CA2400497A1 - Methods and compositions for generating angiostatin - Google Patents

Abstract

The invention provides methods of generating angiostatin in vitro comprising contacting plasminogen with a plasminogen activator and a sulfhydryl donor or contacting plasmin with a sulfhydryl donor. The invention also provides a method of treating angiogenic diseases by administering to an animal suffering from such a disease a sulfhydryl donor, a plasminogen activator, or a combination of a sulfhydryl donor and a plasminogen activator. The invention further comprises a composition for generating angiostatin comprising a sulfhydryl donor and a plasminogen activator. The invention also provides a container holding a sulfhydryl donor and/or a plasminogen activator, said container having a label thereon instructing administration of the sulfhydryl donor and/or plasminogen activator to an animal suffering from an angiogenic disease. The invention further provides plasminogen fragments whose N-terminal amino acid is the same as that of plasmin and whose C-terminal amino acid is located in kringle 5 and which inhibit angiogenesis, antibodies which bind selectively to these fragments, methods and kits for using the antibodies, methods and materials for making the fragments by recombinant DNA techniques, and a method of treating an angiogenic disease comprising administering an effective amount of one of the fragments. Finally, the invention provides a method of treating an angiogenic disease comprising administering a transgene coding for one of the fragments.

Description

WO O1h8921 PCT/USO1/04021 METHODS~AND COMPOSITIONS FOR GENERATING ANGIOSTATIN
This application is a continuation-in-part of pending application Serial No.
08/991,761, filed December 15, 1997, which was a continuation-in-part of pending s application Serial No. 08/710;305, filed September 17, 1996. Beneft of PCT
application PCT/US97116539, filed September 17,1997 is also claimed.
This invention was made, in part, with Government support received from the National Cancer Institute, grant number CA71875. The Government may have fights in the invention.
io FIELD OF THE INVENTION
This invention relates to angiostatin, an inhibitor of angiogenesis.
BACKGROUND OF THE INVENTION
is Angiostatin, a proteolytic fragment of plasminogen believed to consist of lcringles 1 through 3 and all or part of kringle 4, is a potent inhibitor of angiogenesis and the growth of tumor cell metastases. O'Reilly et al., Cell, 79, 31 S-328 (1994); PCT
application WO
95/29242. Angiostatin is found in vivo in tumor-bearing mice. O'Reilly et al., Cell, 79, 315 328 (1994); O'Reilly et aL, Nature Med. 2, 689-692 (I996). The enzymatic mechanism by 2o which angiostatin is generated in vivo remains unknown.
Angiostatin activity can be generated in vitro by limited elastase proteolysis of plasminogen. Sottrup-Jensen et al., in Progress in Chemical Fibrinolysis and Thrombolysis, 3, 191-209 (Davidson et al. eds. 1978). A recent abstract proposes that angiostatin is generated by macrophages infiltrating primary tumors and releasing elastase activity, which is then cleaves plasminogen to form a protein having angiostatin activity.
Dong et al., Proc.
Am. Assoc. Cancer Res., 37 58 (1996). However, while .limited elastase cleavage of plasminogen will yield a fragment or fragments having angiostatin activity, elastase will further digest the fiagment(s) to inactive peptides, and therefore, is probably not the enzyme that generates angiostatin in vivo.

WO 01/58921 PCT/USO1/0:1021 As noted above, angiostatin may be generated in vitro by limited elastase proteolysis of plasminogen: This method has several disadvantages. First, while elastase cleaves plasminogen to generate a fragment containing kringles 1-3, it is not known if this cleavage is at the normal sites where cleavage occurs to produce angiostatin in vivo.
Therefore, the s elastase=derived angiostatin may have altered in vivo processing with altered activity in humans. It may also be immunogenic if the sites of peptide cleavage are different from normal angiostatin.
A second means of producing angiostatin is by expressing the desired kringle domains of the plasminogen cDNA or gene in an expression vector in prokaryotic or io eukaryotic cells. See PCT application WO 95/29242. This approach is also limited since the appropriate domains to express are not known. The product may also be immunogenic and may not be processed in humans as would be the product generated by cleavage of plasminogen by the normal in vivo enzymes.
Finally, angiostatin can be isolated from the body fluids of animals in which it is is produced. See PCT application WO 95!29242. However, angiostatin cannot be produced in sufficient quantities for disease treatment in this manner, and the angiostatin may be contaminated with infectious agents when isolated from such sources.
Clearly a need exists for a method of producing native angiostatin in large quantities.
"Native angiostatin" is defined herein to be the angiostatin produced in vivo or angiostatin, 2o no matter how produced, which is the same as the angiostatin produced in vivo.
SUMMARY OF THE INVENTION
The present invention provides such methods. These methods are based on the discovery that a conditioned culture medium (CCM) produced by culturing cancer cells, 2s primary endothelial cells, smooth muscle cells or fibroblasts produces angiostatin when , contacted with plasminogen or plasmin. The active factors in the CCM have been identified to be a plasminogen activator and a sulfhydryl donor. Thus, the angiostatin produced by the WO O1/~8921 PCT/USO1/04021 use of a plasminogen activator and sulfhydryl donor is the same as angiostatin produced in vivo, i.e., it is native angiostatin.
In one method of the invention for producing angiostatin ~in vitro, plasmin is contacted with a sulfhydryl donor to produce the angiostatin. The plasmin may be generated s -by contacting plasminogen with a plasminogen activator. Most conveniently, all of the reactants (plasminogen, plasminogen activator and sulfhydryl donor) can be contacted simultaneously to produce the angiostatin.
The angiostatin produced by this method, along with any remaining reactants, or angiostatin purified or partially purified from the reactants, may be administered to an io animal, including a human, in need thereof. Animals in need of angiostatin are animals suffering from an angiogenic disease.
The invention further provides a composition for generating angiostatin. The composition comprises a sulfliydryl donor and a plasminogen activator. Two embodiments of the composition are CCM produced by culturing cells capable of producing plasminogen is activator and a lysate of such cells.
The invention also provides a method of treating an angiogenic disease comprising administering to an animal suffering from such a disease an amount of a sulfhydryl donor effective to cause the conversion of plasmin to angiostatin. The plasmin may be that produced by endogenous plasminogen activators) from endogenous plasminogen.
zo Alternatively, the method may further comprise administering an effective amount of plasmin. In yet other embodiments, a plasminogen activator may be administered to the animal to produce the plasmin from endogenous plasminogen or from an effective amount of administered plasminogen.
The invention also provides a method of treating an angiogenic disease comprising is administering to an animal suffering from such a disease an amount of a plasminogen activator effective to cause the conversion of plasminogen (endogenous plasminogen or administered plasminogen) to plasmin. Endogenous or administered sulfhydryl donors then cause the conversion of the plasmin to angiostatin.

WO O1/~8921 PCT/USO1/04021 The invention further provides a container holding a plasminogen activator, alone or in combination with sulfhydryl donor. The container has a label thereon instructing administration of the plasminogen activator or the combination of the plasminogen activator and sulfhydryl donor to an animal suffering from an angiogenic disease. The invention also s provides a container holding a sulfhydryl donor with a label thereon instructing administration of the sulffiydryl donor in an amount effective to cause the conversion of plasmin to angiostatin.
The invention also provides a protein having the following characteristics:
(a) it is a fragment of plasminogen; (b) its N-terminal amino acid is the same as the N-terminal lo amino acid of piasmin; (c) its C-terminal amino acid is in kringle 5; and (d) it inhibits angiogenesis. In one embodiment, the protein is native angiostatin. The invention further provides a DNA molecule coding for the protein, the DNA molecule operatively linked to expression control sequences, a host cell comprising the DNA molecule operatively linked to expression control sequences, and a method of producing the protein comprising culturing ~s ~ the host cell.
The protein may be used to treat angiogenic diseases by administering an effective amount of the protein to an animal suffering from such a disease. An animal suffering from such a disease may also be treated by administering to it a transgene coding for the protein.
Preferably, the protein coded for by the transgene is native angiostatin.
so Finally, the invention provides an antibody which binds selectively to the protein.
Such an antibody may be used to purify the protein from materials containing it. Also, such an antibody which binds selectively to native angiostatin may be used in methods and kits to detect or quantitate native angiostatin.
Zs BRTEF DESCRIPTIOI~~ OF THE DRAWINGS
Figure 1 A: Western blot showing conversion of plasminogen and plasmin to angiostatin by serum-free conditioned medium (SFCNI) produced by PC-3 cells.
Lane 1, molecular weight standard; lane 2, human plasminogen; lane 3, human plasminogen WO O1h8921 PCT/USO1/0=4021 incubated overnight at 37°C in non-conditioned RPMI; lane 4, human plasminogen incubated overnight at 37~C in SFCM from PC-3 cells; lane 5, human plasmin incubated in non-conditioned RPMI; lane 6, human plasmin incubated in SFCM produced by PC-3 cells.
.Figure 1B: Western blot showing that the generation of angiostatin from s plasminogen was time dependent. PC-3 SFCM was incubated with plasminogen and, at the time-points indicated, aliquots were removed and snap frozen prior to western blot analysis.
Trace generation of angiostatin was first observed at 3 hours, and complete conversion was noted at 24 hours.
Figure 1 C: Western blots showing that the generation of angiostatin by PC-3 SFCM
io was concentration dependent. SFCM was diluted with various amounts of fresh RPMI as indicated and incubated with plasminogen for 24 hours.
Figure 1D: Graph illustratingthe relationship of angiostatin generation to the amount of SFCM. The relative angiostatin signal was quantitated by scanning densitometer with background subtraction. At 18 hours incubation, there was a linear relationship between the is amount of angiostatin generated and the amount of PC-3 SFCM present in the reaction mixture.
Figure 2: Western blots after affinity purification of angiostatin generated by incubation of plasminogen with SCFM produced by PC-3 cells. Lane 1, molecular weight standards; lane 2, human plasminogen incubated overnight at 37~C in non-conditioned ao RPMI; lane 3, angiostatin produced by incubation of plasminogen with PC-3 S
CFM and then affinity purified on lysine-sepharose and detected on western blot by staining with Coomassie blue; lane 4, angiostatin produced by incubation of plasminogen with SCFM and then affinity purified on lysine-sepharose and detected on western blot using the monoclonal antibody Kl-3 to kringles 1-3.
Zs~- Fi_~ures 3A-B: Graphs showing that angiostatin produced by incubating plasminogen with PC-3 SCFM inhibits in vitro steps critical for angiogenesis. Figure 3A:
Endothelial cell proliferation. The data are mean ~ standard deviation. Figure 3B: Basic fibroblast growth factor (bFGF)-induced migration. Background migration without the inducer and in the WO O1/~8921 PCT/USO1/04021 presence of stimulatory bFGF are indicated. Toxicity was measured in parallel by trypan blue exclusion and was <10% at all concentrations.
Figures 4A-B: Photographs showing that angiostatin produced by incubating plasminogen with PC-3 SCFM inhibits human endothelial cell tube formation in vitro.
s Human umbilical vein endothelial cells (HWEC) were plated on gels of Matrigel in 24 well dishes and then were treated with 15 pg/ml of angiostatin produced using PC-3 SFCM in non-conditioned RPMI. Figure 4A: Control HLTVEC form branching, interconnecting networks. Figure 4B: By contrast angiostatin produced using PC-3 SFCM caused a significant disruption of the tube network.
lo Figures SA-B: Photographs showing the inhibition of angiogenesis in vivo by angiostatin produced using PC-3 SCFM. Figure SA: A hydron pellet (indicated by the arrow) containing bFGF induced a positive neovascular response 7 days after implantation.
Figure SB: By contrast, no vessels are observed approaching a hydron pellet containing bFGF and 10 ~.g/ml angiostatin produced using PC-3 SFCM (indicated by the arrow).
is Figure 6: Western blat showing that the batch eluate from Reactive Red 120-Agarose generates angiostatin when combined with Reactive Red 120-Agarose flow-through, RPMI
or RPMI amino acids. Lane 1- SFCM + plasminogen; Lane 2 - Reactive Red 120-Agarose flow-through + plasminogen; Lane 3 - Reactive Red 120-Agarose batch eluate after dialysis to TBS + plasminogen; Lane 4 - dialyzed batch eluate + Reactive Red 320-Agarose flow Zo through + plasminogen; .Lane 5 - dialyzed batch eluate + RPMI +
plasminogen; Lane b -dialyzed batch eluate + RPMI vitamin mix + plasminogen; Lane 7 - dialyzed batch eluate +
RPMI amino acid mix + plasminogen; Lane 8 - dialyzed batch eluate + RPMI
vitamin mix and amino acid mix + plasminogen; Lane 9 - plasminogen + unconditioned RPMI.
Figure 7: Graph showing that urokinase-type plasminogen activator (u-PA) activity 2s and plasminogen-angiostatin converting activity (PACA) co-elute on a gradient elution'from Hi-Q anion exchange column. Optical density readings at 280 nm demonstrated several protein peaks. u-PA activity was determined by measuring the cleavage of a chromogenic peptide substrate for plasmin (Val-Leu-Lys p NA) at 405 nm. The peak fractions were assayed for PACA by western blot.
Figure 8: Western blot showing that addition of u-PA and plasminogen to boiled Reactive Red 120-Agarose flow-through or fresh RPMI medium generated angiostatin. Lane s 1 - Reactive Red 120-Agarose flow-through + plasminogen; Lane 2 - Reactive Red 120 Agarose flow-through + plasminogen + u-PA; Lane 3 - Reactive Red 120-Agarose boiled flow-through + plasminogen; Lane 4 - Reactive Red 120-Agarose boiled flow-through +
plasminogen + u-PA; Lane 5 - unconditioned RPMI + plasminogen; Lane 6 -unconditioned RPMI + plasminogen + u-PA.
so F. inure 9: Western blot showing that the Reactive Red 120-Agarose flow-through produces angiostatin in the presence of plasminogen activators. Lane 1-Reactive Red 120-Agarose flow-through + plasminogen; Lane 2 - Reactive Red 120-Agarose flow-through +
plasminogen + u-PA; Lane 3 - Reactive Red 120-Agarose flow-through +
plasminogen + t-. PA. .
~s ~ Figure 10: Western blot showing the production of angiostatin by u-PA and glutathione. Lane 1 - plasminogen + u-PA; Lane 2 - plasminogen + u-PA + 5 ~.M
glutathione; Lane 3 - plasminogen + u-PA + 50 ~M glutathione; Lane 4 -plasminogen + u pA + 100 ~.M glutathione; Lane 5 - plasminogen + u-PA + boiled 5 ~.M
glutathione; Lane 6 - plasminogen + u-PA + boiled 50 ~.M glutathione; Lane 7 - plasminogen + u-PA + boiled Zo 100 ~M glutathione.
Figure 11: Western blot showing that the combination of u-PA and D-penicillamine produces angiostatin. Lane 1 - plasminogen + .100 ~.M D-penicillamine; Lane 2 -plasminogen + u-PA + 100 pM D-penicillamine; Lane 3 - plasminogen + u-PA + ~
1.0 mM
D-penicillamine.
is Figure 12: Western blot'showing the production of angiostatin by u-PA, t-PA
and . streptokinase. The abbreviations used in the figure have the following meanings:
PLG - human plasminogen;
uPA - urokinase-type plasminogen activator;

tPA - tissue-type plasminogen activator;
SK - streptokinase;
+ - with N-acetyl-L-cysteine; and - - without N-acetyl-L-cysteine.
Fi iue 13 : Western blot showing the production of plasmin from plasminogen and the production of angiostatin from the pre-formed, purified plasmin. Lane 1-plasminogen + u-PA-Sepharose; Lane 2 - purified plasmin + 100 uM N-acetyl-L-cysteine.
Figure 14: Graph of mean primary tumor size (mm3) for days 0-21 for control mice and mice treated with N-acetyl-L-cysteine (NAC) or NAC + urokinase-type plasminogen io inhibitor (uPA).
Figure 15: Western blot showing the production of angiostatin by N-acetyl-L-cysteine (NAC) in vivo. Lane 1 - plasma (diluted 1:20) from control mouse #2;
Lane 2 -plasma (diluted 1:20) from control mouse #3; Lane 3 - plasma (diluted 1:20) from first mouse receiving affinity purified, cell-free angiostatin; Lane 4 - plasma (diluted 1:20) from is second mouse receiving affinity-purified, cell-free angiostatin; Lane S -plasma (diluted 1:20) from NAC-treated mouse #1; Lane 6 - plasma (diluted 1:20) from NAC-treated mouse #2;
Lane 7 - plasma (diluted 1:20) from NAC-treated mouse #3; and Lane 8 -affinity-purified, cell-free angiostatin.
Figtue 16: A diagram of human plasminogen showing the amino acid sequence ofthe Zo complete molecule after cleavage of the signal peptide (not shown) (taken from Molecular Basis of Thrombosis and Hemostasis (High and Roberts, eds.1995)). Kringles 1-5 (K1 KS) are indicated. The cleavage sites between residues 77 and 78 and residues 561 and 562 needed for activation of plasminogen to plasmin are indicated by filled arrows. The unfilled arrows represent the positions of introns in the gene. The locations of the N=linked as oligosaccharide at position 289 and the O-linked glycan at position 346 are also indicated.
The * indicate members of the catalytic triad of plasmin (His603, Asp646 and Ser741).
Figure 17A. A Western blot of plasma samples from a patient with a mesothelioma of the right hemi-thorax (Case #2 - see Table 4 below) treated with urokinase alone (Cycle WO O1/~8921 PCT/USOl/0~021 2) and subsequently with captopril and urokinase (Cycle 5). Lane 1: Cycle 2, Day 1, Pre-Treatment. Lane 2: Cycle 2, Day 3, Post-Treatment. Lane 3: Cycle 5, Day 1, Pre-Treatment.
Lane 4: Cycle 5, Day 3, Post-Treatment. A "Cycle" for this patient refers to the three days of treatment plus the days off treatment until the therapy was begun again (see Table 4 s below).
Figure 17B. Graph of cell numbers versus various amounts of the lysine-binding fractions of platelet-poor plasma samples taken from Case #2 in Cycle 2, Day 3 (solid bars) and Cycle S, Day 3 (stippled bars) in a cell proliferation assay.
DETAILED DESCRIPTION OF THE
PRESENTLY PREFERRED EMBODIMENTS
The invention provides in vitro methods of generating native angiostatin. One such method comprises contacting plasminogen with a plasminogen activator and a sulthydryl donor. All three of the reactants may be combined simultaneously.
Alternatively, the is plasminogen may be contacted with a plasmiriogen activator to produce plasmin, and the plasmin then contacted with a sulfliydryl donor to produce the angiostatin.
The plasmin may be at least partially purified prior to contacting it with the sulfhydryi donor. Indeed, angiostatin can be produced directly from plasmin, however made, by contacting the plasmin with a sulfhydryl donor.
2o The plasminogen may be from any animal species. Preferably, however, plasminogen from the species of animal to be treated with the angiostatin is used to avoid immune reactions upon administration of the angiostatin. Thus, if a human is to be treated with the angiostatin, human plasminogen is preferably.used.
Methods of making plasminogen are well known in the art. Plasminogen may also as be purchased commercially. Preferably the plasminogen is prepared by recombinant DNA
or other techniques that avoid the inclusion of infectious agents in the plasminogen preparation.

WO O1/s8921 PCT/USO1/04021 All types of plasminogen activators may be used, including urokinase-type plasminogen activators, tissue-type plasminogen activators and streptokinase.
The plasminogen activator may be from any animal species. Methods of making plasminogen activators are well known in the art, and many plasminogen activators are available s commercially. Preferably the plasminogen activator is prepared by recombinant-DNA or other techniques that avoid the inclusion of infectious agents in the plasminogen activator preparation.
The plasminogen is contacted with the plasminogen activator in amounts and under conditions effective to cause the conversion of the plasminogen to plasmin.
These amounts and conditions are known or can be determined empirically as is known in the art. In particular, from about 1 ng/mI to about 1 ~glml of urokinase plasminogen activator for each microgram of plasminogen in a 1 ml reaction have been found to give complete conversion of plasminogen to plasmin after about 24 hours of incubation at 37°C.
Any sulflrydryl donor may be used. Sulfhydryl donors are well known and are is available commercially. Suitable sulfllydryl donors include L-cysteine, D-cysteine, DL
cysteine, N-acetyl-L-cysteine, reduced glutathione, D-penicillamine and captopril. The sulfhydryl donor is believed to reduce or alter disulfide bond formation in the plasminogen, and/or the plasmin, and/or the angiostatin, and/or an intermediate product.
The sulfliydryl donor is contacted with the plasmin, alone or in the presence of the zo plasminogen and plasminogen activator, in amounts and under conditions effective to cause the conversion of the plasmin to angiostatin. These amounts and conditions can be determined empirically as is known in the art. In particular, from about 10 ~M
to about 1 mM of sulfhydryl donor for each microgram of plasmin in a 1 ml reaction have been found to give complete conversion of plasmin to angiostatin after about 24 hours of incubation at is 37°C.
Plasmin may be generated from plasminogenby a plasminogen activator as described above. The plasmin may be purified from the reactants prior to contacting it with the sulflrydryl donor. Methods of purifying plasmin are known in the art {see, e.g., Example 4).

WO 01/58921 PCT/USOi/0~021 Plasmin purchased commercially or prepared in other ways may also be used to produce angiostatin by contacting the plasmin with a sulfliydryl donor as described above.
The invention further provides a composition for generating angiostatin. The composition comprises a plasminogen~activator and a sulfhydryl donor as described above.
s The plasminogen activator and sulthydryl donor may be contained in any physiologically acceptable solution (e.g., saline, buffers, culture medium) or may be present in crystalline or lyophilized form. Compositions suitable for therapeutic use are described below.
The composition may be a conditioned culture medium (CCM) prepared by culturing cells capable of producing plasminogen activator. Malignant animal cells, human and non to human, which express a plasminogen activator can produce CCM capable of converting plasminogen and plasmin into angiostatin. Suitable malignant cells include human prostate carcinoma cell lines PC-3, DU-145, LN-CaP, human breast carcinoma cell lines MDA-MB-231 and MCF-7, human glioma cell lines U-373, U-118, A-172, and U-87, and mouse melanoma cell line B 16F I Q. Many non-malignant animal cells are known to produce is plasminogen activator. Suitable non-malignant cells include primary endothelial cells (e.g., bovine aortic endothelial cells), smooth muscle cells (e.g., bovine smooth muscle cells), and fibroblasts. In addition, bacterial cells are known which produce plasminogen activator (e.g., streptokinase), and cells of any type can be transformed by recombinant DNA
techniques to produce plasminogen activator. Suitable cells and cell lines are well known in the art and Zo may be obtained commercially, from cell. depositories, and by methods well known in the art.
Suitable culture conditions for these cells are also well known in the art.
The culture medium used must contain a sulfliydryl donor, or a sulfhydryl donor may be added to the CCM after it is produced. Suitable culture media include those available commercially, such . as RPMI, DMEM, etc. The CCM may be produced by simply culttuing the cells under 2s normal culture conditions for a sufficient time to produce CCM capable of converting plasminogen or plasmin to angiostatin. This time can be determined empirically. In particular, it has been found that culturing the mammalian cells for 24-72 hours. after a monolayer has formed at 37°C is sufficient.

WO O1/~8921 PCT/USO1/04021 Alternatively, or in addition, the cells can be lysed after culturing for a time sufficient to allow synthesis of plasminogen activator. This time can be determined empirically, but culturing the cells until a monolayer has formed should be sufficient. The lysate can be used to convert plasminogen and plasmin to angiostatin.
s The angiostatin produced by these methods may be purified from the reaction mixture. Methods of protein purification are well known in the art. In particular, angiostatin may be purified by affinity chromatography using lysine-Sepharose. Residual plasmin activity should be removed with, e.g., soybean trypsin inhibitor-Sepharose, aprotinin-Sepharose, or other affinity chromatography procedures that remove serine proteases or the to plasmin catalytic domain. The angiostatin may also be purified from the reaction mixture using an antibody that binds selectively to it (see below).
The angiostatin produced by these methods (native angiostatin) has been characterized. It reacts with a monoclonal antibody specific for kringles 1-3 of plasminogen and has been found to inhibit angiogenesis as assessed by a variety of tests in vitro and in 15 VIVO.
It has also been found to have the N-terminal sequence of plasmin. For angiostatin produced from human plasminogen, the N-terminal sequence has been found to be Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly [SEQ ID NO:1]. The sequences of the plasmin of other animals are known. Thus, native angiostatin of a particular animal would have the same N
20 . terminal sequence as the plasmin of that animal.
Quite surprisingly, native angiostatin has been found to have its C-terminal amino acid located in kringle 5. In particular, angiostatin produced from human plasminogen has been found to have the C-terminal sequence Cys Tyr Thr Thr Asn Pro Arg [SEQ ID
N0:4]
or Cys Tyr Thr Thr Asn Pro Arg Lys [SEQ ID NO:S] (see Example 6). These C-terminal as sequences would result from a cleavage after amino acid 529 or 530 of plasminogen (see Figure 16), which are known plasmin cleavage sites. Thus, native human angiostatin comprises most of kringle 5 (see Figure 16), which is consistent with its molecular weight of 50-60 kD on polyacrylamide gel electrophoresis under non-reducing conditions.

These findings were surprising because ithad .been thought that angiostatin contained kringles 1-3 and part or all of kringle 4 (see Background section). Also, it has been shown that a molecule consisting of kringles 1-4, while active, was less active than a molecule consisting of kringles 1-3 (see PCT application WO 96/35774). Thus, it was unanticipated s that native angiostatin would include any portion of kringle 5. It was particularly unanticipated that native angiostatin would include most of kringie S.
It is now expected that plasminogen fragments, other than native angiostatin, including at least a portion of kringle 5 will possess angiostatin activity (i.e., will inhibit angiogenesis). Preferably the plasminogen fragment comprises the majority of kringle 5.
~o More preferably the plasminogen fragment comprises most of kringle 5. As used herein "majority of kringle 5" means at least 50% of kringle 5 (e.g., at least 40 amino acids for human kringle 5), and "most of kringle 5" means at least 75% of kringle 5 (e.g., at least 60 amino acids for human kringle 5). Of course, the plasminogen fragment is most preferably native angiostatin for the reasons given above.
~s The sequences of plasminogens from other animals are known (available from, e.g., GenBank). The sequences of human [SEQ ID N0:6], bovine [SEQ ID N0:7], canine [SEQ
ID N0:8], western European hedgehog [SEQ ID N0:9], horse [SEQ ID NO:10], rhesus monkey [SEQ ID NO:11], mouse [SEQ ID N0:12], and pig jSEQ ID N0:13]
plasminogen are given below in the Sequence Listing (downloaded from SWISS-PROT Protein Sequence Zo Database). Native angiostatin for a particular animal would include most of kringle S of that animal's plasminogen and would have a C-terminal sequence corresponding to the C-terminal sequences of human native angiostatin given above. Indeed, a review of these sequences showed that the sequence immediately after the cleavage sites in human plasminogen that produce native angiostatin (SEQ ID N0:2 and SEQ ID N0:3; see Example is ~S below) is conserved in all ofthese plasminogen sequences (see the amino acids highlighted in bold in the Sequence Listing).
As can be observed from the Sequence Listing, the sequences of canine [SEQ ID
N0:8] and horse [SEQ ID NO:10] plasminogens contain only a single kringle domain. This WO O1/~8921 PCT/USO1/04021 single kringle domain is considered a kringle 5 domain by homology to other kringle S
domains, and it contains the conserved sequence (see highlighted amino acids in the Sequence Listing) found in the kringle S domains of the other plasminogens.
Thus, the invention includes plasminogen fragments of canine and horse plasminogens and of any s other plasminogen containing a kringle 5 domain.
Plasminogen fragments of the invention (those having the N-terminal sequence of plasmin and having their C-terminal amino acids located in kringle 5) can be produced by recombinant DNA methods. Preferably the piasminogen fragment is native angiostatin.
Most preferably the plasminogen fragment is native human angiostatin.
Recombinant DNA
~o methods and suitable host cells, vectors and other reagents for use therein, are well known in the art.
The selection of a particular host cell for production of a plasminogen fragment of the invention is dependent upon a number of factors recognized by the art.
These include, for example, compatibility with the chosen expression vector, toxicity of the plasminogen .is fragment to the cell, rate of transformation, expression characteristics, bio-safety, and costs.
A balance of these factois must be struck with the understanding that not all hosts may be equally effective for the expression of a particular plasminogen fragment.
Eukaryotic host cells are preferred for making the plasminogen fragments of the invention. Within the above guidelines, useful eukaryotic host cells include yeast and other ao fungi, animal cell lines, animal cells in an intact animal, insect cells, and other eukaryotic host cells known in the art.
The host cells may be transformed with a vector comprising DNA encoding a plasminogen fragment of the invention. On the vector, the coding sequence.
must be operatively linked to expression control sequences.
zs As used herein "operatively linked" refers to the linking of DNA sequences in such a manner that the plasminogen fragment will be expressed. Preferably the linking, including the order of the sequences, the orientation of the sequences, and the relative spacing of the various sequences, is performed so that optimum expression is obtained.

WO O1/s8921 PCT/USO1/04021 The expression control sequences must include a promoter. The promoter used in the vector may be any sequence which shows transcriptional activity in the host cell and may be derived from genes encoding homologous or heterologous proteins and either extracellular or intracellular proteins. However, the promoter need not be identical to any naturally-s occurnng promoter. It may be composed of portions of various promoters or may be partially or totally synthetic. Guidance for the design of promoters is provided by studies of promoter structure such as that of Harley and Reynolds, Nucleic Acids Res..
15, 2343-61 (1987). Also, the location of the promoter relative to -the transcription start may be optimized. See Roberts, -et al., Proc Natl Acad. Sci. USA, 76, 760-4 (1979).
The promoter lo may be inducible or constitutive, and is preferably a strong promoter. By "strong," it is meant that the promoter provides for a high rate of transcription in the host cell.
In the vector, the coding sequences must be operatively Linked to transcription termination sequences, as well as to the promoter. The coding sequence may also be .
operatively linked to expression control sequences other than the promoters and transcription ~s termination sequences. These additional expression control sequences include activators, enhancers, operators, stop signals, cap signals, polyadenylation signals, 5' untranslated sequences, and other sequences and signals involved with the control of transcription or translation.
The consensus sequence for the translation start sequence of eukaryotes has been Zo defined by Kozak (Cell, 44 283-292 (1986)) to be: C(A/G)CCAUGG. Deviations from this sequence, particularly at the -3 position (A or G), have a large effect on translation of a particular mRNA: Virtually all highly expressed .mammalian genes use this sequence.
Highly expressed yeast mRNAs, .on the other hand, differ from this sequence and instead use the sequence (A/~A(A/U)AAUGUCU (Cigar and Donahue, Gene, 9 1-18 (1987)). These is sequences may be altered empirically to determine the optimal sequence for use in a particular host cell.
DNA coding for a plasminogen fragment of the invention may prepared using standard methods such as those described in Maniatis et aL, Molecular Cloning:
A

WO 01/58921 PCTIUSOI/0~021 LaboratoryManual, Cold Spring Harbor, NY (1982), Sambrook et al., Molecular Cloning:
~! Laboratory Manual, Cold Spring Harbor, NY (1989). In particular, clones coding for plasminogen are known. See, e.g., GenBank PCT application WO 95/29242; Browne et al., Fibrinolysis, 5, 257-260 (1991). Other clones may be identified by methods known in the s art. The clones, whether known or newly-identif ed, may be modified to code for a plasminogen fragment of the invention by methods known in the art.
'The coding sequences may, alternatively, be synthesized using standard techniques that are well known in the art using the known plasminogen sequences. For instance, DNA
sequences may be synthesized by phosphoamidite chemistry in an automated DNA
m synthesizer, purified, annealed, Iigated and cloned into suitable vectors.
Chemical synthesis is preferable for several reasons.
First, chemical synthesis is desirable because codons preferred by the host in which the DNA sequence will be expressed maybe used to optimize expression. Not all of the codons need to be altered to obtain improved expression, but greater than SO%, most is preferably at least about 80%, of the codons should be changed to host-preferred codons.
The codon preferences of many host cells are known. See Maximizing Gene E~:oression, pages 225-85 (Reznikoff & Gold, eds.,1986). The codon preferences of other host cells can be deduced by methods known in the art.
The use of chemically synthesized DNA also allows for the selection of codons with 2o a view to providing unique or nearly unique restriction sites at convenient points in the sequence. The use of these sites provides a convenient means of constructing the synthetic coding sequences. In addition, if secondary structures formed by the messenger RNA
transcript or other destabilizing ~ sequences interfere with transcription or translation, they may be eliminated by altering the codon selections.
2s Chemical synthesis also allows for the use of optimized expression control sequences with the DNA sequence coding for a plasminogen fragment. In this manner, optimal expression of the plasminogen fragments can be obtained. For instance, as noted above, WO Ol/~8921 PCT/USO1/O:J021 promoters can be chemically synthesized and their location relative to the transcription start optimized.
DNA coding for a signal or signal-leader sequence may be located upstream of the DNA sequence encoding the plasminogen fragment. A signal or signal-leader sequence is an amino acid sequence at the amino terminus of a protein which allows the protein to which it is attached to be secreted from the cell in which it is produced. Suitable signal and signal-leader sequences are well known. Although secreted proteins are often 'easier to purify, expression levels are generally lower than those that can be obtained in the absence of secretion.
io Vectors for expressing the plasminogen fragments may be any vector which may conveniently be subjected to recombinant DNA procedures and which is capable of expressing a plasminogen fragment in the selected host cell. The vector used to transform the host cells may have one or more replication systems which allow it to replicate in the host cells. In particular, when the host is a yeast, the vector should contain the yeast 2u is replication genes REP 1-3 and origin of replication.
Alternatively, an integrating vector may be used which allows the integration into the host cell's chromosome of the sequence coding for a plasminogen fragment of the invention.
Although the copy number of the coding sequence in the host cells would be lower than when self replicating vectors are used, transformants having sequences integrated into their Zo chromosomes are generally quite stable.
When the vector is a self replicating vector, it is preferably a high copy number plasmid so that high levels of expression are obtained. As used herein, a "high copy number plasmid"is one which is present at about 100 copies or more per cell. Many suitable high copy number plasmids are known.
Zs The vector desirably also has unique restriction sites for the insei lion of DNA
sequences and a sequence coding for a selectable or identifiable phenotypic trait which is manifested when the vector is present in the host cell ("a selection marker").
If a vector does WO O1/s8921 PCT/USO1/04021 not have unique restriction sites, it may be modified to introduce or eliminate restriction sites to make it more suitable for further manipulations.
After the vector comprising a DNA sequence coding for a plasminogen fragment of the invention is prepared, it is used to transform the host cells. Methods of transforming host s cells are well known in the art, and any of these methods may be used.
Transformed host cells are selected in known ways and then cultured under conditions effective to produce the plasminogen fragment. The methods of culture are those well known in the art for the chosen host cell.
The expressed plasminogen fragment may be recovered using methods of recovering io and purifying proteins from recombinant cell cultures which are well known in the art. In particular, antibodies which bind selectively to the plasminogen fragments of the invention may be used to purify the fragments (see below).
The invention also provides methods of treating an angiogenic disease. An angiogenic disease is one caused by, involving or dependent on angiogenesis.
Angiogenic ~s diseases include neoplastic diseases (e.g:, tumors andtumormetastasis), benign tumors (e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, pyrogenic granulomas), connective tissue disorders (e.g., rheumatoid arthritis and atherosclerosis), ocular angiogenic diseases (e.g., diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis), cardiovascular Zo diseases, cerebral vascular diseases, diabetes-associated diseases and immune disorders (e.g., chronic inflammation and autoimmunity).
The angiogenic disease may be treated by administering an effective amount of native angiostatin or,of another plasminogen fragment having the N-terminal sequence of plasmin and containing at least a portion of kringle S. Native angiostatin is preferred for the reasons is given above.
The angiogenic disease may also be treated by administering an amount of a sulfhydryl donor sufficient to cause conversion of plasmin to angiostatin. An effective amount of a plasminogen activator may also be administered to the animal to produce WO O1h8921 PCT/USO1/0~021 plasmin from plasminogen. The plasminogen or plasmin may be those found endogenously in the animal or effective amounts of plasminogen or plasmin are also administered to the animal. .
In addition, the angiogenic disease may be treated by administering an amount of a plasminogen activator sufficient to cause conversion of .plasminogen to plasmin. the plasminogen may be that found endogenously or an effective amount of plasminogen may also be administered to the animal. The plasmin is converted to angiostatin by a sulfhydryl donor. The sulfhydryl donor may be one found endogenously in the animal or an effective amounts of sulfhydryl donor can also be administered to the animal. One example of an to endogenous free sulfhydryl donor which can facilitate conversion of plasmin to angiostatin is giutathione. Glutathione is a well-known endogenous chemical, present in normal and cancer tissues, which is released by the cells into the extracellular environment. In this extracellular environment, glutathione may serve as a free sulfhydryl donor to mediate conversion of plasmin to angiostatin. Melloni et al. demonstrated that the levels of ,5 glutathione in epithelial lining fluid (ELF), obtained by bronchoalveolar lavage, was significantly greater from patients with lung cancer (1,485.5 +/- 208 ~M) when compared with smokers (544 +/- 97.6 p,M) and nonsmokers (339.3 +/- 112 ~M) without cancer (p <
0.05) ~Melloni et al., Am. J. Respir. Crit. Care Med., 154 (6 Pt 1): 1706-11, 1996.). Of note, as shown by Soff and colleagues, l001zM levels of glutathione can serve as a cofactor Zo for angiostatin generation, supporting the paradigm that endogenous levels of glutathione or other free sulfhydryl donors can be sufficient to convert endogenous plasmin to angiostatin (Gately et al., Proc. Natl. Acad. Sci. USA, 94 (20): 10868-72, 1997).
Any animal suffering from an angiogenic disease can be treated. Suitable animals treatable according to the invention include mammals, such as dogs, cats, horses, other is domestic animals, and humans.
Effective dosage forms, modes of administration and dosage amounts for the various compounds for treating angiogenic diseases may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that WO O1/~8921 PCT/USOl/0=X021 the dosage amount will vary with the activity of the particular compound employed, the severity of the angiogenic disease, the route of administration, the rate of excretion of the compound, the duration of the treatment, the identify of any other drugs being administered to the animal, the age, size and species of the animal, and like factors known in the medical and veterinary arts. In general, a suitable daily dose of a compound of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. However, the daily dosage will be determined by an attending physician or veterinarian within the scope of sound medical judgment. If desired, the effective daily dose may be administered as two, three, four, five, six or more sub-doses, administered io separately at appropriate intervals throughout the day.
The compounds of the present invention may be administered to an animal patient for therapy by any .suitable route of administration, including orally, nasally, rectally, vaginally, parenterally (e.g., intravenously, intraspinally, intraperitoneally, subcutaneously, or intramuscularly), intracisternally, transdermally, intracranially, intracerebrally, and is topically (including buccally and sublingually). The preferred routes of administration are subcutaneous, orally and intravenously. The use of biodegradable polymers similar to that described by Brem, et al., Lancet, 345, 1571 (1995) for the local sustained release of pharmacological agents following incorporation into the biodegradable polymers is also a preferred method of administration. Implantation of the drug-impregnated polymer at, e.g., Zo a tumor site, allows prolonged local exposure with minimal systemic exposure.
While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
The pharmaceutical compositions of the invention comprise a compound of the invention as an active ingredient in admixture with one or more pharmaceutically-acceptable carriers and, zs -optionally, with one or more other compounds, drugs or other materials.
Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

WO O1/~8921 PCT/USO1/0:~021 Pharmaceutical formulations of the present invention include those suitable for oral, nasal, ophthalinic, topical, rectal, vaginal and/or parenteral administration.
Regardless ofthe route of administration selected, the compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
The amount of active ingredient which will be combined with a carrier material ~to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration and all of the other factors described above. The amount of active ingredient which will be combined with a carrier material to produce a single dosage form lo will generally be that amount of the compound which is the lowest dose effective to produce a therapeutic effect or the maximally-tolerated dose that yields a therapeutic increment for life-threatening illnesses, such as cancer.
Methods of preparing pharmaceutical formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, is .optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound o-f the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the form of 2o capsules, cachets, pills, tablets, powders, granules or as a solution or a suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin,.or sucrose arid acacia), and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be is administered as bolus, electuary or paste.
Tn solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers,_such as sodium citrate or dicalcium phosphate, and/or WO 01/58921 PCT/USO1/O:1021 any of the following: (1) fillers or extenders,_such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monosterate;
(8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene giycols, sodium lauryl sulfate, and mixtures thereof; and (10) ~o coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. .
A tablet may be made by compression or molding optionally with one or more ua accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
2o The tablets, and other solid dosage forms of the pharmaceutical compositibns of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells; such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so, as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl as cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient only, WO 01/58921 PCT/i1S01/0~021 or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form.
Liquid dosage forms for oral administration of the compounds of the invention s include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in io particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, .
tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, is perfuming and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
zo Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or.more suitable nonirntating excipients .
or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate, and which is solid at room temperature, but liquid at body temperature and, is _therefore, will melt in the rectum or vaginal cavity and release the active compound.
Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

WO O1h8921 PCT/USO1/04021 Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any buffers, or propellants which may be s required.
The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
io Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
ua Transdermal patches have the added advantage of providing controlled delivery of a comF:>und of the invention to the body. Such dosage forms can be made by dissolving, dispersing or otherwise incorporating a compound of the invention in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing ao a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.
Pharmaceutical compositions of this invention suitable for parenteral administrations comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile 2s injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

WO 01/58921 PCT/USO1/0~021 Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as ~o sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monosterate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be ~s .accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally-administered drug is accomplished by dissolving or suspending the drug in an oil vehicle.
2o Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer_ employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug . .
is in liposomes or microemulsions which are compatible with body tissue.
The'injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
The formulations may be presented in unit-dose or mufti-dose sealed containers, for example, ampoules and vials, and may be stored in a lyophilized condition requiring only the WO Ol/~8921 PCT/USO1/0~021 addition of the sterile liquid carrier, for example water for inj ection, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from sterile powders;
granules and tablets of the type described above.
An angiogenic disease can also be treated by gene therapy. In particular, a transgene comprising DNA coding for a plasminogen .fragment having the N-terminal sequence of plasmin and containing at least a portion of kringle 5 operatively linked to expression control sequences is administered to an animal suffering from such a disease.
Preferably the plasminogen fragment coded for by the transgene is native angiostatin. The preparation of DNA coding for the plasminogen fragments of the invention, including native angiostatin, ~o operatively linked to expression control sequences is described above.
Expression of the transgene in the animal results in the production of the plasminogen fragment, which inhibits angiogenesis in the animal.
Methods and materials for gene therapy are well known in the art. See Culver, Gene Therapy: A Primer for Physicians (Revised 2nd ed., 1996), U.S. Patents Nos.

5,521,291, is 5,460,831 and 5,559,099, PCT applications WO 95/29242, WO 96/14876, and WO
96/35774, all~of which are incorporated herein by reference in their entirety.
See also, Kirshenbaum, et al., J. Clin. Invest., 92, 381-387 (1993) and Drazner et al., J. Clin. Invest., 99, 288-296 (1997). In particular, suitable methods and vehicles for delivery of transgenes are known and may be used to deliver the transgenes of the invention.
2o For instance, the transgene can be transfected into desired cells in vitro, and the transformed cells injected into an animal suffering from an angiogenic disease, preferably after expansion of the number of transformed cells. Methods of transfecting a transgene into cells in vitro are well known and include electroporation, direct injection of naked DNA into cells, particle bombardment, delivery by liposomes or other lipid-based carriers, delivery by 2s viral vectors, etc.
Alternatively, the transgene can be administered to the animal in such a manner that it transforms cells within the animal. Methods of delivering transgenes in vivo are also well known and include direct inj ection of naked DNA into desired tissues, organs, or tumors, use of liposomes and other lipid-based carriers to deliver the transgene, use of a noninfectious viral vector (e.g., a replication-deficient adenoviral vector) to deliver the transgene, use of targeted vehicles (a .vehicle that allows the vehicle to bind to, and deliver the transgene to, a specific cell, tissue, organ or tumor such as liposomes having a tumor-specific-antibody attached to them) to deliver the transgene, etc.
The invention also provides antibodies which bind selectively to a plasminogen fragment of the invention, including antibodies which bind selectively to native angiostatin.
"Binds selectively" means that the antibody binds to a plasminogen fragment of the invention, such as native angiostatin, in preference to plasminogen or plasmin.
io Antibodies coming within the scope of the invention include polyclonal antibodies, affinity-purified antisera, monoclonal antibodies, fragments of antibodies (such as Fab, F(ab') or F(ab')2) that are capable of binding antigen, any known isotype or subclass of antibody, and engineered antibodies (such a single-chain antibody prepared by recombinant DNA
techniques). The only requirements are that the final antibody preparation have specificity is for the plasminogen fragment and be capable of binding selectively to the fragment.
Methods of making antibodies and fragments of antibodies are well known in the art.
For instance, the antibodies of the present invention may be prepared by injecting a suitable host animal (such as a rabbit, goat, horse or other mammal) with a plasminogen fragment of the invention in admixture with an adjuvant. The injections of the fragment are continued ao until an antiserum of suitable titer is obtained. The antiserum is harvested and may be further purified using known techniques if needed or desired. For instance, the antibodies may be affinity purified or may be fractioned such as by DE-S2 chromatography.
Preferably, however, the antibodies of the invention are prepared by somatic cell.
hybridization by fusing cells from an immunized animal (such as rats, hamsters, mice or Zs other mammal) with an immortal cell line such as myeloma cells. The fused cells are cloned, and monoclonal antibodies of appropriate specificity can be isolated by screening the cloned fused cells. Techniques of preparing monoclonal antibodies are well-known.

WO 01/58921 PCT/USO1/0~021 Antibodies which bind selectively to a plasminogen fragment of the invention can be used to purify the plasminogen fragments from fluids containing them. Such fluids include culture media, such as those resulting from practice of the methods of the invention for producing native angiostatin and the plasminogen fragments of the invention (see above).
s Native angiostatin would be found, in addition, in body fluids {e.g., blood, plasma, serum, saliva, urine and fluids produced by tumors).
To purify a plasminogen fragment, the fluid containing it is contacted with an antibody specific for the particular fragment. Preferably, the antibody is attached to a solid surface before being contacted with the fluid containing the fragment.
Suitable solid surfaces lo are well known in the art and are available commercially. Examples .include glass, polyacrylamide, polymethylmethacrylate, polycarbonate, polyacrylonitrile, polyethylene, polypropylene, polystyrene, latex beads, agarose beads, and nylon.
The antibody is preferably attached covalently to the solid surface. Methods and agents for attaching antibodies covalently to solid surfaces. are well known in the art.
is Suitable agents include carbodiimide, cyanoborohydride, diimidoesters, periodate, alkylhalides, succinimides, dimethylpimelimidiate and dimaleimides. See Blair et al.; J.
fmmunol. Methods, 59, 129 (1993); Blair et al., Cancer Res., 41, 2700 (1981);
Gautheier et al., J. Expr. Med.,156, 766 (1982).
The specific concentrations of reactants, the temperature and time of incubation, as Zo well as other conditions for obtaining binding of the antibody to the plasminogen fragment, can be varied depending on such factors as the concentration of the plasminogen fragment in the fluid, the nature of the fluid and the like. Those skilled in the art will be able to determine operative and optimal conditions while employing routine experimentation.
After the antibody has bound to the plasminogen fragment, the remainder of the fluid as -is separated from the bound plasminogen fragment. The plasmii~ogen fragment is then released from the antibody by known methods.
Most preferably, a solid surface with the antibody attached to it is located in a column. For instance, a column filled with agarose beads having antibody attached to them.

WO O1h8921 PCT/USO1/04021 The fluid containing the plasminogen fragments is simply passed through the column, and the plasminogen fragments in the fluid bind to the antibody in the column and are retained in the column, while the remainder of the fluid passes through the column.
After the column is washed, the plasminogen fragments are released from the antibody.
s Antibodies of the invention which bind selectively to native angiostatin can also be used to detect or quantitate native angiostatin for the diagnosis of an angiogenic disease or to monitor for the recurrence of such a disease. Such antibodies can also be used to study the mechanism of action of angiostatin in the body.
Native angiostatin may be detected in materials such as body fluids (see above), cells ~o and tissues (tumor tissue, placenta, uterus, brain, liver and intestines).
Native angiostatin may be released from the cells or tissues by known extraction techniques, or intact cells or tissue sections may be used.
The native angiostatin in fluids or extracts can be detected or quantitated using conventional immunoassay techniques. Such techniques include agglutination, i~ radioimmunoassay, enzyme immunoassays, fluorescence assays, colorimetric assays, etc.
The immunoassay may be performed in the competitive binding format or may be an immunometric assay. It may be a homogenous or heterogenous assay. Suitable homogenous techniques are fluorescence quenching and enhancement, energy transfer immunoassay, double antibody steric hinderance immunoassay, substrate-labeled immunoassay, prosthetic Zo group-labeled immunoassay and enzyme modulator-labeled immunoassay.
The native angiostatin on cells or tissues can be detected by standard immunohistochemical techniques well known in the, art. For example, tumors are biopsied or collected, and tissue sections cut with a microtome to examine sites of native angiostatin production. Such information is useful for diagnostic and possibly therapeutic purposes in Zs the detectic.n and treatment of cancer and is useful for research purposes for studying the mode of action of angiostatin.
The native angiostatin can be detected or quantified using a labeled antibody which binds selectively to native angiostatin (primary antibody) or a labeled component that binds WO O1h8921 PCT/USO1/04021 to immunoglobulin, such as another antibody (secondary antibody) or protein A.
Suitable labels for either the primary antibody or for the component which binds to the primary antibody are well-known in the art. They include: 1 ) enzymes (e.g., horseradish peroxidase;
malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol s dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholine esterase);
2) fluorophores (such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phtaldehyde and fluorescamine), 3) radionucleotides (such as 125i);
io 4) bioluminescent labels (such as luciferin, luciferase and aequorin); 5) chemiluminescent labels (such as luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt and oxalate ester); 6) particulate lables (such as gold nanoparticles); and 7) biotin/avidin or biotin/streptavidin. The binding and defection of these labels can be performed using standard techniques known to those skilled in the art.
is The specific concentrations of reactants, the temperature and time of incubation, as well as other conditions, can be varied in whatever immunoassay or immunohistochemical technique is employed, depending on such factors as the concentration of the native angiostatin in the sample, the nature of the sample and the like. Those skilled in the art will be able to determine operative and optimal conditions for each determination while Zo employing routine experimentation.
A test kit for detecting or quantitating native angiostatin is also part of the invention.
The kit is a packaged combination of one or more containers holding reagents useful in performing the immunoassays or immunohistochemical techniques of the invention. Suit able containers for the reagents of the kit include bottles, vials, test tubes, microtiter plates, is dipsticks, strips, and other solid surfaces.
The kit will comprise a container of an antibody which binds selectively to native angiostatin. These antibodies are those described above. The antibody may be in solution, may be lyophilized, or attached to a solid surface, and may be labeled or unlabeled. The solid surfaces are the types described above, and the antibody is attached as described above.
The kit may further comprise a container holding the above-described labeled component that binds to the primary antibody. The labels are those described above.
s Finally, the kit may also contain other materials which are known in the art and which may be desirable from a commercial and user standpoint. Such materials may include a sample of native angiostatin (for standardizing immunoassays or for binding to cells or tissues in immunohistochemical 'techniques), buffers, enzyme substrates, diluents, and equipment for performing the immunoassay or immuriohistochemical technique.
io EXAMPLES
Example 1: Preparation of Conditioned Medium Containing PIasminogen-An~~iostatin Converting Activity (PACA) ~ s This example demonstrates that a variety of cells express enzymatic activity that can generate bioactive angiostatin from purified human plasminogen or plasmin.
Affinity-purified angiostatin generated by incubating plasminogen or plasmin with serum-free conditioned medium (SFCM) inhibited human endothelial. cell proliferation, migration induced by angiogenic factor basic fibroblast growth factor (bFGF), endothelial cell tube ao formation, and bFGF-induced corneal angiogenesis. Serine proteinase inhibitors, but not inhibitors of metallo-, cysteine, or aspartic proteinases, blocked angiostatin generation.
Elastatinal, a specific inhibitor of elastase, failed to block angiostatin generation, indicating that an elastase is not responsible for the conversion of plasminogen to angiostatin. Instead, the data show that serine proteinase activity is necessary for angiostatin generation.
Zs A. Methods 1. Cell Culture. The human umbilical vein endothelial cells (HUVEC), were grown in RPMI supplemented with 20% bovine calf serum (Hyclone Laboratories Inc., Logan Utah #A-2151-L), 100 U/ml penicillin G, 100 mg/ml streptomycin, L-glutamine, WO O1/~8921 PCT/USO1/04021 (Gibco BRL), 2500 U Sodium heparin (Fisher Scientific, Itasca, II), and SO
mglml endothelial cell growth supplement (Collaborative Biomedical Research, Bedford, MA). The other cells listed in Table 1 were grown in RPMI-1640 supplemented with 10%
fetal bovine serum, 100 U/mi penicillin G, 100 mg/ml streptomycin (Gibco BRL, Gaithersburg, MD).
s Cells were maintained at 37~C in a humidified incubator in an atmosphere of 5% COZ. To generate SFCM, confluent cell monolayers were washed twice with phosphate buffered saline, then serum-free RPMI was added. The next day the SFCM was collected and centrifuged at 3000 rpm for 15 minutes to remove insoluble cellular debris.
2. Ans~iostatin Generation. Two micrograms of human plasminogen, obtained ~o by lysine-sepharose affinity chromatography of human plasma (Castellino &
Powell, Methods Enzymol, 80, 365-78 (1981)), or human plasmin {#527624, Calbiochem-Novabiochem Corp., La Jolla, CA) were added to 100 ~1 aliquots of SFCM and the mixture incubated at 37°C overnight. Aliquots were analyzed for angiostatin generation by western blot (see below). Plasminogen cleavage by SFCM was also assessed in the presence of ~s proteinase inhibitors {Boehringer Mannheim, Indianapolis, IN).
3. Western Blot. Samples were electrophoresed under non-reducing conditions on 12% polyacrylamide gels (NOVEX, San Diego, CA) in Tris-Giycine running buffer (Laemmli, ATature, 227, 680-685 (1970)), and electrotransferred to a 0.45 p.M
polyvinylene difluoride {PVDF) membrane (Immobilon, Millipore, Bedford, M.A.). The membrane was Zo then blocked for 30 minutes in blocking buffer (1 % bovine serum albumin in Tris-buffered saline) and probed with a 1:1000 dilution of a monoclonal antibody to the kringles 1-3 (K1-3) fragment ofhumanplasminogen (VAP 230L, Enzyme ResearchLaboratories, Inc., South Bend, Il~. Following washing, the membrane was incubated for 30 minutes with.an alkaline phosphatase conjugated goat anti-mouse IgG secondary antibody (Kirkegaard &
Perry is Laboratories (KPL), Gaithersburg, MD) and developed using 5-bromo-4-chloro-3-indoyl-phosphatelnitroblue tetrazolium (KPL).

4. Z my o~araphic Analysis. Zymograms to detect matrix metalloproteinase activity were performed as described previously. Heussen & Dowdle, Anal.
Biochem.,102, 196-202 (I980).
5. Chromo en~ic Pegtide Substrates. To determine if an elastase was present, s ~1 of SFCM were incubated with 0.3 mM of chromogenic peptide substrates specific for ela.stase (substrate I, MeOSuc-Ala-Ala-Pro-Val pNA, SEQ ID NO:15; substrate II, Boc-Ala-Ala-Pro-Ala pNA, SEQ ID N0:16); substrate III,pGIu-Pro-Val pNA; substrate IV, Suc-Ala-Ala-Pro-Abu pNA) (Calbiochem Novabiochem Corp.), at 37~C for 2-18 hours.
Substrate cleavage was determined by monitoring the absorbance at 405 nm (Molecular Devices, ~o Menlo Park, CA).

6. Lvsine-SeQharose Purification of An~iostatin. To generate purified angiostatin for bioactivity analyses, human plasminogen was incubated with PC-3 SFCM at 20 ~.g/ml overnight at 37°C. The reaction product was applied to a lysine-sepharose column (Pharmacia Biotech), pre-equilibrated with TBS (50 mM Tris, pH 7.5, and 150 mM
NaCI) ~s Following washes with TBS to remove non-specifically bound protein, angiostatin was eluted in 0.2 M epsilon aminocaproic acid (EACA) in TBS. The eluted fraction was dialyzed (molecular weight cut off 12,000-14,000) to phosphate buffered saline. To remove residual plasmin, the angiostatin was applied to a soybean trypsin inhibitor agarose (Sigma Chemical Co., St. Louis, MO) column, and the flow-through collected, filter-sterilized and stored at Zo -80oC until used. Angiostatin was quantitated by measuring the absorbance at 280 nm, using an A'°~°/1~~ of 8Ø Sottrup-3ensen et al., in Progress in Chemical Fibrinolysis and Thrombolysis, vol. 3, pages 191-209 (Davidson et al:.eds. 1978). The purified angiostatin was also examined by Coomassie brilliant blue staining of polyacrylamide gels, and immunodetection by western blot. Elastase-generated arigiostatin, purified from human is plasma as described in O'Reilly, et al., Nature Med., 2, 689-692 (1996), was a generous gilt from M.S. O'Reilly, Children's Hospital, Harvard University, Boston, MA.

7. Microse~uence_Analysis of Angiostatin. To determine the NH2-terminus of the angiostatin bands, 10 p.g/ml of the affinity-purified angiostatin prepared by incubating plasminogen with PC-3 SFCM was electrophoresed on a 12% SDS polyacrylamide gel, electroblotted to a PVDF membrane, and stained with Coomassie blue. The bands were excised, placed on Porton sample support discs, and sequenced using a pulse liquid-phase sequencer with phenylthiohydantoin analysis.
s 8. Endothelial Cell proliferation Assay. Cell proliferation was determined utilizing the CeIlTiter 96T"'' AQ Non-Radioactive Cell Proliferation Assay (Promega Corp., Madison, W>7. The human endothelial cells were plated in a 96-well tissue culture plates (Becton Dickinson, Lincoln Park, N~ at a concentration of 5.0 x i 03 cells/well. The following day, 1, S, 8, or 10 ~g/ml of angiostatin in fresh medium was added to triplicate io wells. Wells without angiostatin served as control. The cells were incubated for 72 hours, and an absorbance read at 490 nm, reflecting the number ofproliferating cells, was measured using an automated microplate reader (Molecular Devices). The results are reported as the percent of non-treated control cell number.
9. Endothelial Cell Migration Assay. To determine the ability of angiostatin is prepared by incubation of plasminogen with PC-3 SFCM to block migration of endothelial cells towards an angiogenic factor, bFGF, migration assays were performed in a modified Boyden chamber using bovine capillary endothelial cells (a kind gift of Dr.
Folkman, Harvard Medical School, Boston, MA) as described previously. Dameron et al., Science, 265,1582-84 (1994). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) 2o with 10% donor calf serum and 100 mg/ml endothelial cell mitogen and used at passage I5.
To assess migration, the cells were serum starved overnight in DMEM
supplemented with 0.1 % bovine serum albumin (BSA), harvested, suspended in DMEM/BSA, plated at l Oblml on the lower surface of a~gelatinized membrane (Nucleopore Corp., Plesanton, CA) in an inverted Boyden chamber, and incubated for 1.5-2 hours to allow cell attachment. The is chambers were reinvented, test maierial was added to the top well, and the chamber incubated for an additional 3-4 hours. Membranes were then fixed and stained and the number of cells that migrated to the top of the filter in 10 high-power fields was determined.
DMEM with 0.1% BSA was used as a negative control, and bFGF (provided by Dr. Noel Bouck and WO 01/58921 PCT/USO1/0~021 prepared as described in Dameron et al., Science, 265, 1582-1584 (I994)) at 10 ng/ml was used as a positive control.
10. Endothelial Cell Tube Formation. HUVEC were plated on gels of Matrigel (kindly provided by Hynda Kleinman, National Institute of Dental Research) in 24-well tissue culture plates as described previously. Schnaper et al., J. Cell.
Physiol,156, 23 S-246 (I993). Angiostatin, prepared by incubation with PC-3 SFCM, in non-conditioned RPMI
was added to the wells, followed by cells at a final concentration of 4.0 x 104 cells in 1 ml of 50% HUVEC culture medium, 50% RPMI. Each angiostatin or control condition was assayed in triplicate. The cultures were incubated for 16-18 hours at 37°C, in a 5% C02 ~o humidified atmosphere, then fixed with Diff Quick Solution II (Banter, McGraw Park, IL).
A representative area of the tube network was photographed using a Polaroid MicroCam camera at a final magnification of 35X. The photographs were then quantitated by a blinded observer who measured the length of each tube, correcting for portions of tubes that were incomplete. The total length of the tubes was determined for each photograph and the mean ~s tube length was determined. The results were expressed as the mean ~
standard error of the mean.
11. Corneal Anaioaenesis Assay. The corneal assay was performed as described previously. Polverini et al., ~I~Iethods Enzymol,198, 440-450 (1991). Briefly, 5 ~.1 hydron pellets (Hydron Laboratories, New Brunswick, N~ containing 10 ~g/ml bFGF or bFGF plus ao 1 or 10 ug/ml angiostatin were implanted into the cornea of anesthetized rats. A$er 7 days, the animals were sacrificed and corneal vessels were stained with colloidal carbon and corneas were examined for angiogenic activity.
B. Results is 1. Angiostatin Generation By Conditioned Culture Medium. Incubation of human plasminogen with the SFCM produced by PC-3 cells resulted in the generation of multiple immunoreactive bands at approximately 50 kD (Fig. 1A), similar to those observed by O'Reilly et al. Cell, 79, 315-328 (1994). Examination of SFCM from additional cell lines WO Ol/~8921 PCT/USOi/04021 also revealed the generation of the multiple bands, similar to the PC-3 SFCM
(data not shown). These cell lines are listed in Table 1 below.
The initial indication that the product was ~ angiostatin was based on the immunoreactivity with the monoclonal antibody specific for kringles 1-3 (K1-3) of s plasminogen and the size of the cleavage product. Subsequent confirmation that the plasminogen cleavage product was bioactive angiostatin is described below.
Angiostatin generation by PC-3 SFCM was time-dependent. There was a significant decrease in the plasminogen substrate and a corresponding increase in angiostatin beginning -at 3 hours, with complete conversion to angiostatin by 24 hours (Figure 1 B).
Dilution of the ro PC-3 SFCM resulted in a proportional decrease in angiostatin generation (Figures 1 C and 1 D).
To determine whether plasmin, the activated form of the zymogen plasminogen, could also be converted to angiostatin, plasmin was evaluated as a potential substrate.
Incubation of plasmin With PC-3 SFCM yielded a product indistinguishable from the is plasminogen-derived angiostatin (Figure 1 A). in kinetic studies, plasmin was converted to angiostatin at a comparable rate to the plasminogen; SO% conversion by 8 hours, with complete conversion by 24 hours (data not shown). These data suggest that in vitro both plasminogen and plasmin are substrates from which angiostatin can be generated.
3b WO Ol/s8921 PCT/USO1/0~021 Cell :_ines Tested for Generating Activity Angiostatin-Human Prostate Carcinoma Activity s pC_3 +++
DU-145 ~ ++
Ln-CaP +

Human Breast Carcinoma MDA-MB-231 ++
~o MCF-7 +/

Human Giioma U-373 +
U-118 +
A-172 ++
s U-87 +

Mouse Melanoma B16F10 ++

Bovine Smooth Muscle Prima cell line ++

20 Bovine Aortic Endothelia! I
Ce!!s BAEC ++

2s 2. Enzymatic Class Of Plasminogen-Ang~iostatin Converting Activity. To determine the proteolytic class of the angiostatin generating activity, PC-3 SFCM was incubated with plasminogen in the presence of various,proteinase inhibitors.
The proteinase inhibitors were added to the SFCM/plasminogen mix prior to the overnight incubation.
Samples were analyzed by western blot for evidence of inhibition of angiostatin generation.
3o Only serine'proteinase inhibitors blocked angiostatin generation (see Table 2 below).
By contrast none of the other classes of proteinase inhibitors were effective.
Angiostatin can be generated in vitro by limited proteolysis of plasminogen by elastase. Sottrup-Jensen et al. in Progress in Chemical Fibrinolysis and Thrombolysis, 3, WO 01/58921 PCT/USOl/0~1021 191-209 (Davidson et al. eds. 1978); O'Reilly et al., Nature Med., 2, 689-692 (1996); Dong et al., Proc. Am. Assoc. Cancer Res., 37, 58 (1996). In the present study, angiostatin generation was not inhibited by elastatinal, a specific inhibitor of elastase (see Table 2 below). Additionally, no elastase activity was detected in PC-3 SFCM based on co-s incubation of SFCM with 4 elastase-sensitive chromogenic substrates for 24 hours (data not shown). These data indicate that the human plasminogen-angiostatin converting activity is unlikely to depend on the action of an elastase. Furthermore, gelatin zymograms revealed no evidence of active or latent nietalloproteinases in the PC-3 SFCM (not shown).

WO O1h8921 PCT/USO1/04021 Proteinase Inhibitor Concentration Ciass Inhibitory . ~ Activity Pefabloc 4.0 mM Serine ProteinasesComplete s Aprotinin 0.3 NM Serine ProteinasesComplete Soybean Trypsin 2.0 mM Serine ProteinasesComplete Inhibitor Benzamidine 1-10mM Serine ProteinasesWeak Elastatinal 50-100 NM Elastase None io Antipain 100 NM Limited Serine None dihydrochloride Proteinases Leupeptin 100 NM Serine and ThiolNone Proteinases Chymostatin 100 NM Chymotrypsin None Bestatin 10 NM Aminopeptidases Weak is E-64 10 pM Cysteine ProteinasesNone Pepstatin .1.0 NM Aspartic ProteinasesNone EDTA 1-10 mM MetalloproteinasesNone 1-10 Phenanthroline 10 NM MetalloproteinasesNone Phosphoramidon 100 uM MetalloproteinasesNone * Complete inhibition is defined as no immunoreactive angiostatin bands; weak inhibition results in the development of faint angiostatin immunoreactive bands; and none refers to the full generation of angiostatin.
2s 3. Purification Of An iostatin. Angiostatin generated by PC-3 SFCM was affinity purified on lysine-sepharose (O'Reilly et al., Nature ll%fed., 2, 689-692 (1996)), and the resulting product examined by western blot and Coomassie blue staining (Figure 2). The amino-terminal sequence of all three bands was KVYLSE_CKTG [SEQ. ID NO:I ]
that corresponds to residues 78-87 of the plasminogen molecule, co~rming that the product was 3o an internal fragment of plasminogen.

WO O1h8921 PCT/USOl/O~tU21 4. An~iostatin Generated BY, PC-3 SFCM Inhibits An~io enesis. Because angiogenesis represents a cascade of cellular processes that includes endothelial cell proliferation, migration, and tube formation, (Folkman & Shing, J. Biol. Chem, 267, 10931-10934 (1992)), multiple in vitro and in vivo assays related to angiogenesis were s utilized to confirm that the product generated by incubating plasminogen with PC-3 SFCM
was bioactive angiostatin.
Affinity purified angiostatin generated by PC-3 SFCM inhibited human endothelial cell proliferation in a concentration-dependent manner, with significant inhibition observed at 10 pg/ml (P<0.05) in comparison to the non-treated control cell proliferation (Figure 3A):
io Angiostatin generated by PC-3 SFCM also inhibited the bFGF-induced migration of bovine capillary endothelial cells (Figure 3B) with an ED54 of 0.35 ~eg/mI.
The dose/response curve of angiostatin generated by PC-3 SFCM was indistinguishable from that of elastase-generated angiostatin. Inhibition of migration occurred at a 10-fold lower concentration than requir ~d to inhibit proliferation, a f nding that has been reported for other inhibitors of is angiogenesis. Takano et al., Cancer Res., 54, 2654-2660 (1994). This may be due to the fact that the proliferation assay, in contrast to the migration assay, was conducted in RPMI
supplemented with 20% calf serum and endothelial cell growth supplement, and therefore contained multiple stimulatory factors.
Endothelial cell tube formation on Matrigel was significantly inhibited at 15 p.g/ml 20 (Figures 4A and B); the mean length of tubes in non-treated control was 674.5 ~ 54 mm in comparison to angiostatin produced by PC-3 SFCM, 287.7 ~ 47 mm (P<0.005).
To determine the effect of angiostatin generated by PC-3 SFCM on corneal angiogenesis in vivo, its ability to block bFGF-induced angiogenesis in the corneal angiogenesis assay was tested. The bFGF pellet induced angiogenesis in 100% of implanted 25 COiIIP~S (Figure SA). In contrast, angiostatin at 10 ug/ml completely inhibited the bFGF-induced angiogenic response in 3 of 3 animals (Figure SB). At a lower dose of 1.0 ug/ml, angiostatin completely blocked angiogenesis in 2 of 3 animals, with partial inhibition in the third animal.

WO O1/~8921 PCT/USO1/0~021 Taken together, these data indicate that the angiostatin generated by the PC-3 SFCM
is a potent inhibitor of both in vitro and in vivo angiogenesis.
Example 2: Identification of Factors Responsible For Converting s Plasmino~en to An iostatin The human prostate carcinoma cell Line PC-3 was grown and PC-3 SFCM was prepared as described in Example 1. Angiostatin was generated by incubation with PC-3 SFCM or other materials identified below as described in Example i. Western blots were performed as described in Example 1.
~o PC-3 SFCM was applied to a Reactive Red 120-Agarose column (Sigma Chemical Co.). The flow-through had no residual plasminogen-angiostatin generating activity (PACA) as demonstrated by western blot analysis (Figure 6). The bound material was eluted with 1 M KCI according to the manufacturer's protocol, then dialyzed to Tris-buffered saline (TBS, 20 mM Tris, pH 7.4, 100 mM NaCI), with a molecular cut-off of 6000-8000 Dalton. PACA
is was not detected in the dialyzed fraction (Figure 6). The. observation that PACA was not detected in either the flow-through or the eluate led to the hypothesis that two or more factors are necessary to generate angiostatin from plasminogen or plasmin, and that the factors were separated by the Reactive Red 120-Agarose chromatography, with one or more factors being present in the elaute and one more factors being contained in the flow-through.
2o To test this hypothesis, the dialyzed eluate was recombined with the flow-through.
The recombined materials were able to convert plasminogen into angiostatin.
Supplementation of the eluate with fresh RPMI culture medium, as well as the Reactive Red 120-Agarose flow-through, restored the capacity of the eluate to generate angiostatin, suggesting that the necessary factor was a component of RPMI, and not a protein or other is factor unique to the SFCM.
To further define the putative cofactor, the individual components of RPMI
were evaluated for the ability to complement the Reactive Red 120-Agarose eluate.
The cofactor was present in the RPMI amino acid mix (Figure 6).

WO O1/~8921 PCT/i1S01/04021 To determine which amino acid was capable of restoring PACA to the Reactive Red 120-Agarose eluate, the 20 amino acids found in RPMI were tested individually.
L-cysteine was the only amino acid capable of restoring PACA to the Reactive Red 120-Agarose eluate (data not shown).
s Because the addition of L-cysteine to the Reactive Red 120-Agarose eluate restored angiostatin generating activity, it was hypothesized that the cofactor was a sulfhydryl donor.
Pharmacological reducing agents, D-penicillamine and captopril were therefore examined for the ability to restore PACA to the Reactive Red 12-agarose eluate. Addition of 100 ~M
n-penicillamine to the Reactive Red 120-Agarose eluate restored angiostatin generating io activity. Captopril also restored angiostatin generating activity to the Reactive Red 120-Agarose eluate.
PC-3 SFCM was diluted to 50 mM Tris, pH 10.0, 20 mM NaCl and applied to a Hi-Q
Sepharose anion exchange resin (Bio Rad). No PACA was detected in the flow-through.
Preliminary experiments indicated that PACA eluted from the Hi-Q Sepharose is column with 300 mM NaCI. Therefore, the bound material was eluted utilizing a linear gradient from 20 mM to 300 mM NaCI. PACA and urokinase-type plasminogen activator (u-PA) activity were measured in the fractions (after dilution to restore physiological NaCI
concentrations). The u-PA activity and PACA co-purified (Figure 7).
Examination of the Reactive Red 120-Agarose eluate revealed it also contained u-PA.
2o As noted in Example 1, the NH2-terminal cleavage of angiostatin is at Lys", a site that results from cleavage of Glu-plasminogen by plasmin. This suggests that plasmin generation may be a necessary intermediate step in angiostatin generation from plasminogen.
To determine if the factor in the Reactive Red 120-Agarose eluate was u-PA, u-PA
was tested as a substitute for the Reactive Red 120-Agarose eluate. As illustrated in Figure 2s 8, u-PA was capable of generating angiostatin in the presence of boiled Reactive Red 120-Agarose flow-through or RPMI, both sources of sulfhydryl donors. This indicates that the only protein necessary for conversion of plasminogen to angiostatin is u-PA.

WO Ol/~8921 PCT/USO1/04021 Next, u-PA, tissue-type plasminogen activator (t-PA), and streptokinase were tested in combination with the Reactive Red 120-Agarose flow-through for PACA. The plasminogen activators alone failed to generate angiostatin from plasminogen but, in the presence of the flow-through, angiostatin was produced (Figure 9). These data suggest that s plasmin generation is an intermediate for angiostatin generation, and that angiostatin generation is not dependent on which plasminogen activator is present.
Example 3: Generation Of Angiostatin Using Plasminogen Activators And Sulfll~ryl Donors io Having demonstrated that the only protein necessary for conversion ofplasminogen to angiostatin is a plasminogen activator and that a sulflrydryl donor is a necessary cofactor, it was next determined if these components are sufficient for angiostatin generation. All incubations were performed at 37°C for 18 hours in TBS, and the resulting samples were analyzed for angiostatin by V~estern blot (performed as described in Example 1).
is Incubation ofu-PA with plasminogen and at least 5 ~M reduced glutathione produced angiostatin (Figure 10). No angiostatin was produced in the absence of the glutathione.
Use of 100 uM or 1 mM D-penicillamine in combination with u-PA was also capable of generating angiostatin (Figure 11).
Finally, incubation of plasminogen (0.2 uM) with u-PA (0.2 nM), t-PA (1.0 nNn, or 2o streptokinase (8.0 nM) with 100 ~.M N-acetyl-L-cysteine resulted in the production of angiostatin (Figure 12). Plasminogen was not converted to angiostatin in the absence of N-acetyl-L-cysteine.
These data show that plasminogen is converted to angiostatin by each of the classic plasminogen activators in the presence of a sulfliydryl donor, but not in the absence of the zs sulflrydryl donor. Further, these data and the data in Example 2 demonstrate that angiostatin is produced in the presence of physiological (L-cysteine, reduced glutathione) and pharmacological (captopril, D-penicillamine, N-acetyl-L-cysteine) reducing agents.

Example 4: Use Of Plasmin For An~iostatin Generation Two micrograms of human plasminogen in 100 ~l of TBS was incubated with 10 ~1 of uPA-Sepharose (Calbiochem, La Jolla, CA) for 2 hours at ~37°C.
Following this incubation, the sample was centrifuged to sediment the uPA-Sepharose, and the supernatant s containing plasmin was collected. The complete conversion of plasminogen to plasmin was confirmed by analysis of the supernatant on Coomassie-stained reduced polyacrylamide gels.
The purified plasmin was then incubated for 18 hours at 37°C with 100 p.M N-acetyl-L-cysteine, and samples analyzed for angiostatin generation by Western blot (performed as described in Example 1 ).
io The results are shown in Figure 13. These results demonstrate that plasmin is a necessary intermediate in the generation of angiostatin from plasminogen, and that angiostatin can be produced by incubation of purified plasmin with a sulfhydryl donor.
Example 5: Treatment Of Tumors In Vivo With Sulfhydryl Is Donor. With And Without Plasmino~en Activator Eleven female beige nude mice (Taconic Labs, Germantown, NIA 6-8 weeks of age were injected subcutaneously inthe right flankwith 1.0 x 106 marine hemangioendothelioma.
(EOMA) cells (generously provided by Dr. Robert Auerbach, Madison, WI) in 100 u1 phosphate-buffered saline. The EOMA tumor cells were grown in Dulbecco's Modified ao Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mI
penicillin G, and I00 mg/ml streptomycin (Life Technologies Inc., Gaithersburg, MD) and maintained at 37°C in a humidified incubator in an atmosphere of 10%
CO2. The day of injection of the tumor cells was designated day 0.
Beginning on day I, each of the mice was injected subcutaneously twice a day with zs the following:

WO O1/~8921 PCT/USO1/Oa021 Group And Number Of Mice Treatment Control (S) ~ Saline NAC (4) N-acetyl-L-cysteine in saline (6 mg per injection) uPA+NAC (4) urokinase-typeplasminogen activator in saline '° (250 Units per injection) + N-acetyl-L
cysteine in saline (6 mg per injection) The size of the primary tumor in each mouse was measured three times weekly using tissue calipers, and tumor volume was determined using the formula (widths x length x 0.52) is (O'Reilly et al., Nature Medicine, 2, 689-692 (1996)). The results are presented in Figure 14. As can be seen both treatment with NAC and treatment with uPA +NAC
effectively and significantly decreased the mean tumor size as compared to the control group.
It should be noted that one control mouse died on day 10 and another control mouse died on day 17.
None of the mice treated with NAC or uPA + NAC died during the 21-day duration of the ao . experiment.
Plasma samples taken from two of the control mice and three of the NAC-treated mice on the day of sacrifice were assayed for angiostatin by Western blot (performed as described in Example 1). As a control, two mice were injected subcutaneously with 1.00 mg of affinity-purified, cell-free angiostatin twice a day beginning on day 1 until 24 hours prior Zs to sacrifice. Affinity-purified, cell-free angiostatin was generated as described in Example 3 and affinity purified on a lysine-sepharose.column as described in Example 1. ,The results are shown in Figure 1 S. ~As can be seen, administration of NAC to the mice caused the production of angiostatin in vivo (Lanes 5, 6 and 7). No angiostatin production was detected in control mice (Lanes l and 2).

WO 01/58921 PCT/IJSO1/0~021 Example 6: Determination Of The C-Terminal Sequence Of Native An~ iostatin Native angiostatin was generated by incubating human plasminogen (0.21zM) with recombinant human u-PA (0.2 nM) (Abbott Laboratories, North Chicago, IL) and 100 ~M
N-acetyl-L-cysteine at 37°C overnight. The material was then applied to a lysine-sepharose s column (see Example 1), and the flow-through material was collected and concentrated.
Aliquots ofthe concentrated flow-through material were electropheresed under non-reducing conditions on 12% polyacrylamide gels (NOVEX, San Diego, CA) in Tris-Glycine running buffer, eleetrotransferred to a 0.45 p.m polyvinylene difluoride (PVDF) membrane (Immobilon, Millipore, Bedford, MA), and proteins stained with Coomassie blue.
io The stained membrane showed two very prominent bands from the flow-through at approximately 30 kD. Although other bands were observed, the staining of these bands was considerably less than the staining of the two 30 kD bands, indicating that the two 30 kD
bands contained the predominant constituents of the flow-through.
The N-terminal sequences of the proteins in the two 30 kD bands were determined i s by microsequence analysis as described in Example 1. The N-terminal sequence of the most prominent of the two bands was Lys Leu Tyr Asp Tyr Cys Asp Val [SEQ ID N0:2], while the sequence of the other band was Leu Tyr Asp Tyr Cys Asp Val [SEQ ID N0:3].
The location of these sequences in kringle 5 of plasminogen (see Figure 16) and the prominence of the two bands provided extremely strong evidence that these were the fragments released zo as a result of the cleavage of plasminogen to form the C-terminal of native angiostatin.
From the N-terminal sequences of the two 30 kD fragments, it was deduced that the C-terminal sequence of the native angiostatin was Cys Tyr Thr Thr Asn Pro Arg [SEQ ID
NO:4] or Cys Tyr Thr Thr Asn Pro Arg Lys [SEQ ID NO:S]. These C-terminal sequences would be formed by cleavage after amino acid 529 (Arg) or 530 (Lys) of human 2s plasminogen, which is in kringle 5 (see Figure 16), which are known plasmin cleavage sites.
Cleavage at this point would give a plasminogen fragment of about the molecular weight observed for native angiostatin on polyacrylamide gel electrophoresis under non-reducing conditions (SO-60 kD).

WO Ol1S8921 PCT/USO1/0~021 The N-terminal sequence of human native angiostatin [SEQ ID NO:1 ] is given above in Example 1. Thus, human native angiostatin was deduced to be a plasminogen fragment including most of kringle 5 with the N-terminal sequence Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly s [SEQ ID NO:1]
and the C-terminal sequence Cys Tyr Thr Thr Asn Pro Arg [SEQ ID N0:4]
or Cys Tyr Thr Thr Asn Pro Arg Lys [SEQ ID NO:S].
lo Example 7: Antibody To Native An iostatin An antibody to the C-terminal of native human angiostatin (see Example 6) was prepared by immunizing three New Zealand white rabbits with a peptide having the sequence:
is Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg [SEQ ID N0:17]
(purified by RP-HPLC to >95% purity; Multiple Peptide Systems, San Diego, CA) conjugated 1:1 (weight/weight) to Keyhole Limpet Hemocyanin (KLH) using gluteraldehyde Zo as the cross-linking agent. The rabbits were immunized with the peptide-KLH
conjugate, suspended in phosphate buffered saline and emulsified by mixing with an equal volume of complete Freund's Adjuvant, injected into four subcutaneous dorsal sites.
Subsequent immunizations were performed using incomplete Freund's Adjuvant. The rabbits were bled from the auricular artery, the blood allowed to clot, and the serum collected by 2s centrifugation. Affinity purification was carried out using the'synthetic peptide [SEQ ID
N0:17] coupled to agarose. Crude serum was applied to the resin and, after washes, the adsorbed protein was eluted with a low pH glycine-HCl buffer. The antiserum recognized WO O1/s8921 PCT/USO1/0:1021 reduced and non-reduced plasminogen, plasmin and native angiostatin produced as described in Example 6, but not elastase-derived angiostatin (consisting of kringles 1-3).
Example 8: Treatment of Human Cancer Patients s . Four human cancer patients were treated with a combination of a sulfliydryl donor and a plasminogen activator. Captopril was used as the sulfhydryl donor.
Urokinase (uPA}
or tissue plasminogen activator (tPA) was used as the plasminogen activator.
The compounds administered and their doses and schedules of administration are summarized in Table 4 below. In addition, one of the patients (Case #2) received a single treatment with io uPA alone (i.e., without the captopril).
Blood samples, in sodium citrate anticoagulant, were taken before, and at various times after, treatment was begun. Platelet-poor plasma was isolated from the blood, and stored frozen until tested.
Western blots were performed on the plasma samples (diluted 1:20) in Tris-Buffered is saline (20 mM Tris, pH 8.0,1 SO mM NaCI) as described in Example 1. The results of the Western blots performed on some of the plasma samples taken from Case #2 are shown in Figure 17A. As noted above, this patient received uPA alone and in combination with captopril. As can be seen in Figure 17A, a marked increase in angiostatin levels was observed as a result of either treatment. This indicates that captopril, or other sulfhydryl 2o donors, may not be necessary to convert plasmin to angiostatin in some situations. Such situations may include the presence of mesothelioma or other cancers that can release endogenous sulfhydryl donors or that, in some other way, mediate conversion of plasmin to angiostatin.
The lysine-binding proteins were purified from platelet-poor plasma samples obtained 2s after the administration of uPA alone or uPA plus captopril to Case #2.
First, 1 ml of platelet-poor plasma was diluted I :40 with 20 mM Tris, pH 8.0, and incubated with 8 ml of Lysine-Sepharose (pre-equilibrated in 20 mM Tris, pH 8.0) for >12 hours with gentle shaking at 4°C. (In preliminary experiments, the 8:1 ratio of resin:plasma bound >99% of WO O1/~8921 PCT/USOl/0~021 angiostatin and plasma). The plasma supernatant was separated by gentle centrifugation (2000 x g), and the resin was washed 3 times with 40 ml of 100 mM NaCI, 20 mM
Tris, SmM EDTA, pH 8Ø After washing and repeat centrifugation, the bound proteins were eluted with 4 ml of 200 mM EACA, 20 mM Tris, 5 mM EDTA, pH 8.0, and the elution dialyzed to 40 mM NaCI, 20 mM Hepes, SmM EDTA, pH 8.0, and spin concentrated (MW
cutoff=10,000) to S00 microliters. On Western blot and Coomasie-stained polyacrylamide gel electrophoresis, the lysine-binding fractions contained plasminogen, angiostatin, and complexed angiostatin only.
To evaluate the antiangiogenic activity of the lysine-binding fractions of the plasma a o samples taken from Case #2, a celluIarproliferation assay was performed as follows. Bovine aortic endothelial cells were plated in 24-well culture dishes at 1.0 x 104 cellsJwell in DMEM
supplemented with 2.5 % heat-inactivated calf serum,100 Units/ml penicillin 6,100 mg/ml streptomycin, and the cells were incubated overnight at 37°C in a humidified incubator. On each of the following three days, fresh medium containing 3 ng/ml human bFGF
(R&D
~s Systems, Minneapolis,~MN) alone or with various amounts of the lysine-binding fractions was added. As a positive control, 100 nM affinity-purified, cell-free angiostatin (produced as described in Example 6) was used. After 72 hours of treatment, cells were washed with phosphate buffered saline, dispersed with trypsin-EDTA, and the cell number was determined by counting from duplicate wells using a Coulter counter. The results are shown ao in Figure 17B. As shown in that figure, administration of urokinase alone induced antiangiogenic activity as measured by this endothelial cell proliferation assay. While more antiangiogenic activity was induced by the administration ofthe combination of captopril and urokinase, the urokinase alone induced antiangiogenic activity. These data indicate that antiangiogenic activity is induced by plasminogen activators alone, although not as potently is as by the plasminogen activators and free sulfhydryl donors in combination.
Case #1 was a 14 year old girl with recurrent Ewing's sarcoma of the left pelvis.
After she could not tolerate chemotherapy or radiation therapy due to her extensive prior treatment, she received multiple cycles of the combination of captopril and urokinase. The WO 01/58921 PCT/USO1/0~021 doses and schedules of administration are summarized in Table 4. For the first several months, she received the captopril-urokinase combination for 3 consecutive days every 2 weeks. Subsequently, she received the combination 2 consecutive days every~3 weeks for a total of one year. A "cycle" refers to the 2 or 3 days of treatment, plus the days off s treatrnent, until the therapy was begun again 2 or 3 weeks later. The Western blots revealed generation of angiostatin-related protein, which included free angiostatin as well as a complex of angiostatin with another protein or proteins. The other proteins) has(have) not yet been identified. The large immunoreactive bands observed on the Western blot are believed to contain angiostatin because: (a) they cross-react with several antibodies to io angiostatin, including kringie-dependent antibodies and COON-terminus specific antibodies;
(b) when affinity purified with lysine-Sepharose, and disulfide reduced, the complex yields monomeric angiostatin; and (c) the complex can be amity purified with a resin comprising monoclonal antibodies to angiostatin coupled to Sepharose. Over three months of treatment, the patient achieved a complete remission. The therapy was continued for a full year, and ~s the patient has remained in remission during the treatment period, as well as for 6 months after completion of treatment.
All of the cases, their treatments, and the results achieved are summarized in Table 4.

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~1 WO O1/~8921 PCT/USO1/0~1021 SEQUENCE LISTING
<120> NORTHWESTERN UNIVERSITY
<120> METHODS AND COMPOSITIONS FOR GENERATING ANGIOSTATIN
<130> 10651-005-228 (4228-1-1-1) <140> Not Yet Assigned <141> 2000-02-08 <150> 08/710,305 <151> 1996-09-17 <150> 08/991,761 <151> 1997-12-15 <150> PCT/US97/16539 <151> 1997-09-17 <160> 17 <170> PatentIn Ver. 2.1 <210> 1 <211> 10 <212> PRT
<213> Homo Sapiens <400> 1 Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly <210> 2 <211> 8 <212> PRT
<213> Homo Sapiens <400> 2 Lys Leu Tyr Asp Tyr Cys Asp Val <210> 3 <211> 7 <212> PRT
<213> Homo Sapiens <400> 3 Leu Tyr Asp Tyr Cys Asp Val <210> 4 <211> 7 WO O1/~8921 PCTIUSO1/04021 <212> PRT
<213> Homo Sapiens <400> 3 Leu Tyr Asp Tyr Cys Asp Val <210>4 <211>7 <212>PRT

<213>Homo sapiens <400> 4 Cys Tyr Thr Thr Asn Pro Arg <210>5 <211>8 <212>PRT

<213>Homo sapiens <400> 5 Cys Tyr Thr Thr Asn Pro Arg Lys <210> 6 <211> 791 <212> PRT
<213> Homo sapiens <400> 6 Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser 1 ' 5 10 ~ 15 Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala 20 ~ 25 30 Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser 50 55 n 60 Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr WO O1/~8921 PCT/USO1/04021 Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser 100 105 l10 Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp l30 . 135 140 Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val 260 265 ~ 270 Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His Asn Arg Thr Pro Glu Asn Phe Pro C'ys Lys Asn Leu Asp G1u Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp G'ys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser WO O1h8921 PCT/USO1/0=4021 Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr_ Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser 370 375 ~ 380 Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys-Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu.Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile 485 490 ~ 495 Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu LysIAsn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Fro Trp Cys Tyr Thr Thr Asn 515 520 ' 525 Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro.Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln 56'5 '570 575 Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu WO O1h8921 PCT/LJSO1/04021 Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu GIy Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu G'ys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn <210> 7 <211> 812 <212> PRT
<213> Bos taurus <400> 7 Met Leu Pro Ala Ser Pro Lys Met Glu His Lys Ala Val Val Phe Leu Leu Leu Leu Phe Leu Lys Ser Gly Leu Gly Asp Leu Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Leu Ser Leu Ser Arg Lys Asn Leu 35 40 . 45 Ala Gly Arg Ser Val Glu Asp Cys Ala Ala Lys Cys Glu Glu Glu Thr Asp Phe Val Cys Arg Ala Phe Gln Tyr His Ser Lys Glu Gln Gln Cys Val Val Met Ala Glu Asn Ser Lys Asn Thr Pro Val Phe Arg Met Arg Asp Val Ile Leu Tyr Glu Lys Arg Ile Tyr Leu Leu Glu Cys Lys Thr Gly Asn Gly Gln Thr Tyr Arg Gly Thr Thr Ala Glu Thr Lys Ser Gly Val Thr Cys Gln Lys Trp Ser Ala Thr Ser Pro His Val Pro Lys Phe Ser Pro Glu Lys~Phe Pro Leu Ala Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Asn Gly Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asp Ile Pro Glu Cys Glu Asp Lys Cys Met His Cys Ser Gly Glu Asn Tyr Glu Gly Lys Ile Ala Lys Thr Met Ser Gly Arg Asp Cys Gln Ala Trp Asp Sex Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg Pro Trp Cya Phe Thr Thr Asp Pro Gln Lys Arg Trp Glu Phe Cys Asp Ile Pro Arg Cys Thr Thr Pro WO 01!8921 PCT/US01104021 Pro Pro Ser Ser Gly Pro Lys Tyr Gln Cys Leu Lys Gly Thr Gly Lys Asn Tyr Gly Gly Thr Val Ala Val Thr Glu Ser Gly His Thr Cys Gln 290 . 295 300 Arg Trp Ser Glu Gln Thr Pro His Lys His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asn Gly Glu Lys Ala Pro Trp Cys Tyr Thr Thr Asn Ser Glu Val Arg Trp Glu Tyr Cya Thr Ile Pro Ser Cys Glu Ser Ser Pro Leu Ser Thr Glu Arg Met Asp Val Pro Val Pro Pro Glu Gln Thr Pro Val Pro Gln Asp Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Arg Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His Arg His Leu Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Ser Pro Trp Cys Tyr Thr Thr Asp Pro Arg Val Arg Trp Glu Phe Cys Asn Leu Lys Lys Cys Ser Glu Thr Pro Glu Gln Val Pro Ala Ala~Pro Gln Ala Pro Gly Val Glu Asn Pro 4?0 475 480 Pro Glu Ala Asp Cys Met Ile Gly Thr Gly Lys Ser Tyr Arg Gly Lys Lys Ala Thr Thr Val Ala Gly Val Pro Cys Gln Glu Trp Ala Ala Gln Glu Pro Hia Gln His Ser I1e Phe Thr Pro G1u Thr Asn Pro Gln Ser Gly Leu Glu Arg Asn Tyr Cys Arg Asn Pro_Asp Gly Asp Val Asn Gly Pro Trp Cys Tyr Thr.Met Asn Pro Arg Lys Pro Phe Asp Tyr Cys Asp Val Pro Gln Cys Glu Ser Ser Phe Asp Cys Gly Lys Pro Lys Val Glu Pro Lys Lys Cys Ser Gly Arg Ile Val Gly Gly Cys Val Ser Lys Pro His Ser Trp Pro Trp Gln Va1 Ser Leu Arg Arg Ser Ser Arg His Phe Cys Gly Gly Thr Leu Ile Ser Pro Lys Trp Val Leu Thr Ala Ala His Cys Leu Asp Asn Ile Leu A1a Leu Ser Phe Tyr Lys Val Ile Leu Gly Ala His Asn Glu Lys Val Arg Glu Gln Ser Val Gln Glu Ile Pro Val Ser Arg Leu Phe Arg Glu Pro Ser Gln Ala Asp Ile Ala Leu Leu Lys Leu Ser Arg Pro Ala Ile Ile Thr Lys Glu Val Ile Pro Ala Cys Leu Pro Pro Pro Asn Tyr Met Val Ala Ala Arg Thr Glu Cys Tyr Ile Thr Gly Trp Gly G1u Thr Gln Gly Thr Phe Gly Glu G3.y Leu Leu Lys Glu Ala His Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Asn Glu Tyr Leu Asp Gly Arg Val Lys Pro Thr Glu Leu Cys Ala Gly His Leu Ile Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu WO Ol/~8921 PCT/iJS01/04021 Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Pro Tyr Val Pro Trp Ile Glu Glu Thr Met Arg Arg Asn <220> 8 <211> 333 <212> PFtT
<213> Canis familiaris <400> 8 Ala Ser Asp Cys Met Phe G1y Asn Gly Lys Gly Tyr Arg Gly Lys Lys Ala Thr Thr Val Met Gly Ile Pro Cys Gln Glu Trp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Gln Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Asn Gly Pro.
50 55 ' 60 Trp Cys Tyr Thr Met Asn Gln Arg Lys Leu Phe Asp Tyr Cys Asp Val pro Gln Cys Val Ser Thr Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly G'ys Val Ala Asn Fro His Ser Trp Pro Trp Gln Ile Ser Leu Arg Thr Arg Tyr Gly Lys His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Arg Ser Ser Arg Pro Ala Ser Tyr Lys Val Iie Leu Gly Ala His Lys Glu Val Asn Leu Glu Ser Asp Val Gln Glu Ile Glu Val Tyr Lys Leu Phe Leu Glu Pro Thr Arg Ala Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Ser Lys Val Ile Pro Ala Cys Leu Pro Pro Pro Asn.Tyr Val Val Ala Asp Arg Thr Leu Cys Tyr Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Tyr Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val G'ys Asn Arg Tyr Glu 245 250 __ . 255.
Tyr Leu Asn Gly Arg Val Lys Ser Thr Glu Leu'Cys Ala Gly Asn Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Ile Met Arg Asn Asn <210> 9 <211> 809 <212> PRT
<213> Erinaceus europaeus <400> 9 Met Gln Arg Lys G1u Leu Val Leu Leu Phe Leu Leu Phe Leu Gln Pro Gly His Gly Ile Pro Leu Asg Asp Tyr Val Thr T,hr Gln Gly Ala Ser Leu Cys Ser Ser Thr Lys Lys Gln Leu Ser Val Gly Ser Thr Glu Glu . 35 - 40 45 Cys Ala Val Lys G'ys Glu Lys Glu Thr Ser Phe Ile Cys Arg Ser Phe WO O1h8921 PCT/USO1/04021 Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Ser Lys Ser Thr Pro Val Leu Arg Met Arg Asp Val Ile Leu Phe Glu Lys Lys Met Tyr Leu Ser Glu Cys Lys Val Gly Asn Gly Lys Tyr Tyr Arg Gly Thr Va1 Ser Lys Thr Lys Thr Gly Leu Thr Cys Gln Lys Trp Ser Ala Glu Thr Pro His Lys Pro Arg Phe Ser Pro Asp Glu Asn Pro Ser Glu Gly Leu Asp Gln Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Lys Gly Pro Trp Cys Tyr Thr Met Asp Pro Glu Val Arg Tyr Glu Tyr Cys Glu Ile Ile Gln Cys Glu Asp Glu Cys Met His Cys Ser Gly Gln Asn 180 185 . 190 Tyr Val Gly Lys Ile Ser Arg Thr Met Ser Gly Leu Glu Cys Gln Pro 1g5 200 205 Trp Asp Ser Gln Ile Pro His Pro His Gly Phe Ile Pro Ser Lys Phe Pro Ser Lys Asn Leu Lys Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Fro Arg Pro Trp G'ys Phe Thr Met Asp Arg Asn Lys Arg Trp Glu Tyr 245 250 . 255 Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro.Pro Pro Ser Gly Fro Thr Tyr Gln Cys Leu Met Gly Asn Gly Glu His Tyr Gln Gly Asn Val Ala Val Thr Val Ser Gly Leu Thr Cys Gln Arg Trp Gly Glu Gln Ser Pro 290 295 ~ 300 His Arg His Asp Arg Thr Pro Glu Asn Tyr Pro Cys Lys Asn Leu Asp WO O1/~8921 PCT/USO1/04021 Glu Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro Ala Pro Trp Cys Phe Thr Thr Asn Ser-Ser Val Arg Trp Glu Phe Cys Lys I1e Pro Asp Cys Val Ser Ser Ala Ser Glu Thr Glu His Ser Asp,Ala Pro Val Ile Val Pro Pro Glu Gln Thr Pro Val Val Gln Glu Cys Tyr Gln Gly Asn Gly Gln Thr Tyr Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Lys Lys Cys Gln Pro Trp Thr Ser Met Arg Pro His Arg His Ser Lys Thr Pro Glu Asn Tyr Pro Asp Ala Asp Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Gly Asp Lys Gly Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu Phe Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Met Ser Ala Thr Asn Ser Ser Pro Va1 Gln Val Ser Ser Ala Ser Glu Ser Ser Glu Gln Asp Cys Ile Ile Asp Asn Gly Lys Gly Tyr Arg Gly Thr Lys Ala Thr Thr Gly Ala Gly Thr Pro Cys Gln Ala Trp Ala Ala Gln Glu Pro His 500 505 . 510 Arg His.Ser Ile Pha Thr Pro Glu Thr Asn Pro Arg Ala Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Asp Gly Asp Ala Asn Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Phe Asp Tyr Cys Asp Ile Pro His Cys Val Ser Pro Ser Ser Ala Asp G'ys Gly Lys Pro Lys Val Glu Pro 565 5?0 575 WO 01/58921 PCT/iJS01/0~4021 Lys Lys Cys Pro Gly Arg Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Arg Phe Gly Gln His Phe Cys Gly Gly Thr Leu Ile Sex' Pro Glu Trp Val Val Thr Ala Ala His Cys Leu Glu Lys Phe Ser Asn Pro Ala Ile Tyr Lys Val Val Leu Gly Ala His Gln Glu Thr Arg Leu Glu Arg Asp Val Gln Ile Lys Gly Val Thr Lys Met Phe Leu Glu Pro Tyr Arg Ala Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Ile I1e Thr Asp Lys Asp His Pro Ala Cys Leu Pro Asn Ser Asn Tyr Met Val Ala Asp Arg Ser Leu Cys Tyr Ile Thr Gly Trp Gly Glu Thr Lys Gly Thr Tyr Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Lys Val Cys Asn Arg Gln Ser Phe Leu Asn Gly Arg Yal Arg Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Val Asp Ser Cys Gln Gly Asp Sex Gly Gly Pro Leu Val Cys Phe Glu Lys 755 760 ' 765 Asp Arg Tyr Ile Leu Gln Gly Val Thr Ser.Ti~p Gly Leu Gly Cys.Ala 770 ~ ~ 775 780 Arg Leu Thr Arg Pro Gly Val Tyr Val Arg Val Ser Arg Tyr Val Ser Trp Leu Gln Asp Val Met Arg Asn Asn <210> 10 7.3 WO 01/58921 PCT/USO1/0~021 <211> 338 <212> PRT
<213> Equus caballus <400> 10 Val Gln Glu Pro Ser Glu Pro Asp Cys Met Leu Gly Ile Gly Lys Gly Tyr Gln Gly Lys Lys Ala Thr Thr Val Thr Gly Thr Arg Cys Gln Ala Trp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Ala Asn Pro firp Ala Asn Leu Glu Lys Asn Tyr Cys Arg Asn Fro Asp Gly Asp Val Asn Gly Pro Trp Cys Tyr Thr Met Asn Pro Gln Lys Leu Phe 65' 70 75 80 Asp Tyr Cys Asp Val Pro Gln Cys Glu Ser Ser Pro Phe Asp Cys Gly Lys Pro Lys Val Glu Pro Lys Lys Cys Ser Gly Arg Ile Val Gly Gly 100 ~ 105 110 Cys Val Ala Ile Ala His Ser Trp Pro Trp Gln Ile Ser Leu Arg Thr Arg Phe Gly Arg His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Arg Ser Ser Arg Pro Ser Thr Tyr Lys Val Val Leu Gly Thr His His Glu Leu Arg~Leu Ala Ala Gly Ala Gln Gln Ile Asp Val Ser Lys Leu Fhe Leu Glu Pro Ser Arg Ala Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Ile Ile Thr Gln Asn Val Ile Pro AIa Cys Leu Pro Pro Ala Asp Tyr Val Val Ala Asn Trp Ala Glu Cys Phe Val Thr Gly Trp Gly Glu Thr Gln Asp Ser Sex Asn WO Ol/~8921 PCT/USO1/04021 Ala Gly Val Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Tyr Leu Asn Gly Arg Val Lys Ser Thr Glu Leu.

Cys Ala Gly His Leu Val Gly Gly Val Asp Ser Cys Gln Gly Asp Ser ' Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Ser Phe Ile Asn Trp Ile Glu Arg Ile Met Gln Ser Asn <210> 11 <211> 810 <212> PRT
<213> Macaca mulatta <400> 11 Mat Glu His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser Gly Gln Gly Glu Pro Leu Asp Asp Tyr Val Asn Thr Lys G1y Ala Ser Leu Phe Ser Ile Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu 35 40 ' 45 Cys Ala Ala Lys Cys Glu Glu Glu Glu Glu Phe Thr Cys Arg Ser Phe Gln Tyr His Ser Lys G1u Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser Ile Val Phe Arg Met Arg Asp Val Val Leu Phe Glu Lys WO Ol/~8921 PCT/USO1/0~1021 Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Sex Lys Thr Arg Thr Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro Thr Phe Ser Pro Ala Thr His Pro Ser 130 135 ~ 140 Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Gly Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Glu Arg Phe Asp Tyr Cys 165 170 1~5 Asp Ile Pro Glu Cys Glu Asp Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Txp Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asp Val Ala Val Thr Val Ser Gly His Thr Cys His Gly Txp Ser Ala Gln Thr Pro 290 . 295 300 His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Glu Lys Ala Pro Trp Cys Tyr Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys WO O1h8921 PCT/USO1/04021 Glu Ser Ser Pro Val Ser Thr Glu Pro Leu Asp Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val Gln Glu Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His Trp His Glu Lys Thr Pro Glu Asn Phe Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr G1u Gly Ser Val Ala Ala Pro Pro Pro Val Ala Gln Leu Pro Asp Ala Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Lys Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Glu Trp Ala Ala Gln Glu Pro His Ser His Arg Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Phe Asp Tyr Cys Asp Val Pro Gln Cys 545 550 555 ' S60 Ala Ala Ser Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala Tyr Pro His Ser Trp Pro Trp Gln Ile Ser Leu Arg Thr Arg Leu Gly Met His Phe Cys Gly WO O1h8921 PCT/USO1/04021 Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Ser Arg Pro Ser Phe Tyr.Lys Val Ile Leu Gly Ala His Arg Glu Val His Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Lys Met Phe Ser Glu Pro Ala Arg Ala Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Ile Ile Thr Asp Lys Val Ile Pro Ala Gys Leu Pro Ser Pro Asn-Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Tyr Gly Ala Gly Leu Leu Lys Glu Ala Arg Leu Pro Vai Ile Glu Asn Lys Val Cys Aan Arg Tyr Glu Phe Leu Asn Gly Thr Val Lys Thr Thr Glu Leu Cys Ala Gly His Leu Ala Gly G1y . 740 745 750 Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Va1 Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn <210> 12 <211> 812 <212> PRT
<213> Mus musculus <400> 12 Met Asp His Lys Glu Val Ile Leu Leu Phe Leu Leu Leu Leu Lys Pro WO 01/58921 PCT/iTSOlIO=1021 Gly Gln Gly Asp Ser Leu Asp Gly Tyr Ile Ser Thr Gln Gly Ala Ser Leu Phe Ser Leu Thr Lys Lys Gln Leu Ala Ala Gly Gly Val Ser Asp Cys Leu A1a Lys Cys Glu Gly Glu Thr Asp Phe Val Cys Arg Ser Phe G1n Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Ser Lys Thr Ser Ser Ile Tle Arg Met Arg Asp Val Ile Leu Phe Glu Lys Arg Val Tyr Leu Ser Glu Cys Lys Thr Gly Ile Gly Asn Gly Tyr Arg 10p 105 110 Gly Thr Met Ser Arg Thr Lys Ser Gly Val Ala Cys Gln Lys Trp Gly Ala Thr Phe Pro His Val Pro Asn Tyr Sex Pro Ser Thr His Pro Asn Glu Gly Leu Glu Glu Asn Tyr~Cys Arg Asn Pro Asp Asn Asp Glu Gln Gly Pro Trp G'ys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asn Ile Pro Glu Cys Glu Glu Glu Cys Met Tyr Cys Ser Gly Glu Lys Tyr Glu G1y Lys Ile Ser Lys Thr Met Ser Gly Leu Asp Cys Gln Ala 195 200 .-205 Trp Asp Sex Gln Ser Pro His Ala His Gly Tyr Ile Pro Ala Lys Phe Pro Ser Lys Asn Leu Lys Met Asn Tyr Cys His Asn Pro Asp Gly Glu Pro Arg Pro Trp Gds Phe Thr Thr Asp Pro Thr Lys Arg Trp Glu Tyr Gars Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Pro Pro Ser Pro Thr WO O1/~8921 PCT/USO1/04021 Tyr Gln Cys Leu Lys Gly Arg Gly Glu Asn Tyr Arg Gly Thr Val Ser Val Thr Val Ser Gly Lys Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro His Arg His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp G1y Glu Thr Ala Pro Trp Cys Tyr Thr Thr Asg Ser Gln Leu Arg Trp Glu Tyr Cys Glu Ile Pro Ser Cys Glu Ser Ser Ala Ser Pro Asp G1n Ser Asp Ser Ser Val Pro Pro Glu C..u Gln Thr Pro Val Val Gln Glu Cys Tyr Gln Ser Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Lys Lys Cys Gln Ser Trp Ala Ala Met Phe Pro His Arg His Ser Lys Thr Pro Glu Asn Phe Pro Asp Ala Gly Leu Glu Met Asn Tyr Cys Arg Asn Pro Asp Gly Asp Lys Gly Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Arg Cys Ser Glu Thr Gly Gly Ser Val Val Glu Leu 450 455 ~ 460 Pro Thr Val Ser Gln Glu Pro Ser G1y Pro Ser Asp Ser Glu Thr.Asp 465 470 . 475 480 Cys Met Tyr Gly Asn Gly Lys Asp Tyr Arg Gly Lys Thr Ala Val Thr Ala Ala Gly Thr Pro Cys Gln Gly Txp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Gln Thr Asn Pro Arg Ala Asp Leu Giu Lys WO 01/58921 PCT/USO1/0~021 Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Asn Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Ile Pro Leu Cys Ala Ser Ala Ser Ser Phe Glu Cys Gly Lys Pro Gln Val Glu Pro Lys 565 ~ 570 575 Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala Asn Pro His Ser Trp Pro Trp Gln Ile Ser Leu Arg Thr Arg Phe Thr Gly Gln His Phe Cys Gly Gly Thr Leu Ile Ala Pro.Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Ser Arg Pro Glu Phe Tyr Lys Val Ile Leu Gly Ala His Glu Glu Tyr Ile Arg Gly Leu Asp Val Gln Glu Ile Ser Val Ala Lys Leu Ile Leu Glu Pro Asn Asn Arg Asp Ile Ala Leu Leu Lys Leu Ser Arg Pro Ala Thr Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Met Val Ala Asp Arg Thr Ile Cys Tyr Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Arg Leu Lys Glu 705 710 7I5 . 720 Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Val Glu Tyr Leu Asn Asn Arg Val Lys Ser Thr Glu Leu Cys Ala Gly Gln Leu Ala Gly GIy Val Asp Ser Gars Gln Gly Asp Ser Gly Gly Fro Leu Val Cys Phe Giu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Txp Gly Leu WO Ol/~8921 PCT/USO1/04021 Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Asp Trp Ile Glu Arg Glu Met Arg Asn Asn 805 ~ 810 <210> 13 <211> 790 <212> PRT
<213> Sus scrofa <400> 13 Asp Sex Leu Asp Asp.Tyr Val Asn Thr Gln G1y Ala Phe Leu Fhe Ser Leu Ser Arg Lys Gln Val Ala Ala Arg Ser Val Glu Glu Cys Ala Ala Lys Cys Glu Ala Glu Thr Asn Phe Ile Cys Arg Ala Phe Gln Tyr His Ser Lys Asp Gln Gln Cys Val Val Met Ala Glu Asn Sex Lys Thr Ser Pro Ile Ala Arg Met Arg Asp Val Val Leu Phe Glu Lys Arg Ile Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Thr Ser Lys Thr Lys Ser Gly Val Ile Cys Gln Lys Trp Ser Val Ser Ser Pro His Ile Pro Lys Tyr Ser Pro Glu Lys Phe Pro Leu Ala Gly Leu Glu Glu Asn~Tyr Cys Arg Asn Pro Asp Asn Asp Glu Lys Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Thr Arg Phe Asp Tyr Cys Asp Ile Pro Glu Cys Glu Asp Glu Cys Met His Cys Ser Gly Glu His Tyr Glu Gly Lys Ile Ser Lys Thr Met Ser Gly Ile Glu Cys Gln Ser Trp Gly Ser WO 01/58921 PCT/USO1/0~021 Gln Ser Pro His Ala His Gly Tyr Leu Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys biet Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Phe G'ys Asp Ile ' Pro Arg Cys Thr Thr Pro Pro Fro Thr Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Arg Gly Glu Asn Tyr Arg Gly Thr Val Ser Val Thr Ala Ser Gly His Thr Cys Gln Arg Trp Ser Ala Gln Ser Pro His Lys His 275 280 . 285 Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr Thr Asp 305 310 . ~ 315' 320 Sex Glu Val Arg Trp Asp Tyr Cys Lys Ile Pro Ser Cys Gly Ser Ser Thr Thr Ser Thr Glu His Leu Asp Ala Pro Val Pro Pro Glu Gln Thr Pro Val Ala Gln Asp Cys Tyr Arg Gly Asn Gly Glu Ser Tyr Arg Gly Thr Ser Sex Thr Thr Ile Thr Gly Arg Lys Cys Gln~Ser Trp Val Ser 370. 375 380 Met,Thr Pro His Arg His Glu Lys Thr Pro Gly Asn Phe Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Ser Pro 405 ~ 410 415 Trp Cys Tyr Thr Thr Asp Pro Arg Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Glu Thr Glu Gln Gln Val Thr Asn Phe Fro Ala Ile WO 01/58921 PCT/USOl/0~021 Ala Gln Val Pro Ser Val Glu Asp Leu Ser Glu Asp Cys Met Phe Gly Asn Gly Lys Arg Tyr Arg Gly Lys Arg Ala Thr Thx Val Ala Gly Val Pro Cys Gln Glu Trp Ala Ala Gln Glu Pro His Arg His Ser Tle Phe 485 490 '195 Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Asp Asn Gly Pro Trp Cys Tyr Thr Thr Asn Pro Gln Lys Leu Phe Asp Tyr Cys Asp Val Pro Gln Cys Val Thr Ser Ser Phe Asp Cys Gly Lys Pro Lys Val Glu Pro Lys Lys Cys Pro Ala Arg Val Val Gly Gly Cys Val Ser Ile Pro His Sex Trp Pro Trp Gln Ile, 565 570 5,75 Ser Leu Arg Tyr Arg Tyr Arg Gly His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Lys His Cys Leu Glu Lys Ser Ser Ser Pro Ser Ser Tyr Lys Val Ile Leu °~ly Ala His G1u Glu Tyr His Leu Gly Glu Gly Val Gln Glu Ile Asp Val Ser Lys~Leu Phe Lys Glu Pro~Ser Glu Ala Asp Ile A1a Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Va1 Ile Pro Ala Cys Leu Pro Thr Pro Asn Tyr Val 660 665 670 , Val Ala Asp Arg Thr Ala Cys Tyr Ile Thr Gly Trp Gly Glu Thr Lys 675 680 . 685 Gly Thr Tyr Gly Ala Gly Leu Leu Lys Glu Ala Arg Leu Pro Val Ile WO O1/~8921 PCT/LTSO1/0-1021 Glu Asn Lys Val Cys Asn Arg Tyr Glu Tyr Leu Gly Gly Lys Val Ser Pro Asn GIu Leu Cys Ala Gly His Leu Ala Gly Gly Ile Asp Ser Cys 725 730 ~ 735 Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val'Thr Ser Trp Gly Leu Gly Cys Ala Leu Pro Asn Lys Pro Gly Val Tyr Val Arg Va1 Ser Arg Phe Val Thr Trp Ile Glu Glu Ile Met Arg Arg Asn <210> 14 <211> 10 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthetic, hypothetical <400> 14 hawaaugucu 10 <210> 15 <211> 4 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Seqnence:synthetie, substrate <400> 15 Ala Ala Pro Val WO Ol/~8921 PCT/USO1/04021 <210> 16 <211> 4 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthetic, substrate <400> 16 Ala Ala Pro Ala <210> 17 <211> 18 <212> PRT
<213> Homo sapiens <400> 17 Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr fihr Asn Pro Arg

Claims (58)

WE CLAIM:

1. A method of generating angiostatin in vitro comprising contacting plasminogen with a plasminogen activator and a sulfhydryl donor.

2. The method of Claim 1 wherein the plasminogen activator is selected from the group consisting of urokinase, streptokinase, and tissue plasminogen activator.

3. The method of Claim 1 wherein the sulfhydryl donor is selected from the group consisting of cysteine, N-acetyl cysteine, captopril, D-penicillamine, and reduced glutathione.

4. The method of Claim 1 wherein the angiostatin is at least partially purified from the reaction mixture.

5. The method of Claim 1 further comprising administering an effective amount of the angiostatin to an animal in need thereof.

6. The method of Claim 4 further comprising administering an effective amount of the angiostatin to an animal in need thereof.

7. A method of generating angiostatin in vitro comprising:
contacting plasminogen with a plasminogen activator to produce plasmin; and contacting the plasmin with a sulfhydryl donor to produce the angiostatin.

8. The method of Claim 7 wherein the plasminogen activator is selected from the group consisting of urokinase, streptokinase, and tissue plasminogen activator.

9. The method of Claim 7 wherein the sulfhydryl donor is selected from the group consisting of cysteine, N-acetyl cysteine, captopril, D-penicillamine, and reduced glutathione.

10. The method of Claim 7 wherein the plasmin is at least partially purified prior to contacting it with the sulfhydryl donor.

11. The method of Claim 7 wherein the angiostatin is at least partially purified from the reaction mixture.

12. The method of Claim 7 further comprising administering an effective amount of the angiostatin to an animal in need thereof.

13. The method of Claim 11 further comprising administering an effective amount of the angiostatin to an animal in need thereof.

14. A method of generating angiostatin in vitro comprising contacting plasmin with a sulfhydryl donor.

15. The method of Claim 14 wherein the sulfhydryl donor is selected from the group consisting of cysteine, N-acetyl cysteine, captopril, D-penicillamine, and reduced glutathione.

16. The method of Claim 14 wherein the angiostatin is at least partially purified from the reaction mixture.

17. The method of Claim 14 further comprising administering an effective amount of the angiostatin to an animal in need thereof.

18. The method of Claim 16 further comprising administering an effective amount of the angiostatin to an animal in need thereof.

19. A method of treating an angiogenic disease comprising administering to an animal suffering from such a disease an amount of a sulfhydryl donor effective to cause the conversion plasmin to angiostatin.

20. The method of Claim 19 wherein the sulfhydryl donor is selected from the group consisting of cysteine, N-acetyl cysteine, captopril, D-penicillamine and reduced glutathione.

21. The method of Claim 19 wherein an effective amount of plasmin is also administered to the animal.

22. The method of Claim 19 further comprising administering an effective amount of a plasminogen activator to the animal to convert plasminogen to plasmin.

23. The method of Claim 22 wherein the plasminogen activator is selected from the group consisting of urokinase,streptokinase and tissue plasminogen activator.

24. The method of Claim 22 wherein an effective amount of plasminogen is also administered to the animal.

25. A composition for generating angiostatin comprising a sulfhydryl donor and a plasminogen activator.

26. The composition of Claim 25 wherein the sulfhydryl donor is selected from the group consisting of cysteine, N-acetyl cysteine, captopril, D-penicillamine and reduced glutathione.

27. The composition of Claim 25 wherein the plasminogen activator is selected from the group consisting of urokinase, streptokinase and tissue plasminogen activator.

28. The composition of Claim 25 which is a conditioned culture medium produced by culturing cells capable of producing plasminogen activator in a culture medium or is a lysate of such cells.

29. A container holding a plasminogen activator, said container having a label thereon instructing administration of the plasminogen activator to an animal suffering from an angiogenic disease.

30. The container of Claim 29 further holding a sulfhydryl donor and said label on said container instructing administration of the combination of the sulfhydryl donor and plasminogen activator to an animal suffering from an angiogenic disease.

31. A container holding a sulfhydryl donor, said container having a label thereon instructing administration of the sulfhydryl donor to an animal suffering from an angiogenic disease in an amount effective to cause conversion of plasmin to angiostatin.

32. A method of generating angiostatin comprising:
culturing cells capable of producing plasminogen activator in a culture medium for a time sufficient to produce conditioned culture medium (CCM) capable of converting plasminogen into angiostatin; and contacting the CCM with plasminogen to produce the angiostatin.

33. The method of Claim 32 wherein the cells are selected from the group consisting of cancer cells, primary endothelial cells, smooth muscle cells and fibroblasts.

34. The method of Claim 32 wherein the angiostatin is at least partially purified from the CCM.

35. The method of Claim 32 further comprising administering the angiostatin to an animal in need thereof.

36. The method of Claim 34 further comprising administering the angiostatin to an animal in need thereof.

37. A method of generating angiostatin comprising:
culturing and thereafter lysing cells capable of producing plasminogen activator; and contacting the lysate with plasminogen to produce the angiostatin.

38. A protein having the following characteristics:
(a) it is a fragment of plasminogen;
(b) its N-terminal amino acid is the same as the N-terminal amino acid of plasmin;
(c) its C-terminal amino acid is in kringle 5; and (d) it inhibits angiogenesis.

39. The protein of Claim 38 which comprises at least 50% of kringle 5.

40. The protein of Claim 39 which comprises at least 75% of kringle 5.

41. The protein of Claim 38 which is a fragment of human plasminogen and which has the following additional characteristic:
(e) it has an approximate molecular weight of 50-60 kD on polyacrylamide gel electropheresis under non-reducing conditions.

42. The protein of Claim 41 having the following additional characteristics:
(f) it has the N-terminal sequence:
Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly [SEQ ID NO:1]; and (g) it has the C-terminal sequence:
Cys Tyr Thr Thr Asn Pro Arg [SEQ ID NO:4]; or Cys Tyr Thr Thr Asn Pro Arg Lys [SEQ ID NO:5].

43. A DNA molecule comprising a sequence which codes for the protein of any one of Claims 38-42.

44. The DNA molecule of Claim 43 wherein the coding sequence is operatively linked to expression control sequences.

45. A host cell comprising the DNA molecule of Claim 44.

46. A method of producing a plasminogen fragment which inhibits angiogenesis comprising culturing the host cell of Claim 45.

47. An antibody which binds selectively to native angiostatin.

48. A method of detecting or quantitating native angiostatin in a material suspected of containing native angiostatin, the method comprising:
contacting the material with the antibody of Claim 47; and detecting or quantitating any native angiostatin present in the material.

49. A kit for detecting or quantitating native angiostatin comprising a container holding the antibody of Claim 47.

50. An antibody which binds selectively to the protein of Claim 38.

51. A method of purifying a protein of Claim 38 from a material containing it, the method comprising:
contacting the material with the antibody of Claim 50 so that the antibody binds to the protein; and separating the protein bound to the antibody from the remainder of the material.

52. A method of treating an angiogenic disease comprising administering to an animal suffering from such a disease an effective amount of the protein of any one of Claims 38-42.

53. The method of Claim 52 wherein the protein is native angiostatin.

54. A method of treating an angiogenic disease comprising administering to an animal suffering from such a disease a transgene comprising DNA coding for the protein of Claim 38 operatively linked to expression control sequences.

55. The method of Claim 54 wherein the protein coded for by the transgene is native angiostatin.

56. A method of treating an angiogenic disease comprising administering to an animal suffering from such a disease an amount of a plasminogen activator effective to cause the conversion plasminogen to plasmin.

57. The method of Claim 56 wherein an effective amount of plasminogen is also administered to the animal.

58. The method of Claim 56 wherein the plasminogen activator is selected from the group consisting of urokinase, streptokinase and tissue plasminogen activator.