USOO6592865B2 (12) United States Patent (10) Patent No.: US 6,592,865 B2 Parry et al. (45) Date of Patent: Jul. 15, 2003
(54) METHODS AND COMPOSITIONS FOR Drummer et al., Effects of Chronic Enalapril Treatment on MODULATING ACE-2 ACTIVITY Enzymes Resposible for the Catabolism of Angiotensin I and Formation of Angiotensin II, Biochemical Pharmacol (75) Inventors: Tom J. Parry, Walkersville, MD (US); ogy, 39(3):513–518 (1990). Les Sekut, Ijamsville, MD (US) Ito et al., “Pharmacological profile of depressor response elicited by Sarthran in rat ventrolateral medulla, Am. J. (73) Assignee: Human Genome Sciences, Inc., Physiol. Heart Circ. Physiol., 279:H2961-H2966 (2000). Rockville, MD (US) Johnson et al., “Radioimmunoassay for Immunoreactive (*) Notice: Subject to any disclaimer, the term of this des-Leu'HAngiotensin I," Peptides, 10:489–492 (1989). patent is extended or adjusted under 35 Lembeck et al., “Demonstration of extrapulmonary activity U.S.C. 154(b) by 0 days. of angiotensin converting enzyme in intact tissue prepara tions.” Br. J. Pharmacol., 100:49-54 (1990). (21) Appl. No.: 10/158,847 Miano et al., “Different modulation of aromatase activity in frog testis in vitro by ACE and ANG II,” Am. J. Physiol., 277 (22) Filed: Jun. 3, 2002 (Regulatory Integrative Comp. Physiol. 46):R1261-R1267 (65) Prior Publication Data (1999). Oparil et al., “Mechanism of Pulmonary Conversion of US 2003/009 1557 A1 May 15, 2003 Angiotensin I to Angiotensin II in the Dog,” Circ. Res., Related U.S. Application Data 29:682-690 (Dec. 1971). (60) Provisional application No. 60/295,004, filed on Jun. 4, Sibinga et al., “A Pair of ACES for Openers’?,’ Circ. Res., 2001. 87:523-525 (2000). Snyder et al., “Inhibition of angiotensin-converting enzyme (51) Int. Cl." ...... A61K 38/00; A61K 38/08 (52) U.S. Cl...... 424/94, 514/2; 514/15 by des-Leu"-angiotensin I: a potential mechanism of (58) Field of Search ...... 514/2, 15 endogenous angiotensin-converting enzyme regulation,” Biochimica et Biophysica Acta, 871:1–5 (1996). (56) References Cited Tipnis et al., “A Human Homolog of Angiotensin-convert ing Enzyme,” J. Biol. Chem., 275(43):33238–33243 (Oct. U.S. PATENT DOCUMENTS 27, 2000). 5,444,067 A 8/1995 Kivlighn et al. Turner et al., “The angiotensin-converting enzyme gene 5,508.266 A 4/1996 Fink family: genomics and pharmacology,” TiPS, 23(4): 177-183 6,194,556 B1 2/2001 Acton et al. (Apr. 2002). FOREIGN PATENT DOCUMENTS Turner et al., “ACEH/ACE2 is a novel mammalian metal WO WO 99/52540 A1 10/1999 locarboxypeptidase and a homologue of angiotensin-con WO WO OO/18899 A2 4/2000 verting enzyme insensitive to ACE inhibitors,” Can. J. WO WOOO/O66104 A9 11/2000 Physiol. Pharmacol., 80:346–353 (2002). WO WO O2/12471 A2 2/2002 Vickers et al., “Hydrolysis of Biological Peptides by Human WO WO O2/39997 A2 5/2002 Angiotensin-converting Enzyme-related Carboxypepti OTHER PUBLICATIONS dase,” J. Biol. Chem., 277(17): 14838–14843 (Apr. 26, 2002). Bramucci et al., “Bradykinin is not involved in angiotensin converting enzyme modulation of ovarian Steroidogenesis and prostaglandin production in frog Rana eSculenta, "Acta. Primary Examiner Rebecca E. Prouty Physiol. Scand., 175:123-128 (2002). Assistant Examiner. Sheridan L. Swope Bramucci et al., “Different modulation of Steroidogenesis (74) Attorney, Agent, or Firm-Human Genome Sciences, and prostaglandin production in frog ovary in vitro by ACE Inc. and ANG II,” Am. J. Physiol., 273 (Regulatory Integrative (57) ABSTRACT Comp. Physiol. 42:R2089–R2096 (1997). Dasarathy et al., “Calcium ionophore A23.187 elevates The invention relates to the use of angiotensin II in combi angiotensin-converting enzyme in cultured bovine endothe nation with angiotesin 1-9 to potentiate angiotensin II activ lial cells,” Biochinica et Biophysica Acta, 1010:16-19 ity. The invention also relates to the use of combinations of (1989). angiotensin II and angiotensin 1-9 to increase vasoconstric Donoghue, et al., “A Novel Angiotensin-Covnerting tion and to treat disorders associated with low blood pres Enzyme-Related Carboxypeptidase (ADE2) Converts SUC. Angiotensin I to Angiotensin 1-9, Circ. Res., 87:e1-e9 (2000). 5 Claims, 4 Drawing Sheets U.S. Patent Jul. 15, 2003 Sheet 1 of 4 US 6,592,865 B2
Figure 1
All Effect (experimental) A All + A1-9 Combination
All + A1-9 additivity (predicted regr, line) 20 A Combination Regression Line
15
10
O 0.5 1 15 log Peptide Concentration (nM) U.S. Patent Jul. 15, 2003 Sheet 2 of 4 US 6,592,865 B2
Figure 2
1SO
140
120
100 (ôHuuuu)dVIN
A1-9 All U.S. Patent Jul. 15, 2003 Sheet 3 of 4 US 6,592,865 B2
Figure 3
-O- A 1-9 160 -- A & A 1-9 1ug -0- A
a. 150 s C E 140 5 a 30 d
120 ; : . . Vehicle
110 1. 10 1OO 1000 Angiotensin Il Dose (ng/kg/min) U.S. Patent Jul. 15, 2003 Sheet 4 of 4 US 6,592,865 B2
Figure 4
180
160
140
120
100 -O- inactive Peptide -- ACE2 inhibitor peptide - (0- - Peptide vehicle/inactive cohort 80 A Peptide vehicle/ACE2 inhib. cohort V ACE2 Control for Inactive Cohort
60 O 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Peptide Dose (mg/kg) US 6,592,865 B2 1 2 METHODS AND COMPOSITIONS FOR (1985); Snyder et al., Biochemica et Biophysica Acta MODULATING ACE-2 ACTIVITY 871:1–5 (1986)). Circulating A1-9 has been detected in vivo at levels twice that of angiotensin II (Oparil et al., Circula This application claims benefit under 35 U.S.C. S119(e) tion Research 29 682-690 (1971); Johnson et al., Peptides of U.S. Patent Application No. 60/295,004, filed Jun. 4, 10:489–492) (1989)). In the case of ACE-2, the above 2001, which is hereby incorporated by reference in its reaction is not blocked by captopril, lisinopril or enalaprilat entirety. (Donoghue et al., Circulation Research 87:e1-e9 (2000); Tipinis et al., The Journal of Biological Chemistry FIELD OF THE INVENTION 275:33238-33243 (2000)). The unique expression profile of The present invention relates to polypeptides that regulate ACE-2, Spectum of its enzymatic activity and inhibitory production of Angiotensin 1-9 via Such mechanisms as, for effects of its product A1-9 on ACE have led to the specu example, inhibition of ACE-2. Such polypeptides have uses lation that ACE-2 functions to affect circulatory homeostasis for example, in the detection, isolation, and/or purification by promoting vasodilation (Donoghue et al., Circulation of ACE-2 and/or Angiotensin 1-9. The invention also relates Research 87:e 1-e9 (2000); Snyder et al., The Journal of 15 Biological Chemistry 260:7857-7860 (1985)). However, to nucleic acid molecules encoding these ACE-2 binding A1-9 has been shown to be a weak vasoconstrictor in polypeptides, vectors and host cells containing these nucleic isolated rat aorta and have weak pressor activity in anesthe acids, and methods for producing the Same. The present tized rats and dogs (Oparil et al., Circulation Research invention also relates to methods and compositions for 29:682-690 (1971)). Therefore, we hypothesized that one of detecting, diagnosing, or prognosing a disease or disorder the physiologic roles of ACE-2 is to increase arterial pres asSociated with aberrant ACE-2 or Angiotensin 1-9 expres Sure through the actions of its catabolic product, A1-9. AS Sion or inappropriate function of ACE-2.or Angiotensin 1-9, Such, ACE-2 might be a valid target for drug development in comprising ACE-2 binding polypeptides or fragments or hypertension. variants thereof, that specifically regulate ACE-2 action. The present invention further relates to methods and composi Accordingly, molecules that specifically bind ACE-2 tions for preventing, treating or ameliorating a disease or 25 would find a variety of uses in the Study of ACE-2, angio disorder associated with aberrant ACE-2 or Angiotensin 1-9 tensin 1-9) (SEQ ID NO:145), and angiotensin, as well as expression or inappropriate ACE-2 function or Angiotensin ACE, and its known substrates: Angiotensin II (SEQ ID 1-9 function, comprising administering to an animal, pref NO: 144), Angiotensin 1-7, des-Asp, bradykinin, erably a human, an effective amount of one or more ACE-2 neurotensin, and Substance P. Further, molecules that Spe binding polypeptides or fragments or variants thereof, that cifically bind ACE-2 would also find a variety of uses in the Specifically regulate ACE-2. manufacture and purification of ACE-2, ACE, angiotensin, angiotensin II, and/or Angiotensin 1-9 in commercial and BACKGROUND OF THE INVENTION medically pure quantities, and in the development new therapeutic or diagnostic reagents. ACE-2 binding polypep The renin-angiotensin System (RAS) plays an important 35 tides may also find medical utility in, for example, the role in circulatory homeostasis at both Systemic and local treatment of cardiovascular disorders (e.g., hypertension, levels. Angiotensin converting enzyme (ACE), a 175 kD chronic heart failure, left ventricular failure, Stroke, cerebral protein known to be widely distributed throughout the vasospasm after Subarachnoid injury, atherOSclerotic heart cardiovascular System, has been long recognized as the key disease, and retinal hemorrhage), renal disorders (e.g., renal enzyme in the generation of angiotensin II, a peptide that 40 vein thrombosis, kidney infarction, renal artery embolism, regulates fluid balance, blood pressure and local blood flow renal artery Stenosis, myocardial hypertrophy, hypertrophy in a number of tissues (Peach, M. J. Physiological Reviews and/or hyperplasia of conduit and/or resistance vessels, 57:313-370 (1997)). As part of an ongoing strategy to myocyte hypertrophy, and fibroblast proliferative diseases), establish genes associated with cardiovascular function via inflammatory diseases (e.g., SIRS (systemic Inflammatory high throughput cDNA sequencing, we identified a member 45 Response Syndromes), Sepsis, polytrauma, inflammatory of the RAS family of enzymes, ACE-2, from human kidney. bowl disease, acute and chronic pain, rheumatoid arthritis, This enzyme was also identified in a variety of tissues by and osteoarthritis), allergic disorders (e.g., asthma, adult others (Donoghue et al., Circulation Research 87:e1-e9 respiratory distress Syndrome, wound healing, and Scar (2000), Tipinis et al., The Journal of Biological Chemistry formation), as well as several other disorders and/or diseases 275:33238-33243 (2000)). The unmodified ACE-2 protein 50 (e.g., periodontal disease, dysmenorrhea, premature labor, contains transmembrane and Signal peptide domains, but brain edema following focal injury, diffuse axonal injury, unlike ACE, ACE-2 contains just one single extracellular Zn' binding metalloprotease domain (Tipinis et al., The and reperfusion injury). Journal of Biological Chemistry 275:33238–33243 (2000)). SUMMARY OF THE INVENTION ACE-2 mRNA has a more limited expression pattern than 55 The present invention provides new polypeptides and ACE (Donoghue et al., Circulation Research 87:e1-e9 families of polypeptides that specifically bind ACE-2 and/or (2000)) and, remarkably, no detectable expression in lungs ACE-2-like polypeptides. In particular, the invention (unpublished data). encompasses polypeptides that Specifically bind to a ACE-2 and related carboxypeptidases (Snyder et al., The polypeptide or polypeptide fragment of human ACE-2 (SEQ Journal of Biological Chemistry 260:7857-7860 (1985); 60 ID NOs: 138 and/or 142). Kokkonen et al., Circulation 95:1455-1463 (1997)) catalyze In particular, the invention relates to ACE-2 binding the removal of the C-terminal leucine from angiotensin I to polypeptides comprising, or alternatively consisting of, an form the nonapeptide angiotensin 1-9 (A1-9) (SEQ ID amino acid Sequence Selected from the group consisting of NO:145) or des-Leu"-angiotensin I (Donoghue et al., Cir SEQ ID NOs: 1-136, preferably SEQ ID NOs: 11-39, more culation Research 87:e1-e9 (2000); Tipinis et al., The Jour 65 preferably SEQ ID NOS:23–24 and 36–39, as referred to nal of Biological Chemistry 260:7857-7860 (2000); Snyder below, in Tables 1-2 and Example 1 below, and fragments et al., The Journal of Biological Chemistry 260: 7857-7860 and variants thereof. US 6,592,865 B2 3 4 In specific preferred embodiments, the ACE-2 binding X is D, E, G, N, R, Q, S, or V (preferably D or E); polypeptides of the invention bind ACE-2 and/or ACE-2- X is N, T, or W (preferably W); like polypeptides with high affinity. In other embodiments, the ACE-2 binding polypeptides of the invention reversibly X is any amino acid except cysteine; bind ACE-2 and/or ACE-2-like polypeptides. In still other X, is any amino acid except cysteine; embodiments, the ACE-2 binding polypeptides of the inven X is F, W, or Y (preferably F); tion irreversibly bind ACE-2 and/or ACE-2-like polypep X is any amino acid except cysteine; tides. Xo is any amino acid except cysteine; The cysteine residues in certain polypeptides according to X is any amino acid except cysteine; the invention are believed to form a disulfide bond, which 1O would cause the polypeptide containing these cysteine resi X2 is any amino acid except cysteine; dues to form a stable loop Structure under non-reducing X is any amino acid except cysteine; and conditions. Especially preferred ACE-2 binding polypep Z is a polypeptide of at least one amino acid or is absent. tides of the invention are polypeptide molecules that com prise amino acid Sequences that form stable loop Structures 15 or other stable structures that bind ACE-2 or ACE-2-like polypeptides. wherein, Z is a polypeptide of at least one amino acid or is absent; Preferred binding polypeptides specific for ACE-2 include two separated, invariant cyteine residues and are X is any amino acid except cysteine; thus capable of forming a cyclic Strucure under non X is any amino acid except cysteine; reducing conditions via a disulfide bond formed between the X is C, E, or S; cysteine side chains. Specific ACE-2 binding polypeptides X is K, L, or R (preferably R); according to the present invention include polypeptides comprising amino acid Sequences of the following general Xs is A, R, or S (preferably R); formulae I-X: 25 Z is a polypeptide of at least one amino acid or is absent; and wherein, if X is cysteine (C), then Z contains a C-terminal cysteine residue. wherein, Z is a polypeptide of at least 2 amino acids, X is any amino acid except cysteine; wherein, X is L or M (preferably L); Z is a polypeptide of at least one amino acid or is absent; X is For Y (preferably F); X is any amino acid except cysteine (preferably L, H, or X is F, L, M, or V (preferably V); 35 M); Xs is D or E; X is N or T (preferably T); Z is a polypeptide of at least one amino acid or is absent; X is any amino acid except cystein (preferably D, M, N, and or S); Z contains at least one cysteine residue. Such that forma 40 X is V or I (preferably V); tion of a disulfide bond with the invariant cysteine Z is a polypeptide of at least one amino acid or is absent. residue (C) forms a cyclic peptide of six or ten amino acids. wherein, 45 Z is a polypeptide of at least one amino acid or is absent; wherein, X is D or E; Z is a polypeptide of at least Six amino acids; X is D or E; X is F, M, W, or Y (preferably For Y); Z is a polypeptide of at least two amino acids and X is F, I, L, M, or V (preferably F or L); 50 contains at least one cysteine residue Such that forma X is D, E, or T (preferably D); tion of a disulfide bond with the invariant cysteine X is For M (preferably F); residue (C) forms a cyclic peptide of Seven, eight or Z is a polypeptide of at least one amino acid or is absent; twelve amino acids. and Z contains at least one cysteine residue. Such that forma 55 tion of a disulfide bond with the invariant cysteine wherein, residue (C) forms a cyclic peptide of eight or ten amino Z is a polypeptide of at least one amino acid or is absent; acids. X is W or Y; X is any amino acid except cysteine; Z-X-X-X-C-X-Xs-X-X7-X-Xo-X-X-X-X-C-Z (SEQ 60 ID NO:3) III. X is W or Y; X is any amino acid except cysteine; wherein, Xs is any amino acid except cysteine; Z is a polypeptide of at least one amino acid or is absent; X is any amino acid except cysteine; and X is A, D, F, G, H, L, N, P, or S (preferably D); 65 X is A, D, F, G, H, N, S, W, or Y (preferably D); Z is a polypeptide of at least one amino acid or is absent. X is D, E, H, L, M, or V (preferably D or E); US 6,592,865 B2 wherein, Z is a polypeptide of at least one amino acid or is absent; -continued X is any amino acid except cysteine; SEQ ID NO US 6,592,865 B2 8 according to the present invention is a polypeptide compris -continued ing the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly Lys (SEQ ID NO:146). Most preferably, the affinity chro SEQ ID NO : matography material of the invention comprises an ACE-2 IL H A C R P W R G D P W. W. A C T L G 134 binding polypeptide comprising an amino acid Sequence G F T C S P I R. M. F. P. W. F. R. C. D. L. G 135 selected from the group consisting of SEQ ID NOS:11-136, F S P C. K. A. L R H S P W. W. W C P S G 136 which is linked to a chromatography material by a polypep tide linker molecule having the amino acid Sequence Pro Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO:146). ACE-2 ACE-2 binding polypeptide molecules of the invention binding polypeptides of the invention attached, coupled, may also have an amino terminal (N-terminal) capping or linked or adhered to a matrix or resin or other Solid Support functional group, Such as an acetyl group, which, for are useful for methods of detecting, isolating and purifying example, blocks the amino terminal amino group from ACE-2 and/or ACE-2-like polypeptides as well as Angio undesirable reactions or is useful in linking the ACE-2 tensin 1-9 and/or Angeliotensin 1-9-like polypeptides, par binding polypeptide to another molecule, matrix, resin, or ticularly for purification of ACE-2 and/or ACE-2-like Solid Support. ACE-2 binding polypeptides of the invention 15 polypeptides as well as Angiotensin 1-9 and/or Angeliotensin may also have a carboxy terminal (C-terminal) capping or 1-9-like polypeptides by affinity chromatography. functional group, Such as an amide group, which, for In certain preferred embodiments, the ACE-2 binding example, blocks the C-terminal carboxyl group from unde polypeptides of the present invention or phage displaying Sirable reactions or provides a functional group useful in such binding polypeptides, irreversibly bind the ACE-2 conjugating the binding polypeptide to other molecules, protein in its native form. matrices, resins, or Solid Supports. Preferably, the N- and/or In certain preferred embodiments, the ACE-2 binding C-terminal capping groups are polypeptide linker molecules. polypeptides of the present invention or phage displaying An especially preferred C-terminal linker molecule that is Such binding polypeptides, reversibly bind the ACE-2 pro useful for immobilizing an ACE-2 binding polypeptide of tein in its native form. the invention to a Solid Support or chromatographic matrix 25 In a further embodiment, the present invention encom material comprises the amino acid Sequence Pro-Gly-Pro passes a composition of matter comprising isolated nucleic Glu-Gly-Gly-Gly-Lys (SEQ ID NO:146). acids, preferably DNA, encoding an ACE-2 binding The invention also encompasses ACE-2 binding polypep polypeptide of the invention. In a specific embodiment, tides that have been modified, for example, to increase or nucleic acid molecules of the invention encode an ACE-2 decrease the Stability of the molecule, while retaining the binding polypeptide of the invention as provided in SEQ ID ability to bind ACE-2 and/or ACE-2-like polypeptides. An NOs: 1-136. In additional embodiments, nucleic acid mol example of a modified ACE-2 binding polypeptide of the ecules of the invention encode a polypeptide variant or invention is a polypeptide in which one of two cysteine fragment of a polypeptide comprising an amino acid residues is Substituted with a non-naturally occurring amino sequence of SEQ ID NOs: 1-136. In a further additional acid that is capable of condensing with the remaining 35 embodiment, nucleic acid molecules of the invention encode cysteine Side chain to form a stable thioether bridge, thereby an ACE-2 binding polypeptide, the complementary Strand of generating a cyclic BLyS binding polypeptide. Such cyclic which nucleic acid hybridizes to a polynucleotide Sequence thioether molecules of Synthetic peptides may be routinely encoding a polypeptide described in Tables 1-2 and in generated using techniques known in the art, e.g., as Example 1 (SEQ ID NOs: 1-136), under stringent described in PCT publication WO 97/46251, incorporated 40 conditions, e.g., hybridization to filter-bound DNA in herein by reference. 6xsodium chloride/sodium citrate (SSC) at about 45 C. In another embodiment, the invention provides ACE-2 followed by one or more washes in 0.2xSSC/0.1% SDS at binding polypeptides of the invention attached, coupled, about 50–65 C., under highly Stringent conditions, e.g., linked or adhered to a matrix or resin or Solid Support. hybridization to filter-bound nucleic acid in 6xSSC at about Techniques for attaching, linking or adhering polypeptides 45 45° C. followed by one or more washes in 0.1XSSC/0.2% to matrices, resins and Solid Supports are well known in the SDS at about 68 C., or under other stringent hybridization art. Suitable matrices, resins or Solid Supports for these conditions which are known to those of skill in the art (see, materials may be any composition known in the art to which for example, Ausubel, F. M. et al., eds. , 1989, Current an ACE-2 binding polypeptide of the invention could be Protocols in Molecular Biology, Vol. 1, Green Publishing attached, coupled, linked, or adhered, including but not 50 Associates, Inc. and John Wiley & Sons, Inc., New York at limited to, a chromatographic resin or matrix, Such as pages 6.3.1-6.3.6 and 2.10.3). SEPHAROSE-4 FF agarose beads, the wall or floor of a well The present invention also relates to recombinant vectors, in a plastic microtiter dish, Such as used in an enzyme-liked which include the isolated nucleic acid molecules encoding immunosorbent assay (ELISA), or a silica based biochip. the ACE-2 binding polypeptides of the present invention (as Materials useful as solid supports on which to immobilize 55 well as fragments and variants thereof), and to host cells binding polypeptides of the invention include, but are not containing the recombinant vectors, as well as to methods of limited to, polyacrylamide, agarose, Silica, nitrocellulose, making Such vectors and host cells. The invention further paper, plastic, nylon, metal, and combinations thereof. An provides for the use of Such recombinant vectors in the ACE-2 binding polypeptide of the invention may be immo production of ACE-2 binding polypeptides by recombinant bilized on a matrix, resin or Solid Support material by a 60 techniques. non-covalent association or by covalent bonding, using The ACE-2 binding polypeptides, nucleic acids, trans techniques known in the art. Preferably, an ACE-2 binding formed host cells, and genetically engineered viruses and polypeptide of the invention is immobilized on a chroma phage of the invention (e.g., recombinant phage), have uses tography material such as SEPHAROSE-4 FF agarose. In an that include, but are not limited to, the detection, isolation, even more preferred embodiment, an ACE-2 binding 65 and purification of ACE-2. polypeptide of the invention is coupled to a chromatography In another embodiment of the invention, recombinant material using a linker molecule. A preferred linker molecule bacteriophage displaying ACE-2 binding polypeptides on US 6,592,865 B2 10 their Surfaces are also provided. Such phage may be rou present invention (including molecules comprising, or alter tinely generated using techniques known in the art and are natively consisting of, ACE-2 binding polypeptide frag useful, for example, as Screening reagents and reagents for ments or variants thereof). A composition of the invention detecting ACE-2. may comprise, or alternatively consist of, one, two, three, In another embodiment, an ACE-2 binding polypeptide of four, five, ten, fifteen, twenty, or more amino acid Sequences the invention is used to detect or isolate ACE-2 or ACE-2- of one or more ACE-2 binding polypeptides or fragments or variants thereof. Alternatively, a composition of the inven like polypeptides in a Solution. Such Solutions include, but tion may comprise, or alternatively consist of, nucleic acid are not limited to, ACE-2 or ACE-2-like polypeptides Sus molecules encoding one or more ACE-2 binding polypep pended or dissolved in water or a buffer solution as well as tides of the invention. any fluid and/or cell obtained from an individual, biological The present invention further provides for fusion proteins fluid, body tissue, body cell, cell line, tissue culture, or other comprising an ACE-2 binding polypeptide (including mol source which may contain ACE-2 or ACE-2-like ecules comprising, or alternatively consisting of, ACE-2 polypeptides, Such as, cell culture medium, cell extracts, and binding polypeptide fragments or variants thereof) of the tissue homogenates. Biological fluids include, but are not invention, and a heterologous polypeptide. Nucleic acid limited to, Sera, plasma, lymph, blood, blood fractions, 15 molecules encoding these fusion proteins are also encom urine, Synovial fluid, Spinal fluid, Saliva, and mucous. passed by the invention. A composition of the present In another embodiment, the present invention provides a invention may comprise, or alternatively consist of, one, method for detecting ACE-2 protein and/or ACE-2-like two, three, four, five, ten, fifteen, twenty or more fusion polypeptide in a Solution comprising, contacting the Solution proteins of the invention. Alternatively, a composition of the with an ACE-2 binding polypeptide of the invention and invention may comprise, or alternatively consist of, nucleic detecting binding of ACE-2 or ACE-2-like polypeptide to acid molecules encoding one, two, three, four, five, ten, the ACE-2 binding polypeptide. The ACE-2 binding fifteen, twenty or more fusion proteins of the invention. polypeptide may be either free or immobilized. Preferably, The present invention also encompasses methods and the ACE-2 binding polypeptide is a polypeptide immobi compositions for detecting, diagnosing, prognosing, and/or lized on a Solid Surface or chromatographic material or the 25 monitoring diseases or disorders associated with aberrant well of a plastic microtiter assay dish. ACE-2 or ACE expression or inappropriate ACE-2 or ACE Another embodiment of the present invention is a method receptor function in an animal, preferably a mammal, and for isolating ACE-2 protein and/or an ACE-2-like polypep most preferably a human, comprising, or alternatively con tide from a Solution, comprising: sisting of, use of ACE-2 binding polypeptides (including (a) contacting the Solution with an ACE-2 binding molecules which comprise, or alternatively consist of, polypeptide under conditions that permit binding of the ACE-2 binding polypeptide fragments or variants thereof) ACE-2 and/or ACE-2-like polypeptides to ACE-2 bind that specifically bind ACE-2. Diseases and disorders which ing polypeptides, and can be detected, diagnosed, prognosed and/or monitored (b) recovering the ACE-2 and/or ACE-2-like polypep with the ACE-2 binding polypeptides of the invention tides. 35 include, but are not limited to, cardiovascular disorders (e.g., A further embodiment of the present invention is a hypertension, chronic heart failure, left ventricular failure, method for isolating ACE-2 protein and/or an ACE-2-like Stroke, cerebral vasospasm after Subarachnoid injury, ath polypeptide from a Solution, comprising: erosclerotic heart disease, and retinal hemorrhage), renal disorders (e.g., renal vein thrombosis, kidney infarction, (a) contacting the Solution with an ACE-2 binding 40 renal artery embolism, renal artery Stenosis, and edema, polypeptide under conditions that permit binding of the hydronephritis), proliferative diseases or disorders (e.g., ACE-2 and/or ACE-2-like polypeptides to ACE-2 bind vascular Stenosis, myocardial hypertrophy, hypertrophy and/ ing polypeptides, or hyperplasia of conduit and/or resistance vessels, myocyte (b) separating the complex(es) formed by the ACE-2 hypertrophy, and fibroblast proliferative diseases), inflam binding polypeptide and ACE-2 and/or ACE-2-like 45 matory diseases (e.g., SIRS (Systemic Inflammatory polypeptides from other components of the Solution, Response Syndromes), Sepsis, polytrauma, inflammatory (c) dissociating the ACE-2 binding polypeptide from the bowl disease, acute and chronic pain, rheumatoid arthritis, ACE-2 and/or ACE-2 -like polypeptides, and and osteoarthritis), allergic disorders (e.g., asthma, adult (d) recovering the dissociated ACE-2 and/or ACE-2-like respiratory distress Syndrome, wound healing, and Scar polypeptides. 50 formation), as well as Several other disoders and/or diseases In another embodiment, the invention provides kits con (e.g., periodontal disease, dysmenorrhea, premature labor, taining a binding polypeptide of the invention for use in brain edema following focal injury, diffuse axonal injury, methods of detecting or isolating ACE-2 and/or ACE-2-like and reperfusion injury). polypeptides. In Specific embodiments, the present invention encom The present invention also provides panels of ACE-2 55 passes methods and compositions for detecting, diagnosing, binding polypeptides (including molecules comprising, or prognosing and/or monitoring diseases or disorders for alternatively consisting of, ACE-2 binding polypeptide frag preventing, treating and/or ameliorating diseases or disor ments or variants) wherein the panel members correspond to ders associated with hypertension (e.g., accelerated one, two, three, four, five, ten, fifteen, twenty, or more hypertension, renal failure, vascular accidents, myocaridal different ACE-2 binding polypeptides of the invention. The 60 infarction, and stroke). present invention further provides mixtures of ACE-2 bind In other specific embodiments, the present invention ing polypeptides, wherein the mixture corresponds to one, encompasses methods and compositions for detecting, two, three, four, five, ten, fifteen, twenty, or more different diagnosing, prognosing and/or monitoring diseases or dis ACE-2 binding polypeptides of the invention. The present orders associated with hypotension (e.g., shock, intracranial invention also provides for compositions comprising, or 65 hypotension, and Syncope). alternatively consisting of, one, two, three, four, five, ten, The present invention further encompasses methods and fifteen, twenty, or more ACE-2 binding polypeptides of the compositions for preventing, treating and/or ameliorating US 6,592,865 B2 11 12 diseases or disorders associated with aberrant ACE-2 or effective amount of ACE-2 binding polypeptide, wherein the ACE expression or inappropriate ACE-2 or ACE function in effective amount of ACE-2 binding polypeptide inhibits or an animal, preferably a mammal, and most preferably a reduces ACE-2 mediated enzymatic action. human, comprising, or alternatively consisting of, adminis The present invention further encompasses methods and tering to an animal in which Such treatment, prevention or compositions for inhibiting or reducing pain, comprising, or amelioration is desired one or more ACE-2 binding polypep alternatively consisting of, administering to an animal in tides (including molecules which comprise, or alternatively which Such inhibition or reduction is desired, an ACE-2 consist of, ACE-2 binding polypeptide fragments or variants binding polypeptide in an amount effective to inhibit or thereof) in an amount effective to treat, prevent or ameliorate reduce ACE-2 enzymatic activity. the disease or disorder. Diseases and disorders which can be The present invention further encompasses methods and prevented, treated, and/or ameliorated with the ACE-2 bind compositions for inhibiting or reducing inflammatory reac ing polypeptides of the invention include, but are not limited tions in various tissues comprising, or alternatively consist to, cardiovascular disorders (e.g., hypertension, chronic ing of, contacting an effective amount of ACE-2 binding heart failure, left ventricular failure, Stroke, cerebral vasos polypeptide, wherein the effective amount of ACE-2 binding pasm after Subarachnoid injury, atherosclerotic heart 15 polypeptide inhibits or reduces ACE-2 enzymatic activity. disease, and retinal hemorrhage), renal disorders (e.g., renal In a Specific embodiment, the present invention encom vein thrombosis, kidney infarction, renal artery embolism, passes methods and compositions for inhibiting or reducing renal artery Stenosis, and edema, hydronephritis), prolifera inflammatory reactions in Smooth muscle tissues tive diseases or disorders (e.g., vascular Stenosis, myocardial comprising, or alternatively consisting of, contacting an hypertrophy, hypertrophy and/or hyperplasia of conduit and/ effective amount of ACE-2 binding polypeptide, wherein the or resistance vessels, myocyte hypertrophy, and fibroblast effective amount of ACE-2 binding polypeptide inhibits or proliferative diseases), inflammatory diseases (e.g., SIRS reduces ACE-2 enzymatic activity. (Systemic Inflammatory Response Syndromes), Sepsis, The present invention further encompasses methods and polytrauma, inflammatory bowl disease, acute and chronic compositions for inhibiting or reducing abnormal histamine pain, rheumatoid arthritis, and osteoarthritis), allergic dis 25 release comprising, or alternatively consisting of, adminis orders (e.g., asthma, adult respiratory distress Syndrome, tering to an animal in which Such inhibition or reduction is wound healing, and Scar formation), as well as Several other desired, an ACE-2 binding polypeptide in an amount effec disoders and/or diseases (e.g., periodontal disease, tive to inhibit or reduce ACE-2 enzymatic activity. dySmenorrhea, premature labor, brain edema following focal In another embodiment, the present invention encom injury, diffuse axonal injury, and reperfusion injury). passes methods and compositions for inhibiting or reducing In Specific embodiments, the present invention encom vasoconstriction and/or other diseases or disorders associ passes methods and compositions (e.g., ACE-2 binding ated with vasoconstriction comprising, or alternatively con polypeptides that antagonize ACE-2 activity) for preventing, sisting of, administering to an animal in which Such inhibi treating and/or ameliorating diseases or disorders associated tion or reduction is desired, an ACE-2 binding polypeptide with hypertension (e.g., accelerated hypertension, renal 35 in an amount effective to inhibit or reduce ACE-2 enzymatic failure, Vascular accidents, myocaridal infarction, and activity. Stroke). In yet another embodiment, this invention also provides a In a specific embodiment, this invention also provides a method for reducing or inhibiting diseases and/or disorders method for preventing, ameliorating, or treating diseases asSociated with aberrant action of ACE-2 comprising, or and/or disorders associated with hypotension (e.g., shock, 40 alternatively consisting of, administering to an animal in Syncope, and intracranial hypotension) comprising, or alter which Such inhibition or reduction is desired, an ACE-2 natively consisting of, administering to an animal in which binding polypeptide and an effective amount of an ACE Such inhibition or reduction is desired, an effective amount inhibiting compound. of angiotensin H, or an angiotensin II-like compound, and an Additionally, this invention also provides a method for effective amount of angiotensin 1-9, or an angiotensin 45 reducing or inhibiting diseases and/or disorders associated 1-9-like compound. with aberrant action of ACE comprising, or alternatively In Specific embodiments, the present invention encom consisting of, administering to an animal in which Such passes methods and compositions (e.g., ACE-2 binding inhibition or reduction is desired, an ACE-2 binding polypeptides that antagonize ACE-2 or ACE activity) for polypeptide and an effective amount of an ACE inhibiting preventing, treating and/or ameliorating other diseases or 50 compound. disorders associated with vasoconstriction, comprising, or alternatively consisting of, administering to an animal in BRIEF DESCRIPTION OF THE DRAWINGS which Such treatment, prevention, and/or amelioration is FIG. 1. Synergistic effects of Angiotensin1-9 (A1-9) on desired, an ACE-2 binding polypeptide in an amount effec angiotensin II (AII)-induced vasoconstriction, representa tive to treat, prevent and/or ameliorate the disease or disor 55 tive experiment (experiment 9 from Table 3). Data are der. presented as the mean percentage of the maximal contractile The present invention further encompasses methods and response as defined by full KCl depolarization following compositions for inhibiting or reducing Stenosis, including treatment with angiotensin peptides for four separate rings aortic Stenosis, buttonhole Stenosis, coronary Ostial Stenosis, used to determine average response at each concentration. double aortic Stenosis, fish-mouth mitral Stenosis, bronchial 60 Generated tension was plotted against peptide concentration Stenosis, hypertrophic pyloric Stenosis, pyloric Stenosis, and the resulting concentration-response curves were Sub infundibular Stenosis, idiopathic hypertrophic Subaortic jected to regression analysis to determine whether the effects Stenosis, idiopathic Subglottic Stenosis, pulmonary Stenosis, of the combination of A1-9 and angiotensin II produced muscular Subaortic Stenosis, laryngeal Stenosis, mitral additivity or Synergy. A1-9 had no effect on vascular tone in Stenosis, Supravalvar and Subvalvar Stenosis, Subvalvular 65 this experiment, hence the predicted effect of the combina and Supravalvular Stenosis, and tricuspid Stenosis, tion that would be expected if A1-9 additively potentiated comprising, or alternatively consisiting of, contacting an AII-mediated vasoconstriction is effectively that of AII US 6,592,865 B2 13 14 alone (Solid line). The Synergistic or Supraadditive potentia are not limited to, the ACE-2 polypeptide as shown in SEQ tion of AII-mediated vasoconstriction by A1-9 is shown ID NO:138, the ACE-2 polypeptide as shown in SEQ ID (Squares and dotted line) as a leftward shift. NO:142, the ACE-2 extracellular domain (SEQID NO:140), FIG. 2. The effect of A1-9 on (A) mean arterial and the ACE-2 transmembrane domain (SEQ ID NO:139). pressure-tSEM in the awake and freely-ranging rat (n=6 5 ACE-2 and ACE-2-like polypeptides retain at least one rats). Average mean arterial and pulse pressure as well as functional activity of the natural or full-length ACE-2, heart rate data were derived from the last 5 min of a 20 min including but not limited to the following activities: cleaving continuous intravenous infusion period. * indicates a Sig angiotensin to angiotensin 1-9 and regulating the cleavage nificant difference compared to vehicle-treated rats follow and/or Synthesis of bradykinin, kinetensin, tachykinin, ing analysis of variance and Dunnett's post-hoc test. A1-9 or neurotensin, Substance P, and endothelin. In a preferred angiotensin II (AII) continuous infusion doses are in lug/kg/ embodiment, the ACE-2 and ACE-2-like polypeptides retain min. the ability to cleave angiotensin to angiotesin 1-9. ASSayS that can be used to determine the functional activities of FIG. 3. The effect of A1-9 on angiotensin II-mediated ACE-2 or ACE-2 like polypeptides can readily be deter preSSorresponses in normotensive awake male rats. Data are 15 mined by one skilled in the art (e.g., See assays disclosed in reported as average mean arterial pressure-tSEM (n=6 rats). Moore et al., 1999, Supra) “ACE-2-like polypeptides” also FIG. 4. Arterial pressure responses to ACE-2 inhibitor or include fusion polypeptides in which all or a portion of inactive peptide in Spontaneously hypertensive rats for 2.5 ACE-2 is fused or conjugated to another polypeptide. ACE min following bolus injection. Data are reported as mean 2-like polypeptides that are fusion polypeptides retain at MAP-SEM. Since ACE-2 and inactive peptides were tested least one functional activity of ACE-2, preferably the ability in different cohorts of rats, Separate vehicle treatment groups to cleave angiotensin to angiotensin 1-9 and regulate the are plotted. The inactive peptide treated group was also cleavage and/or Synthesis of bradykinin, kinetensin, treated with ACE-2 inhibitory peptide at the end of the tachykinin, neurotensin, Substance P, and endothelin. In a experiment for control. * indicates Significant difference preferred embodiment, the ACE-2 and ACE-2-like polypep between ACE-2 inhibitor and vehicle treatment (ANOVA tides that are fusion polypeptides retain the ability to cleave followed by Dunnett's). t indicates significant difference 25 angiotensin to angiotensin 1-9. ACE-2.fusion polypeptides between ACE-2 inhibitor and control peptide treated groups. may be made by recombinant DNA techniques in which a The control peptide had no effect on MAP. gene or other polynucleotide coding Sequence for ACE-2 or a fragment thereof is ligated in-frame (recombined) with the DEFINITIONS coding Sequence of another protein or polypeptide. The resulting recombinant DNA molecule is then inserted into In order that the invention may be clearly understood, the any of a variety of plasmid or phage expression vectors, following terms are defined: which enable expression of the fusion protein molecule in an The term "recombinant is used to describe non-naturally appropriate eukaryotic or prokaryotic host cell. ACE-2 altered or manipulated nucleic acids, host cells transfected fusion polypeptides may be generated by Synthetic or Semi with exogenous nucleic acids, or polypeptide molecules that 35 are expressed non-naturally, through manipulation of iso Synthetic procedures as well. lated nucleic acid (typically, DNA) and transformation or The terms “ ACE-2 target” or “ACE-2 target protein’ are transfection of host cells. "Recombinant' is a term that Sometimes used herein and encompass ACE-2 and/or ACE Specifically encompasses nucleic acid molecules that have 2-like polypeptides. Thus, the ACE-2 binding polypeptides 40 of the invention bind “ACE-2 target proteins” and can be been constructed in vitro using genetic engineering used to bind, detect, remove, and/or purify "ACE-2 target techniques, and use of the term “recombinant as an adjec proteins.” tive to describe a molecule, construct, vector, cell, polypep tide or polynucleotide Specifically excludes naturally occur The term “binding polypeptide' is used herein to refer to ring Such molecules, constructs, Vectors, cells, polypeptides any polypeptide capable of forming a binding complex with or polynucleotides. 45 another molecule, polypeptide, peptidomimetic or transfor The term “bacteriophage” is defined as a bacterial virus mant. containing a nucleic acid core and a protective shell built up A “ACE-2 binding polypeptide' is a molecule of the by the aggregation of a number of different protein mol invention that can bind an ACE-2 target protein. Non ecules. The terms “bacteriophage' and “phage' are Synony limiting examples of ACE-2 binding polypeptides of the 50 invention are the polypeptide molecules having an amino mous and are used herein interchangeably. acid sequence described herein (see SEQ ID NOS: 1-136). The term “affinity ligand” is sometimes used herein and is The term ACE-2 binding polypeptide also encompasses Synonymous with ACE-2 binding polypeptides of the inven ACE-2 binding fragments and variants (including tion. derivatives) of polypeptides having the specific amino acid The term “ACE-2 protein' as used herein encompasses 55 sequences described herein (SEQ ID NOs: 1-136). By both the membrane (e.g., SEQ ID NOS: 138 and 142) and “variant' of an amino acid Sequence as described herein is soluble forms (e.g., SEQ ID NO: 140). ACE-2 protein may meant a polypeptide that binds ACE-2, but does not neces be monomeric, dimeric, or trimeric or multivalent, Sarily comprise an identical or Similar amino acid Sequence preferably, ACE-2 proteins are homotrimeric. of an ACE-2 binding polypeptide specified herein. ACE-2 The term “ACE-2-like polypeptide' as used herein 60 binding polypeptides of the invention which are variants of encompasses natural ACE-2 or full-length recombinant an ACE-2 binding polypeptide Specified herein Satisfy at ACE-2 as well as fragments and variants thereof, Such as, a least one of the following: (a) a polypeptide comprising, or modified or truncated form of natural ACE-2 or full-length alternatively consisting of, an amino acid Sequence that is at recombinant ACE-2, which ACE-2 and ACE-2-like least 30%, at least 35%, at least 40%, at least 45%, at least polypeptide retain an ACE-2 functional activity. ACE-2 or 65 50%, at least 55%, at least 60%, at least 65%, at least 70%, ACE-2 fragments that may be specifically bound and/or at least 75%, at least 80%, at least 85%, at least 90%, at least inhibited by the compositions of the invention include, but 95%, least 99%, or 100% identical to the amino acid US 6,592,865 B2 15 16 Sequence of an ACE-2 binding polypeptide Sequence dis nucleic acid Sequences is determined using the algorithm of closed herein (SEQ ID NOs: 1-136), (b) a polypeptide Karlin and Atschul (Proc. Natl. Acad. Sci. USA, 87: encoded by a nucleotide Sequence, the complementary 2264-2268 (1990)), modified as in Karlin and Altschul Sequence of which hybridizes under Stringent conditions to (Proc. Natl. Acad. Sci. USA, 90: 5873–5877 (1993)). Such a nucleotide Sequence encoding an ACE-2 binding polypep an algorithm is incorporated into the NBLAST and tide disclosed herein (e.g., a nucleic acid sequence encoding XBLAST programs of Altschul et al. (J. Mol. Biol., 215: the amino acid sequence of SEQ ID NOs: 1-136), and/or a 403-410 (1990)). BLAST nucleotide searches are per fragment of an ACE-2 binding polypeptide disclosed herein, formed with the NBLAST program to obtain nucleotide of at least 5 amino acid residues, at least 10 amino acid Sequences homologous to a nucleic acid molecule described residues, at least 15 amino acid residues, or at least 20 amino acid residues. ACE-2 binding polypeptides of the invention herein. BLAST protein searches are performed with the also encompass polypeptide Sequences that have been modi XBLAST program to obtain amino acid Sequences homolo fied for various applications provided that Such modifica gous to a reference polypeptide. To obtain gapped align tions do not eliminate the ability to bind an ACE-2 target. ments for comparison purposes, Gapped BLAST is utilized Specific, non-limiting examples of modifications contem as described in Altschul et al. (Nucleic Acids Res., 25: plated include C-terminal or N-terminal amino acid Substi 15 3389–3402 (1997)). When utilizing BLAST and Gapped tutions or peptide chain elongations for the purpose of BLAST programs, the default parameters of the respective linking the ACE-2 bindor to a chromatographic material or programs (e.g., XBLAST and NBLAST) are used. See, other Solid Support. Other Substitutions contemplated herein http://www.ncbi.nlm.nih.gov. Alternatively, the percent include Substitution of one or both of a pair of cysteine identity of two amino acid Sequences or of two nucleic acid residues that normally form disulfide links, for example with Sequences can be determined once the Sequences are aligned non-naturally occurring amino acid residues having reactive for optimal comparison purposes (e.g., gaps can be intro Side chains, for the purpose of forming a more Stable bond duced in the Sequence of a first amino acid or nucleic acid between those amino acid positions than the former disulfide Sequence for optimal alignment with a Second amino acid or bond. All Such modified binding polypeptides are also nucleic acid sequence). The amino acid residues or nucle considered ACE-2 binding polypeptides according to this 25 otides at corresponding amino acid positions or nucleotide invention So long as the modified polypeptides retain the positions are then compared. When a position in the first ability to bind ACE-2 and/or ACE-2-like polypeptides, and Sequence is occupied by the same amino acid residue or therefore, may be used in one or more of the various nucleotide at the corresponding position in the Second methods described herein, Such as, to detect, purify, or Sequence, then the molecules are identical at that position. isolate ACE-2 or ACE-2-like polypeptides in or from a The percent identity between the two Sequences is a function Solution. ACE-2 binding polypeptides of the invention also of the number of identical positions shared by the Sequences include variants of the Specific ACE-2 binding polypeptide (i.e., 7% identity=number of identical overlapping positions/ sequences disclosed herein (e.g., SEQ ID NOS: 1-136) total number of positionsx100%). In one embodiment, the which have an amino acid Sequence corresponding to one of two Sequences are the same length. these polypeptide Sequences, but in which the polypeptide 35 The term “polypeptide', as used herein, refers to a linear, Sequence is altered by Substitutions, additions or deletions branched, or cyclic (e.g., containing a loop structure) poly that provide for moleucles that bind ACE-2. Thus, the mer of two or more amino acid residues linked with a ACE-2 binding polypeptides include polypeptides peptide bond. The term “polypeptide' is not restricted to any containing, as a primary amino acid Sequence, all or part of particular upper limit of amino acid residues. Thus, the the particular ACE-2 binding polypeptide Sequence includ 40 ACE-2 affinity ligands of the invention that comprise an ing altered Sequences in which functionally equivalent amino acid Sequence described herein are properly referred amino acid residues are Substituted for residues within the to as "ACE-2 binding polypeptides' because Such binding Sequence, resulting in a peptide which is functionally active. polypeptides contain at least two amino acid residues held For example, one or more amino acid residues within the together by a peptide bond, even though Such molecules may Sequence can be Substituted by another amino acid of a 45 also contain one or more additional moieties or groups that Similar polarity which acts as a functional equivalent, result are not amino acids, Such as N-terminal and/or C-terminal ing in a Silent alteration. Conservative Substitutions for an capping or functional groups, and that may or may not be amino acid within the Sequence may be Selected from other involved in a peptide bond. The polypeptides of the inven members of the class to which the amino acid belongs. For tion may be monovalent, divalent, trivalent, or multivalent example, the nonpolar (hydrophobic) amino acids include 50 and may comprise one or more of the ACE-2 binding alanine, leucine, isoleucine, Valine, proline, phenylalanine, polypeptides having the amino acid Sequence of SEQ ID tryptophan and methionine. The polar neutral amino acids NOs: 1-136 and/or fragments or variants thereof. The term include glycine, Serine, threonine, cysteine, tyrosine, "peptide' is used herein to have the same meaning as asparagine, and glutamine. The positively charged (basic) “polypeptide.” The term “antibody,” as used herein, refers to amino acids include arginine, lysine and histidine. The 55 immunoglobulin molecules and immunologically active negatively charged (acidic) amino acids include aspartic portions of immunoglobulin molecules, i.e., molecules that acid and glutamic acid. Such ACE-2 binding polypeptides contain an antigen binding Site that immunospecifically can be made either by chemical peptide Synthesis or by binds an antigen. AS Such, the term antibody encompasses recombinant production from a nucleic acid encoding the not only whole antibody molecules, but also antibody frag ACE-2 binding polypeptide which nucleic acid has been 60 ments as well as variants (including derivatives) of antibod mutated. Any technique for mutagenesis known in the art ies and antibody fragments. Examples of molecules which can be used, including but not limited to, chemical are described by the term “antibody” in this application mutagenesis, in vitro Site-directed mutagenesis (Hutchinson include, but are not limited to: single chain Fvs (scFVs), Fab et al., J. Biol. Chem., 253:6551 (1978)), use of TAB.RTM. fragments, Fab' fragments, F(ab'), disulfide linked Fvs linkers (Pharmacia), etc. 65 (SdPVs), FVs, and fragments comprising or alternatively AS used and understood herein, percent homology or consisting of, either a VL or a VH domain. The term “single percent identity of two amino acid Sequences or of two chain Fv' or “scFv” as used herein refers to a polypeptide US 6,592,865 B2 17 18 comprising a VL domain of antibody linked to a VH domain progeny or potential progeny of Such a cell. Progeny may of an antibody. not be identical to the parent cell transfected with the nucleic “Feed stream”: ACE-2 and ACE-2-like polypeptides that acid molecule due to mutations or environmental influences are bound by an ACE-2 binding polypeptide of this inven that may occur in Succeeding generations or integration of tion may be produced by any method known in the art, the nucleic acid molecule into the host cell genome. including, but not limited to, chemical Synthesis, production Other terms are defined as necessary in the text below. in transformed host cells, Secretion into culture medium by naturally occurring cells or recombinantly transformed DETAILED DESCRIPTION OF THE bacteria, yeasts, fungi, insect cells, plant cells, and mam INVENTION malian cells, production in genetically engineered organ isms (for example, transgenic mammals); and production in The present invention provides novel binding moieties for non-genetically engineered organisms. The Solution, ACE-2. Such binding moieties make possible the efficient Sample, or mixture that contains an ACE-2 or ACE-2-like detection and isolation of ACE-2 or ACE-2-like polypep polypeptide as it is produced or is found present in a tides in tissueS or in a Solution or System that contains 15 ACE-2 or ACE-2-like polypeptides. The ACE-2 binding production Solution will Sometimes be referred to as the polypeptides disclosed herein can also be used to immobi “feed stream'. lize ACE-2 targets and provide a means of removing ACE-2 The term “binding” refers to the determination by stan target proteins from Solutions or Systems containing them. dard techniques that a binding polypeptide recognizes and The preferred binding moieties of the present invention bind binds to a given target. Such Standard techniques include, but are not limited to, affinity chromatography, equilibrium ACE-2 with high affinity, i.e., acting at low concentrations. dialysis, gel filtration, enzyme linked immunosorbent assay The present invention also encompasses methods and (ELISA), FACS analysis, and the monitoring of spectro compositions for detecting, diagnosing, prognosing, and/or Scopic changes that result from binding, e.g., using fluores monitoring diseases or disorders associated with aberrant cence anisotropy, either by direct binding measurements or ACE-2 or Angiotensin 1-9 expression or inappropriate 25 ACE-2 or Angiotensin 1-9 function in an animal, preferably competition assays with another binder. a mammal, and most preferably a human, comprising, or The term “specificity” refers to a binding polypeptide of alternatively consisting of, use of ACE-2 binding polypep the invention that has a higher binding affinity for one target tides (including molecules which comprise, or alternatively over another. Thus, the term "ACE-2 target protein Speci consist of, ACE-2 binding polypeptide fragments or variants ficity' refers to a molecule having a higher affinity for thereof) that specifically bind ACE-2. Diseases and disor ACE-2 target protein as compared with another molecule ders which can be detected, diagnosed, prognosed and/or that is not an ACE-2 target protein. monitored with the ACE-2 binding polypeptides of the The term “epitopes” as used herein refers to portions of invention include, but are not limited to, cardiovascular ACE-2 having antigenic or immunogenic activity in an disorders (e.g., hypertension, chronic heart failure, left ven animal, preferably a mammal. An epitope having immuno 35 tricular failure, Stroke, cerebral vasospasm after Subarach genic activity is a portion of ACE-2 that elicits an antibody noid injury, atherosclerotic heart disease, and retinal response in an animal. An eptiope having antigenic activity hemorrhage), renal disorders (e.g., renal vein thrombosis, is a portion of ACE-2 to which an antibody or ACE-2 kidney infarction, renal artery embolism, renal artery binding polypeptide specifically binds as determined by any Stenosis, and edema, hydronephritis), proliferative diseases method known in the art, for example, by the immunoassays 40 or disorders (e.g., vascular Stenosis, myocardial described herein. Antigenic epitopes need not necessarily be hypertrophy, hypertrophy and/or hyperplasia of conduit and/ immunogenic. or resistance vessels, myocyte hypertrophy, and fibroblast The term "fragment' as used herein refers to a polypep proliferative diseases), inflammatory diseases (e.g., SIRS tide comprising an amino acid Sequence of at least 5 amino (Systemic Inflammatory Response Syndromes), Sepsis, acid residues, at least 6 amino acid residues, at least 7 amino 45 polytrauma, inflammatory bowl disease, acute and chronic acid residues, at least 8 amino acid residues, at least 9 amino pain, rheumatoid arthritis, and osteoarthritis), allergic dis acid residues, at least 10 amino acid residues, at least 11 orders (e.g., asthma, adult respiratory distress Syndrome; amino acid residues, at least 12 amino acid residues, at least wound healing, and Scar formation), as well as Several other 13 amino acid residues, at least 14 amino acid residues, at disoders and/or diseases (e.g., periodontal disease, least 15 amino acid residues, at least 16 amino acid residues, 50 dySmenorrhea, premature labor, brain edema following focal at least 17 amino acid residues, at least 18 amino acid injury, diffuse axonal injury, and reperfusion injury). residues, at least 19 amino acid residues, at least 20 amino Preferably, the present invention also encompasses meth acid residues, at least 21 amino acid residues, at least 22 ods and compositions for detecting, diagnosing, prognosing, amino acid residues, at least 23 amino acid residues, at least and/or monitoring diseases or disorders including, but not 24 amino acid residues, or at least 25 amino acid residues of 55 limited to, hypertension and diseases and/or disorders asso the amino acid Sequence of ACE-2, or an ACE-2 binding ciated with hypertension, Such as accelerated hypertension, polypeptide (including molecules that comprise, or alterna episodic hypertension, paroxysmal hypertension, portal tively consist of, ACE-2 binding polypeptide fragments or hypertension, primary hypertension, Secondary variants thereof). hypertensoin, Systemic venous hypertension, borderline The term “fusion protein” as used herein refers to a 60 hypertension, adrenal hypertension, benign hypertension, polypeptide that comprises, or alternatively consists of, an idiopathic hypertension, pale hypertension, postpartm amino acid Sequence of an ACE-2 binding polypeptide of hypertension, pregnancy-induced hypertension (gestational the invention and an amino acid Sequence of a heterologous hypertension), essential hypertension, labile hypertension, polypeptide (i.e., a polypeptide unrelated to the ACE-2 pulmonary hypertension, renal and renovascular binding polypeptide). 65 hypertension, and Goldblatt hypertension, left ventricular The term “host cell as used herein refers to the particular failure, atherosclerotic heart disease, Stroke, retinal hemor Subject cell transfected with a nucleic acid molecule and the rhage or infarction (Keith-Wagener-Barker changes), renal US 6,592,865 B2 19 20 failure, renovascular disease, exudates, papilledema, Vascu (Angiotensin converting enzyme-2) and/or ACE-2-like lar accidents, myocardial infarction, dissecting aneurysm. polypeptides. In particular, the invention encompasses The present invention further encompasses methods and polypeptides that Specifically bind to a polypeptide or compositions for preventing, treating and/or ameliorating polypeptide fragment of human ACE-2 (SEQID NO: AAA). diseases or disorders, especially diseases and disorders of 5 In specific preferred embodiments, the ACE-2 binding vasoconstriction, or alternatively consisting of, administer polypeptides of the invention bind ACE-2 and/or ACE-2- ing to an animal in which Such treatment, prevention or like polypeptides with high affinity. In other embodiments, amelioration is desired one or more ACE-2 binding polypep the ACE-2 binding polypeptides of the invention reversibly tides (including molecules which comprise, or alternatively bind ACE-2 and/or ACE-2-like polypeptides. In still other consist of, ACE-2 binding polypeptide fragments or variants embodiments, the ACE-2 binding polypeptides of the inven thereof) in an amount effective to treat, prevent or ameliorate tion irreversibly bind ACE-2 and/or ACE-2-like polypep the disease or disorder. The present invention further encom tides. passes methods and compositions for preventing, treating In preferred embodiments, the ACE-2 binding polypep and/or ameliorating diseases or disorders associated with tides of the present invention (including molecules aberrant ACE-2 or Angiotensin 1-9 expression or inappro 15 comprising, or alternatively consisting of, ACE-2 binding priate ACE-2 or Angiotensin 1-9 function in an animal, polypeptide fragments or variants thereof), Specifically bind preferably a mammal, and most preferably a human, to ACE-2 and do not croSS-react with any other antigens. comprising, or alternatively consisting of, administering to The cysteine residues in polypeptides are believed to form an animal in which Such treatment, prevention or ameliora a disulfide bond, which would cause the polypeptide con tion is desired one or more ACE-2 binding polypeptides taining these cysteine residues to form a stable loop Structure (including molecules which comprise, or alternatively con under non-reducing conditions. Especially preferred ACE-2 sist of, ACE-2 binding polypeptide fragments or variants binding polypeptides of the invention are polypeptide thereof) in an amount effective to treat, prevent or ameliorate molecules, which comprise amino acid Sequences that form the disease or disorder. stable loop structures or other stable structures that bind Diseases and disorders which can be prevented, treated, 25 ACE-2 or ACE-2-like polypeptides. and/or ameliorated with the ACE-2 binding polypeptides of In specific embodiments, the invention relates to ACE-2 the invention include, but are not limited to, cardiovascular binding polypeptides comprising, or alternatively consisting disorders (e.g., hypertension, chronic heart failure, left ven of, an amino acid Sequence Selected from the group con tricular failure, Stroke, cerebral vasospasm after Subarach sisting of SEQ ID NOs: 1-136, preferably SEQ ID noid injury, atherosclerotic heart disease, and retinal NOS:11–39, and most preferably SEQ ID NOs: 23–24 and hemorrhage), renal disorders (e.g., renal vein thrombosis, 36-39, as referred to in Tables 1-2 and in Example 1 below. kidney infarction, renal artery embolism, renal artery Ten consensus sequences (SEQ ID NOS:1-10) have been Stenosis, and edema, hydronephritis), proliferative diseases determined based on the Specific ACE-2 binding polypep or disorders (e.g., vascular Stenosis, myocardial tides shown in paragraph 530 of Example 1. In specific hypertrophy, hypertrophy and/or hyperplasia of conduit and/ 35 embodiments, ACE-2 binding polypeptides of the invention or resistance vessels, myocyte hypertrophy, and fibroblast comprise one or more of these Sequences. Such preferred proliferative diseases), inflammatory diseases (e.g., SIRS ACE-2 binding polypeptides include a polypeptide with the (Systemic Inflammatory Response Syndromes), Sepsis, potential to form a loop Structure comprising, or alterna polytrauma, inflammatory bowl disease, acute and chronic tively consisting of, an amino acid Sequence Selected from pain, rheumatoid arthritis, and osteoarthritis), allergic dis 40 A-J (SEQ ID NOs: 1–10): orders (e.g., asthma, adult respiratory distress Syndrome, wound healing, and Scar formation), as well as Several other disoders and/or diseases (e.g., periodontal disease, wherein, dySmenorrhea, premature labor, brain edema following focal Z is a polypeptide of at least 2 amino acids, injury, diffuse axonal injury, and reperfusion injury). 45 X is any amino acid except cysteine; Preferably, the present invention also encompasses meth ods and compositions for preventing, treating and/or ame X is L or M (preferably L); liorating diseases or disorders including, but not limited to, X is For Y (preferably F); hypertension and diseases and/or disorders associated with X is F, L, M, or V (preferably V); hypertension, Such as accelerated hypertension, episodic 50 Xs is D or E; hypertension, paroxy Small hypertension, portal Z is a polypeptide of at least one amino acid or is absent; hypertension, primary hypertension, Secondary and hypertensoin, Systemic venous hypertension, borderline Z contains at least one cysteine residue Such that forma hypertension, adrenal hypertension, benign hypertension, tion of a disulfide bond with the invariant cysteine idiopathic hypertension, pale hypertension, postpartm 55 residue (C) forms a cyclic peptide of six or ten amino hypertension, pregnancy-induced hypertension (gestational acids. hypertension), essential hypertension, labile hypertension, pulmonary hypertension, renal and renovascular hypertension, and Goldblatt hypertension, left ventricular wherein, failure, atherosclerotic heart disease, Stroke, retinal hemor 60 rhage or infarction (Keith-Wagener-Barker changes), renal Z is a polypeptide of at least Six amino acids, failure, renovascular disease, exudates, papilledema, X is F, M, W, or Y (preferably For Y); .Vascular accidents, myocardial infarction, dissecting aneu X is F, I, L, M, or V (preferably F or L); rySm. X is D, E, or T (preferably D); ACE-2 Binding Polypeptides 65 X is For M (preferably F); The present invention provides new polypeptides and Z is a polypeptide of at least one amino acid or is absent; families of polypeptides that specifically bind to ACE-2 and US 6,592,865 B2 21 22 Z contains at least one cysteine residue Such that forma Z is a polypeptide of at least one amino acid or is absent; tion of a disulfide bond with the invariant cysteine X is W or Y; residue (C) forms a cyclic peptide of eight or ten amino acids. X is any amino acid except cysteine; X is W or Y; X is any amino acid except cysteine; Xs is any amino acid except cysteine; X is any amino acid except cysteine; and Z is a polypeptide of at least one amino acid or is absent; Z is a polypeptide of at least one amino acid or is absent. X is A, D, F, G, H, L, N, P, or S (preferably D); X is A, D, F, G, H, N, S, W, or Y (preferably D); X is D, E, H, L, M, or V (preferably D or E); wherein, X is D, E, G, N, R, Q, S, or V (preferably D or E); Z is a polypeptide of at least one amino acid or is absent; X is N, T, or W (preferably W); 15 X is any amino acid except cysteine; X is any amino acid except cysteine; X is any amino acid except cysteine; X-7 is any amino acid except cysteine; X is any amino acid except cysteine; and X is F, W, or Y (preferably F); Z is a polypeptide of at least one amino acid or is absent. X is any amino acid except cysteine; Xo is any amino acid except cysteine; X is any amino acid except cysteine; wherein, X2 is any amino acid except cysteine; Z is a polypeptide of at least one amino acid or is absent; X is any amino acid except cysteine; and 25 X is D or H (preferably D); Z is a polypeptide of at least one amino acid or is absent. X is H, N, or W; X is G or is absent; X is T or N (preferably T); wherein, X is A, N, W, or Y; Z is a polypeptide of at least one amino acid or is absent; Xis H, N, or Q; and X is any amino acid except cysteine; Z is a polypeptide of at least one amino acid or is absent. X is any amino acid except cysteine; X is C, E, or S; 35 X is K, L, or R (preferably R); wherein, Xs is A, R, or S (preferably R); Z is a polypeptide of at least one amino acid or is absent; Z is a polypeptide of at least one amino acid or is absent; X is K, L, R, or S; and wherein, if X3 is cysteine (C), then Z contains a X is A or P (preferably P); C-terminal cysteine residue. 40 X is 1, L, Q, or V, X is D, G, H, M, Q, or Y; X is D, F, K, S, or Y, wherein, X is F, K, M, or W (preferably W); Z is a polypeptide of at least one amino acid or is absent; 45 X, is A, F, K, R, or V; and Z is a polypeptide of at least one amino acid or is absent. X is L, H, or M; wherein said polypeptides bind ACE-2 and/or ACE-2-like X is N or T (preferably T); polypeptides. X is D, M, N, or S; ACE-2 binding polypeptide molecules of the invention X is V or I (preferably V); 50 may also have an amino terminal (N-terminal) capping or Z is a polypeptide of at least one amino acid or is absent. functional group, Such as an acetyl group, which, for example, blocks the amino terminal amino group from Z-C-F-X-W-X-Z, (SEQ ID NO:6): F. undesirable reactions or is useful in linking the ACE-2 binding polypeptide to another molecule, matrix, resin, or wherein, 55 Solid Support. The nature of the Solid Support, process for Z is a polypeptide of at least one amino acid or is absent; attachment of the ACE-2 binding polypeptide to the solid X is D or E; Support, Solvent, and conditions of the affinity isolation or X is D or E; Selection procedure are largely conventional and well known Z is a polypeptide of at least two amino acids and to those skilled in the art. ACE-2 binding polypeptides of the 60 invention may also have a carboxy terminal (C-terminal) contains at least one cysteine residue Such that forma capping or functional group, Such as an amide group, which, tion of a disulfide bond with the invariant cysteine for example, blocks the C-terminal carboxyl group from residue (C) form S a cyclic peptide of Seven, eight or undesirable reactions or provides a functional group useful twelve amino acids. in conjugating the binding polypeptide to other molecules, 65 matrices, resins, or Solid Supports. Preferably, the N- and/or C-terminal capping groups are polypeptide linker molecules. wherein, An especially preferred C-terminal linker molecule that is US 6,592,865 B2 23 24 useful for immobilizing an ACE-2 binding polypeptide of such inhibiting polypeptides, reversibly inhibit the ACE-2 the invention to a Solid Support or chromatographic matrix protein in its native form. material comprises the amino acid Sequence Pro-Gly-Pro ACE-2 binding polypeptides of the invention inhibit Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 146). ACE-2 target protein with high affinity. In Specific The invention also encompasses, ACE-2 binding polypep embodiments, ACE-2 binding polypeptides of the invention tides that have been modified, for example, to increase or bind ACE-2 target proteins with a dissociation constant or decrease the Stability of the molecule, while retaining the K of less than or equal to 5x10 M, 10 M, 5x10 M, ability to bind ACE-2 and/or ACE-2-like polypeptides. An 10 M, 5x10' M, 10 M, 5x10 M, or 10 M. More example of a modified ACE-2 binding polypeptide of the preferably, ACE-2 binding polypeptides of the invention invention is a polypeptide in which one of two cysteine bind ACE-2 target proteins with a dissociation constant or residues is Substituted with a non-naturally occurring amino K, less than or equal to 5x10M, 10 M., 5x107M, 107 acid that is capable of condensing with the remaining M, 5x10 M, or 10 M. Even more preferably, ACE-2 cysteine Side chain to form a stable thioether bridge, thereby binding polypeptides of the invention bind ACE-2 target generating a cyclic ACE-2 binding polypeptide. Such cyclic proteins with a dissociation constant or K, less than or equal thioether molecules of Synthetic peptides may be routinely 15 to 5x10 M, 10 M, 5x100 M, 10° 19 M, 5x10 M, generated using techniques known in the art and described, 10-11 M, 5x10-12 M, 10-12 M, 5x10-13 M, 10-13 M, e.g., in PCT publication WO 97/46251, incorporated herein 5x10' M, 10' M, 5x10's M, or 10 M. by reference. In certain preferred embodiments, ACE-2 binding In another embodiment, the invention provides ACE-2 polypeptides of the invention reversibly bind ACE-2 and/or binding polypeptides of the invention attached, coupled, ACE-2-like polypeptides and release bound ACE-2 protein linked or adhered to a matrix or resin or Solid Support. in an active form, preferably in the native form, under Techniques for attaching linking or adhering polypeptides to Specific release conditions. In Specific embodiments, ACE-2 matrices, resins and Solid Supports are well known in the art. binding polypeptides of the invention bind ACE-2 target Suitable matrices, resins or Solid Supports for these materials proteins with off-rates or k, less than or equal to 101s, may be any composition known in the art to which binding 25 5x10 s, 10° S, 5x10° S, or 5x10 s. More polypeptides are commonly attached, coupled, linked, or preferably, ACE-2 binding polypeptides of the invention adhered, including but not limited to, a chromatographic bind ACE-2 target proteins with off-rates or k, less than or resin or matrix, such as SEPHAROSE-4 FF agarose beads, equal to, 10's", 5x10's, 10's", 5x10's, 10s, the wall or floor of a well in a plastic microtiter dish, such 5x10s, or 5x107 s. as used in an enzyme-liked immunosorbent assay (ELISA), Binding experiments to determine K, and off-rates can be or a Silica based biochip. Materials useful as Solid Supports performed in a number of conditions including, but not on which to immobilize binding polypeptides of the inven limited to, pH 6.0, 0.01% Tween 20, pH 6.0, 0.1% tion include, but are not limited to, polyacrylamide, agarose, gelatin), pH5.0, 0.01% Tween 20, pH9.0, 0.1% Tween Silica, nitrocellulose, paper, plastic, nylon, metal, and com 20), pH6.0, 15% ethylene glycol, 0.01% Tween20), pH5.0, binations thereof. An ACE-2 binding polypeptide of the 35 15% ethylene glycol, 0.01% Tween 20), and pH9.0, 15% invention may be immobilized on a matrix, resin or Solid ethylene glycol, 0.01% Tween 20 The buffers in which to Support material by a non-covalent association or by cova make these Solutions can readily be determined by one of lent bonding, using techniques known in the art. Preferably, skill in the art, and depend largely on the desired pH of the an ACE-2 binding polypeptide of the invention is immobi final solution. Low pH solutions (fat milk), washing the membrane in washing buffer ACE-2 binding polypeptides of the present invention 65 (e.g., PBS-Tween 20), incubating the membrane with ACE-2 (including molecules comprising, or alternatively consisting binding polypeptide (the ACE-2 binding polypeptide of of, ACE-2 binding polypeptide fragments or variants interest) diluted in blocking buffer, washing the membrane US 6,592,865 B2 47 48 in Washing buffer, incubating the membrane with a Second tide antibody has a higher affinity for the ACE-2 binding ary antibody (which recognizes the ACE-2 binding polypeptide than the anti-ACE-2 antibody may have for polypeptide) conjugated to an enzyme (e.g., horseradish ACE-2. peroxidase or alkaline phosphatase) or radioactive molecule The effectiveness of incorporating an ACE-2 binding (e.g., P or 'I) diluted in blocking buffer, washing the 5 polypeptide in an immunoprecipitation protocol to precipi membrane in wash buffer, and detecting the presence of the tate ACE-2 can be assessed by, e.g., Western blot analysis. antigen. Alternatively, the ACE-2 binding polypeptide may One of skill in the art would be knowledgeable as to the be directly conjugated to a detection molecule (e.g., an parameters that can be modified to increase the binding of enzyme or radiolabel), thereby omitting the need for a the ACE-2 binding polypeptide to an antigen and decrease Secondary anti-ACE-2 binding polypeptide antibody. One of the background (e.g., pre-clearing the cell lysate with skill in the art would be knowledgeable as to the parameters Sepharose beads). For further discussion regarding immu that can be modified to increase the Signal detected and to noprecipitation protocols See, e.g., Current Protocols in reduce the background noise. For further discussion regard Molecular Biology, Vol. 1, Ausubeletal, eds. (John Wiley & ing western blot protocols See, e.g., Current Protocols in Sons, Inc., New York 1994) at 10.16.1. Molecular Biology, Vol. 1, Ausubeletal, eds. (John Wiley & 15 The binding affinity of an ACE-2 binding polypeptide Sons, Inc., New York 1994) at 10.8.1. (including molecules comprising, or alternatively consisting ELISAS comprise preparing antigen (e.g., ACE-2 target), of, ACE-2 binding polypeptide fragments or variants coating the well of a 96-well microtiter plate with the thereof) to an antigen and the off-rate of an ACE-2 binding antigen, Washing away antigen that did not bind the Wells, polypeptide-antigen interaction can be determined by com adding the ACE-2 binding polypeptide of interest conju petitive binding assays. One example of a competitive gated to a detectable compound Such as an enzyme (e.g., binding assay is a modified radioimmunoassay comprising horseradish peroxidase or alkaline phosphatase) to the wells the incubation of labeled antigen (e.g., H- or 'I-labeled and incubating for a period of time, Washing away unbound ACE-2 target) with the ACE-2 binding polypeptide of ACE-2 binding polypeptides or non-specifically bound interest in the presence of increasing amounts of unlabeled ACE-2 binding polypeptides, and detecting the presence of 25 antigen, followed by detection of the ACE-2 binding the ACE-2 binding polypeptides Specifically bound to the polypeptide bound to the labeled antigen. The affinity of the antigen coating the well. In ELISAS the ACE-2 binding ACE-2 binding polypeptide of the present invention for polypeptide employed in the assay does not have to be ACE-2 and the binding off-rates can be determined from the conjugated to a detectable compound; instead, an antibody data by Scatchard plot analysis. Competition with an anti that recognizes the ACE-2 binding polypeptide and that is ACE-2 antibody or ACE-2 binding polypeptide can also be conjugated to a detectable compound may be added to the determined using radioimmunoassays. In this case, ACE-2 is well. Further, instead of coating the well with the antigen, incubated with an ACE-2 binding polypeptide of the present the ACE-2 binding polypeptide. may be coated to the well. invention conjugated to a labeled compound (e.g., with H In this case, the detectable molecule could be the antigen or "I) in the presence of increasing amounts of an unla conjugated to a detectable compound Such as an enzyme 35 beled ACE-2 binding polypeptide or anti-ACE-2 antibody. (e.g., horseradish peroxidase or alkaline phosphatase). One In a preferred embodiment, BIAcore kinetic analysis is of skill in the art would be knowledgeable as to the param used to determine the binding on and off rates of ACE-2 eters that can be modified to increase the Signal detected as binding polypeptides (including molecules comprising, or well as other variations of ELISAS known in the art. For alternatively consisting of, ACE-2 binding polypeptide frag further discussion regarding ELISAS See, e.g., Current Pro 40 ments or variants thereof) to ACE-2, or fragments of ACE-2. tocols in Molecular Biology, Vol. 1, Ausubel et al, eds. (John BIAcore kinetic analysis comprises analyzing the binding Wiley & Sons, Inc., New York 1994) at 11.2.1. and dissociation of ACE-2 from chips with immobilized Immunoprecipitation protocols generally use antibody ACE-2 binding polypeptides on their Surface (see Example molecules to imunopreciptate a protein of interest. An 3, infra). ACE-2 preciptation protocol could easily be modified to use 45 The ACE-2 binding polypeptides of the invention an ACE-2 binding polypeptide in place of an anti-ACE-2 (including molecules comprising, or alternatively consisting antibody. Immunopreciptation protocols generally comprise of, ACE-2 binding polypeptide fragments or variants lysing a population of cells in a lysis buffer such as RIPA thereof) can also be assayed for their ability to inhibit, buffer (1% NP-40 or Triton X-100, 1% sodium increase, or not significantly alter, the enzymatic activity of deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium 50 ACE-2 using techniques known to those skilled in the art. phosphate at pH 7.2, 1% Trasylol) supplemented with pro For example, cells expressing a Substrate for ACE-2 (e.g., tein phosphatase and/or protease inhibitors (e.g., EDTA, angiotensin, bradykinin, tachykinin, neurotensin, Substance PMSF, aprotinin, sodium vanadate), adding the antibody of P, endothelin, and/or kinetensin) can be contacted with interest to the cell lysate, incubating for a period of time ACE-2 in the presence or absence of an ACE-2 binding (e.g., 1 to 4 hours) at 40 degrees C., adding protein A and/or 55 polypeptide, and the ability of the ACE-2 binding polypep protein G Sepharose beads to the cell lysate, incubating for tide to inhibit, increase, or not significantly alter, ACE-2 about an hour or more at 40 degrees C., washing the beads binding to the cells can be measured. Alternatively, the in lysis buffer and resuspending the beads in SDS/sample ACE-2 binding polypeptide may be preincubated with buffer. If one wanted to substitute an ACE-2 binding ACE-2 prior to exposure of ACE-2 to cells expressing the polypeptide for the anti-ACE-2 antibody one could readily 60 ACE-2 receptor. ACE-2 binding to cells can be measured by, do So, and then isolate the ACE-2-ACE-2 binding polypep for example, flow cytometry or a Scintillation assay. ACE-2 tide complexes with an antibody that recognizes the ACE-2 or the ACE-2 binding polypeptide can be labeled with a binding polypeptide. Then the triple complex of ACE-2, detectable compound Such as a radioactive label (e.g., P. ACE-2 binding polypeptide, and anti-ACE-2 binding S, and I) or a fluorescent label (e.g., fluorescein polypeptide antibody could be isolated using protein A 65 isothiocyanate, rhodamine, phycoerythrin, phycocyanin, and/or Protein G as described above. Such a protocol may be allophycocyanin, o-phthaldehyde and fluorescamine) to desirable if, for example, the anti-ACE-2 binding polypep enable detection of an interaction between ACE-2 and its US 6,592,865 B2 49 SO Substrates and/or ACE-2 and an ACE-2 binding polypeptide or fragments or variants thereof, may be routinely tested for of the invention. their ability to inhibit ACE-2 from enzymatically acting on The ability of ACE-2 binding polypeptides of the inven any of its Substrates (e.g., Angiotensin, bradykinin, tion to inhibit, increase, or not significantly alter, ACE-2 tachykinin, neurotensin, Substance P, or endothelin). binding to a Substrate can also be determined in cell-free Uses of the Binding Polypeptides and Recombinant Bacte assays. For example, native or recombinant ACE-2 (e.g., riophage of the Invention having the amino acid sequence of SEQ ID NOs: 138 and/or The ACE-2 binding polypeptides described herein are 142) or a fragment thereof can be contacted with a ACE-2 especially useful to detect, isolate, or remove ACE-2 target binding polypeptide and the ability of the ACE-2 binding proteins in Solutions. Such Solutions may be simple disper polypeptide to inhibit, increase, or not significantly alter, sions or solutions of ACE-2 and/or ACE-2-like polypeptide ACE-2 from binding to a substrate can be determined. For in water or aqueous buffer or more complex Solutions, Such example, one could use an ELISA, or other Suitable assay to as, blood and other biological fluids, tissue homogenates cell test the ability of ACE-2 binding polypeptides of the inven extracts, or biopsy Samples, and cell culture media contain tion to inhibit, increase, or not significantly alter, ACE-2 ing ACE-2 or ACE-2-like polypeptides. Biological fluids from binding to a Substrate. One way to do Such an assay 15 include, but are not limited to Sera, plasma, lymph, blood, would be to immobilize the ACE-2 receptor on a solid blood fractions urine, Synovial fluid, Spinal fluid, Saliva, and support. Then ACE-2 or ACE-2 fragments labeled with a UCOUS. detectable compound which had been preincubated with an In one embodiment, the present invention provides a ACE-2 binding polypetide of the invention are tested for method for detecting an ACE-2 protein and/or an ACE-2- their ability to bind the ACE-2 substrate immobilized on the like polypeptide in a Solution comprising contacting the Solid Support. ACE-2 may be partially or completely purified solution with an ACE-2 binding polypeptide of the invention (e.g., partially or completely free of other polypeptides) or and detecting binding of ACE-2 or ACE-2-like polypeptide part of a cell lysate. Further, the ACE-2 polypeptide may be to the ACE-2 binding polypeptide. The ACE-2 binding a fusion protein comprising ACE-2 or a biologically active polypeptide may be either free or immobilized. Preferably, portion thereof and a domain Such as an Immunoglobulin Fc 25 the ACE-2 binding polypeptide is a polypeptide immobi or glutathione-S-transferase. Additionally, the ACE-2 bind lized on a Solid Surface or chromatographic material or the ing polypeptide and/or ACE-2 Substrate may be a fusion well of a plastic microtiter assay dish. protein comprising an ACE-2 binding portion of the Another embodiment of the present invention is a method polypeptide Substrate and a domain Such as an Immunoglo for isolating ACE-2 protein and/or ACE-2-like polypeptide bulin Fc or glutathionine-S-transferase. For example, amino from a Solution containing it, comprising: acid residues 1-154 of TACI (GenBank accesion number contacting the Solution with an ACE-2 binding polypep AAC51790), or 1-48 of BCMA (GenBank accession num tide under conditions that permit binding of ACE-2 ber NP 001183) may be fused to the Fc region of an IgG and/or ACE-2-like polypeptides to ACE-2 binding molecule and used in a cell free assay to determine the polypeptide, and ability of ACE-2 binding polypeptides of the invention to 35 recovering the ACE-2 and/or ACE-2-like polypeptides. inhibit, increase, or not significantly alter, ACE-2 binding to A further embodiment of the present invention is a an ACE-2 substrate. Alternatively, ACE-2 can be biotiny method for isolating ACE-2 protein and/or ACE-2-like lated using techniques well known to those skilled in the art polypeptide from a Solution containing it, comprising: (e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.). The ACE-2 binding polypeptides of the invention 40 contacting the Solution with an ACE-2 binding polypep (including molecules comprising, or alternatively consisting tide under conditions that permit binding of ACE-2 of, ACE-2 binding polypeptide fragments or variants and/or ACE-2-like polypeptides to ACE-2 binding thereof), can also be assayed for their ability to inhibit, polypeptide, and Stimulate, or not significantly alter, ACE-2-induced enzy Separating the complex(es) formed by the ACE-2 binding matic activity using techniques known to those of skill in the 45 polypeptide and ACE-2 and/or ACE-2-like polypep art. For example, ACE-2 activity can be assayed by tides from other components of the Solution. H-thymidine incorporation assays and trypan blue cell Preferably such method also includes the further steps of: counts (see, e.g., Moore et al., Science, 285: 260-263 dissociating the ACE-2 binding polypeptide from the (1999)). Additionally, the ACE-2 binding polypeptides of ACE-2 and/or ACE-2-like polypeptides, and the invention, or fragments or variants thereof, can be 50 recovering the dissociated, ACE-2 and/or ACE-2-like assayed for their ability to inhibit, Stimulate, or not signifi polypeptide. cantly alter, ACE-2-induced cleavage of angiotensin using The invention also provides for kits containing a binding techniques known to those of skill in the art (see, e.g., Tipnis polypeptide of the invention for use in methods of detecting et al., Journal of Biological Chemistry 275:33238–33243 or isolating ACE-2 and/or ACE-2-like polypeptides. (2000)). For example, hydrolysis of angiotensin can be 55 According to the invention, detection or isolation of determined by analyzing cleavage products detected by high ACE-2 target proteins comprises contacting a Solution con performance liquid chromatography (HPLC). Further, the taining an ACE-2 target protein with an ACE-2 binding ACE-2 binding polypeptides of the invention, or fragments polypeptide. Depending on the particular application, the or varients thereof, can be assayed for their ability to inhibit, ACE-2 binding polypeptide may be free in Solution or Stimulate, or not significantly alter ACE-2 regulation of 60 immobilized on a Solid Support or chromatographic material. bradykinin, tachykinin, neurotensin, Substance P, and endot Sufficient time is allowed to permit binding between the helin Synthesis and/or cleavage using the same or similar ACE-2 target protein and the binding polypeptides, and techniques known to those of skill in the art. non-binding components in the Solution or mixture are The ACE-2 binding polypeptides of the invention, or removed or washed away. The formation of a binding fragments or variants thereof can also be assayed for their 65 complex between the binding polypeptide and the ACE-2 ability to neutralize, enhance, or not significantly alter, target protein can then be detected, for example, by detecting ACE-2 activity. For example, ACE-2 binding polypeptides the Signal from a label on the binding polypeptide, which is US 6,592,865 B2 S1 52 one component of the binding complex. A label may be any from the binding polypeptide under elution conditions and label that generates a signal that can be detected by Standard recovered in a purified form. methods, Such as a fluorescent label, a radioactive Methods of producing ACE-2 or ACE-2-like polypeptides compound, or an enzyme that reacts with a Substrate to usually yield ACE-2 or ACE-2-like polypeptides in a feed generate a detectable Signal. Suitable Such labels are dis Stream that additionally contains impurities (with respect to cussed above. A phage binding polypeptide according to the the ACE-2 target). One purpose of the present invention is invention, that is, a recombinant phage displaying an ACE-2 to produce ACE-2 binding polypeptides and preparations binding polypeptide on its Surface, may form a complex with (Such as affinity chromatography media or Surfaces) com ACE-2 and/or ACE-2-like polypeptides that is detectable as prising ACE-2 binding polypeptides that allow rapid and a precipitate or Sediment in a reaction tube, which can be highly specific purification of ACE-2 target proteins from a detected Visually after Settling or centrifugation. feed Stream. ACE-2 binding polypeptides obtained herein Alternatively, a Sandwich-type assay may be used, wherein may easily be tailored to isolate ACE-2 target protein from an ACE-2 binding polypeptide is immobilized on a Solid a particular feed Stream, using or routinely modifying con Support Such as a plastic tube or well, or a chromatographic ditions and techniques known in the art. If an alternate Support matrix Such as agarose beads, then the Solution 15 production method for ACE-2 is used, producing a different Suspected of containing the ACE-2 target is contacted with feed Stream, a different Set of ACE-2 binding polypeptides the immobilized binding polypeptide and non-binding mate and/or conditions may be necessary to achieve the same rials or components are removed or washed away. level of purification. The new set of ACE-2 binding polypep The binding polypeptides according to this invention are tides and/or conditions can be readily obtained following or particularly useful for detection and/or isolation of ACE-2 modifying procedures outlined herein, or otherwise known and/or ACE-2-like polypeptides by affinity chromatography in the art. methods. Any conventional method of chromatography may Use of ACE-2 Binding Polypeptides for Epitope Mapping be employed. Preferably, an ACE-2 binding polypeptide of The present invention provides ACE-2 binding polypep the invention will be immobilized on a solid support tides (including molecules comprising, or alternatively con Suitable, for example, for packing a chromatography col 25 Sisting of, ACE-2 binding polypeptide fragments or variants umn. The immobilized ACE-2 binding polypeptide affinity thereof), that can be used to identify epitopes of ACE-2. In ligand can then be loaded or contacted with a feed Stream particular, the ACE-2 binding polypeptides of the present under conditions favorable to formation of binding invention can be used to identify epitopes of human ACE-2 polypeptide/ ACE-2 (or ACE-2-like polypeptide) com (SEQ ID NOs: 138 and/or 142) or ACE-2 expressed on plexes. Non-binding materials can be washed away. human myocytes and/or proximal tubules and/or epithelial Examples of Suitable wash conditions can readily be deter cells using techniques described herein or otherwise known mined by one of skill in the art and include but are not in the art. Fragments which function as epitopes may be limited to PBS/0.01% Tween 20, pH7.2 and 1M NaCl/10 produced by any conventional means. (See, e.g., Houghten, mM Tris, pH7.5). Tris wash buffers may be preferable since Proc. Natl. Acad. Sci. USA, 82:5131–5135 (1985), further phosphates can preciptate in 50% ethylene glycol. In 35 described in U.S. Pat. No. 4,631,211.) general, non-limiting terms, wash buffers are pH 7.0, option Diagnostic Uses of ACE-2 Binding Polypeptides ally containing 0.0 to 1.5 M NaCl, more preferably 1M Labeled and non-labelled ACE-2 binding polypeptides of NaCl. Additionally, wash buffers may optionally contain a the invention (including molecules comprising, or alterna mild detrgenet, Such as, for example, Tween 20, Tween 80, tively consisting of, ACE-2 binding polypeptide fragments or NP-80. ACE-2 or ACE-2-like polypeptide can be eluted 40 or variants thereof) which specifically bind to ACE-2 can be from the ACE-2 binding polypeptide by introducing Solution used for diagnostic purposes to detect, diagnose, prognose, conditions that favor dissociation of the binding complex. or monitor diseases and/or disorders associated with the Suitable elution solutions can readily be determined by one aberrant expression and/or activity of ACE-2. The invention of skill in the art and include but are not limited to 50% provides for the detection of aberrant expression of ACE-2 ethylene glycol/100 mM NaOAc). By way of non-limiting 45 comprising: (a) assaying the expression of ACE-2 in a example, useful elution buffers, for the purposes of the biological Sample from an individual using one or more present invention contain 40-60% ethylene glycol, prefer ACE-2 binding polypeptides of the invention that Specifi ably 50% ethylene glycol.; and 50–100 mM NaOAc with a cally binds to ACE-2; and (b) comparing the level of ACE-2 pH in the range of pH 4-pH7, more preferably, pH 4-pH 6 with a Standard level of ACE-2, e.g., in normal biological and most preferably pH 4.5-pH 5.5. Preferably, a fast flow 50 Samples, whereby an increase or decrease in the assayed affinity chromatographic technique is used to bind the mol level of ACE-2 compared to the standard level of ACE-2 is ecules and from which purified ACE-2 or ACE-2-like indicative of aberrant expression. polypeptides are eluted. By “biological sample” is intended any fluids and/or cells Alternatively, batch chromatography can be carried out by obtained from an individual, body fluid, body tissue, body mixing a Solution containing the ACE-2 target and the 55 cell, cell line, tissue culture, or other Source which may ACE-2 binding polypeptide, then isolating complexes of the contain ACE-2 protein or mRNA. Body fluids include, but ACE-2 target and the binding polypeptides. For this type of are not limited to, Sera, plasma, urine, Synovial fluid, Spinal Separation, many methods are known. For example, the fluid, Saliva, and mucous. Tissues Samples may be taken binding polypeptide may be immobilized on a Solid Support from Virtually any tissue in the body. Tissue Samples may Such as beads, then Separated from the feed Stream along 60 also be obtained from autopsy material. Methods for obtain with the ACE-2 target by filtration. In another example, the ing tissue biopsies and body fluids from mammals are well ACE-2 binding polypeptide may be modified with its own known in the art. Where the biological sample is to include affinity tag, Such as a polyHis tail or Streptavidin binding mRNA, a tissue biopsy is the preferred Source. region, which can be used to isolate the binding polypeptide The invention also provides for the detection of aberrant after complexes have formed using an immobilized metal 65 expression and/or activity of ACE-2 Substrates (e.g., affinity chromatographic resin or Steptavidin-coated Sub angiotensin, bradykinin, tachykinin, neurotensin, Substance Strate. Once Separated, the ACE-2 target can be released P, and endothelin) comprising (a) assaying the expression of US 6,592,865 B2 S3 S4 ACE-2 Substrates in a biological Sample from an individual to ACE-2; and (b) comparing the level of ACE-2 substrate using one or more ACE-2 binding polypeptides or fragments with a Standard level of ACE-2 Substrate, e.g., in normal or variants thereof that specifically binds only to soluble biological Samples, whereby an increase or decrease in the ACE-2, but does not inhibit ACE-2/ACE-2 Substrate bind assayed level of ACE-2 Substrate compared to the Standard ing. Such an ACE-2 binding polypeptide, by way of an level of ACE-2 Substrate is indicative of a cardiovascular example that is not to be construed as limiting, would be one disease, disorder, or condition. In specific embodiments, an that is able to capture a biotinylated ACE-2 from solution, increase in the assayed level of ACE-2 Substrate is indicative but that would not prevent ACE-2 from binding to it receptor of a cardiovascular disease, disorder, or condition. In other expressed, for example on IM-9 cells, and (b) comparing the Specific embodiments, a decrease in the assayed level of level of ACE-2 Substrate with a standard level of ACE-2 ACE-2 Substrate is indicative of a cardiovascular disease, Substrate, e.g., in normal tissue or cell Samples, whereby an disorder, or condition. increase or decrease in the assayed level of ACE-2 Substrate Cardiac and cardiovascular disorders, diseases, or condi compared to the standard level of ACE-2 substrate is indica tions that may be detected, diagnosed, prognosed, or moni tive of aberrant expression. tored using the ACE-2 binding polypeptides of the invention Angiotensin II is a known potent vasoconstrictor, high 15 include, but are not limited to, arrhythmias, carcinoid heart concentrations of which are associated with Such diseaseS as disease, high cardiac output, low cardiac output, cardiac hypertension, congestive heart failure, and Several other tamponade, endocarditis (including bacterial), heart cardiovascular disorders. Production of angiotensin II is aneurysm, cardiac arrest, congestive heart failure, conges under control of the ACE enzyme, which competes with tive cardiomyopathy, paroxysmal dyspnea, cardiac edema, ACE-2 for a common Substrate, angiotensin. Thus, an an heart hypertrophy, congestive cardiomyopathy, left ventricu abnomally high agiotensin II level could result from abnor lar hypertrophy, right ventricular hypertrophy, post mally low activity of ACE-2, thereby allowing most of all of infarction heart rupture, Ventricular Septal rupture, heart the angiotensin to be converted to angiotensin II rather than Valve diseases, myocardial diseases, myocardial ischemia, angiotensin 1-9. Conversely, inappropriately low concentra pericardial effusion, pericarditis (including constrictive and tions of angiotensin II, which may play a role in hypotensive 25 tuberculous), pneumopericardium, postpericardiotomy disorders and shock, may result from inappropriately high Syndrome, pulmonary heart disease, rheumatic heart disease, activity of ACE-2, whereby most or all angiotensin is Ventricular dysfunction, hyperemia, cardiovascular preg converted to angiotensin 1-9 rather than angiotensin II. nancy complications, Scimitar Syndrome, cardiovascular Furthermore, angiotensin 1-9 potentiates the vasoconstrictor Syphilis, and cardiovascular tuberculosis. and pressor action of angiotensin II, indicating a role for Additional cardiovascular disorders, diseases, or condi angtiotensin 1-9 in the precise regulation of blood pressure tions that may be detected, diagnosed, prognosed, or moni and vascular constriction (see Example 9). tored by the ACE-2 binding polypeptides of the invention Thus, the ACE-2 binding polypeptides of the invention include, but are not limited to, arrhythmias, Such as Sinus (including molecules comprising, or alternatively consisting arrhythmia, atrial fibrillation, atrial flutter, bradycardia, of, ACE-2 binding polypeptide fragments or variants 35 extrasyStole, Adams-Stokes Syndrome, bundle-branch thereof) which specifically bind to ACE-2 can be used for block, Sinoatrial block, long QT Syndrome, parasyStole, diagnostic purposes to detect, diagnose, prognose, or moni Lown-Ganong-Levine Syndrome, Mahaim-type pre tor cardiovascular diseases and disorders, including but not excitation syndrome, Wolff-Parkinson-White syndrome, limited to hypertension, hypotension, and/or diseases, Sick Sinus Syndrome, tachycardias, and Ventricular fibrilla disorders, or conditions associated therewith. The invention 40 tion. Tachycardias include paroxy Small tachycardia, provides for the detection of aberrant expression of ACE-2 Supraventricular tachycardia, accelerated idioVentricular comprising: (a) assaying the expression of ACE-2 in a rhythm, atrioventricular nodal reentry tachycardia, ectopic biological Sample from an individual using one or more atrial tachycardia, ectopic junctional tachycardia, Sinoatrial ACE-2 binding polypeptides of the invention that Specifi nodal reentry tachycardia, Sinus tachycardia, TorSades de cally binds to ACE-2; and (b) comparing the level of ACE-2 45 Pointes, and Ventricular tachycardia. with a Standard level of ACE-2, e.g., in normal biological Additional cardiovascular disorders, diseases, or condi Samples, whereby an increase or decrease in the assayed tions that may be detected, diagnosed, prognosed, or moni level of ACE-2 compared to the standard level of ACE-2 is tored by the ACE-2 binding polypeptides of the invention indicative of a cardiovascular disease, disorder, or condition. include, but are not limited to heart Valve diseases, Such as In Specific embodiments, an increase in the assayed level of 50 aortic valve insufficiency, aortic valve Stenosis, hear ACE-2 is indicative of a cardiovascular disease, disorder, or murmurs, aortic valve prolapse, mitral valve prolapse, tri condition. In other Specific embodiments, a decrease in the cuspid valve prolapse, mitral valve insufficiency, mitral assayed level of ACE-2 is indicative of a cardiovascular Valve Stenosis, pulmonary atresia, pulmonary valve disease, disorder, or condition. insufficiency, pulmonary valve Stenosis, tricuspid atresia, ACE-2 binding polypeptides of the invention (including 55 tricuspid valve insufficiency, and tricuspid valve Stenosis. molecules comprising, or alternatively consisting of, ACE-2 Myocardial diseases that may be detected, diagnosed, binding polypeptide fragments or variants thereof) which prognosed, or monitored, using the ACE-2 binding polypep specifically bind to ACE-2 but do not inhibit ACE-2/ACE-2 tides of the invention include, but are not limited to alcoholic Substrate binding can be used for diagnostic purposes to cardiomyopathy, congestive cardiomyopathy, hypertrophic detect, diagnose, prognose, or monitor cardiovascular dis 60 cardiomyopathy, aortic Subvalvular Stenosis, pulmonary eases and disorders, including but not limited to hyperten Subvalvular Stenosis, restrictive cardiomyopathy, Chagas Sion and/or diseases, disorders, or conditions associated cardiomyopathy, endocardial fibroelastosis, endomyocardial therewith. The invention provides for the detection of aber fibrosis, Kearns Syndrome, myocardial reperfusion injury, rant expression of an ACE-2 Substrate comprising: (a) and myocarditis. assaying the expression of an ACE-2 Substrate in a biologi 65 Myocardial ischemias that may be detected, diagnosed, cal Sample from an individual using one or more ACE-2 prognosed, or monitored, using the ACE-2 binding polypep binding polypeptides of the invention that Specifically binds tides of the invention include, but are not limited to coronary US 6,592,865 B2 SS S6 disease, Such as angina pectoris, coronary aneurysm, coro node Syndrome, thromboangiitis obliterans, hyperSensitivity nary arteriosclerosis, coronary thrombosis, coronary vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasospasm, myocardial infarction and myocardial Stunning. vasculitis, and Wegener's granulomatosis. Additional cardiovascular diseases that may be detected, In a specific embodiment, the compositions of the present diagnosed, prognosed, ormonitored using the ACE-2 bind invention are used to detect, diagnose, prognose, or monitor ing polypeptides of the invention also include vascular hypertension. diseaseS Such as aneurysms, angiodysplasia, angiomatosis, In another specific embodiment, the compositions of the bacillary angiomatosis, Hippel-Lindau Disease, Klippel present invention are used to detect, diagnose, prognose, or Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, monitor congestive heart failure. angioneurotic edema, aortic diseases, Takayasu's Arteritis, In a further specific embodiment, the compositions of the aortitis, Leriche's Syndrome, arterial occlusive diseases, present invention are used to detect, diagnose, prognose, or arteritis, enarteritis, polyarteritis nodosa, cerebrovascular monitor hypotension. disorders, diabetic angiopathies, diabetic retinopathy, In an even further Specific embodiment, the compositions embolisms, thrombosis, erythromelalgia, hemorrhoids, of the present invention are used to detect, diagnose, hepatic veno-occlusive disease, hypertension, hypotension, 15 prognose, or monitor Shock. ischemia, peripheral vascular diseases, phlebitis, pulmonary Angiotensin II also Stimulates the release of aldosterone. veno-occlusive disease, Raynaud's disease, CREST Aldosterone is an adrenal cortex hormone that promotes Syndrome, retinal vein occlusion, Scimitar Syndrome, Supe retention of Salt and water by the kidneys, which increases rior Vena cava Syndrome, tel angiectasia, ataciatelangiectasi plasma Volume and, thereby, increases blood pressure. In a, hereditary hemorrhagic telangiectasia, Varicocele, Vari addition to chronic or acute hypertension and hypotension, cose veins, varicose ulcer, Vasculitis, and Venous insuffi aberrant action of aldosterone is known to cause Several ciency. renal disorders. AS discussed previously herein, angiotensin Aneurysms that may be detected, diagnosed, prognosed, II concentration can be correlated with activity or presence or monitored using the ACE-2 binding polypeptides of the of ACE-2. Thus, the ACE-2 binding polypeptides of the invention include, but are not limited to dissecting 25 invention (including molecules comprising, or alternatively aneurysms, false aneurysms, infected aneurysms, ruptured consisting of, ACE-2 binding polypeptide fragments or aneurysms, aortic aneurysms, cerebral aneurysms, coronary variants thereof) which specifically bind to ACE-2 can be aneurysms, heart aneurysms, and iliac aneurysms. used for diagnostic purposes to detect, diagnose, prognose, Arterial occlusive diseases that may be detected, or monitor diseases and disorders associated with aberrant diagnosed, prognosed, or monitored using the ACE-2 bind aldosterone action, including but not limited to renal dis ing polypeptides of the invention include, but are not limited eases and disorders, hypertension, hypotension, and/or to include, but are not limited to, arteriosclerosis, intermit diseases, disorders, or conditions associated there with. The tent claudication, carotid Stenosis, fibromuscular dysplasias, invention provides for the detection of aberrant expression mesenteric vascular occlusion, Moyamoya disease, renal of ACE-2 comprising: (a) assaying the expression of ACE-2 artery obstruction, retinal artery occlusion, and throm 35 in a biological Sample from an individual using one or more boangiitis obliterans. ACE-2 binding polypeptides of the invention that Specifi Cerebrovascular disorders that may be detected, cally binds to ACE-2; and (b) comparing the level of ACE-2 diagnosed, prognosed, or monitored using the ACE-2 bind with a Standard level of ACE-2, e.g., in normal biological ing polypeptides of the invention include, but are not limited Samples, whereby an increase or decrease in the assayed to, carotid artery diseases, cerebral amyloid angiopathy, 40 level of ACE-2 compared to the standard level of ACE-2 is ce rebral aneurysm, cerebral anoxia, cerebral indicative of a renal disease, disorder, or condition. In arteriosclerosis, cerebral arteriovenous malformation, cere Specific embodiments, an increase in the assayed level of bral artery diseases, cerebral embolism and thrombosis, ACE-2 is indicative of a renal disease, disorder, or condition. carotid artery thrombosis, sinus thrombosis, Wallenberg's In other specific embodiments, a decrease in the assayed Syndrome, cerebral hemorrhage, epidural hematoma, Sub 45 level of ACE-2 is indicative of a renal disease, disorder, or dural hematoma, SubaraXhnoid hemorrhage, cerebral condition. infarction, cerebral ischemia (including transient), Subcla ACE-2 binding polypeptides of the invention (including vian Steal Syndrome, periventricular leukomalacia, Vascular molecules comprising, or alternatively consisting of, ACE-2 headache, cluster headache, migraine, and vertebrobasilar binding polypeptide fragments or variants thereof) which insufficiency. 50 specifically bind to ACE-2 but do not inhibit ACE-2/ACE-2 Embolisms that may be detected, diagnosed, prognosed, Substrate binding can be used for diagnostic purposes to or monitored using the ACE-2 binding polypeptides of the detect, diagnose, prognose, or monitor diseases and disor invention include, but are not limited to air embolisms, derS associated with aberrant aldosterone activity, including amniotic fluid embolisms, cholesterol embolisms, blue toe but not limited to renal diseases and/or disorders, hyperten Syndrome, fat embolisms, pulmonary embolisms, and thro 55 Sion and/or diseases, disorders, or conditions associated moboembolisms. Thrombosis include, but are not limited to, therewith. The invention provides for the detection of aber coronary thrombosis, hepatic vein thrombosis, retinal vein rant expression of an ACE-2 Substrate comprising: (a) occlusion, carotid artery thrombosis, Sinus thrombosis, Wal assaying the expression of an ACE-2 Substrate in a biologi lenberg's Syndrome, and thrombophlebitis. cal Sample from an individual using one or more ACE-2 Ischemic disorders that may be detected, diagnosed, 60 binding polypeptides of the invention that Specifically binds prognosed, or monitored using the ACE-2 binding polypep to ACE-2; and (b) comparing the level of ACE-2 substrate tides of the invention include, but are not limited to, cerebral with a Standard level of ACE-2 Substrate, e.g., in normal ischemia, ischemic colitis, compartment Syndromes, ante biological Samples, whereby an increase or decrease in the rior compartment Syndrome, myocardial ischemia, reperfu assayed level of ACE-2 Substrate compared to the Standard Sion injuries, and peripheral limb ischemia. Vasculitis 65 level of ACE-2 Substrate is indicative of a renal disease, includes, but is not limited to, aortitis, arteritis, Behcet's disorder, or condition. In Specific embodiments, an increase Syndrome, Churg-StrauSS Syndrome, mucocutaneous lymph in the assayed level of ACE-2 Substrate is indicative of a US 6,592,865 B2 57 58 renal disease, disorder, or condition. In other Specific In another specific embodiment, the present invention embodiments, a decrease in the assayed level of ACE-2 encompasses methods and compositions for detecting, diag Substrate is indicative of a renal disease, disorder, or con nosing and/or prognosing diseases or disorders of epithelial dition. cells. Renal disorders, diseases, and/or conditions that may be In further embodiments, the present invention encom detected, diagnosed, prognosed, monitored, treated, passes methods and compositions for detecting, diagnosing, prevented, and/or ameliorated using the ACE-2 binding prognosing and or monitoring growth, progression, and/or polypeptides of the invention include, but are not limited to metastases of malignancies and proliferative diseases or acute kidney failure, chronic kidney failure, atheroembolic renal failure, end-stage renal disease, inflammatory diseases disorders associated with increased cell Survival, or the of the kidney (e.g., acute glomerulonephritis, postinfectious inhibition of apoptosis. For a review of Such disorders, See glomerulonephritis, rapidly progressive glomerulonephritis, Fishman et al., Medicine, 2d Ed. (J. B. Lippincott Co., nephrotic Syndrome, membranous glomerulonephritis, Philadelphia 1985). Proliferative diseases and disorders is familial nephrotic Syndrome, membranoproliferative glom also extended to include premalignant conditions (e.g., erulo nephritis I and II, me Sangial prolife rative benign tumors, hyperproliferative disorders, and benign glomerulonephritis, chronic glomerulonephritis, acute tubu 15 proliferative disorders-see below) as well as proliferative lointerstitial nephritis, chronic tubulointerstitial nephritis, disorders of Smooth muscle cells and endothelial cells. Other acute post-Streptococcal glomerulonephritis (PSGN), abnormal growth conditions that may be treated, diagnosed, pyelonephritis, lupus nephritis, chronic nephritis, interstitial prognosed or monitored include, but are not limited to, nephritis, and post-Streptococcal glomerulonephritis), blood hyperplasia, metaplasia, or most particularly, dysplasia has vessel disorders of the kidneys (e.g., kidney infarction, occurred (for review of Such abnormal growth conditions, atheroembolic kidney disease, cortical necrosis, malignant see Robbins and Angell, Basic Pathology, 2d Ed. (W. B. nephrosclerosis, renal vein thrombosis, renal Saunders Co., Philadelphia 1976), pp. 68-79.) Hyperplasia underperfusion, renal retinopathy, renal ischemia is a form of controlled cell proliferation involving an reperfusion, renal artery embolism, and renal artery increase in cell number in a tissue or organ, without signifi Stenosis), and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria, hyponatremia, 25 cant alteration in Structure or function. AS but one example, hypernatremia, hypokalemia, hyperkalemia, hypocalcemia, endometrial hyperplasia often precedes endometrial cancer. hyper calcemia, hypophosphate mia, and Metaplasia is a form of controlled cell growth in which one hyperphosphatemia). type of adult or fully differentiated cell substitutes for In a further embodiment, the ACE-2 binding polypeptides another type of adult cell. Metaplasia can occur in epithelial of the present invention (including molecules comprising, or or connective tissue cells. Atypical metaplasia involves a alternatively consisting of, ACE-2 binding polypeptide frag Somewhat disorderly metaplastic epithelium. Dysplasia is ments or variants thereof) which specifically bind to ACE-2 frequently a forerunner of cancer, and is found mainly in the can be used for diagnostic purposes to detect, diagnose, epithelia; it is the most disorderly form of non-neoplastic prognose, or monitor diseases and/or disorders associated cell growth, involving a loSS in individual cell uniformity with cell proliferation. Smooth muscle cell proliferation in 35 and in the architectural orientation of cells. Dysplastic cells the intima of muscular arteries is the primary cause of often have abnormally large, deeply Stained nuclei, and restenosis after vascular Surgery (e.g., angioplasty) and in exhibit pleomorphism. Dysplasia characteristically occurs atherosclerosis. Several animal Studies have indicated that where there exists chronic irritation or inflammation, and is the renin-angiotensin System plays an important role in this often found in the cervix, respiratory passages, oral cavity, vascular response. Specifically, it has been shown that 40 and gallbladder. chronic treatment with inhibitors of ACE (e.g., compositions In another specific embodiment, the present invention analagous to Enalapril, Ramipril, and Captopril) reduces encompasses methods and compositions for detecting, diag myometrial thickening after balloon injury in rat carotid nosing and/or prognosing growth, progression, and/or artery or aorta (Powell et al., Journal of the American metastases of Smooth muscle cells. College of Cardiology 17: 137B-142B (1991)). Further, it is 45 In another specific embodiment, the present invention known that angiotensin II Stimulates cell growth and repli encompasses methods and compositions for detecting, diag cation in the cardiovascular System through binding angio nosing and/or prognosing growth, progression, and/or tensin II receptors (Rosendorff, Journal of the American metastases of epithelial cells. College of Cardiology 28:803 (1996)). Thus, the composi AS discussed elsewhere herein, bradykinin are believed to tions of the present invention may be used to detect, 50 also be peptide substrates of ACE-2. Bradykinin are diagnose, prognose, or montior diseases and disorders asso involved in inflammatory reactions of various tissues. For ciated with cell proliferation including, but not limited to, example, in the intestine bradykinin Stimulate contraction of Senosis, (e.g., buttonhole Stenosis, coronary ostial Stenosis, Smooth muscle and Secretion of ions and fluid in response to double aortic Stenosis, fish-mouth mitral Stenosis, bronchial injury (Manning et al., Nature 229: 256 (1982)). Thus, in a Stenosis, hypertrophic pyloric Stenosis, pyloric Stenosis, 55 another embodiment, the ACE-2 binding polypeptides of the infundibular Stenosis, idiopathic hypertrophic Subaortic present invention (including molecules comprising, or alter Stenosis, idiopathic Subglottic Stenosis, pulmonary Stenosis, natively consisting of, ACE-2 binding polypeptide frag muscular Subaortic Stenosis, laryngeal Stenosis, mitral ments or variants thereof) which specifically bind to ACE-2 Stenosis, Supravalvar and Subvalvar Stenosis, Subvalvular can be used for diagnostic purposes to detect, diagnose, and Supravalvular Stenosis, and tricuspid stenosis), myome 60 prognose, or monitor inflammation and diseases and/or trial hypertrophy, hypertrophy or hyperplasia of conduit and disorders associated therewith. Such conditions include, but resistance vessels, atherOSclerosis, and Several forms of are in no way limited to, inflammation associated with cancer and neoplastic disorders. infection (e.g., Septic shock, Sepsis, or Systemic inflamma In a Specific embodiment, the present invention encom tory response Syndrome (SIRS)), ischemia-reperfusion passes methods and compositions for detecting, diagnosing 65 injury, acute idiopathic inflammation, alterative and/or prognosing diseases or disorders of Smooth muscle inflammation, atrophic inflammation, catarrhal cells. inflammation, chronic and chronic active inflammation, US 6,592,865 B2 59 60 fibrinopurulent inflammation, graulomatous inflammation, detecting the labeled ACE-2 binding polypeptide in the immune inflammation, interstitial inflammation, necrotic subject, such that detection of labeled ACE-2 binding inflammation, proliferative inflammation, pseudomembra polypeptide or fragment thereof above the background level nous inflammation, purulent inflammation, Serofibrinous and above or below the level observed in a person without inflammation, polytrauma, pain, endotoxin lethality, arthritis the disease or disorder indicates that the Subject has a (e.g., osteoarthritis and rheumatoid arthritis), corhplement particular disease or disorder associated with aberrant mediated hyperacute rejection, nephritis, cytokine or expression of ACE-2 or ACE-2 substrate. Background level chemokine induced lung injury, inflammatory bowel can be determined by various methods, including comparing disease, Crohn's disease, and resulting from Over production the amount of labeled molecule detected to a Standard value of cytokines (e.g., TNF or IL-1). previously determined for a particular System. The invention provides a diagnostic assay for diagnosing It will be understood by those skilled in the art that the or prognosing a disease or disorder, comprising: (a) assaying Size of the Subject and the imaging System used will deter for the level of ACE-2 in a biological sample of an indi mine the quantity of imaging moiety needed to produce vidual using one or more ACE-2 binding polypeptides of the diagnostic images. In the case of a radioisotope moiety, for invention that specifically bind to ACE-2, and (b) comparing 15 a human Subject, the quantity of radioactivity injected will the level of ACE-2 with a standard ACE-2 level, e.g., in a normally range from about 5 to 20 millicuries of 'Tc. The biological Sample from a patient without the disease or labeled ACE-2 binding polypeptide will then preferentially disorder, whereby an increase or decrease in the assayed accumulate at the location of cells which contain the Specific ACE-2 level compared to the standard level of ACE-2 is protein. In Vivo tumor imaging is described in Burchiel et indicative of a particular disease or disorder. With respect to al., “Immunopharmacokinetics of Radiolabeled Antibodies cancer, the presence of a relatively high amount of ACE-2 in and Their Fragments,” Chapter 13 in Tumor Imaging. The biopsied tissue from an individual may indicate a predispo Radiochemical Detection of Cancer, S. W. Burchiel and B. Sition for the development of the disease, or may provide a A. Rhodes, eds., Masson Publishing Inc. (1982). means for detecting the disease prior to the appearance of Depending on Several variables, including the type of actual clinical Symptoms. A more definitive diagnosis of this 25 label used and the mode of administration, the time interval type may allow health professionals to employ preventative following the administration for permitting the labeled mol measures or aggressive treatment earlier thereby preventing ecule to preferentially concentrate at Sites in the Subject and the development or further progression of the cancer. for unbound labeled molecule to be cleared to background ACE-2 binding polypeptides of the invention (including level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In molecules comprising, or alternatively consisting of, ACE-2 another embodiment the time interval following administra binding polypeptide fragments or variants thereof) can be tion is 5 to 20 days or 5 to 10 days. used to assay protein levels in a biological Sample using In an embodiment for monitoring of the disease or classical immunohistological methods as described herein or disorder, the method is carried out by repeating the method as known to those of skill in the art (e.g., see Jalkanen et al., for diagnosing the disease or disorder, for example, one J. Cell. Biol., 101:976–985 (1985); Jalkanen et al., J. Cell. 35 month after initial diagnosis, Six months after initial Biol., 105:3087–3096 (1987)). Other methods that can be diagnosis, one year after initial diagnosis, etc. and compar used for detecting protein gene expression that might utilize ing the results of the Successive tests. ACE-2 binding polypeptides or fragments or variants Presence of the labeled molecule can be detected in the thereof include, but are not limited to, the enzyme linked patient using methods known in the art for in Vivo Scanning. immunosorbent assay (ELISA) and the radioimmunoassay 40 These methods depend upon the type of label used. Skilled (RIA). Suitable antibody assay labels are known in the art artisans will be able to determine the appropriate method for and include enzyme labels, Such as, glucose oxidase, alka detecting a particular label. Methods and devices that may line phophatase, and horseradish peroxidase; radioisotopes, be used in the diagnostic methods of the invention include, such as iodine (I, I, I, ''I), carbon (''C), sulfur but are not limited to, computed tomography (CT), whole (S), tritium (H), indium ('In, ''In, In, "In), 45 body Scan Such as position emission tomography (PET), technetium ('Tc.'"Tc), thallium ('Ti), gallium (Ga, magnetic resonance imaging (MRI), and Sonography. 7Ga), palladium (Pd), molybdenum (Mo), xenon In a specific embodiment, the molecule is labeled with a (*Xe), fluorine (F), Sm, 77Lu, Gd, 'Pm, 'La, radioisotope and is detected in the patient using a radiation 175Yb. 166Ho, 90Y, *7Sc, 8Re, 88Re, 42Pr, 105Rh, and 97Ru; responsive Surgical instrument (see, e.g., Thurston et al., luminescent labels, Such as luminol; and fluorescent labels, 50 U.S. Pat. No. 5,441,050). In another embodiment, the mol Such as fluorescein and rhodamine, and biotin. ecule is labeled with a fluorescent compound and is detected Certain embodiments of the invention are directed to the in the patient using a fluorescence responsive Scanning detection and diagnosis of a disease or disorder associated instrument. In another embodiment, the molecule is labeled with aberrant expression of ACE-2 or ACE-2 substrate in an with a positron emitting metal and is detected in the patient animal, preferably a mammal and most preferably a human. 55 using positron emission-tomography. In yet another In one embodiment, diagnosis comprises: (a) administering embodiment, the molecule is labeled with a paramagnetic (for example, parenterally, Subcutaneously, or label and is detected in a patient using magnetic resonance intraperitoneally) to a Subject an effective amount of a imaging (MRI). labeled ACE-2 binding polypeptide of the invention Immunophenotyping Using ACE-2 Binding Polypeptides (including molecules comprising, or alternatively consisting 60 The ACE-2 binding polypeptides of the invention of, ACE-2,binding polypeptide fragments or variants (including molecules comprising, or alternatively consisting thereof) that specifically binds to ACE-2; (b) waiting for a of, ACE-2 binding polypeptide fragments or variants time interval following the administering for permitting the thereof) may be utilized for immunophenotyping of cell labeled ACE-2 binding polypeptide to preferentially con lines and biological Samples by their ACE-2 expression or centrate at Sites in the Subject where ACE-2 is expressed 65 ACE-2 Substrate expression. Various techniques can be (and for unbound labeled molecule to be cleared to back employed utilizing ACE-2 binding polypeptides, fragments, ground level); (c) determining background level; and (d) or variants of the invention to Screen for cellular populations US 6,592,865 B2 61 62 (i.e., cardiac myocytes, proximal convoluted tubules, endot includes, but is not limited to, alleviating Symptoms asso helial cells, and epithelial cells of Bowman's capsule) ciated with those diseases, disorders or conditions. ACE-2 expressing ACE-2 or ACE-2 Substrate. Such techniques binding polypeptides of the invention may be provided in include magnetic Separation using ACE-2 binding pharmaceutically acceptable compositions as known in the polypeptide-coated magnetic beads, “panning with ACE-2 art or as described herein. binding polypeptide attached to a Solid matrix (i.e., plate), ACE-2 binding polypeptides of the present invention and flow cytometry (see, e.g., U.S. Pat. No. 5,985,660; and (including molecules comprising, or alternatively consisting Morrison et al., Cell, 96:737-49 (1999)). These techniques of, ACE-2 binding polypeptide fragments or variants allow for the Screening of particular populations of cells. thereof) that function as agonists or antagonists of ACE-2, In one embodiment, ACE-2 binding polypeptides of the preferably of ACE-2-induced signal transduction, can be invention (including molecules comprising, or alternatively administered to an animal to treat, prevent or ameliorate a consisting of, ACE-2 binding polypeptide fragments or disease or disorder associated with aberrant ACE-2 variants thereof) are used to identify cells, Such as cardiac expression, lack of ACE-2 function, aberrant ACE-2 Sub myocytes, proximal convoluted tubules, endothelial cells, Strate expression, or lack of ACE-2 Substrate function. For and epithelial cells of Bowman's capsule. 15 example, ACE-2 binding polypeptides of the invention Therapeutic Uses of Peptide Compositions of the Invention which disrupt the interaction between ACE-2 and one or Co-administration of Angiotensin 1-9 and Angiotensin II more of its Substrates may be administered to an animal to had the Surprising result of increasing vasoconstriction to a treat, prevent or ameliorate a disease or disorder associated Significantly greater amount than the Sum of the individual with aberrant ACE-2 expression, excessive ACE-2 function, peptides administered alone, Suggesting a Superadditive or aberrant ACE-2 Substrate expression, or excessive ACE-2 Synergistic interaction (see Example 9). This effect has great Substrate function. ACE-2 binding polypeptides of the therapeutic potential in treating diseases and disorders invention which do not prevent ACE-2 from binding its related to inappropriate low blood pressure. Thus, in pre Substrate but inhibit or downregulate ACE-2-induced signal ferred embodiments for raising blood pressure, peptide transduction can be administered to an animal to treat, compositions of the invention comprise angiotensin 1-9 and 25 prevent or ameliorate a disease or disorder associated with angiotensin II used, for example, in combination with aberrant ACE-2 expression, excessive ACE-2 function, aber eachother, either Sequentially or Simultaneously. Thus, the rant ACE-2 Substrate expression, or excessive ACE-2 Sub Therapeutic/Prophylactic Compositions and Administration Strate function. In particular, ACE-2 binding polypeptides of described below for ACE-2 binding peptides (e.g., dosage the present invention which prevent ACE-2-induced signal amounts and regimens, routes of adminSitration, and transduction by Specifically recognizing the unbound ACE co-administered agents), are applied, in accordance with the 2, Substrate-bound ACE-2, or both unbound and Substrate invention, to Angiotensin 1-9 and Angiotensin II as well, in bound ACE-2 can be administered to an animal to treat, embodiments relating to raising blood pressure. prevent or ameliorate a disease or disorder associated with The present invention is further directed to therapies with aberrant ACE-2 expression, excessive ACE-2 function, aber involve administering angiotensin 1-9/angiotensin 1-9-like 35 rant ACE-2 Substrates expression, or excessive ACE-2 Sub polypeptides in conjunction with angiotensin II/angiotensin Strates function. II-like polypeptides to an animal, preferably a mammal, and The ability of an ACE-2 binding polypeptide of the most preferably a human, patient for treating one or more of invention to inhibit or downregulate ACE-2-induced signal the disclosed diseases, disorders, or conditions. transduction may be determined by techniques described In a preferred embodiment, angiotensin 1-9/angiotensin 40 herein or otherwise known in the art. For example, ACE-2- 1-9-like polypeptides in conjunction with angiotensin induced cleavage of ACE-2 Substrates can be determined by II/angiotensin II-like polypeptides is administered to an detecting cleavage products via high performance liquid animal, preferably a mammal, and most preferably a human, chromatography (HPLC). patient for treating diseases and/or disorders associated with In a specific embodiment, an ACE-2 binding polypeptide hypotension, including, for example, shock, Syncope, and 45 of the present invention (including molecules comprising, or others as listed herein. alternatively consisting of, ACE-2 binding polypeptide frag The present invention is further directed to ACE-2 bind ments or variants thereof) that inhibits or reduces ACE-2 ing polypeptide-based therapies which involve administer activity by at least 95%, at least 90%, at least 85%, at least ing ACE-2 binding polypeptides of the invention (including 80%, at least 75%, at least 70%, at least 60%, at least 50%, molecules comprising, or alternatively consisting of, ACE-2 50 at least 45%, at least 40%, at least 45%, at least 35%, at least binding polypeptide fragments or variants thereof) to an 30%, at least 25%, at least 20%, or at least 10% relative to animal, preferably a mammal, and most preferably a human, ACE-2 activity in the absence of the ACE-2 binding patient for treating one or more of the disclosed diseases, polypeptide, is administered to an animal to treat, prevent or disorders, or conditions. Therapeutic compounds of the ameliorate a disease or disorder associated with aberrant invention include, but are not limited to, ACE-2 binding 55 ACE-2 expression, excessive ACE-2 function, aberrant polypeptides of the invention and nucleic acids encoding ACE-2 receptor expression, or excessive ACE-2 receptor ACE-2 binding polypeptides of the invention and antibodies function. In another embodiment, a combination of ACE-2 that bind ACE-2 binding polypeptides of the invention as binding polypeptides, a combination of ACE-2 binding described herein. The ACE-2 binding polypeptides of the polypeptide fragments, a combination of ACE-2 binding invention can be used to treat, ameliorate or prevent 60 polypeptide variants, or a combination of ACE-2 binding diseases, disorders or conditions associated with aberrant polypeptides, ACE-2 binding polypeptide fragments, and/or expression and/or activity of ACE-2 or ACE-2 substrates, variants that inhibit or reduce ACE-2 activity by at least including, but not limited to, any one or more of the diseases, 95%, at least 90%, at least 85%, at least 80%, at least 75%, disorders, or conditions described herein. The treatment at least 70%, at least 65%, at least 60%, at least 55%, at least and/or prevention of diseases, disorders, or conditions asso 65 50%, at least 45%, at least 40%, at least 45%, at least 35%, ciated with aberrant ACE-2 expression and/or activity or at least 30%, at least 25%, at least 20%, or at least 10% aberrant ACE-2 Substrate expression and/or activity relative to ACE-2 activity in absence of said ACE-2 binding US 6,592,865 B2 63 64 polypeptides, ACE-2 binding polypeptide fragments, and/or proliferative diseases), inflammatory diseases (e.g., SIRS ACE-2 binding polypeptide variants are administered to an (Systemic Inflammatory Response Syndromes), Sepsis, animal to treat, prevent or ameliorate a disease or disorder polytrauma, inflammatory bowl disease, acute and chronic asSociated with aberrant ACE-2 expression, excessive pain, rheumatoid arthritis, and osteoarthritis), allergic dis ACE-2 function, aberrant ACE-2 Substrate expression, or orders (e.g., asthma, adult respiratory distress Syndrome, excessive ACE-2 Substrate function. wound healing, and Scar formation), as well as Several other Further, ACE-2 binding polypeptides of the present inven disorders and/or diseases (e.g., periodontal disease, dySmenorrhea, premature labor, brain edema following focal tion (including molecules comprising, or alternatively con injury, diffuse axonal injury, and reperfusion injury). Sisting of, ACE-2 binding polypeptide fragments or variants In a specific embodiment, the present invention provides thereof) which activate ACE-2-induced signal transduction a method of treating, preventing or ameliorating diseases or can be administered to an animal to treat, prevent or ame disorders associated with hypertension, comprising admin liorate a disease or disorder associated with aberrant ACE-2 istering to an animal in which Such treatment, prevention, or expression, lack of ACE-2 function, aberrant ACE-2 Sub amelioration is desired, an ACE-2 binding polypeptide in an Strate expression, or lack of ACE-2 Substrate function. These amount effective to treat, prevent or ameliorate the disease ACE-2 binding polypeptides may potentiate or activate 15 or disorder. Diseases and disorders associated with hyper either all or a subset of the biological activities of ACE-2- tension include, for example, accelerated hypertension, epi mediated Substrate action, for example, by regulating cleav Sodic hypertension, paroxysmal hypertension, portal age or synthesis of ACE-2 substrates. The ACE-2 binding hypertension, primary hypertension, Secondary polypeptides of the invention may be administered with or hypertensoin, Systemic venous hypertension, borderline without being pre-complexed with ACE-2. In a specific hypertension, adrenal hypertension, benign hypertension, embodiment, an ACE-2 binding polypeptide of the present idiopathic hypertension, pale hypertension, postpartm invention that increases ACE-2 activity by at least 5%, at hypertension, pregnancy-induced hypertension (gestational least 10%, at least 15%, at least 20%, at least 25%, at least hypertension), essential hypertension, labile hypertension, 30%, at least 35%, at least 40%, at least 45%, at least 50%, pulmonary hypertension, renal and renovascular at least 55%, at least 60%, at least 65%, at least 70%, at least 25 hypertension, and Goldblatt hypertension, left ventricular failure, atherosclerotic heart disease, Stroke, retinal hemor 75%, at least 80%, at least 85%, at least 90%, at least 95%, rhage or infarction (Keith-Wagener-Barker changes), renal at least 99%, or 100% or more relative to ACE-2 activity in failure, renovascular disease, chronic heart failure, exudates, absence of the ACE-2 binding polypeptide is administered to papilledema, Vascular accidents, myocardial infarction, dis an animal to treat, prevent or ameliorate a disease or disorder Secting aneurysm associated with aberrant ACE-2 expression, lack of ACE-2 In a specific embodiment, the present invention provides function, aberrant ACE-2 Substrate expression, or lack of a method of treating, preventing or ameliorating diseases or ACE-2 Substrate function. In another embodiment, a com disorders associated with hypotension, comprising admin bination of ACE-2 binding polypeptides, a combination of istering to an animal in which such treatment, prevention, or ACE-2 binding polypeptide fragments, a combination of amelioration is desired, an ACE-2 binding polypeptide in an ACE-2 binding polypeptide variants, or a combination of 35 amount effective to treat, prevent or ameliorate the disease ACE-2 binding polypeptides, ACE-2 binding polypeptide or disorder. Diseases and disorders associated with hypoten fragments and/or ACE-2 binding polypeptide variants that Sion include, for example, arterial hypotension, idiopathic increase ACE-2 activity by at least 5%, at least 10%, at least orthostatic hypotension, intracranial hypotension, orthos 15%, at least 20%, at least 25%, at least 30%, at least 35%, tatic hypotension, induced or controlled hypotension, shock at least 40%, at least 45%, at least 50%, at least 55%, at least 40 (e.g., anaphylactic shock, anaphylactiod shock, anestetic 60%, at least 65%, at least 70%, at least 75%, at least 80%, Shock, cardiogenic shock, chronic Shock, deferred or at least 85%, at least 90%, at least 95%, at least 99%, or delayed shock, hemorrhagic Shock, hypovolemic shock, 100% or more relative to ACE-2 activity in absence of the oligemic shock, Septic Shock, and vasogenic shock), and said ACE-2 binding polypeptides or ACE-2 binding Syncope (e.g., local Syncope, postural Syncope, tussive polypeptide fragments and/or ACE-2 binding polypeptide 45 Syncope, and vasodepressor Syncope). variants is administered to an animal to treat, prevent or One or more ACE-2 binding polypeptides of the present ameliorate a disease or disorder associated with aberrant invention (including molecules comprising, or alternatively ACE-2 expression, lack of ACE-2 function, aberrant ACE-2 consisting of, ACE-2 binding polypeptide fragments or Substrate expression, or lack of ACE-2 Substrate function. variants thereof) that specifically bind to ACE-2 may be In a specific embodiment, the present invention provides 50 used locally or Systemically in the body as a therapeutic. The a method of treating, preventing or ameliorating a disease or ACE-2 binding polypeptides of the invention (including disorder associated with aberrant ACE-2 or ACE-2 Substrate molecules comprising, or alternatively consisting of, ACE-2 expression or activity, comprising administering to an ani binding polypeptide fragments or variants thereof) may also mal in which Such treatment, prevention or amelioration is be advantageously utilized in combination with monoclonal desired, an ACE-2 binding polypeptide in an amount effec 55 or chimeric antibodies, lymphokines and/or hematopoietic tive to treat, prevent or ameliorate the disease or disorder. growth factors (Such as, e.g., IL-2, IL-3 and IL-7), for Diseases and disorders which may be treated, prevented or example, which Serve to increase the number or activity of ameliorated by this method include, but are not limited to, effector cells which interact with the ACE-2 binding cardiovascular disorders (e.g., hypertension, chronic heart polypeptides. failure, left Ventricular failure, Stroke, cerebral vasospasm 60 The ACE-2 binding polypeptides of the invention after Subarachnoid injury, atherosclerotic heart disease, and (including molecules comprising, or alternatively consisting retinal hemorrhage), renal disorders (e.g., renal vein of, ACE-2 binding polypeptide fragments or variants thrombosis, kidney infarction, renal artery embolism, renal thereof) may be administered alone or in combination with artery Stenosis, and edema, hydronephritis), proliferative other types of treatments (e.g., radiation therapy, diseases or disorders (e.g., vascular Stenosis, myocardial 65 chemotherapy, hormonal therapy, immunotherapy, anti hypertrophy, hypertrophy and/or hyperplasia of conduit and/ tumor agents, anti-angiogenesis and anti-inflammatory or resistance vessels, myocyte hypertrophy, and fibroblast agents). US 6,592,865 B2 65 66 It is preferred to use high affinity and/or potent in vivo forms of ACE-2. In another preferred embodiment, ACE-2 inhibiting and/or neutralizing ACE-2 binding polypeptides binding polypeptides of the invention inhibit or reduce the of the invention (including molecules comprising, or alter production of Angiotensin 1-9 and other products of ACE-2 natively consisting of, ACE-2 binding polypeptide frag action induced by either or both forms of ACE-2. ments or variants thereof) that specifically bind to ACE-2, or In one embodiment, the invention provides a method of polynucleotides encoding ACE-2 binding polypeptides that delivering radiolabelled ACE-2 binding polypeptide and/or specifically bind to ACE-2, for both immunoassays directed ACE-2 binding polypeptide conjugates of the invention to to and therapy of disorders related to ACE-2 polynucleotides targeted cells, Such as, for example, cardiac myocytes cells or polypeptides, including fragments thereof. Such ACE-2 expressing the membrane-bound form of ACE-2, or proxi binding polypeptides will preferably have an affinity for mal tubules expressing an ACE-2 Substrate. ACE-2 and/or ACE-2 fragments. Preferred binding affinities In one embodiment, the invention provides methods and include those with a dissociation constant or K of less than compositions for inhibiting or reducing angiotensin 1-9 or equal to 5x10° M, 10 M, 5x10 M, 10 M, 5x10' production, comprising, or alternatively consisting of, con M, 10 M, 5x10 M, or 10 M. More preferably, ACE-2 tacting an effective amount of ACE-2 binding polypeptide binding polypeptides of the invention bind ACE-2 target 15 with ACE-2, wherein the effective amount of ACE-2 binding proteins with a dissociation constant or K, less than or equal polypeptide inhibits or reduces ACE-2 mediated production to 5x10 M, 10 M, 5x107M, 107M,5x10 M, or 10 of angiotensin 1-9. In another embodiment, the invention M. Even more preferably, ACE-2 binding polypeptides of provides methods and compositions for inhibiting or reduc the invention bind ACE-2 target proteins with a dissociation ing angiotensin 1-9 production, comprising, or alternatively constant or K, less than or equal to 5x10 M, 10M, consisting of, administering to an animal in which Such 5x100 M, 5x10-11 M, 10-11 M, 5x10-12 M, 10-12 M, inhibition or reduction is desired, an ACE-2 binding 5x-13M, 10-13M, 5x10-14 M, 101* M, 5x10-15 M, 10-15 polypeptide in an amount effective to inhibit or reduce M. production of angiotensin 1-9. In a preferred embodiment, ACE-2 binding polypeptides Additionally, angiotensin 1-9 can be hydrolyzed by ACE of the invention neutralize ACE-2 activity. In another pre 25 into angiotensin 1-5. Thus, in another embodiment, the ferred embodiment, ACE-2 binding polypeptides of the present invention provides methods and compositions for invention inhibit the activity of ACE-2 substrates, including, inhibiting or reducing angiotensin 1-5 production, for example, angiotensin, bradykinin, tachykinin, comprising, or alternatively consisting of, contacting an neurotensin, Substance P, and endothelin. In a further effective amount of ACE-2 binding polypeptide with ACE embodiment, the ACE-2 binding polypeptides of the inven 2, wherein the effective amount of ACE-2 binding polypep tion are administered in conjunction with an agent known to tide inhibits or reduces ACE-2 mediated production of inhibit ACE (e.g., Enalapril(R), Captopril (R, Fosinopril(R), angiotensin 1-5. In another embodiment, the invention pro and Pramipril(B) to inhibit activity of ACE and/or ACE-2 vides methods and compositions for inhibiting or reducing Substrates, including, for example, angiotensin, bradykinin, angiotensin 1-5 production, comprising, or alternatively tachykinin, neurotensin, Substance P, and endothelin. 35 consisting of, administering to an animal in which Such In a preferred embodiment, ACE-2 binding polypeptides inhibition or reduction is desired, an ACE-2 binding of the invention (including molecules comprising, or alter polypeptide in an amount effective to inhibit or reduce natively consisting of, ACE-2 binding polypeptide frag production of angiotensin 1-5. ments or variants thereof) inhibit or reduce binding of the Further, ACE-2 and ACE compete for the substrate Soluble form of ACE-2 to an ACE-2 Substrate. In another 40 angiotensin, hydrolyzing angiotensin to angiotensin 1-9 and preferred embodiment ACE-2 binding polypeptides of the angiotensin II, respectively. Thus, the present invention invention inhibit or reduce cleavage of ACE-2 substrates provides for methods and compositions for enhancing or induced by the soluble form of ACE-2. In another preferred increasing angiotensin II production, comprising, or alter embodiment ACE-2 binding polypeptides of the invention natively consisting of, contacting an effective amount of inhibit or reduce the production of Angiotensin 1-9 and other 45 ACE-2 binding polypeptide with ACE-2, wherein the effec products of ACE-2 action induced by the soluble form of tive amount of ACE-2 binding polypeptide enhances or ACE-2. increases ACE mediated production of angiotensin II. In In a preferred embodiment, ACE-2 binding polypeptides another embodiment, the invention provides methods and of the invention (including molecules comprising, or alter compositions for enhancing or increasing angiotensin II natively consisting of, ACE-2 binding polypeptide frag 50 production, comprising, or alternatively consisting of, ments or variants thereof) inhibit or reduce binding of the administering to an animal in which Such enhancing or membrane-bound form of ACE-2 to an ACE-2 Substrate. In increasing is desired, an ACE-2 binding polypeptide in an another preferred embodiment ACE-2 binding polypeptides amount effective to enhance or increase production of angio of the invention inhibit or reduce cleavage of ACE-2 Sub tensin II. Determination of angiotenin II levels are most strates induced by the membrane-bound form of ACE-2. In 55 often performed by comparing the level of angiotensin II in another preferred embodiment ACE-2 binding polypeptides a Sample to a Standard containing a known amount of of the invention inhibit or reduce the production of Angio angiotensin II using ELISA assayS. Determination of angio tensin 1-9 and other products of ACE-2 action induced by tensin II levels in a given Sample, can readily be determined the membrane-bound form of ACE-2. using ELISA or other method known in the art. In a preferred embodiment, ACE-2 binding polypeptides 60 Additionally, ACE-2 has significant Sequence homologies of the invention (including molecules comprising, or alter with ACE functional domains, Suggesting that both types of natively consisting of, ACE-2 binding polypeptide frag enzymes Share additional Similar Substrates beyond angio ments or variants thereof) inhibit or reduce binding of both tensin. Thus, the present invention provides for methods and the Soluble and membrane-bound forms of ACE-2 to an compositions for enhancing or increasing bradykinin ACE-2 substrate. In another preferred embodiment, ACE-2 65 activity, comprising, or alternatively consisting of, contact binding polypeptides of the invention inhibit or reduce ing an effective amount of ACE-2 binding polypeptide with cleavage of ACE-2 substrates induced by either or both ACE-2, wherein the effective amount of ACE-2 binding US 6,592,865 B2 67 68 polypeptide inhibits or reduces ACE-2 mediated degradation In addition, the present invention provides for methods of bradykinin. In another embodiment, the invention pro and compositions for enhancing or increasing endothelin vides methods and compositions for enhancing or increasing activity, comprising, or alternatively consisting of, contact bradykinin activity, comprising, or alternatively consisting ing an effective amount of ACE-2 binding polypeptide with of, administering to an animal in which Such enhancing or ACE-2, wherein the effective amount of ACE-2 binding increasing is desired, an ACE-2 binding polypeptide in an polypeptide inhibits or reduces ACE-2 mediated degradation of endothelin. In another embodiment, the invention pro amount effective to enhance or increase activity of brady vides methods and compositions for enhancing or increasing kinin. Determination of bradykinin levels are most often endothelin activity, comprising, or alternatively consisting performed by comparing the level of bradykinin in a Sample of, administering to an animal in which Such enhancing or to a Standard containing a known amount of bradykinin 1O increasing is desired, an ACE-2 binding polypeptide in an using ELISA assays. Determination of bradykinin levels in amount effective to enhance or increase activity of endot a given Sample, can readily be determined using ELISA or helin. Determination of endothelin levels are most often other method known in the art. performed by comparing the level of endothelin in a Sample Additionally, the present invention provides for methods to a Standard containing a known amount of endothelin and compositions for enhancing or increasing tachykinin 15 using ELISA assays. Determination of endothelin levels in activity, comprising, or alternatively consisting of, contact a given Sample, can readily be determined using ELISA or ing an effective amount of ACE-2 binding polypeptide with other method known in the art. ACE-2, wherein the effective amount of ACE-2 binding Angiotensin, angiotenin II, bradykinin, tachykinin, polypeptide inhibits or reduces ACE-2 mediated degradation neurotensin, Substance P, and endothelin all are well known of tachykinin. In another embodiment, the invention pro in the art to regulate blood preSSure, Sodium homeostasis, vides methods and compositions for enhancing or increasing and inflammatory processes (for review see Kramer et al., tachykinin activity, comprising, or alternatively consisting Journal of Cardiovascular Pharmacology 15 Suppl. 6: of, administering to an animal in which Such enhancing or 591-598 (1990); Johnson et al., Journal of Hypertension increasing is desired, an ACE-2 binding polypeptide in an Supplement 15: S3-S6 (1997); Regoli et al., Regulatory amount effective to enhance or increase activity of tachyki 25 Peptides 45:323-340 (1993); Textor et al., Liver Transplant nin. Determination of tachykinin levels are most often 6:521-530 (2000)). Errant regulation of blood pressure, performed by comparing the level of tachykinin in a Sample Sodium homeostasis, or inflammatory responses affects Sev to a Standard containing a known amount of tachykinin eral physiological Systems, including the cardiovascular using ELISA assayS. Determination of tachykinin levels in System, renal System, and the immune System. By modulat a given Sample, can readily be determined using ELISA or ing activity of angiotensin, angiotenin II, bradykinin, other method known in the art. tachykinin, neurotensin, Substance P, and endothelin, com Additionally, the present invention provides for methods positions of the present invention can be used as therapeutic and compositions for enhancing or increasing neurotensin or pharmaceutical agent to treat, prevent, or ameliorate activity, comprising, or alternatively consisting of, contact diseases and/or disorders associated with aberrant blood ing an effective amount of ACE-2 binding polypeptide with 35 preSSure, Sodium homeostasis, or inflammatory processes. ACE-2, wherein the effective amount of ACE-2 binding In one embodiment, therapeutic or pharmaceutical com polypeptide inhibits or reduces ACE-2 mediated degradation positions of the present invention are administered to an of neurotensin. In another embodiment, the invention pro animal to treat, prevent, or ameliorate diseases and/or dis vides methods and compositions for enhancing or increasing orders associated with hypertension including, but not lim neurotensin activity, comprising, or alternatively consisting 40 ited to, accelerated hypertension, episodic hypertension, of, administering to an animal in which Such enhancing or paroxysmal hypertension, portal hypertension, primary increasing is desired, an ACE-2 binding polypeptide in an hypertension, Secondary hypertensoin, Systemic venous amount effective to enhance or increase activity of neuro hypertension, borderline hypertension, adrenal tensin. Determination of neurotensin levels are most often hypertension, benign hypertension, idiopathic hypertension, performed by comparing the level of neurotensin in a Sample 45 pale hypertension, postpartm hypertension, pregnancy to a Standard containing a known amount of neurotensin induced hypertension (gestational hypertension), essential using ELISA assayS. Determination of neurotensin levels in hypertension, labile hypertension, pulmonary hypertension, a given Sample, can readily be determined using ELISA or renal and renovascular hypertension, and Goldblatt other method known in the art. hypertension, left ventricular failure, atherOSclerotic heart Moreover, the present invention provides for methods and 50 disease, Stroke, retinal hemorrhage or infarction (Keith compositions for enhancing or increasing Substance P Wagener-Barker changes), renal failure, renovascular activity, comprising, or alternatively consisting of, contact disease, exudates, papilledema, Vascular accidents, myocar ing an effective amount of ACE-2 binding polypeptide with dial infarction, dissecting aneurysm. ACE-2, wherein the effective amount of ACE-2 binding In another embodiment, therapeutic or pharmaceutical polypeptide inhibits or reduces ACE-2 mediated degradation 55 compositions of the present invention are administered to an of Substance P. In another embodiment, the invention pro animal to treat, prevent, or ameliorate diseases and/or dis vides methods and compositions for enhancing or increasing orders associated with hypotension including, but not lim Substance Pactivity, comprising, or alternatively consisting ited to, arterial hypotension, idiopathic orthostatic of, administering to an animal in which Such enhancing or hypotension, induced or controlled hypotension, shock (e.g., increasing is desired, an ACE-2 binding polypeptide in an 60 anaphylactic shock, anaphylactoid shock, anestetic Shock, amount effective to enhance or increase activity of Sub cardiogenic Shock, chronic Shock, deferred or delayed stance P. Determination of Substance Plevels are most often Shock, hemorrhagic shock, hypovolemic shock, oligemic performed by comparing the level of Substance P in a Shock, Septic shock, and vasogenic shock), and Syncope Sample to a Standard containing a known amount of Sub (e.g., local Syncope, postural Syncope, tussive Syncope, and stance P using ELISA assays. Determination of Substance P 65 vasodepressor Syncope). levels in a given Sample, can readily be determined using In a preferred embodiment, therapeutic compositions of ELISA or other method known in the art. the present invention are administered to an animal US 6,592,865 B2 69 70 (preferrably administered to a human) to treat, prevent, or dysplasias, mesenteric vascular occlusion, Moyamoya ameliorate diseases and/or disorders associated with shock. disease, renal artery obstruction, retinal artery occlusion, In a further preferred embodiment, shock is treated, and thromboangiitis obliterans), arteritis, enarteritis, pol prevented, or ameliorated by administering to an animal yarteritis nodosa, cerebrovascular disorders (e.g., carotid (preferrably administered to a human) angiotensin 1-9/ 5 artery diseases, cerebral amyloid angiopathy, cerebral angiotensin 1-9-like polypeptides in conjunction with angio aneurysm, cerebral anoxia, cerebral arterioSclerosis, cere tensin II/angiotensin II-like polypeptides (see Example 9). bral arteriovenous malformation, cerebral artery diseases, In another preferred embodiment, shock is treated, cerebral embolism and thrombosis, carotid artery prevented, or ameliorated by administering to an animal thrombosis, Sinus thrombosis, Wallenberg's Syndrome, cere (preferrably administered to a human) compositions of the bral hemorrhage, epidural hematoma, Subdural. hematoma, invention that increase concentrations of angiotensin 1-9/ SubaraXhnoid hemorrhage, cerebral infarction, cerebral angiotensin 1-9-like polypeptides and/or angiotensin ischemia (including transient), Subclavian Steal Syndrome, II/angiotensin II-like polypeptides. periventricular leukomalacia, Vascular headache, cluster In another embodiment, therapeutic or pharmaceutical headache, migraine, and vertebrobasilar insufficiency), dia compositions of the present invention are administered to an 15 betic angiopathies, diabetic retinopathy, embolisms (e.g., air animal to treat, prevent, or ameliorate diseases and/or dis embolisms, amniotic fluid embolisms, cholesterol orders associated with cardiovascular disease including, but embolisms, blue toe Syndrome, fat embolisms, pulmonary not limited to, arrhythmias (e.g., Sinus arrhythmia, atrial embolisms, and thromoboembolisms), thrombosis (e.g., fibrillation, atrial flutter, bradycardia, extrasyStole, Adams coronary thrombosis, hepatic vein thrombosis, retinal vein Stokes Syndrome, bundle-branch block, sinoatrial block, occlusion, carotid artery thrombosis, Sinus thrombosis, Wal long QT syndrome, parasystole, Lown-Ganong-Levine lenberg’s Syndrome, and thrombophlebitis), Syndrome, Mahaim-type pre-excitation syndrome, Wolff erythromelalgia, hemorrhoids, hepatic veno-occlusive Parkinson-White Syndrome, Sick Sinus Syndrome, tachycar disease, hypertension, hypotension, ischemia (e.g., cerebral dias (e.g., paroxysmal tachycardia, Supraventricular ischemia, ischemic colitis, compartment Syndromes, ante tachycardia, accelerated idioVentricular rhythm, atrioven 25 rior compartment Syndrome, myocardial ischemia, reperfu tricular nodal reentry tachycardia, ectopic atrial tachycardia, Sion injuries, and peripheral limb ischemia), peripheral .ectopic junctional tachycardia, Sinoatrial nodal reentry vascular diseases, phlebitis, pulmonary veno-occlusive tachycardia, Sinus tachycardia, Torsades de Pointes, and disease, Raynaud's disease, CREST syndrome, retinal vein ventricular tachycardia), and ventricular fibrillation), carci occlusion, Scimitar Syndrome, Superior Vena cava noid heart disease, high cardiac output, low cardiac output, Syndrome, telangiectasia, atacia telangiectasia, hereditary cardiac tamponade, endocarditis (including bacterial), heart hemorrhagic telangiectasia, Varicocele, Varicose veins, Vari aneurysm, cardiac arrest, congestive heart failure, conges cose ulcer, vasculitis (e.g., aortitis, arteritis, Behcet’s tive cardiomyopathy, paroxySmal dyspnea, cardiac edema, Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph heart hypertrophy, congestive cardiomyopathy, left ventricu node Syndrome, thromboangiitis obliterans, hyperSensitivity lar hypertrophy, right ventricular hypertrophy, post 35 vasculitis, Schoenlein-Henoch purpura, allergic cutaneous infarction heart rupture, Ventricular Septal rupture, heart vasculitis, and Wegener's granulomatosis), and venous valve diseases (e.g., aortic valve insufficiency, aortic valve insufficiency. Stenosis, hear murmurs, aortic valve prolapse, mitral valve In a further embodiment, therapeutic or pharmaceutical prolapse, tricuspid valve prolapse, mitral valve insufficiency, compositions of the present invention are administered to an mitral valve Stenosis, pulmonary atresia, pulmonary valve 40 animal to treat, prevent, or ameliorate diseases and/or dis insufficiency, pulmonary valve Stenosis, tricuspid atresia, orders associated with the renal System including, but not tricuspid valve insufficiency, and tricuspid valve Stenosis), limited to, acute kidney failure, chronic kidney failure, myocardial diseases (e.g., alcoholic cardiomyopathy, con atheroembolic renal failure, end-stage renal disease, inflam gestive cardiomyopathy, hypertrophic cardiomyopathy, aor matory diseases of the kidney (e.g., acute tic Subvalvular Stenosis, pulmonary Subvalvular Stenosis, 45 glomerulonephritis, postinfectious glomerulonephritis, rap restrictive cardiomyopathy, Chagas cardiomyopathy, idly progressive glomerulonephritis, nephrotic Syndrome, endocardial fibroelastosis, endomyocardial fibrosis, Kearns membranous glomerulonephritis, familial nephrotic Syndrome, myocardial reperfusion injury, and myocarditis), Syndrome, membranoproliferative glomerulonephritis I and myocardial ischemia (e.g., coronary disease, Such as angina II, mesangial proliferative glomerulonephritis, chronic pectoris, coronary aneurysm, coronary arteriosclerosis, 50 glomerulonephritis, acute tubulointerstitial nephritis, coronary thrombosis, coronary vasospasm, myocardial inf chronic tubulointerstitial nephritis, acute post-Streptococcal arction and myocardial stunning), pericardial effusion, peri glomerulonephritis (PSGN), pyelonephritis, lupus nephritis, carditis (including constrictive and tuberculous), chronic nephritis, interstitial nephritis, and post pneumopericardium, postpericardiotomy Syndrome, pulmo Streptococcal glomerulonephritis), blood vessel disorders of nary heart disease, rheumatic heart disease, Ventricular 55 the kidneys (e.g., kidney infarction, atheroembolic kidney dysfunction, hyperemia, cardiovascular pregnancy disease, cortical necrosis, malignant nephrosclerosis, renal complications, Scimitar Syndrome, cardiovascular Syphilis, vein thrombosis, renal underperfusion, renal retinopathy, cardiovascular tuberculosis, aneurysms (e.g., dissecting renal ischemia-reperfusion, renal artery embolism, and renal aneurysms, false aneurysms, infected aneurysms, ruptured artery Stenosis), and electrolyte imbalances (e.g., aneurysms, aortic aneurysms, cerebral aneurysms, coronary 60 nephro calcinosis, pyuria, edema, hydronephritis, aneurysms, heart aneurysms, and iliac aneurysms), proteinuria, hyponatremia, hypernatremia, hypokalemia, angiodysplasia, angiomatosis, bacillary angiomatosis, hyperkale mia, hypo calcemia, hyper calcemia, Hippel-Lindau Disease, Klippel-Trenaunay-Weber hypophosphatemia, and hyperphosphatemia). Syndrome, Sturge-Weber Syndrome, angioneurotic edema, In an even further embodiment, therapeutic or pharma aortic diseases, Takayasu's Arteritis, aortitis, Leriche's 65 ceutical compositions of the present invention are adminis Syndrome, arterial occlusive diseases (e.g., arteriosclerosis, tered to an animal to treat, prevent, or ameliorate diseases intermittent claudication, carotid Stenosis, fibromuscular and/or disorders associated with inflammatory responses. US 6,592,865 B2 71 72 Such inflammatory conditions include, but are not limited failure, Vitiligo, Vasculitis, post-MI, cardiotomy Syndrome, to, for example, respiratory disorders (such as, e.g., asthma urticaria, atopic dermatitis, asthma, inflammatory and allergy); gastrointestinal disorders (such as, e.g., inflam myopathies, and other inflammatory, granulamatous, matory bowel disease); cancers (Such as, e.g., gastric, degenerative, and atrophic disorders. ovarian, lung, bladder, liver, and breast); CNS disorders Similarly, in Specific embodiments, polypeptides, (Such as, e.g., multiple Sclerosis, blood-brain barrier antibodies, or polynucleotides of the invention, and/or ago permeability, ischemic brain injury and/or Stroke, traumatic nists or antagonists thereof, are useful to treat, diagnose, brain injury, neurodegenerative disorders (Such as, e.g., and/or prevent transplantation rejections, graft-Versus-host Parkinson's disease and Alzheimer's disease), AIDS-related disease, autoimmune and inflammatory diseases (e.g., dementia, and prion disease); cardiovascular disorders (Such immune complex-induced vasculitis, glomerulonephritis, as, e.g., atherosclerosis, myocarditis, cardiovascular disease, hemolytic anemia, myasthenia gravis, type II collagen and cardiopulmonary bypass complications); as well as induced arthritis, experimental allergic and hyperacute many additional diseases, conditions, and disorders that are Xenograft rejection, rheumatoid arthritis, and Systemic lupus characterized by inflammation (Such as, e.g., chronic hepa erythematosus (SLE). Organ rejection occurs by host titis (B and C), rheumatoid arthritis, gout, trauma, Septic 15 immune cell destruction of the transplanted tissue through Shock, pancreatitis, Sarcoidosis, dermatitis, renal ischemia an immune response. Similarly, an immune response is also reperfusion injury, Graves disease, Systemic lupus involved in GVHD, but, in this case, the foreign transplanted erythematosis, diabetes mellitus (i.e., type 1 diabetes), and immune cells destroy the host tissues. Polypeptides, allogenic transplant rejection). antibodies, or polynucleotides of the invention, and/or ago Similarly, polynucleotides, polypeptides, antibodies, and/ nists or antagonists thereof, that inhibit an immune response, or agonists or antagonists of the present invention may also particularly the activation, proliferation, differentiation, or be used to treat, prevent, or ameliorate inflammation, chemotaxis of T-cells, may be an effective therapy in pre including, but not limited to, inflammation associated with venting organ rejection or GVHD. infection (e.g., Septic shock, Sepsis, or Systemic inflamma Further, allergic reactions and conditions, Such as asthma tory response Syndrome (SIRS)), ischemia-reperfusion 25 (particularly allergic asthma) or other respiratory problems, injury, polytrauma, pain, endotoxin lethality, arthritis (e.g., may also be treated, prevented, and/or diagnosed using osteoarthritis and rheumatoid arthritis), complement polypeptides, antibodies, or polynucleotides of the mediated hyperacute rejection, nephritis, cytokine or invention, and/or agonists or antagonists thereof. Moreover, chemokine induced lung injury, inflammatory bowel these molecules can be used to treat, prevent, and/or diag disease, Crohn's disease, and resulting from Over production nose anaphylaxis, hyperSensitivity to an antigenic molecule, of cytokines (e.g., TNF or IL-1). or blood group incompatibility. Additionally, many autoimmune disorders are in part Beyond its role as a neuromodulator, neurotensin is found manifested as inappropriate inflammation resulting from in high concentrations in gut endocrine cells of the ileum and inappropriate recognition of Self as foreign material by is released following ingestion of food. Since ACE-2 has immune cells. Therefore, the administration of polynucle 35 been found to hydrolyze neurotensin (as described herein), otides and polypeptides of the invention that can inhibit an the invention provides for methods and compositions that inappropriate inflammatory response may be an effective can be used for digestive purposes. therapy in preventing autoimmune disorders. Thus, in one embodiment, the invention can be used to Autoimmune diseases and/or disorders that may be treat, prevent, or ameliorate diseases and disorders of the treated, prevented, or ameliorated by compositions of the 40 digestive System including, but not limited to, disorders of present invention include, but not limited to, autoimmune the Small intestine, Such as malabsorption Syndromes, hemolytic anemia, autoimmune neonatal thrombocytopenia, distension, irritable bowel Syndrome, Sugar intolerance, idiopathic thrombo cytope nia purpura, celiac disease, duodenal ulcers, duodenitis, tropical Sprue, autoimmunocytopenia, hemolytic anemia, antiphospholipid Whipple's disease, intestinal lymphangiectasia, Crohn's Syndrome, dermatitis, allergic encephalomyelitis, 45 disease, appendicitis, obstructions of the ileum, Meckel's myocarditis, relapsing polychondritis, rheumatic heart diverticulum, multiple diverticula, failure of complete rota disease, glomerulonephritis (e.g., IgA nephropathy), Mul tion of the Small and large intestine, lymphoma, and bacte tiple Sclerosis, Neuritis, Uveitis Ophthalmia, rial and parasitic diseases (Such as Traveler's diarrhea, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein typhoid and paratyphoid, cholera, infection by Roundworms purpura), Reiter's Disease, Stiff-Man Syndrome, Autoim 50 (Ascariasis lumbricoides), Hookworms (Ancylostoma mune Pulmonary Inflammation, Autism, Guillain-Barre duodenale), Threadworms (Enterobius vermicularis), Tape Syndrome, insulin dependent diabetes mellitis, and autoim worms (Taenia Saginata, EchinococcuS granulosus, Diphyl mune inflammatory eye, autoimmune thyroiditis, hypothy lobothrium spp., and T. Solium). roidism (i.e., Hashimoto's thyroiditis, Systemic lupus The invention can also be used to treat, prevent, or erhythematosus, Goodpasture's Syndrome, Pemphigus, 55 ameliorate diseases and disorders disorders of the large Receptor autoimmunities Such as, for example, (a) Graves intestine, including antibiotic-associated colitis, Disease, (b) Myasthenia Gravis, and (c) insulin resistance, diverticulitis, ulcerative colitis, acquired megacolon, autoimmune hemolytic anemia, autoimmune thrombocy abscesses, fungal and bacterial infections, anorectal disor topenic purpura, rheumatoid arthritis, Schleroderma with ders (e.g., fissures, hemorrhoids), colonic diseases (colitis, anti-collagen antibodies, mixed connective tissue disease, 60 colonic neoplasms colon cancer, adenomatous colon polyps polymyositis/dermatomyositis, pernicious anemia, idio (e.g., Villous adenoma), colon carcinoma, colorectal cancer, pathic Addison's disease, infertility, glomerulonephritis colonic diverticulitis, colonic diverticulosis, megacolon Such as primary glomerulonephritis and IgA nephropathy, Hirschsprung disease, toxic megacolon; Sigmoid diseases bullous pemphigoid, Sjogren's Syndrome, diabetes millitus, proctocolitis, Sigmoin neoplasms), constipation, Crohn's and adrenergic drug resistance (including adrenergic drug 65 disease, diarrhea (infantile diarrhea, dysentery), duodenal resistance with asthma or cystic fibrosis), chronic active diseases (duodenal neoplasms, duodenal obstruction, duode hepatitis, primary biliary cirrhosis, other endocrine gland nal ulcer, duodenitis), enteritis (enterocolitis), HIV US 6,592,865 B2 73 74 enteropathy, ileal diseases (ileal neoplasms, ileitis), immu hepatis, Lipomas, Inflammatory pseudotumor, noproliferative Small intestinal disease, inflammatory bowel Miscellaneous), Epithelial tumors Bile duct epithelium disease (ulcerative colitis, Crohn's disease), intestinal (Bile duct hamartoma, Bile duct adenoma), Hepatocyte atresia, parasitic diseases (anisakiasis, balantidiasis, blasto (Adenoma, Focal nodular hyperplasia, Nodular regenerative cystis infections, cryptosporidiosis, dientamoebiasis, amebic hyperplasia), malignant liver tumors hepatocellular, dysentery, giardiasis), intestinal fistula (rectal fistula), intes he patobla Stoma, he pato cellular carcinoma, tinal neoplasms (cecal neoplasms, colonic neoplasms, cho langio cellular, cho langio carcinoma, duodenal neoplasms, ileal neoplasms, intestinal polyps, jeju cy Stade no carcinoma, tumors of blood vessels, nal neoplasms, rectal neoplasms), intestinal obstruction angiosarcoma, Karposi's Sarcoma, he mangioendothelioma, (afferent loop Syndrome, duodenal obstruction, impacted other tumors, embryonal Sarcoma, fibrosarcoma, feces, intestinal pseudo-obstruction Icecal Volvulus), leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, intussusception), intestinal perforation, intestinal polyps teratoma, carcinoid, Squamous carcinoma, primary (colonic polyps, gardner Syndrome, peutz-jeghers lymphoma), peliosis hepatis, erythrohepatic porphyria, Syndrome), jejunal diseases (jejunal neoplasms), malabsorp hepatic porphyria (acute intermittent porphyria, porphyria tion Syndromes (blind loop Syndrome, celiac disease, lactose 15 cutanea tarda), Zellweger Syndrome). intolerance, short bowl Syndrome, tropical Sprue, whipple's Moreover, the invention can be used to treat, prevent, or disease), mesenteric vascular occlusion, pneumatosis cys ameliorate diseases and disorders disorders of the toides intestinalis, protein-losing enteropathies (intestinal gallbladder, Such as gallstones (chole lithiasis and lymphagiectasis), rectal diseases (anus diseases, fecal choledocholithiasis), postcholecystectomy Syndrome, diver incontinence, hemorrhoids, proctitis, rectal fistula, rectal ticulosis of the gallbladder, acute cholecystitis, chronic prolapse, rectocele), peptic ulcer (duodenal ulcer, peptic cholecystitis, bile duct tumors, and mucocele. esophagitis, hemorrhage, perforation, Stomach ulcer, Further, the invention can be used to treat, prevent, or Zollinger-Ellison Syndrome), postgastrectomy syndromes ameliorate diseases and disorders disorders of the pancreas (dumping Syndrome), Stomach diseases (e.g., achlorhydria, including, but not limited to, acute pancreatitis, chronic duodenogastric reflux (bile reflux), gastric antral vascular 25 pancreatitis (acute necrotizing pancreatitis, alcoholic ectasia, gastric fistula, gastric outlet obstruction, gastritis pancreatitis), neoplasms (adenocarcinoma of the pancreas, (atrophic or hypertrophic), gastroparesis, Stomach dilatation, cy Stadenocarcinoma, insulinoma, gastrinoma, and Stomach diverticulum, Stomach neoplasms (gastric cancer, glucagonoma, cystic neoplasms, islet-cell tumors, gastric polyps, gastric adenocarcinoma, hyperplastic gastric pancreoblastoma), and other pancreatic diseases (e.g., cystic polyp), Stomach rupture, Stomach ulcer, Stomach Volvulus), fibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, tuberculosis, visceroptosis, vomiting (e.g., hematemesis, insufficiency). hyperemesis gravidarum, postoperative nausea and Other diseases and disoders of the gastroinstestinal Sys Vomiting) and hemorrhagic colitis. tem that can be treated, prevented, or ameliorated by com Additionally, the invention can be used to treat, prevent, positions of the present invention include dysphagia, or ameliorate diseases and disorders disorders of the liver, 35 odynophagia, inflammation of the esophagus, peptic Such as intrahepatic cholestasis (alagille Syndrome, biliary esophagitis, gastric reflux, Submucosal fibrosis and liver cirrhosis), fatty liver (alcoholic fatty liver, reye Stricturing, Mallory-Weiss lesions, leiomyomas, lipomas, Syndrome), hepatic vein thrombosis, hepatolentricular epidermal cancers, adeoncarcinomas, gastric retention degeneration, hepatomegaly, hepatopulmonary Syndrome, disorders, gastroenteritis, gastric atrophy, gastric/stomach hepatorenal Syndrome, portal hypertension (esophageal and 40 cancers, polyps of the Stomach, autoimmune disorderS Such gastric varices), liver abscess (amebic liver abscess), liver as pernicious anemia, pyloric Stenosis, gastritis (bacterial, cirrhosis (alcoholic, biliary and experimental), alcoholic Viral, eosinophilic, StreSS-induced, chronic erosive, atrophic, liver diseases (fatty liver, hepatitis, cirrhosis), parasitic plasma cell, and Ménétriers), peritoneal diseases (e.g., (hepatic echinococcosis, fascioliasis, amebic liver abscess), chyloperioneum, hemoperitoneum, mesenteric cyst, mesen jaundice (hemolytic, hepatocellular, and cholestatic), 45 teric lymphadenitis, mesenteric vascular occlusion, cholestasis, portal hypertension, liver enlargement, ascites, panniculitis, neoplasms, peritonitis, pneumoperitoneum, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepa bubphrenic abscess.), biliary tract diseases, Such as, titis (autoimmune, hepatitis B, hepatitis C, hepatitis D, drug gastroschisis, fistula (e.g., biliary fistula, esophageal fistula, induced), toxic hepatitis, viral human hepatitis (hepatitis A, gastric fistula, intestinal fistula, pancreatic fistula), neo hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson's 50 plasms (e.g., biliary tract neoplasms, esophageal neoplasms, disease, granulomatous hepatitis, Secondary biliary Such as adenocarcinoma of the esophagus, esophageal Squa cirrhosis, hepatic encephalopathy, portal hypertension, mous cell carcinoma, gastrointestinal neoplasms, pancreatic Varices, hepatic encephalopathy, primary biliary cirrhosis, neoplasms, Such as adenocarcinoma of the pancreas, muci primary Sclerosing cholangitis, hepatocellular adenoma, nous cystic neoplasm of the pancreas, pancreatic cystic he mangiomas, bile Stones, liver failure (hepatic 55 neoplasms, pancreatoblastoma, and peritoneal neoplasms), encephalopathy, acute liver failure), and liver neoplasms esophageal disease (e.g., bullous diseases, candidiasis, gly (angiomyolipoma, calcified liver metastases, cystic liver cogenic acanthosis, ulceration, barrett esophagus varices, me tastase S, epithelial tumors, fibrolamellar atresia, cyst, diverticulum (e.g., Zenker's diverticulum), hepatocarcinoma, focal nodular hyperplasia, hepatic fistula (e.g., tracheoesophageal fistula), motility disorders adenoma, hepatobiliary cyStadenoma, hepatoblastoma, 60 (e.g., CREST Syndrome, deglutition disorders, achalasia, hepatocellular carcinoma, hepatoma, liver cancer, liver Spasm, gastroesophageal reflux), neoplasms, perforation hemangioendothelioma, mesenchymal hamartoma, mesen (e.g., Boerhaave Syndrome, Mallory-Weiss Syndrome), chymal tumors of liver, nodular regenerative hyperplasia, Stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal benign liver tumors (Hepatic cysts Simple cysts, Polycystic hernia); gastrointestinal diseases, Such as, gastroenteritis liver disease, Hepatobiliary cyStadenoma, Choledochal 65 (e.g., cholera morbus, norwalk virus infection), hemorrhage cyst, Mesenchymal tumors Mesenchymal hamartoma, (e.g., hematemesis, melena, peptic ulcer hemorrhage), Stom Infantile hemangioendothelioma, Hemangioma, Peliosis ach neoplasms (gastric cancer, gastric polyps, gastric US 6,592,865 B2 75 76 adenocarcinoma, Stomach cancer)), hernia (e.g., congenital In one embodiment, therapeutic or pharmaceutical com diaphragmatic hernia, femoral hernia, inguinal hernia, obtu positions of the present invention can be used as analgesics rator hernia, umbilical hernia, Ventral hernia), and intestinal to reduce or inhibit pain. For example, therapeutic or phar diseases (e.g., cecal diseases (appendicitis, cecal maceutical compositions of the present invention can be neoplasms)). used to treat, prevent, or ameliorate perioperative pain Several in vivo studies have indicated that the renin and/or for use in Surgical procedures and labor. angiotensin System plays an important role in the prolifera Additionally, therapeutic or pharmaceutical compositions tion of Smooth muscle cells. For example, recent evidence of the present invention can be used to treat, prevent, or indicates that Angiotensin II Stimulates the growth of Vas ameliorate pain associated with cancer and treatment of cular Smooth muscle cells in response to injury (see Inagami cancer (e.g., radiation therapy, Surgery, and/or and Eguchi, Brazilian Journal of Medical and Biological chemotherapy). Research 33: 619–624 (2000)). Specifically, endogenous Moreover, therapeutic or pharmaceutical compositions of angiotensin II Stimulates the progression from G1 to S phase the present invention can be used to treat, prevent, or in vascular Smooth muscle cells (Kubo et al., American ameliorate neuropathic pain disorders including, but not Journal of Hypertension, 13:1117-1124 (2000)). This poses 15 limited to, refleX Sympathetic dystrophy, causalgia, phantom a particular problem relative to many Surgical procedures, limb pain, trigeminal neuralgia, atypical trigeminal Such as angioplasty, where Smooth muscle cell proliferation neuralgia, geniculate neuralgia, gloSSopharyngeal neuralgia, is a primary cause of artery restenosis. hallucinatory neuralgia, idiopathic neuralgia, intercoStal ACE-2 directly competes with ACE for angiotensin to neuralgia, mammary neuralgia, Morton neuralgia, occipital produce angiotensin 1-9 and angiotensin II, respectively. neuralgia, periodic migrainous neuralgia, Sciatic neuralgia, Thus, it is logical to assume that regulation of ACE-2 Sphenopalatine neuralgia, Suboccipital neuralgia, Supraor activity affects production of angiotensin II. Specifically, bital neuralgia, and Symptomatic neuralgia. inhibition of ACE-2 prevents the conversion of angiotenin to Therapeutic or pharmaceutical compositions of the angiotensin 1-9, making available an increased concentra present invention can also be used to treat, prevent, or tion of angiotenin for conversion to angiotensin II. 25 ameliorate idiopathic pain. Conversely, stimulation of ACE-2 increases utilization of Additionally, therapeutic or pharmaceutical compositions angiotensin, decreasing the amount available for conversion of the present invention can be used to treat, prevent, or to angiotensin II. Thus, the invention provides methods and ameliorate pain associated with diseases and/or disorders compositions for regulating angiotenin II-mediated cell pro including, but in no way limited to, burns, angina, myocar liferation. dial ischemia, minor or Severe trauma, migraine, shock, Hence, in another embodiment, therapeutic or pharma arthritis, rheumatoid arthritis, infection (e.g., herpes Zoster, ceutical compositions of the present invention are adminis AIDS and AIDS-related conditions, Sepsis, and pneumonia), tered to an animal to treat, prevent, or ameliorate diseases and rhinitis. and/or disorders associated with cell proliferation including, Further, therapeutic or pharmaceutical compositions of but not limited to, neoplasms located in the: colon, abdomen, 35 the present invention can be used to treat pain of the body bone, breast, digestive System, liver, pancreas, peritoneum, including, for example, abdominal pain, back pain endocrine glands (adrenal, parathyroid, pituitary, testicles, (particularly lower back pain), pelvic pain, joint pain, ovary, thymus, thyroid), eye, head and neck, nervous headache, facial pain, and muscular pain, as well as bodily (central and peripheral), lymphatic System, pelvic, skin, Soft pain due to trauma, Stings, bites, and central nervous System tissue, Spleen, thoracic, and urogenital tissues, and lymphop 40 injury. roliferative disorders, paraproteine mias, purpura, In another embodiment, the presence of ACE-2 in the sarcoidosis, Se Zary Syndrome, Walden strons testis Suggests that the present invention may also have Macroglobulinemia, Gaucher's Disease, histiocytosis, and utility in treating infertility or other disorders relating to any other hyperproliferative disease, besides neoplasia, male reproduction (e.g., erectile dysfunction) and/or gamete located in an aforementioned organ System. 45 formation and maturation. In a preferred embodiment, the compositions of the In a further embodiment, compositions of the present present invention can be used to treat, prevent, or ameliorate invention can be useful in treating, preventing, or amelio Stenosis, restenosis, myocardial hypertrophy, hypertrophy or rating cognitive diseases. hyperplasia of conduit and resistance vessels, and athero In another embodiment, the invention provides a method Sclerosis. 50 for the specific delivery of ACE-2 binding polypeptides and Pain and hyperalgesia, the perceptual companions of ACE-2 binding polypeptide conjugates of the invention to tissue injury and inflammation, are in part attributable to the cells by administering molecules of the invention that are Sensitization of primary afferent nociceptors by endog asSociated with heterologous polypeptides or nucleic acids. enously released chemicals, Such as bradykinin. Application In one example, the invention provides for a method for of exogenous bradykinin or Stimulation of endogenous 55 delivering a therapeutic protein into the targeted cell. In bradykinin production has been demonstrated to cause hype another example, the invention provides a method for deliv ralgesia in animal models of pain (for review see Burch and ering a single Strand nucleic acid (e.g., antisense or Kyle, Life Sciences 50: 829–838 (1992)). Additionally, a ribozymes) or double Stranded nucleic acid (e.g., DNA that number of Studies have shown that bradykinin antagonists can integrate into the cell's genome or replicate episomally are capable of blocking or ameliorating pain in both animal 60 and that can be transcribed) in the target cell. models and humans (for review see Burch et al., Medical Gene Therapy Research Review 10:237-269 (1990); Sharma, Genetic In a specific embodiment, nucleic acids comprising Pharmacology 24: 267-274 (1993)). As discussed elsewhere Sequences encoding ACE-2 binding polypeptides or func herein, the compositions of the invention may be used to tional derivatives thereof, are administered to treat, inhibit or regulate the hydrolysis (and, therefore, activity) of bradyki 65 prevent a disease or disorder associated with aberrant nin. Thus, the invention provides methods and compositions expression and/or activity of ACE-2 and/or its Substrates, by for regulating bradykinin-mediated pain and hyperalgesia. way of gene therapy. Gene therapy refers to therapy per US 6,592,865 B2 77 78 formed by the administration to a Subject of an expressed or expression, by targeting a specific receptor (See, e.g., PCT expressible nucleic acid. In this embodiment of the publications WO 92/06180; WO92/22635; WO92/20316; invention, the nucleic acids produce their encoded protein WO 93/14188, WO 93/20221). Alternatively, the nucleic that mediates a therapeutic effect. acid can be introduced intracellularly and incorporated Any of the methods for gene therapy available in the art within host cell DNA for expression, by homologous recom can be used according to the present invention. Exemplary bination (Koller and Smithies, Proc. Natl. Acad. Sci. USA, methods are described below. 86:8932-8935 (1989); Zijlstra et al., Nature, 342:435–438 For general reviews of the methods of gene therapy, See (1989)). Goldspiel et al., Clinical Pharmacy, 12:488-505 (1993); Wu In a Specific embodiment, Viral vectors that contains and Wu, Biotherapy, 3:87–95 (1991); Tolstoshev, Ann. Rev. nucleic acid Sequences encoding an ACE-2 binding polypep Pharmacol. Toxicol., 32:573–596 (1993); Mulligan, tide of the invention or fragments or variants thereof are Science, 260:926-932 (1993); and Morgan and Anderson, used. For example, a retroviral vector can be used (see Ann. Rev. Biochem, 62:191-217 (1993); May, TIBTECH, 1 Miller et al., Meth. Enzymol., 217:581-599 (1993)). These 1(5):155-215 (1993). Methods commonly known in the art retroviral vectors contain the components necessary for the of recombinant DNA technology which can be used are 15 correct packaging of the viral genome and integration into described in Current Protocols in Molecular Biology, the host cell DNA. The nucleic acid Sequences encoding the Ausubel et al., eds. (John Wiley & Sons, NY 1993); and ACE-2 binding polypeptide to be used in gene therapy are Kriegler, Gene Transfer and Expression, A Laboratory cloned into one or more vectors, which facilitates delivery of Manual (Stockton Press, NY 1990). the gene into a patient. Additional details concerning retro In a preferred aspect, a composition of the invention viral vectors can be found in Boesen et al., Biotherapy, 6:29 comprises, or alternatively consists of, nucleic acids encod 1-302 (1994), which describes the use of a retroviral vector ing an ACE-2 binding polypeptide, Said nucleic acids being to deliver the mdr 1 gene to hematopoietic Stem cells in part of an expression vector that expresses the ACE-2 order to make the Stem cells more resistant to chemotherapy. binding polypeptide or fragment thereof or chimeric protein Other references illustrating the use of retroviral vectors in including it in a Suitable host. In particular, Such nucleic 25 gene therapy are: Clowes et al., J. Clin. Invest., 93:644-651 acids have promoters, preferably heterologous promoters, (1994); Klein et al., Blood, 83:1467–1473 (1994); Salmons operably linked to the ACE-2 binding polypeptide coding and Gunzberg, Human Gene Therapy, 4:129-141 (1993); region, Said promoter being inducible or constitutive, and, and Grossman and Wilson, Curr. Opin. in Genetics and optionally, tissue-Specific. In another particular Devel, 3:110-114 (1993). embodiment, nucleic acid molecules are used in which the Other viral vectors that can be used in gene therapy are ACE-2 binding polypeptide coding Sequences and any other adenoviruses. Adenoviruses are especially attractive desired Sequences are flanked by regions that promote vehicles for delivering genes to respiratory epithelia. Aden homologous recombination at a desired Site in the genome, oviruses naturally infect respiratory epithelia, where they thus providing for intrachromosomal expression of the cause a mild disease. Other targets for adenovirus-based ACE-2 binding polypeptide encoding nucleic acids (Koller 35 delivery Systems are liver, the central nervous System, and Smithies, Proc. Natl. Acad. Sci. USA, 86:8932-8935 endothelial cells, and muscle. Adenoviruses have the advan (1989); Zijlstra et al., Nature, 342:435–438 (1989). tage of being capable of infecting non-dividing cells. See, Delivery of the nucleic acids into a patient may be either Kozarsky and Wilson, Current Opinion in Genetics and direct, in which case the patient is directly exposed to the Development, 3:499-503 (1993), presenting a review of nucleic acid or nucleic acid-carrying vectors, or indirect, in 40 adenovirus-based gene therapy. Bout et al., Human Gene which case, cells are first transformed with the nucleic acids Therapy, 5:3-10 (1994) demonstrated the use of adenovirus in vitro, then transplanted into the patient. These two vectors to transfer genes to the respiratory epithelia of rhesus approaches are known, respectively, as in Vivo or ex vivo monkeys. Other instances of the use of adenoviruses in gene gene therapy. therapy can be found in Rosenfeld et al., Science, In a specific embodiment, the nucleic acid Sequences are 45 252:431–434 (1991); Rosenfeld et al., Cell, 68:143–155 directly administered in Vivo, where it is expressed to (1992); Mastrangeli et al., J. Clin. Invest., 91:225-234 produce the encoded product. This can be accomplished by (1993); PCT publication WO 94/12649; and Wang et al., any of numerous methods known in the art, e.g., by con Gene Therapy, 2:775–783 (1995). In a preferred Structing them as part of an appropriate nucleic acid expres embodiment, adenovirus vectors are used. Sion vector and administering it So that they become 50 Adeno-associated virus (AAV) has also been proposed for intracellular, e.g., by infection using defective or attenuated use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med., retrovirals or other viral vectors (see U.S. Pat. No. 4,980, 204:289–300 (1993); U.S. Pat. No. 5,436,146). 286), or by direct injection of naked DNA, or by use of Another approach to gene therapy involves transferring a microparticle bombardment (e.g., a gene gun; Biolistic, gene to cells in tissue culture by Such methods as Dupont), or coating with lipids or cell-Surface receptors or 55 electroporation, lipofection, calcium phosphate mediated transfecting agents, encapsulation in liposome S, transfection, or viral infection. Usually, the method of trans microparticles, or microcapsules, or by administering them fer includes the transfer of a selectable marker to the cells. in linkage to a peptide which is known to enter the nucleus, The cells are then placed under Selection to isolate those by administering it in linkage to a ligand Subject to receptor cells that have taken up and are expressing the transferred mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem., 60 gene. Those cells are then delivered to a patient. 262:4429–4432 (1987)) (which can be used to target cell In this embodiment, the nucleic acid is introduced into a types specifically expressing the receptors), etc. In another cell prior to administration in Vivo of the resulting recom embodiment, nucleic acid-ligand complexes can be formed binant cell. Such introduction can be carried out by any in which the ligand comprises a fusogenic viral peptide to method known in the art, including but not limited to disrupt endoSomes, allowing the nucleic acid to avoid lyso 65 transfection, electroporation, microinjection, infection with Somal degradation. In yet another embodiment, the nucleic a viral or bacteriophage vector containing the nucleic acid acid can be targeted in Vivo for cell Specific uptake and Sequences, cell fusion, chromosome-mediated gene transfer, US 6,592,865 B2 79 80 microcell-mediated gene transfer, Spheroplast fusion, etc. invention has a desired effect upon Such cell types. Numerous techniques are known in the art for the introduc Preferably, the ACE-2 binding polypeptides or compositions tion of foreign genes into cells (see, e.g., Loeffler and Behr, of the invention are also tested in in vitro assays and animal Meth. Enzymol., 217:599–618 (1993); Cohen et al., Meth. model Systems prior to administration to humans. Enzymol., 217:618–644 (1993); Clin. Pharma. Ther., ACE-2 binding polypeptides or compositions of the 29:69-92m (1985)) and may be used in accordance with the present invention for use in therapy can be tested for their present invention, provided that the necessary developmen toxicity in Suitable animal model Systems, including but not tal and physiological functions of the recipient cells are not limited to rats, mice, chicken, cows, monkeys, and rabbits. disrupted. The technique should provide for the stable For in Vivo testing of an ACE-2 binding polypeptide or transfer of the nucleic acid to the cell, So that the nucleic acid composition's toxicity any animal model System known in is expressible by the cell and preferably heritable and the art may be used. expressible by its cell progeny. Efficacy in treating or preventing vasoconstriction may be The resulting recombinant cells can be delivered to a demonstrated by detecting the ability of an ACE-2 binding patient by various methods known in the art. Recombinant polypeptide or composition of the invention to prevent or blood cells (e.g., hematopoietic stem or progenitor cells) are 15 treat hypertension, which can be defined as a diastolic blood preferably administered intravenously. The amount of cells preSSure greater than 90 mmHg and/or a Systolic blood envisioned for use depends on the desired effect, patient preSSure of greater than 140 mmHg, as measured in an adult State, etc., and can be determined by one skilled in the art. over 18 years of age. Since children and pregnant women Cells into which a nucleic acid can be introduced for have a lower avaerage blood pressure, a mean diastolic purposes of gene therapy encompass any desired, available blood pressure over 80 mmHg and systolic blood pressure cell type, and include but are not limited to cardiac over 120 mmHg is considered indicative of hypertension. myocytes, proximal tubules, endothelial cells, epithelial ACE-2 binding polypeptides or compositions of the cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, invention can be tested for the ability to inhibit or prevent blood cells Such as T lymphocytes, B lymphocytes, Smooth muscle cell proliferation, Such as occurs in Vascular monocytes, macrophages, neutrophils, eosinophils, 25 Stenosis and tumor formation, in in vitro, ex vivo, and in megakaryocytes, granulocytes, various Stem or progenitor Vivo assayS. For example, cellular proliferation can be cells, in particular hematopoietic Stem or progenitor cells, assayed by H-thymidine incorporation assays and trypan e.g., as obtained from bone marrow, umbilical cord blood, blue cell counts. peripheral blood, fetal liver, etc. ACE-2 binding polypeptides or compositions of the In a preferred embodiment, the cell used for gene therapy invention can be tested for the ability to modulate inflam is autologous to the patient. matory responses by contacting cells involved in inflamma In an embodiment in which recombinant cells are used in tory responses, preferably human cells involved inflamma gene therapy, nucleic acid Sequences encoding an ACE-2 tory responses (e.g., basophils and mast cells), With an binding polypeptide or fragment thereof are introduced into ACE-2 binding polypeptide or composition of the invention the cells such that they are expressible by the cells or their 35 to modulate (i.e., increase or decrease) the inflammatory progeny, and the recombinant cells are then administered in response. The ability of an ACE-2 binding polypeptide or Vivo for therapeutic effect. In a specific embodiment, Stem or composition of the invention to modulate inflammatory progenitor cells are used. Any Stem and/or progenitor cells responses can be assessed by detecting the proliferation of that can be isolated and maintained in vitro can potentially cells involved in the immune response (e.g., basophile and be used in accordance with this embodiment of the present 40 mast cells), detecting the activation of signalling molecules invention (see, e.g., PCT publication WO 94/08598; (e.g., bradykinin), detecting Secretion of fluid and/or ions, Stemple and Anderson, Cell, 7 1:973-985 (1992); detecting functio laesa, detecting changes in calor or rubor Rheinwald, Meth. Cell Bio., 21A:229 (1980); and Pittelkow at affected Site, detecting the expression of antigens, or and Scott, Mayo Clinic Proc., 61:771 (1986)). detecting changes in blood flow to the affected Site. Tech In a Specific embodiment, the nucleic acid to be intro 45 niques known to those of Skill in the art can be used for duced for purposes of gene therapy comprises an inducible measuring these activities. For example, antigen expression promoter operably linked to the coding region, Such that can be assayed, for example, by immunoassays including, expression of the nucleic acid is controllable by controlling but not limited to, competitive and non-competitive assay the presence or absence of the appropriate inducer of tran Systems using techniqueS Such as western blots, immuno Scription. 50 histochemistry radioimmunoassays, ELISA (enzyme linked Demonstration of Therapeutic or Prophylactic Utility of a immunosorbent assay), “Sandwich immunoassays, immu Composition noprecipitation assays, precipitin reactions, gel diffusion The compounds of the invention are preferably tested in precipitin reactions, immunodiffusion assays, agglutination Vitro, and then in Vivo for the desired therapeutic or pro assays, complement-fixation assays, immunoradiometric phylactic activity, prior to use in humans. For example, in 55 assays, fluorescent immunoassays, protein A immunoassays Vitro assays which can be used to determine whether admin and FACS analysis. The activation of Signaling molecules istration of a Specific ACE-2 binding polypeptide or com can be assayed, for example, by kinase assays and electro position of the present invention is indicated, include in vitro phoretic shift assays (EMSAs). In a preferred embodiment, cell culture assays in which a patient tissue Sample is grown the ability of an ACE-2 binding polypeptide or composition in culture, and exposed to, or otherwise administered, an 60 of the invention to enhance or increase bradykinin concen ACE-2 binding polypeptide or composition of the present trations and/or activity is measured. invention, and the effect of Such an ACE-2 binding polypep ACE-2 binding polypeptides or compositions of the tide or composition of the present invention upon the tissue invention can also be tested for their ability to alleviate of Sample is observed. In various Specific embodiments, in one or more Symptoms associated with cancer, a cardiovas Vitro assays can be carried out with representative cells of 65 cular disorder (e.g., hypertension or hypotension), a neuro cell types involved in a patient's disorder, to determine if an logical disorder, or a digestive disorder. Further, ACE-2 ACE-2 binding polypeptide or composition of the present binding polypeptides or compositions of the invention can US 6,592,865 B2 81 82 be tested for their ability to increase the survival period of and Fidler, eds. (Liss, New York 1989), pp. 353–365; animals Suffering from disease or disorder, including cancer, Lopez-Berestein, ibid., pp. 317-327; See, generally, ibid.). a cardiovascular disorder, a neurological disorder, or a In yet another embodiment, the composition can be deliv digestive disorder. Techniques known to those of skill in the ered in a controlled release System. In one embodiment, a art can be used to analyze the function of the ACE-2 binding pump may be used (see Langer, Supra; Sefton, CRC Crit. polypeptides or compositions of the invention in Vivo. Ref. Biomed. Eng., 14:201 (1987); Buchwald et al., Surgery, Therapeutic/Prophylactic Compositions and Administration 88:507 (1980); Saudek et al., N. Engl. J. Med., 321:574 The invention provides methods of treatment, inhibition (1989)). In another embodiment, polymeric materials can be and prophylaxis by administration to a Subject of an effective used (see, Medical Applications of Controlled Release, amount of ACE-2 binding polypeptide (or fragment or Langer and Wise, eds. (CRC Press, Boca Raton, Fla. 1974); variant thereof) or pharmaceutical composition of the Controlled Drug Bioavailability, Drug Product Design and invention, preferably an ACE-2 binding polypeptide of the Performance, Smolen and Ball, eds. (Wiley, New York invention. In a preferred aspect, an ACE-2 binding polypep 1984); Ranger and Peppas, Macromol. Sci. Rev. Macromol. tide or fragment or variant thereof is Substantially purified Chem., 23:61 (1983); see also Levy et al., Science, 228:190 (i.e., Substantially free from Substances that limit its effect or 15 (1985); During et al., Ann. Neurol., 25:351 (1989); Howard produce undesired side-effects). The Subject is preferably an et al., J. NeuroSurg., 7 1:105 (1989)). In yet another animal, including but not limited to, animals. Such as cows, embodiment, a controlled release System can be placed in pigs, horses, chickens, cats, dogs, etc., and is preferably a proximity of the therapeutic target, e.g., the brain, thus mammal, and most preferably a human. requiring only a fraction of the Systemic dose (see, e.g., Formulations and methods of administration that can be Goodson, in Medical Applications of Controlled Release, employed when the compound comprises a nucleic acid or supra, vol. 2, pp. 115-138 (1984)). Other controlled release an immunoglobulin are described above, additional appro Systems are discussed in the review by Langer (Science, priate formulations and routes of administration can be 249:1527-1533 (1990)). Selected from among those described herein below. In a Specific embodiment where the composition of the Various delivery Systems are known and can be used to 25 invention is a nucleic acid encoding a protein, the nucleic administer ACE-2 binding polypeptide or fragment or vari acid can be administered in Vivo to promote expression of its ant thereof of the invention, e.g., encapsulation in encoded protein, by constructing it as part of an appropriate liposomes, microparticles, microcapsules, recombinant cells nucleic acid expression vector and administering it So that it capable of expressing the ACE-2 binding polypeptide or becomes intracellular, e.g., by use of a retroviral vector (see ACE-2 binding polypeptide fragment, receptor-mediated U.S. Pat. No. 4,980,286), or by direct injection, or by use of endocytosis (see, e.g., Wu and Wu, J. Biol. Chem., microparticle bombardment (e.g., a gene gun; Biolistic, 262:4429–4432 (1987)), construction of a nucleic acid as Dupont), or coating with lipids or cell-Surface receptors or. part of a retroviral or other vector, etc. Methods of intro transfecting agents, or by administering it in linkage to a duction include, but are not limited to, intradermal, homeobox-like peptide which is known to enter the nucleus intramuscular, intraperitoneal, intravenous, Subcutaneous, 35 (see, e.g., Joliot et al., Proc. Natl. Acad. Sci. USA, intranasal, epidural, and oral routes. The compositions may 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be administered by any convenient route, for example by be introduced intracellularly and incorporated within host infusion or bolus injection, by absorption through epithelial cell DNA for expression, by homologous recombination. or mucocutaneous linings (e.g., oral mucosa, rectal and The present invention also provides pharmaceutical com intestinal mucosa, etc.) and may be administered together 40 positions. Such compositions comprise a therapeutically with other biologically active agents. Administration can be effective amount of an ACE-2 binding polypeptide or a Systemic or local. In addition, it may be desirable to intro fragment thereof, and a pharmaceutically acceptable carrier. duce the pharmaceutical compositions of the invention into In a specific embodiment, the term "pharmaceutically the central nervous System by any Suitable route, including acceptable” means approved by a regulatory agency of the intraventricular and intrathecal injection; intraventricular 45 Federal or a state government or listed in the U.S. Pharma injection may be facilitated by an intraventricular catheter, copeia or other generally recognized pharmacopeia for use for example, attached to a reservoir, Such as an Ommaya in animals, and more particularly in humans. The term reservoir. Pulmonary administration can also be employed, “carrier” refers to a diluent, adjuvant, excipient, or vehicle e.g., by use of an inhaler or nebulizer, and formulation with with which the therapeutic is administered. Such pharma an aeroSolizing agent. 50 ceutical carriers can be Sterile liquids, Such as water and oils, In a specific embodiment, it may be desirable to admin including those of petroleum, animal, vegetable or Synthetic ister the pharmaceutical compositions of the invention origin, Such as peanut oil, Soybean oil, mineral oil, Sesame locally to the area in need of treatment; this may be achieved oil and the like. Water is a preferred carrier when the by, for example, and not by way of limitation, local infusion pharmaceutical composition is administered intravenously. during Surgery, topical application, e.g., in conjunction with 55 Saline Solutions and aqueous dextrose and glycerol Solutions a wound dressing after Surgery, by injection, by means of a can also be employed as liquid carriers, particularly for catheter, by means of a Suppository, or by means of an injectable Solutions. Suitable pharmaceutical excipients implant, Said implant being of a porous, non-porous, or include Starch, glucose, lactose, Sucrose, gelatin, malt, rice, gelatinous material, including membranes, Such as Sialastic flour, chalk, Silica gel, Sodium Stearate, glycerol membranes, or fibers. Preferably, when administering a 60 monoStearate, talc, Sodium chloride, dried skim milk, protein, including an ACE-2 binding polypeptide, of the glycerol, propylene, glycol, water, ethanol and the like. The invention, care must be taken to use materials to which the composition, if desired, can also contain minor amounts of protein does not absorb. wetting or emulsifying agents, or pH buffering agents. These In another embodiment, the composition can be delivered compositions can take the form of Solutions, Suspensions, in a vesicle, in particular a liposome (see, Langer, Science, 65 emulsion, tablets, pills, capsules, powders, Sustained-release 249:1527–1533 (1990); Treat et al., in Liposomes in the formulations, and the like. The composition can be formu Therapy of Infectious Disease and Cancer, Lopez-Berestein lated as a Suppository, with traditional binders and carriers US 6,592,865 B2 83 84 Such as triglycerides. Oral formulation can include Standard ACE-2 binding polypeptides are administered in complex carrierS Such as pharmaceutical grades of mannitol, lactose, with ACE-2. Preferably the ACE-2 binding polypeptide is Starch, magnesium Stearate, Sodium Saccharine, cellulose, radiolabelled or in complex with a radioisotope, toxin, or magnesium carbonate, etc. Examples of Suitable pharma prodrug. Combinations may be administered either ceutical carriers are described in Remington's Pharmaceu concomitantly, e.g., as an admixture, Separately but Simul tical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co., taneously or concurrently, or Sequentially. This includes 1990). Such compositions will contain a therapeutically presentations in which the combined agents are administered effective amount of the ACE-2 binding polypeptide or together as a therapeutic mixture, and also procedures in fragment thereof, preferably in purified form, together with which the combined agents are administered separately but a Suitable amount of carrier So as to provide the form for Simultaneously, e.g., as through Separate intravenous lines proper administration to the patient. The formulation should into the same individual. Administration “in combination” Suit the mode of administration. further includes the Separate administration of one of the In a preferred embodiment, the composition is formulated compounds or agents given first, followed by the Second. in accordance with routine procedures as a pharmaceutical The ACE-2 binding polypeptides and ACE-2 binding composition adapted for intravenous administration to 15 polypeptide compositions of the invention may be admin human beings. Typically, compositions for intravenous istered alone or in combination with other adjuvants. Adju administration are Solutions in Sterile isotonic aqueous vants that may be administered with the ACE-2 binding buffer. Where necessary, the composition may also include polypeptides and ACE-2 binding polypeptide compositions a Solubilizing agent and a local anesthetic Such as ligno of the invention include, but are not limited to, alum, alum camne to ease pain at the Site of the injection. Generally, the plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), ingredients are Supplied either Separately or mixed together QS21 (Genentech, Inc.), BCG, and MPL. In a specific in unit dosage form, for example, as a dry lyophilized embodiment, ACE-2 binding polypeptides and ACE-2 bind powder or water free concentrate in. a hermetically Sealed ing polypeptide compositions of the invention are adminis container Such as an ampoule or Sachette indicating the tered in combination with alum. In another Specific quantity of active agent. Where the composition is to be 25 embodiment, ACE-2 binding polypeptides and ACE-2 bind administered by infusion, it can be dispensed with an ing polypeptide compositions of the invention are adminis infusion bottle containing Sterile pharmaceutical grade water tered in combination with QS-21. Further adjuvants that or saline. Where the composition is administered by may be administered with the ACE-2 binding polypeptides injection, an ampoule of Sterile water for injection or Saline and ACE-2 binding polypeptide compositions of the inven can be provided So that the ingredients may be mixed prior tion include, but are not limited to, Monophosphoryl lipid to administration. immunomodulator, AdjuVax 100a, QS-21, QS-18, The compositions of the invention can be formulated as CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant neutral or Salt forms. Pharmaceutically acceptable Salts technology. Vaccines that may be administered with the include those formed with anions such as those derived from ACE-2 binding polypeptides and ACE-2 binding polypep hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., 35 tide compositions of the invention include, but are not and those formed with cations Such as those derived from limited to, Vaccines directed toward protection against Sodium, potassium, ammonium, calcium, ferric hydroxides, MMR (measles, mumps, rubella), polio, varicella, tetanus/ isopropylamine, triethylamine, 2-ethylaminoethanol, diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, histidine, procaine, etc. whooping cough, pneumonia, influenza, Lyme’s Disease, The amount of the composition of the invention which 40 rotavirus, cholera, yellow fever, Japanese encephalitis, will be effective in the treatment, inhibition and prevention poliomyelitis, rabies, typhoid fever, and pertussis, and/or of a disease or disorder associated with aberrant expression PNEUMOVAX-23 TM. and/or activity of a polypeptide of the invention can be The ACE-2 binding polypeptides and ACE-2 binding determined by Standard clinical techniques. In addition, in polypeptide compositions of the invention may be admin Vitro assays may optionally be employed to help identify 45 istered alone or in combination with other therapeutic optimal dosage ranges. The precise dose to be employed in agents, including but not limited to, chemotherapeutic the formulation will also depend on the route of agents, antibiotics, antivirals, Steroidal and non-Steroidal administration, and the Seriousness of the disease or anti-inflammatories, conventional immunotherapeutic disorder, and should be decided according to the judgment agents and cytokines. Combinations may be administered of the practitioner and each patient's circumstances. Effec 50 either concomitantly, e.g., as an admixture, Separately but tive doses may be extrapolated from dose-response curves Simultaneously or concurrently, or Sequentially. This derived from in vitro or animal model test Systems. includes presentations in which the combined agents are For ACE-2 binding polypeptides, the dosage administered administered together as a therapeutic mixture, and also to a patient is typically 0.1 mg/kg to 100 mg/kg of the procedures in which the combined agents are administered patient's body weight. Preferably, the dosage administered 55 Separately but simultaneously, e.g., as through Separate to a patient is between 0.1 mg/kg and 20 mg/kg of the intravenous lines into the same individual. Administration patient's body weight, more preferably 1 mg/kg to 10 mg/kg "in combination' further includes the Separate administra of the patient's body weight. Further, the dosage and fre tion of one of the compounds or agents given first, followed quency of administration of therapeutic or pharmaceutical by the Second. compositions of the invention may be reduced by enhancing 60 In one embodiment, the ACE-2 binding polypeptides and uptake and tissue penetration (e.g., into the brain) of the ACE-2 binding polypeptide compositions of the invention ACE-2 binding polypeptides by modifications Such as, for are administered in combination with antihypertensives example, lipidation. including, but not limited to, diuretics, beta-blockers, cal The ACE-2 binding polypeptides and ACE-2 binding cium channel blockers, ACE inhibitors, angiotensin II recep polypeptide compositions of the invention may be admin 65 tor blockers, alpha blockers, combined alpha and beta istered alone or in combination with other molecules includ blockers, central agonists, peripheral adrenergic inhibitors, ing ACE-2. In further embodiments of the invention, the and blood vessel dilators. US 6,592,865 B2 85 86 In a preferred embodiment, the ACE-2 binding polypep oxide gas (INOmax(E), nitroglycerin (glyceryl trinitrate, tides and ACE-2 binding polypeptide compositions of the Nitro-Dur(R), Nitroligual(R), Nitrostat(R), NTG, Transderm invention are administered in combination with diuretics Nirto(R), papaverine, and tolazoline (Priscoline(R). including, but not limited to, bumetanide (BumeX(R), chlo Antilipemic agents that may be administered in combi rothiazide (Diuril(R), chlorthalidone (Hygroton(R), furo nation with the ACE-2 binding polypeptides and ACE-2 semide (Lasix (B), hydrochlorothiazide (Esidrix (R), binding polypeptide compositions of the present invention Hydro diuril(R), mannitol, metola Zone (Diulo (R), include, but are not limited to, atorvastatin (Lipitor(R), ZaroxolynoR), amiloride and hydrochlorothiazide mix cholestyramine(Questran(R), colestipol (Colestid(R), fluvas (Moduretic(R), triamterene and hydrochlorothiazide mix tatin (Lescole), gemfibrozil (LopidE), niacin (nicotinic (Dyazide(E, MaXZide(R), and potassium-sparring diuretics, acid), prevastatin (Pravachol?R), and simvastatin (Zocor(R). Such as a miloride (Midamor (R), Spironolactone In another preferred embodiment, a therapeutically effec (Aldactone(R), and triamterene (Dyrenium(R). tive amount of angiotensin 1-9 is administered in combina In a preferred embodiment, the ACE-2 binding polypep tion with a therapeutically effective amount of angiotensin II tides and ACE-2 binding polypeptide compositions of the or other hypotensive agents (see Example 9). invention are administered in combination with beta block 15 Additionally, in another embodiment, the ACE-2 binding ers including, but not limited to, atenolol (Tenormin(E), polypeptides and ACE-2 binding polypeptide compositions esmolol (Brevibloc(R), labetalol (Normodyne(E,Trandate(R), of the invention are administered in combination with intra metoprolol (Lopressor(R), propranolol (Inderal(R), Sotalol vascular radiation to prevent, treat, or ameliorate restenosis. (Betapace(R), acebutolol (Sectral(R), nadolol (Corgard(R), In a preferred embodiment, the ACE-2 binding polypeptides pindolol (Visken(R), and timolol (Blocadren(R). and ACE-2 binding polypeptide compositions of the inven In a preferred embodiment, the ACE-2 binding polypep tion are administered in combination with beta-emitting tides and ACE-2 binding polypeptide compositions of the phosphorus-32. In another preferred ambodiment, the invention are administered in combination with ACE inhibi ACE-2 binding polypeptides and ACE-2 binding polypep tors including, but not limited to, benazepril (Lotensin(E), tide compositions of the invention are administered in captopril (Capoten(R), enalapril (Vasotec(R), fosinopril 25 combination with iridium-192. (Monopril(R), lisinopril (Zestril?R, Prinivil(R), and quinapril In a further embodiment, the ACE-2 binding polypeptides (Accupril(R). and ACE-2 binding polypeptide compositions of the inven In a preferred embodiment, the ACE-2 binding polypep tion are administered in combination with analgesics and tides and ACE-2 binding polypeptide compositions of the anti-inflammatory agents. Analgesic agents that may be invention are administered in combination with angiotensin administered in combination with the ACE-2 binding II blockers including, but not limited to losartan (Cozaar(B). polypeptides and ACE-2 binding polypeptide compositions In a preferred embodiment, the ACE-2 binding polypep of the invention include, but are not limited to, opioids, Such tides and ACE-2 binding polypeptide compositions of the as codeine, fentanyl (ActigE, Duragesic(R), Oralet (E), invention are administered in combination with calcium Sublimaze(R), hydromorphone (Dilaudid(R), meperidine channel blockers including, but not limited to, amlodipine 35 (Demerol(R), methadone (Dolophine (R), morphine (Norvasc(R), diltiazem (Cardizemce), felodipine (Plendil(R), (Duramorph(R), Infomorph(R), morphine sulfate, MSO4), isradipine (Dynacirc(R), nicardipine (Cardenef), nifedipine oxycodone, remifentanil (Ultiva(E), and Sufentanil (Procardia(R), and Verapamil (Calan(R), Isoptin(R). (Sufenta.E), opiate partial agonists, Such as butorphanol In a preferred embodiment, the ACE-2 binding polypep (Stadol(R), nalbuphine (Nubain(R), and tramadol (Ultram(R). tides and ACE-2 binding polypeptide compositions of the 40 Anti-inflammatory agents that may be administered in com invention are administered in combination with alpha block bination with the ACE-2 binding polypeptides and ACE-2 ers including, but not limited to, doxazosin (Cardura.E), binding peptide compositions of the invention include, but prazosin (Minipress(R), and tamsulosin (Flomax(R). are not limited to, NSAIDs (non-steroidal anti-inflammatory In a preferred embodiment, the ACE-2 binding polypep drugs), Such as aspirin (ASACE), Empirine), choline magne tides and ACE-2 binding polypeptide compositions of the 45 sium trisalicylate (Trilisate(E), diclofenac, diflunisal, invention are administered in combination with central fenoprofen, flurbiprofin (Ocufen(R), ibuprofin (Advil(R), agonists including, but not limited to, clonidine Motrin(R), Rufen(R), indomethacin (IndocincB), ketoprofen, (Catapres(E), methyldopa (Aldomet(R), guanabenz meclofenamate, nabu metone (Relafen(R), naproxen (Wytensin(R), and guanfacine (Tenex(R). (NaprosynE), naproxen Sodium (Aleve(E), AnaproXCE), In a preferred embodiment, the ACE-2 binding polypep 50 Oxaprozin, phenylbutazone, piroxicam (Feldene(R), Salsalate tides and ACE-2 binding polypeptide compositions of the (Disalcid(R), sulindac (Clinoril(R), tolmetin, celecoxib invention are administered in combination with peripheral (Celebrex(R), and ketorolac (Toradol(R). adrenergic inhibitors including, but not limited to, reserpine, In an even further embodiment, the ACE-2 binding guanadrel (Hylorel(F), and guanethidine (ISmelin(R). polypeptides and ACE-2 binding polypeptide compositions In a preferred embodiment, the ACE-2 binding polypep 55 of the invention are administered alone or in combination tides and ACE-2 binding polypeptide compositions of the with other anti-inflammatory agents. Anti-inflammatory invention are administered in combination with blood vessel agents that may be administered with the ACE-2 binding dilators including, but not limited to, hydralZine polypeptides and ACE-2 binding polypeptide compositions (Apresoline(R) and minoxidil (Loniten(R). of the invention include, but are not limited to, glucocorti In another preferred embodiment, the ACE-2 binding 60 coids and the nonsteroidal anti-inflammatories, aminoaryl polypeptides and ACE-2 binding polypeptide compositions carboxylic acid derivatives, arylacetic acid derivatives, aryl of the invention are administered in combination with butyric acid derivatives, arylcarboxylic acids, arylpropionic vasodilating agents including, but not limited to, alproStadil acid derivatives, pyrazoles, pyrazolones, Salicylic acid (PGE-1, prostaglandin E-1, Prostin VR Pediatric(R), amyl derivatives, thiazinecarboxamides, e-acetamidocaproic acid, nitrite, dipyridamole (Persantin(R), epoprostenol (Flolance), 65 S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, isosorbide dinitrate (Isordil(R), Sorbitrate(R), isosorbide a mixe trine, benda Zac, ben Zy damine, bucolome, monomitrate (IMDURCR), nimodipine (nimotop(R), nitric difen piramide, dita Zol, emorfa Zone, guaia Zulene, US 6,592,865 B2 87 88 nimeSulide, orgotein, oxaceprol, paranyline, periSOXal, Kits for Detecting and/or Quantitating ACE-2 or ACE-2-like pifoXime, produaZone, proxazole, and tenidap. Polypeptides In another embodiment, compostions of the invention are The present invention is also directed to an assay kit administered in combination with a chemotherapeutic agent. which can be useful in Screening for the presence of ACE-2 Chemotherapeutic agents that may be administered with the and/or quantitating ACE-2 concentrations in a fluid, Such as, ACE-2 binding polypeptides and ACE-2 binding polypep for example, a biological fluid (e.g., blood, Serum, Synovial fluid). tide compositions of the invention include, but are not In a particular embodiment of the present invention, an limited to, antibiotic derivatives (e.g., doxorubicin, assay kit is contemplated which comprises in one or more bleomycin, daunorubicin, and dactinomycin); antiestrogens containers one or more ACE-2 binding polypeptides of the (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, invention and optionally, a detection means for determining methotrexate, floxuridine, interferon alpha-2b, glutamic the presence of an ACE-2-ACE-2 binding polypeptide inter acid, plicamycin, mercaptopurine, and 6-thioguanine); cyto action or the absence thereof. The kit further optionally toxic agents (e.g., carmustine, BCNU, lomustine, CCNU, contains ACE-2 protein that may be used, for example as a cytosine arabinoside, cyclophosphamide, estramustine, 15 control. The ACE-2 binding polypeptide may be free or hydroxyurea, procarbazine, mitomycin, buSulfan, cis-platin, expressed on the Surface of a phage. and V in cristine Sulfate); horm one S (e.g., In a specific embodiment, either the ACE-2 binding medroxyprogesterone, estramustine phosphate Sodium, ethi polypeptide or the ACE-2 protein is labeled. As further nyl e Stradiol, e Stradiol, mege Strol acetate, discussed herein, a wide range of labels can be used accor methylte Stosterone, diethylstilbestrol diphosphate, dance with the present invention, including but not limited chlorotrianisene, and testolactone), nitrogen mustard deriva to conjugating the recognition unit to biotin by conventional tives (e.g., mephalen, chorambucil, mechlorethamine means. Alternatively, the label may comprise, e.g., a (nitrogen mustard) and thiotepa); Steroids and combinations fluorogen, an enzyme, an epitope, a chromogen, or a radio (e.g., bethamethasone Sodium phosphate); and others (e.g., nuclide. Preferably, the biotin is conjugated by covalent 25 attachment to either the ACE-2 binding polypeptide or the dicarbazine, asparaginase, mitotane, Vincristine Sulfate, Vin ACE-2 protein. Preferably, the ACE-2 binding polypeptide blastine Sulfate, and etoposide). is immobilized on a Solid Support. The detection means In a Specific embodiment, ACE-2 binding polypeptides employed to detect the label will depend on the nature of the and ACE-2 binding polypeptide compositions of the inven label and can be any known in the art, e.g., film to detect a tion are administered in combination with CHOP radionuclide, an enzyme Substrate that gives rise to a detect (cyclophosphamide, doxorubicin, Vincristine, and able signal to detect the presence of an enzyme, antibody to prednisone) or any combination of the components of detect the presence of an epitope, etc. CHOP. In one embodiment, the compositions of the inven The invention also provides a pharmaceutical pack or kit tion are administered in combination with anti-CD20 comprising one or more containers filled with one or more antibodies, human monoclonal anti-CD20 antibodies. In 35 of the ingredients of the pharmaceutical compositions of the another embodiment, the compositions of the invention are invention. Optionally associated with Such container(s) can administered in combination with anti-CD20 antibodies and be a notice in the form prescribed by a governmental agency CHOP, or anti-CD20 antibodies and any combination of one regulating the manufacture, use or Sale of pharmaceuticals or or more of the components of CHOP, particularly cyclo biological products, which notice reflects approval by the phosphamide and/or prednisone. In a Specific embodiment, 40 agency of manufacture, use or Sale for human administra compositions of the invention are administered in combina tion. In one preferred embodiment the kit comprises a vial tion with Rituximab. In a further embodiment, compositions containing ACE-2 binding polypeptides conjugated to a of the invention are administered with Rituximab and toxin or a label (as described herein). Such conjugated CHOP, or Rituximab and any combination of one or more of binding polypeptide may be used to kill a particular popu the components of CHOP, particularly cyclophosphamide 45 lation of cells or to quantitate a particular population of cells. and/or prednisone. In a specific embodiment, compositions In a preferred embodiment, Such conjugated ACE-2 binding of the invention are administered in combination with tosi polypeptides are used to kill cells expressing the membrane tumomab. In a further embodiment, compositions of the bound form of ACE-2. In another preferred embodiment, invention are administered with tositumomab and CHOP, or Such conjugated ACE-2 binding polypeptides are used to toSitumomab and any combination of one or more of the 50 quantitate cells expressing the membrane-bound form of components of CHOP, particularly cyclophosphamide and/ ACE-2. or prednisone. The anti-CD20 antibodies may optionally be In one embodiment, the present invention provides a asSociated with radioisotopes, toxins or cytotoxic prodrugs. pharmaceutical pack or kit comprising a first container In another specific embodiment, the compositions of the containing ACE-2 binding polypetides attached to a macro invention are administered in combination Zevalin TM. In a 55 cyclic chelator (e.g., DOTA), a second container containing further embodiment, compositions of the invention are ACE-2 polypeptide, and a third container containing radio administered with ZevalinTM and CHOP, or Zevalin TM and metal ions (e.g., 'Y, 'In, or I.). The contents of these any combination of one or more of the components of containers could then be mixed, and incubated for a period CHOP, particularly cyclophosphamide and/or prednisone. of time to allow the reagents to associate with one another, Zevalin TM may be associated with one or more radisotopes. 60 prior to administration to a patient. In addition it may also Particularly preferred isotopes are 'Y and 'In. be desirable to purify the ACE-2/ACE-2 binding Additionally, the ACE-2 binding polypeptides and ACE-2 polypeptide-macrocyclic chelator/radiometalion complexes binding polypeptide compositions of the invention may be from exceSS reagents that did not form into complexes, for administered alone or in combination with other therapeutic example, by filtration in a spin column based on a size regimens, including but not limited to, radiation therapy. 65 exclusion principle (e.g. Bio-Spin columns available from Such combinatorial therapy may be administered Sequen Biorad Laboratories, Inc.) Thus in another embodiment, the tially and/or concomitantly. present invention provides a pharmaceutical pack or kit US 6,592,865 B2 89 90 comprising a first container containing ACE-2 binding poly In one diagnostic configuration, test Serum is reacted with petides attached to a macrocyclic chelator (e.g., DOTA), a a Solid phase reagent having a Surface-bound ACE-2 binding Second container containing ACE-2 polypeptide, and a third polypeptide according to the present invention. After ACE-2 container containing radiometal ions (e.g., 'Y, 'In, or binds to a Specific ACE-2 binding polypeptide, the unbound 131I), and a means for purifying ACE-2/ACE-2 binding Serum components are removed by Washing, reporter polypeptide-macrocyclic chelator/radiometal ion com labeled anti-ACE-2 binding polypeptide antibody is added, plexes. unbound anti-ACE-2 binding polypeptide antibody is In other embodiments, the present invention provides a removed by Washing, and a reagent is reacted with reporter pharmaceutical pack or kit comprising a first container labeled anti-ACE-2 binding polypeptide antibody to bind containing ACE-2 and ACE-2 binding poypeptides, and a second container containing radiometalions (e.g., 'Y, 'In, 1O reporter to the reagent in proportion to the amount of bound or ''I). In a specific embodiment, the present invention ACE-2 binding polypeptide on the Solid Support. Typically, provides a pharmaceutical pack or kit comprising a first the reporter is an enzyme which is detected by incubating the container containing ACE-2 and ACE-2 binding Solid phase in the presence of a Suitable fluorometric, poypeptides, a Second container containing radiometal ions luminescent or colorimetric Substrate. (e.g., 'Y, 'In, or ''I), and a means for purifying ACE 15 The Solid Surface reagent in the above assay is prepared 2/ACE-2 binding polypeptide-macrocyclic chelator/ by known techniques for attaching protein material to Solid radiometal ion complexes. Support material, Such as polymeric beads, dip Sticks, In Still other embodiments, the present invention provides 96-well plate or filter material. These attachment methods a pharmaceutical pack or kit comprising a first container generally include non-Specific adsorption of the protein to containing ACE-2 binding polypetides attached to a macro the Support or covalent attachment of the protein, typically cyclic chelator (e.g., DOTA) and a second container con through a free amine group, to a chemically reactive group taining radiometal ions (e.g., 'Y, 'In, or I.). In a on the Solid Support, Such as an activated carboxyl, Specific embodiment, the present invention provides a phar hydroxyl, or aldehyde group. Alternatively, Streptavidin maceutical pack or kit comprising a first container ACE-2 coated plates can be used in conjunction with biotinylated binding polypetides attached to a macrocyclic chelator (e.g., 25 ACE-2 binding polypeptides. DOTA), a second container containing radiometal ions (e.g., Thus, the invention provides an assay System or kit for 'Y, 'In, or I.) and a means for purifying ACE-2 binding carrying out this diagnostic method. The kit generally polypeptide-macrocyclic chelator/radiometal ion com includes a Support with Surface-bound recombinant ACE-2, plexes. and a reporter-labeled anti-ACE-2 binding polypeptide anti The present invention provides kits that can be used in the body for detecting surface-bound anti-ACE-2 binding above. methods. In one embodiment, a kit comprises an polypeptide. ACE-2 binding polypeptide of the invention, preferably a Methods of Screening for ACE-2 Binding Molecules purified ACE-2 binding polypeptide, in one or more con The invention also encompasses Screening methods for tainers. In an alterative embodiment, a kit comprises an identifying polypeptides and nonpolypeptides that bind ACE-2 binding polypeptide fragment that Specifically binds 35 ACE-2, and the ACE-2 binding molecules identified thereby. to ACE-2. In a specific embodiment, the kits of the present This method comprises the Steps of: invention contain a Substantially isolated ACE-2 polypep (a) contacting ACE-2 or ACE-2-like polypeptide with a tide as a control. Preferably, the kits of the present invention plurality of molecules, and further comprise a control binding polypeptide which does (b) identifying molecule(s) that binds the ACE-2 or ACE not react with ACE-2. In another specific embodiment, the 40 2-like polypeptide. kits of the present invention contain a means for detecting The step of contacting the ACE-2 protein or ACE-2-like the binding of an ACE-2 binding polypeptide to ACE-2 (e.g., protein with the plurality of molecules may be effected in a the ACE-2 binding polypeptide may be conjugated to a number of ways. For example, one may contemplate immo detectable Substrate Such as a fluorescent compound, an bilizing ACE-2 target on a Solid Support and bringing a enzymatic Substrate, a radioactive compound or a lumines 45 solution of the plurality of molecules in contact with the cent compound, or a Second antibody which recognizes the immobilized ACE-2 target. Such a procedure would be akin ACE-2 binding polypeptide may be conjugated to a detect to an affinity chromatographic process, with the affinity able Substrate). In specific embodiments, the kit may include matrix being comprised of the immobilized ACE-2 protein a recombinantly produced or chemically Synthesized ACE or ACE-2-like polypeptide. The molecules having a Selec 2. The ACE-2 provided in the kit may also be attached to a 50 tive affinity for the ACE-2 or ACE-2-like polypeptide can Solid Support. In a more specific embodiment the detecting then be purified by affinity selection. The nature of the solid means of the above-described kit includes a Solid Support to support, process for attachment of the ACE-2 or ACE-2-like which ACE-2 is attached. Such a kit may also include a polypeptide to the Solid Support, Solvent, and conditions of non-attached reporter-labeled anti-ACE-2 binding polypep the affinity isolation or Selection are largely conventional tide antibody. In this embodiment, binding of the ACE-2 55 and well known to those of ordinary skill in the art. binding polypeptide to ACE-2 can be detected by binding of Alternatively, one may also separate a plurality of the Said reporter-labeled antibody. Alternatively, or in polypeptides into Substantially Separate fractions comprising addition, the detecting means may include a labeled, com a Subset of or individual polypeptides. For instance, one can peting antigen. Separate the plurality of polypeptides by gel electrophoresis, In an additional embodiment, the invention includes a 60 column chromatography, or like method known to those of diagnostic kit for use in Screening Serum containing ACE-2 ordinary skill for the Separation of polypeptides. The indi or ACE-2-like polypeptides. The diagnostic kit includes a vidual polypeptides can also be produced by a transformed Substantially isolated ACE-2 binding polypeptide Specifi host cell in Such a way as to be expressed on or about its cally reactive with ACE-2 target, and means for detecting outer Surface (e.g., a recombinant phage). Individual isolates the binding of ACE-2 target to the ACE-2 binding polypep 65 can then be "probed” using an ACE-2 target protein, option tide. In one embodiment, the ACE-2 binding polypeptide is ally in the presence of an inducer should one be required for attached to a Solid Support. expression, to determine if any Selective affinity interaction US 6,592,865 B2 91 92 takes place between the ACE-2 target protein and the 89:9367–9371 (1992)) can also be used. Another example of individual clone. Prior to contacting the ACE-2 target pro a library that can be used, in which the amide functionalities tein with each fraction comprising individual polypeptides, in peptides have been permethylated to generate a chemi the polypeptides could first be transferred to a Solid Support cally transformed combinatorial library, is described by for additional convenience. Such a Solid Support may simply Ostresh et al. (Proc. Natl. Acad. Sci. USA, 91:11138-11142 be a piece of filter membrane, Such as one made of nitro (1994)). cellulose or nylon. In this manner, positive clones could be The variety of non-peptide libraries that are useful in the identified from a collection of transformed host cells of an present invention is great. For example, Ecker and Crooke, expression library, which harbor a DNA construct encoding Bio/Technology, 13:351-360 (1995) list benzodiazepines, a polypeptide having a Selective affinity for ACE-2 or hydantoins, piperazinediones, biphenyls, Sugar analogs, ACE-2-like polypeptide. Furthermore, the amino acid beta-mercaptoketones, arylacetic acids, acylpiperidines, Sequence of the polypeptide having a Selective affinity for benzopyrans, cubanes, Xanthines, aminimides, and oxazo the ACE-2 protein or ACE-2-like protein can be determined lones as among the chemical Species that form the basis of directly by conventional means, or the coding Sequence of various libraries. the DNA encoding the polypeptide can frequently be deter 15 Non-peptide libraries can be classified broadly into two mined more conveniently. The primary amino acid Sequence types: decorated monomers and oligomers. Decorated can then be deduced from the corresponding DNA sequence. monomer libraries employ a relatively simple Scaffold struc If the amino acid Sequence is to be determined from the ture upon which a variety functional groupS is added. Often polypeptide itself, one may use microSequencing tech the scaffold will be a molecule with a known useful phar niques. The Sequencing technique may include mass Spec macological activity. For example, the Scaffold might be the troScopy. benzodiazepine Structure. In certain situations, it may be desirable to wash away any Non-peptide oligomer libraries utilize a large number of ACE-2 or ACE-2-like polypeptide, or alterntatively, monomers that are assembled together in ways that create unbound polypeptides, from a mixture of ACE-2 or ACE new shapes that depend on the order of the monomers. 2-like polypeptide and the plurality of polypeptides prior to 25 Among the monomer units that have been used are attempting to determine or to detect the presence of a carbamates, pyrrolinones, and morpholinos. Peptoids, Selective affinity interaction. One or more Such a wash Steps peptide-like oligomers in which the Side chain is attached to may be particularly desirable when the ACE-2 or ACE-2- the alpha amino group rather than the alpha carbon, form the like polypeptide or the plurality of polypeptides is bound to basis of another version of non-peptide oligomer libraries. a Solid Support. The first non-peptide oligomer libraries utilized a Single type The plurality of molecules provided according to this of monomer and thus contained a repeating backbone. method may be provided by way of diversity libraries, such Recent libraries have utilized more than one monomer, as random or combinatorial peptide or non-peptide libraries giving the libraries added flexibility. which can be Screened for molecules that Specifically bind to Screening the libraries can be accomplished by any of a ACE-2. Peptide libraries may be designed such that the 35 variety of commonly known methods. See, e.g., the follow polypeptides encoded by the libraries are automatically ing references, which disclose Screening of peptide libraries: fused to a polypeptide linker moiety, for example. Many Parmley and Smith, Ady Exp. Med. Biol., 251:215-218 libraries are known in the art that can be used, e.g., chemi (1989); Scott and Smith, Science, 249:386–390 (1990); cally Synthesized libraries, recombinant (e.g., phage display Fowlkes et al., BioTechniques, 13:422-427 (1992); Olden libraries), and in vitro translation-based libraries. Examples 40 burg et al., Proc. Natl. Acad. Sci. USA, 89:5393–5397 of chemically synthesized libraries are described in Fodor et (1992); Yu et al., Cell, 76:933–945 (1994); Staudt et al., al., Science, 251:767-773 (1991); Houghten et al., Nature, Science, 241:577–580 (1988); Bock et al., Nature, 354:84–86 (1991); Lam et al., Nature, 354:82–84 (1991); 355:564–566 (1992); Tuerk et al., Proc. Natl. Acad. Sci. Medynski, Bio/Technology, 12:709–710 (1994); Gallop et USA, 89:6988-6992 (1992); Ellington et al., Nature, al., J. Medicinal Chemistry, 37(9): 1233–1251 (1994); Ohlm 45 355:850–852 (1992); U.S. Pat. Nos. 5,096,815; 5,223,409; eyer et al., Proc. Natl. Acad. Sci. USA, 90:10922–10926 and 5,198.346, all to Ladner et al.; Rebar and Pabo, Science, (1993); Erb et al., Proc. Natl. Acad. Sci. USA, 263:671-673 (1993); and PCT publication WO 94/18318. 91:11422-11426 (1994); Houghten et al., Biotechniques, In a specific embodiment, Screening to identify a mol 13:412 (1992); Jayawickreme et al., Proc. Natl. Acad. Sci. ecule that binds ACE-2 can be carried out by contacting the USA, 91:1614–1618 (1994); Salmon et al., Proc. Natl. Acad. 50 library members with ACE-2 or ACE-2-like polypeptide Sci. USA, 90:11708–11712 (1993); PCT publication WO immobilized on a Solid phase and harvesting those library 93/20242; and Brenner and Lerner, Proc. Natl. Acad. Sci. members that bind to the ACE-2 or ACE-2-like polypeptide. USA, 89:5381-5383 (1992). Examples of Such Screening methods, termed “panning Examples of phage display libraries are described in Scott techniques are described by way of example in Parmley and and Smith, Science, 249:386–390 (1990); Devlin et al., 55 Smith, Gene, 73:305-318 (1998); Fowlkes et al., Science, 249:404–406 (1990); Christian et al., J. Mol. Biol., BioTechniques, 13:422–427 (1992); PCT publication WO 227:711–718 (1992); Lenstra, J. Immunol. Meth., 94/18318; and in references cited therein. 152:149-157 (1992); Kay et al., Gene, 128:59–65 (1993); In another embodiment, the two-hybrid system for select and PCT publication WO 94/18318. ing interacting proteins in yeast (Fields and Song, Nature, In vitro translation-based libraries include but are not 60 340:245–246 (1989); Chien et al., Proc. Natl. Acad. Sci. limited to those described in PCT publication WO91/05058 USA, 88: 9578-9582 (1991)) can be used to identify mol and Mattheakis et al., Proc. Natl. Acad. Sci. USA, ecules that specifically bind to ACE-2 or ACE-2-like 91:9022-9026 (1994). polypeptides. By way of examples of non-peptide libraries, a benzodi Where the ACE-2 binding molecule is a polypeptide, the azepine library (see, e.g., Bunin et al., Proc. Natl. Acad. Sci. 65 polypeptide can be conveniently Selected from any peptide USA, 91:4708–4712 (1994) can be adapted for use. Peptoid library, including random peptide libraries, combinatorial libraries (Simon et al., Proc. Natl. Acad. Sci. USA, peptide libraries, or biased peptide libraries. The term US 6,592,865 B2 93 94 “biased” is used herein to mean that the method of gener proteins as well as the conditions under which ACE-2 ating the library is manipulated So as to restrict one or more binding polypeptides optimally release ACE-2 target pro parameters that govern the diversity of the resulting collec teins. Similarly, the binding and/or release conditions may tion of molecules, in this case peptides. be selected with regard to known interactions between a Thus, a truly random peptide library would generate a binding domain and the ACE-2 target protein, for example, collection of peptides in which the probability of finding a to favor the interaction under the binding and/or release particular amino acid at a given position of the peptide is the conditions, or they may be selected without regard to Such Same for all 20 amino acids. A bias can be introduced into known interactions. Likewise, the parental binding domain the library, however, by Specifying, for example, that a can be Selected taking into account a desired binding and/or lysine occur every fifth amino acid, that certain amino acid release condition or not. It is understood that if the binding positions in a peptide remain fixed (e.g., as cysteine), or that domain analogues of a library are unstable under a proposed positions 4, 8, and 9, for example, of a decapeptide library or desired binding or release condition, no useful binding be limited to permit several but less than all of the twenty polypeptides may be obtained. naturally-occurring amino acids. Clearly, many types of In Selecting the parental binding domain, the most impor biases can be contemplated, and the present invention is not 15 tant consideration is how the analogue domains will be restricted to any particular bias. Furthermore, the present presented to the ACE-2 target protein, that is, in what invention contemplates Specific types of peptide libraries, conformations the ACE-2 target and the polypeptide ana Such as phage displayed peptide libraries and those that logues will contact one another. In preferred embodiments, utilize a DNA construct comprising a lambda phage vector for example, the polypeptide analogues will be generated by with a DNA insert. insertion of Synthetic DNA encoding the polypeptide ana AS mentioned above, in the case of an ACE-2 binding logue into a replicable genetic package, resulting in display molecule that is a polypeptide, the polypeptide may have of the domain on the Surface of a microorganism, Such as about 6 to less than about 60 amino acid residues, preferably M13 phage, using techniques as described in Kay et al., about 6 to about 10 amino acid residues, and most Phage Display of Peptides and Proteins. A Laboratory preferably, about 6 to about 22 amino acids. In another 25 Manual (Academic Press, Inc.; San Diego 1996) and U.S. embodiment, an ACE-2 binding polypeptide has in the range Pat. No. 5,223,409 (Ladner et al.), incorporated herein by of 15-100 amino acids, or 20-50 amino acids. reference. For formation of phage display libraries, it is The selected ACE-2 binding polypeptide can be obtained preferred to use Structured polypeptides as the parental by chemical Synthesis or recombinant expression. binding domain or template, as opposed to unstructured, The Specific ACE-2 binding polypeptides disclosed herein linear peptides. Mutation of Surface residues in a protein were isolated using phage display technology, to identify domain or polypeptide molecule will usually have little ACE-2 binding polypeptides exhibiting particular prese effect on the overall structure or general properties (Such as lected binding properties. These ACE-2 binding polypep size, stability, and temperature of denaturation) of the pro tides were isolated initially by Screening nine phage display tein, while at the same time mutation of Surface residues libraries, that is, populations of recombinant bacteriophage 35 may profoundly affect the binding properties of the mol transformed to express an exogenous recombinant polypep ecule. The more tightly a polypeptide Segment is tide on their Surface. In order to isolate new polypeptide constrained, the less likely it is to bind to any particular binding moieties for a particular target, Such as ACE-2, target. If it does bind, however, the binding is likely to be Screening of peptide libraries, for example using phage tighter and more specific. Thus, it is preferred to Select a display techniques, is especially advantageous, in that very 40 parental binding domain wherein the parental polypetide has large numbers (e.g.,5x10) of potential binders can be tested Structure and, thereby in turn, Select a structure for the and Successful binders isolated in a short period of time. polypeptide analogues of the library, which is constrained In order to prepare a phage library of potential binding within a framework having Some degree of rigidity. polypeptides to Screen for members of the library that are Preferably the protein domain that is used as the template ACE-2 binding polypeptides, a candidate binding domain is 45 or parental domain for generating the library of domain Selected to Serve as a structural template for the polypeptides analogues will be a peptide molecule that is a relatively to be displayed in the library. The phage library is made up Small protein or polypeptide. Small polypeptides offer Sev of polypeptide analogues of this template or "parental bind eral advantages over large proteins: First, the mass per ing domain.” The parental binding domain is a polypeptide binding site is reduced. Highly Stable protein domains molecule that may be a naturally occurring or Synthetic 50 having low molecular weights, for example, Kunitz domains protein or polypeptide, or polypeptide region or domain of (-7 kilodaltons, kDa), Kazal domains (-7 kDa), Cucurbida a protein. The parental binding domain may be Selected maxima trypsin inhibitor (CMTI) domains (-3.5 kDa), and based on knowledge of a known interaction between the endothelin (~2 kDa), can show much higher binding per parental binding domain and a target protein, but this is not gram than do antibodies (150 kDa) or single chain scFv critical. In fact, it is not essential that the parental binding 55 antibodies (30 kDa). Second, the possibility of non-specific domain have any affinity for a target at all because its binding is reduced because there is leSS molecular Surface purpose is to provide a structure from which a multiplicity available for nonspecific binding. Third, Small polypeptides of polypeptide analogues (a "library”) can be generated, can be engineered to have unique tethering Sites in a way which multiplicity of polypeptide analogues will include one that is impracticable for larger proteins or antibodies. For or more binding polypeptides that exhibit the desired bind 60 example, Small proteins and polypeptides can be engineered ing and release properties with respect to ACE-2 target to have lysines only at Sites Suitable for tethering to a proteins (and any other properties selected). chromatography matrix. This is not feasible for antibodies. Knowledge of the exact polypeptide that will Serve as the Fourth, a constrained polypeptide Structure is more likely to parental binding domain, or knowledge of a class of proteins retain its functionality when transferred (with the structural or domains to which the parental binding domain belongs 65 domain intact) from one framework to another. For instance, can be useful in determining the conditions under which the binding domainstructure is likely to be transferable from ACE-2 binding polypeptides optimally bind ACE-2 target the framework used for presentation in a library, Such as US 6,592,865 B2 95 96 displayed on a phage, to an isolated protein removed from these techniques, a polypeptide analogue biased library may the presentation framework or immobilized on a chromato be Screened to reveal members that bind tightly, that is, have graphic Substrate. high affinity for ACE-2 target protein, under the Screening In Specific embodiments, the ACE-2 binding polypeptides conditions. of the invention are immobilized. ACE-2 binding polypep In the present invention target ACE-2 protein molecules tide molecules according to the invention may be were biotinylated and then bound to Streptavidin-coated immobilized, for example, on chromatographic Support magnetic particles. Eight phage display libraries of different materials to form efficient ACE-2 separation or affinity design were screened for the ability to bind the immobilized chromatographic media. Immobilized ACE-2 binding ACE-2. Six of the libraries were characterized by M13 polypeptides of the invention have uses that include, but are phage displaying a variegated exogenous peptide loop of not limited to, detecting, isolating or removing ACE-2 target different lengths and overall structure: The TN6/6 library proteins from Solutions. One Strategy for generating ACE-2 was constructed to display a single microprotein binding binding polypeptide molecules that can be immobilized, for loop contained in a 12-amino acid template. The TN7/4 example, on matrices, resins, or Supports, involves Selecting library was constructed to display a single microprotein appropriate binding domain templates Such that ACE-2 15 binding loop contained in a 13-amino acid template. The binding polypeptide molecules are generated that have one TN8/9 library was constructed to display a single micropro or more amino acids that may be used to covalently link the tein binding loop contained in a 14-amino acid template. The ACE-2 binding polypeptide to a chromatographic resin or TN9/4 library was constructed to display a single micropro substrate to form an affinity resin. Similarly, the N-terminal tein binding loop contained in a 15-amino acid template. The amino group or the C-terminal carboxyl group of a peptide TN10/9 library was constructed to display a single micro molecule may be modified by adding a capping group to protein binding loop contained in a 16-amino acid template. render it inert or a functional group, which permits linkage The TN12/1 library was constructed to display a single to a Support medium. For example, the C-terminal carboxyl microprotein binding loop contained in an 18-amino acid group of a protein domain may be converted to an amide or template. Two commercially available linear phage display a hydrazide (-NH-NH) group for reaction with an 25 libraries were also screened, designated PhD 7 and PhD 12 aldehyde-functional Substrate or other amine-reactive Sub (New England Biolabs). The PhD 7 library displayed a Strate. This technique is preferred. Another preferred modi linear random-sequence 7-mer and the PhD 12 library fication of ACE-2 binding polypeptides useful for linking an displayed a random-Sequence 12-mer. ACE-2 binding polypeptide molecule of the invention to a ACE-2 binding phage were not isolated from either of the chromatography material is a polypeptide linker comprising, comercially available libraries, PhD 7 and PhD 12. or alternatively consisting of, the amino acid Sequence After analysis of the Sequences isolated from the library Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO:13). Screenings, Several families of ACE-2 binding peptides were In one non-limiting example of a Screening procedure to defined. The amino acid sequences of the ACE-2-binding obtain ACE-2 binding polypeptides encompassed by the “hits” are set forth in Example 1 (infra). invention, the phage in a phage display library are contacted 35 AS it within the Scope of the present invention to Screen with and allowed to bind an ACE-2 target protein that is phage libraries that bind one or more of the various forms of immobilized on a Solid Support. Those phage that display ACE-2, the following outlines Some assays that may be used non-binding polypeptides are Separated from those that bind in Screening for ACE-2 binding polypeptides that bind the the ACE-2 target protein. Any of various techniques known Soluble form of ACE-2, the membrane-bound form of ACE in the art may be applied to dissociate the bound phage from 40 2, or both the Soluble and the membrane-bound forms of the immobilized ACE-2 protein, and to collect and/or ACE-2. ASSays to determine the Specificity of binding amplify the phage and/or their nucleic acid contents. Using polypeptides for different forms of a protein are commonly these techniques it is possible to identify a ACE-2 binding known in the art and may be readily adapted for determining phage that is about 1 in 20 million in the population. the specificity of ACE-2 binding polypeptides for different Libraries, displaying 10-20 million or more potential bind 45 forms of ACE-2. ing peptide molecules each, are rapidly Screened to find ACE-2 binding polypeptides of the invention (including high-affinity ACE-2 binding polypeptides. molecules comprising, or alternatively consisting of, ACE-2 In each round of Screening, the diversity of a population binding polypeptide fragments or variants thereof) may be falls until only efficient binding polypeptides remain, that is, Screened in a variety of assays to identify those ACE-2 the process converges. Typically, a phage display library will 50 binding polypeptides that Specifically bind to the Soluble contain several closely related binding polypeptides (10 to form of ACE-2. ACE-2 binding polypeptides may be 50 different binding polypeptides out of 10 million). Indi assayed in neutralization assays described herein (see cations of convergence include increased binding (measured Example 4) or otherwise known in the art. For example, by phage titers) and recovery of closely related Sequences. ACE-2 binding polypeptides may be tested for their ability After a first Set of binding polypeptide molecules is 55 to inhibit soluble ACE-2 from binding an ACE-2 substrate. identified, the Sequence information can be used to design ACE-2 binding polypeptides of the invention (including other libraries biased for members having additional desired molecules comprising, or alternatively consisting of, ACE-2 properties, for example, discrimination between different binding polypeptide fragments or variants thereof) may be forms of ACE-2 (e.g., the membrane form and the soluble Screened in a variety of assays commonly known in the art form of ACE-2) and fragments thereof, or discrimination 60 to identify those ACE-2 binding polypeptides that Specifi between ACE-2 and closely related impurities in a feed cally bind to the membrane-bound form of ACE-2. For Stream. example, ACE-2 binding polypeptides may be assayed for Such techniques make it possible not only to Screen a binding ACE-2 protein present on cell membranes of cells large number of potential binding polypeptides, but make it that express ACE-2. practical to repeat the binding and elution cycles and to build 65 ACE-2 binding polypeptides of the invention (including Secondary, biased libraries for Screening polypeptide molecules comprising, or alternatively consisting of, ACE-2 analogue-displaying phage that meet Specific criteria. Using binding polypeptide fragments or variants) may be screened US 6,592,865 B2 97 98 in a variety of assays to identify those ACE-2 binding Specific for both a polypeptide of the present invention as polypeptides or ACE-2 binding polypeptide fragments or well as for a heterologous epitope, Such as a heterologous variants that specifically bind to the soluble form and polypeptide or Solid Support material. See, e.g., PCT pub membrane-bound form of ACE-1. This can readily be deter lications WO 93/17715, WO 92/08802, WO 91/00360, WO mined by performing assays to distinguish binding to the 92/05793; Tutt et al., J. Immunol., 147:60–69 (1991); U.S. Soluble form and assays to distinguish binding to the Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; membrane-bound form (Such as the assays described herein 5,601,819; Kostelny et al., J. Immunol., 148:1547–1553 or otherwise known in the art), and identifying the ACE-2 (1992). binding polypeptides that bind both forms. Antibodies of the present invention may be described or Additionally, ACE-2 binding polypeptides of the inven Specified in terms of the epitope(s) or portion(s) of an ACE-2 tion may be screened for the ability to inhibit, stimulate or binding polypeptide of the present invention which they not significantly alter ACE-2 activity, e.g., the ability of recognize or specifically bind. Antibodies which specifically ACE-2 to bind to its Substrate (e.g., angiotensin). bind any epitope or polypeptide of the present invention may Anti-ACE-2 Binding Polypeptide Antibodies also be excluded. Therefore, the present invention includes Further polypeptides of the invention relate to antibodies 15 antibodies that Specifically bind ACE-2 binding polypep and T-cell antigen receptors (TCR) which immunospecifi tides of the present invention, and allows for the exclusion cally bind an ACE-2 binding polypeptide of the present of the Same. invention (as determined by immunoassays well known in In further preferred, nonexclusive embodiments, the anti the art for assaying specific antibody-antigen binding). Anti bodies of the invention (e.g., anti-idiotypic antibodies) bodies of the invention include, but are not limited to, inhibit one or more biological activities of ACE-2 through polyclonal, monoclonal, multi Specific, human, humanized Specific binding to ACE-2. In another preferred or chimeric antibodies, Single chain antibodies, Fab embodiment, the antibody of the invention inhibits ACE-2- fragments, F(ab') fragments, fragments produced by a Fab mediated vasoconstriction. expression library, anti-idiotypic (anti-id) antibodies Antibodies of the present invention may also be described (including, e.g., anti-id antibodies to antibodies of the 25 or Specified in terms of their croSS-reactivity. Antibodies that invention), and epitope-binding fragments of any of the do not bind any other ACE-2 binding polypeptide of the above. The term “antibody,” as used herein, refers to immu present invention are included. Antibodies that bind noglobulin molecules and immunologically active portions polypeptides with at least 95%, at least 90%, at least 85%, of immunoglobulin molecules, i.e., molecules that contain at least 80%, at least 75%, at least 70%, at least 65%, at least an antigen binding site that immunospecifically binds an 60%, at least 55%, and at least 50% identity (as calculated antigen. The immunoglobulin molecules of the invention using methods known in the art and described herein) to an can be of any type (e.g., IgG, IgE, IgM, Ig), IgA and IgY), ACE-2 binding polypeptide of the present invention are also class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or included in the present invention. Antibodies that do not Subclass of immunoglobulin molecule. Immunoglobulins bind polypeptides with less than 95%, less than 90%, less may have both a heavy and light chain. In Specific 35 than 85%, less than 80%, less than 75%, less than 70%, less embodiments, the immunoglobulin molecules of the inven than 65%, less than 60%, less than 55%, and less than 50% tion are IgG1. In other specific embodiments, the immuno identity (as calculated using methods known in the art and globulin molecules of the invention are IgG4. An array of described herein) to an ACE-2 binding polypeptide of the IgG, IgE, IgM, Ig), IgA, and IgY heavy chains may be present invention are also included in the present invention. paired with a light chain of the kappa or lambda forms. 40 Further included in the present invention are antibodies Most preferably the antibodies are human antigen-binding which bind polypeptides encoded by polynucleotides, the antibody fragments of the present invention and include, but complement of which hybridize to a polynucleotides of the are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs present invention under Stringent hybridization conditions (scFv), single-chain antibodies, disulfide-linked FVs (sdFv) (as described herein). Antibodies of the present invention and fragments comprising either a VL or VH domain. 45 may also be described or Specified in terms of their binding Antigen-binding antibody fragments, including Single-chain affinity to an ACE-2 binding polypeptide of the invention. antibodies, may comprise the variable region(s) alone or in Preferred binding affinities include those with a dissociation combination with the entirety or a portion of the following: constant or Kd less than 5x10 M, 10 M., 5x10 M, hinge region, CH1, CH2, and CH3 domains. Also included 10M, 5x107 M, 107 M., 5x10 M, 10 M, 5x10 M, in the invention are antigen-binding fragments also com 50 10 M,5x100 M, 100 M,5x10 M, 10 M,5x10° prising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the 1015 M. invention may be from any animal origin including birds and The invention also provides antibodies that competitively mammals. Preferably, the antibodies are human, murine inhibit binding of an antibody to an ACE-2 binding polypep (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, 55 tide of the invention as determined by any method known in camel, horse, or chicken. AS used herein, “human' antibod the art for determining competitive binding. In preferred ies include antibodies having the amino acid Sequence of a embodiments, the antibody competitively inhibits binding to human. immunoglobulin and include antibodies isolated the ACE-2 binding polypeptide by at least 95%, at least from human immunoglobulin libraries or from animals 90%, at least 85%, at least 80%, at least 75%, at least 70%, transgenic for one or more human immunoglobulin and that 60 at least 60%, or at least 50%. do not express endogenous immunoglobulins, as described Antibodies of the present invention (e.g., anti-idiotypic infra and, for example in, U.S. Pat. No. 5,939,598 to antibodies) may act as agonists or antagonists of ACE-2 or Kucherlapati et al. alternatively may not significantly alter ACE-2 mediated The antibodies of the present invention may be activity. For example, the present invention includes anti monospecific, bispecific, trispecific or of greater multispeci 65 bodies (e.g., anti-idiotypic antibodies) which disrupt ACE ficity. Multispecific antibodies may be specific for different 2/ACE-2 Substrate (e.g., angiotensin, bradykinin, epitopes of a polypeptide of the present invention or may be tachykinin, endothelin, neurotensin, or Substance P) inter US 6,592,865 B2 99 100 actions either partially or fully. In another example, anti cleavage, acetylation, formylation, metabolic Synthesis of bodies of the invention enhance ACE-2/ACE-2 receptor tunicamycin, etc. Additionally, the derivative may contain interactions either partially or fully. Such activity may be the one or more non-classical amino acids. result of, for example, the antibody binding to an ACE-2 The antibodies of the present invention may be generated binding polypeptide of the invention, or alternatively as a by any suitable method known in the art. Polyclonal anti result of direct binding of the antibody (e.g., an anti bodies to an antigen-of-interest can be produced by various idiotypic antibody to ACE-2). procedures well known in the art. For example, a polypep Preferrably, antibodies of the present invention bind an tide of the invention can be administered to various host ACE-2 binding polypeptide disclosed herein, a portion animals including, but not limited to, rabbits, mice, rats, etc. thereof, or an antibody that binds an ACE-2 binding to induce the production of Sera containing polyclonal polypeptide disclosed herein, or a portion thereof. The antibodies Specific for the antigen. Various adjuvants may be invention features both ACE-2 binding polypeptide-specific used to increase the immunological response, depending on antibodies and antibodies that are Specific to ACE-2 binding the host species, and include but are not limited to, Freund's polypeptide/ACE-2 complexes. The invention features anti (complete and incomplete), mineral gels Such as aluminum bodies that enhance ACE-2/ACE-2 binding polypeptide 15 hydroxide, Surface active Substances Such as lySolecithin, binding and/or ACE-2/ACE-2 substrate binding. The inven pluronic polyols, polyanions, peptides, oil emulsions, key tion also features antibodies that do not inhibit or reduce hole limpet hemocyanins, dinitrophenol, and potentially ACE-2/ACE-2 binding polypeptide binding and/or ACE-2/ useful human adjuvants such as BCG (bacille Calmette ACE-2 substrate binding. The invention also features ACE-2 Guerin) and corynebacterium parvum. Such adjuvants are binding polypeptide Specific antibodies that inhibit binding also well known in the art. of the ACE-2 binding polypeptide to ACE-2 or ACE-2 According to certain embodiments of the invention, mul binding to an ACE-2 Substrate. In Specific embodiments, tivalent ACE-2 binding polypeptides are administered to the antibodies are provided that inhibit ACE-2 activity or ACE-2 host animal. Multivalent ACE-2 binding polypeptide com substrate activity by at least 95%, at least 90%, at least 85%, plexes may be prepared using techniques and materials at least 80%, at least 75%, at least 70%, at least 60%, or at 25 known in the art Such as, for example, by croSS-linking the least 50% of the activity in absence of the antibody. Recep polypeptide to a carrier protein (e.g., bovine Serum albumin tor activation (i.e., Signaling) may be determined by tech (BSA), human albumin, keyhole limpet hemocyanin (KLH), niques described herein or otherwise known in the art. For or Succinylated KLH) by use of conventional cross-linking example, receptor activation can be determined by detecting reagents. the phosphorylation (e.g., tyrosine or serine/threonine) of In specific embodiments multivalent ACE-2 binding the receptor or its Substrate by immunoprecipitation fol polypeptides of the invention are administered in the form of lowed by western blot analysis (for example, as described multiple antigen peptides (MAP) (Tam, J. Imm. Meth., Supra). 124:53–61 (1989); Tam, Proc. Natl. Acad. Sci. USA, The antibodies of the present invention may be used, for 85:5409-5413 (1988)). In this form, the multivalent ACE-2 purposes including, but not limited to, purify, detect, and 35 binding polypeptide is Synthesized on a branching lysyl target the ACE-2 binding polypeptides of the present matrix using Solid-phase peptide Synthesis methods. Recog invention, including both in vitro and in Vivo diagnostic and nition units in the form of MAP may be prepared by methods therapeutic methods. For example, the antibodies have use known in the art (Tam, 1989, Supra; Tam, 1988, Supra), or, in immunoassays for qualitatively and quantitatively mea for example, by a stepwise Solid-phase procedure on MAP Suring levels of ACE-2 in biological Samples. See, e.g., 40 resins (Applied BioSystems), utilizing methodology estab Harlow et al., Antibodies: A Laboratory Manual (Cold lished by the manufacturer. MAP peptides may be synthe Spring Harbor Laboratory Press, Cold Spring Harbor 1988). sized comprising (ACE-2 binding polypeptide) LyS, AS discussed in more detail below, the antibodies of the (ACE-2 binding polypeptide) LyS, (ACE-2 binding present invention may be used either alone or in combina polypeptide) LyS, or more levels of branching. tion with other compositions. The antibodies may further be 45 Monoclonal antibodies can be prepared using a wide recombinantly fused to a heterologous polypeptide at the N variety of techniques known in the art including the use of or C-terminus or chemically conjugated (including hybridoma, recombinant, and phage display technologies, or covalently and non-covalently conjugated) to polypeptides a combination thereof. For example, monoclonal antibodies or other compositions. For example, antibodies of the can be produced using hybridoma techniques including present invention may be recombinantly fused or conjugated 50 those known in the art and taught, for example, in Harlow et to molecules useful as labels in detection assays and effector al., Antibodies: A Laboratory Manual (Cold Spring Harbor molecules Such as heterologous polypeptides, drugs, Laboratory Press, Cold Spring Harbor 1988); Hammerling radionuclides, or toxins. See, e.g., PCT publications WO et al., in Monoclonal Antibodies and T-Cell Hybridomas 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. (Elsevier, N.Y. 1981), pp. 563-681 (said references incor 5,314,995; and EP 396 387. 55 porated by reference in their entireties). The term “mono The antibodies of the invention include derivatives that clonal antibody” as used herein is not limited to antibodies are modified, i.e., by the covalent attachment of any type of produced through hybridoma technology. The term "mono molecule to the antibody Such that covalent attachment does clonal antibody” refers to an antibody that is derived from a not prevent the antibody from generating an anti-idiotypic Single clone, including any eukaryotic, prokaryotic, or phage response. For example, but not by way of limitation, the 60 clone, and not the method by which it is produced. antibody derivatives include antibodies that have been A "monoclonal antibody' may comprise, or alternatively modified, e.g., by glycosylation, acetylation, pegylation, consist of, two proteins, i.e., a heavy and a light chain. phosphorylation, amidation, derivatization by known Methods for producing and Screening for Specific anti protecting/blocking groups, proteolytic cleavage, linkage to bodies using hybridoma technology are routine and well a cellular ligand or other protein, etc. Any of numerous 65 known in the art and are discussed in detail in the Examples chemical modifications may be carried out by known (e.g., Degradation). In a non-limiting example, mice can be techniques, including, but not limited to, Specific chemical immunized with a polypeptide of the invention or a cell US 6,592,865 B2 101 102 expressing Such peptide. Once an immune response is mammalian cells, insect cells, plant cells, yeast, and detected, e.g., antibodies Specific for the antigen are detected bacteria, e.g., as described in detail below. For example, in the mouse Serum, the mouse spleen is harvested and techniques to recombinantly produce Fab, Fab' and F(ab') Splenocytes isolated. The Splenocytes are then fused by fragments can also be employed using methods known in the well-known techniques to any Suitable myeloma cells, for art such as those disclosed in PCT publication WO example cells from cell line SP20 available from the Ameri 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 can Type Culture Collection (ATCC), to form hybridoma (1992); and Sawai et al., AJRI, 34:26-34 (1995); and Better cells. Hybridomas are selected and cloned by limited dilu et al., Science, 240: 1041-1043 (1988) (said references tion. The hybridoma clones are then assayed by methods incorporated herein by reference in their entireties). known in the art for cells that Secrete antibodies capable of Examples of techniques which can be used to produce binding a polypeptide of the invention. AScites fluid, which Single-chain FVS and antibodies include those described in generally contains high levels of antibodies, can be gener U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., ated by immunizing mice with positive hybridoma clones. Methods in Enzymology, 203:46-88 (1991); Shu et al., Proc. Accordingly, the present invention provides methods of Natl. Acad. Sci. USA, 90:7995–7999 (1993); and Skerra et generating monoclonal antibodies as well as antibodies 15 al., Science, 240:1038-1040 (1988). For some uses, includ produced by the method comprising culturing a hybridoma ing in Vivo use of antibodies in humans and in vitro detection cell Secreting an antibody of the invention wherein, assays, it may be preferable to use chimeric, humanized, or preferably, the hybridoma is generated by fusing Splenocytes human antibodies. A chimeric antibody is a molecule in isolated from a mouse immunized with an antigen of the which different portions of the antibody are derived from invention with myeloma cells and then Screening the hybri different animal Species, Such as antibodies having a variable domas resulting from the fusion for hybridoma clones that region derived from a murine monoclonal antibody and a Secrete an antibody able to bind a polypeptide of the human immunoglobulin constant region. Methods for pro invention. ducing chimeric antibodies are known in the art. See e.g., Antibody fragments that recognize Specific epitopes may Morrison, Science, 229:1202 (1985); Oi et al., be generated by known techniques. For example, Fab and 25 BioTechniques, 4:214 (1986); Gillies et al., J. Immunol. F(ab')2 fragments of the invention may be produced by Methods, 125:191-202 (1989); U.S. Pat. Nos. 5,807,715; proteolytic cleavage of immunoglobulin molecules, using 4,816,567; and 4,816,397, which are incorporated herein by enzymes Such as papain (to produce Fab fragments) or reference in their entirety. A humanized antibody is an pepsin (to produce F(ab')2 fragments). F(ab')2 fragments antibody molecule made using one or more complementarity contain the variable region, the light chain constant region determining regions (CDRS) from a non-human species and the CH1 domain of the heavy chain. antibody that binds the desired antigen and framework For example, the antibodies of the present invention can regions from a human immunoglobulin molecule. Often, also be generated using Various phage display methods framework residues in the human framework regions will be known in the art. In phage display methods, functional substituted with the corresponding residue from the CDR antibody domains are displayed on the Surface of phage 35 donor antibody to alter, preferably improve, antigenbinding. particles that carry the polynucleotide Sequences encoding These framework substitutions are identified by methods them. In a particular embodiment, Such phage can be utilized well known in the art, e.g., by modeling of the interactions to display antigen-binding domains expressed from a rep of the CDR and framework residues to identify framework ertoire or combinatorial antibody library (e.g., human or residues important for antigen binding and Sequence com murine). Phage expressing an antigen binding domain that 40 parison to identify unusual framework residues at particular binds the antigen of interest can be Selected or identified positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; with antigen, e.g., using labeled antigen or antigen bound or Riechmann et al., Nature, 332:323 (1988), which are incor captured to a Solid Surface or bead. Phage used in these porated herein by reference in their entireties.) Antibodies methods are typically filamentous phage including fa and can be humanized using a variety of techniques known in the M13 binding domains expressed from phage with Fab, Fv or 45 art including, for example, CDR-grafting (EP239 400; PCT disulfide stabilized Fv antibody domains recombinantly publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530, fused to either the phage gene III or gene VIII protein. 101; and 5,585,089), veneering or resurfacing (EP592 106; Examples of phage display methods that can be used to EP 519 596; Padlan, Molecular Immunology, 28(4/5) make the antibodies of the present invention include those :489-498 (1991); Studnicka et al., Protein Engineering, disclosed in Brinkman et al., J. Immunol. Methods, 50 7(6):805–814 (1994); Roguska. et al., Proc. Natl. Acad. Sci. 182:41-50 (1995); Ames et al., J. Immunol. Methods, USA, 91:969–973 (1994)), and chain shuffling (U.S. Pat. No. 184:177-186 (1995); Kettleborough et al., Eur: J. Immunol., 5,565,332). 24:952-958 (1994); Persic et al., Gene, 1879–18 (1997); Completely human antibodies are particularly desirable Burton et al., Advances in Immunology, 57:191-280 (1994); for therapeutic treatment of human patients. Human anti PCT international application No. PCT/GB91/01134, PCT 55 bodies can be made by a variety of methods known in the art publications WO 90/02809; WO 91/10737; WO92/01047; including phage display methods described above using WO 92/18619; WO 93/11236; WO 95/15982; WO antibody libraries derived from human immunoglobulin 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403, sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716, 484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571, 111; and PCT publications WO 98/46645, WO 98/50433, 698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 60 WO 98/24893, WO 98/16654, WO 96/34096, WO and 5,969,108; each of which is incorporated herein by 96/33735, and WO 91/10741; each of which is incorporated reference in its entirety. herein by reference in its entirety. AS described in the above references, after phage Human antibodies can also be produced using transgenic Selection, the antibody coding regions from the phage can be mice which are incapable of expressing functional endog isolated and used to generate whole antibodies, including 65 enous immunoglobulins, but which can express human human antibodies, or any other desired antigen binding immunoglobulin genes. For example, the human heavy and fragment, and expressed in any desired host, including light chain immunoglobulin gene complexes may be intro US 6,592,865 B2 103 104 duced randomly or by homologous recombination into Polynucleotides Encoding Antibodies mouse embryonic Stem cells. Alternatively, the human Vari The invention further provides polynucleotides compris able region, constant region, and diversity region may be ing a nucleotide Sequence encoding an antibody of the invention and fragments thereof. The invention also encom introduced into mouse embryonic Stem cells in addition to passes polynucleotides that hybridize under Stringent the human heavy and light chain genes. The mouse heavy hybridization conditions, e.g., as defined Supra, to poly and light chain immunoglobulin genes may be rendered nucleotides that encode an antibody, preferably, that Specifi non-functional Separately or Simultaneously with the intro cally binds to ACE-2 or an ACE-2 binding polypeptide of duction of human immunoglobulin loci by homologous the invention. recombination. In particular, homozygous deletion of the JH The polynucleotides may be obtained, and the nucleotide region prevents endogenous antibody production. The modi Sequence of the polynucleotides determined, by any method fied embryonic Stem cells are expanded and microinjected known in the art. For example, if the nucleotide Sequence of into blastocysts to produce chimeric mice. The chimeric the antibody is known, a polynucleotide encoding the anti body may be assembled from chemically synthesized oli mice are then bred to produce homozygous offspring that gonucleotides (e.g., as described in Kutmeier et al., express human antibodies. The transgenic mice are immu BioTechniques, 17:242 (1994)), which, briefly, involves the nized in the normal fashion with a Selected antigen, e.g., all 15 Synthesis of overlapping oligonucleotides containing por or a portion of a polypeptide of the invention. Monoclonal tions of the Sequence encoding the antibody, annealing and antibodies directed against the antigen can be obtained from ligating of those oligonucleotides, and then amplification of the immunized, transgenic mice using conventional hybri the ligated oligonucleotides by PCR. doma technology. The human immunoglobulin transgenes Alternatively, a polynucleotide encoding an antibody may harbored by the transgenic mice rearrange during B cell be generated from nucleic acid from a Suitable Source. If a differentiation, and Subsequently undergo class Switching clone containing a nucleic acid encoding a particular anti and Somatic mutation. Thus, using Such a technique, it is body is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immuno possible to produce therapeutically useful IgG, IgA, IgM globulin may be chemically Synthesized or obtained from a and IgE antibodies. For an overview of this technology for suitable source (e.g., an antibody clDNA library, or a cDNA producing human antibodies, See Lonberg and HuSZar, Int. 25 library generated from, or nucleic acid, preferably poly A+ Rev. Immunol., 13:65-93 (1995). For a detailed discussion RNA, isolated from, any tissue or cells expressing the of this technology for producing human antibodies and antibody, Such as hybridoma cells Selected to express an human monoclonal antibodies and protocols for producing antibody of the invention) by PCR amplification using such antibodies, see, e.g., PCT publications WO 98/24893; synthetic primers hybridizable to the 3' and 5' ends of the WO 92/01047; WO 96/34096; WO 96/33735; European Sequence or by cloning using an oligonucleotide probe Patent 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633, Specific for the particular gene Sequence to identify, e.g., a 425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885, cDNA clone from a cDNA library that encodes the antibody. 793; 5,916,771; and 5,939,598, each of which is incorpo Amplified nucleic acids generated by PCR may then be rated by reference herein in its entirety. In addition, cloned into replicable cloning vectors using any method known in the art. companies Such as Abgenix, Inc. (Freemont, Calif.) and 35 Once the nucleotide Sequence and corresponding amino GenPharm (San Jose, Calif.) can be engaged to provide acid Sequence of the antibody is determined, the nucleotide human antibodies directed against a Selected antigen using Sequence of the antibody may be manipulated using methods technology Similar to that described above. well known in the art for the manipulation of nucleotide Completely human antibodies that recognize a Selected Sequences, e.g., recombinant DNA techniques, Site directed epitope can be generated using a technique referred to as 40 mutagenesis, PCR, etc. (See, for example, the techniques "guided Selection.” In this approach, a Selected non-human described in Sambrook et al., Molecular Cloning. A Labo monoclonal antibody, e.g., a mouse antibody, is used to ratory Manual, 2d Ed. (Cold Spring Harbor Laboratory, guide the Selection of a completely human antibody recog Cold Spring Harbor, N.Y. 1990) and Current Protocols in nizing the same epitope. (See, Jespers et al., Bio/technology, Molecular Biology, Ausubel et al., eds. (John Wiley & Sons, 12:899–903 (1988).) 45 NY 1993), which are both incorporated by reference herein Further, antibodies to the ACE-2 binding polypeptides of in their entireties), to generate antibodies having a different the invention can, in turn, be utilized to generate anti amino acid Sequence, for example to create amino acid idiotype antibodies that “mimic' polypeptides of the inven Substitutions, deletions, and/or insertions. tion using techniques well known to those skilled in the art. In a specific embodiment, the amino acid Sequence of the (See, e.g., Greenspan & Bona, FASEB J., 7(5):437-444 50 heavy and/or light chain variable domains may be inspected (1989) and Nissinoff, J. Immunol., 147(8):2429–2438 to identify the Sequences of the complementarity determin (1991).) For example, antibodies which bind to and com ing regions (CDRs) by methods that are well known in the petitively inhibit the binding of ACE-2 binding polypeptide art, e.g., by comparison to known amino acid Sequences of to ACE-2 can be used to generate anti-idiotypes that other heavy and light chain variable regions to determine the “mimic' the ACE-2/ACE-2 binding polypeptide binding 55 regions of Sequence hyperVariability. Using routine recom binant DNA techniques, one or more of the CDRs may be domain and, as a consequence, bind to and neutralize or inserted within framework regions, e.g., into human frame enhance ACE-2 binding to an ACE-2 Substrate (e.g., work regions to humanize a non-human antibody, as angiotensin, bradykinin, tachykinin, endothelin, described Supra. The framework regions may be naturally neurotensin, or Substance P). Such neutralizing anti occurring or consensus framework regions, and preferably idiotypes or Fab fragments of Such anti-idiotypes can be 60 human framework regions (See, e.g., Chothia et al., J. Mol. used in therapeutic regimens to bind ACE-2 and/or neutral Biol., 278: 457-479 (1998) for a listing of human framework ize or enhance ACE-2 mediated acitivity. In a specific regions). Preferably, the polynucleotide generated by the embodiment, anti-idiotypic antibodies can be used to bind combination of the framework regions and CDRs encodes ACE-2, and thereby block its biological activity. In another an antibody that specifically binds ACE-2 or an ACE-2 Specific embodiment, anti-idiotypic antibodies can be used 65 binding polypeptide of the invention. Preferably, as dis to bind ACE-2, and thereby enhance its biological activity cussed Supra, one or more amino acid Substitutions may be (e.g., via multimerization of ACE-2). made within the framework regions, and, preferably, the US 6,592,865 B2 105 106 amino acid Substitutions improve binding of the antibody to cultured by conventional techniques to produce an antibody its antigen. Additionally, Such methods may be used to make of the invention. Thus, the invention includes host cells amino acid Substitutions or deletions of one or more variable containing a polynucleotide encoding an antibody of the region cysteine residues participating in an intrachain dis invention, or a heavy or light chain thereof, or a single chain ulfide bond to generate antibody molecules lacking one or antibody of the invention, operably linked to a heterologous more intrachain disulfide bonds. Other alterations to the promoter. In preferred embodiments for the expression of polynucleotide are encompassed by the present invention double-chained antibodies, vectors encoding both the heavy and within the skill of the art. and light chains may be co-expressed in the host cell for In addition, techniques developed for the production of expression of the entire immunoglobulin molecule, as “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:851-855 (1984); Neuberger et al., Nature, detailed below. 312:604-608 (1984); Takeda et al., Nature, 314:452–454 A variety of host-expression vector Systems may be (1985)) by Splicing genes from a mouse antibody molecule utilized to express the antibody molecules of the invention. of appropriate antigen Specificity together with genes from Such host-expression Systems represent vehicles by which a human antibody molecule of appropriate biological activ the coding Sequences of interest may be produced and ity can be used. AS described Supra, a chimeric antibody is 15 Subsequently purified, but also represent cells which may, a molecule in which different portions are derived from when transformed or transfected with the appropriate nucle different animal Species, Such as those having a variable otide coding Sequences, express an antibody molecule of the region derived from a murine antibody and a human immu invention in situ. These include but are not limited to noglobulin constant region, e.g., humanized antibodies. microorganisms Such as bacteria (e.g., E. coli, B. Subtilis) Alternatively, techniques described for the production of transformed with recombinant bacteriophage DNA, plasmid single chain antibodies (U.S. Pat. No. 4,946,778; Bird, DNA or cosmid DNA expression vectors containing anti Science, 242:423-42 (1988); Huston et al., Proc. Natl. Acad. body coding sequences; yeast (e.g., Saccharomyces, Pichia) Sci. USA, 85:5879–5883 (1988); and Ward et al., Nature, transformed with recombinant yeast expression vectors con 334:544-54 (1989)) can be adapted to produce single chain taining antibody coding Sequences, insect cell Systems antibodies. Single chain. antibodies are formed by linking 25 infected with recombinant virus expression vectors (e.g., the heavy and light chain fragments of the Fv region via an baculovirus) containing antibody coding sequences, plant amino acid bridge, resulting in a Single chain polypeptide. cell Systems infected with recombinant virus expression Techniques for the assembly of functional Fv fragments in vectors (e.g., cauliflower mosaic virus, CaMV; tobacco E. coli may also be used (Skerra et al., Science, mosaic virus, TMV) or transformed with recombinant plas 242:1038–1041 (1988)). mid expression vectors (e.g., Ti plasmid) containing anti Methods of Producing Antibodies body coding sequences, or mammalian cell Systems (e.g., The antibodies of the invention can be produced by any COS, CHO, BHK, 293, 3T3 cells) harboring recombinant method known in the art for the Synthesis of antibodies, in expression constructs containing promoters derived from the particular, by chemical Synthesis or preferably, by recom genome of mammalian cells (e.g., metallothionein binant expression techniques. 35 promoter) or from mammalian viruses (e.g., the adenovirus Recombinant expression of an antibody of the invention, late promoter; the vaccinia virus 7.5K promoter). Preferably, or fragment, derivative or analog thereof, (e.g., a heavy or bacterial cells Such as Escherichia coli, and more preferably, light chain of an antibody of the invention or a single chain eukaryotic cells, especially for the expression of whole antibody of the invention), requires construction of an recombinant antibody molecule, are used for the expression expression vector containing a polynucleotide that encodes 40 of a recombinant antibody molecule. For example, mamma the antibody. Once a polynucleotide encoding an antibody lian cells such as Chinese hamster ovary cells (CHO), in molecule or a heavy or light chain of an antibody or portion conjunction with a vector Such as the major intermediate thereof (preferably containing the heavy or light chain early gene promoter element from human cytomegalovirus variable domain) of the invention has been obtained, the is an effective expression System for antibodies (Foecking et vector for the production of the antibody molecule may be 45 al., Gene, 45:101 (1986); Cockett et al., Bio/Technology, 8:2 produced by recombinant DNA technology using techniques (1990)). well known in the art. Thus, methods for preparing a protein In bacterial Systems, a number of expression vectors may by expressing a polynucleotide containing an antibody be advantageously Selected depending upon the use intended encoding nucleotide Sequence are described herein. Methods for the antibody molecule being expressed. For example, which are well known to those skilled in the art can be used 50 when a large quantity of Such a protein is to be produced, for to construct expression vectors containing antibody coding the generation of pharmaceutical compositions of an anti Sequences and appropriate transcriptional and translational body molecule, vectors which direct the expression of high control Signals. These methods include, for example, in Vitro levels of fusion protein products that are readily purified recombinant DNA techniques, Synthetic techniques, and in may be desirable. Such vectors include, but are not limited, Vivo genetic recombination. The invention, thus, provides 55 to the E. coli expression vector puR278 (Ruther et al., replicable vectors comprising a nucleotide Sequence encod EMBO J., 2:1791 (1983)), in which the antibody coding ing an antibody molecule of the invention, or a heavy or light Sequence may be ligated individually into the vector in chain thereof, or a heavy or light chain variable domain, frame with the lacZ coding region So that a fusion protein is operably linked to a promoter. Such vectors may include the produced; plN vectors (Inouye & Inouye, Nucleic Acids nucleotide Sequence encoding the constant region of the 60 Res., 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. antibody molecule (see, e.g., PCT publication WO Chem., 24:5503–5509 (1989)); and the like. pCEX vectors 86/05807; PCT publication WO 89/01036; and U.S. Pat. No. may also be used to express foreign polypeptides as fusion 5,122,464) and the variable domain of the antibody may be proteins with glutathione S-transferase (GST). In general, cloned into Such a vector for expression of the entire heavy Such fusion proteins are Soluble and can easily be purified or light chain. 65 from lysed cells by adsorption and binding to matrix The expression vector is transferred to a host cell by glutathione-agarose beads followed by elution in the pres conventional techniques and the transfected cells are then ence of free glutathione. The pGEX vectors are designed to US 6,592,865 B2 107 108 include thrombin or factor Xa protease cleavage Sites So that integrate the plasmid into their chromosomes and grow to the cloned target gene product can be released from the GST form foci which in turn can be cloned and expanded into cell moiety. lines. This method may advantageously be used to engineer In an insect System, Autographa Californica nuclear poly cell lines which express the antibody molecule. Such engi hedrosis virus (AcNPV) is used as a vector to express neered cell lines may be particularly useful in Screening and foreign genes. The virus grows in Spodoptera frugiperda evaluation of compounds that interact directly or indirectly cells. The antibody coding Sequence may be cloned indi with the antibody molecule. vidually into non-essential regions (for example the poly A number of Selection Systems may be used, including but hedrin gene) of the virus and placed under control of an not limited to the herpes simplex virus thymidine kinase AcNPV promoter (for example the polyhedrin promoter). (Wigler et al., Cell, 11:223 (1977)), hypoxanthine-guanine In mammalian host cells, a number of viral-based expres phosphoribosyltransferase (Szybalska & Szybalski, Proc. Sion Systems may be utilized. In cases where an adenovirus Natl. Acad. Sci. USA, 48:202 (1992)), and adenine phos is used as an expression vector, the antibody coding phoribosyltransferase (Lowy et al., Cell, 22:817 (1980)) Sequence of interest may be ligated to an adenovirus genes can be employed in th:-, hgprt- or aprt-cells, respec transcription/translation control complex, e.g., the late pro 15 tively. Also, antimetabolite resistance can be used as the moter and tripartite leader Sequence. This chimeric gene basis of selection for the following genes: dhfr, which may then be inserted in the adenovirus genome by in Vitro confers resistance to methotrexate (Wigler et al., Proc. Natl. or in Vivo recombination. Insertion in a non-essential region Acad. Sci. USA, 77.357 (1980); O'Hare et al., Proc. Natl. of the viral genome (e.g., region E1 or E3) will result in a Acad. Sci. USA, 78:1527 (1981)); gpt, which confers resis recombinant virus that is viable and capable of expressing tance to mycophenolic acid (Mulligan & Berg, Proc. Natl. the antibody molecule in infected hosts. See, e.g., Logan & Acad. Sci. USA, 78.2072 (1981)); neo, which confers resis Shenk, Proc. Natl. Acad. Sci. USA, 81:355–359 (1984). tance to the aminoglycoside G-418; Wu and Wu, Biotherapy, Specific initiation Signals may also be required for efficient 3:87–95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol., translation of inserted antibody coding Sequences. These 32:573–596 (1993); Mulligan, Science, 260:926-932 Signals include the ATG initiation codon and adjacent 25 (1993); and Morgan and Anderson, Ann. Rev. Biochem., Sequences. Furthermore, the initiation codon must be in 62:191-217 (1993); May, 1993, TIBTECH 11(5):155–215); phase with the reading frame of the desired coding Sequence and hygro, which conferS resistance to hygromycin to ensure translation of the entire insert. These exogenous (Santerre et al., Gene, 30:147 (1984)). Methods commonly translational control Signals and initiation codons can be of known in the art of recombinant DNA technology may be a variety of origins, both natural and Synthetic. The effi routinely applied to Select the desired recombinant clone, ciency of expression may be enhanced by the inclusion of and Such methods are described, for example, in Current appropriate transcription enhancer elements, transcription Protocols in Molecular Biology, Ausubel et al., eds. (John terminators, etc. (see, Bittner et al., Methods in Enzymol, Wiley & Sons, NY 1993); Kriegler, Gene Transfer and 153:51–544 (1987)). Expression, A Laboratory Manual (Stockton Press, NY In addition, a host cell Strain may be chosen which 35 1990); and Current Protocols in Human Genetics, Dracopoli modulates the expression of the inserted Sequences, or et al., eds. (John Wiley & Sons, NY 1994), Chapters 12 and modifies and processes the gene product in the Specific 13; Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981), fashion desired. Such modifications (e.g., glycosylation) and which are incorporated by reference herein in their entire processing (e.g., cleavage) of protein products may be ties. important for the function of the protein. Different host cells 40 The expression levels of an antibody molecule can be have characteristic and Specific mechanisms for the post increased by vector amplification (for a review, see Beb translational processing and modification of proteins and bington and Hentschel, The use of vectors based on gene gene products. Appropriate cell lines or host Systems can be amplification for the expression of cloned genes in mam chosen to ensure the correct modification and processing of malian cells in DNA cloning, Vol.3. (Academic Press, New the foreign protein expressed. To this end, eukaryotic host 45 York, 1987)). When a marker in the vector system express cells which possess the cellular machinery for proper pro ing antibody is amplifiable, increase in the level of inhibitor cessing of the primary transcript, glycosylation, and phos present in culture of host cell will increase the number of phorylation of the gene product may be used. Such mam copies of the marker gene. Since the amplified region is malian host cells include but are not limited to CHO, VERY, asSociated with the antibody gene, production of the anti BHK, Hela, COS, MDCK, NSO, 293, 3T3, WI38, and in 50 body will also increase (Crouse et al., Mol. Cell. Biol, 3:257 particular, breast cancer cell lines Such as, for example, (1983)). BT483, HSS78T, HTB2, BT20 and T47D, and normal mam The host cell may be co-transfected with two expression mary gland cell line such as, for example, CRL7030 and vectors of the invention, the first vector encoding a heavy HS578BSt. chain derived polypeptide and the Second vector encoding a For long-term, high-yield production of recombinant 55 light chain derived polypeptide. The two vectors may con proteins, stable expression is preferred. For example, cell tain identical Selectable markers which enable equal expres lines which stably express the antibody molecule may be Sion of heavy and light chain polypeptides. Alternatively, a engineered. Rather than using expression vectors which Single vector may be used which encodes, and is capable of contain viral origins of replication, host cells can be trans expressing, both heavy and light chain polypeptides. In Such formed with DNA controlled by appropriate expression 60 Situations, the light chain should be placed before the heavy control elements (e.g., promoter, enhancer, Sequences, tran chain to avoid an excess of toxic free heavy chain Scription terminators, polyadenylation sites, etc.), and a (Proudfoot, Nature, 322:52 (1986); Kohler, Proc. Natl. Selectable marker. Following the introduction of the foreign Acad. Sci. USA, 77:2197 (1980)). The coding sequences for DNA, engineered cells may be allowed to grow for 1-2 days the heavy and light chains may comprise cDNA or genomic in an enriched media, and then are Switched to a Selective 65 DNA media. The Selectable marker in the recombinant plasmid Once an antibody molecule of the invention has been conferS resistance to the Selection and allows cells to Stably produced by an animal, chemically Synthesized, or recom US 6,592,865 B2 109 110 binantly expressed, it may be purified by any method known chains of mammalian immunoglobulins. (EP394827; Trau in the art for purification of an immunoglobulin molecule, necker et al., Nature, 331:84-86 (1988). The polypeptides of for example, by chromatography (e.g., ion exchange, the present invention fused or conjugated to an antibody affinity, particularly by affinity for the Specific antigen after having disulfide-linked dimeric structures (due to the IgG) Prote in A, and sizing column chromatography), may also be more efficient in binding and neutralizing other centrifugation, differential Solubility, or by any other Stan molecules, than the monomeric Secreted protein or protein dard technique for the purification of proteins. In addition, fragment alone. (Fountoulakis et al., J. Biochem., the antibodies of the present invention or fragments thereof 270:3958-3964 (1995)). In many cases, the Fc part in a can be fused to heterologous polypeptide Sequences fusion protein is beneficial in therapy and diagnosis, and described herein or otherwise known in the art, to facilitate thus can result in, for example, improved pharmacokinetic purification. properties (see, EP-A-232 262). Alternatively, deleting the The present invention encompasses antibodies recombi Fc part after the fusion protein has been expressed, detected, nantly fused or chemically conjugated (including both cova and purified, would be desired. For example, the Fc portion lent and non-covalent conjugations) to a polypeptide (or may hinder therapy and diagnosis if the fusion protein is portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 15 used as an antigen for immunizations. In drug discovery, for 80, 90 or 100 amino acids of the polypeptide) of the present example, human proteins, Such as hL-5, have been fused invention to generate fusion proteins. The fusion does not with Fc portions for the purpose of high-throughput Screen necessarily need to be direct, but may occur through linker ing assays to identify antagonists of hL-5. (See, Bennett et Sequences. The antibodies may be specific for antigens other al., J. Molecular Recognition, 8:52-58 (1995); Johanson et than ACE-2 binding polypeptides of the present invention. al., J. Biol. Chem., 270:9459–9471 (1995). For example, antibodies may be used to target the polypep Moreover, the antibodies or fragments thereof of the tides of the present invention to particular cell types, either present invention can be fused to marker Sequences, Such as in vitro or in Vivo, by fusing or conjugating the polypeptides a peptide to facilitate purification. In preferred of the present invention to antibodies Specific for particular embodiments, the marker amino acid Sequence is a hexa cell Surface receptors. Antibodies fused or conjugated to the 25 histidine peptide, Such as the tag provided in a pCE vector polypeptides of the present invention may also be used in in (QIAGEN, Inc., 92.59 Eton Avenue, Chatsworth, Calif., Vitro immunoassays and purification methods using methods 91311), among others, many of which are commercially known in the art. See e.g., Harbor et al., Supra, and PCT available. As described in Gentz et al., Proc. Natl. Acad. Sci. publication WO 93/21232; EP 439,095; Naramura et al., USA, 86:821-824 (1989), for instance, hexa-histidine pro Immunol. Lett., 39:91-99 (1994); U.S. Pat. No. 5,474,981; vides for convenient purification of the fusion protein. Other Gillies et al., Proc. Natl. Acad. Sci. USA, 89:1428–1432 peptide tags useful for purification include, but are not (1992); Fell et al., J. Immunol., 146:2446-2452(1991), limited to, the "HA' tag, which corresponds to an epitope which are incorporated by reference in their entireties. derived from the influenza hemagglutinin protein (Wilson et The present invention further includes compositions com al., Cell, 37:767 (1984)) and the “flag” tag. prising the polypeptides of the present invention fused or 35 The present invention further encompasses antibodies or conjugated to antibody domains other than the variable fragments thereof conjugated to a diagnostic or therapeutic regions. For example, the polypeptides of the present inven agent. The antibodies can be used diagnostically to, for tion may be fused or conjugated to an antibody Fc region, or example, monitor the development or progression of a tumor portion thereof. The antibody portion fused to a polypeptide as part of a clinical testing procedure to, e.g., determine the of the present invention may comprise the constant region, 40 efficacy of a given treatment regimen. Detection can be hinge region, CH1 domain, CH2 domain, and CH3 domain facilitated by coupling the antibody to a detectable Sub or any combination of whole domains or portions thereof. stance. Examples of detectable Substances include various The polypeptides may also be fused or conjugated to the enzymes, prosthetic groups, fluorescent materials, lumines above antibody portions to form multimers. For example, Fc cent materials, bioluminescent materials, radioactive portions fused to the polypeptides of the present invention 45 materials, positron emitting metals using various positron can form dimers through disulfide bonding between the Fc emission tomographies, and nonradioactive paramagnetic portions. Higher multimeric forms can be made by fusing metal ions. The detectable Substance may be coupled or the polypeptides to portions of IgA and IgM. Methods for conjugated either directly to the antibody (or fragment fusing or conjugating the polypeptides of the present inven thereof) or indirectly, through an intermediate (Such as, for tion to antibody portions are known in the art. See, e.g., U.S. 50 example, a linker known in the art) using techniques known Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; in the art. See, for example, U.S. Pat. No. 4,741,900 for 5.447.851; 5,112,946; EP307434; EP 367 166; PCT pub metal ions which can be conjugated to antibodies for use as lications WO 96/04388; WO 91/06570; Ashkenazi et al., diagnostics according to the present invention. Examples of Proc. Natl. Acad. Sci. USA, 88:10535-10539 (1991); Zheng Suitable enzymes include horseradish peroxidase, alkaline et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., 55 phosphatase, beta-galactosidase, or acetylcholinesterase, Proc. Natl. Acad. Sci. USA, 89:11337-11341 (1992) (said examples of Suitable prosthetic group complexes include references incorporated by reference in their entireties). streptavidin/biotin and avidin/biotin; examples of suitable AS discussed, Supra, the polypeptides corresponding to an fluorescent materials include umbelliferone, fluorescein, ACE-2 binding polypeptide of the invention may be fused or fluorescein isothiocyanate, rhodamine, dichlorotriaziny conjugated to the above antibody portions to increase the in 60 lamine fluorescein, dansyl chloride or phycoerythrin; an Vivo half life of the polypeptides or for use in immunoassays example of a luminescent material includes luminol, using methods known in the art. Further, the ACE-2 binding examples of bioluminescent materials include luciferase, polypeptides of the invention may be fused or conjugated to luciferin, and aequorin; and examples of Suitable radioactive the above antibody portions to facilitate purification. One material include 'I, I, '''In or 'Tc. reported example describes chimeric proteins consisting of 65 Further, an antibody or fragment thereof may be conju the first two domains of the human CD4-polypeptide and gated to a therapeutic moiety Such as a cytotoxin, e.g., a various domains of the constant regions of the heavy or light cytostatic or cytocidal agent, a therapeutic agent or a radio US 6,592,865 B2 111 112 active metal ion, e.g., alpha-emitterS Such as, for example, by Segal in U.S. Pat. No. 4,676,980, which is incorporated 'Bi. A cytotoxin or cytotoxic agent includes any agent that herein by reference in its entirety. is detrimental to cells. Examples include paclitaXol, cytocha An antibody, with or without a therapeutic moiety con lasin B, gramicidin D, ethidium bromide, emetine, jugated to it, administered alone or in combination with mitomycin, etoposide, tenoposide, Vincristine, vinblastine, cytotoxic factor(s) and/or cytokine(s) can be used as a colchicin, doxorubicin, daunorubicin, dihydroxy anthracin therapeutic. dione, mitoxantrone, mithramycin, actinomycin D, Assays For Antibody Binding 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, The antibodies of the invention may be assayed for lidocaine, propranolol, and puromycin and analogs or immunospecific binding by any method known in the art. homologs thereof. Therapeutic agents include, but are not The immunoassays which can be used include but are not limited to, antimetabolites (e.g., methotrexate, limited to competitive and non-competitive assay Systems 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil using techniqueS Such as western blots, radioimmunoassays, decarbazine), alkylating agents (e.g., mechlorethamine, ELISA (enzyme linked immunosorbent assay), “sandwich.” thioepa chlorambucil, melphalan, carmustine (BSNU) and immunoassays, immunoprecipitation assays, precipitin lomustine (CCNU), cyclothosphamide, busulfan, reactions, gel diffusion precipitin reactions, immunodiffu dibromomannitol, Streptozotocin, mitomycin C, and cis 15 Sion assays, agglutination assays, complement-fixation dichlorodiamine platinum (II) (DDP) cisplatin), anthracy assays, immunoradiometric assays, fluorescent clines (e.g., daunorubicin (formerly daunomycin) and immunoassays, protein A immunoassays, to name but a few. doxorubicin), antibiotics (e.g., dactinomycin (formerly Such assays are routine and well known in the art (see, e.g., actinomycin), bleomycin, mithramycin, and anthramycin Current Protocols in Molecular Biology, Ausubel et al., eds. (AMC)), and anti-mitotic agents (e.g., Vincristine and (John Wiley & Sons, NY 1993), which is incorporated by vinblastine). reference herein in its entirety). Exemplary immunoassays The conjugates of the invention can be used for modifying are described briefly below (but are not intended by way of a given biological response, the therapeutic agent or drug limitation). moiety is not to be construed as limited to classical chemical Immunoprecipitation protocols generally comprise lysing therapeutic agents. For example, the drug moiety may be a 25 a population of cells in a lysis buffer such as RIPA buffer protein or polypeptide possessing a desired biological activ (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% ity. Such proteins may include, for example, a toxin Such as SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% abrin, ricin A, pseudomonas eXotoxin, or diphtheria toxin; a Trasylol) Supplemented with protein phosphatase and/or protein Such as tumor necrosis factor, alpha-interferon, beta protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium interferon, nerve growth factor, platelet derived growth Vanadate), adding the antibody of interest to the cell lysate, factor, tissue plasminogen activator, an apoptotic agent, e.g., incubating for a period of time (e.g., 1–4 hours) at 4 C., TNF-alpha, TNF-beta, AIM I (See, PCT publication WO adding protein A and/or protein G Sepharose beads to the cell 97/33899), AIM II (See, PCT publication WO 97/34911), lysate, incubating for about an hour or more at 4 C., Fas Ligand (Takahashi et al., Int. Immunol, 6:1567–1574 Washing the beads in lysis buffer and resuspending the beads (1994)), VEGI (See, PCT publication WO99/23105), CD40 35 in SDS/sample buffer. The ability of the antibody of interest Ligand, a thrombotic agent or an anti-angiogenic agent, e.g., to immunoprecipitate a particular antigen can be assessed angiostatin or endostatin; or, biological response modifiers by, e.g., Western blot analysis. One of skill in the art would Such as, for example, lymphokines, interleukin-1 (“IL-1), be knowledgeable as to the parameters that can be modified interleukin-2 (“IL-2), interleukin-6 (“IL-6”), granulocyte to increase the binding of the antibody to an antigen and macrophage colony Stimulating factor (“GM-CSF), granu 40 decrease the background (e.g., pre-clearing the cell lysate locyte colony stimulating factor (“G-CSF), or other growth with Sepharose beads). For further discussion regarding factors. immunoprecipitation protocols See, e.g., Current Protocols Techniques for conjugating Such therapeutic moiety to in Molecular Biology, Ausubel et al., eds. (John Wiley & antibodies are well known, See, e.g., Arnon et al., “Mono Sons, NY 1993) at 10.16.1. clonal Antibodies For Immunotargeting Of Drugs. In Cancer 45 Western blot analysis generally comprises preparing pro Therapy”, in Monoclonal Antibodies And Cancer Therapy, tein Samples, electrophoresis of the protein Samples in a Reisfeld et al., eds. (Alan R. Liss, Inc. 1985), pp. 243-56; polyacrylamide gel (e.g., 8%–20% SDS-PAGE depending Hellstrom et al., “Antibodies For Drug Delivery', in Con on the molecular weight of the antigen), transferring the trolled Drug Delivery (2nd Ed.), Robinson et al., eds. protein Sample from the polyacrylamide gel to a membrane (Marcel Dekker, Inc. 1987), pp. 623-53; Thorpe, “Antibody 50 such as nitrocellulose, PVDF or nylon, blocking the mem Carriers Of Cytotoxic Agents. In Cancer Therapy: A brane in blocking solution (e.g., PBS with 3% BSA or Review', in Monoclonal Antibodies '84. Biological And non-fat milk), washing the membrane in washing buffer Clinical Applications, Pinchera et al., eds., pp. 475-506 (e.g., PBS-Tween 20), blocking the membrane with primary (1985); “Analysis, Results, And Future Prospective Of The antibody (the antibody of interest) diluted in blocking buffer, Therapeutic Use Of Radiolabeled Antibody in Cancer 55 Washing the membrane in Washing buffer, blocking the Therapy”, in Monoclonal Antibodies For Cancer Detection membrane with a secondary antibody (which recognizes the And Therapy, Baldwin et al., eds. (Academic Press 1985), primary antibody, e.g., an anti-human antibody) conjugated pp. 303-16; and Thorpe et al., “The Preparation And Cyto to an enzymatic Substrate (e.g., horseradish peroxidase or toxic Properties Of Antibody-Toxin Conjugates”, Immunol. alkaline phosphatase) or radioactive molecule (e.g., P or Rev., 62:119–58 (1982). 60 'I) diluted in blocking buffer, washing the membrane in Antibodies may also be attached to Solid Supports, which wash buffer, and detecting the presence of the antigen. One are particularly useful for immunoassays or purification of of skill in the art would be knowledgeable as to the param the ACE-2 binding polypeptide. Such Solid Supports include, eters that can be modified to increase the Signal detected and but are not limited to, glass, cellulose, polyacrylamide, to reduce the background noise. For further discussion nylon, polystyrene, polyvinyl chloride or polypropylene. 65 regarding western blot protocols See, e.g., Current Protocols Alternatively, an antibody can be conjugated to a Second in Molecular Biology, Ausubel et al., eds. (John Wiley & antibody to form an antibody heteroconjugate as described Sons, NY 1993) at 10.8.1. US 6,592,865 B2 113 114 ELISAS comprise preparing antigen, coating the well of a includes binding ACE-2 binding polypeptides. of the present 96-well microtiter plate with the antigen, adding the anti invention that have been coadministered in order to bind or body of interest conjugated to a detectable compound Such neutralize ACE-2, or by direct cytotoxicity of the antibody, as an enzymatic Substrate (e.g., horseradish peroxidase or e.g., as mediated by complement (CDC) or by effector cells alkaline phosphatase) to the well and incubating for a period (ADCC). ACE-2 binding polypeptides and anti-ACE-2 of time, and detecting the presence of the antigen. In ELISAS binding polypeptide antibodies may be administered either the antibody of interest does not have to be conjugated to a locally or Systemically. Some of these approaches are detectable compound; instead, a second antibody (which described in more detail below. Armed with the teachings recognizes the antibody of interest) conjugated to a detect provided herein, one of ordinary skill in the art will know able compound may be added to the well. Further, instead of how to use the antibodies of the present invention for coating the well with the antigen, the antibody may be diagnostic, monitoring or therapeutic purposes without coated to the well. In this case, a Second antibody conjugated undue experimentation. to a detectable compound may be added following the The antibodies of this invention may be advantageously addition of the antigen of interest to the coated well. One of utilized in combination with other monoclonal or chimeric skill in the art would be knowledgeable as to the parameters 15 antibodies, or with lymphokines or hematopoietic growth that can be modified to increase the Signal detected as well factors (Such as, e.g., IL-2, IL-3 and IL-7), for example, as other variations of ELISAS known in the art. For further which serve to increase the number or activity of effector discussion regarding ELISAS See, e.g., Current Protocols in cells which interact with the antibodies. Molecular Biology, Ausubel et al., eds. (John Wiley & Sons, The antibodies of the invention may be administered NY 1993) at 11.2.1. alone or in combination with other types of treatments (e.g., The binding affinity of an antibody to an antigen and the radiation therapy, chemotherapy, hormonal therapy, off-rate of an antibody-antigen interaction can be determined immunotherapy, anti-tumor agents, antibiotics, and by competitive binding assays. One example of a competi immunoglobulin). Generally, administration of products of a tive binding assay is a radioimmunoassay comprising the Species origin or species reactivity (in the case of antibodies) incubation of labeled antigen (e.g., H or 'I) with the 25 that is the same Species as that of the patient is preferred. antibody of interest in the presence of increasing amounts of Thus, in a preferred embodiment, human antibodies, frag unlabeled antigen, and the detection of the antibody bound ments derivatives, analogs, or nucleic acids, are adminis to the labeled antigen. The affinity of the antibody of interest tered to a human patient for therapy or prophylaxis. for a particular antigen and the binding off-rates can be It is preferred to use high affinity and/or potent in vivo determined from the data by Scatchard plot analysis. Com inhibiting and/or neutralizing antibodies against polypep petition with a Second antibody can also be determined using tides of the present invention, fragments or regions thereof, radioimmunoassays. In this case, the antigen is incubated for both immunoassays directed to and therapy of disorders with antibody of interest conjugated to a labeled compound related to polypeptides, including fragments thereof, of the (e.g., H or 'I) in the presence of increasing amounts of an present invention. Such antibodies, fragments, or regions, unlabeled Second antibody. 35 will preferably have an affinity for polypeptides of the Therapeutic Uses of Antibodies invention, including fragments thereof. Preferred binding The present invention is further directed to antibody affinities include those with a dissociation constant or KD based therapies which involve administering antibodies of less than 5x10 M, 10 M, 5x10 M, 10 M, 5x107M, the invention to an animal, preferably a mammal, and most 107 M, 5x10M, 10 M, 5x10 M, 10 M,5x100 M, preferably a human, patient for treating one or more of the 40 100 M, 5x10-11 M, 10-11 M, 5x10-12 M, 10-12 M, diseases, disorders, or conditions disclosed herein. Thera 5x10 M, 10 M, 5x10' M, 10' M, 5x10's M, and peutic compounds of the invention include, but are not 1015 M. limited to, antibodies of the invention (including fragments, Demonstration of Therapeutic or Prophylactic Activity of analogs and derivatives thereof as described herein) and Antibodies nucleic acids encoding antibodies of the invention 45 The compounds or pharmaceutical compositions of the (including fragments, analogs and derivatives thereof and invention are preferably tested in vitro, and then in vivo for anti-idiotypic antibodies as described herein). The antibod the desired therapeutic or prophylactic activity, prior to use ies of the invention can be used to treat, inhibit or prevent in humans. For example, in vitro assays to demonstrate the diseases, disorders or conditions associated with aberrant therapeutic or prophylactic utility of a compound or phar ACE-2 expression and/or activity, including, but not limited 50 maceutical composition include, the effect of a compound on to, any one or more of the diseases, disorders, or conditions a cell line or a patient tissue Sample. The effect of the described herein. compound or composition on the cell line and/or tissue The treatment and/or prevention of diseases, disorders, or Sample can be determined utilizing techniques known to conditions associated with aberrant expression and/or activ those of Skill in the art including, but not limited to, rosette ity of ACE-2 or an ACE-2 substrate includes, but is not 55 formation assays and cell lysis assays. In accordance with limited to, alleviating Symptoms associated with those the invention, in vitro assays which can be used to determine diseases, disorders or conditions. The antibodies of the whether administration of a Specific compound is indicated, invention may also be used to target and kill cells expressing include in vitro cell culture assays in which a patient tissue ACE-2 on their surface and/or cells having ACE-2 bound to Sample is grown in culture, and exposed to or otherwise their Surface. This targeting may be the result of binding of 60 administered a compound, and the effect of Such compound the antibody to ACE-2 binding polypeptides of the invention upon the tissue sample is observed. that have been coadministered, or alternatively, the result of Therapeutic and/or Prophylactic Administration and Com direct binding of the antibody to ACE-2. Antibodies of the position invention may be provided in pharmaceutically acceptable The invention provides methods of treatment, inhibition compositions as known in the art or as described herein. 65 and prophylaxis by administration to a Subject of an effective Non-limiting examples of the ways in which the antibod amount of a compound or pharmaceutical composition of ies of the present invention may be used therapeutically the invention, preferably an antibody of the invention. In a US 6,592,865 B2 115 116 preferred embodiment, the compound is Substantially puri romol. Sci. Rev. Macromol. Chem., 23:61 (1983); see also fied (e.g., Substantially free from Substances that limit its Levy et al., Science, 228:190 (1985); During et al., Ann. effect or produce undesired side effects). The subject is Neurol., 25:351 (1989); Howard et al., J. Neurosurg, 71:105 preferably an animal, including but not limited to animals (1989)). In yet another embodiment, a controlled release Such as cows, pigs, horses, chickens, cats, dogs, etc., and is System can be placed in proximity of the therapeutic target, preferably a mammal, and most preferably human. thus requiring only a fraction of the Systemic dose (see, e.g., Formulations and methods of administration that can be Goodson, in Medical Applications of Controlled Release, employed when the compound comprises a nucleic acid or Langer and Wise, eds. (CRC Press, Boca Raton, Fla. 1974), an immunoglobulin are described above, additional appro vol. 2, pp. 115–138 (1984)). priate formulations and routes of administration can be Other controlled release Systems are discussed in the Selected from among those described herein below. review by Langer (Science 249:1527–1533 (1990)). Various delivery Systems are known and can be used to In a specific embodiment where the compound of the administer a compound of the invention, e.g., encapsulation invention is a nucleic acid encoding a protein, the nucleic in liposomes, microparticles, microcapsules, recombinant acid can be administered in Vivo to promote expression of its cells capable of expressing the compound, receptor 15 encoded protein, by constructing it as part of an appropriate mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem., nucleic acid expression vector and administering it So that it 262:4429–4432 (1987)), construction of a nucleic acid as becomes intracellular, e.g., by use of a retroviral vector (see part of a retroviral or other vector, etc. Methods of intro U.S. Pat. No. 4,980,286), or by direct injection, or by use of duction include but are not limited to intradermal, microparticle bombardment (e.g., a gene gun; Biolistic, intramuscular, intraperitoneal, intravenous, Subcutaneous, Dupont), or coating with lipids or cell-Surface receptors or intranasal, epidural, and oral routes. The compounds or transfecting agents, or by administering it in linkage to a compositions may be administered by any convenient route, homeobox-like peptide which is known to enter the nucleus for example by infusion or bolus injection, by absorption (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA, through epithelial or mucocutaneous linings (e.g., oral 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can mucosa, rectal and intestinal mucosa, etc.) and may be 25 be introduced intracellularly and incorporated within host administered together with other biologically active agents. cell DNA for expression, by homologous recombination. Administration can be Systemic or local. In addition, it may The present invention also provides pharmaceutical com be desirable to introduce the pharmaceutical compounds or positions. Such compositions comprise a therapeutically compositions of the invention into the central nervous effective amount of a compound, and a pharmaceutically System by any Suitable route, including intraventricular and acceptable carrier. In a specific embodiment, the term "phar intrathecal injection; intraventricular injection may be facili maceutically acceptable” means approved by a regulatory tated by an intraventricular catheter, for example, attached to agency of the Federal or a State government or listed in the a reservoir, Such as an Ommaya reservoir. Pulmonary U.S. Pharmacopeia or other generally recognized pharma administration can also be employed, e.g., by use of an copeia for use in animals, and more particularly in humans. inhaler or nebulizer, and formulation with an aeroSolizing 35 The term “carrier' refers to a diluent, adjuvant, excipient, or agent. vehicle with which the therapeutic is administered. Such In a specific embodiment, it may be desirable to admin pharmaceutical carriers can be Sterile liquids, Such as water ister the pharmaceutical compounds or compositions of the and oils, including those of petroleum, animal, vegetable or invention locally to the area in need of treatment; this may Synthetic origin, Such as peanut oil, Soybean oil, mineral oil, be achieved by, for example, and not by way of limitation, 40 sesame oil and the like. Water is a preferred carrier when the local infusion during Surgery, topical application, e.g., in pharmaceutical composition is administered intravenously. conjunction with a wound dressing after Surgery, by Saline Solutions and aqueous dextrose and glycerol Solutions injection, by means of a catheter, by means of a Suppository, can also be employed as liquid carriers, particularly for or by means of an implant, Said implant being of a porous, injectable Solutions. Suitable pharmaceutical excipients non-porous, or gelatinous material, including membranes, 45 include Starch, glucose, lactose, Sucrose, gelatin, malt, rice, Such as Sialastic membranes, or fibers. Preferably, when flour, chalk, Silica gel, Sodium Stearate, glycerol administering a protein, including an antibody, of the monoStearate, talc, Sodium chloride, dried skim milk, invention, care must be taken to use materials to which the glycerol, propylene, glycol, water, ethanol and the like. The protein does not absorb. composition, if desired, can also contain minor amounts of In another embodiment, the compound or composition 50 wetting or emulsifying agents, or pH buffering agents. These can be delivered in a vesicle, in particular a liposome (see compositions can take the form of Solutions, Suspensions, Langer, Science, 249:1527–1533 (1990); Treat et al., in emulsion, tablets, pills, capsules, powders, Sustained-release Liposomes in the Therapy of Infectious Disease and Cancer, formulations and the like. The composition can be formu Lopez-Berestein and Fidler, eds. (Liss, New York 1989), pp. lated as a Suppository, with traditional binders and carriers 353–365; Lopez-Berestein, ibid., pp. 317-327; see generally 55 Such as triglycerides. Oral formulation can include Standard ibid.) carrierS Such as pharmaceutical grades of mannitol, lactose, In yet another embodiment, the compound or composition Starch, magnesium Stearate, Sodium Saccharine, cellulose, can be delivered in a controlled release System. In one magnesium carbonate, etc. Examples of Suitable pharma embodiment, a pump may be used (see Langer, Supra; ceutical carriers are described in Remington's Pharmaceu Sefton, CRC Crit. Ref. Biomed. Eng., 14:201 (1987); Buch 60 tical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co., wald et al., Surgery, 88:507 (1980); Saudek et al., N. Engl. 1990). Such compositions will contain a therapeutically J. Med., 321:574 (1989)). In another embodiment, poly effective amount of the compound, preferably in purified meric materials can be used (see Medical Applications of form, together with a Suitable amount of carrier So as to Controlled Release, Langer and Wise, eds. (CRC Press, provide the form for proper administration to the patient. Boca Raton, Fla. 1974); Controlled Drug Bioavailability, 65 The formulation should suit the mode of administration. Drug Product Design and Performance, Smolen and Ball, In a preferred embodiment, the composition is formulated eds. (Wiley, New York 1984); Ranger and Peppas, J. Mac in accordance with routine procedures as a pharmaceutical US 6,592,865 B2 117 118 composition adapted for intravenous administration to invention provides for the detection of aberrant expression human beings. Typically, compositions for intravenous of ACE-2, comprising (a) contacting cells or body fluid with administration are Solutions in Sterile isotonic aqueous an ACE-2 binding polypeptide; (b) assaying the expression buffer. Where necessary, the composition may also include of ACE-2 in cells or body fluid of an individual using one or a Solubilizing agent and a local anesthetic Such as lignocaine more antibodies Specific to the ACE-2 binding polypeptide to ease pain at the Site of the injection. Generally, the and (c) comparing the level of ACE-2 expression with a ingredients are Supplied either Separately or mixed together Standard ACE-2 expression level, whereby an increase or in unit dosage form, for example, as a dry lyophilized decrease in the assayed ACE-2 expression level compared to powder or water free concentrate in a hermetically Sealed the Standard expression level is indicative of aberrant container Such as an ampoule or Sachette indicating the expression. quantity of active agent. Where the composition is to be The invention provides a diagnostic assay for diagnosing administered by infusion, it can be dispensed with an a disorder, comprising (a) contacting cells or body fluid with infusion bottle containing Sterile pharmaceutical grade water an ACE-2 binding polypeptide; (b) assaying the expression or saline. Where the composition is administered by of ACE-2 in cells or body fluid of an individual using one or injection, an ampoule of Sterile water for injection or Saline 15 more antibodies Specific to the ACE-2 binding polypeptide can be provided So that the ingredients may be mixed prior of interest and (c) comparing the level of ACE-2 expression to administration. with a standard ACE-2 expression level, whereby an The compounds of the invention can be formulated as increase or decrease in the assayed ACE-2 expression level neutral or Salt forms. Pharmaceutically acceptable Salts compared to the Standard expression level is indicative of a include those formed with anions such as those derived from particular disorder. With respect to cancer, the presence of a hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., relatively high amount of ACE-2 in biopsied tissue from an and those formed with cations Such as those derived from individual may indicate a predisposition for the development Sodium, potassium, ammonium, calcium, ferric hydroxides, of the disease, or may provide a means for detecting the isopropylamine, triethylamine, 2-ethylamino ethanol, disease prior to the appearance of actual clinical Symptoms. histidine, procaine, etc. 25 A more definitive diagnosis of this type may allow health The amount of the compound of the invention which will professionals to employ preventative measures or aggressive be effective in the treatment, inhibition and prevention of a treatment earlier thereby preventing the development or disease or disorder associated with aberrant expression further progression of the cancer. and/or activity of a polypeptide of the invention can be Antibodies of the invention can be used to assay ACE-2 determined by Standard clinical techniques. In addition, in protein levels in a biological Sample using or routinely Vitro assays may optionally be employed to help identify modifying classical immunohistological methods known to optimal dosage ranges. The precise dose to be employed in those of skill in the art (e.g., see Jalkanen et al., J. Cell. Biol., the formulation will also depend on the route of 101:976–985 (1985); Jalkanen et al., J. Cell. Biol., administration, and the Seriousness of the disease or 105:3087-3096 (1987)). Other antibody-based methods use disorder, and should be decided according to the judgment 35 ful for detecting protein gene expression include of the practitioner and each patient's circumstances. Effec immunoassays, Such as the enzyme linked immunosorbent tive doses. may be extrapolated from dose-response curves assay (ELISA) and the radioimmunoassay (RIA). Suitable derived from in vitro or animal model test Systems. antibody assay labels are known in the art and include For antibodies, the dosage administered to a patient is enzyme labels, Such as, glucose oxidase; radioisotopes, Such typically 0.1 mg/kg to 100 mg/kg of the patient's body 40 as iodine (I, I, I, 'I), carbon ('C), sulfur (S), weight. Preferably, the dosage administered to a patient is tritium (H), indium ("In, '"In, In, '''In), and tech between 0.1 mg/kg and 20 mg/kg of the patient's body netium ('Tc, 'Tc), thallium ('Ti), gallium (Ga, 7Ga), weight, more preferably 1 mg/kg to 10 mg/kg of the palladium ('Pd), molybdenum ('Mo), xenon (Xe), patient's body weight. Generally, human antibodies have a fluorine (F), 15°Sm, 177Lu, 159Gd, 1“Pm, 140La, 175Yb, longer half-life within the human body than antibodies from 45 Ho, 90Y, 7Sc, 18Re, Re, 'Pr, 105Rh, 97Ru; lumi other Species due to the immune response to the foreign neScent labels, Such as luminol, and fluorescent labels, Such polypeptides. Thus, lower dosages of human antibodies and as fluorescein and rhodamine, and biotin. less frequent administration is often possible. Further, the Techniques known in the art may be applied to label dosage and frequency of administration of antibodies of the antibodies of the invention. Such techniques include, but are invention may be reduced by enhancing uptake and tissue 50 not limited to, the use of bifunctional conjugating agents penetration (e.g., into the brain) of the antibodies by modi (see, e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; fications Such as, for example, lipidation. 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; The invention also provides a pharmaceutical pack or kit 5,342,604, 5,274,119, 4,994,560; and 5,808.003; the con comprising one or more containers filled with one or more tents of each of which are hereby incorporated by reference of the ingredients of the pharmaceutical compositions of the 55 in its entirety). invention. Optionally associated with Such container(s) can One embodiment of the invention is the detection and be a notice in the form prescribed by a governmental agency diagnosis of a disease or disorder associated with aberrant regulating the manufacture, use or Sale of pharmaceuticals or expression of ACE-2 in an animal, preferably a mammal and biological products, which notice reflects approval by the most preferably a human. In one embodiment, diagnosis agency of manufacture, use or Sale for human administra 60 comprises: (a) administering (for example, parenterally, tion. Subcutaneously, or intraperitoneally) to a Subject an effective Diagnosis and Imaging amount of a labeled molecule which specifically binds to Labeled antibodies, and derivatives and analogs thereof, ACE-2 (e.g., an ACE-2 binding polyptide of the invention) which specifically bind to an ACE-2 binding polypeptide of or which specifically binds to a molecule that Specifically interest can be used for diagnostic purposes to detect, 65 binds to ACE-2 (e.g., an anti- ACE-2 binding polypeptide diagnose, or monitor diseases and/or disorders associated antibody of the invention); (b) waiting for a time interval with the aberrant expression and/or activity of ACE-2. The following the administering for permitting the labeled mol US 6,592,865 B2 119 120 ecule to preferentially concentrate at Sites in the Subject a control antibody which does not react with the polypeptide where the polypeptide is expressed (and for unbound labeled of interest. In another Specific embodiment, the kits of the molecule to be cleared to background level); (c) determining present invention comprise two or more antibodies background level; and (d) detecting the labeled molecule in (monoclonal and/or polyclonal) that recognize the same the subject, such that detection of labeled molecule above and/or different Sequences or regions of a polypeptide the background level indicates that the Subject has a par according to the invention. In another Specific embodiment, ticular disease or disorder associated with aberrant expres the kits of the present invention contain a means for detect Sion of the polypeptide of interest. Background level can be ing the binding of an antibody to a polypeptide of interest determined by various methods including, comparing the (e.g., the antibody may be conjugated to a detectable Sub amount of labeled molecule detected to a Standard value Strate Such as a fluorescent compound, an enzymatic previously determined for a particular System. Substrate, a radioactive compound or a luminescent It will be understood by those skilled in the art that the compound, or a Second antibody which recognizes the first Size of the Subject and the imaging System used will deter antibody may be conjugated to a detectable Substrate). mine the quantity of imaging moiety needed to produce In another specific embodiment of the present invention, diagnostic images. In the case of a radioisotope moiety, for 15 the kit is a diagnostic kit for use in Screening Serum a human Subject, the quantity of radioactivity injected will containing antibodies Specific against proliferative and/or normally range from about 5 to 20 millicuries of 'mTc. The cancerous polynucleotides and polypeptides. Such a kit may labeled antibody or antibody fragment will then preferen include a control antibody that does not react with the tially accumulate at the location of cells which contain the polypeptide of interest. Such a kit may include a Substan Specific polypeptide. In Vivo tumor imaging is described in tially isolated polypeptide antigen comprising an epitope S. W. Burchiel et al., “Immunopharmacokinetics of Radio which is specifically immunoreactive with at least one labeled Antibodies and Their Fragments.” (Chapter 13 in anti-polypeptide antigen antibody. Further, Such a kit Tumor Imaging. The Radiochemical Detection of Cancer, S. includes means for detecting the binding of Said antibody to W. Burchiel and B. A. Rhodes, eds. (Masson Publishing Inc. the antigen (e.g., the antibody may be conjugated to a 1982). 25 fluorescent compound Such as fluorescein or rhodamine Depending on Several variables, including the type of which can be detected by flow cytometry). In specific label used and the mode of administration, the time interval embodiments, the kit may include a recombinantly produced following the administration for permitting the labeled mol or chemically Synthesized polypeptide antigen. The ecule to preferentially concentrate at Sites in the Subject and polypeptide antigen of the kit may also be attached to a Solid for unbound labeled molecule to be cleared to background Support. level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In In a more specific embodiment the detecting means of the another embodiment the time interval following administra above-described kit includes a Solid Support to which Said tion is 5 to 20 days or 5 to 10 days. polypeptide antigen is attached. Such a kit may also include In a further embodiment, monitoring of the disease or a non-attached reporter-labeled anti-human antibody. In this disorder is carried out by repeating the method for diagnos 35 embodiment, binding of the antibody to the polypeptide ing the disease or disorder, for example, one month after antigen can be detected by binding of the Said reporter initial diagnosis, Six months after initial diagnosis, one year labeled antibody. after initial diagnosis, etc. and comparing the results. In an additional embodiment, the invention includes a Presence of the labeled molecule can be detected in the diagnostic kit for use in Screening Serum containing antigens patient using methods known in the art for in Vivo Scanning. 40 of the polypeptide of the invention. The diagnostic kit These methods depend upon the type of label used. Skilled includes a Substantially isolated antibody Specifically immu artisans will be able to determine the appropriate method for noreactive with polypeptide or polynucleotide antigens, and detecting a particular label. Methods and devices that may means for detecting the binding of the polynucleotide or be used in the diagnostic methods of the invention include polypeptide antigen to the antibody. In one embodiment, the but are not limited to computed tomography (CT), whole 45 antibody is attached to a Solid Support. In a specific body Scan Such as position emission tomography (PET), embodiment, the antibody may be a monoclonal antibody. magnetic resonance imaging (MRI), and Sonography. The detecting means of the kit may include a Second, labeled In a specific embodiment, the molecule is labeled with a monoclonal antibody. Alternatively, or in addition, the radioisotope and is detected in the patient using a radiation detecting means may include a labeled, competing antigen. responsive surgical instrument (Thurston et al., U.S. Pat. No. 50 In one diagnostic configuration, test Serum is reacted with 5,441,050). In another embodiment, the molecule is labeled a Solid phase reagent having a Surface-bound antigen with a fluorescent compound and is detected in the patient obtained by the methods of the present invention. After using a fluorescence responsive Scanning instrument. In binding with Specific antigen antibody to the reagent and another embodiment, the molecule is labeled with a positron removing unbound Serum components by Washing, the emitting metal and is detected in the patent using positron 55 reagent is reacted with reporter-labeled anti-human antibody emission-tomography. In yet another embodiment, the mol to bind reporter to the reagent in proportion to the amount of ecule is labeled with a paramagnetic label and is detected in bound anti-antigen antibody on the Solid Support. The a patient using magnetic resonance imaging (MRI). reagent is again washed to remove unbound labeled Antibody Kits antibody, and the amount of reporter associated with the The present invention provides kits that can be used in the 60 reagent is determined. Typically, the reporter is an enzyme above methods. In one embodiment, a kit comprises an which is detected by incubating the Solid phase in the antibody of the invention, preferably a purified antibody, in presence of a Suitable fluorometric, luminescent or calori one or more containers. In a specific embodiment, the kits of metric Substrate (Sigma, St. Louis, Mo.). the present invention contain a Substantially isolated The Solid Surface reagent in the above assay is prepared polypeptide comprising an epitope which is specifically 65 by known techniques for attaching protein material to Solid immunoreactive with an antibody included in the kit. Support material, Such as polymeric beads, dip Sticks, Preferably, the kits of the present invention further comprise 96-well plate or filter material. These attachment methods US 6,592,865 B2 121 122 generally include non-Specific adsorption of the protein to bind to and, for example, neutralize ACE-2. Such neutral the Support or covalent attachment of the protein, typically izing anti-idiotypes or Fab fragments of Such anti-idiotypes through a free amine group, to a chemically reactive group can be used in therapeutic regimens to neutralize ACE-2. For on the Solid Support, Such as an activated carboxyl, example, Such anti-idiotypic antibodies can be used to bind hydroxyl, or aldehyde group. Alternatively, Streptavidin ACE-2 and thereby block ACE-2 mediated vasoconstriction. coated plates can be used in conjunction with biotinylated protein(s). EXAMPLES Thus, the invention provides an assay System or kit for carrying out this diagnostic method. The kit generally Isolation of ACE-2 binding polypeptides and their use in includes a Support with Surface-bound recombinant accordance with this invention will be further illustrated antigens, and a reporter-labeled anti-human antibody for below. The Specific parameters included in the following detecting Surface-bound anti-antigen antibody. examples are intended to illustrate the practice of the In another specific embodiment, any of the antibodies invention, and they are not presented to in any way limit the listed above are conjugated to a toxin or a label (as described Scope of the invention. Supra). Such conjugated antibodies are used to kill a par ticular population of cells or to quantitate a particular 15 Example 1 population of cells. In a preferred embodiment, Such con jugated antibodies are used to kill Smooth muscle cells Screening of Phage Display Libraries expressing ACE-2 on their Surface. In another preferred The Specific polypeptides according to this invention were embodiment, Such conjugated antibodies are used to quan Selected from Screening eight phage display libraries. Six of titate Smooth muscle cells expressing ACE-2 on their Sur the libraries each displayed a short, variegated exogenous face. In a further preferred embodiment, Such conjugated peptide loop of 6, 7, 8, 9, 10, or 12 amino acids on the antibodies are used to kill endothelial cells expressing Surface of M13 phage, at the amino terminus of protein III. ACE-2 on their surface. In a further preferred embodiment, The libraries are designated TN6/6 (having a potential Such conjugated antibodies are used to quantitate endothelial 3.3x10' amino acid sequence diversity); TN7/4 (having a cells expressing ACE-2 on their Surface. 25 potential 1.2x10" amino acid sequence diversity), TN8/9 In another specific embodiment, any of the antibodies (having a potential 2.2x10" amino acid sequence diversity), listed above are conjugated to a toxin or a label (as described TN9/4 (having a potential 4.2x10" amino acid sequence Supra). Such conjugated antibodies are used to kill a par diversity, TN10/9 (having a potential 3.0x10' amino acid ticular population of cells or to quantitate a particular Sequence diversity), and TN12/1 (having a sequence diver population of cells. In a preferred embodiment, Such con sity of 4.6x10'). jugated antibodies are used to kill Smooth muscle cells The TN6/6 library was constructed to display a single expressing the membrane-bound form of ACE-2. In another microprotein binding loop contained in a 12-amino acid preferred embodiment, Such conjugated antibodies are used template. The TN6/6 library utilized a template sequence of to quantitate Smooth muscle cells expressing the membrane Xaa-Xaa-Xaa-CyS-Xaa-Xaa-Xaa-Xaa-CyS-Xaa-Xaa-Xaa bound form of ACE-2. In a further preferred embodiment, 35 (SEQ ID NO:153). The amino acids at positions 2, 3, 5, 6, Such conjugated antibodies are used to kill endothelial cells 7, 8, 10, and 11 of the template were varied to permit any expressing the membrane-bound form of ACE-2. In a further amino acid except cysteine (C). The amino acids at positions preferred embodiment, Such conjugated antibodies are used 1 and 12 of the template were varied to permit any amino to quantitate endothelial cells expressing the membrane acid except cysteine (C), glutamic acid (E), isoleucine (I), bound form of ACE-2. 40 Lysine (K), methionine (M), and threonine (T). The antibodies of the invention also have uses as thera The TNT/4 library was constructed to display a single peutics and/or prophylactics which include, but are not microprotein binding loop contained in a 13-amino acid limited to, regulation of vasoconstriction, as an analgesic template. The TN7/4 library utilized a template sequence of agent, regulation of Smooth muscle cell proliferation, and in Xaa-Xaa-Xaa-CyS-Xaa-Xaa-Xaa-Xaa-Xaa-CyS-Xaa-Xaa activating cells or blocking cell activation and/or killing cell 45 lineages that express the membrane bound form of ACE-2 Xaa (SEQ ID NO:154). The amino acids at amino acid on their cell Surfaces (e.g., to treat, prevent, and/or diagnose positions 1, 2, 3, 5, 6, 7, 8, 9, 11, 12, and 13 of the template atherosclerosis, restenosis, and other diseases or conditions). were varied to permit any amino acid except cysteine (C). In a specific embodiment, the antibodies of the invention fix The TN8/9 library was constructed to display a single complement. In other Specific embodiments, as further 50 microprotein binding loop contained in a 14-amino acid described herein, the antibodies of the invention (or frag template. The TN8/9 library utilized a template sequence of ments thereof) are associated with heterologous polypep Xaa-Xaa-Xaa-CyS-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-CyS-Xaa tides or nucleic acids (e.g. toxins, Such as, compounds that Xaa-Xaa (SEQID NO:155). The amino acids at positions 1, bind and activate endogenous cytotoxic effecter Systems, 2, 3, 5, 6, 7, 8, 9, 10, 12, 13, and 14 in the template were and radioisotopes; and cytotoxic prodrugs). 55 varied to permit any amino acid except cysteine (C). As discussed above, antibodies to the ACE-2 binding The TN9/4 library was constructed to display a single polypeptides of the invention can, in turn, be utilized to microprotein binding loop contained in an 15-amino acid generate anti-idiotype antibodies that “mimic” the ACE-2 template. The TN9/1 library utilized a template sequence binding polypeptide, using techniques well known to those Xaa-Xaa-Xaa-CyS-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-CyS skilled in the art. (See, e.g., Greenspan & Bona, FASEB.J., 60 Xaa-Xaa-Xaa (SEQ ID NO:156). The amino acids at posi 7(5):437–444 (1989), and Nissinoff, J. Immunol., tions 1,2,3,5,6,7,8,9, 10, 11, 13, 14 and 15 in the template 147(8):2429–2438 (1991)). For example, antibodies which were varied to permit any amino acid except cysteine (C). bind to ACE-2 binding polypeptides and competitively The TN10/9 library was constructed to display a single inhibit ACE-2/ACE-2 binding polypeptide binding can be microprotein binding loop contained in a 16-amino acid used to generate anti-idiotypes that "mimic' the ACE-2 65 template. The TN10/9 library utilized a template sequence binding polypeptide/ACE-2 binding domain and, as a Xaa-Xaa-Xaa-CyS-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa consequence, Cys-Xaa-Xaa-Xaa (SEQ ID NO:157). The amino acids at US 6,592,865 B2 123 124 positions 1, 2, 15, and 16 in the template were varied to (Kirkegaad & Perry Laboratories, Gaithersburg, Md.), and permit any amino acid Selected from a group of 10 amino read at 630 nm on an ELISA plate reader. acids: D, F, H, L, N, P, R, S, W, or Y). The amino acids at DNA sequences encoding peptides from positive phage positions 3 and 14 in the template were varied to permit any binders were amplified by PCR and sequenced by automatic amino acid Selected from a group of 14 amino acids: A, D, 5 F, G, H, L, N, P, Q, R, S, V, W, or Y). The amino acids at Sequencing. A number of ACE-2-binding polypeptides were positions 5, 6, 7, 8, 9, 10, 11, and 12 in the template were identified, with ACE-2 binders being isolated from five of varied to permit any amino acid except cysteine (C). the cyclic peptide libraries screened (i.e., all except TN9/4). The TN12/1 library was constructed to display a single Analysis of recurring amino acid Sequences among the microprotein binding loop contained in an 18-amino acid ACE-2 binding polypeptides revealed a Series of polypep template. The TN12/1 library utilized a template sequence tide “families' exhibiting common core structures: Xaa-Xaa-Xaa-CyS-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO:158). The amino acids at position 1, 2, 17, and 18 in the template were varied SEQ ID NO : to permit any amino acid Selected from a group of 12 amino 15 acids: A, D, F, G, H, L, N, P, R, S, W, or Y). The amino acids Sequence Family I at positions 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 16 were 20 varied to permit any amino acid except cysteine (C). 21 We also endeavored to select ACE-2 binding polypeptides 22 from two commercially available linear phage display 23 24 libraries, designated PhD 7 and PhD 12, respectively (New 25 England Biolabs). The PhD 7 library displays a linear 26 random-sequence 7-mer; the PhD 12 libary displays a 27 random-Sequence 12-mer. No ACE-2 binding phage were 28 isolated from these two linear libraries. 29 25 Sequence Family I The libraries were Selected against a FLAG-tagged ACE-2 target, which was immobilized to streptoavidin 30 31 coated paramagnetic beads (Seradyn, Ramsey, Minn.) via 32 biotinylated anti-FLAG antibody (M2 antibody, Sigma, St. 33 Louis, Mo.). Before Selection against the target, the libraries 34 were depleted 5 times with FLAG peptide/anti-FLAG 35 36 antibody-immobilized beads, to remove binders to the strep 37 toavidin beads, the anti-FLAG antibody and the FLAG 38 peptide. The depleted libraries were incubated with 6 ug 39 FLAG-ACE-2 (obtained from Human Genome Sciences, 35 40 41 Inc.) in Solution for 1 hour at room temperature (RT), and 42 then incubated with anti-FLAG antibody-immobilized beads 43 for 1 hour. The beads were washed 7 times with PBS, 0.2% 44 Tween 20 (PBST) to remove unbound phage. After washing, 45 46 40 the bound phage were eluted by introducing free FLAG 47 peptide (100 ug/ml). Eluted phage were amplified, and 48 underwent two more Similar rounds of Selection. In round 1, 49 the six constrained peptide libraries, TN6/6, TN7/4, TN8/9, 5 O 51 TN9/4, TN 10/9, and TN12/1, were selected separately. To 52 accelerate the Selection procedures, in the Subsequent rounds 45 53 of Selection, these Six libraries were combined into two equence Family I pools: TN6/6, TN7/4, and TN8/9 were combined to form 54 pool A, whereas TN9/4, TN 10/9, and TN12/1 were com 55 bined to form pool B. The two linear peptide libraries, PhD7 56 and PhD12 (New England BioLabs) were combined 50 57 together at round 1. 58 59 Phage enriched from the third round of selection were 60 screened by ELISA for strong ACE-2 binders. Immulon 2 61 96-well plates (Dynatech) were coated with streptavidin for 62 55 63 1 hour at 37 C. and subsequently coated with anti-FLAG 64 antibody for 1 hour at room temperature. Half of the plates 65 were further coated with FLAG-ACE-2 as the target plates, 66 and the other half of the plates were coated with FLAG 67 peptide as background plates. The amount of each protein or C 68 equence Family W peptide coated was 100 ng per well. The coated plates were 60 then incubated for 1 hour at room temperature with 1:2 69 diluted overnight phage cultures that were made by inocu g 70 lating phage from individual plaques into bacterial host 71. 72 cells. After being washed 7 times with PBST, the plates were 73 incubated with HRP-conjugated anti-M13 antibody 65 74 (Pharmacia) for 1 hour at room temperature, washed 5 times, 75 developed with TMB peroxidase substrate solution
US 6,592,865 B2 127 128 Lead peptide inhibitor candidates were identified from in tyrosine) for one another, the exchange of hydrophobic vitro studies utilizing peptide M-2195 as a substrate. Assay residues (e.g., leucine, isoleucine, and valine) for one mixture was prepared first by pre-incubating (30 minutes, another, the exchange of polar residues (e.g., glutamine and room temperature) the enzyme with the inhibitor (0.45 ug asparagine) for one another, the exchange of acidic residues (50 pmoles) ACE-2, 0-0.5 uM inhibitor rage, up to 90 uL 5 (e.g., arginine, lysine, and histidine) for one another, and the volume with 100 mM TRIS pH 7.4, 0.1% Tween-20, 0.4% exchange of Small residues (e.g., alanine, Serine, threonine, DMSO) in each well, in triplicate, by dispensing volumes methionine, and glycine) for one another, the exchange of using a repeat pipetter (Rainin) into a 96-well clear-bottom aromatic residues for one another. Additionally, nonclassical back plate (Costar, 07-200-590). The layout of the plate was amino acids, chemical amino acid analogs, or chemically such that M-2195 substrate concentration were varied by modified classical amino acids can be introduced as a column and inhibitor concentrations were varied by row. Substitution or addition to an ACE-2 binding polypeptide of Substrate stock (10 ul of 10x concentration) was added to the invention. Non-classical amino acids include, but are not each well to be tested in blocks of three vertically down the limited to, the D-isomers of the common amino acids, plate. The reaction was monitored on a Spectrafluor Plus 2,4-diaminobutyric acid (Dbu), 4-aminobutyric acid (bAbu), (Tecan) at an excitation wavelength of 340 nm and an 15 2-aminobutyric acid (Abu), 6-amino hexanoic acid (epsilon emission wavelength of 400 nm (+/-35 nm) every 30 Ahx), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid Seconds for a time period of 10 minutes. All data points were (bAib), 3-aminopropanoic acid (bAla), ornithine (Orn), nor transferred to and analysed by Prism 2.01 software. The leucine (Nle), norvaline (Nva), 3-hydroxyproline (3Hyp), results are shown in table 2. 4-hydroxyproline (4Hyp), sarcosine (MeCly), citrulline,
TABLE 2
Clone #of Inhibi- IC50 Ki SEO ID DX-No. Name Sequence Res. tion puM nM NO:
DX-SOO A-B5 Ac-GSNRECHALFCMDFAPGEGGG-NH2 21 -- 11 DX-501 A-H2 Ac-GSSPTCRALFCVDFAPGEGGG-NH2 21 -- 12 DX-502 A-B6 Ac-GSLEMCEALFCVEFAPGEGGG-NH2 21 13 DX-503 A-C11 Ac-GSDONCFAMYCFEFAPGEGGG-NH2 21 14 DX-504 A-F12 Ac-AGEGNCFLIGPWCFEFGTEGGG-NH2 22 15 DX-505 A3-E12 Ac-AGYEDCIGHALFCMTFGTEGGG-NH2 22 -- 16 *DX-506 ACEH1O-A6 Ac-AGWELCNGVMALFCVEFGTEGGG-NH2 23 n.fd. 17 DX-507 ACEHS-H8 Ac-GSNDYCTVFTGALFCLDFAPEGGG-NH2 24 18 DX-508 ACEH1-C1 Ac-GSYDNCLGLANLNFCFDFAPEGGG-NH2 24 -- 19 DX-509 ACEH6-D12 Ac-GDDDHCEWASYWKWDLCLHDDPEGGG-NH2 26 -- 2O DX-510 ACEH4-H9 Ac-GDDDDCGWIGFANFHLCLHGDPEGGG-NH2 26 21 DX-511 ACEH1-D3 Ac-GDPFECDWGPWTLEMLCGPPDPEGGG-NH2 26 -- 22 DX-512 B-H, Ac-GDRLHCKPOROSPWMKCOHLDPEGGG-NH2 26 ------O.O6 150 23 DX-513 ACEH2-D8 Ac-GDLHACRPVRGDPWWACTLGDPEGGG-NH2 26 ------O.O9 150 24 DX-514 ACEH2-A2 Ac-GSPNOCGVDIWALFCVDFAPEGGGK-NEH2 25 -- 25 DX-515 ACEH2-A2 Ac-GSPNQCGVDIWALFCVDFAPEGGGK(fitc)-NH2 25 26 DX-524 360c-7-G10 Ac-GSRGCRDSRCNWWAPGEGGG-NH2 21 ------O.6 27 DX-525 36Oc-8-H9 Ac-GSRGFCRDSSCSFPAPGEGGG-NH2 21 ------1. 28 DX-526 36Oc-7-C3 Ac-GSWPTCLTMDCWYNAPGEGGG-NH2 21 -- 29 DX-527 36Oc-7-D4 Ac-AGWVLCFEWEDCDEKGTEGGG-NH2 21 3O DX-528 360c-2-A12 Ac-AGVYFCFDWEODCDEMGTEGGG-NH2 22 31 DX-529 36Oc-4-ES Ac-AGWEVCHWAPMMCKHGGTEGGG-NH2 22 ------0.4 32 DX-53O 36Oc-7-D8 Ac-AGOKECKFGYPHCLPWGTEGGG-NH2 22 ---- 3O 33 DX-531 36Oc-8-G11 Ac-AGSDWCGTWNNPCFHOGTEGGG-NH2 22 ------0.5 34 DX-537 ACEH2-D8 Ac-GDLHACRPVRGDPWWACTLGDPEGGGK(fitc)-NH2 26 35 DX-599 ACEH2-F6 Ac-GDRYLCLPORDKPWKFCNWFDPEGGG-NH2 26 ------O.14 36 DX-6OO ACEH1-F11 Ac-GDYSHCSPLRYYPWWKCTYPDPEGGG-NH2 26 ------O.O25 37 DX-601 ACEH1-G10 Ac-GDGFTCSPIRMFPWFRCDLGDPEGGG-NH2 26 ------O.O68 38 DX-602 B-GO9 Ac-GDFSPCKALRHSPWWVCPSGDPEGGG-NH2 26 ------O.12 39 -: signifies no inhibition on ACE-2 activity +: signifies weak inhibition (20-60% inhibition at ~100 uM) ++: signifies moderate inhibition (at least 80% inhibition at ~100 uM; with IC50 of ~30 uM) +++: signifies strong inhibition (-99% inhibition at ~100 uM; with IC50 between 0.4-1 uM) ++++: signifies very strong inhibition (~100% inhibition at 100 uM; with IC50 0.15 uM) n.fd.: not determined, due to difficulties in sythesizing DX515: fluorescence-labeled version of DX514 DX537: fluorescence-labeled version of DX513.
Example 2 homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, fluoro-amino acids, Synthesis of Further ACE-2 Binding Peptides designer amino acids Such as B-methyl amino acids, 60 Co-methylamino acids, NC.-methylamino acids, and amino Once a promising ACE-2 binding polypeptide has been acid analogs in general. isolated, improvements to that polypeptide can be made by changing, adding or removing individual or multiple amino Example 3 acid residues from the polypeptide. Amino acid Substitutions Biacore Analysis of the Affinity of ACE-2 Binding can be conservative or non conservative. Conservative 65 Polypeptides amino acid exchanges include, for example, the exchange of Binding of ACE-2 binding polypeptides to ACE-2, for aromatic residues (e.g., phenylalanine, tryptophan, and example, can be analyzed by BIAcore analysis. Either US 6,592,865 B2 129 130 ACE-2 (or another antigen for which one wants to know the ACE-2 binding polypeptides to His-tag, HA-tag, protein A, affinity of an ACE-2 binding polypeptide) or ACE-2 binding IgG domains, and maltose binding protein facilitates puri polpeptide can be covalently immobilized to a BIAcore fication. (See, EP A 394 827; Traunecker et al., Nature, sensor chip (CM5 chip) via amine groups using N-ethyl-N'- 331:84-86 (1988)). Similarly, fusion to IgG-1, IgG-3, and (dimethylamino propyl)carbodiimide/N- albumin increases the half-life time in vivo. Nuclear local hydroxysuccinimide chemistry. Various dilutions of ACE-2 ization signals fused to ACE-2 binding polypeptides can target the protein to a specific Subcellular localization, while binding polypeptides or ACE-2 (or other antigen for which covalent heterodimer or homodimers can increase or one wants to know the affinity of an ACE-2 binding decrease the activity of a fusion protein. Fusion proteins can polypeptide), respectively are flowed over the derivatized also create chimeric molecules having more than one func CM5 chip in flow cells at 15 microlters/min. for a total tion. Finally, fusion proteins can increase Solubility and/or volume of 50 microliters. The amount of bound protein is Stability of the fused protein compared to the non-fused determined during washing of the flow cell with HBS buffer protein. All of the types of fusion proteins described above (10 mM HEPES, pH7.4, 150 mM NaCl, 3.4 mM EDTA, can be made using techniques known in the art or by using 0.005% surfactant P20). Binding specificty for the protein of or routinely modifying the following protocol, which out inerest is determined by competition with Soluble competitor 15 lines the fusion of a polypeptide to an IgG molecule. in the presence the protein of ineterest. Briefly, the human Fc portion of the IgG molecule can be The flow cell Surface can be regenerated by displacing PCR amplified, using primers that span the 5' and 3' ends of bound protein by washing with 20 microliters of 10 mM the sequence described below (SEQ ID NO:184). These glycine-HCl, pH2.3. For kinetic analysis, the flow cells are primerS also preferably contain convenient restriction tested at different flow rates and different polypetide densi enzyme Sites that will facilitate cloning into an expression ties on the CM5 chip. The on-rates and off-rates can be vector, preferably a mammalian expression vector. determined using the kinetic evaluation program in BIAe For example, if the pC4 (Accession No. 209646) expres valuation 3 Software. Sion vector is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3' BamHI site should Example 4 25 be destroyed. Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, In Vitro Screening of ACE-2 Antagonists and ACE-2 binding polynucleotide is ligated into this BamHI site. Note that the polynucleotide is cloned without The bioassay for assessing the effects of putative ACE-2 a stop codon, otherwise a fusion protein will not be pro antagonistS is performed in triplicate in 96 well format by duced. mixing equal Volumes of ACE-2, responder cells, and puta If the naturally occurring Signal Sequence is used to tive antagonist each of which is prepared as a 3x Stock produce the Secreted protein, pC4 does not need a Second reagent. Signal peptide. Alternatively, if the naturally occurring Signal Endothelial cells of coronary vessels are washed and Sequence is not used, the vector can be modified to include resuspended in complete medium (CM) (RPMI 1640 with a heterologous signal sequence. (See, e.g., WO 96/34891) 10% FBS containing 100 U/ml penicillin, 100 tug/ml 35 Human IgG Fc region: streptomycin, 4 mM glutamine, 5x10E-5 M beta GGG AT C C G GAGCC CAAATCTTC TGA mercaptoethanol) at a concentration of 3x10e6 cells/mL. CAAAA CT CACA CAT GCC CA C C G T G C C CAG CAC CTGAATTCGAGGGTG CACC GT Staphylococcus aureus, Cowan I (SAC, Cal Biochem) is CAGTCTTCCTCTTCCCCCCAAAACCCAAGGAC added to cells at 3x concentration (3x=1:33,333 dilution of 40 A C C C T CAT GAT C T C C C G GAC T C C T G A G Stock). GT CAC AT GC GTG G T G G T G GAC GTAAG C Meanwhile, eight serial dilutions (3-fold) of potential CAC GAAG ACC CT GAG GT CAA GT antagonists are prepared in CM Such that the diluted antago TCAACTGGTACGTGGACGGCGTGGAGGTGCAT nists are at 3x the final concentrations to be tested in the AATG CCAAG ACAAA G CCGCGG GAG GAG assay. ACE-2 binding polypeptides are routinely tested 45 CA GTA CAA CAG CAC GTA C C G T G T G GT Starting at a final concentration of 10.ug/mL and going down CAGC GT C C T CAC C GT C C T G CAC CAG to about 1.5 ng/mL. GACTGGCTGAATGGCAAGGAGTACAAGTGCAA Human raCE-2 is prepared in CM to 3x concentration G GT CTCCAA CAAA G C C C T C C CAA C (3x=300 ng/mL, 30 ng/mL, and 3 ng/mL) in CM. Potential C C C CAT CGAGAAAA C CATCTCCAAA G C 50 CAAAGGGCAGCCCCGAGA ACCACAGGTG inhibitors are routinely tested at Several concentrations of TACACCCTGCCCCCATCCCGGGATGAGCTGACC ACE-2 to avoid false negatives due to unexpectedly low AAGAA C CAG GT CAGCCTGAC CTG CCTG affinity or antagonist concentration. GT CAAAG G CTTCTATCCAA G CGA Fifty microliters of diluted antagonist and 50 it of diluted CAT C GCC GTG GA GTGG GAGA G ACE-2 are added to the putative antagonist dilution Series. CAATGGGCAGCCGGAGAACAACTACAAGACCA Cells are then incubated for 72 hours (37° C., 5% CO) in 55 CGC CTCCC GTG CTG GA CTCC GACGG CTC a fully humidified chamber. After 72 hrs., the cells are CTTCTTC CTCTACA G CAAG CT CACC GT G supplemented with 0.5 uCi/well 3H-thymidine (e.g., 6.7 GACAAG A G CAG G T G GCA G CAGGG Ci/mmol) and incubated for an additional 24 hours. Plates GAACGTCTTCTCATGCTCCGTGATGCATGAGGC are harvested using a Tomtec Cell Harvester and filters TCTG CACA ACCACTACAC GCAGAAGAGC counted in a TopCount Scintillation counter (Packard). 60 CTCTCC CT G TCTCCGGGTAAATGAG T G C GACGGCCGCGACTCTAGAGGAT (SEQ ID NO:150) Example 5 Example 6 Protein Fusions of ACE-2 Binding Polypeptides Isolation of scEV Molecules Recognizing ACE-2 ACE-2 binding polypeptides of the invention are option 65 Binding Polypeptides ally fused to other proteins. These fusion proteins can be Naturally occuring V-genes isolated from human PBLS used for a variety of applications. For example, fusion of are constructed into a large library of antibody fragments US 6,592,865 B2 131 132 which contain reactivities against polypeptides of the bicarbonate, pH 9.6. Clones positive in ELISA are further present invention to which the donor may or may not have characterized by PCR fingerprinting (see, e.g., WO been exposed (see, e.g., U.S. Pat. No. 5,885,793, incorpo 92/01047) and then by sequencing. rated herein by reference in its entirety). Additionally, ScFVS may be converted to complete Ig Rescue of the Library molecules using techniques which are commonly known in A library of ScFVs is constructed from the RNA of human the art. PBLs as described in WO 92/01047. To rescue phage displaying antibody fragments, approximately 10° E. coli Example 7 harbouring the phagemid are used to inoculate 50 ml of 2xTY containing 1% glucose and 100 lug/ml of amplicillin Production of an anti-ACE-2 Binding Polypeptide (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with Antibody Hybridoma Technology shaking. Five ml of this culture is used to innoculate 50 ml The antibodies of the present invention can be prepared of 2xTY-AMP-GLU, 2x108 TU of A gene 3 helper phage by a variety of methods. (See, Current Protocols, Chapter 2.) (M13 A gene III, see WO92/01047) are added and the AS one example of Such methods, cells expressing ACE-2 culture incubated at 37 C. for 45 minutes without shaking 15 binding polypeptides are administered to an animal to and then at 37 C. for 45 minutes with shaking. The culture induce the production of Sera containing polyclonal anti is centrifuged at 4000 rp.m. for 10 minutes and the pellet bodies. In a preferred method, a preparation of ACE-2 resuspended in 2 liters of 2xTY containing 100 tug/ml binding polypeptide is prepared and purified to render it ampicillin and 50 ug/ml kanamycin and grown overnight. Substantially free of natural contaminants which is then Phage are prepared as described in WO92/01047. conjugated to a carrier molecule Such as keyhole limpet M13 A gene III is prepared as follows: M13 A gene III hemocyanin (KLH), Suucinylated KLH, or chicken gamma helper phage does not encode gene III protein, hence the globulin (CGG). Such a preparation is then introduced into phage(mid) displaying antibody fragments have a greater an animal in order to produce polyclonal antisera of greater avidity of binding to antigen. Infectious M13 A gene III Specific activity. particles are made by growing the helper phage in cells 25 harboring a pUC19 derivative Supplying the wild type gene In the most preferred method, the antibodies of the present III protein during phage morphogenesis. The culture is invention are monoclonal antibodies (or ACE-2 protein incubated for 1 hour at 37 C. without shaking and then for binding fragments thereof). Such monoclonal antibodies can a further hour at 37 C. with shaking. Cells are pelleted be prepared using hybridoma technology. (Kohler et al., (EC-Centra 8, 4000 revS/min. for 10 min.), resuspended in Nature, 256:495 (1975); Kohler et al., Eur. J. Immunol., 300 ml 2xTY broth containing 100 lug ampicillin/ml and 25 6:511 (1976); Kohler et al., Eur: J. Immunol. 6:292 (1976); ug kanamycin/mi (2xTY-AMP-KAN) and grown overnight, Hammerling et al., in Monoclonal Antibodies and T-Cell Shaking at 37 C. Phage particles are purified and concen Hybridomas (Elsevier, N.Y. 1981), pp. 563-681.) In general, trated from the culture medium by two PEG-precipitations Such procedures involve immunizing an animal (preferably (Sambrook et al., 1990), resuspended in 2 ml PBS and 35 a mouse) with ACE-2 binding polypeptide or, more passed through a 0.45um filter (Minisart NML; Sartorius) to preferably, with a Secreted ACE-2 binding polypeptide give a final concentration of approximately 1013 transduc expressing cell. Such cells may be cultured in any Suitable ing units/ml (ampicillin-resistant clones). tissue culture medium; however, it is preferable to culture Panning of the Library cells in Earle's modified Eagle's medium Supplemented with Immunotubes (Nunc) are coated overnight in PBS with 4 40 10% fetal bovine serum (inactivated at about 56° C), and ml of either 100 mg/ml or 10 mg/ml of a polypeptide of the Supplemented with about 10 g/l of nonessential amino acids, present invention. Tubes are blocked with 2% Marvel-PBS about 1,000 U/ml of penicillin, and about 100 tug/ml of for 2 hours at 37° C. and then washed 3 times in PBS. Streptomycin. Approximately 1013 TU of phage are applied to the tube and The Splenocytes of Such mice are extracted and fused with incubated for 30 minutes at room temperature tumbling on 45 a Suitable myeloma cell line. Any Suitable myeloma cell line an over and under turntable and then left to stand for another may be employed in accordance with the present invention; 1.5 hours. Tubes are washed 10 times with PBS 0.1% however, it is preferable to employ the parent myeloma cell Tween-20 and 10 times with PBS. Phage are eluted by line (SP2/0), available from the ATCC. After fusion, the adding 1 ml of 100 mM triethylamine and rotating 15 resulting hybridoma cells are selectively maintained in HAT minutes on an under and over turntable after which the 50 medium, and then cloned by limiting dilution as described solution is immediately neutralized with 0.5 ml of 1.0 M by Wands et al. (Gastroenterology, 80:225–232 (1981).) The Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of hybridoma cells obtained through Such a Selection are then mid-log E. coli TG1 by incubating eluted phage with bac assayed to identify clones which Secrete antibodies capable teria for 30 minutes at 37 C. The E. coli are then plated on of binding the ACE-2 binding polypeptide. TYE plates containing 1% glucose and 100 lug/ml amplicil 55 Alternatively, additional antibodies capable of binding to lin. The resulting bacterial library is then rescued with A ACE-2 binding polypeptide can be produced in a two-step gene III helper phage as described above to prepare phage procedure using anti-idiotypic antibodies. Such a method for a Subsequent round of Selection. This proceSS is then makes use of the fact that antibodies are themselves repeated for a total of 4 rounds of affinity purification with antigens, and therefore, it is possible to obtain an antibody tube-washing increased to 20 times with PBS, 0.1% Tween 60 which binds to a second antibody. In accordance with this 20 and 20 times with PBS for rounds 3 and 4. method, protein specific antibodies are used to immunize an Characterization of Binders animal, preferably a mouse. The Splenocytes of Such an Eluted phage from the 3rd and 4th rounds of selection are animal are then used to produce hybridoma cells, and the used to infect E. coli HB 2151 and soluble scFv is produced hybridoma cells are Screened to identify clones which pro (Marks et al., 1991) from single colonies for assay. ELISAS 65 duce an antibody whose ability to bind to the ACE-2 binding are performed with microtitre plates coated with either 10 polypeptide-specific antibody can be blocked by ACE-2 pg/ml of the polypeptide of the present invention in 50 mM binding polypeptide. Such antibodies comprise anti US 6,592,865 B2 133 134 idiotypic antibodies to the ACE-2 binding protein-Specific combination of the two over a dose range from 1-30 nM antibody and can be used to immunize an animal to induce (alone or in combination) in mature rat aortic rings. The formation of further ACE-2 binding polypeptide-specific maximum tension developed by A1-9 animal and always antibodies. less than that of angiotensin II. When A1-9 was combined with angiotensin II, the resulting Emax was greater than the It will be appreciated that Fab and F(ab') and other Sum of the individual peptide Emax values, Suggesting a fragments of the antibodies of the present invention may be Superadditive or Synergistic interaction. Because A1-9 is used according to the methods disclosed herein. Such frag Virtually devoid of activity, the quantitation of its ments are typically produced by proteolytic cleavage, using constrictor-enhancing action on angiotensin II was derived enzymes Such as papain (to produce Fab fragments) or from the shift in the angiotensin II dose-effect curve as Seen pepsin (to produce F(ab')2 fragments). Alternatively, in FIG.1. The slopes of the dose-effect curves did not differ; Secreted ACE-2 binding protein-binding fragments can be however, the relative potency of angiotensin II was produced through the application of recombinant DNA increased by a factor of 11.6 (95% confidence limits of technology or through Synthetic chemistry. 3.02-44.7) beyond that of simple additive potentiation, For in vivo use of antibodies in humans, it may be indicative of Synergism for the combination. In control preferable to use “humanized’ chimeric monoclonal anti 15 experiments, angiotensin I failed to potentiate angiotensin bodies. Such antibodies can be produced using genetic II-mediated vasoconstriction. This indicates that the Synergy constructs derived from hybridoma cells producing the observed between A1-9 and angiotensin II is specific for monoclonal antibodies described above. Methods for pro A1-9 and is not a of the ACE and ACE-2 substrate, angio ducing chimeric antibodies are known in the art. (See, for tensin I. review, Morrison, Science, 229:1202 (1985); Oi et al., BioTechniques, 4:214 (1986); Cabilly et al., U.S. Pat. No. TABLE 3 4,816,567; Taniguchi et al., EP171496; Morrison et al., EP A1-9 AI AI-9 + AII 173 494; Neuberger et al., WO 86/01533; Robinson et al., %. Max. %. Max. %. Max. WO 87/02671; Boulianne et al., Nature, 312:643 (1984); Experiment Developed Force Developed Force Developed Force 25 Neuberger et al., Nature, 314:268 (1985)). 1. 3.5 22 82 Example 8 2 O 8.6 18 3 7.5 17 24 4 6.4 9.9 34 Fluorometric Assays for ACE-2 5 3.2 41 48 The activity of ACE-2 was measured utilizing a fluoro metric assay in which peptide M-2195 (BACHEM) is used To investigate the effects of A1-9 on arterial pressure, as a substrate. ACE-2 cleaves off the C-terminal lysine from A1-9 was administered to awake, free-ranging Sprague the peptide with a concomitant increase in fluorescence Dawley rats via indwelling intravenous and arterial cath Signal. eters. Intravenous infusion of A1-9 (1–3 ug/kg/min) in the The assays were performed in 96-well clear-bottom black 35 awake rat produced a dose-dependent tonic increase in mean plates (Costar,07-200-590). In each well, 0.45ug (5 pmoles) arterial pressure to a maximum of approximately 30 mmHg of ACE-2 is added to 90 it of an assay Solution containing (FIG. 2). Pulse pressure (systolic-diastolic) was also 100 mM TRIS at pH 7.4, 0.1% Tween, and 0.4% DMSO. elevated and heart rate was decreased in a dose-dependent Substrate stock at 10 till of 10x concentration is added to manner by A1-9 infusion (data not shown). These responses each well (for example, 10 uL of 1 mM stock for a final 100 40 Seen with A1-9 were similar in magnitude to those induced AlM concentration). The reaction was monitored on a Spec by angiotensin II. The observed bradycardia (presumed to be trafluor Plus (Tecan) at an excitation wavelength of 340 nm reflexive) in association with the increase in mean arterial and an emission wavelength of 400 nm (+/-35 nm) every 30 and pulse pressure Suggests that A1-9 increases vascular Seconds up to 10 minutes. All Samples were measured in tone in Vivo, perhaps through potentiating the activity of triplicate. The data points were transferred to and analysed 45 angiotensin II. on Prism 2.01 Software. To examine any interactions induced by coadministration Degradation: Identification and Analysis of the Functional of A1-9 and angiotensin II, arterial preSSure was monitored Roles of ACE-2 Inhibitory Peptides in the awake rat after they received either A1-9 (1 lug/kg/ The effects of graded doses of A1-9 (Fields et al., Peptide min) or Saline coinfused with angiotensin II (1-100 ng/kg/ Research 4: 95-101 (1991)) or A1-10 (substrate control) 50 min). The submaximal infusion of A1-9 potentiated the alone or in combination (Tallarida, Drug Synergism and preSSor response elicted by angiotensin II in an additive Dose-Effect Data Analysis, Chapman and Hall/CRC, New fashion (FIG. 3). Angiotensin II coinfused with a control York (2000), Tallarida et al., Life Science 61: PL417–425 nonapeptide with no intrinsic hemodynamic activity in (1997)) with angiotensin II were examined on isolated aortic awake rats produced a dose-effect curve similar to that of vascular rings developed tension in response to vasoactive 55 angiotensin II alone (data not shown). Substances. Aortas were removed from mature male To test further the hypothesis that downregulating ACE-2 Sprague-Dawley rats, cleared of connective tissue, and cut activity would decrease arterial pressure, tagged human into 1 mm rings. Rings were mounted to an isometric force ACE-2 was generated in order to identify potential peptide transducer and placed in Krebs-Henseleit solution bubbled ACE-2 inhibitors whose Sequences were derived from phage with 95% O2/5% CO2 at 37° C. Rings were exposed to a 2 60 display. The human ACE-2 cdNA was used as template for g preload, relaxed and then exposed to various concentra amplification by PCR of the nucleotides corresponding to tions of angiotensin peptides alone or in combination. Fol the extracellular domain (M1-S740). The 5' primer: lowing angiotensin peptide dosing, rings were fully con 5 ATGGATGATCAGCCATCATGTCAAGCTCTTCCTG stricted with KCl to determine the maximum degree of 3' (SEQ ID NO:151) vasoconstriction. 65 and 3' primer: Table 3 shows the maximum developed isometric force 5' GTATG CTCTA GATTAGGAAA (Emax) following treatment with A1-9, angiotensin II or the CAGGGGGCTGGTTAG 3' (SEQ ID NO:152) US 6,592,865 B2 135 136 generate a 2200 bp fragment which was digested with BclI MAP lasting approximately 1-2 min at the lower doses and and Xbal and cloned into the BamHI and Xbal sites of a approximately 6 min in duration at the 3 mg/kg dose level. baculovirus transfer vector p A2. Following DNA sequence The maximal average depressor response at a dose of 3 confirmation, the plasmid (A2: Ace-H) was transfected into mg/kg was 70.5+4.6 mmHg from an average MAP of Sf9 cells to generate a recombinant baculovirus (Coleman et 155-t10 mmHg. Transient tachycardia (average maximal al, Gene 190:163–170 (1997)). Metabolic labeling was used change between 50–70 bpm at a dose level of 2-3 mg/kg) to confirm the presence of a novel band of -85 Kd corre was observed to coincide with the depressor response (data sponding to the human Ace-H protein in conditioned media not shown). At no dose level was the MAP or HR altered by from Sf9 cells. For protein production, Sf9 cells were seeded the control peptide. in Serum-free media and infected at a multiplicity of infec In terms of cardiovascular function, the expression pattern tion of 1-2 with the recombinant baculovirus. Conditioned of ACE-2 Suggests a potential role in the regulation of local media was harvested, clarified by filtration, and used for circulation in the kidney and heart. However, our results Subsequent enzyme purification. Initially, FLAG peptide indicate that ACE-2 may play a role in regulating Systemic was attached to Streptavidin beads using bead-immobilized circulatory homeostasis. A1-9, known to be produced from biotinylated anti-FLAG antibody. Proprietary phage display 15 ACE-2 mediated catabolism of angiotensin I, potentiated libraries were depleted on these beads 5 times to remove angiotensin II-mediated vasoconstriction in isolated rat aor phages bound to the FLAG peptide. Depleted libraries were tic rings and pressor responses in the awake rat. Although incubated with FLAG-ACE-2 and then immobilized on there has been speculation that A1-9 might serve to inhibit Streptavidin beads. The beads were washed Stringently and ACE and lead to vasodilation (Donoghue, Circulation the bound phage was eluted with FLAG peptide. Eluted Research 87:e 1-e9 (2000), Snyder et al., The Journal of phages were amplified and characterized by ELISA using Biological Chemistry 260:7857-7860 (1985)), the present FLAG-ACE-2 coated in microtiter plates. Positive binders results Support the hypothesis that ACE-2 upregulates Sys were Sequenced and collapsed into Several families based on temic arterial pressure under conditions where intact car amino acid Sequences. diovascular reflexes are present. An unlikely possibility is A phage display technology was used to identify families 25 that the observed effects of A1-9 were due to its conversion of peptide inhibitors of rhaCE-2. FLAG-ACE-2 (above) was used for panning peptides binding to ACE-2. Peptide to angiotensin II in Vivo Since biochemical and in vivo binders were Sequenced and collapsed into Several families evidence Supports that this mechanism is not a major con based upon amino acid Sequences. Several members from tributor to the normal formation of angiotensin II (Johnson the peptide families were synthesized and their inhibitory et al., Peptides 10:489–492 (1989); Donoghue et al., Cir activities were tested using recombinant human-ACE-2. culation Research 87:e1-e9 (2000); Oparil et al., Circulation Using M-2195 peptide as Substrate, multiple inhibitors were Research 29.682-690 (1971)). identified and the most active ACE-2 inhibitor peptide was The identification of a peptide inhibitor of ACE-2 has chosen for further studies. Another ACE-2 binding peptide enabled the study of the effects of ACE-2 inhibition in a that failed to inhibit rhACE-2 was identified, synthesized 35 model of Spontaneous hypertension in Vivo. The current and used as a control for in vivo experiments. The Ki for the results indicate that the inhibition of ACE-2 reduces arterial ACE-2 inhibitor peptide was 250 nM, acting as a mixed-type preSSure through decreasing circulating and/or levels of inhibitor for ACE-2 when M-2195 was used as a Substrate. A1-9, an angiotensin II Synergizing peptide. Supporting this is the demonstration that blockade of ACE-2 causes a This peptide also inhibited conversion of angiotensin I to depressor response in awake Spontaneously hypertensive A1-9 by ACE-2. The ACE-2 inhibitor peptide was specific 40 for ACE-2 and did not inhibit the activity of ACE. For ratS. example, the inhibitor did not affect the hydrolysis of the Following the foregoing description, the characteristics substrate, hip-his-leu (250 mM) by ACE (1.5 nM), even at important for affinity binding polypeptides permitting detec 25 mM. In comparison, the hydrolytic activity of ACE was tion or Separation of ACE-2 or ACE-2-like polypeptides completely inhibited by 25 mM captopril. Thus, the ACE-2 45 (ACE-2 target protein) in or from any Solution can be inhibitor peptide was found to be specific for ACE-2 and appreciated. Additional binding polypeptide embodiments blocked conversion of angiotensin I to A1-9. of the invention and alternative methods adapted to a A lead inhibitor peptide candidate identified from in vitro particular solution or feed stream will be evident from Studies caused a dose-dependent depressor response upon iv Studying the foregoing description. All Such embodiments bolus administration in the awake SHR (FIG. 4). As the 50 and obvious alternatives are intended to be within the Scope peptide inhibitor dose was increased, the magnitude and of this invention, as defined by the claims that follow. duration of the depressor response increased. This depressor Publications referred to above are hereby incorporated by response was characterized by an initial transient fall in reference.
SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS : 158 <21 Oc SEQ ID NO 1 <211 LENGTH 10 <212> TYPE PRT <213> ORGANISM: homo sapiens <22O > FEATURE <221 NAME/KEY: MISC FEATURE LOCATION: (2) ... (2) US 6,592,865 B2 137 138
-continued OTHER INFORMATION: X equals any amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (4) . . (5) OTHER INFORMATION: X equals any amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (7) . . (8) OTHER INFORMATION: X equals any amino acid <400 SEQUENCE: 1 Glx Xaa Ala Xaa Xaa Cys Xaa Xaa Phe Glx 1 5 10
SEQ ID NO 2 LENGTH 7 TYPE PRT ORGANISM: homo sapiens FEATURE: NAME/KEY: MISC FEATURE LOCATION: (2) ... (2) OTHER INFORMATION: X equals any amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (4) . . (6) OTHER INFORMATION: X equals any amino acid <400 SEQUENCE: 2 Glx Xaa Cys Xaa Xaa Xaa Glx 1 5
SEQ ID NO 3 LENGTH 17 TYPE PRT ORGANISM: homo sapiens FEATURE: NAME/KEY: MISC FEATURE LOCATION: (2) ... (4) OTHER INFORMATION: X equals any amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (6) . . (15) OTHER INFORMATION: X equals any amino acid <400 SEQUENCE: 3 Glx Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 15
Glx
SEQ ID NO 4 LENGTH 11 TYPE PRT ORGANISM: homo sapiens FEATURE: NAME/KEY: MISC FEATURE LOCATION: (3) . . (6) OTHER INFORMATION: X equals any amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (9) ... (9) OTHER INFORMATION: X equals any amino acid <400 SEQUENCE: 4 Glx Arg Xaa Xala Xala Xaa Asp Ser Xaa Cys Glx 1 5 10
SEQ ID NO 5 LENGTH 9 TYPE PRT ORGANISM: homo sapiens US 6,592,865 B2 139 140
-continued
FEATURE: NAME/KEY: MISC FEATURE LOCATION: (3) . . (5) OTHER INFORMATION: X equals any amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (8) ... (8) OTHER INFORMATION: X equals any amino acid <400 SEQUENCE: 5 Glx Cys Xaa Xala Xaa Asp Cys Xaa Glx 1 5
SEQ ID NO 6 LENGTH 7 TYPE PRT ORGANISM: homo sapiens FEATURE: NAME/KEY: MISC FEATURE LOCATION: (4) ... (4) OTHER INFORMATION: X equals any amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (6) . . (6) OTHER INFORMATION: X equals any amino acid <400 SEQUENCE: 6 Glx Cys Phe Xaa Trp Xaa Glx 1 5
SEQ ID NO 7 LENGTH 13 TYPE PRT ORGANISM: homo sapiens FEATURE NAME/KEY: MISC FEATURE LOCATION: (2) ... (2) OTHER INFORMATION: X equals any amino acid FEATURE NAME/KEY: MISC FEATURE LOCATION: (4) . . (4) OTHER INFORMATION: X equals any amino acid FEATURE NAME/KEY: MISC FEATURE LOCATION: (7) . . (8) OTHER INFORMATION: X equals any amino acid FEATURE NAME/KEY: MISC FEATURE LOCATION: (10 ) . . (11) OTHER INFORMATION: X equals any amino acid <400 SEQUENCE: 7 Glx Xaa Glu Xaa Cys His Xaa Xaa Pro Xaa Xaa Cys Glx 1 5 10
SEQ ID NO 8 LENGTH 15 TYPE PRT ORGANISM: homo sapiens FEATURE: NAME/KEY: MISC FEATURE LOCATION: (9) . . (10) OTHER INFORMATION: X equals any amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (13) . . (13) OTHER INFORMATION: X equals any amino acid <400 SEQUENCE: 8 Glx Lys Glu Cys Lys Phe Gly Tyr Xaa Xala Cys Lieu Xaa Trp Glx 1 5 10 US 6,592,865 B2 141 142
-continued
<210 SEQ ID NO 9 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens &220s FEATURE <221 NAME/KEY: MISC FEATURE <222> LOCATION: (2) ... (3) <223> OTHER INFORMATION: X equals any amino acid &220s FEATURE <221 NAME/KEY: MISC FEATURE <222> LOCATION: (5) . . (6) <223> OTHER INFORMATION: X equals any amino acid &220s FEATURE <221 NAME/KEY: MISC FEATURE <222> LOCATION: (8) ... (9) <223> OTHER INFORMATION: X equals any amino acid <400 SEQUENCE: 9 Glx Xaa Xaa Cys Xaa Xaa Trp Xaa Xaa Pro Cys Glx 1 5 10
<210> SEQ ID NO 10 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens &220s FEATURE <221 NAME/KEY: MISC FEATURE <222> LOCATION: (3) . . (5) <223> OTHER INFORMATION: X equals any amino acid &220s FEATURE <221 NAME/KEY: MISC FEATURE <222> LOCATION: (7) . . (8) <223> OTHER INFORMATION: X equals any amino acid &220s FEATURE <221> NAME/KEY: MISC FEATURE <222> LOCATION: (11) . . (12) <223> OTHER INFORMATION: X equals any amino acid <400 SEQUENCE: 10 Glx Cys Xaa Xaa Xaa Arg Xaa Xaa Pro Trp Xaa Xaa Cys Glx 1 5 10
<210> SEQ ID NO 11 <211& LENGTH 21 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 11 Gly Ser Asn Arg Glu Cys His Ala Lieu Phe Cys Met Asp Phe Ala Pro 1 5 10 15 Gly Glu Gly Gly Gly 2O
<210> SEQ ID NO 12 <211& LENGTH 21 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 12 Gly Ser Ser Pro Thr Cys Arg Ala Leu Phe Cys Val Asp Phe Ala Pro 1 5 10 15 Gly Glu Gly Gly Gly 2O
<210> SEQ ID NO 13 <211& LENGTH 21 &212> TYPE PRT <213> ORGANISM: homo sapiens US 6,592,865 B2 143 144
-continued
<400 SEQUENCE: 13 Gly Ser Leu Glu Met Cys Glu Ala Leu Phe Cys Val Glu Phe Ala Pro 1 5 10 15 Gly Glu Gly Gly Gly 2O
<210> SEQ ID NO 14 <211& LENGTH 21 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 14 Gly Ser Asp Glin Asn Cys Phe Ala Met Tyr Cys Phe Glu Phe Ala Pro 1 5 10 15 Gly Glu Gly Gly Gly 2O
<210 SEQ ID NO 15 <211& LENGTH 22 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 15 Ala Gly Glu Gly Asn Cys Phe Leu Ile Gly Pro Trp Cys Phe Glu Phe 1 5 10 15 Gly Thr Glu Gly Gly Gly 2O
<210> SEQ ID NO 16 <211& LENGTH 21 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 16 Gly Ser Asp Glin Asn Cys Phe Ala Met Tyr Cys Phe Glu Phe Ala Pro 1 5 10 15 Gly Glu Gly Gly Gly 2O
<210 SEQ ID NO 17 <211& LENGTH 22 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 17 Ala Gly Glu Gly Asn Cys Phe Leu Ile Gly Pro Trp Cys Phe Glu Phe 1 5 10 15 Gly Thr Glu Gly Gly Gly 2O
<210> SEQ ID NO 18 <211& LENGTH 24 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 18 Gly Ser Asn Asp Tyr Cys Thr Val Phe Thr Gly Ala Leu Phe Cys Leu 1 5 10 15 Asp Phe Ala Pro Glu Gly Gly Gly 2O US 6,592,865 B2 145 146
-continued
<210 SEQ ID NO 19 <211& LENGTH 24 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 19 Gly Ser Tyr Asp Asn. Cys Lieu Gly Lieu Ala Asn Lieu. Asn. Phe Cys Phe 1 5 10 15 Asp Phe Ala Pro Glu Gly Gly Gly 2O
<210> SEQ ID NO 20 &2 11s LENGTH 26 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 20 Gly Asp Asp Asp His Cys Glu Trp Ala Ser Tyr Trp Lys Trp Asp Lieu 1 5 10 15 Cys Lieu. His Asp Asp Pro Glu Gly Gly Gly 2O 25
<210> SEQ ID NO 21 &2 11s LENGTH 26 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 21 Gly Asp Asp Asp Asp Cys Gly Trp Ile Gly Phe Ala Asn. Phe His Lieu 1. 5 10 15 Cys Lieu. His Gly Asp Pro Glu Gly Gly Gly 2O 25
<210> SEQ ID NO 22 &2 11s LENGTH 26 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 22 Gly Asp Pro Phe Glu Cys Asp Trp Gly Pro Trp Thr Leu Glu Met Leu 1 5 10 15 Cys Gly Pro Pro Asp Pro Glu Gly Gly Gly 2O 25
<210> SEQ ID NO 23 &2 11s LENGTH 26 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 23 Gly Asp Arg Lieu. His Cys Lys Pro Glin Arg Glin Ser Pro Trp Met Lys 1 5 10 15 Cys Gln His Lieu. Asp Pro Glu Gly Gly Gly 2O 25
<210> SEQ ID NO 24 &2 11s LENGTH 26 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 24 Gly Asp Lieu. His Ala Cys Arg Pro Val Arg Gly Asp Pro Trp Trp Ala US 6,592,865 B2 147 148
-continued
1 5 10 15 Cys Thr Lieu Gly Asp Pro Glu Gly Gly Gly 2O 25
<210> SEQ ID NO 25 &2 11s LENGTH 25 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 25 Gly Ser Pro Asn. Glin Cys Gly Val Asp Ile Trp Ala Lieu Phe Cys Wal 1 5 10 15 Asp Phe Ala Pro Glu Gly Gly Gly Lys 2O 25
<210> SEQ ID NO 26 &2 11s LENGTH 25 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 26 Gly Ser Pro Asn. Glin Cys Gly Val Asp Ile Trp Ala Lieu Phe Cys Wal 1 5 10 15 Asp Phe Ala Pro Glu Gly Gly Gly Lys 2O 25
<210 SEQ ID NO 27 <211& LENGTH 21 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 27 Gly Ser Arg Ile Gly Cys Arg Asp Ser Arg Cys Asn Trp Trp Ala Pro 1 5 10 15 Gly Glu Gly Gly Gly 2O
<210> SEQ ID NO 28 <211& LENGTH 21 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 28 Gly Ser Arg Gly Phe Cys Arg Asp Ser Ser Cys Ser Phe Pro Ala Pro 1 5 10 15 Gly Glu Gly Gly Gly 2O
<210 SEQ ID NO 29 <211& LENGTH 21 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 29 Gly Ser Trp Pro Thr Cys Leu Thr Met Asp Cys Val Tyr Asn Ala Pro 1 5 10 15 Gly Glu Gly Gly Gly 2O
<210 SEQ ID NO 30 <211& LENGTH 21 &212> TYPE PRT US 6,592,865 B2 149 150
-continued <213> ORGANISM: homo sapiens <400 SEQUENCE: 30 Ala Gly Trp Val Lieu. Cys Phe Glu Trp Glu Asp Cys Asp Glu Lys Gly 1 5 10 15 Thr Glu Gly Gly Gly 2O
<210> SEQ ID NO 31 <211& LENGTH 22 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 31 Ala Gly Val Tyr Phe Cys Phe Asp Trp Glu Glin Asp Cys Asp Glu Met 1 5 10 15 Gly Thr Glu Gly Gly Gly 2O
<210> SEQ ID NO 32 <211& LENGTH 22 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 32 Ala Gly Trp Glu Val Cys His Trp Ala Pro Met Met Cys Lys His Gly 1 5 10 15 Gly Thr Glu Gly Gly Gly 2O
<210 SEQ ID NO 33 <211& LENGTH 22 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 33 Ala Gly Glin Lys Glu Cys Lys Phe Gly Tyr Pro His Cys Lieu Pro Trp 1 5 10 15 Gly Thr Glu Gly Gly Gly 2O
<210> SEQ ID NO 34 <211& LENGTH 22 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 34 Ala Gly Ser Asp Trp Cys Gly Thir Trp Asn. Asn Pro Cys Phe His Glin 1 5 10 15 Gly Thr Glu Gly Gly Gly 2O
<210 SEQ ID NO 35 &2 11s LENGTH 27 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 35 Gly Asp Lieu. His Ala Cys Arg Pro Val Arg Gly Asp Pro Trp Trp Ala 1 5 10 15 Cys Thr Lieu Gly Asp Pro Glu Gly Gly Gly Lys 2O 25 US 6,592,865 B2 151 152
-continued
<210 SEQ ID NO 36 &2 11s LENGTH 26 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 36 Gly Asp Arg Tyr Lieu. Cys Lieu Pro Glin Arg Asp Llys Pro Trp Llys Phe 1 5 10 15 Cys Asn Trp Phe Asp Pro Glu Gly Gly Gly 2O 25
<210 SEQ ID NO 37 &2 11s LENGTH 26 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 37 Gly Asp Tyr Ser His Cys Ser Pro Leu Arg Tyr Tyr Pro Trp Trp Lys 1 5 10 15 Cys Thr Tyr Pro Asp Pro Glu Gly Gly Gly 2O 25
<210 SEQ ID NO 38 &2 11s LENGTH 26 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 38 Gly Asp Gly Phe Thr Cys Ser Pro Ile Arg Met Phe Pro Trp Phe Arg 1 5 10 15 Cys Asp Leu Gly Asp Pro Glu Gly Gly Gly 2O 25
<210 SEQ ID NO 39 &2 11s LENGTH 26 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 39 Gly Asp Phe Ser Pro Cys Lys Ala Lieu Arg His Ser Pro Trp Trp Val 1 5 10 15 Cys Pro Ser Gly Asp Pro Glu Gly Gly Gly 2O 25
<210> SEQ ID NO 40 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 40 Asn Arg Glu Cys His Ala Leu Phe Cys Met Asp Phe 1 5 10
<210> SEQ ID NO 41 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 41 Ser Pro Thr Cys Arg Ala Leu Phe Cys Val Asp Phe 1 5 10 US 6,592,865 B2 153 154
-continued
<210> SEQ ID NO 42 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 42 Ser Glu Asn. Cys Glin Ala Lieu Phe Cys Val Asp Phe 1 5 10
<210> SEQ ID NO 43 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 43 Ser Pro Thr Cys Arg Ala Leu Phe Cys Val Asp Phe 1 5 10
<210> SEQ ID NO 44 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 44 Leu Glu Met Cys Glu Ala Leu Phe Cys Val Glu Phe 1 5 10
<210> SEQ ID NO 45 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 45 Asn Pro Glu Cys Gly Ala Leu Phe Cys Met Glu Phe 1 5 10
<210> SEQ ID NO 46 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 46 Asp Phe Gly Cys Asn Ala Met Phe Cys Val Glu Phe 1 5 10
<210> SEQ ID NO 47 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 47 Asp Glin Asn Cys Phe Ala Met Tyr Cys Phe Glu Phe 1 5 10
<210> SEQ ID NO 48 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 48 Asn Asp Tyr Cys Thr Val Phe Thr Gly Ala Leu Phe Cys Leu Asp Phe 1 5 10 15
<210 SEQ ID NO 49 US 6,592,865 B2 15S 156
-continued
&2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 49 Pro Asn Glin Cys Gly Val Asp Ile Trp Ala Lieu Phe Cys Wall Asp Phe 1 5 10 15
<210 SEQ ID NO 50 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 50 Glu Gly Asn Cys Phe Leu Ile Gly Pro Trp Cys Phe Glu Phe 1 5 10
<210 SEQ ID NO 51 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 51 Glu Gly Asn Cys Phe Leu Ile Gly Pro Trp Cys Phe Glu Phe 1 5 10
<210> SEQ ID NO 52 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400s. SEQUENCE: 52 His Ile Glu Cys Glu Glu Trp Gly Tyr Trp Cys Ile Glu Met 1 5 10
<210 SEQ ID NO 53 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 53 Trp Glu Asp Cys Leu Trp Ile Gly Met Met Cys Val Glu Phe 1 5 10
<210> SEQ ID NO 54 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 54 Tyr Glu Asp Cys Ile Gly. His Ala Leu Phe Cys Met Thr Phe 1 5 10
<210 SEQ ID NO 55 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 55 Asp Asp Lys Cys Phe Gly Trp Ala His Phe Cys Phe Asp Phe 1 5 10
<210 SEQ ID NO 56 <211& LENGTH: 14 &212> TYPE PRT US 6,592,865 B2 157 158
-continued <213> ORGANISM: homo sapiens <400 SEQUENCE: 56 Gly Gly Glin Cys Gly Thr Ser Tyr Leu Phe Cys Ile Asp Phe 1 5 10
<210 SEQ ID NO 57 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 57 Tyr Ser Gly Cys Ala Asp Met Tyr Met Phe Cys Ile Asp Phe 1 5 10
<210 SEQ ID NO 58 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 58 Gly Gly Glin Cys Gly Thr Ser Tyr Leu Phe Cys Ile Asp Phe 1 5 10
<210 SEQ ID NO 59 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 59 Lys Phe Glu Cys Met Pro Ser Ser Leu Phe Cys Val Asp Phe 1 5 10
<210 SEQ ID NO 60 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 60 Asp Asp Tyr Cys Phe Asn Ile Ser Ser Tyr Ser Tyr Cys Phe Asp Phe 1 5 10 15
<210> SEQ ID NO 61 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 61 Lieu. His Asp Cys Phe Ile Tyr Ala Asp Tyr Glu Tyr Cys Phe Asp Phe 1 5 10 15
<210> SEQ ID NO 62 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 62 Asn His His Cys Leu Glu Phe Ser Ser Phe Glu Tyr Cys Phe Asp Phe 1 5 10 15
<210 SEQ ID NO 63 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens US 6,592,865 B2 159 160
-continued <400 SEQUENCE: 63 Asp Asn Lieu. Cys Met Ser Gly Gly Ser Phe Asp Tyr Cys Phe Asp Phe 1 5 10 15
<210> SEQ ID NO 64 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 64 Ser Asp Tyr Cys Val Gly Asn Asn Ala Val Thr Tyr Cys Phe Asp Phe 1 5 10 15
<210 SEQ ID NO 65 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 65 Asn Lieu. Asp Cys Ile Tyr Lieu Glin Asn His Ser Tyr Cys Phe Asp Phe 1 5 10 15
<210 SEQ ID NO 66 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 66 Asp Asp Asp Cys Met Met Leu Pro Leu Thr Met Phe Cys Phe Asp Phe 1 5 10 15
<210 SEQ ID NO 67 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 67 Tyr Asp Asn. Cys Lieu Gly Lieu Ala Asn Lieu. Asn. Phe Cys Phe Asp Phe 1 5 10 15
<210 SEQ ID NO 68 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 68 His Lieu. Asp Cys Tyr Asn Lieu Val Asp Asn Met Phe Cys Phe Asp Phe 1 5 10 15
<210 SEQ ID NO 69 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 69 Asn Trp Asn. Cys Lieu Gly Thr Asn. Glu Lieu Glin Phe Cys Lieu. Asp Phe 1 5 10 15
<210 SEQ ID NO 70 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 70 US 6,592,865 B2 161 162
-continued Tyr Phe Ala Cys Thr Asn. Asn Asp Ser Tyr Lieu Phe Cys Lieu. Asp Phe 1 5 10 15
<210 SEQ ID NO 71 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 71 Tyr Asn. Phe Cys Met Lieu. Ile Gly Glu Arg Asp Tyr Cys Lieu. Asp Phe 1 5 10 15
<210 SEQ ID NO 72 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 72 Asp Asp Val Cys Tyr Ser Lieu. Ile Met Ala Asp Tyr Cys Lieu. Asp Phe 1 5 10 15
<210 SEQ ID NO 73 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 73 Tyr Phe Ala Cys Thr Asn. Asn Asp Ser Tyr Lieu Phe Cys Lieu. Asp Phe 1 5 10 15
<210> SEQ ID NO 74 &2 11s LENGTH 17 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 74 Asp Asp Met Cys Arg Trp Tyr Pro Phe Ala Ser Phe Tyr Met Cys Leu 1 5 10 15
Phe
<210 SEQ ID NO 75 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 75 Asp Asp His Cys Glu Trp Ala Ser Tyr Trp Llys Trp Asp Lieu. Cys Lieu 1 5 10 15 His Asp
<210 SEQ ID NO 76 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 76 Asp Asp Val Cys Glu Asn Ala Asp Phe Ala Trp Lieu Gly Trp Cys Met 1 5 10 15
His Phe
<210 SEQ ID NO 77 &2 11s LENGTH 18 &212> TYPE PRT US 6,592,865 B2 163 164
-continued <213> ORGANISM: homo sapiens <400 SEQUENCE: 77 Asp Asp Asp Cys Gly Trp Ile Gly Phe Ala Asn. Phe His Lieu. Cys Lieu 1 5 10 15 His Gly
<210 SEQ ID NO 78 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 78 Phe Asp Asp Cys Gln Thr Ser Trp Phe Glin Gly Phe Trp Leu Cys Ile 1 5 10 15 Asp Asp
<210 SEQ ID NO 79 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 79 Phe His Asp Cys Ser Trp Gly Pro Trp Gly Pro Trp Glu Ile Cys Thr 1 5 10 15 Arg Lieu
<210 SEQ ID NO 80 &211's LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 80 Ser Asn Asp Cys Val Trp Lieu Glin Phe Trp Gly Gly Asp Met Cys Phe 1 5 10 15
Leu Pro
<210> SEQ ID NO 81 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 81 Asn Ala Asp Cys Glu Trp Val Asn. Phe Asn His Val Asp Lieu. Cys Met 1 5 10 15 Trp Asn
<210> SEQ ID NO 82 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 82 Gly Ser Asp Cys Glu Trp Val Asin Phe Thr Met Phe Gln Met Cys Ile 1 5 10 15
Ser Asn
<210 SEQ ID NO 83 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens US 6,592,865 B2 165 166
-continued
<400 SEQUENCE: 83 Ala Trp Asp Cys Glu Trp Asn Leu Phe Asp Ser Thr Phe Phe Cys Pro 1 5 10 15 Gly Phe
<210> SEQ ID NO 84 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 84 Leu Tyr Glu Cys Glu Trp Lys Glin Phe Gly Pro Val Glu Met Cys Leu 1 5 10 15
Asn Phe
<210 SEQ ID NO 85 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 85 His Ser Glu Cys Arg Trp Glu Trp Phe Gly Arg Thr Met Ile Cys Met 1 5 10 15
Ser Phe
<210 SEQ ID NO 86 &2 11s LENGTH 18 212s. TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 86 Ser Gly Glu Cys Asn Trp Glin Glin Phe Ser Gly Trp Glu Ile Cys Lieu 1 5 10 15 Arg Asp
<210 SEQ ID NO 87 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 87 Ala Tyr Lieu. Cys Asp Trp Ile Leu Phe Asp Ser Phe Glu Met Cys Lieu 1 5 10 15
Ala Pro
<210 SEQ ID NO 88 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 88 Pro Phe Glu Cys Asp Trp Gly Pro Trp Thr Leu Glu Met Leu Cys Gly 1 5 10 15
Pro Pro
<210 SEQ ID NO 89 &2 11s LENGTH 15 &212> TYPE PRT <213> ORGANISM: homo sapiens US 6,592,865 B2 167 168
-continued <400 SEQUENCE: 89 Arg Gly His Cys Arg Asp Ser Arg Cys Met Met Asn Ala Pro Gly 1 5 10 15
<210 SEQ ID NO 90 &2 11s LENGTH 15 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 90 Arg Ile Gly Cys Arg Asp Ser Arg Cys Asn Trp Trp Ala Pro Gly 1 5 10 15
<210 SEQ ID NO 91 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 91 Arg Gly Phe Cys Arg Asp Ser Ser Cys Ser Phe Pro 1 5 10
<210 SEQ ID NO 92 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 92 Arg Gly Trp Cys Lieu. Asp Ser Arg Cys Lys Val Phe 1 5 10
<210 SEQ ID NO 93 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 93 Phe Leu Phe Cys Arg Lieu Ala Ser Arg Asp Ser Arg Cys Ala Ser Pro 1 5 10 15
<210 SEQ ID NO 94 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 94 Phe Asn Pro Cys Arg Lieu Glin Ser Arg Asp Ser Ala Cys Arg Phe Arg 1 5 10 15
<210 SEQ ID NO 95 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 95 Phe Phe Pro Cys Arg Ala Leu Glu Lys Asp Ser Arg Cys Ser Phe Phe 1 5 10 15
<210 SEQ ID NO 96 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 96 US 6,592,865 B2 169 170
-continued His Phe Ser Cys Arg Lieu Pro Ser Lieu. Asp Ser Arg Cys Glin Leu Trp 1 5 10 15
<210 SEQ ID NO 97 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 97 Asn Asp Val Cys Lieu. Asn Asp Asp Cys Val Tyr Gly 1 5 10
<210 SEQ ID NO 98 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 98 Trp Pro Thr Cys Leu Thr Met Asp Cys Val Tyr Asn 1 5 10
<210 SEQ ID NO 99 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 99 His Tyr Asn. Cys His Thr Asn Asp Cys Val Val Lieu 1 5 10
<210> SEQ ID NO 100 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 100 His Leu Arg Cys Met Thr Ser Asp Cys Ile His Phe 1 5 10
<210> SEQ ID NO 101 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 101 Trp Val Lieu. Cys Phe Glu Trp Glu Asp Cys Asp Glu Lys 1 5 10
<210> SEQ ID NO 102 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 102 Tyr Glu Tyr Cys Phe Glu Trp Glu Gln Cys Trp Glu Lys 1 5 10
<210> SEQ ID NO 103 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 103 Gly Ile Phe Cys Phe Glu Trp Glu Thr Cys Tyr Glin Ala 1 5 10 US 6,592,865 B2 171 172
-continued
<210> SEQ ID NO 104 <211& LENGTH: 11 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 104 Pro Glin Phe Cys Phe Glu Trp Glu Pro Cys Phe 1 5 10
<210 SEQ ID NO 105 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 105 Ile Gly Phe Cys Phe Glu Trp Glu Val Cys Tyr Glu Gly 1 5 10
<210> SEQ ID NO 106 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 106 Ser Ile Tyr Cys Phe Asp Trp Glu Asp Cys Trp Asp Glu 1 5 10
<210 SEQ ID NO 107 &2 11s LENGTH 13 212s. TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 107 Tyr Asp Trp Cys Phe Asp Trp Glu Gln Cys Trp Asp Glin 1 5 10
<210 SEQ ID NO 108 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 108 Val Gly Phe Cys Phe Asp Trp Glu Pro Cys Asp Glu Lieu 1 5 10
<210 SEQ ID NO 109 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 109 Met Asp Phe Cys Phe Asp Trp Glu Glu Cys Trp Thr Asn 1 5 10
<210> SEQ ID NO 110 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 110 Asn Ile Phe Cys Phe Asp Trp Glu Pro Cys His Phe Gly 1 5 10 US 6,592,865 B2 173 174
-continued <210> SEQ ID NO 111 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 111 Phe Glu Ile Cys Phe Asp Trp Glu Val Cys His Glu Gln 1 5 10
<210> SEQ ID NO 112 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 112 Asp Tyr Lieu. Cys Phe Asp Trp Glu Ala Cys Trp Leu Ser 1 5 10
<210> SEQ ID NO 113 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 113 Tyr Ala Met Cys Phe Asp Trp Asp Glu Cys Phe Leu Gly 1 5 10
<210> SEQ ID NO 114 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens &220s. FEATURE <221 NAME/KEY: MISC FEATURE <222> LOCATION: (2) ... (2) <223> OTHER INFORMATION: X equals any amino acid <400 SEQUENCE: 114 Trp Xaa Trp Cys Phe Glu Trp Glu Asp Trp Cys Lieu Val Glu 1 5 10
<210> SEQ ID NO 115 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 115 Tyr Glin Phe Cys Phe Asp Trp Glu Thir Thr Cys Trp Leu Asp 1 5 10
<210> SEQ ID NO 116 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 116 Val Tyr Phe Cys Phe Asp Trp Glu Glin Asp Cys Asp Glu Met 1 5 10
<210> SEQ ID NO 117 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 117 Phe Glin Lieu. Cys Phe Asp Trp Glu Glu Glu Cys Glu Glu Ser 1 5 10 US 6,592,865 B2 175 176
-continued
<210> SEQ ID NO 118 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 118 Trp Ala Val Cys Phe Asp Trp Glu Asn. Cys Gly Asp Lys 1 5 10
<210 SEQ ID NO 119 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 119 Trp Glin Phe Cys Phe Asp Trp Asp Lieu. Asn. Cys Asp Lieu Arg 1 5 10
<210> SEQ ID NO 120 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 120 Tyr Trp Phe Cys Phe Asp Trp Glu Glu Asp Ala Asn Gly His Cys Gly 1 5 10 15 Gly Asn
<210> SEQ ID NO 121 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 121 Phe Lieu Lieu. Cys Phe Asp Trp Asp Ile Asp Trp Glu Tyr Gly Cys Glin 1 5 10 15
His His
<210> SEQ ID NO 122 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 122 Tyr Glu Glu Cys His Trp Arg Pro Met Ala Cys Ser Thr His 1 5 10
<210> SEQ ID NO 123 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 123 Trp Glu Val Cys His Trp Ala Pro Met Met Cys Lys His Gly 1 5 10
<210> SEQ ID NO 124 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 124 US 6,592,865 B2 177 178
-continued Tyr Glu Phe Cys His Tyr Ala Pro Glin Glu Cys Lys His Met 1 5 10
<210> SEQ ID NO 125 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens &220s FEATURE <221 NAME/KEY: MISC FEATURE <222> LOCATION: (1) . . (1) <223> OTHER INFORMATION: X equals any amino acid &220s FEATURE <221 NAME/KEY: MISC FEATURE <222> LOCATION: (10) . . (10) <223> OTHER INFORMATION: X equals any amino acid <400 SEQUENCE: 125 Xaa Lys Glu Cys Lys Phe Gly Tyr Ser Xaa Cys Lieu Ala Trp 1 5 10
<210> SEQ ID NO 126 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 126 Glin Lys Glu Cys Lys Phe Gly Tyr Pro His Cys Lieu Pro Trp 1 5 10
<210> SEQ ID NO 127 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 127 Glu His Asn Cys Thr Trp Trp Asin Pro Cys Trp Thr Thr 1 5 10
<210> SEQ ID NO 128 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 128 Met Asp His Cys Thr Trp Tyr Glin Pro Cys Val Leu Lys 1 5 10
<210> SEQ ID NO 129 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 129 Trp Asp His Cys Asn Trp Ala His Pro Cys Ser Arg Lys 1 5 10
<210> SEQ ID NO 130 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 130 Ser Asp Trp Cys Gly. Thir Trp Asn Asn Pro Cys Phe His Glin 1 5 10
<210> SEQ ID NO 131 US 6,592,865 B2 179 18O
-continued
&2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 131 Arg Tyr Lieu. Cys Lieu Pro Glin Arg Asp Llys Pro Trp Llys Phe Cys Asn 1 5 10 15 Trp Phe
<210> SEQ ID NO 132 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 132 Arg Lieu. His Cys Lys Pro Glin Arg Glin Ser Pro Trp Met Lys Cys Glin 1 5 10 15
His Lieu
<210> SEQ ID NO 133 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 133 Tyr Ser His Cys Ser Pro Leu Arg Tyr Tyr Pro Trp Trp Lys Cys Thr 1 5 10 15
Tyr Pro
<210> SEQ ID NO 134 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 134 Lieu. His Ala Cys Arg Pro Val Arg Gly Asp Pro Trp Trp Ala Cys Thr 1 5 10 15 Leu Gly
<210 SEQ ID NO 135 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 135 Gly Phe Thr Cys Ser Pro Ile Arg Met Phe Pro Trp Phe Arg Cys Asp 1 5 10 15 Leu Gly
<210> SEQ ID NO 136 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 136 Phe Ser Pro Cys Lys Ala Leu Arg His Ser Pro Trp Trp Val Cys Pro 1 5 10 15 Ser Gly
<210 SEQ ID NO 137 &2 11s LENGTH 2920 US 6,592,865 B2 181 182
-continued
DNA RGANISM: homo sapiens &220s FEATURE EAK EY: misc feature ATION: (1707) . . (1707) R INFORMATION n ecua any amino acid misc feature (2702). ... (2702) ORMATION: n ecua any amino acid misc feature (27.49). . (2749) ORMATION: n ecua any amino acid misc feature (2757) . . (2757 ) ORMATION: n ecua any amino acid misc feature (2788) . . (2789) O RMATION: n ecua any amino acid misc feature 9) . . (2819) ORMATION: n ecua any amino acid misc feature (2835) . . (2835) O RMATION: n ecua any amino acid &220s FEATURE <221> NAME misc feature <222> LOCATION (2856) . . (2856) &223> OTHE ORMATION: n ecua any amino acid <400> SEQU ENCE 137 gtggatgttga tottggctcc cc.ggggacga tgtcagotct toctggctoc ttctoagcct 60 tgttgctgta actgctgcto agtccaccat tgaggalacag gccaaga cat ttittgggaca 120 agtttaacca cgaag.ccgaa gacctgttct atcaaagttc acttgcttct tggaattata 18O acaccalatat tactgaagag aatgtccaaa acatgaataa tgctggggac aaatggtotg 240 ccitttittaala ggaacagticc acacttgccc aaatgitatcc actacaagaa attcagaatc to acagtcaa gcttcagotg caggctottc agcaaaatgg gtottcagtg citcto agaag 360 acaagagcaa. acggttgaac acaattictaa atacaatgag caccatctac agtactggaa 420 aagtttgtaa cccagataat ccacaagaat gcttattact tgaaccaggt ttgaatgaaa 480 taatggcaaa cagtttagac tacaatgaga ggctctgggC ttgggaaag.c tggagatctg 540 aggtoggcaa. gCagct gagg ccattatatg aagagtatgt ggtottgaaa aatgagatgg 600 caagagcaaa to attatgag gacitatgggg attattggag aggagacitat gaagtaaatg 660 gggtagatgg citatgacitac agcc.gcggCC agttgattga agatgtggaa catacctittg 720 aagagattaa accattatat gaacatctitc atgccitatgt gaggccaaag ttgatgaatg ccitatic ctitc. citatatoagt cca attggat gccitccctg.c toatttgctt ggtgatatgt 840 ggggtagatt ttggacaaat ytgtacwstt tgacagttcc citttggacag a.a.a. C.C. aaa.ca 9 OO tagatgttac tgatgcaatg gtggacCagr cctgg gatgc acagagaata ttcaaggagg 96.O cc.gagaagtt citttgttatct gttggtottc ctaatatgac tdaaggattic tgggaaaatt 1020 ccatgctaac ggaccCagga aatgttcaga aag cagtctg ccatcc.caca gcttggg acc 1080 tggggaaggg cg actticagg atcctitatgt gcacaaaggit gacaatggac gactitcc toga 1140 cagotcatca tgagatgggg catatocagt atgatatggc atatgctgca caaccttitt.c 1200 tgctaagaaa tggagctaat gaaggatticc atgaagctdt tggggaaatc atgtcactitt 1260 US 6,592,865 B2 183 184
-continued citgcagocac acctaag cat ttaaaatcca ttggtottct gtcacco gat tittcaagaag 320 acaatgaaac agaaataaac titcctgctica aacaag cact cacgattgtt goggacitctgc catttacitta catgttagag aagtggaggt ggatggtott taaaggggala attcc caaag 4 40 accagtggat gaaaaagtgg togggagatga agc gagagat agttggggtg gtggaac Ctg 5 OO tg.ccccatga tigaaacatac tatgaccocg catctotgtt coatgtttct aatgattact 560 cattcattcg atattacaca agg accottt accaatticca gtttcaagaa goactttgtc aag cagctaa acatgaaggc cct citgcaca aatgtgacat citcaaactot acagaagctg 680 gacagaaact gttcaatatgctgaggnittg gaaaatcaga accotgg acc ctago attgg 740 aaaatgttgt aggagcaaag alacatgaatg taaggcc act gctcaactac tittgagcc.ct 800 tatttacct g gctgaaagac cagaacaaga attcttttgt gggatggagt accgactgga 860 gtoccatatgc agaccaaag.c atcaaagtga ggataagcct aaaatcagot cittggagata 920 aag catatga atggaacgac aatgaaatgt acctgttccg atcatctgtt gcatatgcta tgaggcagta citttittaaaa gtaaaaaatc agatgattot ttittggggag gaggatgtgc 20 40 gagtggctaa tittgaaacca agaatctoct ttaatttctt totcactg.ca cctaaaaatg 2100 tgtctgatat cattcc taga actgaagttgaaaaggc.cat caggatgtcc cqgagcc.gta 216 O tdaatgatgc titt.ccgtotg aatgacgaca gcc tagagtt totggggata cagocaa.cac 2220 ttgg acctico talaccagocc cct gtttcca tatggctgat tdtttittgga gttgttgatgg 228O gagtgatagt ggttgg catt gtcatcct ga tottcactgg gatcagagat cqgaagaagg 234. O gcctgtaaat ggaatticcitg cattgctota accatgtaca accittgg act tagcttttac 24 OO citgtaactgg cittctgagag acaaagagga gaalacct tca citcc tagtac accitattaca 2460 gctgcagagg tagaggagac agttgcagaa citagttacaa toacgataag aaacaatact 252O ttgttattoc atagdaccitt taatgttcat gtgitattatc toagctagoc titgaaccgcc 258O taagta aggt gatgaggacg ggtttaa.gcc ccactgatat tittaaaagcc cagagaaaag 264 O tgttcgttcc totact aacc togttcttitta gag cagg gat citgcatact a ggcctgcago 27 OO cnaaatgagt aggtag ccca citacctattt ttgtatagoc agagggctna gaatggnttt 276 O. tacattittaa gtggttttac atttalagninc aaaagaagga taatatttca to acaagtna 282O aaattatatgaactnaaaat totatgaatt titatignattt atatttcagt attcataatt 2880 aaagttitt at tdaactacaa aaaaaaaaaa aaaaaaaaaa 2920
SEQ ID NO 138 LENGTH 711 TYPE PRT ORGANISM: homo sapiens FEATURE: NAME/KEY: MISC FEATURE LOCATION: (219) . . (219) OTHER INFORMATION Xaa equals any amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (240) . . (240) OTHER INFORMATION Xaa equals any amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (499).. (499) OTHER INFORMATION Xaa equals any amino acid <400 SEQUENCE: 138 Met Asn. Asn Ala Gly Asp Lys Trp Ser Ala Phe Lieu Lys Glu Glin Ser 1 5 10 15 US 6,592,865 B2 18S 186
-continued
Thr Telu Ala Glin Met Pro Telu Glin Glu Ile Glin Asn Lieu. Thr Wall 25 30
Telu Glin Telu Glin Ala Teu Glin Glin Asn Gly Ser Ser Wall Telu Ser 35 40 45
Glu Asp Ser Arg Teu Asn Thr Ile Teu Asn Thr Met Ser Thr 50 55 60
Ile Ser Thr Gly Lys Wall Asn Pro Asp Asn Pro Glin Glu Cys 65 70 75
Teu Telu Telu Glu Pro Gly Teu Asn Glu Ile Met Ala Asn Ser Telu Asp 85 90 95
Asn Glu Arg Teu Trp Ala Trp Glu Ser Trp Arg Ser Glu Wall Gly 100 105 110
Glin Telu Arg Pro Teu Glu Glu Wall Wall Teu Asn Glu 115 120 125
Met Ala Arg Ala Asn His Tyr Glu Asp Gly Asp Trp Gly 130 135 1 4 0
Asp Glu Wall Asn Gly Wall Asp Gly Tyr Asp Ser Arg Gly Glin 145 15 O 155 160
Teu Ile Glu Asp Wall Glu His Thr Phe Glu Glu Ile Pro Telu Tyr 1.65 170 175
Glu His Telu His Ala Wall Arg Pro Teu Met Asn Ala Tyr Pro 18O 185 190
Ser Ile Ser Pro Ile Gly Cys Telu Pro Ala His Teu Teu Gly Asp 195 200
Met Trp Gly Phe Trp Thr Asn Telu Xaa Teu Thr Wall Pro Phe 210 215 220
Gly Glin Pro Asn Ile Asp Wall Thr Asp Ala Met Wall Asp Glin Xaa 225 230 235 240
Trp Asp Ala Glin Arg Ile Phe Glu Ala Glu Phe Phe Wall Ser 245 250 255
Wall Gly Telu Pro Asn Met Thr Glin Gly Phe Trp Glu Asn Ser Met Telu 260 265 27 O
Thr Asp Pro Gly Asn Wall Glin Lys Ala Wall His Pro Thr Ala Trp 275 280 285
Asp Telu Gly Lys Gly Asp Phe Arg Ile Telu Met Cys Thr Wall Thr 29 O 295
Met Asp Asp Phe Teu Thr Ala His His Glu Met Gly His Glin Tyr 305 310 315 320
Asp Met Ala Ala Ala Glin Pro Phe Telu Teu Arg Asn Ala Asn 325 330 335
Glu Gly Phe His Glu Ala Wall Gly Glu Ile Met Ser Teu Ser Ala Ala 340 345 350
Thr Pro Lys His Teu Ser Ile Gly Telu Teu Ser Pro Asp Phe Glin 355 360 365
Glu Asp Asn Glu Thr Glu Ile Asn Phe Telu Teu Lys Glin Ala Telu Thr 370 375
Ile Wall Gly Thr Teu Pro Phe Thr Met Teu Glu Trp Trp 385 390 395 400
Met Wall Phe Gly Glu Ile Pro Asp Glin Trp Met Lys Trp 405 410 415
Trp Glu Met Lys Arg Glu Ile Wall Gly Wall Wall Glu Pro Wall Pro His 420 425 430
Asp Glu Thr Asp Pro Ala Ser Telu Phe His Wall Ser Asn Asp US 6,592,865 B2 187 188
-continued
435 4 40 4 45
Ser Phe Ile Arg Tyr Tyr Thr Thr Teu Tyr Glin Phe Glin Phe 450 455 460
Glin Glu Ala Telu Glin Ala Ala Lys His Glu Gly Pro Teu His Lys 465 470 475 480
Asp Ile Ser Asn Ser Thr Glu Ala Gly Glin Teu Phe Asn Met 485 490 495
Teu Xaa Gly Lys Ser Glu Pro Trp Thr Teu Ala Teu Glu Asn Wall 5 OO 505 510
Wall Gly Ala Asn Met Asn Wall Arg Pro Teu Teu Asn Phe Glu 515 525
Pro Telu Phe Thr Trp Teu Lys Asp Glin Asn Asn Ser Val Gly 530 535 540
Trp Ser Thr Asp Trp Ser Pro Ala Asp Glin Ser Ile Val Arg 545 550 555 560
Ile Ser Telu Ser Ala Teu Gly Asp Lys Ala Glu Trp Asn Asp 565 570 575
Asn Glu Met Teu Phe Arg Ser Ser Wall Ala Ala Met Arg Glin 585 59 O
Phe Telu Wall Asn Glin Met Ile Teu Phe Gly Glu Glu Asp 595 600 605
Wall Arg Wall Asn Teu Lys Pro Ile Ser Phe Asn Phe Phe Wall 610 615
Thr Ala Pro Asn Wall Ser Asp Ile Pro Arg Thr Glu Wall Glu 625 630 635 640
Ala Ile Arg Met Ser Arg Ser Ile Asn Asp Ala Phe Arg Lieu 645 650 655
Asn Asp Asp Ser Teu Glu Phe Telu Gly Ile Glin Pro Thr Teu Gly Pro 660 665 670
Pro Asn Glin Pro Pro Wall Ser Ile Trp Telu Ile Wall Phe Wal Wall 675 680 685
Met Gly Wall Ile Wall Wall Gly Ile Wall Ile Teu Ile Phe Thr Gly Ile 69 O. 695 7 OO
Arg Asp Arg Gly Teu 705 710
SEQ ID NO 139 LENGTH 17 TYPE PRT ORGANISM: homo sapiens <400 SEQUENCE: 139 Leu Ile Val Phe Gly Val Val Met Gly Val Ile Val Val Gly Ile Val 1 5 10 15
Ile
<210> SEQ ID NO 140 &2 11s LENGTH 681 &212> TYPE PRT <213> ORGANISM: homo sapiens &220s FEATURE <221 NAME/KEY: MISC FEATURE <222> LOCATION: (219) . . (219) <223> OTHER INFORMATION: Xaa equals any amino acid &220s FEATURE <221 NAME/KEY: MISC FEATURE <222> LOCATION: (240)... (240) <223> OTHER INFORMATION: Xaa equals any amino acid US 6,592,865 B2 189 190
-continued
FEATURE: NAME/KEY: MISC FEATURE LOCATION: (499).. (499) OTHER INFORMATION: Xaa equals any amino acid <400 SEQUENCE: 1 4 0
Met Asn. Asn Ala Gly Asp Trp Ser Ala Phe Teu Glu Glin Ser 1 5 10 15
Thr Telu Ala Glin Met Pro Telu Glin Glu Ile Glin Asn Teu Thr Wall 25 30
Telu Glin Telu Glin Ala Teu Glin Glin Asn Gly Ser Ser Wall Telu Ser 35 40 45
Glu Asp Ser Arg Teu Asn Thr Ile Teu Asn Thr Met Ser Thr 50 55 60
Ile Ser Thr Gly Lys Wall Asn Pro Asp Asn Pro Glin Glu Cys 65 70 75
Teu Telu Telu Glu Pro Gly Teu Asn Glu Ile Met Ala Asn Ser Telu Asp 85 90 95
Asn Glu Arg Teu Trp Ala Trp Glu Ser Trp Arg Ser Glu Wall Gly 100 105 110
Glin Telu Arg Pro Teu Glu Glu Wall Wall Teu Asn Glu 115 120 125
Met Ala Arg Ala Asn His Tyr Glu Asp Gly Asp Trp Gly 130 135 1 4 0
Asp Glu Wall Asn Gly Wall Asp Gly Asp Ser Arg Gly Glin 145 15 O 155 160
Leu Ile Glu Asp Wall Glu His Thr Phe Glu Glu Ile Pro Leu Tyr 1.65 170 175
Glu His Telu His Ala Wall Arg Pro Teu Met Asn Ala Tyr Pro 18O 185 190
Ser Ile Ser Pro Ile Gly Cys Telu Pro Ala His Teu Teu Gly Asp 195 200
Met Trp Gly Arg Phe Trp Thr Asn Telu Xaa Teu Thr Wall Pro Phe 210 215 220
Gly Glin Pro Asn Ile Asp Wall Thr Asp Ala Met Wall Asp Glin Xaa 225 230 235 240
Trp Asp Ala Glin Arg Ile Phe Glu Ala Glu Phe Phe Wall Ser 245 250 255
Wall Gly Telu Pro Asn Met Thr Glin Gly Phe Trp Glu Asn Ser Met Telu 260 265 27 O
Thr Asp Pro Gly Asn Wall Glin Lys Ala Wall His Pro Thr Ala Trp 275 280 285
Asp Telu Gly Gly Asp Phe Arg Ile Telu Met Cys Thr Wall Thr 29 O 295
Met Asp Asp Teu Thr Ala His His Glu Met Gly His Glin Tyr 305 310 315 320
Asp Met Ala Ala Ala Glin Pro Phe Telu Teu Arg Asn Ala Asn 325 330 335
Glu Gly Phe His Glu Ala Wall Gly Glu Ile Met Ser Teu Ser Ala Ala 340 345 350
Thr Pro Lys His Teu Ser Ile Gly Telu Teu Ser Pro Asp Phe Glin 355 360 365
Glu Asp Asn Glu Thr Glu Ile Asn Phe Telu Teu Lys Glin Ala Telu Thr 370 375 38O