamartomas (Fafucci’s syndrome), hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome) and Von Hipple Lindau AMENDED SHEET

   -138- PCT/US00/31803 syndrome ; the chronic inflammatory diseases are selected from the group consisting of diabetes mellitus, hemophiliac joints, inflammatory bowel disease, nonhealing fractures, periodontitis (rapidly progressing and juvenile), psoriasis, rheumatoid arthritis, venous stasis ulcers, granulations-burns, hypertrophic scars, liver cirrhosis, osteoradionecrosis, postoperative adhesions, pyogenic granuloma and systemic sclerosis; the circulatory disorder is Raynaud's phenomenon; the crest syndromes are selected from the group consisting of calcinosis, esophageal, dyomotiloty, sclerodactyly and teangiectasis; the dermatological disorders are selected from the group consisting of systemic vasculitis, scleroderma, pyoderma gangrenosum, vasculopathy, venous, arterial ulcers, Sturge-Weber syndrome, Port-wine stains, blue rubber bleb nevus syndrome, Klippel-Trenaunay-Weber syndrome and Osler-Weber-Rendu syndrome; the ocular disorders are selected from the group consisting of blindness caused by ocular neovascular disease, corneal graft neovascularization, macular degeneration in the eye, neovascular glaucoma, trachoma, diabetic retinopathy, myopic degeneration, retinopathy of prematurity, retrolental fibroplasia and corneal neovascularization.

   75. A protein according to claim 1, or claim 2, or claim 5, substantially as herein described and illustrated.

   76. A molecule according to claim 9, or claim 10, or claim 11, substantially as herein described and illustrated. AMENDED SHEET

   ’ -139- PCT/US00/31803

   77. A vector according to claim 12, substantially as herein described and illustrated.

   78. A cell according to claim 16, substantially as herein described and illustrated.

   79. A method according to claim 24, substantially as herein described and illustrated.

   80. A molecule according to claim 25, substantially as herein described and illustrated.

   81. A kit according to claim 29, substantially as herein described and illustrated.

   82. An antibody according to claim 28, substantially as herein described and illustrated.

   83. A conjugate according to claim 30, substantially as herein described and illustrated.

   84. A combination according to claim 32, substantially as herein described and illustrated.

   85. A support according to claim 36, substantially as herein described and illustrated.

   86. A method according to claim 38, or claim 39, substantially as herein described and illustrated.

   87. A method according to claim 49, substantially as herein described and illustrated.

   88. Use according to claim 47, or claim 61, substantially as herein described and illustrated. AMENDED SHEET

   -140- PCT/US00/31803

   89. A substance or composition for use in a method of treatment according to claim 68, substantially as herein described and illustrated.

   90. An animal according to claim 56, substantially as herein described and illustrated.

   91. A new protein, a new molecule, a new vector, a new cell, a new method for producing a protein, a new molecule, a new kit, a new antibody, a new conjugate, a new combination, a new solid support, a new method of identifying a compound, a new non-therapeutic method, a new use of an inhibitor of an endotheliase, a new recombinant non-human animal, or a substance or composition for a new use in a method of treatment, substantially as herein described. AMENDED SHEET