USOO8946.156B2
(12) United States Patent (10) Patent No.: US 8,946,156 B2 Ballance et al. (45) Date of Patent: *Feb. 3, 2015
(54) ALBUMINFUSION PROTEINS 2039/54 (2013.01); C07K 2319/00 (2013.01); (75) Inventors: David J. Ballance, Berwyn, PA (US); C07K 2319/21 (2013.01); C07K 2319/31 Darrell Sleep, West Bridgford (GB); (2013.01); C07K 2319/50 (2013.01); C07K Christopher P. Prior, Rosemont, PA 2319/75 (2013.01) (US); Homayoun Sadeghi, Doylestown, USPC ...... 514/12: 514/2:530/350, 530/300 PA (US); Andrew J. Turner, King of (58) Field of Classification Search Prussia, PA (US) None (73) Assignees: Human Genome Sciences, Inc., See application file for complete search history. Rockville, MD (US); Novozymes Biopharma DK A/S (DK) (56) References Cited (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S. PATENT DOCUMENTS U.S.C. 154(b) by 55 days. 4,456,748 A 6, 1984 Goeddel 4,530,901 A 7, 1985 Weissmann This patent is Subject to a terminal dis 4,569,908 A 2f1986 Market al. claimer. 4,716,217 A 12/1987 Caruthers et al. 4,734.491 A 3, 1988 Caruthers et al. (21) Appl. No.: 13/464,248 4,758,428 A 7, 1988 Market al. (22) Filed: May 4, 2012 4,966,843. A 10, 1990 McCormicket al. (65) Prior Publication Data 5,002,764 A 3, 1991 PeetSet al. 5,326,859 A 7, 1994 Sugano et al. US 2013/0266553 A1 Oct. 10, 2013 5,376,567 A 12/1994 McCormicket al. 5,378,823. A 1/1995 Martal et al. Related U.S. Application Data 5,480,640 A 1/1996 Morales et al. (60) Division of application No. 1 1/927,602, filed on Oct. 5,514,567 A 5/1996 Sugano et al. 29, 2007, which is a division of application No. (Continued) 11/078,914, filed on Mar. 14, 2005, now Pat. No. 7,482,013, which is a continuation of application No. FOREIGN PATENT DOCUMENTS 09/832,501, filed on Apr. 12, 2001, now abandoned. CA 2O7O781 2, 1991 (60) Provisional application No. 60/229,358, filed on Apr. EP O 147 193 A2 7, 1985 12, 2000, provisional application No. 60/199,384, (Continued) filed on Apr. 25, 2000, provisional application No. OTHER PUBLICATIONS 60/256,931, filed on Dec. 21, 2000. Cohen et al. (51) Int. Cl. Abstract of Nossent et al. (2006), J. Thromb. Haem. 4:12, pp. 2556 CI2N 9/96 (2006.01) 2562. A6 IK38/48 (2006.01) Doctor, B.P. et al., “Enzymes as Pretreatment Drugs for A6 IK9/00 (2006.01) Organophosphate Toxicity.” Neuroscience & Biobehavioral Reviews A6 IK38/2I (2006.01) 15:123-128 (1991). A6 IK38/38 (2006.01) European Supplemental Search Report and Opinion for EP06813242 C07K I4/56 (2006.01) dated Jun. 19, 2009. Extract from U.S. Food and Drug Administration—derived package C07K I4/6 (2006.01) insert for Abbokinase (Urokinase), Nov. 25, 2008 http://www.fda. C07K I4/62 (2006.01) gov/cder/foi/label/2002/urokabb101002LB.htm. C07K I4/65 (2006.01) Extract from Johns Hopkins product information regarding G-CSF C07K 4/705 (2006.01) Nov. 25, 2008 http://prodhopkins-abxguide.org/antibiotics/biologi C07K I4/75 (2006.01) cal/g-csf.html?contentInstanceId--. . . . C07K 4/76 (2006.01) (Continued) C07K 4/765 (2006.01) CI2N 5/62 (2006.01) A6 IK 47/48 (2006.01) Primary Examiner — Hope Robinson A61 K 47/42 (2006.01) (74) Attorney, Agent, or Firm — Baker & Hostetler LLP A61 K 4.8/00 (2006.01) A61 K39/00 (2006.01) (57) ABSTRACT (52) U.S. Cl. The present invention encompasses albumin fusion proteins. CPC ...... A61K 38/4846 (2013.01); A61 K9/0019 Nucleic acid molecules encoding the albumin fusion proteins (2013.01); A61 K38/21 (2013.01); A61 K are also encompassed by the invention, as are vectors con taining these nucleic acids, host cells transformed with these 38/212 (2013.01); A61K38/38 (2013.01); nucleic acid vectors, and methods of making the albumin C07K 14/56 (2013.01); C07K 14/61 (2013.01); fusion proteins of the invention and using these nucleic acids, C07K 14/62 (2013.01); C07K 14/65 (2013.01); vectors, and/or host cells. Additionally the present invention C07K 14/705 (2013.01); C07K 14/7151 encompasses pharmaceutical compositions comprising albu (2013.01); C07K 14/76 (2013.01); C07K min fusion proteins and methods of treating, preventing, or 14/765 (2013.01); C12N 15/62 (2013.01); ameliorating diseases, disorders or conditions using albumin CI2N 9/96 (2013.01); A61K47/48338 fusion proteins of the invention. (2013.01); A0IK 2217/05 (2013.01); A61 K 47/42 (2013.01); A61 K48/00 (2013.01); A61 K 19 Claims, 20 Drawing Sheets US 8,946,156 B2 Page 2
(56) References Cited Osborn B.L., et al., “Pharmacokinetic and Pharmacodynamic Studies of a Human Serum Albumin-Interferon-O, Fusion Protein in U.S. PATENT DOCUMENTS Cynomolgus Monkeys.” The Journal of Pharmacology and Experi mental Therapeutics, vol. 303, No. 2, pp. 540-548 (2002). 5,540,923 A 7, 1996 Ebbesen et al. Huang Y.-J., et al., “Substantially Improved Pharmacokinetics of 5,695,750 A 12/1997 Doctor et al. Recombinant Human Butyrylcholinesterase by Fusion to Human 5,795,779 A 8, 1998 McCormicket al. Serum Albumin.” BMC Biotechnology, vol. 8, No. 50, 11 pages 5,876,969 A * 3/1999 Fleer et al...... 435/69.7 6,001,625 A 12/1999 Broomfield et al. (2008). 6,069,133 A 5, 2000 Chiou et al. Extended European Search Report mailed Oct. 15, 2010, for Euro 6,905,688 B2 6, 2005 Rosen et al. pean Application No. 08843705.8. 7,138,371 B2 11/2006 DeFrees et al. Hidaka et al., “Genetic Analysis of a Japanese Patient With 7,271,149 B2 9, 2007 Glaesner Butyrylcholinesterase Deficiency.” Ann. Hum. Genet. 61:491-496 7,482,013 B2 1/2009 Ballance et al. (1997). 7,731,957 B1 6, 2010 Zhan et al. GeneID: 590, “BCHE butyrylcholinesterase.” Entrez Gene (updated 2002/01 19489 A1 8/2002 Lockridge et al. Jan. 10, 2006). 2003/0147918 A1 8, 2003 Maertens et al. GeneID: 1110, "CHE2 cholinesterase (serum) 2. Entrez Gene 2003. O187226 A1 10/2003 Goodey et al. (updated Nov. 29, 2005). 2003. O199043 A1 10/2003 Ballance et al. NP 000046, “butyrylcholinesterase precursor Homo sapiens.” 2003/0219875 A1 11/2003 Rosen et al. 2004, OO16005 A1 1/2004 Karatzas et al. (updated Dec. 25, 2005). 2005/023.9167 A1 10, 2005 Fleer Belfiore, F. et al., "Serum Enzymes in Diabetes Mellitus.” Clin. 2005/0266533 A1 12/2005 Ballance et al. Chem. 19:447-452 (1973). 2006, OO14254 A1 1/2006 Haseltine et al. Blong, R.M. et al., “Tetramerization Domain of Human 2006/0051859 A1 3, 2006 Fu et al. Butyrylcholinesterase is at the C-Terminus.” Biochem. J. 327:747 2007/0162986 A1 7/2007 De Bold et al. 757 (1997). 2008. O194481 A1 8, 2008 Rosen et al. Brimijoin, S. et al., “Immunochemistry of Mammalian 2008/0267962 A1 10/2008 Ballance et al. Cholinesterases.” FASEB 45:2960-2964 (1986). 2009 OO29914 A1 1/2009 Rosen et al. Brimijoin, S. et al., “Immunology and Molecular Biology of the 2011/0002888 A1 1/2011 Rosen et al. Cholinesterases: Current Results and Prospects.” International Review of Neurobiology 28:363-410 (1986). FOREIGN PATENT DOCUMENTS Das, P.K. et al., “On Genetically Determined Human Serum Cholinesterases.” Enzyme 21:253-274 (1976). EP O 130967 A1 9, 1985 Delbruck, A. et al., “A Rare Genetically Determined Variant of EP O 215 658 A2 3, 1987 EP O 591 524 4f1994 Pseudocholinesterase in Two German Families With High Plasma EP 1832 599 A2 9, 2007 Enzyme Activity.” Eur: J. Biochem.99:65-69 (1979). 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OTHER PUBLICATIONS Massoulie, J. et al., “Structure and Functions of Acetylcholinesterase International Search Report PCT/US07/13383 dated Jul. 30, 2008. and Butyrylcholinesterase.” Progress in Brain Research 98: 139-146 Lee, K.O., et al., Improved intracellular delivery of (1993). glucocerebrosidase mediated by the HIV-1 TAT protein transduction McTiernan, C. et al., "Brain cDNA Clone for Human domain, Biochemical and Biophysical Research Communications Cholinesterase.” Proc. Natl. Acad. Sci. USA 84.6682-6686 (1987). 337:701-701 (2005). Neville, L.F. et al., “Intramolecular Relationships in Cholinesterases Murray, G.J., et al., Gaucher's disease: lack of antibody response in Revealed by Oocyte Expression of Site-Directed and Natural Vari 12 patients following repeated intravenous infusions of mannose ants of Human BCHE” EMBO.J. 11:1641-1649 (1992). terminal glucocerebrosidase, Journal of Immunological Methods, Panteghini, M. et al., “Methods for Serum Cholinesterase Assay and 137: 113-120 (1991). Classification of Genetic Variants.” Clinica Chimica Acta 183:87-90 European Search Report EP06076852 dated Oct. 8, 2007. (1989). Yeh, P. etal. Design of yeast-secreted albumin derivatives for human Prody, C.A. et al., “Isolation and Characterization of Full-Length therapy: Biological and antiviral properties of a serum albumin-CD4 cDNA Clones Coding for Cholinesterase From Fetal Human Tis genetic conjugate, Proc. Natl. Acad. Sci. USA 80: 1904-1908 (1992). sues.” Proc. Natl. Acad. Sci. USA 84:3555-3559 (1987). Halpern W., et al., “Albugranin"M, a Recombinant Human Granulo La Du, B.N. et al., “Molecular Biology of Human Serum cyte Colony Stimulating Factor (G-CSF) Genetically Fused to Cholinesterase.” FASEB 45:2965-2969 (1986). Recombinant Human Albumin Induces Prolonged Myelopoietic La Du, B.N., “Identification of Human Serum Cholinesterase Vari Effects in Mice and Monkeys.” Pharmaceutical Research, vol. 19. ants. Using the Polymerase Chain Reaction Amplification Tech No. 11, pp. 1720-1729 (2002). nique.” TiPS 10:309-313 (1989). Osborn B.L., et al., “Albutropin: A Growth Hormone-Albumin LaMotta, R.V. et al., “Molecular Heterogeneity of Human Serum Fusion with Improved Pharmacokinetics and Pharmacodynamics in Cholinesterase.” Clin. Chem. 17:135-144 (1971). Rats and Monkeys.” European Journal of Pharmacology, vol. 456, pp. Lockridge, O. et al., “Comparison of Atypical and Usual Human 149-158 (2002). Serum Cholinesterase.” J. Biol. Chem. 253:361-366 (1978). US 8,946,156 B2 Page 3
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(1982). for EP Application No. 06076852.0 (7 pages). Lockridge, O. et al., “Complete Amino Acid Sequence of Human Galliano M. et al., FEBS Letters 208:364-368 (1986). Serum Cholinesterase.” J. Biol. Chem. 262:549-557 (1987). Galliano M. et al., FEBS Letters 233:100-104 (1988). Lockridge, O. et al., “Location of Disulfide Bonds Within the Galliano M. et al., Proc. Natl. Acad. Sci. USA 87:8721-8725 (1990). Sequence of Human Serum Cholinesterase.” J. Biol. Chem. Galliano M. et al., J. Biol. Chem. 261:4283-4287 (1986). Hershfield MS et al., Proc. Natl. Acad. Sci. USA 88:7185-7189 262:12945-12952 (1987). (1991). Lockridge, O., "Structure of Human Serum Cholinesterase.” BioEs Minchiotti L et al., Biochimica et Biophysica Acta 1 119:232-238 says 9:125-128 (1988). (1992). Whittaker, V.P. et al., “The Cholinesterases: A Discussion of Some Peters T. Clin. Chem. 23:5-12 (1977). Unanswered Questions.” Progress in Brain Research 98: 155-159 Reed RGet al., Clin. Chem. 34:1992-1994 (1988). (1993). Weitkamp LR et al., Ann. Hum. Genet. 36:381-392 (1973). Bellon et al., "Chemical Structure of Two Fragments of Human Weitkamp LR et al., Ann. Hum. Genet. 37:219-226 (1973). Serum Albumin and their Location in the Albumin Molecule.” International Search Report for PCT/US06/29391 mailed on Jun. 1, Biochem. J. 147:585-592 (1975). 2007. Carmona et al., “Plasma Butyrylcholinesterase Activity and Cocaine International Search Report for PCT/US08/12306 mailed on Mar. 26, Half-Life Differ Significantly in Rhesus and Squirrel Monkeys.” Life 2009. Sciences 59:939-943 (1996). Supplementary European Search Report for EP 06813242 dated Jun. Carmona et al., “Butyrylcholinesterase Accelerates Cocaine Metabo 19, 2009. lism: In Vitro and In Vivo Effects in Nonhuman Primates and Supplementary European Search Report for EP 01934875 dated Jun. Humans.” Drug Metabolism and Disposition 28:367-371 (Mar. 4, 2003. 2000). International Search Report for PCT/US01/12009 mailedon Aug. 22. Dockal et al., “Five Recombinant Fragments of Human Serum Albu 2001. min-Tools for the Characterization of the Warfarin Binding Site.” International Preliminary Report on Patentability issued May 14, Protein Science 9:1455-1465 (2000). 2010 and Written Opinion mailed Mar. 26, 2009, for International Hoffman et al., “Administration of Purified Human Plasma Patent Application No. PCT/US2008/012306, filed Oct. 30, 2008. Cholinesterase Protects Against CocaineToxicity in Mice.” Clinical Nachon F., et al., “Engineering of a monomeric and low-glycosylated Toxicology 34:259-266 (1996). form of human butyrylcholinesterase.” Eur, J. Biochem... vol. 269, pp. Mattes et al., "Cocaine and Butyrylcholinesterase(BChE): Determi 630-637 (2002). nation of Enzymatic Parameters.” Life Sciences 58:PL257-261 Sun H., et al., “Cocaine Metabolism Accelerated by a Re-Engineered (1996). Human Butyrylcholinesterase.” Journal of Pharmacology and Minchiotti et al., “The Molecular Defectin a COOH-Terminal-Modi Experimental Therapeutics, vol. 302, No. 2, pp. 710-716 (2002). fied and Shortened Mutant of Human Serum Albumin, J. Biol. Duysen E.G., et al., “Wild-type and A328W mutant human Chem. 264:3385-3389 (1989). butyrylcholinesterase tetramers expressed in Chinese hamster ovary Peach et al., “Albumin Rugby Park: A Truncated Albumin Variant cells have a 16-hour half-life in the circulation and protect mice from Caused by a G-> C Splice-Site Mutation in Intron 13.” Biochimica et cocaine toxicity.” J. Pharmacol. Exp. Ther. 302(2): 751-8 (2002). Biophysica Acta 1180: 107-110 (1992). Gao Y., et al., “An albumin-butyrylcholinesterase for cocaine toxicity Takahashi et al., “Structural Changes and Metal Binding by and addiction: catalytic and pharmacokinetic properties.” Chem. Proalbumins and Other Amino-Terminal Genetic Variants of Human Biol. Interact. 175(1-3): 83-7 (2008) (Author Manuscript). Serum Albumin.” PNAS 84:7403-7407 (1987). Extended European Search Report mailed Sep. 14, 2009, for Euro Watkins et al., “cDNA and Protein Sequence of Polymorphic pean Application No. 078.09374.7. Macaque Albumins That Differ in Bilirubin Binding.” PNAS 90:2409-2413 (1993). * cited by examiner U.S. Patent Feb. 3, 2015 Sheet 1 of 20 US 8,946,156 B2
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Figure 1 U.S. Patent Feb. 3, 2015 Sheet 2 of 20 US 8,946,156 B2
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Figure 2 U.S. Patent Feb. 3, 2015 Sheet 3 of 20 US 8,946,156 B2
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Figure 3A
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DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA HHHHH HHH HHH HHHHHHHHHH HHHHH HHHHHHHHHH
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X K 45 DYLSVVLNOL CVLHEKTPVS DRVTKCCTES LVNRRPPCFSA LEVDETYWPK HHHHHHHHHH HHHHH HHHHHHHHH HHHHHHHH 50 EFNAETFTFH ADICTLSEKE ROIKKQTLV ELVKHKPKAT KEOLKAVMDD HHH HHH HHHHMMEE HHH HHHHHHHH
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Figure 10
a. Randomisation of Loop IV. y 151. APELLFFAKR YKAAFTECC AADKAACLLP KLDELRDEGK ASSAKQRLKC HHHHHHHHHH HHHHHHHHH HHHHH HHHHHHHHHH HHHHHHHHHH
V 151. APELLFFAKR YKAAFTECCX XXCCCCXCLLP KLDELRDEGK ASSAKQRLKC HHHHHHHHHH HHHHHHHHH HHHHH HHHHHHHHHH HHHHHHHHHH
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US 8,946,156 B2 1. 2 ALBUMINFUSION PROTEINS on a Suitable plasmid as to express a fusion polypeptide. The expression may be effected in vitro from, for example, This is a divisional of application Ser. No. 1 1/927,602, filed prokaryotic or eukaryotic cells, or in vivo e.g. from a trans Oct. 29, 2007, which is a divisional of application Ser. No. genic organism. 11/078,914, filed Mar. 14, 2005, now U.S. Pat. No. 7,482,013, Therapeutic proteins in their native state or when recom which is a continuation of application Ser. No. 09/832,501, binantly produced. Such as interferons and growth hormones, filed Apr. 12, 2001, now abandoned, which are incorporated are typically labile molecules exhibiting short shelf-lives, herein by reference in their entirety, and which claim the particularly when formulated in aqueous solutions. The insta benefit of priority under 35 U.S.C. S 119(e) based on the bility in these molecules when formulated for administration following U.S. provisional applications: 60/229.358 filed on 10 dictates that many of the molecules must be lyophilized and Apr. 12, 2000; 60/199,384 filed on Apr. 25, 2000; and 60/256, refrigerated at all times during storage, thereby rendering the 931 filed on Dec. 21, 2000. Each of the provisional applica molecules difficult to transport and/or store. Storage prob tions is hereby incorporated herein by reference in its entirety. lems are particularly acute when pharmaceutical formula tions must be stored and dispensed outside of the hospital SEQUENCE LISTING 15 environment. Many protein and peptide drugs also require the addition of high concentrations of other protein Such as albu The instant application contains a Sequence Listing which minto reduce or prevent loss of protein due to binding to the has been submitted electronically in ASCII format and is container. This is a major concern with respect to proteins hereby incorporated by reference in its entirety. Said ASCII such as IFN. For this reason, many Therapeutic proteins are copy, created on May 28, 2014, is named formulated in combination with large proportion of albumin 101179.000066SL.txt and is 52,756 bytes in size. carrier molecule (100-1000 fold excess), though this is an undesirable and expensive feature of the formulation. BACKGROUND OF THE INVENTION Few practical solutions to the storage problems of labile protein molecules have been proposed. Accordingly, there is The invention relates generally to Therapeutic proteins 25 a need for stabilized, long lasting formulations of proteina (including, but not limited to a polypeptide, antibody, or ceous therapeutic molecules that are easily dispensed, pref peptide, or fragments and variants thereof) fused to albumin erably with a simple formulation requiring minimal post or fragments or variants of albumin. The invention further storage manipulation. relates to Therapeutic proteins (including, but not limited to, a polypeptide, antibody, or peptide, or fragments and variants 30 SUMMARY OF THE INVENTION thereof) fused to albuminor fragments or variants of albumin, that exhibit extended shelf-life and/or extended ortherapeutic The present invention is based, in part, on the discovery activity in Solution. These fusion proteins are herein collec that Therapeutic proteins may be stabilized to extend the tively referred to as “albumin fusion proteins of the inven shelf-life, and/or to retain the Therapeutic protein’s activity tion.” The invention encompasses therapeutic albumin fusion 35 for extended periods of time in solution, in vitro and or in proteins, compositions, pharmaceutical compositions, for Vivo, by genetically or chemically fusing or conjugating the mulations and kits. Nucleic acid molecules encoding the Therapeutic protein to albumin or a fragment (portion) or albumin fusion proteins of the invention are also encom variant of albumin, that is sufficient to stabilize the protein passed by the invention, as are vectors containing these and or its activity. In addition it has been determined that the nucleic acids, host cells transformed with these nucleic acids 40 use of albumin-fusion proteins or albumin conjugated pro vectors, and methods of making the albumin fusion proteins teins may reduce the need to formulate protein solutions with of the invention using these nucleic acids, vectors, and/or host large excesses of carrier proteins (such as albumin, unfused) cells. to prevent loss of Therapeutic proteins due to factors such as The invention is also directed to methods of in vitro stabi binding to the container. lizing a Therapeutic protein via fusion or conjugation of the 45 The present invention encompasses albumin fusion pro Therapeutic protein to albumin or fragments or variants of teins comprising a Therapeutic protein (e.g., a polypeptide, albumin. antibody, or peptide, or fragments and variants thereof) fused Human serum albumin (HSA, or HA), a protein of 585 to albuminor a fragment (portion) or variant of albumin. The amino acids in its mature form (as shown in FIG. 15 or in SEQ present invention also encompasses albumin fusion proteins ID NO:18), is responsible for a significant proportion of the 50 comprising a Therapeutic protein (e.g. a polypeptide, anti osmotic pressure of serum and also functions as a carrier of body, or peptide, or fragments and variants thereof) fused to endogenous and exogenous ligands. At present, HA for clini albuminor a fragment (portion) or variant of albumin, that is cal use is produced by extraction from human blood. The sufficient to prolong the shelf life of the Therapeutic protein, production of recombinant HA (rRIA) in microorganisms has and/or stabilize the Therapeutic protein and/or its activity in been disclosed in EP 330 451 and EP 361991. 55 Solution (or in a pharmaceutical composition) in vitro and/or The role of albumin as a carrier molecule and its inert in vivo. Nucleic acid molecules encoding the albumin fusion nature are desirable properties for use as a carrier and trans proteins of the invention are also encompassed by the inven porter of polypeptides in vivo. The use of albumin as a com tion, as are vectors containing these nucleic acids, host cells ponent of an albumin fusion protein as a carrier for various transformed with these nucleic acids vectors, and methods of proteins has been suggested in WO 93/15199, WO 93/15200, 60 making the albuminfusion proteins of the invention and using and EP 413 622. The use of N-terminal fragments of HA for these nucleic acids, vectors, and/or host cells. fusions to polypeptides has also been proposed (EP399 666). The invention also encompasses pharmaceutical formula Fusion of albuminto the Therapeutic protein may beachieved tions comprising an albumin fusion protein of the invention by genetic manipulation, such that the DNA coding for HA, and a pharmaceutically acceptable diluent or carrier. Such or a fragment thereof, is joined to the DNA coding for the 65 formulations may be in a kit or container. Such kit or con Therapeutic protein. A suitable host is then transformed or tainer may be packaged with instructions pertaining to the transfected with the fused nucleotide sequences, so arranged extended shelf life of the Therapeutic protein. Such formula US 8,946,156 B2 3 4 tions may be used in methods of treating, preventing, ame FIG. 4 shows a map of a plasmid (ppPC0005) that can be liorating or diagnosing a disease or disease symptom in a used as the base vector into which polynucleotides encoding patient, preferably a mammal, most preferably a human, com the Therapeutic proteins (including polypeptides and frag prising the step of administering the pharmaceutical formu ments and variants thereof) may be cloned to form HA lation to the patient. fusions. Plasmid Map key: PRB1p: PRB1 S. cerevisiae pro In other embodiments, the present invention encompasses moter, FL: Fusion leader sequence: rhA. cDNA encoding methods of preventing treating, or ameliorating a disease or HAADH1t: ADH1 S. cerevisiae terminator: T3: T3 sequenc disorder. In preferred embodiments, the present invention ing primer site; T7: T7 sequencing primer site; Amp R: B-lac encompasses a method of treating a disease or disorder listed tamase gene: ori: origin of replication. Please note that in the in the “Preferred Indication Y column of Table 1 comprising 10 provisional applications to which this application claims pri administering to a patient in which Such treatment, prevention ority, the plasmid in FIG. 4 was labeled pPPC0006, instead of or amelioration is desired an albumin fusion protein of the pPPC0005. In addition the drawing of this plasmid did not invention that comprises a Therapeutic protein portion corre show certain pertinent restriction sites in this vector. Thus in sponding to a Therapeutic protein (or fragment or variant the present application, the drawing is labeled pPPC0005 and thereof) disclosed in the “Therapeutic Protein X column of 15 more restriction sites of the same vector are shown. Table 1 (in the same row as the disease or disorder to be FIG. 5 compares the recovery of vial-stored HA-IFN solu treated is listed in the "Preferred Indication Y column of tions of various concentrations with a stock solution after 48 Table 1) in an amount effective to treat prevent or ameliorate or 72 hours of storage. the disease or disorder. FIG. 6 compares the activity of an HA-C-IFN fusion pro In another embodiment, the invention includes a method of tein after administration to monkeys via IV or SC. extending the shelf life of a Therapeutic protein (e.g. a FIG. 7 describes the bioavailability and stability of an polypeptide, antibody, or peptide, or fragments and variants HA-C-IFN fusion protein. thereof) comprising the step of fusing or conjugating the FIG. 8 is a map of an expression vector for the production Therapeutic protein to albumin or a fragment (portion) or of HA-C-IFN. variant of albumin, that is sufficient to extend the shelf-life of 25 FIG.9 shows the location of loops in HA. FIG.9 discloses the Therapeutic protein. In a preferred embodiment, the SEQID NO: 18. Therapeutic protein used according to this method is fused to FIG. 10 is an example of the modification of an HA loop. the albumin, or the fragment or variant of albumin. In a most FIG. 10 discloses residues 151-200 of SEQID NO: 18, SEQ preferred embodiment, the Therapeutic protein used accord ID NO:51, and residues 151-200 of SEQID NO: 18, respec ing to this method is fused to albumin, or a fragment or variant 30 tively, in order of appearance. of albumin, via recombinant DNA technology or genetic FIG. 11, comprising FIG. 11A-C, is a representation of the engineering. HA loops. FIG. 11A-C discloses SEQID NO:18. In another embodiment, the invention includes a method of FIG. 12 shows the HA loop IV. stabilizing a Therapeutic protein (e.g. a polypeptide, anti FIG. 13 shows the tertiary structure of HA. body, or peptide, or fragments and variants thereof) in solu 35 FIG. 14 shows an example of a scFv-HA fusion tion, comprising the step of fusing or conjugating the Thera FIG. 15, comprising FIG. 15A-D, shows the amino acid peutic protein to albuminor a fragment (portion) or variant of sequence of the mature form of human albumin (SEQ ID albumin, that is sufficient to stabilize the Therapeutic protein. NO:18) and a polynucleotide encoding it (SEQ ID NO:17). In a preferred embodiment, the Therapeutic protein used according to this method is fused to the albumin, or the 40 DETAILED DESCRIPTION fragment or variant of albumin. In a most preferred embodi ment, the Therapeutic protein used according to this method As described above, the present invention is based, in part, is fused to albumin, or a fragment or variant of albumin, via on the discovery that a Therapeutic protein (e.g., a polypep recombinant DNA technology or genetic engineering. tide, antibody, or peptide, or fragments and variants thereof) The present invention further includes transgenic organ 45 may be stabilized to extend the shelf-life and/or retain the isms modified to contain the nucleic acid molecules of the Therapeutic protein’s activity for extended periods of time in invention, preferably modified to express the albumin fusion Solution (or in a pharmaceutical composition) in vitro and/or proteins encoded by the nucleic acid molecules. in Vivo, by genetically fusing or chemically conjugating the Therapeutic protein, polypeptide or peptide to all or a portion BRIEF DESCRIPTION OF THE FIGURES 50 of albumin sufficient to stabilize the protein and its activity. The present invention relates generally to albumin fusion FIG. 1 depicts the extended shelf-life of an HA fusion proteins and methods of treating, preventing, or ameliorating protein in terms of the biological activity (Nb2 cell prolifera diseases or disorders. As used herein, “albumin fusion pro tion) of HA-hGH remaining after incubation in cell culture tein’ refers to a protein formed by the fusion of at least one media for up to 5 weeks at 37°C. Under these conditions, 55 molecule of albumin (or a fragment or variant thereof) to at hGH has no observed activity by week 2. least one molecule of a Therapeutic protein (or fragment or FIG. 2 depicts the extended shelf-life of an HA fusion variant thereof). An albumin fusion protein of the invention protein in terms of the stable biological activity (Nb2 cell comprises at least a fragment or variant of a Therapeutic proliferation) of HA-hGH remaining after incubation in cell protein and at least a fragment or variant of human serum culture media for up to 3 weeks at 4, 37, or 50° C. Data is 60 albumin, which are associated with one another, preferably normalized to the biological activity of hCGH at time Zero. by genetic fusion (i.e. the albumin fusion protein is generated FIGS. 3A and 3B compare the biological activity of HA by translation of a nucleic acid in which a polynucleotide hCH with hCH in the Nb2 cell proliferation assay. FIG. 3A encoding all or a portion of a Therapeutic protein is joined shows proliferation after 24 hours of incubation with various in-frame with a polynucleotide encoding all or a portion of concentrations of hCH or the albumin fusion protein, and 65 albumin) or chemical conjugation to one another. The Thera FIG.3B shows proliferation after 48 hours of incubation with peutic protein and albumin protein, once part of the albumin various concentrations of hCH or the albumin fusion protein. fusion protein, may be referred to as a "portion”, “region” or US 8,946,156 B2 5 6 “moiety' of the albumin fusion protein (e.g. a “Therapeutic ameliorate a disease, condition or disorder. As a non-limiting protein portion” or an “albumin protein portion'). example, a “Therapeutic protein' may be one that binds spe In one embodiment, the invention provides an albumin cifically to aparticular cell type (normal (e.g. lymphocytes) or fusion protein comprising, or alternatively consisting of a abnormal e.g. (cancer cells)) and therefore may be used to Therapeutic protein (e.g. as described in Table 1) and a serum target a compound (drug, or cytotoxic agent) to that cell type albumin protein. In other embodiments, the invention pro specifically. vides an albumin fusion protein comprising, or alternatively In another non-limiting example, a “Therapeutic protein’ consisting of a biologically active and/or therapeutically is a protein that has a biological activity, and in particular, a active fragment of a Therapeutic protein and a serum albumin biological activity that is useful for treating preventing or protein. In other embodiments, the invention provides an 10 ameliorating a disease. A non-inclusive list of biological albumin fusion protein comprising, or alternatively consist activities that may be possessed by a Therapeutic protein ing of a biologically active and/or therapeutically active vari includes, enhancing the immune response, promoting angio ant of a Therapeutic protein and a serum albumin protein. In genesis, inhibiting angiogenesis, regulating hematopoietic preferred embodiments, the serum albumin protein compo functions, stimulating nerve growth, enhancing an immune nent of the albumin fusion protein is the mature portion of 15 response, inhibiting an immune response, or any one or more serum albumin. of the biological activities described in the “Biological In further embodiments, the invention provides an albumin Activities' section below. fusion protein comprising, or alternatively consisting of a As used herein, “therapeutic activity” or “activity” may Therapeutic protein, and a biologically active and/or thera refer to an activity whose effect is consistent with a desirable peutically active fragment of serum albumin. In further therapeutic outcome in humans, or to desired effects in non embodiments, the invention provides an albumin fusion pro human mammals or in other species or organisms. Therapeu tein comprising, or alternatively consisting of a Therapeutic tic activity may be measured in vivo or in vitro. For example, protein and a biologically active and/or therapeutically active a desirable effect may be assayed in cell culture. As an variant of serum albumin. In preferred embodiments, the example, when hCGH is the Therapeutic protein, the effects of Therapeutic protein portion of the albumin fusion protein is 25 hGH on cell proliferation as described in Example 1 may be the mature portion of the Therapeutic protein. In a further used as the endpoint for which therapeutic activity is mea preferred embodiment, the Therapeutic protein portion of the Sured. Such in vitro or cell culture assays are commonly albumin fusion protein is the extracellular soluble domain of available for many Therapeutic proteins as described in the the Therapeutic protein. In an alternative embodiment, the art. Therapeutic protein portion of the albumin fusion protein is 30 Examples of useful assays for particular Therapeutic pro the active form of the Therapeutic protein. teins include, but are not limited to, GMCSF (Eaves, A.C. and Infurther embodiments, the invention provides an albumin Eaves C. J., Erythropoiesis in culture. In: McCullock E. A fusion protein comprising, or alternatively consisting of a (edt) Cell culture techniques—Clinics in hematology, WB biologically active and/or therapeutically active fragment or Saunders, Eastbourne, pp. 371-91 (1984), Metcalf, D., Inter variant of a Therapeutic protein and a biologically active 35 national Journal of Cell Cloning 10: 116-25 (1992); Testa, N. and/or therapeutically active fragment or variant of serum G. et al., Assays for hematopoietic growth factors. In: Balk albumin. In preferred embodiments, the invention provides will FR (edt) Cytokines. A practical Approach, pp. 229-44: an albumin fusion protein comprising, or alternatively con IRL Press Oxford 1991) EPO (bioassay: Kitamura et al. J. sisting of the mature portion of a Therapeutic protein and the Cell. Physiol. 140 p 323 (1989)); Hirudin (platelet aggrega mature portion of serum albumin. 40 tion assay: Blood Coagul Fibrinolysis 7(2):259-61 (1996)); Therapeutic Proteins IFNC (anti-viral assay: Rubinstein et al. J. Virol. 37(2):755-8 As Stated above, an albumin fusion protein of the invention (1981); anti-proliferative assay: Gao Y. et al Mol Cell Biol. comprises at least a fragment or variant of a Therapeutic 19(11):7305-13 (1999); and bioassay: Czarniecki et al. J. protein and at least a fragment or variant of human serum Virol. 49 p.490 (1984)). GCSF (bioassay: Shirafuji et al., Exp. albumin. which are associated with one another, preferably 45 Hematol. 17 p.116 (1989): proliferation of murine NFS-60 by genetic fusion or chemical conjugation. cells (Weinsteinet al., Proc Natl AcadSci 83:5010-4 (1986)): As used herein. “Therapeutic protein’ refers to proteins, insulin (H-glucose uptake assay: Steppan et al., Nature 409 polypeptides, antibodies, peptides or fragments or variants (6818):307-12 (2001)): hCGH (Ba/F3-hGHR proliferation thereof, having one or more therapeutic and/or biological assay: J Clin Endocrinol Metab 85(11):4274-9 (2000): Inter activities. Therapeutic proteins encompassed by the invention 50 national standard for growth hormone: Horm Res. 51 Suppl include but are not limited to, proteins, polypeptides, pep 1:7-12 (1999)); factor X (factor X activity assay: Van Wijket tides, antibodies, and biologics. (The terms peptides, pro al. Thromb Res 22:681-686 (1981)); factor VII (coagulation teins, and polypeptides are used interchangeably herein.) It is assay using prothrombin clotting time: Belaaouai et al., J. specifically contemplated that the term “Therapeutic protein’ Biol. Chem. 275:27123-8 (2000); Diaz-Collier et al. Thromb encompasses antibodies and fragments and variants thereof. 55 Haemost 71:339-46 (1994)), or as shown in Table 1 in the Thus an albumin fusion protein of the invention may contain “Exemplary Activity Assay” column. at least a fragment or variant of a Therapeutic protein, and/or Therapeutic proteins corresponding to a Therapeutic pro at least a fragment or variant of an antibody. Additionally, the tein portion of an albumin fusion protein of the invention, term “Therapeutic protein’ may refer to the endogenous or Such as cell Surface and secretory proteins, are often modified naturally occurring correlate of a Therapeutic protein. 60 by the attachment of one or more oligosaccharide groups. The By a polypeptide displaying a “therapeutic activity” or a modification, referred to as glycosylation, can dramatically protein that is “therapeutically active' is meant a polypeptide affect the physical properties of proteins and can be important that possesses one or more known biological and/or therapeu in protein stability, secretion, and localization. Glycosylation tic activities associated with a Therapeutic protein such as one occurs at specific locations along the polypeptide backbone. or more of the Therapeutic proteins described herein or oth 65 There are usually two major types of glycosylation: glycosy erwise known in the art. As a non-limiting example, a “Thera lation characterized by O-linked oligosaccharides, which are peutic protein' is a protein that is useful to treat, prevent or attached to serine or threonine residues; and glycosylation US 8,946,156 B2 7 8 characterized by N-linked oligosaccharides, which are (GMCSF), insulin, single chain antibodies, autocrine motility attached to asparagine residues in an Asn-X-Ser/Thr factor, Scatter factor, laminin, hirudin, applaggin, monocyte sequence, where X can be any amino acid except proline. chemotactic protein (MCP/MCAF), macrophage colony N-acetylneuramic acid (also known as sialic acid) is usually stimulating factor (M-CSF), osteopontin, platelet factor 4. the terminal residue of both N-linked and 0-linked oligosac- 5 tenascin, vitronectin, in addition to those described in Table 1. charides. Variables Such as protein structure and cell type These proteins and nucleic acid sequences encoding these influence the number and nature of the carbohydrate units proteins are well known and available in public databases such as Chemical Abstracts Services Databases (e.g. the CAS within the chains at different glycosylation sites. Glycosyla Registry), GenBank, and GenSeq as shown in Table 1. tion isomers are also common at the same site within a given Additional Therapeutic proteins corresponding to a Thera cell type. 10 peutic protein portion of an albumin fusion protein of the For example, several types of human interferon are glyco invention include, but are not limited to one or more of the sylated. Natural human interferon-C2 is O-glycosylated at Therapeutic proteins or peptides disclosed in the “Therapeu threonine 106, and N-glycosylation occurs at asparagine 72 in tic Protein X column of Table 1, or fragment or variable interferon-C.14 (Adolf et al., J. Biochem 276:511 (1991); thereof. Nyman TAet al., J. Biochem329:295 (1998)). The oligosac- 15 Table 1 provides a non-exhaustive list of Therapeutic pro charides at asparagine 80 in natural interferon-B1C. may play teins that correspond to a Therapeutic protein portion of an an important factor in the solubility and stability of the pro albumin fusion protein of the invention. The “Therapeutic tein, but may not be essential for its biological activity. This permits the production of an unglycosylated analog (inter Protein X column discloses Therapeutic protein molecules feron-B1b) engineered with sequence modifications to 20 followed by parentheses containing scientific and brand enhance stability (Hosoi et al. J. Interferon Res. 8:375 (1988: names that comprise, or alternatively consist of that Thera Karpusas et al., Cell Mol Life Sci54: 1203 (1998): Knight.J. peutic protein molecule or a fragment or variant thereof Interferon Res. 2:421 (1982): Runkeletal. Pharm Res 15:641 “Therapeutic protein X’ as used herein may refer either to an (1998): Lin, Dev. Biol. Stand. 96.97 (1998))1. Interferon-y individual Therapeutic protein molecule (as defined by the contains two N-linked oligosaccharide chains at positions 25 25 amino acid sequence obtainable from the CAS and Genbank and 97, both important for the efficient formation of the accession numbers), or to the entire group of Therapeutic bioactive recombinant protein, and having an influence on the proteins associated with a given Therapeutic protein mol pharmacokinetic properties of the protein (Sareneva et al. ecule disclosed in this column. The “Exemplary Identifier” Eur. J. Biochem 242:191 (1996): Sareneva et al., Biochem J. column provides Chemical Abstracts Services (CAS) Regis 303:831 (1994); Sareneva et al., J. Interferon Res. 13:267 30 try Numbers (published by the American Chemical Society) (1993)). Mixed O-linked and N-linked glycosylation also and/or Genbank Accession Numbers (e.g., Locus ID, occurs, for example in human erythropoietin, N-linked gly NP XXXXX (Reference Sequence Protein), and cosylation occurs at asparagine residues located at positions XP XXXXX (Model Protein) identifiers available through 24, 38 and 83 while O-linked glycosylation occurs at a serine the national Center for Biotechnology Information (NCBI) residue located at position 126 (Lai et al., J. Biol. Chem. 35 webpage at www.ncbi.nlm.nih.gov) that correspond to 261:3116 (1986): Broudy et al. Arch. Biochem. Biophys. entries in the CAS Registry or Genbank database which con 265:329 (1988)). tain an amino acid sequence of the Therapeutic Protein Mol Therapeutic proteins corresponding to a Therapeutic pro ecule or of a fragment or variant of the Therapeutic Protein tein portion of an albumin fusion protein of the invention, as well as analogs and variants thereof, may be modified so that 40 Molecule. The Summary pages associated with each of these glycosylation at one or more sites is altered as a result of CAS and Genbank Accession Numbers are each incorporated manipulation(s) of their nucleic acid sequence, by the host by reference in their entireties, particularly with respect to the cell in which they are expressed, or due to other conditions of amino acid sequences described therein. The “PCT/Patent their expression. For example, glycosylation isomers may be Reference' column provides U.S. Patent numbers, or PCT produced by abolishing or introducing glycosylation sites, 45 International Publication Numbers corresponding to patents e.g. by Substitution or deletion of amino acid residues, such as and/or published patent applications that describe the Thera Substitution of glutamine for asparagine, or unglycosylated peutic protein molecule. Each of the patents and/or published recombinant proteins may be produced by expressing the patent applications cited in the “PCT/Patent Reference’ col proteins in host cells that will not glycosylate them, e.g. in E. umn are herein incorporated by reference in their entireties. In coli or glycosylation-deficient yeast. These approaches are 50 particular, the amino acid sequences of the specified polypep described in more detail below and are known in the art. tide set forth in the sequence listing of each cited “PCT/Patent Therapeutic proteins corresponding to a Therapeutic pro Reference', the variants of these amino acid sequences (mu tein portion of an albumin fusion protein of the invention tations, fragments, etc.) set forth, for example, in the detailed include, but are not limited to, plasma proteins. More specifi description of each cited “PCT/Patent Reference', the thera cally, such Therapeutic proteins include, but are not limited 55 peutic indications set forth, for example, in the detailed to, immunoglobulins, serum cholinesterase, alpha-1 antit description of each cited “PCT/Patent Reference', and the rypsin, aprotinin, coagulation factors in both pre and active activity assays for the specified polypeptide set forth in the forms including but not limited to, von Willebrand factor, detailed description, and more particularly, the examples of fibrinogen, factor II, factor VII, factor VIIA activated factor, each cited “PCT/Patent Reference” are incorporated herein factor VIII, factor IX, factor X, factor XIII, c1 inactivator, 60 antithrombin III, thrombin, prothrombin, apo-lipoprotein, by reference. As an example, the amino acid sequence of c-reactive protein, and protein C. Therapeutic proteins corre full-length human butyrylcholinesterase (BChE) is disclosed sponding to a Therapeutic protein portion of an albumin as Seq #1 (SEQID NO:1) in U.S. Pat. No. 6,001,625, which fusion protein of the invention further include, but are not is cited in the 'PCT/Patent Reference’ column for the listed limited to, human growth hormone (hGH), C.-interferon, 65 “Therapeutic Protein X Serum Cholinesterase. The incorpo erythropoietin (EPO), granulocyte-colony stimulating factor rated amino acid sequence of full-length BChE (SEQ ID (GCSF), granulocyte-macrophage colony-stimulating factor NO:52) is as follows: US 8,946,156 B2 10
Met His Ser Wall Thir Ile Ile Ile Arg Phe Lell Phe Trp Phe 15
Lell Luell Luell Cys Met Lell Ile Gly Lys Ser His Thir Glu Asp Asp Ile 25
Ile Ile Thir Asn Gly Lys Wall Arg Gly Met Asn Luell Thir Wall 3 5 4 O 45
Phe Gly Thir Wall Thir Ala Phe Luell Gly Ile Pro Ala Glin Pro SO 55 6 O
Pro Luell Arg Lell Arg Phe Pro Glin Ser Lell Thir Trp 65 70
Ser Asp Trp Asn Ala Thir Ala ASn Ser Glin Asn 85 90 95
Ile Asp Ser Phe Pro Gly Phe His Gly Ser Glu Met Trp Asn Pro 105 11 O
Asn Thir Luell Ser Glu Asp Cys Luell Tyr Luell Asn Wall Trp Ile Pro 12 O 125
Ala Pro Pro Asn Ala Thir Wall Luell Ile Trp Ile Gly Gly 13 O 135 14 O
Gly Phe Glin Thir Gly Thir Ser Ser Luell His Wall Asp Gly Phe 145 150 155 160
Lell Ala Arg Wall Glu Arg Wall Ile Wall Wall Ser Met Asn Tyr Arg Wall 1.65 17O
Gly Ala Luell Gly Phe Lell Ala Luell Pro Gly ASn Pro Glu Ala Pro Gly 18O 185 19 O
Asn Met Gly Luell Phe Asp Glin Glin Luell Ala Luell Glin Trp Wall Glin 195
Asn Ile Ala Ala Phe Gly Gly Asn Pro Ser Wall Thir Luell Phe Gly 21 O 215 22O
Glu Ser Ala Gly Ala Ala Ser Wall Ser Luell His Lell Lell Ser Pro Gly 225 23 O 235 24 O
Ser His Ser Luell Phe Thir Arg Ala Ile Luell Glin Ser Gly Ser Phe Asn 245 250 255
Ala Pro Trp Ala Wall Thir Ser Luell Tyr Glu Ala Arg Asn Arg Thir Luell 26 O 265 27 O
Asn Luell Ala Lell Thir Gly Cys Ser Arg Glu Asn Glu Thir Glu Ile 28O 285
Ile Lys Luell Arg Asn Lys Asp Pro Glin Glu Ile Lell Luell Asn Glu 29 O 295 3 OO
Ala Phe Wall Wall Pro Tyr Gly Thir Pro Luell Ser Wall Asn Phe Gly Pro 3. OS 310 315
Thir Wall Asp Gly Asp Phe Lell Thir Asp Met Pro Asp Ile Luell Luell Glu 3.25 330 335
Lell Gly Glin Phe Thir Glin Ile Luell Wall Gly Wall Asn Asp 34 O 345 35. O
Glu Gly Thir Ala Phe Lell Wall Tyr Gly Ala Pro Gly Phe Ser Asp 355 360 365
Asn Asn Ser Ile Ile Thir Arg Glu Phe Glin Glu Gly Luell Ile 37 O 375
Phe Phe Pro Gly Wall Ser Glu Phe Gly Glu Ser Ile Luell Phe His 385 390 395 4 OO
Thir Asp Trp Wall Asp Asp Glin Arg Pro Glu Asn Tyr Arg Glu Ala 4 OS 41O 415
Lell Gly Asp Wall Wall Gly Asp Asn Phe Ile Pro Ala Luell Glu 42O 425 43 O US 8,946,156 B2 11 12 - Continued Phe Thr Lys Lys Phe Ser Glu Trp Gly Asn. Asn Ala Phe Phe 435 44 O 445
Phe Glu His Arg Ser Ser Llys Lieu Pro Trp Pro Glu Trp Met Gly Val 450 45.5 460
Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Lieu Pro Luell Glu Arg 465 470
Arg Asp Asn Tyr Thir Lys Ala Glu Glu Ile Lieu. Ser Arg Ser Ile Wall 485 490 495
Arg Trp Ala ASn Phe Ala Tyr Gly ASn Pro ASn Glu Thr Gin SOO 505
Asn. Asn Ser Thir Ser Trp Pro Wall Phe Ser Thir Glu Glin 515 525
Lell Thir Luell Asn Thir Glu Ser Thir Arg Ile Met Thr Luell Arg Ala 53 O 535 54 O
Glin Glin Arg Phe Trp Thir Ser Phe Phe Pro Wall Luell Glu Met 5.45 550 555 560
Thir Gly Asn Ile Asp Glu Ala Glu Trp Glu Trp Ala Gly Phe His 565 st O sts
Arg Trp Asn Asn Tyr Met Met Asp Trp Asn. Glin Phe Asn Asp 585 59 O
Thir Ser Lys Glu Ser Wall Gly Luell 595
The “Biological activity” column describes Biological activi description of the respective activity assay described in the ties associated with the Therapeutic protein molecule. The 30 reference (see Methods section, for example) for assaying the “Exemplary Activity Assay” column provides references that corresponding biological activity set forth in the “Biological describe assays which may be used to test the therapeutic and/or biological activity of a Therapeutic protein oran albu Activity” column of Table 1. The “Preferred Indication Y” min fusion protein of the invention comprising a Therapeutic column describes disease, disorders, and/or conditions that protein X portion. Each of the references cited in the “Exem 35 may be treated, prevented, diagnosed, or ameliorated by plary Activity Assay” column are herein incorporated by Therapeutic protein X or an albumin fusion protein of the reference in their entireties, particularly with respect to the invention comprising a Therapeutic protein X portion.
US 8,946,156 B2 35 36
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:CIISnooT
US 8,946,156 B2 45 46 In preferred embodiments, the albumin fusion proteins of from the amino terminus of the albumin fusion protein. In the invention are capable of a therapeutic activity and/or particular. N-terminal deletions may be described by the gen biologic activity corresponding to the therapeutic activity eral formula m-q, where q is a whole integer representing the and/or biologic activity of the Therapeutic protein corre total number of amino acid residues in the albumin fusion sponding to the Therapeutic protein portion of the albumin 5 protein, and m is defined as any integer ranging from 2 to q-6. fusion protein listed in the corresponding row of Table 1. Polynucleotides encoding these polypeptides are also encom (See, e.g. the “Biological Activity” and “Therapeutic Protein passed by the invention. X' columns of Table 1.) In further preferred embodiments, Also as mentioned above, even if deletion of one or more the therapeutically active protein portions of the albumin amino acids from the N-terminus or C-terminus of a reference fusion proteins of the invention are fragments or variants of 10 polypeptide (e.g., a Therapeutic protein and/or serum albu the reference sequence cited in the “Exemplary Identifier min protein) results in modification or loss of one or more column of Table 1., and are capable of the therapeutic activity biological functions of the protein, other functional activities and/or biologic activity of the corresponding Therapeutic (e.g. biological activities, ability to multimerize, ability to protein disclosed in “Biological Activity” column of Table 1. bind a ligand) and/or Therapeutic activities may still be Polypeptide and Polynucleotide Fragments and Variants 15 retained. For example the ability of polypeptides with C-ter Fragments minal deletions to induce and or bind to antibodies which The present invention is further directed to fragments of the recognize the complete or mature forms of the polypeptide Therapeutic proteins described in Table 1, albumin proteins, generally will be retained when less than the majority of the and/or albumin fusion proteins of the invention. residues of the complete or mature polypeptide are removed Even if deletion of one or more amino acids from the from the C-terminus. Whether a particular polypeptide lack N-terminus of a protein results in modification or loss of one ing the N-terminal and/or C-terminal residues of a reference or more biological functions of the Therapeutic protein, albu polypeptide retains Therapeutic activity can readily be deter min protein, and/or albumin fusion protein, other Therapeutic mined by routine methods described herein and/or otherwise activities and/or functional activities (e.g., biological activi known in the art. ties, ability to multimerize, ability to bind a ligand) may still 25 The present invention further provides polypeptides hav be retained. For example, the ability of polypeptides with ing one or more residues deleted from the carboxy terminus of N-terminal deletions to induce and/or bind to antibodies the amino acid sequence of a Therapeutic protein correspond which recognize the complete or mature forms of the ing to a Therapeutic protein portion of an albumin fusion polypeptides generally will be retained when less than the protein of the invention (e.g., a Therapeutic protein referred to majority of the residues of the complete polypeptide are 30 in Table 1). In particular, C-terminal deletions may be removed from the N-terminus. Whether a particular polypep described by the general formula 1-n, where n is any whole tide lacking N-terminal residues of a complete polypeptide integer ranging from 6 to q-1, and where q is a whole integer retains such immunologic activities can readily be deter representing the total number of amino acid residues in a mined by routine methods described herein and otherwise reference polypeptide (e.g., a Therapeutic protein referred to known in the art. It is not unlikely that a mutein with a large 35 in Table 1). Polynucleotides encoding these polypeptides are number of deleted N-terminal amino acid residues may retain also encompassed by the invention. Some biological or immunogenic activities. In fact, peptides In addition, the present invention provides polypeptides composed of as few as six amino acid residues may often having one or more residues deleted from the carboxy termi evoke an immune response. nus of the amino acid sequence of an albumin protein corre Accordingly, fragments of a Therapeutic protein corre 40 sponding to an albumin protein portion of an albumin fusion sponding to a Therapeutic protein portion of an albumin protein of the invention (e.g., serum albumin). In particular, fusion protein of the invention, include the full length protein C-terminal deletions may be described by the general formula as well as polypeptides having one or more residues deleted 1-n, where n is any whole integer ranging from 6 to 584, from the amino terminus of the amino acid sequence of the where 584 is the whole integer representing the total number reference polypeptide (e.g. a Therapeutic protein as disclosed 45 of amino acid residues in serum albumin (SEQ ID NO:18) in Table 1). In particular, N-terminal deletions may be minus 1. Polynucleotides encoding these polypeptides are described by the general formula m-q, where q is a whole also encompassed by the invention. integer representing the total number of amino acid residues Moreover, the present invention provides polypeptides in a reference polypeptide (e.g. a Therapeutic protein referred having one or more residues deleted from the carboxy termi to in Table 1), and m is defined as any integer ranging from 2 50 nus of an albumin fusion protein of the invention. In particu to q-6. Polynucleotides encoding these polypeptides are also lar, C-terminal deletions may be described by the general encompassed by the invention. formula 1-n, where n is any whole integer ranging from 6 to In addition, fragments of serum albumin polypeptides cor q-1, and where q is a whole integer representing the total responding to an albumin protein portion of an albumin number of amino acid residues in an albuminfusion protein of fusion protein of the invention, include the full length protein 55 the invention. Polynucleotides encoding these polypeptides as well as polypeptides having one or more residues deleted are also encompassed by the invention. from the amino terminus of the amino acid sequence of the In addition, any of the above described N- or C-terminal reference polypeptide (i.e. serum albumin). In particular. deletions can be combined to produce a N- and C-terminal N-terminal deletions may be described by the general for deleted reference polypeptide. The invention also provides mula m-585, where 585 is a whole integer representing the 60 polypeptides having one or more amino acids deleted from total number of amino acid residues in serum albumin (SEQ both the amino and the carboxyl termini, which may be ID NO: 18), and m is defined as any integer ranging from 2 to described generally as having residues m-n of a reference 579. Polynucleotides encoding these polypeptides are also polypeptide (e.g., a Therapeutic protein referred to in Table 1, encompassed by the invention. or serumalbumin (e.g., SEQID NO:18), or an albumin fusion Moreover, fragments of albumin fusion proteins of the 65 protein of the invention) where n and m are integers as invention, include the full length albumin fusion protein as described above. Polynucleotides encoding these polypep well as polypeptides having one or more residues deleted tides are also encompassed by the invention. US 8,946,156 B2 47 48 The present application is also directed to proteins contain bound DNA in 6x Sodium chloride Sodium citrate (SSC) at ing polypeptides at least 80%, 85%, 90%, 95%, 96%. 97%, about 45 degrees Celsius, followed by one or more washes in 98% or 99% identical to a reference polypeptide sequence 0.2xSSC. 0.1% c SDS at about 50-65 degrees Celsius), under (e.g., a Therapeutic protein, serum albumin protein or an highly stringent conditions (e.g. hybridization to filter bound albumin fusion protein of the invention) set forth herein, or DNA in 6x sodium chloride/Sodium citrate (SSC) at about 45 fragments thereof. In preferred embodiments, the application degrees Celsius, followed by one or more washes in 0.1xSSC, is directed to proteins comprising polypeptides at least 80%. 0.2% SDS at about 68 degrees Celsius), or under other strin 85%, 90%, 95%, 96%, 97%. 98% or 99% identical to refer gent hybridization conditions which are known to those of ence polypeptides having the amino acid sequence of N- and skill in the art (see, for example, Ausubel. F. M. et al. eds. C-terminal deletions as described above. Polynucleotides 10 1989 Current protocol in Molecular Biology. Green publish encoding these polypeptides are also encompassed by the ing associates. Inc., and John Wiley & Sons Inc., New York, at invention. pages 6.3.1-6.3.6 and 2.10.3). Polynucleotides encoding Preferred polypeptide fragments of the invention are frag these polypeptides are also encompassed by the invention. ments comprising, or alternatively, consisting of an amino By a polypeptide having an amino acid sequence at least, acid sequence that displays a Therapeutic activity and/or 15 for example, 95% “identical to a query amino acid sequence functional activity (e.g. biological activity) of the polypeptide of the present invention, it is intended that the amino acid sequence of the Therapeutic protein or serum albumin protein sequence of the Subject polypeptide is identical to the query of which the amino acid sequence is a fragment. Other pre sequence except that the Subject polypeptide sequence may ferred polypeptide fragments are biologically active frag include up to five amino acid alterations per each 100 amino ments. Biologically active fragments are those exhibiting acids of the query amino acid sequence. In other words, to activity similar, but not necessarily identical, to an activity of obtain a polypeptide having an amino acid sequence at least the polypeptide of the present invention. The biological activ 95% identical to a query amino acid sequence, up to 5% of the ity of the fragments may include an improved desired activity, amino acid residues in the Subject sequence may be inserted, or a decreased undesirable activity. deleted, or substituted with another amino acid. These alter Variants 25 ations of the reference sequence may occur at the amino- or “Variant” refers to a polynucleotide or nucleic acid differ carboxy-terminal positions of the reference amino acid ing from a reference nucleic acid or polypeptide, but retaining sequence or anywhere between those terminal positions, essential properties thereof. Generally, variants are overall interspersed either individually among residues in the refer closely similar, and, in many regions, identical to the refer ence sequence or in one or more contiguous groups within the ence nucleic acid or polypeptide. 30 reference sequence. As used herein, “variant, refers to a Therapeutic protein As a practical matter, whether any particular polypeptide is portion of an albumin fusion protein of the invention, albumin at least 80%. 85%. 90%, 95%, 96%, 97%, 98% or 99% portion of an albumin fusion protein of the invention, or identical to for instance, the amino acid sequence of an albu albumin fusion protein differing in sequence from a Thera min fusion protein of the invention or a fragment thereof peutic protein (e.g. see “therapeutic' column of Table 1), 35 (such as the Therapeutic protein portion of the albumin fusion albumin protein, and/or albumin fusion protein of the inven protein or the albumin portion of the albumin fusion protein), tion, respectively, but retaining at least one functional and/or can be determined conventionally using known computer therapeutic property thereof (e.g. atherapeutic activity and/or programs. A preferred method for determining the best over biological activity as disclosed in the “Biological Activity” all match between a query sequence (a sequence of the column of Table 1) as described elsewhere herein or other 40 present invention) and a subject sequence, also referred to as wise known in the art. Generally, variants are overall very a global sequence alignment, can be determined using the similar, and, in many regions, identical to the amino acid FASTDB computer program based on the algorithm of Brut sequence of the Therapeutic protein corresponding to a lag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a Therapeutic protein portion of an albumin fusion protein of sequence alignment the query and Subject sequences are the invention, albumin protein corresponding to an albumin 45 either both nucleotide sequences or both amino acid protein portion of an albumin fusion protein of the invention, sequences. The result of said global sequence alignment is and/or albumin fusion protein of the invention. Nucleic acids expressed as percent identity. Preferred parameters used in a encoding these variants are also encompassed by the inven FASTDB amino acid alignment are: Matrix=PAM 0. tion. k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Ran The present invention is also directed to proteins which 50 domization Group Length=0, Cutoff Score=1, Window comprise, or alternatively consist of an amino acid sequence Size=sequence length, Gap Penalty=5, Gap Size Pen which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% alty=0.05, Window Size=500 or the length of the subject or 100%, identical to, for example, the amino acid sequence amino acid sequence, whichever is shorter. of a Therapeutic protein corresponding to a Therapeutic pro If the Subject sequence is shorter than the query sequence tein portion of an albumin fusion protein of the invention 55 due to N- or C-terminal deletions, not because of internal (e.g., an amino acid sequence disclosed in the “Exemplary deletions, a manual correction must be made to the results. Identifier column of Table 1, or fragments or variants This is because the FASTDB program does not account for N thereof), albumin proteins (e.g., SEQID NO:18 or fragments and C-terminal truncations of the Subject sequence when or variants thereof) corresponding to an albumin protein por calculating global percent identity. For Subject sequences tion of an albumin fusion protein of the invention, and/or 60 truncated at the N- and C-termini, relative to the query albumin fusion proteins of the invention. Fragments of these sequence, the percent identity is corrected by calculating the polypeptides are also provided (e.g. those fragments number of residues of the query sequence that are N- and described herein). Further polypeptides encompassed by the C-terminal of the Subject sequence, which are not matched, invention are polypeptides encoded by polynucleotides aligned with a corresponding Subject residue, as a percent of which hybridize to the complement of a nucleic acid molecule 65 the total bases of the query sequence. Whether a residue is encoding an amino acid sequence of the invention under matched/aligned is determined by results of the FASTDB stringent hybridization conditions (e.g. hybridization to filter sequence alignment. This percentage is then subtracted from US 8,946,156 B2 49 50 the percent identity, calculated by the above FASTDB pro parison between sequences, available in the GCG package gram using the specified parameters, to arrive at a final per version 10.0, uses DNA parameters GAP=50 (gap creation cent identity score. This final percent identity score is what is penalty) and LEN=3 (gap extension penalty) and the equiva used for the purposes of the present invention. Only residues lent settings in protein comparisons are GAP-8 and LEN=2. to the N- and C-termini of the subject sequence, which are not The polynucleotide variants of the invention may contain matched/aligned with the query sequence, are considered for alterations in the coding regions, non-coding regions, or both. the purposes of manually adjusting the percent identity score. Especially preferred are polynucleotide variants containing That is, only query residue positions outside the farthest N alterations which produce silent Substitutions, additions, or and C-terminal residues of the Subject sequence. deletions, but do not alter the properties or activities of the For example, a 90 amino acid residue Subject sequence is 10 encoded polypeptide. Nucleotide variants produced by silent aligned with a 100 residue query sequence to determine per Substitutions due to the degeneracy of the genetic code are cent identity. The deletion occurs at the N-terminus of the preferred. Moreover, polypeptide variants in which less than subject sequence and therefore, the FASTDB alignment does 50, less than 40, less than 30, less than 20, less than 10, or not show a matching/alignment of the first 10 residues at the 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, N-terminus. The 10 unpaired residues represent 10% of the 15 deleted, or added in any combination are also preferred. Poly sequence (number of residues at the N- and C-termini not nucleotide variants can be produced for a variety of reasons, matched/total number of residues in the query sequence) so e.g., to optimize codon expression for a particular host 10% is subtracted from the percent identity score calculated (change codons in the human mRNA to those preferred by a by the FASTDB program. If the remaining 90 residues were bacterial host, such as, yeast or E. coli). perfectly matched the final percent identity would be 90%. In In a preferred embodiment, a polynucleotide encoding an another example, a 90 residue subject sequence is compared albumin portion of an albumin fusion protein of the invention with a 100 residue query sequence. This time the deletions are is optimized for expression in yeast or mammalian cells. In internal deletions so there are no residues at the N- or C-ter further preferred embodiment, a polynucleotide encoding a mini of the Subject sequence which are not matched/aligned Therapeutic protein portion of an albumin fusion protein of with the query. In this case the percent identity calculated by 25 the invention is optimized for expression in yeast or mamma FASTDB is not manually corrected. Once again, only residue lian cells. In a still further preferred embodiment, a poly positions outside the N- and C-terminal ends of the subject nucleotide encoding an albumin fusion protein of the inven sequence, as displayed in the FASTDB alignment, which are tion is optimized for expression in yeast or mammalian cells. not matched/aligned with the query sequence are manually In an alternative embodiment, a codon optimized poly corrected for. No other manual corrections are to made for the 30 nucleotide encoding a Therapeutic protein portion of an albu purposes of the present invention. min fusion protein of the invention does not hybridize to the The variant will usually have at least 75% (preferably at wild type polynucleotide encoding the Therapeutic protein least about 80%, 90%, 95% or 99%) sequence identity with a under stringent hybridization conditions as described herein. length of normal HA or Therapeutic protein which is the same In a further embodiment, a codon optimized polynucleotide length as the variant. Homology or identity at the nucleotide 35 encoding an albumin portion of an albumin fusion protein of oramino acid sequence level is determined by BLAST (Basic the invention does not hybridize to the wild type polynucle Local Alignment Search Tool) analysis using the algorithm otide encoding the albumin protein under Stringent-hybrid employed by the programs blastp, blastin, blastX, thlastn and ization conditions as described herein. In another embodi thlastx (Karlin et al., Proc. Natl. Acad. Sci. USA 87: 2264 ment, a codon optimized polynucleotide encoding an albumin 2268 (1990) and Altschul. J. Mol. Evol. 36: 290-300 (1993), 40 fusion protein of the invention does not hybridize to the wild fully incorporated by reference) which are tailored for type polynucleotide encoding the Therapeutic protein portion sequence similarity searching. or the albumin protein portion under stringent hybridization The approach used by the BLAST program is to first con conditions as described herein. sider similar segments between a query sequence and a data In an additional embodiment, polynucleotides encoding a base sequence, then to evaluate the statistical significance of 45 Therapeutic protein portion of an albumin fusion protein of all matches that are identified and finally to summarize only the invention do not comprise, or alternatively consist of the those matches which satisfy a preselected threshold of sig naturally occurring sequence of that Therapeutic protein. In a nificance. For a discussion of basic issues in similarity search further embodiment, polynucleotides encoding an albumin ing of sequence databases, see Altschulet al., (Nature Genet protein portion of an albumin fusion protein of the invention ics 6: 119-129 (1994)) which is fully incorporated by 50 do not comprise, or alternatively consist of the naturally reference. The search parameters for histogram, descriptions, occurring sequence of albumin protein. In an alternative alignments, expect (i.e. the statistical significance threshold embodiment, polynucleotides encoding an albumin fusion for reporting matches against database sequences), cutoff, protein of the invention do not comprise, or alternatively matrix and filter are at the default settings. The default scoring consist of the naturally occurring sequence of a Therapeutic matrix used by blastp, blastX, thlastin, and thlastx is the BLO 55 protein portion or the albumin protein portion. SUM62 matrix (Henikoffet al., Proc. Natl. Acad. Sci. USA Naturally occurring variants are called “allelic variants.” 89: 10915-10919 (1992), fully incorporated by reference). and refer to one of several alternate forms of agene occupying For blastin, the scoring matrix is set by the ratios of M (i.e. the a given locus on a chromosome of an organism, (Genes II, reward score for a pair of matching residues) to N (i.e. the Lewin, B., ed., John Wiley & Sons, New York (1985)). These penalty score for mismatching residues), wherein the default 60 allelic variants can vary at either the polynucleotide and/or values for M and N are 5 and -4, respectively. Four blastin polypeptide level and are included in the present invention. parameters may be adjusted as follows: Q=10 (gap creation Alternatively, non-naturally occurring variants may be pro penalty); R=10 (gap extension penalty): wink-1 (generates duced by mutagenesis techniques or by direct synthesis. word hits at every wink" position along the query); and Using known methods of protein engineering and recom gapw-16 (sets the window width within which gapped align 65 binant DNA technology, variants may be generated to ments are generated). The equivalent Blastp parameter set improve or alter the characteristics of the polypeptides of the tings were Q=9: R=2: wink=1; and gapw-32. A Bestfit com present invention. For instance, one or more amino acids can US 8,946, 156 B2 51 52 be deleted from the N-terminus or C-terminus of the polypep By comparing amino acid sequences in different species, tide of the present invention without substantial loss of bio conserved amino acids can be identified. These conserved logical function. As an example, Ron et al. (J. Biol. Chem. amino acids are likely important for protein function. In con 268: 2984-2988 (1993)) reported variant KGF proteins hav trast, the amino acid positions where substitutions have been ing heparin binding activity even after deleting 3, 8, or 27 5 tolerated by natural selection indicates that these positions are amino-terminal amino acid residues. Similarly, Interferon not critical for protein function. Thus, positions tolerating gamma exhibited up to tentimes higher activity after deleting amino acid substitution could be modified while still main 8-10amino acid residues from the carboxy terminus of this taining biological activity of the protein. protein, (Dobeliet al., J. Biotechnology 7:199-216 (1988).) The second strategy uses genetic engineering to introduce Moreover, ample evidence demonstrates that variants often 10 amino acid changes at specific positions of a cloned gene to retain a biological activity similar to that of the naturally identify regions critical for protein function. For example, site occurring protein. For example, Gayle and coworkers (J. directed mutagenesis or alanine-scanning mutagenesis (in Biol. Chem. 268:22105-22111 (1993)) conducted extensive troduction of single alanine mutations at every residue in the mutational analysis of human cytokine IL-1a. They used ran molecule) can be used. See Cunningham and Wells, Science dom mutagenesis to generate over 3,500 individual IL-1 a 15 244: 1081-1085 (1989). The resulting mutant molecules can mutants that averaged 2.5 amino acid changes per variant over then be tested for biological activity. the entire length of the molecule. Multiple mutations were As the authors state, these two strategies have revealed that examined at every possible amino acid position. The investi proteins are surprisingly tolerant of amino acid Substitutions. gators found that “most of the molecule could be altered The authors further indicate which amino acid changes are with little effect on either binding or biological activity.” In 20 likely to be permissive at certain amino acid positions in the fact, only 23 unique amino acid sequences, out of more than protein. For example, most buried (within the tertiary struc 3.500 nucleotide sequences examined, produced a protein ture of the protein) amino acid residues require nonpolar side that significantly differed in activity from wild-type. chains, whereas few features of Surface side chains are gen Furthermore, even if deleting one or more amino acids erally conserved. Moreover, tolerated conservative amino from the N-terminus or C-terminus of a polypeptide results in 25 acid substitutions involve replacement of the aliphatic or modification or loss of one or more biological functions, other hydrophobic amino acids Ala, Val, Leu and Ile: replacement biological activities may still be retained. For example, the of the hydroxyl residues Ser and Thr; replacement of the ability of a deletion variant to induce and/or to bindantibodies acidic residues Asp and Glu: replacement of the amide resi which recognize the secreted form will likely be retained dues ASn and Gln, replacement of the basic residues Lys, Arg, when less than the majority of the residues of the secreted 30 and His: replacement of the aromatic residues Phe, Tyr, and form are removed from the N-terminus or C-terminus. Trp, and replacement of the Small-sized amino acids Ala, Ser, Whether a particular polypeptide lacking N- or C-terminal Thr, Met, and Gly. Besides conservative amino acid substitu residues of a protein retains such immunogenic activities can tion, variants of the present invention include (i) polypeptides readily be determined by routine methods described herein containing Substitutions of one or more of the non-conserved and otherwise known in the art. 35 amino acid residues, where the Substituted amino acid resi Thus, the invention further includes polypeptide variants dues may or may not be one encoded by the genetic code, or which have a functional activity (e.g., biological activity and/ (ii) polypeptides containing Substitutions of one or more of or therapeutic activity). In highly preferred embodiments the the amino acid residues having a Substituent group, or (iii) invention provides variants of albumin fusion proteins that polypeptides which have been fused with or chemically con have a functional activity (e.g., biological activity and/or 40 jugated to another compound, such as a compound to increase therapeutic activity, such as that disclosed in the “Biological the stability and/or solubility of the polypeptide (for example, Activity” column in Table 1) that corresponds to one or more polyethylene glycol), (iv) polypeptide containing additional biological and/or therapeutic activities of the Therapeutic amino acids, such as, for example, an IgG Fc fusion region protein corresponding to the Therapeutic protein portion of peptide. Such variant polypeptides are deemed to be within the albumin fusion protein. Such variants include deletions, 45 the scope of those skilled in the art from the teachings herein. insertions, inversions, repeats, and Substitutions selected For example, polypeptide variants containing amino acid according to general rules known in the art so as have little Substitutions of charged amino acids with other charged or effect on activity. neutral amino acids may produce proteins with improved In preferred embodiments, the variants of the invention characteristics, such as less aggregation. Aggregation of phar have conservative substitutions. By "conservative substitu- 50 maceutical formulations both reduces activity and increases tions” is intended Swaps within groups such as replacement of clearance due to the aggregate’s immunogenic activity. See the aliphatic or hydrophobic amino acids Ala, Val, Leu and Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Rob Ile; replacement of the hydroxyl residues Ser and Thr: bins et al. Diabetes 36: 838-845 (1987); Cleland et al., Crit. replacement of the acidic residues Asp and Glu, replacement Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993). of the amide residues Asn and Gln, replacement of the basic 55 In specific embodiments, the polypeptides of the invention residues LyS, Arg, and His; replacement of the aromatic resi comprise, or alternatively, consist of fragments or variants of dues Phe, Tyr, and Trp, and replacement of the small-sized the amino acid sequence of a Therapeutic protein described amino acids Ala, Ser. Thr, Met, and Gly. herein and/or human serum albumin, and/or albumin fusion Guidance concerning how to make phenotypically silent protein of the invention, wherein the fragments or variants amino acid Substitutions is provided, for example, in Bowie et 60 have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid al., “Deciphering the Message in Protein Sequences: Toler residue additions, Substitutions, and or deletions when com ance to Amino Acid Substitutions. Science 247: 1306-1310 pared to the reference amino acid sequence. In preferred (1990), wherein the authors indicate that there are two main embodiments, the amino acid substitutions are conservative. strategies for studying the tolerance of an amino acid Nucleic acids encoding these polypeptides are also encom sequence to change. 65 passed by the invention. The first strategy exploits the tolerance of amino acid Sub The polypeptide of the present invention can be composed stitutions by natural selection during the process of evolution. of amino acids joined to each other by peptide bonds or US 8,946,156 B2 53 54 modified peptide bonds. i.e. peptide isosteres, and may con ity associated with the Therapeutic protein (or fragment or tain amino acids other than the 20 gene-encoded amino acids. variant thereof) when it is not fused to albumin. The polypeptides may be modified by either natural pro The albumin fusion proteins of the invention can be cesses. Such as post-translational processing, or by chemical assayed for functional activity (e.g., biological activity) using modification techniques which are well known in the art. 5 or routinely modifying assays known in the art, as well as Such modifications are well described in basic texts and in assays described herein. Specifically, albumin fusion proteins more detailed monographs, as well as in a Voluminous may be assayed for functional activity (e.g., biological activ research literature. Modifications can occur anywhere in a ity or therapeutic activity) using the assay referenced in the polypeptide, including the peptide backbone, the amino acid “Exemplary Activity Assay” column of Table 1. Additionally, side-chains and the amino or carboxyl termini. It will be 10 one of skill in the art may routinely assay fragments of a appreciated that the same type of modification may be present Therapeutic protein corresponding to a Therapeutic protein in the same or varying degrees at several sites in a given portion of an albumin fusion protein of the invention, for polypeptide. Also, a given polypeptide may contain many activity using assays referenced in its corresponding row of types of modifications. Polypeptides may be branched, for Table 1. Further, one of skill in the art may routinely assay example, as a result of ubiquitination, and they may be cyclic, 15 fragments of an albumin protein corresponding to an albumin with or without branching. Cyclic, branched, and branched protein portion of an albumin fusion protein of the invention, cyclic polypeptides may result from posttranslation natural for activity using assays known in the art and/or as described processes or may be made by synthetic methods. Modifica in the Examples section below. tions include acetylation, acylation, ADP-ribosylation, ami For example, in one embodiment where one is assaying for dation, covalent attachment of flavin, covalent attachment of the ability of an albumin fusion protein of the invention to a heme moiety, covalent attachment of a nucleotide or nucle bind or compete with a Therapeutic protein for binding to an otide derivative, covalent attachment of a lipid or lipid deriva anti-Therapeutic polypeptide antibody and/or anti-albumin tive, covalent attachment of phosphotidylinositol, cross-link antibody, various immunoassays known in the art can be ing, cyclization, disulfide bond formation, demethylation, used, including but not limited to competitive and non-com formation of covalent cross-links, formation of cysteine, for 25 petitive assay systems using techniques such as radioimmu mation of pyroglutamate, formylation, gamma-carboxyla noassays, ELISA (enzyme linked immunosorbent assay), tion, glycosylation, GPI anchor formation, hydroxylation, 'sandwich’ immunoassays, immunoradiometric assays, gel iodination, methylation, myristylation, oxidation, pegylation, diffusion precipitation reactions, immunodiffusion assays, in proteolytic processing, phosphorylation, prenylation, racem situ immunoassays (using colloidal gold, enzyme or radio ization, selenoylation, sulfation, transfer-RNA mediated 30 isotope labels, for example), western blots, precipitation reac addition of amino acids to proteins such as arginylation, and tions, agglutination assays (e.g. gel agglutination assays, ubiquitination, (See, for instance, PROTEINS STRUC hemagglutination assays), complement fixation assays. TURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Immunofluorescence assays, protein A assays, and immuno Creighton, W.H. Freeman and Company, New York (1993); electrophoresis assays, etc. In one embodiment, antibody POST TRANSLATIONAL COVALENT MODIFICATION 35 binding is detected by detecting a label on the primary anti OF PROTEINS, B. C. Johnson, Ed., Academic Press, New body. In another embodiment, the primary antibody is York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182: detected by detecting binding of a secondary antibody or 626-646 (1990); Rattanet al., Ann. N.Y. Acad. Sci. 663:48-62 reagent to the primary antibody. In a further embodiment, the (1992)). secondary antibody is labeled. Many means are known in the Functional Activity 40 art for detecting binding in an immunoassay and are within A polypeptide having functional activity” refers to a the scope of the present invention. polypeptide capable of displaying one or more known func In a preferred embodiment, where a binding partner (e.g. a tional activities associated with the full-length, pro-protein, receptor or a ligand) of a Therapeutic protein is identified, and/or mature form of a Therapeutic protein. Such functional binding to that binding partner by an albumin fusion protein activities include, but are not limited to, biological activity, 45 containing that Therapeutic protein as the Therapeutic pro antigenicity ability to bind (or compete with a polypeptide tein portion of the fusion can be assayed, e.g. by means for binding) to an anti-polypeptide antibody, immunogenic well-known in the art, such as, for example, reducing and ity (ability to generate antibody which binds to a specific non-reducing gel chromatography, protein affinity chroma polypeptide of the invention), ability to form multimers with tography, and affinity blotting. Seegenerally, Phizicky et al., polypeptides of the invention, and ability to bind to a receptor 50 Microbiol. Rev. 59:94-123 (1995). In another embodiment, or ligand for a polypeptide. the ability of physiological correlates of an albumin fusion A polypeptide having biological activity” refers to a protein of the present invention to bind to a substrate(s) of the polypeptide exhibiting activity similar to, but not necessarily Therapeutic polypeptide corresponding to the Therapeutic identical to, an activity of a Therapeutic protein of the present portion of the albumin fusion protein of the invention can be invention, including mature forms, as measured in a particu 55 routinely assayed using techniques known in the art. lar biological assay, with or without dose dependency. In the In an alternative embodiment, where the ability of an albu case where dose dependency does exist, it need not be iden min fusion protein of the invention to multimerize is being tical to that of the polypeptide, but rather substantially similar evaluated, association with other components of the multimer to the dose-dependence in a given activity as compared to the can be assayed, e.g. by means well-known in the art, such as, polypeptide of the present invention (i.e. the candidate 60 for example, reducing and non-reducing gel chromatography, polypeptide will exhibit greater activity or not more than protein affinity chromatography, and affinity blotting. See about 25-fold less and, preferably, not more than about ten generally, Phizicky et al., Supra. fold less activity, and most preferably, not more than about In addition, assays described herein (see Examples and three-fold less activity relative to the polypeptide of the Table 1) and otherwise known in the art may routinely be present invention). 65 applied to measure the ability of albumin fusion proteins of In preferred embodiments, an albumin fusion protein of the the present invention and fragments, variants and derivatives invention has at least one biological and/or therapeutic activ thereof to elicit biological activity and/or Therapeutic activity US 8,946,156 B2 55 56 (either in vitro or in vivo) related to either the Therapeutic human albumin and fragments of human albumin, for protein portion and/or albumin portion of the albumin fusion example those fragments disclosed in EP 322 094 (namely protein of the present invention. Other methods will be known HA (Pn), where n is 369 to 419). The albumin may be derived to the skilled artisan and are within the scope of the invention. from any vertebrate, especially any mammal, for example Albumin human, cow, sheep, or pig. Non-mammalian albumins As described above, an albumin fusion protein of the inven include, but are not limited to lien and salmon. The albumin tion comprises at least a fragment or variant of a Therapeutic portion of the albumin fusion protein may be from a different protein and at least a fragment or variant of human serum animal than the Therapeutic protein portion. albumin, which are associated with one another, preferably Generally speaking, an HA fragment or variant will be at by genetic fusion or chemical conjugation. 10 least 100 amino acids long, preferably at least 150 amino The terms, human serum albumin (HSA) and human albu acids long. The HA variant may consist of or alternatively min (HA) are used interchangeably herein. The terms, “albu comprise at least one whole domain of HA, for example min and "serum albumin' are broader, and encompass human domains 1 (amino acids 1-194 of SEQID NO: 18), 2 (amino serum albumin (and fragments and variants thereof) as well as acids 195-387 of SEQID NO: 18).3 (amino acids 388-585 of albumin from other species (and fragments and variants 15 SEQID NO:18), 1+2(1-387 of SEQID NO:18), 2+3 (195 thereof). 585 of SEQID NO:18) or 1+3 (amino acids 1-194 of SEQID As used herein, “albumin” refers collectively to albumin NO:18+amino acids 388-585 of SEQ ID NO:18). Each protein or amino acid sequence, or an albumin fragment or domain is itself made up of two homologous Subdomains variant, having one or more functional activities (e.g. biologi namely 1-105.120-194, 195-291.316-387,388-491 and 512 cal activities) of albumin. In particular, “albumin” refers to 585, with flexible inter-subdomain linker regions comprising human albuminor fragments thereof (see EP201 239, EP322 residues Lys106 to Glu1 19, Glu292 to Val315 and Glu492 to 094 WO97/24445, WO95/23857) especially the mature form Alas 11. of human albuminas shown in FIG. 15 and SEQID NO: 18, Preferably, the albumin portion of an albumin fusion pro or albumin from other vertebrates or fragments thereof, or tein of the invention comprises at least one subdomain or analogs or variants of these molecules or fragments thereof. 25 domain of HA or conservative modifications thereof. If the In preferred embodiments, the human serum albumin pro fusion is based on Subdomains, some or all of the adjacent tein used in the albumin fusion proteins of the invention linker is preferably used to link to the Therapeutic protein contains one or both of the following sets of point mutations moiety. with reference to SEQID NO: 18: Leu-407 to Ala, Leu-408 to Albumin Fusion Proteins Val, Val-409 to Ala, and Arg-410 to Ala; or Arg-410 to A, 30 The present invention relates generally to albumin fusion Lys-413 to Gln, and Lys-414 to Gln (see, e.g. International proteins and methods of treating, preventing, or ameliorating Publication No, WO95/23857, hereby incorporated in its diseases or disorders. As used herein, "albumin fusion pro entirety by reference herein). In even more preferred embodi tein’ refers to a protein formed by the fusion of at least one ments, albumin fusion proteins of the invention that contain molecule of albumin (or a fragment or variant thereof) to at one or both of above-described sets of point mutations have 35 least one molecule of a Therapeutic protein (or fragment or improved stability resistance to yeast Yap3p proteolytic variant thereof). An albumin fusion protein of the invention cleavage, allowing increased production of recombinant comprises at least a fragment or variant of a Therapeutic albumin fusion proteins expressed in yeast host cells. protein and at least a fragment or variant of human serum As used herein, a portion of albumin Sufficient to prolong albumin, which are associated with one another, preferably the therapeutic activity or shelf-life of the Therapeutic protein 40 by genetic fusion (i.e., the albumin fusion protein is generated refers to a portion of albumin sufficient in length or structure by translation of a nucleic acid in which a polynucleotide to stabilize or prolong the therapeutic activity of the protein so encoding all or a portion of a Therapeutic protein is joined that the shelf life of the Therapeutic protein portion of the in-frame with a polynucleotide encoding all or a portion of albumin fusion protein is prolonged or extended compared to albumin) or chemical conjugation to one another. The Thera the shelf-life in the non-fusion state. The albumin portion of 45 peutic protein and albumin protein, once part of the albumin the albumin fusion proteins may comprise the full length of fusion protein, may be referred to as a "portion”, “region” or the HA sequence as described above or as shown in FIG. 15, “moiety' of the albumin fusion protein. or may include one or more fragments thereofthat are capable In one embodiment, the invention provides an albumin of Stabilizing or prolonging the therapeutic activity. Such fusion protein comprising, or alternatively consisting of a fragments may be of 10 or more amino acids in length or may 50 Therapeutic protein (e.g. as described in Table 1) and a serum include about 15, 20, 25, 30, 50, or more contiguous amino albumin protein. In other embodiments, the invention pro acids from the HA sequence or may include part or all of vides an albumin fusion protein comprising, or alternatively specific domains of HA. For instance, one or more fragments consisting of a biologically active and/or therapeutically of HA spanning the first two immunoglobulin-like domains active fragment of a Therapeutic protein and a serum albumin may be used. 55 protein. In other embodiments, the invention provides an The albumin portion of the albumin fusion proteins of the albumin fusion protein comprising, or alternatively consist invention may be a variant of normal HA. The Therapeutic ing of a biologically active and/ortherapeutically active vari protein portion of the albumin fusion proteins of the invention ant of a Therapeutic protein and a serum albumin protein. In may also be variants of the Therapeutic proteins as described preferred embodiments, the serum albumin protein compo herein. The term “variants' includes insertions, deletions and 60 nent of the albumin fusion protein is the mature portion of Substitutions, either conservative or non conservative, where serum albumin. Such changes do not substantially alter one or more of the In further embodiments, the invention provides an albumin oncotic, useful ligand-binding and non-immunogenic prop fusion protein comprising, or alternatively consisting of a erties of albumin, or the active site, or active domain which Therapeutic protein, and a biologically active and/or thera confers the therapeutic activities of the Therapeutic proteins. 65 peutically active fragment of serum albumin. In further In particular, the albumin fusion proteins of the invention embodiments, the invention provides an albumin fusion pro may include naturally occurring polymorphic variants of tein comprising, or alternatively consisting of a Therapeutic US 8,946,156 B2 57 58 protein and a biologically active and/or therapeutically active tions of randomized peptides into particular loops of the HA variant of serum albumin. In preferred embodiments, the molecule and in which all possible combinations of amino Therapeutic protein portion of the albumin fusion protein is acids are represented. the mature portion of the Therapeutic protein. Such library(s) could be generated on HA or domain frag In further embodiments, the invention provides an albumin ments of HA by one of the following methods: fusion protein comprising, or alternatively consisting of a (a) randomized mutation of amino acids within one or biologically active and or therapeutically active fragment or more peptide loops of HA or HA domain fragments. Either variant of a Therapeutic protein and a biologically active and one, more or all the residues within a loop could be mutated in or therapeutically active fragment or variant of serum albu this manner (for example see FIG. 10a); min. In preferred embodiments, the invention provides an 10 (b) replacement of, or insertion into one or more loops of albumin fusion protein comprising, or alternatively consist HA or HA domain fragments (i.e., internal fusion) of a ran ing of the mature portion of a Therapeutic protein and the domized peptide(s) of length X, (where X is an amino acid mature portion of serum albumin. and n is the number of residues (for example see FIG. 10b): Preferably, the albumin fusion protein comprises HA as the (c) N-, C- or N- and C-terminal peptide?protein fusions in N-terminal portion, and a Therapeutic protein as the C-termi 15 addition to (a) and/or (b). nal portion. Alternatively, an albumin fusion protein compris The HA or HA domain fragment may also be made multi ing HA as the C-terminal portion, and a Therapeutic protein functional by grafting the peptides derived from different as the N-terminal portion may also be used. screens of different loops against different targets into the In other embodiments, the albumin fusion protein has a same HA or HA domain fragment. Therapeutic protein fused to both the N-terminus and the In preferred embodiments, peptides inserted into a loop of C-terminus of albumin. In a preferred embodiment, the human serum albumin are peptide fragments or peptide vari Therapeutic proteins fused at the N- and C-termini are the ants of the Therapeutic proteins disclosed in Table 1. More same Therapeutic proteins. In a preferred embodiment, the particularly, the invention encompasses albumin fusion pro Therapeutic proteins fused at the N- and C-termini are differ teins which comprise peptide fragments or peptide variants at ent Therapeutic proteins. In another preferred embodiment, 25 least 7 at least 8, at least 9, at least 10, at least 11, at least 12, the Therapeutic proteins fused at the N- and C-termini are at least 13, at least 14, at least 15, at least 20, at least 25, at least different Therapeutic proteins which may be used to treat or 30, at least 35, or at least 40 amino acids in length inserted into prevent the same disease, disorder, or condition (e.g. as listed a loop of human serum albumin. The invention also encom in the “Preferred IndicationY column of Table 1). In another passes albumin fusion proteins which comprise peptide frag preferred embodiment, the Therapeutic proteins fused at the 30 ments or peptide variants at least 7 at least 8, at least 9, at least N- and C-termini are different Therapeutic proteins which 10, at least 11, at least 12, at least 13, at least 14, at least 15, may be used to treat or prevent diseases or disorders (e.g. as at least 20, at least 25, at least 30, at least 35, or at least 40 listed in the “Preferred Indication Y column of Table 1) amino acids fused to the N-terminus of human serum albu which are known in the art to commonly occur in patients min. The invention also encompasses albumin fusion proteins simultaneously. 35 which comprise peptide fragments or peptide variants at least In addition to albumin fusion protein in which the albumin 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least portion is fused N-terminal and/or C-terminal of the Thera 13, at least 14, at least 15, at least 20, at least 25, at least 30, peutic protein portion, albumin fusion proteins of the inven at least 35, or at least 40 amino acids fused to the C-terminus tion may also be produced by inserting the Therapeutic pro of human serum albumin. tein or peptide of interest (e.g. Therapeutic protein X as 40 Generally, the albumin fusion proteins of the invention disclosed in Table 1) into an internal region of HA. For may have one HA-derived region and one Therapeutic pro instance, within the protein sequence of the HLA molecule a tein-derived region. Multiple regions of each protein, how number of loops or turns exist between the end and beginning ever, may be used to make an albumin fusion protein of the of C-helices, which are stabilized by disulphide bonds (see invention. Similarly, more than one Therapeutic protein may FIGS. 9-11). The loops, as determined from the crystal struc 45 be used to make an albumin fusion protein of the invention. ture of HA (FIG. 13) (PDB identifiers IAO6, IBJ5, IBKE, For instance, a Therapeutic protein may be fused to both the IBMO, IE7E to IE71 and IUOR) for the most part extendaway N- and C-terminal ends of the HA. In such a configuration, the from the body of the molecule. These loops are useful for the Therapeutic protein portions may be the same or different insertion, or internal fusion, of therapeutically active pep Therapeutic protein molecules. The structure of bifunctional tides, particularly those requiring a secondary structure to be 50 albumin fusion proteins may be represented as: X-HA-Y or functional, or Therapeutic proteins, to essentially generate an Y-HA-X. albumin molecule with specific biological activity. For example, an anti-BLySTM sclv-HA-IFNC-2b fusion Loops in human albumin structure into which peptides or may be prepared to modulate the immune response to IFNC.- polypeptides may be inserted to generate albumin fusion 2b by anti-BLySTM scFv. An alternative is making a bi (or proteins of the invention include: Val54-Asnó1, Thr76 55 even multi) functional dose of HA-fusions e.g. HA-IFNC-2b Asp89, Ala82-Glu100, Gln170-Ala176, His247-Glu252, fusion mixed with HA-anti-BLySTM schv fusion or other Glu266-Glu277, Glu280-His288, Ala362-Glu368. Lys439 HA-fusions in various ratio’s depending on function, half-life Pro447, Val462-Lys475. Thr478-Pro486, and Lys560 etc. Thr566. In more preferred embodiments, peptides or Bi- or multi-functional albumin fusion proteins may also polypeptides are inserted into the Val54-Asnó1, Gln 170 60 be prepared to target the Therapeutic protein portion of a Ala176, and/or Lys560-Thr566 loops of mature human albu fusion to a target organ or cell type via protein or peptide at the min (SEQID NO: 18). opposite terminus of HA. Peptides to be inserted may be derived from either phage As an alternative to the fusion of known therapeutic mol display or synthetic peptide libraries screened for specific ecules, the peptides could be obtained by screening libraries biological activity or from the active portions of a molecule 65 constructed as fusions to the N-, C- or N- and C-termini of with the desired function. Additionally, random peptide HA, or domain fragment of HA, oftypically 6, 8, 12, 20 or 25 libraries may be generated within particular loops or by inser or X, (where X is an amino acid (aa) and n equals the number US 8,946,156 B2 59 60 of residues) randomized amino acids, and in which all pos Expression of Fusion Proteins sible combinations of amino acids were represented. A par The albumin fusion proteins of the invention may be pro ticular advantage of this approach is that the peptides may be duced as recombinant molecules by secretion from yeast, a selected in situ on the HA molecule and the properties of the microorganism Such as a bacterium, or a human oranimal cell peptide would therefore be as selected for rather than, poten 5 line. Preferably, the polypeptide is secreted from the host tially, modified as might be the case for a peptide derived by cells. We have found that, by fusing the hCH coding sequence any other method then being attached to HA. to the HA coding sequence, either to the 5' end or 3' end, it is Additionally, the albumin fusion proteins of the invention possible to secrete the albumin fusion protein from yeast may include a linker peptide between the fused portions to without the requirement for a yeast-derived pro sequence. provide greater physical separation between the moieties and 10 This was Surprising, as other workers have found that a yeast derived pro sequence was needed for efficient secretion of thus maximize the accessibility of the Therapeutic protein hGH in yeast. portion, for instance, for binding to its cognate receptor. The For example, Hiramatsu et al. (Appl Environ Microbiol linker peptide may consist of amino acids Such that it is 56:2125 (1990); Appl Environ Microbiol 57:2052 (1991)) flexible or more rigid. 15 found that the N-terminal portion of the pro sequence in the The linker sequence may be cleavable by a protease or Mucor pusillus rennin pre-pro leader was important. Other chemically to yield the growth hormone related moiety. Pref authors, using the MFO-1 signal, have always included the erably, the protease is one which is produced naturally by the MFC-1 pro sequence when secreting hCH. The pro sequences host, for example the S. cerevisiae proteasekeX2 or equivalent were believed to assist in the folding of the hCH by acting as proteases. an intramolecular chaperone. The present invention shows Therefore, as described above, the albumin fusion proteins that HA or fragments of HA can perform a similar function. of the invention may have the following formula R1-L-R2: Hence, a particular embodiment of the invention comprises R2-L-R1; or R1-L-R2-L-R1, wherein R1 is at least one a DNA construct encoding a signal sequence effective for Therapeutic protein, peptide or polypeptide sequence, and directing secretion in yeast, particularly a yeast-derived sig not necessarily the same Therapeutic protein. L is a linker and 25 nal sequence (especially one which is homologous to the R2 is a serum albumin sequence. yeast host), and the fused molecule of the first aspect of the In preferred embodiments, Albumin fusion proteins of the invention, there being no yeast-derived pro sequence between invention comprising a Therapeutic protein have extended the signal and the mature polypeptide. shelf life compared to the shelf life the same Therapeutic The Saccharomyces cerevisiae invertase signal is a pre protein when not fused to albumin. Shelf-life typically refers 30 ferred example of a yeast-derived signal sequence. to the time period over which the therapeutic activity of a Conjugates of the kind prepared by Poznansky et al., Therapeutic protein in solution or in some other storage for (FEBS Lett. 239:18 (1988)), in which separately-prepared mulation, is stable without undue loss of therapeutic activity. polypeptides are joined by chemical cross-linking, are not Many of the Therapeutic proteins are highly labile in their contemplated. unfused state. As described below, the typical shelf-life of 35 The present invention also includes a cell, preferably a these Therapeutic proteins is markedly prolonged upon incor yeast cell transformed to express an albumin fusion protein of poration into the albumin fusion protein of the invention. the invention. In addition to the transformed host cells them Albumin fusion proteins of the invention with “prolonged selves, the present invention also contemplates a culture of or “extended shelf-life exhibit greater therapeutic activity those cells, preferably a monoclonal (clonally homogeneous) relative to a standard that has been subjected to the same 40 culture, or a culture derived from a monoclonal culture, in a storage and handling conditions. The standard may be the nutrient medium. If the polypeptide is secreted, the medium unfused full-length Therapeutic protein. When the Therapeu will contain the polypeptide, with the cells, or without the tic protein portion of the albumin fusion protein is an analog, cells if they have been filtered or centrifuged away. Many a variant, or is otherwise altered or does not include the expression systems are known and may be used, including complete sequence for that protein, the prolongation of thera 45 bacteria (for example E. coli and Bacillus subtilis), yeasts (for peutic activity may alternatively be compared to the unfused example Saccharomyces cerevisiae, Kluyveromyces lactis equivalent of that analog, variant, altered peptide or incom and Pichiapastoris, filamentous fungi (for example Aspergil plete sequence. As an example, an albumin fusion protein of lus), plant cells, animal cells and insect cells. the invention may retain greater than about 100% of the Preferred yeast strains to be used in the production of therapeutic activity, or greater than about 105%, 110%, 50 albumin fusion proteins are D88, DXY1 and BXP10, D88 120%, 130%. 150% or 200% of the therapeutic activity of a leu2-3, leu2-122, can 1, pra1, ubc4 is a derivative of parent standard when Subjected to the same storage and handling strain AH22his" (also known as DB1: see, e.g. Sleep et al. conditions as the standard when compared at a given time Biotechnology 8:42-46 (1990)). The strain contains a leu2 point. mutation which allows for auxotropic selection of 2 micron Shelf-life may also be assessed in terms of therapeutic 55 based plasmids that contain the LEU2 gene. D88 also exhibits activity remaining after storage, normalized to therapeutic a derepression of PRB1 in glucose excess. The PRB1 pro activity when storage began. Albumin fusion proteins of the moter is normally controlled by two checkpoints that monitor invention with prolonged or extended shelf-life as exhibited glucose levels and growth stage. The promoter is activated in by prolonged or extended therapeutic activity may retain wildtype yeast upon glucose depletion and entry into station greater than about 50% of the therapeutic activity, about 60%, 60 ary phase. Strain D88 exhibits the repression by glucose but 70%, 80%, or 90% or more of the therapeutic activity of the maintains the induction upon entry into stationary phase. The equivalent unfused Therapeutic protein when subjected to the PRA 1 gene encodes a yeast vacuolar protease, YscA same conditions. For example, as discussed in Example 1, an endoprotease A, that is localized in the ER. The UBC4 gene is albumin fusion protein of the invention comprising hCH in the ubiquitination pathway and is involved in targeting fused to the full length HA sequence may retain about 80% or 65 short lived and abnormal proteins for ubiquitin dependant more of its original activity in solution for periods of up to 5 degradation. Isolation of this ubc4 mutation was found to weeks or more under various temperature conditions. increase the copy number of an expression plasmid in the cell US 8,946,156 B2 61 62 and cause an increased level of expression of a desired protein of the signal peptide of human serum albumin (SEQ ID expressed from the plasmid (see, e.g. International Publica NO:29) and the last five amino acids of the mating factor tion No. WO99/00504, hereby incorporated in its entirety by alpha 1 promoter (SLDKR Residues 20-24 of SEQ ID NO: reference herein). 29), see EP-A-387 319 which is hereby incorporated by ref DXY1, a derivative of D88, has the following genotype: erence in its entirety. leu2-3, leu2-122, can1, pral, ubc4, ura3:yap3. In addition The plasmids, pPPC0005, pScCHSA, pScNHSA, and to the mutations isolated in D88, this strain also has a knock pC4:HSA were deposited on Apr. 11, 2001 at the American out of the YAP3 protease. This protease causes cleavage of Type Culture Collection, 10801 University Boulevard, mostly di-basic residues (RR, RK, KR, KK) but can also Manassas, Va. 20110-2209. Another vector useful for promote cleavage at single basic residues in proteins. Isola 10 expressing an albumin fusion protein in yeast the pSAC35 tion of this yap3 mutation resulted in higher levels of full length HSA production (see. e.g., U.S. Pat. No. 5,965,386, vector which is described in Sleep et al., BioTechnology 8:42 and Kerry-Williams et al., Yeast 14:161-169 (1998), hereby (1990) which is hereby incorporated by reference in its incorporated in their entireties by reference herein). entirety. BXP10 has the following genotype: leu2-3, leu2-122. 15 A variety of methods have been developed to operably link can 1, pra1, ubc4, ura3, yap3::URA3, lys2, hsp150::LYS2, DNA to vectors via complementary cohesive termini. For pmr1::URA3. In addition to the mutations isolated in DXY1. instance, complementary homopolymer tacts can be added to this strain also has a knockout of the PMT1 gene and the the DNA segment to be inserted to the vector DNA. The HSP150 gene. The PMT1 gene is a member of the evolution vector and DNA segment are then joined by hydrogen bond arily conserved family of dolichyl-phosphate-D-mannose ing between the complementary homopolymeric tails to form protein O-mannosyltransferases (Pmts). The transmembrane recombinant DNA molecules. topology of Pmt1p Suggests that it is an integral membrane Synthetic linkers containing one or more restriction sites protein of the endoplasmic reticulum with a role in O-linked provide an alternative method of joining the DNA segment to glycosylation. This mutation serves to reduce/eliminate vectors. The DNA segment, generated by endonuclease O-linked glycosylation of HSA fusions (see, e.g., Interna 25 restriction digestion, is treated with bacteriophage T4 DNA tional Publication No. WO00/44772, hereby incorporated in polymerase or E. coli DNA polymerase 1, enzymes that its entirety by reference herein). Studies revealed that the Hsp remove protruding. Y-single-stranded termini with their 3' 150 protein is inefficiently separated from rHA by ion 5'-exonucleolytic activities, and fill in recessed 3'-ends with exchange chromatography. The mutation in the HSP150 gene their polymerizing activities. removes a potential contaminant that has proven difficult to 30 The combination of these activities therefore generates remove by standard purification techniques. See, e.g., U.S. blunt-ended DNA segments. The blunt-ended segments are Pat. No. 5,783,423, hereby incorporated in its entirety by then incubated with a large molar excess of linker molecules reference herein. in the presence of an enzyme that is able to catalyze the The desired protein is produced in conventional ways, for ligation of blunt-ended DNA molecules, such as bacterioph example from a coding sequence inserted in the host chromo 35 age T4 DNA ligase. Thus, the products of the reaction are Some or on a free plasmid. The yeasts are transformed with a DNA segments carrying polymeric linker sequences at their coding sequence for the desired protein in any of the usual ends. These DNA segments are then cleaved with the appro ways, for example electroporation. Methods for transforma priate restriction enzyme and ligated to an expression vector tion of yeast by electroporation are disclosed in Becker & that has been cleaved with an enzyme that produces termini Guarente (1990) Methods Enzymol. 194, 182. 40 compatible with those of the DNA segment. Successfully transformed cells, i.e. cells that contain a Synthetic linkers containing a variety of restriction endo DNA construct of the present invention, can be identified by nuclease sites are commercially available from a number of well known techniques. For example, cells resulting from the Sources including International Biotechnologies Inc, New introduction of an expression construct can be grown to pro Haven, Conn., USA. duce the desired polypeptide. Cells can be harvested and 45 A desirable way to modify the DNA in accordance with the lysed and their DNA content examined for the presence of the invention, if, for example, HA variants are to be prepared, is DNA using a method such as that described by Southern to use the polymerase chain reaction as disclosed by Saiki et (1975).J. Mol. Biol. 98,503 or Berent et al. (1985) Biotech. 3, al. (1988) Science 239, 487-491. In this method the DNA to 208. Alternatively, the presence of the protein in the superna be enzymatically amplified is flanked by two specific oligo tant can be detected using antibodies. 50 nucleotide primers which themselves become incorporated Useful yeast plasmid vectors include pRS403-406 and into the amplified DNA. The specific primers may contain pRS413-416 and are generally available from Stratagene restriction endonuclease recognition sites which can be used Cloning Systems, La Jolla, Calif. 92037, USA. Plasmids for cloning into expression vectors using methods known in pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating the art. plasmids (YIps) and incorporate the yeast selectable markers 55 Exemplary genera of yeast contemplated to be useful in the HIS3, 7RPI. LEU2 and URA3. Plasmids pRS413-416 are practice of the present invention as hosts for expressing the Yeast Centromere plasmids (Ycps). albumin fusion proteins are Pichia (formerly classified as Preferred vectors for making albumin fusion proteins for Hansenula), Saccharomyces, Kluyveromyces, Aspergillus, expression in yeast include pPPC0005, pScCHSA, pSc Candida, Torulopsis, Torulaspora, Schizosaccharomyces, NHSA, and pC4:HSA which are described in detail in 60 Citeromyces, Pachysolen, Zygosaccharomyces, Debaromy Example 2. FIG. 4 shows a map of the pPPC0005plasmid that ces, Trichoderma, Cephalosporium, Humicola, Mucor, Neu can be used as the base vector into which polynucleotides rospora, Yarrowia, Metschunikowia, Rhodosporidium, Leu encoding Therapeutic proteins may be cloned to form HA cosporidium, BorryOascus, Sporidiobolus, Endomycopsis, fusions. It contains a PRB1 S. cerevisiae promoter (PRB1p), and the like. Preferred genera are those selected from the a Fusion leader sequence (FL), DNA encoding HA (rhA) and 65 group consisting of Saccharomyces, Schizosaccharomyces, an ADH1 S. cerevisiae terminator sequence. The sequence of Kluyveronmyces, Pichia and Torulaspora. Examples of Sac the fusion leader sequence consists of the first 19 amino acids charomyces spp., are S. cerevisiae, S. italicus and S. rouxii. US 8,946,156 B2 63 64 Examples of Kluyveromyces spp., are K. fragilis, K. lactis Gen. Microbiol. 132, 3459-3465 include information on and K. marxianus. A Suitable Torulaspora species is T. del Hansenula Vectors and transformation, Suitable promoters brueckii. Examples of Pichia (Hansenula) spp. are Pangusta being MOX1 and FMD1; whilst EP 361991, Fleer et al. (formerly H. polymorpha), P anomala (formerly H. (1991) and other-publications from Rhone-Poulenc Rorer anomala) and P. pastoris. Methods for the transformation of 5 teach how to express foreign proteins in Kluyveromyces spp. S. cerevisiae are taught generally in EP 251744, EP258 067 a suitable promoter being PGK1. and WO 90/01063, all of which are incorporated herein by The transcription termination signal is preferably the 3' reference. flanking sequence of a eukaryotic gene which contains proper Preferred exemplary species of Saccharomyces include S. signals for transcription termination and polyadenylation. cerevisiae, S. italicus, S. diastaticus, and Zygosaccharomyces 10 Suitable 3' flanking sequences may, for example, be those of rouxii. Preferred exemplary species of Kluyveromyces the gene naturally linked to the expression control sequence include K. fragilis and K. lactis. Preferred exemplary species used, i.e. may correspond to the promoter. Alternatively, they of Hansenula include H. polymorpha (now Pichia angusta), may be different in which case the termination signal of the S. H. anomala (now Pichia anomala), and Pichia capsulata. cerevisiae ADH1 gene is preferred. Additional preferred exemplary species of Pichia include P 15 The desired albumin fusion protein may be initially pastoris. Preferred exemplary species of Aspergillus include expressed with a secretion leader sequence, which may be A. niger and A. nidulans. Preferred exemplary species of any leader effective in the yeast chosen. Leaders useful in S. Yarrowia include Y lipolytica. Many preferred yeast species cerevisiae include that from the mating factor C. polypeptide are available from the ATCC(R) (American Type Culture Col (MFO-1) and the hybrid leaders of EP-A-387 319. Such lead lection). For example, the following preferred yeast species ers (or signals) are cleaved by the yeast before the mature are available from the ATCC(R) and are useful in the expres albumin is released into the surrounding medium. Further sion of albumin fusion proteins: Saccharomyces cerevisiae such leaders include those of S. cerevisiae invertase (SUC2) Hansen, teleomorph strain BY4743 yap3 mutant (ATCC(R) disclosed in JP 62-096086 (granted as 91 1036516), acid Accession No. 4022731); Saccharomyces cerevisiae Hansen, phosphatase (PH05), the pre-sequence of MFO-1, 0 gluca teleomorph strain BY4743 hsp150 mutant (ATCCR) Acces 25 nase (BGL2) and killer toxin: S, diastaticus glucoamylase II; sion No. 4021266); Saccharomyces cerevisiae Hansen, teleo S. Carlsbergensis C-galactosidase (MEL1); K. lactis killer morph strain BY4743 pmtl mutant (ATCC(R) Accession No. toxin; and Candida glucoamylase. 4023.792); Saccharomyces cerevisiae Hansen, teleomorph Additional Methods of Recombinant and Synthetic Produc (ATCCR) Accession Nos. 20626; 4.4773: 44774; and 62995); tion of Albumin Fusion Proteins Saccharomyces diastaticus Andrews et Gilliland ex van der 30 The present invention also relates to Vectors containing a Walt, teleomorph (ATCCR) Accession No. 62987); Kluyvero polynucleotide encoding an albumin fusion protein of the myces lactis (Dombrowski) van der Walt, teleomorph present invention, host cells, and the production of albumin (ATCCR) Accession No. 76492); Pichia angusta (Teunisson fusion proteins by synthetic and recombinant techniques. The et al.) Kurtzman, teleomorph deposited as Hansenula poly vector may be, for example, a phage, plasmid, viral, or retro morpha de Morais et Maia, teleomorph (ATCC(R) Accession 35 viral vector. Retroviral vectors may be replication competent No. 26012); Aspergillus niger van Tieghem, anamorph or replication defective. In the latter case, viral propagation (ATCCR) Accession No. 9029); Aspergillus niger van generally will occur only in complementing host cells. Tieghem, anamorph (ATCCR) Accession No. 16404); The polynucleotides encoding albumin fusion proteins of Aspergillus nidulans (Eidam) Winter, anamorph (ATCC(R) the invention may bejoined to a vector containing a selectable Accession No. 48756); and Yarrowia lipolytica (Wickerham 40 marker for propagation in a host. Generally, a plasmid vector et al.) van der Walt et von Arx, teleomorph (ATCCR) Acces is introduced in a precipitate, such as a calcium phosphate sion No. 201847). precipitate, or in a complex with a charged lipid. If the vector Suitable promoters for S. cerevisiae include those associ is a virus, it may be packaged in vitro using an appropriate ated with the PGK1 gene, GAL1 or GAL10 genes, CYC1, packaging cell line and then transduced into host cells. PHOS, TRP1, ADH1, ADH2, the genes for glyceraldehyde 45 The polynucleotide insert should be operatively linked to 3-phosphate dehydrogenase, hexokinase, pyruvate decar an appropriate promoter, Such as the phage lambda PL pro boxylase, phosphofructokinase, triose phosphate isomerase, moter, the E. colilac, trp, phoA and tac promoters, the SV40 phosphoglucose isomerase, glucokinase, alpha-mating factor early and late promoters and promoters of retroviral LTRs, to pheromone, a mating factor pheromone, the PRB1 pro name a few. Other suitable promoters will be known to the moter, the GUT2 promoter, the GPD1 promoter, and hybrid 50 skilled artisan. The expression constructs will further contain promoters involving hybrids of parts of 5' regulatory regions sites for transcription initiation, termination, and, in the tran with parts of 5' regulatory regions of other promoters or with scribed region, a ribosome binding site for translation. The upstream activation sites (e.g. the promoter of EP-A-258 coding portion of the transcripts expressed by the constructs 067). will preferably include a translation initiating codon at the Convenient regulatable promoters for use in Schizosaccha 55 beginning and a termination codon (UAA, UGA or UAG) romyces pombe are the thiamine-repressible promoter from appropriately positioned at the end of the polypeptide to be the nmtgene as described by Maundrell (1990).J. Biol. Chem. translated. 265, 10857-10864 and the glucose repressiblejbpl gene pro As indicated, the expression vectors will preferably moter as described by Hoffman & Winston (1990) Genetics include at least one selectable marker. Such markers include 124, 807-816. 60 dihydrofolate reductase, G418, glutamine synthase, or neo Methods of transforming Pichia for expression of foreign mycin resistance for eukaryotic cell culture, and tetracycline, genes are taught in, for example, Cregg et al. (1993), and kanamycin or amplicillin resistance genes for culturing in E. various Phillips patents (e.g. U.S. Pat. No. 4,857.467, incor coli and other bacteria. Representative examples of appropri porated herein by reference), and Pichia expression kits are ate hosts include, but are not limited to, bacterial cells, such as commercially available from Invitrogen BV. Leek, Nether 65 E. coli, Streptomyces and Salmonella typhimuritum cells: lands, and Invitrogen Corp., San Diego, Calif. Suitable pro fungal cells, such as yeast cells (e.g., Saccharomyces cerevi moters include AOX1 and AOX2. Gleeson et al. (1986) J. siae or Pichia pastoris (ATCC Accession No. 201178)): US 8,946,156 B2 65 66 insect cells such as Drosophila S2 and Spodoptera Sf9 cells: synthase expression system and components thereof are animal cells such as CHO, COS, NSO, 293, and Bowes mela detailed in PCT publications: WO87/04462: WO86/05807; noma cells; and plant cells. Appropriate culture mediums and WO89/01036: WO89/10404; and WO91/06657, which are conditions for the above-described host cells are known in the hereby incorporated in their entireties by reference herein. art. Additionally, glutamine synthase expression vectors can be Among vectors preferred for use in bacteria include obtained from Lonza Biologics, Inc. (Portsmouth, N.H.). pOE70, pGE60 and pQE-9, available from QIAGEN, Inc.: Expression and production of monoclonal antibodies using a pBluescript vectors, Phagescript vectors, pNH8A, pNH 16a, GS expression system in murine myeloma cells is described pNH18A, pNH46A, available from Stratagene Cloning Sys in Bebbington et al. Bio/Technology 10:169 (1992) and in tems, Inc.; and ptrc99a, pKK223-3, pKK233-3, p)R540, 10 pRIT5 available from Pharmacia Biotech, Inc. Among pre Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are ferred eukaryotic vectors are pWLNEO, pSV2CAT, pCG-44, herein incorporated by reference. pXT1 and pSG available from Stratagene; and pSVK3, The present invention also relates to host cells containing pBPV, pMSG and pSVL available from Pharmacia. Preferred the above-described vector constructs described herein, and expression vectors for use in yeast systems include, but are 15 additionally encompasses host cells containing nucleotide not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, sequences of the invention that are operably associated with pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, one or more heterologous control regions (e.g., promoter pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available and/or enhancer) using techniques known of in the art. The from Invitrogen, Carlbad, Calif.). Other suitable vectors will host cell can be a higher eukaryotic cell. Such as a mammalian be readily apparent to the skilled artisan. cell (e.g., a human derived cell), or a lower eukaryotic cell, In one embodiment, polynucleotides encoding an albumin Such as a yeast cell, or the host cell can be a prokaryotic cell, fusion protein of the invention may be fused to signal Such as a bacterial cell. A host strain may be chosen which sequences which will direct the localization of a protein of the modulates the expression of the inserted gene sequences, or invention to particular compartments of a prokaryotic or modifies and processes the gene product in the specific fash eukaryotic cell and/or direct the secretion of a protein of the 25 ion desired. Expression from certain promoters can be invention from a prokaryotic or eukaryotic cell. For example, elevated in the presence of certain inducers; thus expression in E. coli, one may wish to direct the expression of the protein of the genetically engineered polypeptide may be controlled. to the periplasmic space. Examples of signal sequences or Furthermore, different host cells have characteristics and spe proteins (or fragments thereof) to which the albumin fusion cific mechanisms for the translational and post-translational proteins of the invention may be fused in order to direct the 30 processing and modification (e.g. phosphorylation, cleavage) expression of the polypeptide to the periplasmic space of of proteins. Appropriate cell lines can be chosen to ensure the bacteria include, but are not limited to, the pelB signal desired modifications and processing of the foreign protein sequence, the maltose binding protein (MBP) signal expressed. sequence, MBP, the ompA signal sequence, the signal Introduction of the nucleic acids and nucleic acid con sequence of the periplasmic E. coli heat-labile enterotoxin 35 structs of the invention into the host cell can be effected by B-Subunit, and the signal sequence of alkaline phosphatase. calcium phosphate transfection. DEAE-dextran mediated Several vectors are commercially available for the construc transfection, cationic lipid-mediated transfection, electropo tion of fusion proteins which will direct the localization of a ration, transduction, infection, or other methods. Such meth protein, such as the pMAL series of vectors (particularly the ods are described in many standard laboratory manuals, such pMAL-p series) available from New England Biolabs. In a 40 as Davis et al. Basic Methods In Molecular Biology (1986). It specific embodiment, polynucleotides albumin fusion pro is specifically contemplated that the polypeptides of the teins of the invention may be fused to the pelB pectate lyase present invention may in fact be expressed by a host cell signal sequence to increase the efficiency of expression and lacking a recombinant vector. purification of Such polypeptides in Gram-negative bacteria. In addition to encompassing host cells containing the vec See, U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents of 45 tor constructs discussed herein, the invention also encom which are herein incorporated by reference in their entireties. passes primary, secondary, and immortalized host cells of Examples of signal peptides that may be fused to an albu Vertebrate origin, particularly mammalian origin, that have min fusion protein of the invention in order to direct its been engineered to delete or replace endogenous genetic secretion in mammalian cells include, but are not limited to material (e.g. the coding sequence corresponding to a Thera the MPIF-1 signal sequence (e.g. amino acids 1-21 of Gen 50 peutic protein may be replaced with an albuminfusion protein Bank Accession number AAB51134), the stanniocalcin sig corresponding to the Therapeutic protein), and/or to include nal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:34), genetic material (e.g. heterologous polynucleotide sequences and a consensus signal sequence (MPTWAWWLFLVLLLA Such as for example, an albumin fusion protein of the inven LWAPARG, SEQID NO:35). A suitable signal sequence that tion corresponding to the Therapeutic protein may be may be used in conjunction with baculoviral expression sys 55 included). The genetic material operably associated with the tems is the gp67 signal sequence (e.g., amino acids 1-19 of endogenous polynucleotide may activate, alter, and/or GenBank Accession Number AAA72759). amplify endogenous polynucleotides. Vectors which use glutamine synthase (GS) or DHFR as In addition, techniques known in the art may be used to the selectable markers can be amplified in the presence of the operably associate heterologous polynucleotides (e.g. poly drugs methionine SulphoXimine or methotrexate, respec 60 nucleotides encoding an albumin protein, or a fragment or tively. An advantage of glutamine synthase based vectors are variant thereof) and/or heterologous control regions (e.g. pro the availability of cell lines (e.g., the murine myeloma cell moter and or enhancer) with endogenous polynucleotide line, NSO) which are glutamine synthase negative. sequences encoding a Therapeutic protein via homologous Glutamine synthase expression systems can also function in recombination (see, e.g. U.S. Pat. No. 5,641,670, issued Jun. glutamine synthase expressing cells (e.g. Chinese Hamster 65 24, 1997: International Publication Number WO 96/2941.1; Ovary (CHO) cells) by providing additional inhibitor to pre International Publication Number WO 94/12650; Koller et vent the functioning of the endogenous gene. A glutamine al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989): and US 8,946,156 B2 67 68 Zijlstra et al., Nature 342:435-438 (1989), the disclosures of initial modified methionine residue, in some cases as a result each of which are incorporated by reference in their entire of host-mediated processes. Thus, it is well known in the art ties). that the N-terminal methionine encoded by the translation Albumin fusion proteins of the invention can be recovered initiation codon generally is removed with high efficiency and purified from recombinant cell cultures by well-known from any protein after translation in all eukaryotic cells. methods including ammonium sulfate or ethanol precipita While the N-terminal methionine on most proteins also is tion, acid extraction, anion or cation exchange chromatogra efficiently removed in most prokaryotes, for some proteins, phy, phosphocellulose chromatography, hydrophobic inter this prokaryotic removal process is inefficient, depending on action chromatography, affinity chromatography, the nature of the amino acid to which the N-terminal methion hydroxylapatite chromatography, hydrophobic charge inter 10 ine is covalently linked. action chromatography and lectin chromatography. Most In one embodiment, the yeast Pichia pastoris is used to preferably, high performance liquid chromatography express albumin fusion proteins of the invention in a eukary (“HPLC) is employed for purification. otic system. Pichia pastoris is a methylotrophic yeast which In preferred embodiments the albumin fusion proteins of can metabolize methanol as its sole carbon source. A main the invention are purified using Anion Exchange Chromatog 15 step in the methanol metabolization pathway is the oxidation raphy including, but not limited to chromatography on of methanol to formaldehyde using O. This reaction is cata Q-Sepharose, DEAE Sepharose, poros HQ, poros DEAE, lyzed by the enzyme alcohol oxidase. In order to metabolize Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/ methanol as its sole carbon Source. Pichia pastoris must Source Q and DEAE, Fractogel Q and DEAE columns. generate high levels of alcohol oxidase due, in part, to the In specific embodiments the albumin fusion proteins of the relatively low affinity of alcohol oxidase for O. Conse invention are purified using Cation Exchange Chromatogra quently, in a growth medium depending on methanol as a phy including, but not limited to, SP-sepharose, CM main carbon source, the promoter region of one of the two sepharose, poros HS, poros CM, Toyopearl SP. Toyopearl alcohol oxidase genes (AOX1) is highly active. In the pres CM, Resource Source S and CM, Fractogel S and CM col ence of methanol, alcohol oxidase produced from the AOX1 umns and their equivalents and comparables. 25 gene comprises up to approximately 30% of the total soluble In specific embodiments the albumin fusion proteins of the protein in Pichia pastoris. See Ellis, S. B. et al. Mol. Cell. invention are purified using Hydrophobic Interaction Chro Biol. 5:111-21 (1985): Koutz, P. J., et al., Yeast 5:167-77 matography including, but not limited to, Phenyl, Butyl, (1989); Tschopp, J. F. et al., Nucl. Acids Res. 15:3859-76 Methyl, Octyl, Hexyl-sepharose, poros Phenyl, Butyl, (1987). Thus, a heterologous coding sequence, Such as, for Methyl, Octyl, Hexyl, Toyopearl Phenyl, Butyl, Methyl, 30 example, a polynucleotide of the present invention, under the Octyl, Hexyl Resource/Source Phenyl, Butyl, Methyl, Octyl, transcriptional regulation of all or part of the AOX1 regula Hexyl, Fractogel Phenyl, Butyl, Methyl, Octyl, Hexyl col tory sequence is expressed at exceptionally high levels in umns and their equivalents and comparables. Pichia yeast grown in the presence of methanol. In specific embodiments the albumin fusion proteins of the In one example, the plasmid vector pPIC9K is used to invention are purified using Size Exclusion Chromatography 35 express DNA encoding an albumin fusion protein of the including, but not limited to, Sepharose S100, S200, S300, invention, as set forth herein, in a Pichea yeast system essen Superdex resin columns and their equivalents and compa tially as described in “Pichia Protocols: Methods in Molecu rables. lar Biology. D. R. Higgins and J. Cregg, eds. The Humana In specific embodiments the albumin fusion proteins of the Press, Totowa, N.J., 1998. This expression vector allows invention are purified using Affinity Chromatography includ 40 expression and secretion of a polypeptide of the invention by ing, but not limited to, Mimetic Dye affinity, peptide affinity virtue of the strong AOX1 promoter linked to the Pichia and antibody affinity columns that are selective for either the pastoris alkaline phosphatase (PHO) secretory signal peptide HSA or the “fusion target molecules. (i.e., leader) located upstream of a multiple cloning site. In preferred embodiments albumin fusion proteins of the Many other yeast vectors could be used in place of invention are purified using one or more Chromatography 45 pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, methods listed above. In other preferred embodiments, albu pPICZ, pGAPZ pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, min fusion proteins of the invention are purified using one or pHIL-S1, pPIC3.5K, and PAO815, as one skilled in the art more of the following Chromatography columns, Q would readily appreciate, as long as the proposed expression sepharose FF column, SPSepharose FF column, Q Sepharose construct provides appropriately located signals for transcrip High Performance Column, Blue Sepharose FF column, Blue 50 tion, translation, Secretion (if desired), and the like, including Column, Phenyl Sepharose FF column, DEAE Sepharose FF, an in-frame AUG as required. or Methyl Column. In another embodiment, high-level expression of a heter Additionally, albumin fusion proteins of the invention may ologous coding sequence, Such as, for example, a polynucle be purified using the process described in International Pub otide encoding an albumin fusion protein of the present inven lication No. WO00/44772 which is herein incorporated by 55 tion, may be achieved by cloning the heterologous reference in its entirety. One of skill in the art could easily polynucleotide of the invention into an expression vector Such modify the process described therein for use in the purifica as, for example, pGAPZ or pGAPZalpha, and growing the tion of albumin fusion proteins of the invention. yeast culture in the absence of methanol. Albumin fusion proteins of the present invention may be In addition, albumin fusion proteins of the invention can be recovered from: products of chemical synthetic procedures; 60 chemically synthesized using techniques known in the art and products produced by recombinant techniques from a (e.g. see Creighton, 1983, Proteins: Structures and Molecular prokaryotic or eukaryotic host, including, for example, bac Principles, W.H. Freeman & Co. N.Y. and Hunkapiller et al. terial, yeast, higher plant, insect, and mammalian cells. Nature, 310:105-111 (1984)). For example, a polypeptide Depending upon the host employed in a recombinant produc corresponding to a fragment of a polypeptide can be synthe tion procedure, the polypeptides of the present invention may 65 sized by use of a peptide synthesizer. Furthermore, if desired, be glycosylated or may be non-glycosylated. In addition, nonclassical amino acids or chemical amino acid analogs can albumin fusion proteins of the invention may also include an be introduced as a Substitution or addition into the polypep US 8,946,156 B2 69 70 tide sequence. Non-classical amino acids include, but are not Nucl. Med. Biol. 26(8):943-50 (1999), which are hereby limited to, to the D-isomers of the common amino acids, incorporated by reference in their entirety. 2,4-diaminobutyric acid, C.-amino isobutyric acid, 4-ami As mentioned, the albumin fusion proteins of the invention nobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-AhX, may be modified by either natural processes. Such as post 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, translational processing, or by chemical modification tech 3-amino propionic acid, ornithine, norleucine, norvaline, niques which are well known in the art. It will be appreciated hydroxyproline, sarcosine, citrulline, homocitrulline, cystic that the same type of modification may be present in the same acid, t-butylglycine, t-butylalanine, phenylglycine, cyclo or varying degrees at several sites in a given polypeptide. hexylalanine, b-alanine, fluoro-amino acids, designer amino Polypeptides of the invention may be branched, for example, acids Such as b-methylamino acids, Ca-methylamino acids, 10 as a result of ubiquitination, and they may be cyclic, with or Na-methyl amino acids, and amino acid analogs in general. without branching. Cyclic, branched, and branched cyclic Furthermore, the amino acid can be D (dextrorotary) or L polypeptides may result from posttranslation natural pro (levorotary). cesses or may be made by synthetic methods. Modifications The invention encompasses albumin fusion proteins of the include acetylation, acylation. ADP-ribosylation, amidation, present invention which are differentially modified during or 15 covalent attachment of flavin, covalent attachment of a heme after translation, e.g. by glycosylation, acetylation, phospho moiety, covalent attachment of a nucleotide or nucleotide rylation, amidation, derivatization by known protecting/ derivative, covalent attachment of a lipid or lipid derivative, blocking groups, proteolytic cleavage, linkage to an antibody covalent attachment of phosphotidylinositol, cross-linking, molecule or other cellular ligand, etc. Any of numerous cyclization, disulfide bond formation, demethylation, forma chemical modifications may be carried out by known tech tion of covalent cross-links, formation of cysteine, formation niques, including but not limited, to specific chemical cleav of pyroglutamate, formylation, gamma-carboxylation, glyco age by cyanogen bromide, trypsin, chymotrypsin, papain, V8 Sylation, GPI anchor formation, hydroxylation, iodination, protease, NaBH; acetylation, formylation, oxidation, reduc methylation, myristylation, oxidation, pegylation, pro tion; metabolic synthesis in the presence oftunicamycin; etc. teolytic processing, phosphorylation, prenylation, racemiza Additional post-translational modifications encompassed 25 tion, selenoylation, sulfation, transfer-RNA mediated addi by the invention include, for example, e.g., N-linked or tion of amino acids to proteins such as arginylation, and O-linked carbohydrate chains, processing of N-terminal or ubiquitination. (See, for instance, PROTEINS STRUC C-terminal ends), attachment of chemical moieties to the TURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. amino acid backbone, chemical modifications of N-linked or Creighton, W.H. Freeman and Company, New York (1993): O-linked carbohydrate chains, and addition or deletion of an 30 POST TRANSLATIONAL COVALENT MODIFICATION N-terminal methionine residue as a result of procaryotic host OF PROTEINS, B. C. Johnson, Ed., Academic Press, New cell expression. The albumin fusion proteins may also be York, pgs. 1-12 (1983): Seifter et al. Meth. Enzymol. 182: modified with a detectable label, such as an enzymatic, fluo 626-646 (1990); Rattanet al., Ann. N.Y. Acad. Sci. 663:48-62 rescent, isotopic or affinity label to allow for detection and (1992)). isolation of the protein. 35 Albumin fusion proteins of the invention and antibodies Examples of Suitable enzymes include horseradish peroxi that bind a Therapeutic protein or fragments or variants dase, alkaline phosphatase, beta-galactosidase, or acetylcho thereof can be fused to marker sequences, such as a peptide to linesterase; examples of suitable prosthetic group complexes facilitate purification. In preferred embodiments, the marker include streptavidin/biotin and avidin/biotin; examples of amino acid sequence is a hexa-histidine peptide. Such as the suitable fluorescent materials include umbelliferone, fluores 40 tag provided in a pCE vector (QIAGEN, Inc. 9259 Eton cein, fluorescein isothiocyanate, rhodamine, dichlorotriazi Avenue, Chatsworth, Calif. 91311), among others, many of nylamine fluorescein, dansyl chloride or phycoerythrin; an which are commercially available. As described in Gentz et example of a luminescent material includes luminol; al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for examples of bioluminescent materials include luciferase, instance, hexa-histidine provides for convenient purification luciferin, and aequorin; and examples of Suitable radioactive 45 of the fusion protein. Other peptide tags useful for purifica material include iodine ('''I, ‘I, ‘I, ''I), carbon (''C), tion include, but are not limited to the “HA’ tag, which sulfur (S), tritium (H), indium (''' In, In, "In, corresponds to an epitope derived from the influenza hemag ''"In), technetium (Tc, ''"Tc), thallium ('Ti), gallium glutinin protein (Wilson et al. Cell 37:767 (1984)) and the (*Ga, Ga), palladium (Pd), molybdenum (Mo), xenon “flag” tag. (Xe), fluorine (18F), 'Sim, 77Lu, 15°Gd, 14°Pm, 140La, 50 Further, an albumin fusion protein of the invention may be 175Yb, 166Ho, 98y. 7Sc, 18.Re, 188Re, 142Pr 105Rh, and Ru. conjugated to a therapeutic moiety Such as a cytotoxin, e.g. a In specific embodiments, albumin fusion proteins of the cytostatic or cytocidal agent, a therapeutic agent or a radio present invention or fragments or variants thereofare attached active metal ion, e.g. alpha-emitters such as, for example, to macrocyclic chelators that associate with radiometal ions, 213Bi. A cytotoxin or cytotoxic agent includes any agent that including but not limited to, "77Lu, Y. Ho, and 'Sim, to 55 is detrimental to cells. Examples include paclitaxol, cytocha polypeptides. In a preferred embodiment, the radiometal ion lasin B, gramicidin D, ethidium bromide, emetime, mitomy associated with the macrocyclic chelators is '''In. In another cin, etoposide, tenoposide, Vincristine, vinblastine, colchicin, preferred embodiment, the radiometalion associated with the doxorubicin, daunorubicin, dihydroxy anthracin dione, macrocyclic chelator is 'Y. In specific embodiments, the mitoxantrone, mithramycin, actinomycin D, 1-dehydrotest macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N, 60 osterone, glucocorticoids, procaine, tetracaine, lidocaine, N',N',N'-tetraacetic acid (DOTA). In other specific embodi propranolol, and puromycin and analogs or homologs ments, DOTA is attached to an antibody of the invention or thereof. Therapeutic agents include, but are not limited to fragment thereof via linker molecule. Examples of linker antimetabolites (e.g. methotrexate, 6-mercaptopurine, molecules useful for conjugating DOTA to a polypeptide are 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alky commonly known in the art—see, for example, DeNardo et 65 lating agents (e.g., mechlorethamine, thioepa chlorambucil, al. Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al., melphalan, carmustine (BSNU) and lomustine (CCNU), Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al. cyclothosphamide, buSulfan, dibromomannitol, Streptozoto US 8,946,156 B2 71 72 cin, mitomycin C, and cis-dichlorodiamine platinum (II) 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, (DDP) cisplatin), anthracyclines (e.g., daunorubicin (for 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, merly daunomycin) and doxorubicin), antibiotics (e.g., dac 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, tinomycin (formerly actinomycin), bleomycin, mithramycin, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, and anthramycin (AMC)), and anti-mitotic agents (e.g., vin 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, cristine and vinblastine). 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 The conjugates of the invention can be used for modifying kDa. a given biological response, the therapeutic agent or drug As noted above, the polyethylene glycol may have a moiety is not to be construed as limited to classical chemical branched structure. Branched polyethylene glycols are therapeutic agents. For example, the drug moiety may be a 10 described, for example, in U.S. Pat. No. 5,643,575; Morpurgo protein or polypeptide possessing a desired biological activ et al. Appl. Biochem. Biotechnol. 56:59-72 (1996): Vorobjev ity. Such proteins may include, for example, a toxin Such as et al. Nucleosides Nucleotides 18:2745-2750 (1999); and abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a Caliceti et c1. Bioconjug. Chem. 10:638-646 (1999), the dis protein Such as tumor necrosis factor, alpha-interferon, B-in closures of each of which are incorporated herein by refer terferon, nerve growth factor, platelet derived growth factor, 15 CCC. tissue plasminogen activator, an apoptotic agent, e.g., TNF The polyethylene glycol molecules (or other chemical alpha, TNF-beta, AIM I (See, International Publication No. moieties) should be attached to the protein with consideration WO 97/33899), AIM II (See, International Publication No. of effects on functional or antigenic domains of the protein. WO 97/34911), Fas Ligand (Takahashi et al. Int. Immunol. There are a number of attachment methods available to those 6:1567-1574 (1994)), VEGI (See, International Publication skilled in the art, such as, for example, the method disclosed No. WO99/23105), a thrombotic agent oran anti-angiogenic in EP 0401 384 (coupling PEG to G-CSF), herein incorpo agent, e.g., angiostatin or endostatin; or, biological response rated by reference: see also Malik et al., Exp. Hematol. modifiers such as, for example, lymphokines, interleukin-1 20:1028-1035 (1992), reporting pegylation of GM-CSF (“IL-1), interleukin-2 (“IL-2), interleukin-6 (“IL-6’), using tresyl chloride. For example, polyethylene glycol may granulocyte macrophage colony Stimulating factor (“GM 25 be covalently bound through amino acid residues via reactive CSF), granulocyte colony stimulating factor (“G-CSF), or group, Such as a free amino or carboxyl group. Reactive other growth factors. Techniques for conjugating Such thera groups are those to which an activated polyethylene glycol peutic moiety to proteins (e.g. albumin fusion proteins) are molecule may be bound. The amino acid residues having a well known in the art. free amino group may include lysine residues and the N-ter Albumin fusion proteins may also be attached to Solid 30 minal amino acid residues: those having a free carboxyl group Supports, which are particularly useful for immunoassays or may include aspartic acid residues glutamic acid residues and purification of polypeptides that are boundby, that bind to, or the C-terminal amino acid residue. Sulfhydryl groups may associate with albumin fusion proteins of the invention. Such also be used as a reactive group for attaching the polyethylene Solid Supports include, but are not limited to, glass, cellulose, glycol molecules. Preferred for therapeutic purposes is polyacrylamide, nylon, polystyrene, polyvinyl chloride or 35 attachment at an amino group. Such as attachment at the polypropylene. N-terminus or lysine group. Albumin fusion proteins, with or without a therapeutic As Suggested above, polyethylene glycol may be attached moiety conjugated to it, administered alone or in combination to proteins via linkage to any of a number of amino acid with cytotoxic factor(s) and or cytokine(s) can be used as a residues. For example, polyethylene glycol can be linked to therapeutic. 40 proteins via covalent bonds to lysine, histidine, aspartic acid, Also provided by the invention are chemically modified glutamic acid, or cysteine residues. One or more reaction derivatives of the albumin fusion proteins of the invention chemistries may be employed to attach polyethylene glycol to which may provide additional advantages Such as increased specific amino acid residues (e.g., lysine, histidine, aspartic solubility, stability and circulating time of the polypeptide, or acid, glutamic acid, or cysteine) of the protein or to more than decreased immunogenicity (see U.S. Pat. No. 4,179,337). 45 one type of amino acid residue (e.g. lysine, histidine, aspartic The chemical moieties for derivitization may be selected acid, glutamic acid, cysteine and combinations thereof) of the from water soluble polymers such as polyethylene glycol, protein. ethylene glycol propylene glycol copolymers, carboxymeth One may specifically desire proteins chemically modified ylcellulose, dextran, polyvinyl alcohol and the like. The albu at the N-terminus. Using polyethylene glycol as an illustra min fusion proteins may be modified at random positions 50 tion of the present composition, one may select from a variety within the molecule, or at predetermined positions within the of polyethylene glycol molecules (by molecular weight, molecule and may include one, two, three or more attached branching, etc.), the proportion of polyethylene glycol mol chemical moieties. ecules to protein (polypeptide) molecules in the reaction mix, The polymer may be of any molecular weight, and may be the type of pegylation reaction to be performed, and the branched or unbranched. For polyethylene glycol, the pre 55 method of obtaining the selected N-terminally pegylated pro ferred molecular weight is between about 1 kDa and about tein. The method of obtaining the N-terminally pegylated 100 kDa (the term “about indicating that in preparations of preparation (i.e., separating this moiety from other monop polyethylene glycol, Some molecules will weigh more, some egylated moieties if necessary) may be by purification of the less, than the Stated molecular weight) for ease in handling N-terminally pegylated material from a population of pegy and manufacturing. Other sizes may be used, depending on 60 lated protein molecules. Selective proteins chemically modi the desired therapeutic profile (e.g., the duration of Sustained fied at the N-terminus modification may be accomplished by release desired, the effects, if any on biological activity, the reductive alkylation which exploits differential reactivity of ease in handling, the degree or lack of antigenicity and other different types of primary amino groups (lysine versus the known effects of the polyethylene glycol to a Therapeutic N-terminal) available for derivatization in a particular pro protein or analog). For example, the polyethylene glycol may 65 tein. Under the appropriate reaction conditions, Substantially have an average molecular weight of about 200, 500, 1000, selective derivatization of the protein at the N-terminus with 1500, 2000, 2500,3000, 3500,4000, 4500, 5000, 5500, 6000, a carbonyl group containing polymer is achieved. US 8,946,156 B2 73 74 As indicated above, pegylation of the albumin fusion pro proteins of the invention, comprises the steps of coating an teins of the invention may be accomplished by any number of ELISA plate with an anti-human serum albumin antibody, means. For example, polyethylene glycol may be attached to blocking the plate to prevent non-specific binding, washing the albumin fusion protein either directly or by an intervening the ELISA plate, adding a solution containing the albumin linker. Linkerless systems for attaching polyethylene glycol 5 fusion protein of the invention (at one or more different con to proteins are described in Delgado et al. Crit. Rev. Thera. centrations), adding a secondary anti-Therapeutic protein Drug Carrier Sys. 9:249-304 (1992): Francis et al. Intern.J. of specific antibody coupled to a detectable label (as described Hematol. 68:1-18 (1998): U.S. Pat. No. 4,002,531; U.S. Pat. herein or otherwise known in the art), and detecting the pres No. 5,349,052: WO95/06058: and WO 98/32466, the disclo ence of the secondary antibody. In an alternate version of this sures of each of which are incorporated herein by reference. 10 protocol, the ELISA plate might be coated with the anti One system for attaching polyethylene glycol directly to Therapeutic protein specificantibody and the labeled second amino acid residues of proteins without an intervening linker ary reagent might be the anti-human albumin specific anti employs tresylated MPEG, which is produced by the modi body. fication of monmethoxy polyethylene glycol (MPEG) using Uses of the Polynucleotides tresylchloride (CISOCHCF). Upon reaction of protein 15 Each of the polynucleotides identified herein can be used in with tresylated MPEG, polyethylene glycol is directly numerous ways as reagents. The following description should attached to amine groups of the protein. Thus, the invention be considered exemplary and utilizes known techniques. includes protein-polyethylene glycol conjugates produced by The polynucleotides of the present invention are useful to reacting proteins of the invention with a polyethylene glycol produce the albumin fusion proteins of the invention. As molecule having a 2.2.2-trifluoroethane Sulphonyl group. described in more detail below, polynucleotides of the inven Polyethylene glycol can also be attached to proteins using tion (encoding albumin fusion proteins) may be used in a number of different intervening linkers. For example, U.S. recombinant DNA methods useful in genetic engineering to Pat. No. 5,612,460, the entire disclosure of which is incorpo make cells, cell lines, or tissues that express the albumin rated herein by reference, discloses urethane linkers for con fusion protein encoded by the polynucleotides encoding albu necting polyethylene glycol to proteins. Protein-polyethylene 25 min fusion proteins of the invention. glycol conjugates wherein the polyethylene glycol is attached Polynucleotides of the present invention are also useful in to the protein by a linker can also be produced by reaction of gene therapy. One goal of gene therapy is to insert a normal proteins with compounds such as MPEG-succinimidylsucci gene into an organism having a defective gene, in an effort to nate, MPEG activated with 1,1'-carbonyldiimidazole, correct the genetic defect. The polynucleotides disclosed in MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophe 30 the present invention offer a means of targeting Such genetic nolcarbonate, and various MPEG-succinate derivatives. A defects in a highly accurate manner. Another goal is to insert number of additional polyethylene glycol derivatives and a new gene that was not present in the host genome, thereby reaction chemistries for attaching polyethylene glycol to pro producing a new trait in the host cell. Additional non-limiting teins are described in International Publication No. WO examples of gene therapy methods encompassed by the 98/32466, the entire disclosure of which is incorporated 35 present invention are more thoroughly described elsewhere herein by reference. Pegylated protein products produced herein (see, e.g. the sections labeled “Gene Therapy”, and using the reaction chemistries set out herein are included Examples 17 and 18). within the scope of the invention. Uses of the Polypeptides The number of polyethylene glycol moieties attached to Each of the polypeptides identified herein can be used in each albumin fusion protein of the invention (i.e., the degree 40 numerous ways. The following description should be consid of Substitution) may also vary. For example, the pegylated ered exemplary and utilizes known techniques. proteins of the invention may be linked, on average, to 1,2,3, Albumin fusion proteins of the invention are useful to 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol provide immunological probes for differential identification molecules. Similarly, the average degree of Substitution of the tissue(s) (e.g. immunohistochemistry assays Such as, within ranges such as 1-3, 2-4, 3-5, 4-6, 5–7, 6-8, 7-9, 8-10, 45 for example, ABC immunoperoxidase (Hsu et al. J. His 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, tochem. Cytochem. 29:577-580 (1981)) or cell type(s) (e.g., or 18-20 polyethylene glycol moieties per protein molecule. immunocytochemistry assays). Methods for determining the degree of substitution are dis Albumin fusion proteins can be used to assay levels of cussed, for example, in Delgado et al. Crit. Rev. Thera. Drug polypeptides in a biological sample using classical immuno Carrier Sys. 9:249-304 (1992). 50 histological methods known to those of skill in the art (e.g. see The polypeptides of the invention can be recovered and Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, purified from chemical synthesis and recombinant cell cul et al. J. Cell. Biol. 105:3087-3096 (1987)). Other methods tures by standard methods which include, but are not limited useful for detecting protein gene expression include immu to, ammonium sulfate or ethanol precipitation, acid extrac noassays, such as the enzyme linked immunosorbent assay tion, anion or cation exchange chromatography, phosphocel 55 (ELISA) and the radioimmunoassay (RIA). Suitable assay lulose chromatography, hydrophobic interaction chromatog labels are known in the art and include enzyme labels, such as, raphy, affinity chromatography, hydroxylapatite glucose oxidase: radioisotopes, such as iodine ("I, I, I, chromatography and lectin chromatography. Most prefer '*'I), carbon (''C), sulfur (S), tritium (H), indium (''"In, ably, high performance liquid chromatography (“HPLC) is ''"In, In, '''In), and technetium (Tc, "Tc), thallium employed for purification. Well known techniques for refold 60 ('Tc), gallium (Ga, Ga), palladium ('Pd), molybde ing protein may be employed to regenerate active conforma num (Mo), xenon (Xe), fluorine (F), 'Sim, 77Lu, tion when the polypeptide is denatured during isolation and/ 15°Gd, 14°Pm, 140La, 175Yb. 166Ho, 90Y. 7Sc, 18.Re, 188Re, or purification. ''Pr, 'Rh, 7Ru: luminescent labels, such as luminol; and The presence and quantity of albuminfusion proteins of the fluorescent labels, such as fluorescein and rhodamine, and invention may be determined using ELISA, a well known 65 biotin. immunoassay known in the art. In one ELISA protocol that Albumin fusion proteins of the invention can also be would be useful for detecting/quantifying albumin fusion detected in vivo by imaging. Labels or markers for in vivo US 8,946,156 B2 75 76 imaging of protein include those detectable by X-radiogra topes, holotoxins, modified toxins, catalytic Subunits of tox phy, nuclear magnetic resonance (NMR) or electron spin ins, or any molecules or enzymes not normally present in or relaxation (ESR). For X-radiography, suitable labels include on the surface of a cell that under defined conditions cause the radioisotopes such as barium or cesium, which emit detect cell's death. Toxins that may be used according to the meth able radiation but are not overtly harmful to the subject. ods of the invention include, but are not limited to radioiso Suitable markers for NMR and ESR include those with a topes known in the art, compounds such as, for example, detectable characteristic spin, such as deuterium, which may antibodies (or complement fixing containing portions be incorporated into the albumin fusion protein by labeling of thereof) that bind an inherent or induced endogenous cyto nutrients given to a cell line expressing the albumin fusion toxic effector system, thymidine kinase, endonuclease, protein of the invention. 10 RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, An albumin fusion protein which has been labeled with an diphtheria toxin, Saporin, momordin, gelonin, pokeweed anti appropriate detectable imaging moiety, such as a radioisotope viral protein, alpha-sarcin and cholera toxin. "Toxin' also (for example, ''I, In, ''"Tc, (I. 'I, I, ''I), carbon includes a cytostatic or cytocidal agent, atherapeutic agent or (C), sulfur (S), tritium (H), indium (''"In, ''"In, In, a radioactive metal ion, e.g., alpha-emitters such as, for '''In), and technetium (Tc, ''"Tc), thallium ('Ti), gal 15 example, Bi, or other radioisotopes such as, for example, lium (Ga, Ga), palladium ('Pd), molybdenum (Mo), 105Pd, 133Xe, 131I, *Ge, 57Co, 65Zn, 85Sr. 32P 35S, 90Y, xenon (Xe), fluorine (F. Sm, 77Lu, Lu, ''Gd, Sm, 153Gd, 169Yb, 5 Cr, Mn, 7Se, Sn, 'Yttrium, 149Pm, 140La, 175Yb. 15°Ho, 90Y, 7Sc, 186Re, 188Re, 142 Pr. '''Tin. 'Rhenium, 'Holmium, and 'Rhenium: lumines 'Rh, 7Ru), a radio-opaque substance, or a material detect cent labels, such as luminol; and fluorescent labels, such as able by nuclear magnetic resonance, is introduced (for fluorescein and rhodamine, and biotin. In a specific embodi example, parenterally, Subcutaneously or intraperitoneally) ment, the invention provides a method for the specific into the mammal to be examined for immune system disorder. destruction of cells (e.g. the destruction of tumor cells) by It will be understood in the art that the size of the subject and administering polypeptides of the invention or antibodies of the imaging system used will determine the quantity of imag the invention in association with the radioisotope 'Y. In ing moiety needed to produce diagnostic images. In the case 25 another specific embodiment, the invention provides a of a radioisotope moiety, for a human Subject, the quantity of method for the specific destruction of cells (e.g., the destruc radioactivity injected will normally range from about 5 to 20 tion of tumor cells) by administering polypeptides of the millicuries of '"Tc. The labeled albumin fusion protein will invention or antibodies of the invention in association with then preferentially accumulate at locations in the body (e.g. the radioisotope '''In. In a further specific embodiment, the organs, cells, extracellular spaces or matrices) where one or 30 invention provides a method for the specific destruction of more receptors, ligands or Substrates (corresponding to that of cells (e.g., the destruction of tumor cells) by administering the Therapeutic protein used to make the albumin fusion polypeptides of the invention orantibodies of the invention in protein of the invention) are located. Alternatively, in the case association with the radioisotope I. where the albumin fusion protein comprises at least a frag Techniques known in the art may be applied to label ment or variant of a Therapeutic antibody, the labeled albu 35 polypeptides of the invention. Such techniques include, but min fusion protein will then preferentially accumulate at the are not limited to, the use of bifunctional conjugating agents locations in the body (e.g. organs, cells, extracellular spaces (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696.239; or matrices) where the polypeptides epitopes corresponding 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; to those bound by the Therapeutic antibody (used to make the 5,342,604, 5,274,119: 4,994,560; and 5,808.003; the con albumin fusion protein of the invention) are located. In vivo 40 tents of each of which are hereby incorporated by reference in tumor imaging is described in S. W. Burchiel et al. “Immu its entirety). nopharmacokinetics of Radiolabeled Antibodies and Their The albumin fusion proteins of the present invention are Fragments” (Chapter 13 in Tumor Imaging. The Radiochemi useful for diagnosis, treatment, prevention and/or prognosis cal Detection of Cancer, S. W. Burchiel and B. A. Rhodes, of various disorders in mammals, preferably humans. Such eds., Masson Publishing Inc. (1982)). The protocols 45 disorders include, but are not limited to, those described described therein could easily be modified by one of skill in herein under the section heading “Biological Activities.” the art for use with the albumin fusion proteins of the inven below. tion. Thus, the invention provides a diagnostic method of a In one embodiment, the invention provides a method for disorder, which involves (a) assaying the expression level of the specific delivery of albumin fusion proteins of the inven 50 a certain polypeptide in cells or body fluid of an individual tion to cells by administering albumin fusion proteins of the using an albumin fusion protein of the invention; and (b) invention (e.g. polypeptides encoded by polynucleotides comparing the assayed polypeptide expression level with a encoding albumin fusion proteins of the invention and/or standard polypeptide expression level, whereby an increase antibodies) that are associated with heterologous polypep or decrease in the assayed polypeptide expression level com tides or nucleic acids. In one example, the invention provides 55 pared to the standard expression level is indicative of a dis a method for delivering a Therapeutic protein into the targeted order. With respect to cancer, the presence of a relatively high cell. In another example, the invention provides a method for amount of transcript in biopsied tissue from an individual delivering a single stranded nucleic acid (e.g., antisense or may indicate a predisposition for the development of the ribozymes) or double stranded nucleic acid (e.g., DNA that disease, or may provide a means for detecting the disease can integrate into the cells genome or replicate episomally 60 prior to the appearance of actual clinical symptoms. A more and that can be transcribed) into the targeted cell. definitive diagnosis of this type may allow health profession In another embodiment, the invention provides a method als to employ preventative measures or aggressive treatment for the specific destruction of cells (e.g., the destruction of earlier thereby preventing the development or further pro tumor cells) by administering albumin fusion proteins of the gression of the cancer. invention in association with toxins or cytotoxic prodrugs. 65 Moreover, albumin fusion proteins of the present invention By “toxin' is meant one or more compounds that bind and can be used to treat or prevent diseases or conditions such as, activate endogenous cytotoxic effector systems, radioiso for example, neural disorders, immune system disorders, US 8,946,156 B2 77 78 muscular disorders, reproductive disorders, gastrointestinal der, which involves measuring the expression level of the disorders, pulmonary disorders, cardiovascular disorders, gene encoding a polypeptide in tissues, cells or body fluid renal disorders, proliferative disorders, and/or cancerous dis from an individual and comparing the measured gene expres eases and conditions. For example, patients can be adminis sion level with a standard gene expression level, whereby an tered a polypeptide of the present invention in an effort to 5 increase or decrease in the gene expression level(s) compared replace absent or decreased levels of the polypeptide (e.g. to the standard is indicative of a disorder. These diagnostic insulin), to supplement absent or decreased levels of a differ assays may be performed in vivo or in vitro. Such as, for ent polypeptide (e.g. hemoglobin S for hemoglobin B, SOD, example, on blood samples, biopsy tissue or autopsy tissue. catalase, DNA repair proteins), to inhibit the activity of a poly The present invention is also useful as a prognostic indica peptide (e.g. an oncogene or tumor Suppressor), to activate the 10 tor, whereby patients exhibiting enhanced or depressed gene activity of a polypeptide (e.g. by binding to a receptor), to expression will experience a worse clinical outcome reduce the activity of a membrane bound receptor by com By “assaying the expression level of the gene encoding a peting with it for free ligand (e.g. soluble TNF receptors used polypeptide' is intended qualitatively or quantitatively mea in reducing inflammation), or to bring about a desired Suring or estimating the level of a particular polypeptide (e.g. response (e.g. blood vessel growth inhibition, enhancement 15 a polypeptide corresponding to a Therapeutic protein dis of the immune response to proliferative cells or tissues). closed in Table 1) or the level of the mRNA encoding the In particular, albumin fusion proteins comprising of at least polypeptide of the invention in a first biological sample either a fragment or variant of a Therapeutic antibody can also be directly (e.g. by determining or estimating absolute protein used to treat disease (as described Supra, and elsewhere level or mRNA level) or relatively (e.g., by comparing to the herein). For example, administration of an albumin fusion 20 polypeptide level or mRNA level in a second biological protein comprising of at least a fragment or variant of a sample). Preferably, the polypeptide expression level or Therapeutic antibody can bind, and/or neutralize the mRNA level in the first biological sample is measured or polypeptide to which the Therapeutic antibody used to make estimated and compared to a standard polypeptide level or the albumin fusion protein immunospecifically binds, and/or mRNA level, the standard being taken from a second biologi reduce overproduction of the polypeptide to which the Thera- 25 cal sample obtained from an individual not having the disor peutic antibody used to make the albumin fusion protein der or being determined by averaging levels from a popula immunospecifically binds. Similarly, administration of an tion of individuals not having the disorder. As will be albumin fusion protein comprising of at least a fragment or appreciated in the art, once a standard polypeptide level or variant of a Therapeutic antibody can activate the polypeptide mRNA level is known, it can be used repeatedly as a standard to which the Therapeutic antibody used to make the albumin 30 for comparison. fusion protein immunospecifically binds, by binding to the By “biological sample' is intended any biological sample polypeptide bound to a membrane (receptor). obtained from an individual, cell line, tissue culture, or other At the very least, the albumin fusion proteins of the inven Source containing polypeptides of the invention (including tion of the present invention can be used as molecular weight portions thereof) or mRNA. As indicated, biological samples markers on SDS-PAGE gels or on molecular sieve gel filtra- 35 include body fluids (such as sera, plasma, urine, synovial fluid tion columns using methods well known to those of skill in and spinal fluid) and tissue sources found to express the full the art. Albumin fusion proteins of the invention can also be length or fragments thereof of a polypeptide or mRNA. Meth used to raise antibodies, which in turn may be used to measure ods for obtaining tissue biopsies and body fluids from mam protein expression of the Therapeutic protein, albumin pro mals are well known in the art. Where the biological sample tein, and/or the albumin fusion protein of the invention from 40 is to include mRNA, a tissue biopsy is the preferred source. a recombinant cell, as a way of assessing transformation of Total cellular RNA can be isolated from a biological the host cell, or in a biological sample. Moreover, the albumin sample using any Suitable technique Such as the single-step fusion proteins of the present invention can be used to test the guanidinium-thiocyanate-phenol chloroform method biological activities described herein. described in Chomczynski and Sacchi, Anal. Biochem. 162: Diagnostic Assays 45 156-159 (1987). Levels of mRNA encoding the polypeptides The compounds of the present invention are useful for of the invention are then assayed using any appropriate diagnosis, treatment, prevention and/or prognosis of various method. These include Northern blot analysis, S1 nuclease disorders in mammals, preferably humans. Such disorders mapping, the polymerase chain reaction (PCR), reverse tran include, but are not limited to those described for each Thera Scription in combination with the polymerase chain reaction peutic protein in the corresponding row of Table 1 and herein 50 (RT-PCR), and reverse transcription in combination with the under the section headings “Immune Activity.” “Blood ligase chain reaction (RT-LCR). Related Disorders,” “Hyperproliferative Disorders,” “Renal The present invention also relates to diagnostic assays such Disorders. “Cardiovascular Disorders.” “Respiratory Disor as quantitative and diagnostic assays for detecting levels of ders.” “Anti-Angiogenesis Activity.” “Diseases at the Cellular polypeptides that bind to are bound by, or associate with Level.” “Wound Healing and Epithelial Cell Proliferation.” 55 albumin fusion proteins of the invention, in a biological “Neural Activity and Neurological Diseases.” “Endocrine sample (e.g. cells and tissues), including determination of Disorders.” “Reproductive System Disorders.” “Infectious normal and abnormal levels of polypeptides. Thus, for Disease.” “Regeneration,” and/or “Gastrointestinal Disor instance, a diagnostic assay in accordance with the invention ders, infra. for detecting abnormal expression of polypeptides that bind For a number of disorders, substantially altered (increased 60 to, are bound by, or associate with albumin fusion proteins or decreased) levels of gene expression can be detected in compared to normal control tissue samples may be used to tissues, cells or bodily fluids (e.g. Sera, plasma, urine, semen, detect the presence of tumors. Assay techniques that can be synovial fluid or spinal fluid) taken from an individual having used to determine levels of a polypeptide that bind to are Such a disorder, relative to a 'standard” gene expression level. bound by, or associate with albumin fusion proteins of the that is, the expression level in tissues or bodily fluids from an 65 present invention in a sample derived from a host are well individual not having the disorder. Thus, the invention pro known to those of skill in the art. Such assay methods include vides a diagnostic method useful during diagnosis of a disor radioimmunoassays, competitive-binding assays. Western US 8,946,156 B2 79 80 Blot analysis and ELISA assays. Assaying polypeptide levels albumin fusion proteins will typically comprise incubating a in a biological sample can occur using any art-known method. sample, Such as a biological fluid, a tissue extract, freshly Assaying polypeptide levels in a biological sample can harvested cells, or lysates of cells which have been incubated occur using a variety oftechniques. For example, polypeptide in cell culture, in the presence of a detectably labeled anti expression in tissues can be studied with classical immuno body capable of binding gene products or conserved variants histological methods (Jalkanen et al., J. Cell. Biol. 101:976 or peptide fragments thereof, and detecting the bound anti 985 (1985); Jalkanen, M, et al., J. Cell. Biol. 105:3087-3096 body by any of a number of techniques well-known in the art. (1987)). Other methods useful for detecting polypeptide gene The biological sample may be brought in contact with and expression include immunoassays, such as the enzyme linked immobilized onto a solid phase Support or carrier Such as immunosorbent assay (ELISA) and the radioimmunoassay 10 nitrocellulose, or other solid support which is capable of (RIA). Suitable antibody assay labels are known in the art and immobilizing cells, cell particles or soluble proteins. The include enzyme labels, such as, glucose oxidase, and radio support may then be washed with suitable buffers followed by isotopes, such as iodine ('I, 'I), carbon (''C), sulfur (S), treatment with the detectably labeled albumin fusion protein tritium (H), indium ('In), and technetium ("Tc), and of the invention. The solid phase support may then be washed fluorescent labels, such as fluorescein and rhodamine, and 15 with the buffer a second time to remove unbound antibody or biotin. polypeptide. Optionally the antibody is subsequently labeled. The tissue or cell type to be analyzed will generally include The amount of bound label on solid support may then be those which are known, or Suspected, to express the gene of detected by conventional means. interest (such as, for example, cancer). The protein isolation By “solid phase support or carrier' is intended any support methods employed herein may, for example, be such as those capable of binding a polypeptide (e.g., an albumin fusion described in Harlow and Lane (Harlow. E. and Lane, D., protein, or polypeptide that binds, is bound by, or associates 1988, “Antibodies: A Laboratory Manual, Cold Spring Har with an albumin fusion protein of the invention.) Well-known bor Laboratory Press, Cold Spring Harbor, N.Y.), which is Supports or carriers include glass, polystyrene, polypropy incorporated herein by reference in its entirety. The isolated lene, polyethylene, dextran, nylon, amylases, natural and cells can be derived from cell culture or from a patient. The 25 modified celluloses, polyacrylamides, gabbros, and magne analysis of cells taken from culture may be a necessary step in tite. The nature of the carrier can be either soluble to some the assessment of cells that could be used as part of a cell extent or insoluble for the purposes of the present invention. based gene therapy technique or, alternatively, to test the The Support material may have virtually any possible struc effect of compounds on the expression of the gene. tural configuration so long as the coupled molecule is capable For example, albumin fusion proteins may be used to quan 30 of binding to a polypeptide. Thus, the Support configuration titatively or qualitatively detect the presence of polypeptides may be spherical, as in a bead, or cylindrical, as in the inside that bind to are bound by, or associate with albumin fusion surface of a test tube, or the external surface of a rod. Alter proteins of the present invention. This can be accomplished, natively, the Surface may be flat such as a sheet, test Strip, etc. for example, by immunofluorescence techniques employing Preferred supports include polystyrene beads. Those skilled a fluorescently labeled albumin fusion protein coupled with 35 in the art will know many other suitable carriers for binding light microscopic, flow cytometric, or fluorimetric detection. antibody or antigen, or will be able to ascertain the same by In a preferred embodiment, albumin fusion proteins com use of routine experimentation. prising at least a fragment or variant of an antibody that The binding activity of a given lot of albumin fusion pro immunospecifically binds at least a Therapeutic protein dis tein may be determined according to well known methods. closed herein (e.g. the Therapeutic proteins disclosed in Table 40 Those skilled in the art will be able to determine operative and 1) or otherwise known in the art may be used to quantitatively optimal assay conditions for each determination by employ or qualitatively detect the presence of gene products or con ing routine experimentation. served variants or peptide fragments thereof. This can be In addition to assaying polypeptide levels in a biological accomplished, for example, by immunofluorescence tech sample obtained from an individual, polypeptide can also be niques employing a fluorescently labeled antibody coupled 45 detected in Vivo by imaging. For example, in one embodiment with light microscopic, flow cytometric, or fluorimetric of the invention, albumin fusion proteins of the invention are detection. used to image diseased or neoplastic cells. The albumin fusion proteins of the present invention may, Labels or markers for in vivo imaging of albumin fusion additionally, be employed histologically, as in immunofluo proteins of the invention include those detectable by X-radi rescence, immunoelectron microscopy or non-immunologi 50 ography, NMR, MRI, CAT-scans or ESR. For X-radiography, cal assays, for in situ detection of polypeptides that bind to are suitable labels include radioisotopes such as barium or bound by, or associate with an albumin fusion protein of the cesium, which emit detectable radiation but are not overtly present invention. In situ detection may be accomplished by harmful to the subject. Suitable markers for NMR and ESR removing a histological specimen from a patient, and apply include those with a detectable characteristic spin, such as ing thereto a labeled antibody or polypeptide of the present 55 deuterium, which may be incorporated into the albumin invention. The albumin fusion proteins are preferably applied fusion protein by labeling of nutrients of a cell line (or bac by overlaying the labeled albumin fusion proteins onto a terial or yeast strain) engineered. biological sample. Through the use of Such a procedure, it is Additionally, albumin fusion proteins of the invention possible to determine not only the presence of the polypep whose presence can be detected, can be administered. For tides that bind to, are bound by, or associate with albumin 60 example, albumin fusion proteins of the invention labeled fusion proteins, but also its distribution in the examined tis with a radio-opaque or other appropriate compound can be sue. Using the present invention, those of ordinary skill will administered and visualized in Vivo, as discussed, above for readily perceive that any of a wide variety of histological labeled antibodies. Further, such polypeptides can be utilized methods (such as staining procedures) can be modified in for in vitro diagnostic procedures. order to achieve Such in situ detection. 65 A polypeptide-specific antibody or antibody fragment Immunoassays and non-immunoassays that detect which has been labeled with an appropriate detectable imag polypeptides that bind to, are bound by, or associate with ing moiety, such as a radioisotope (for example, ''I, In, US 8,946,156 B2 81 82 "Tc), a radio-opaque substance, or a material detectable by The albumin fusion protein can also be detectably labeled nuclear magnetic resonance, is introduced (for example, using fluorescence emitting metals such as "Eu, or others of parenterally, Subcutaneously or intraperitoneally) into the the lanthanide series. These metals can be attached to the mammal to be examined for a disorder. It will be understood antibody using Such metal chelating groups as diethylenetri in the art that the size of the Subject and the imaging system aminepentacetic acid (DTPA) or ethylenediaminetetraacetic used will determine the quantity of imaging moiety needed to acid (EDTA). produce diagnostic images. In the case of a radioisotope The albumin fusion proteins can also can be detectably moiety, for a human Subject, the quantity of radioactivity labeled by coupling it to a chemiluminescent compound. The injected will normally range from about 5 to 20 millicuries of presence of the chemiluminescent-tagged albumin fusion 10 protein is then determined by detecting the presence of lumi "Tc. The labeled albumin fusion protein will then prefer nescence that arises during the course of a chemical reaction. entially accumulate at the locations in the body which contain Examples of particularly useful chemiluminescent labeling a polypeptide or other substance that binds to is bound by or compounds are luminol, isoluminol, theromatic acridinium associates with an albumin fusion protein of the present ester, imidazole, acridinium salt and oxalate ester. invention. In vivo tumor imaging is described in S. W. Bur 15 Likewise, a bioluminescent compound may be used to chiel et al., “Immunopharmacokinetics of Radiolabeled Anti label albumin fusion proteins of the present invention. Biolu bodies and Their Fragments” (Chapter 13 in Tumor Imaging: minescence is a type of chemiluminescence found in biologi The Radiochemical Detection of Cancer. S. W. Burchiel and cal systems in, which a catalytic protein increases the effi B. A. Rhodes, eds., Masson Publishing Inc. (1982)). ciency of the chemiluminescent reaction. The presence of a One of the ways in which an albumin fusion protein of the bioluminescent protein is determined by detecting the pres present invention can be detectably labeled is by linking the ence of luminescence. Important bioluminescent compounds same to a reporter enzyme and using the linked product in an for purposes of labeling are luciferin, luciferase and aequorin. enzyme immunoassay (EIA) (Voller, A., “The Enzyme Transgenic Organisms Linked Immunosorbent Assay (ELISA)', 1978, Diagnostic Transgenic organisms that express the albumin fusion pro Horizons 2:1-7, Microbiological Associates Quarterly Publi 25 teins of the invention are also included in the invention. Trans cation, Walkersville, Md.): Voller et al., J. Clin. Pathol. genic organisms are genetically modified organisms into 31:507-520 (1978); Butler. J. E., Meth. Enzymol. 73:482-523 which recombinant, exogenous or cloned genetic material has (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC been transferred. Such genetic material is often referred to as Press, Boca Raton, Fla.: Ishikawa, E. et al., (eds.), 1981, a transgene. The nucleic acid sequence of the transgene may Enzyme Immunoassay. Kgaku Shoin, Tokyo). The reporter 30 include one or more transcriptional regulatory sequences and enzyme which is bound to the antibody will react with an other nucleic acid sequences such as introns, that may be appropriate substrate, preferably a chromogenic substrate, in necessary for optimal expression and secretion of the Such a manner as to produce a chemical moiety which can be encoded protein. The transgene may be designed to direct the detected, for example, by spectrophotometric, fluorimetric or expression of the encoded protein in a manner that facilitates by visual means. Reporter enzymes which can be used to 35 its recovery from the organism or from a product produced by detectably label the antibody include, but are not limited to, the organism, e.g. from the milk, blood, urine, eggs, hair or malate dehydrogenase, staphylococcal nuclease, delta-5-ste seeds of the organism. The transgene may consist of nucleic roid isomerase, yeast alcohol dehydrogenase, alpha-glycero acid sequences derived from the genome of the same species phosphate, dehydrogenase, triose phosphate isomerase, or of a different species than the species of the target animal. horseradish peroxidase, alkaline phosphatase, asparaginase, 40 The transgene may be integrated either at a locus of a genome glucose oxidase, beta-galactosidase, ribonuclease, urease, where that particular nucleic acid sequence is not otherwise catalase, glucose-6-phosphate dehydrogenase, glucoamylase normally found or at the normal locus for the transgene. and acetylcholinesterase. Additionally, the detection can be The term “germ cell line transgenic organism' refers to a accomplished by colorimetric methods which employ a chro transgenic organism in which the genetic alteration or genetic mogenic Substrate for the reporter enzyme. Detection may 45 information was introduced into a germ line cell, thereby also be accomplished by visual comparison of the extent of conferring the ability of the transgenic organism to transfer enzymatic reaction of a Substrate in comparison with simi the genetic information to offspring. If such offspring in fact larly prepared Standards. possess Some or all of that alteration or genetic information, Albumin fusion proteins may also be radiolabelled and then they too are transgenic organisms. The alteration or used in any of a variety of other immunoassays. For example, 50 genetic information may beforeign to the species of organism by radioactively labeling the albumin fusion proteins, it is to which the recipient belongs, foreign only to the particular possible to the use the albumin fusion proteins in a radioim individual recipient, or may be genetic information already munoassay (RIA) (see, for example, Weintraub, B., Prin possessed by the recipient. In the last case, the altered or ciples of Radioimmunoassays, Seventh Training Course on introduced gene may be expressed differently than the native Radioligand Assay Techniques, The Endocrine Society, 55 gene. March, 1986, which is incorporated by reference herein). The A transgenic organism may be a transgenic animal or a radioactive isotope can be detected by means including, but transgenic plant. Transgenic animals can be produced by a not limited to, a gamma counter, a Scintillation counter, or variety of different methods including transfection, elec autoradiography. troporation, microinjection, gene targeting in embryonic It is also possible to label the albumin fusion proteins with 60 stem cells and recombinant viral and retroviral infection (see, a fluorescent compound. When the fluorescently labeled anti e.g. U.S. Pat. No. 4.736,866; U.S. Pat. No. 5,602,307; Mullins body is exposed to light of the proper wave length, its pres et al. (1993) Hypertension 22(4):630-633: Brenin et al. ence can then be detected due to fluorescence. Among the (1997) Surg. Oncol. 6(2)99-110; Tuan (ed.). Recombinant most commonly used fluorescent labeling compounds are Gene Expression Protocols, Methods in Molecular Biology fluorescein isothiocyanate, rhodamine, phycoerythrin, phy 65 No. 62, Humana Press (1997)). The method of introduction of cocyanin, allophycocyanin, ophthaldehyde and fluorescam nucleic acid fragments into recombination competent mam 1C. malian cells can be by any method which favors co-transfor US 8,946,156 B2 83 84 mation of multiple nucleic acid molecules. Detailed proce An albumin fusion protein of the invention can also be dures for producing transgenic animals are readily available expressed in a transgenic plant, e.g. a plant in which the DNA to one skilled in the art, including the disclosures in U.S. Pat. transgene is inserted into the nuclear or plastidic genome. Plant transformation procedures used to introduce foreign No. 5,489,743 and U.S. Pat. No. 5,602,307. nucleic acids into plant cells or protoplasts are known in the A number of recombinant or transgenic mice have been art (e.g. see Example 19). See, in general, Methods in Enzy produced, including those which express an activated onco mology Vol. 153 (“Recombinant DNA Part D.) 1987, Wu and gene sequence (U.S. Pat. No. 4.736,866): express simian Grossman Eds. Academic Press and European Patent Appli SV40 T-antigen (U.S. Pat. No. 5,728,915); lack the expres cation EP 693554, Methods for generation of genetically sion of interferon regulatory factor 1 (IRF-1) (U.S. Pat. No. engineered plants are further described in U.S. Pat. No. 5.283, 5,731,490); exhibit dopaminergic dysfunction (U.S. Pat. No. 10 184, U.S. Pat. No. 5,482,852, and European Patent Applica 5,723,719); express at least one human gene which partici- tion EP 693 554, all of which are hereby incorporated by pates in blood pressure control (U.S. Pat. No. 5,731,489); reference. display greater similarity to the conditions existing in natu- Pharmaceutical or Therapeutic Compositions rally occurring Alzheimer's disease (U.S. Pat. No. 5,720, 15 The albumin fusion proteins of the invention or formula 936); have a reduced capacity to mediate cellular adhesion tions thereof may be administered by any conventional (U.S. Pat. No. 5,602,307); possess a bovine growth hormone method including parenteral (e.g. Subcutaneous or intramus gene (Clutter et al. (1996) Genetics 143(4): 1753-1760); or, cular) injection or intravenous infusion. The treatment may are capable of generating a fully human antibody response consist of a single dose or a plurality of doses over a period of (McCarthy (1997) The Lancet 349(9049):405). time. While mice and rats remain the animals of choice for most " While it is possible for an albumin fusion protein of the transgenic experimentation, in some instances it is preferable invention to be administered alone, it is preferable to present or even necessary to use alternative animal species. Trans- it as a pharmaceutical formulation, together with one or more genic procedures have been Successfully utilized in a variety acceptable carriers. The carrier(s) must be “acceptable' in the of non-murine animals, including sheep, goats, pigs, dogs, sense of being compatible with the albumin fusion protein cats, monkeys, chimpanzees, hamsters, rabbits, cows and and not deleterious to the recipients thereof. Typically, the guinea pigs (see, e.g. Kim et al. (1997) Mol. Reprod. Dev. carriers will be water or saline which will be sterile and 46(4):515-526: Houdebine (1995) Reprod. Nutr. Dev. 35(6): pyrogen free. Albumin fusion proteins of the invention are 609-617: Petters (1994) Reprod. Fertil. Dev. 6(5):643-645; particularly well Suited to formulation in aqueous carriers Schnieke et al., (1997) Science 278(5346):2130-2133; and 30 Such as sterile pyrogen free water, Saline or other isotonic Amoah (1997) J. Animal Science 75(2):578-585). solutions because of their extended shelf-life in solution. For theh To 1nvention direct the into ENE. the milk of transgenicE. mammals, protein 1t may be ERNeformulated Physical well in advance compositions 1n aqueous ofR form, ity for is1nstance, may put under the control of a promoter that is preferentially weeks or months or longer time periods before being dis activated in mammary epithelial cells. Promoters that control 35 pensed. the genes encoding milk proteins are preferred, for example For example, wherein the Therapeutic protein is hCGH, the promoter for casein, beta lactoglobulin, whey acid pro- EPO, alpha-IFN or beta-IFN, formulations containing the tein, or lactalbumin (see, e.g., DiTullio (1992) BioTechnol- albumin fusion protein may be prepared taking into account ogy 10:74-77: Clarket al. (1989) BioTechnology 7:487-492; the extended shelf-life of the albumin fusion protein in aque Gorton et al. (1987) BioTechnology 5:1 183-1187; and ous formulations. As exhibited in Table 2, most Therapeutic Soulier et al. (1992) FEBS Letts. 297:13). The transgenic proteins are unstable with short shelf-lives after formulation mammals of choice would produce large Volumes of milk and with an aqueous carrier. As discussed above, the shelf-life of have long lactating periods, for example goats, cows, camels many of these Therapeutic proteins are markedly increased or or sheep. prolonged after fusion to HA. TABLE 2 Tradename, Storage Conditions of Protein Manufacturer Route Formulation Non-Fusion Protein
interferon, Roferon-A, SC Sol in 4-8°C. alpha-2a. Hoffmann-LaRoche im (vial or pre-filled Syringe) interferon, Intron-A, iv Sc im Sol n: 4-8°C. alpha-2b Schering Plough powder + dil. (all preps, before and after dilution) COMBO Rebetron (Intron-A+ po + Rebetol capsule + interferon alpha- Rebetol) SC Intron-A injection 2b + Schering Plough Ribavirin interferon, Infergen SC Sol in 4-8°C. Alphacon-1 Amgen interferon, Wellferon, SC Sol in 4-8°C. alpha-n1, Wellcome im (with albumin as Lympho- stablizer ) blastoid interferon, Avonex. im powder + dil. 4-8°C. beta-1a Biogen (with albumin) (before and after dilution) (Use within 3-6 h of reconstitution) US 8,946,156 B2 85 86 TABLE 2-continued Tradename, Storage Conditions of Protein Manufacturer Route Formulation Non-Fusion Protein Rebif, SC Sol n, Aires-Serono in pre-filled Syringe (Europe only) Interferon, Betaseron, SC powder + dil. 4-8°C. beta-1b Chiron (with albumin) (before and after dilution) (Europe: Betaferon) (Use within 3h of reconstitution) Single use vials. Interferon, Actimmune, SC 4-8°C. Gamma-1b InterMune (before and after dilution) Pharmaceuticals (Use within 3h of reconstitution). Growth Genotropin, powderfdil cartridges 4-8°C. Hormone Pharmacia Upjohn (single or multi-use); (before and after dilution); (somatropin) single use MiniQuick single use MiniQuick injector Delivery Device should be refrigerated until use. Humatrope, SC powder + dil. 4-8°C. Eli Lilly im (Vial or pen cartridge) (before and after dilution) (Use vials within 25 h, cartridges within 28 d, of reconstitution). Norditropin, Novo Nordisk Pharmaceuticals Nutropin, SC powder + dil. 4-8°C. Genentech (stable for 14 d after dil n) (all preps, before and after dilution) Nutropin AQ, SC Sol in 4-8°C. Genentech (Stable for 28 d after 1st use) Nutropin Depot, SC microsphere suspension 4-8°C. Genentech 8S Single use pkges. Dose powder + dil. 1-2X/month (ProLease micro-encapsulation technol.) Saizen, SC powder + dil. Powder should be stored (Serono) im at Rm Temp . After reconstitution store 4-8°C. for up to 14 d. Serostim, Powder should be stored Serono at Rm Temp . After reconstitution store in 4-8°C. for up to 14 d. hCH, with Protropin, SC powder + dil. 4-8°C. N-term. Met Genentech im (all preps, before and (somatrem) after dilution) Erythropoietin Epogen, iv Sol in 4-8°C. (Epoetin alfa) Amgen SC (use within 21 d of first use) (Single & multi-dose vials) Procrit, iv Sol in 4-8°C. Amgen SC (use within 21 d of first use) (Single & multi-dose vials)
In instances where aerosol administration is appropriate, Gonda (1990) Critical Reviews in Therapeutic Drug Carrier the albumin fusion proteins of the invention can be formu- 55 Systems 6:273-313, and Raeburn et al. (1992) Pharmacol. lated as aerosols using standard procedures. The term “aero Toxicol. Methods 27:143-159. Sol' includes any gas-borne Suspended phase of an albumin The formulations of the invention are also typically non fusion protein of the instant invention which is capable of immunogenic, in part, because of the use of the components being inhaled into the bronchioles or nasal passages. Specifi of the albumin fusion protein being derived from the proper cally, aerosol includes a gas-borne Suspension of droplets of 60 species. For instance, for human use, both the Therapeutic an albumin fusion protein of the instant invention, as may be produced in a metered dose inhaler or nebulizer, or in a mist protein and albumin portions of the albumin fusion protein sprayer. Aerosol also includes a dry powder composition of a will typically be human. In some cases, wherein either com compound of the instant invention Suspended in air or other ponent is non human-derived, that component may be carrier gas, which may be delivered by insufflation from an 65 humanized by Substitution of key amino acids so that specific inhaler device, for example, See Ganderton & Jones, Drug epitopes appear to the human immune system to be human in Delivery to the Respiratory Tract, Ellis Horwood (1987); nature rather than foreign. US 8,946,156 B2 87 88 The formulations may conveniently be presented in unit additives, carriers, fillers and diluents. Nutraceuticals are dosage form and may be prepared by any of the methods well described in Scott Hegenhart, Food Product Design, Decem known in the art of pharmacy. Such methods include the step ber 1993. of bringing into association the albumin fusion protein with The invention also provides methods of treatment and/or the carrier that constitutes one or more accessory ingredients. 5 prevention of diseases or disorders (such as, for example, any In general the formulations are prepared by uniformly and one or more of the diseases or disorders disclosed herein) by intimately bringing into association the active ingredient with administration to a subject of an effective amount of an albu liquid carriers or finely divided solid carriers or both, and minfusion protein of the invention or a polynucleotide encod then, if necessary, shaping the product. ing an albumin fusion protein of the invention ("albumin Formulations suitable for parenteral administration 10 fusion polynucleotide’) in a pharmaceutically acceptable car include aqueous and non-aqueous sterile injection solutions 1. which may contain anti-oxidants, buffers, bacteriostats and The albumin fusion protein and/or polynucleotide will be solutes which render the formulation appropriate for the formulated and dosed in a fashion consistent with good medi intended recipient; and aqueous and non-aqueous sterile Sus 15 cal practice, taking into account the clinical condition of the pensions which may include Suspending agents and thicken individual patient (especially the side effects of treatment ing agents. The formulations may be presented in unit-dose or with the albumin fusion protein and/or polynucleotide alone), multi-dose containers, for example sealed ampules, vials or the site of delivery, the method of administration, the sched Syringes, and may be stored in a freeze-dried (lyophilised) uling of administration, and other factors known to practitio condition requiring only the addition of the sterile liquid ners. The “effective amount” for purposes herein is thus deter carrier, for example water for injections, immediately prior to mined by Such considerations. use. Extemporaneous injection Solutions and Suspensions As a general proposition, the total pharmaceutically effec may be prepared from sterile powders. Dosage formulations tive amount of the albumin fusion protein administered may contain the Therapeutic protein portion at a lower molar parenterally perdose will be in the range of about 1 lug/kg day concentration or lower dosage compared to the non-fused 25 to 10 mg/kg/day of patient body weight, although, as noted standard formulation for the Therapeutic protein given the above, this will be subject to therapeutic discretion. More extended serum half-life exhibited by many of the albumin preferably, this dose is at least 0.01 mg/kg day, and most fusion proteins of the invention. preferably for humans between about 0.01 and 1 mg/kg/day As an example, when an albumin fusion protein of the for the hormone. If given continuously, the albumin fusion invention comprises growth hormone as one or more of the 30 protein is typically administered at a dose rate of about 1 Therapeutic protein regions, the dosage form can be calcu ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections lated on the basis of the potency of the albumin fusion protein per day or by continuous Subcutaneous infusions, for relative to the potency of hCGH, while taking into account the example, using a mini-pump. An intravenous bag Solution prolonged serum half-life and shelf-life of the albumin fusion may also be employed. The length of treatment needed to proteins compared to that of native hCH. Growth hormone is 35 observe changes and the interval following treatment for typically administered at 0.3 to 30.0 IU/kg/week, for example responses to occur appears to vary depending on the desired 0.9 to 12.0 IU kg week, given in three or seven divided doses effect. for a year or more. In an albumin fusion protein consisting of Albumin fusion proteins and or polynucleotides can be are full length HA fused to full length GH, an equivalent dose in 40 administered orally, rectally, parenterally, intracisternally, terms of units would represent a greater weight of agent but intravaginally, intraperitoneally, topically (as by powders, the dosage frequency can be reduced, for example to twice a ointments, gels, drops or transdermal patch), bucally, or as an week, once a week or less. oral or nasal spray. “Pharmaceutically acceptable carrier' Formulations or compositions of the invention may be refers to a non-toxic solid, semisolid or liquid filler, diluent, packaged together with, or included in a kit with, instructions 45 encapsulating material or formulation auxiliary of any. The or a package insert referring to the extended shelf-life of the term “parenteral as used herein refers to modes of adminis albumin fusion protein component. For instance. Such tration which include intravenous, intramuscular, intraperito instructions or package inserts may address recommended neal, intrasternal, Subcutaneous and intraarticular injection storage conditions, such as time, temperature and light, taking and infusion. into account the extended or prolonged shelf-life of the albu 50 Albumin fusion proteins and/or polynucleotides of the min fusion proteins of the invention. Such instructions or invention are also suitably administered by Sustained-release package inserts may also address the particular advantages of systems. Examples of Sustained-release albumin fusion pro the albumin fusion proteins of the inventions, such as the ease teins and/or polynucleotides are administered orally, rectally, of storage for formulations that may require use in the field, parenterally, intracistemally, intravaginally, intraperito outside of controlled hospital, clinic or office conditions. As 55 neally, topically (as by powders, ointments, gels, drops or described above, formulations of the invention may be in transdermal patch), bucally, or as an oral or nasal spray. aqueous form and may be stored under less than ideal circum "Pharmaceutically acceptable carrier refers to a non-toxic stances without significant loss of therapeutic activity. Solid, semisolid or liquid filler, diluent, encapsulating mate Albumin fusion proteins of the invention can also be rial or formulation auxiliary of any type. The term included in nutraceuticals. For instance, certain albumin 60 “parenteral as used herein refers to modes of administration fusion proteins of the invention may be administered in natu which include intravenous, intramuscular, intraperitoneal, ral products, including milk or milk product obtained from a intrasternal, Subcutaneous and intraarticular injection and transgenic mammal which expresses albumin fusion protein. infusion. Additional examples of Sustained-release albumin Such compositions can also include plant or plant products fusion proteins and/or polynucleotides include Suitable poly obtained from a transgenic plant which expresses the albumin 65 meric materials (such as, for example, semi-permeable poly fusion protein. The albumin fusion protein can also be pro mer matrices in the form of shaped articles, e.g., films, or vided in powder or tablet form, with or without other known mirocapsules), Suitable hydrophobic materials (for example US 8,946,156 B2 89 90 as an emulsion in an acceptable oil) or ion exchange resins, disaccharides, and other carbohydrates including cellulose or and sparingly soluble derivatives (such as, for example, a its derivatives, glucose, manose, or dextrins; chelating agents sparingly soluble salt). Such as EIDTA; Sugar alcohols such as mannitol or Sorbitol; Sustained-release matrices include polylactides (U.S. Pat. counterions such as sodium: and/or nonionic Surfactants such No. 3,773.919, EP 58.481), copolymers of L-glutamic acid 5 as polysorbates, poloxamers, or PEG. and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers The albumin fusion protein is typically formulated in such 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, (Langeret al., J. Biomed. Mater. Res. 15:167-277 (1981), and preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be Langer. Chem. Tech. 12:98-105 (1982)), ethylene vinyl understood that the use of certain of the foregoing excipients, acetate (Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric 10 carriers, or stabilizers will result in the formation of polypep acid (EP 133,988). tide salts. Sustained-release albumin fusion proteins and/or poly Any pharmaceutical used for therapeutic administration nucleotides also include liposomally entrapped albumin can be sterile. Sterility is readily accomplished by filtration fusion proteins and/or polynucleotides of the invention (see through sterile filtration membranes (e.g., 0.2 micron mem generally, Langer, Science 249:1527-1533 (1990): Treat etal, 15 branes). Albuminfusion proteins and/or polynucleotides gen in Liposomes in the Therapy of Infectious Disease and Can erally are placed into a container having a sterile access port, cer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. for example, an intravenous solution bag or vial having a 317-327 and 353–365 (1989)). Liposomes containing the stopper pierceable by a hypodermic injection needle. albumin fusion protein and/or polynucleotide are prepared by Albumin fusion proteins and/or polynucleotides ordinarily methods known per se: DE 3,218,121; Epstein et al., Proc. will be stored in unit or multi-dose containers, for example, Natl. Acad. Sci. (USA) 82:3688-3692 (1985): Hwang et al. sealed ampoules or vials, as an aqueous solution or as a Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP lyophilized formulation for reconstitution. As an example of 52.322; EP 36,676: EP 88,046: EP 143,949: EP 142,641: a lyophilized formulation, 10-ml vials are filled with 5 ml of Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and sterile-filtered 1% (w/v) aqueous albumin fusion protein and/ 4,544.545: and EP 102,324. Ordinarily, the liposomes are of 25 or polynucleotide solution, and the resulting mixture is lyo the small (about 200-800 Angstroms) unilamellar type in philized. The infusion solution is prepared by reconstituting which the lipid content is greater than about 30 mol, percent the lyophilized albumin fusion protein and/or polynucleotide cholesterol, the selected proportion being adjusted for the using bacteriostatic Water-for-Injection. optimal Therapeutic. In a specific and preferred embodiment, the Albumin In yet an additional embodiment, the albumin fusion pro 30 fusion protein formulations comprises 0.01M sodium phos teins and/or polynucleotides of the invention are delivered by phate, 0.15 mM sodium chloride, 0.16 micromole sodium way of a pump (see Langer, supra: Sefton, CRC Crit. Ref. octanoate, milligram of fusion protein, 15 micrograms/milli Biomed. Eng. 14:201 (1987); Buchwaldetal. Surgery 88:507 liter polysorbate 80, pH 7.2. In another specific and preferred (1980); Saudek et al. N. Engl. J. Med. 321:574 (1989)). embodiment, the Albumin fusion protein formulations con Other controlled release systems are discussed in the 35 sists 0.01 M sodium phosphate, 0.15 mM sodium chloride, review by Langer (Science 249:1527-1533 (1990)). 0.16 micromole Sodium octanoate/milligram of fusion pro For parenteral administration, in one embodiment, the tein, 15 micrograms/milliliter polysorbate 80, pH 7.2. The pH albumin fusion protein and/or polynucleotide is formulated and buffer are chosen to match physiological conditions and generally by mixing it at the desired degree of purity, in a unit the salt is added as a tonicifier. Sodium octanoate has been dosage injectable form (Solution, Suspension, or emulsion), 40 chosen due to its reported ability to increase the thermal with a pharmaceutically acceptable carrier, i.e., one that is stability of the protein in solution. Finally, polysorbate has non-toxic to recipients at the dosages and concentrations been added as a generic Surfactant, which lowers the Surface employed and is compatible with other ingredients of the tension of the solution and lowers non-specific adsorption of formulation. For example, the formulation preferably does the albumin fusion protein to the container closure system. not include oxidizing agents and other compounds that are 45 The invention also provides a pharmaceutical pack or kit known to be deleterious to the Therapeutic. comprising one or more containers filled with one or more of Generally, the formulations are prepared by contacting the the ingredients of the albumin fusion proteins and/or poly albumin fusion protein and/or polynucleotide uniformly and nucleotides of the invention. Associated with Such container intimately with liquid carriers or finely divided solid carriers (s) can be a notice in the form prescribed by a governmental or both. Then, if necessary, the product is shaped into the 50 agency regulating the manufacture, use or sale of pharmaceu desired formulation. Preferably the carrier is a parenteral ticals or biological products, which notice reflects approval carrier, more preferably a solution that is isotonic with the by the agency of manufacture, use or sale for human admin blood of the recipient. Examples of such carrier vehicles istration. In addition, the albumin fusion proteins and/or poly include water, Saline, Ringer's solution, and dextrose solu nucleotides may be employed in conjunction with other tion. Non-aqueous vehicles such as fixed oils and ethyl oleate 55 therapeutic compounds. are also useful herein, as well as liposomes. The albumin fusion proteins and or polynucleotides of the The carrier Suitably contains minor amounts of additives invention may be administered alone or in combination with Such as Substances that enhance isotonicity and chemical adjuvants. Adjuvants that may be administered with the albu stability. Such materials are non-toxic to recipients at the min fusion proteins and/or polynucleotides of the invention dosages and concentrations employed, and include buffers 60 include, but are not limited to, alum, alum plus deoxycholate Such as phosphate, citrate. Succinate, acetic acid, and other (ImmunoAg), IMITP-PE (Biocine Corp.), QS21 (Genentech, organic acids or their salts; antioxidants such as ascorbic acid; Inc.), BCG (e.g. THERACYSR), MPL and nonviable prepa low molecular weight (less than about ten residues) polypep rations of Corynebacterium parvum. In a specific embodi tides, e.g. polyarginine or tripeptides; proteins, such as serum ment, albumin fusion proteins and/or polynucleotides of the albumin, gelatin, or immunoglobulins: hydrophilic polymers 65 invention are administered in combination with alum. In Such as polyvinylpyrrolidone; amino acids, such as glycine, another specific embodiment, albumin fusion proteins and or glutamic acid, aspartic acid, or arginine; monosaccharides, polynucleotides of the invention are administered in combi US 8,946,156 B2 91 92 nation with QS-21. Further adjuvants that may be adminis gen, alpha2-antiplasmin, streptokinae (e.g. KABIKI tered with the albumin fusion proteins and/or polynucleotides NASETM), antiresplace (e.g. EMINASETM), tissue plasmino of the invention include, but are not limited to, Monophos gen activator (t-PA, altevase, ACTIVASETM), urokinase (e.g. phoryl lipid immunomodulator. AdjuVax 100a, QS-21, ABBOKINASETM), sauruplase, (Prourokinase, single chain QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal urokinase), and aminocaproic acid (e.g., AMICARTM). In a adjuvant technology. Vaccines that may be administered with specific embodiment, compositions of the invention are the albumin fusion proteins and/or polynucleotides of the administered in combination with tissue plasminogen activa invention include, but are not limited to, vaccines directed tor and aspirin. toward protection against MMR (measles, mumps, rubella), In another embodiment, the albumin fusion proteins and/or polio, varicella, tetanusidiptheria, hepatitis A, hepatitis B. 10 polynucleotides of the invention are administered in combi Haemophilus influenzae B, whooping cough, pneumonia, nation with antiplatelet drugs. Antiplatelet drugs that may be influenza, Lyme’s Disease, rotavirus, cholera, yellow fever, administered with the compositions of the invention include, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, but are not limited to, aspirin, dipyridamole (e.g., PERSAN and pertussis. Combinations may be administered either con TINETM), and ticlopidine (e.g., TICLIDTM). comitantly, e.g., as an admixture, separately but simulta 15 In specific embodiments, the use of anti-coagulants, neously or concurrently; or sequentially. This includes pre thrombolytic and/or antiplatelet drugs in combination with sentations in which the combined agents are administered albumin fusion proteins and/or polynucleotides of the inven together as a therapeutic mixture, and also procedures in tion is contemplated for the prevention, diagnosis, and/or which the combined agents are administered separately but treatment of thrombosis, arterial thrombosis, venous throm simultaneously, e.g. as through separate intravenous lines bosis, thromboembolism, pulmonary embolism, atheroscle into the same individual. Administration “in combination' rosis, myocardial infarction, transient ischemic attack, further includes the separate administration of one of the unstable angina. In specific embodiments, the use of antico compounds or agents given first, followed by the second. agulants, thrombolytic drugs and/or antiplatelet drugs in The albumin fusion proteins and/or polynucleotides of the combination with albumin fusion proteins and/or polynucle invention may be administered alone or in combination with 25 otides of the invention is contemplated for the prevention of other therapeutic agents. Albumin fusion protein and/or poly occlusion of Saphenous grafts, for reducing the risk of nucleotide agents that may be administered in combination periprocedural thrombosis as might accompany angioplasty with the albumin fusion proteins and/or polynucleotides of procedures, for reducing the risk of stroke in patients with the invention, include but not limited to, chemotherapeutic atrial fibrillation including nonrheumatic atrial fibrillation, agents, antibiotics, steroidal and non-steroidal anti-inflam 30 for reducing the risk of embolism associated with mechanical matories, conventional immunotherapeutic agents, and/or heart valves and or mitral valves disease. Other uses for the therapeutic treatments described below. Combinations may therapeutics of the invention, alone or in combination with be administered either concomitantly, e.g., as an admixture, antiplatelet, anticoagulant, and/or thrombolytic drugs, separately but simultaneously or concurrently; or sequen include, but are not limited to the prevention of occlusions in tially. This includes presentations in which the combined 35 extracorporeal devices (e.g. intravascular canulas, Vascular agents are administered togetheras a therapeutic mixture, and access shunts in hemodialysis patients, hemodialysis also procedures in which the combined agents are adminis machines, and cardiopulmonary bypass machines). tered separately but simultaneously, e.g. as through separate In certain embodiments, albumin fusion proteins and/or intravenous lines into the same individual. Administration “in polynucleotides of the invention are administered in combi combination further includes the separate administration of 40 nation with antiretroviral agents, nucleoside/nucleotide one of the compounds or agents given first, followed by the reverse transcriptase inhibitors (NRTIs), non-nucleoside second. reverse transcriptase inhibitors (NNRTIs), and/or protease In one embodiment, the albumin fusion proteins and, or inhibitors (PIs). NRTIs that may be administered in combi polynucleotides of the invention are administered in combi nation with the albumin fusion proteins and/or polynucle nation with an anticoagulant. Anticonagulants that may be 45 otides of the invention, include, but are not limited to, RET administered with the compositions of the invention include, ROVIRR (zidovudine/AZT), VIDEX(R) (didanosine/ddl), but are not limited to heparin, low molecular weight heparin, HIVIDR) (Zalcitabine/ddC), ZER1TR) (stavudine/d4T), warfarin sodium (e.g. COUMADINR), dicumarol, 4-hy EPIVIR(R) (lamivudine/3TC), and COMBIVIRR (zidovu droxycoumarin, anisindione (e.g. MIRADONTM), acenocou dine/lamivudine). NNRTIs that may be administered in com marol (e.g., nicoumalone, SINTHROMETM), indan-1,3-di 50 bination with the albumin fusion proteins and/or polynucle one, phenprocoumon (e.g. MARCUMARTM), ethyl otides of the invention, include, but are not limited to, biscoumacetate (e.g. TROMEXANTM), and aspirin. In a spe VIRAMUNE(R) (nevirapine), RESCRIPTOR(R) (delavirdine), cific embodiment, compositions of the invention are admin and SUSTIVAR (efavirenz). Protease inhibitors that may be istered in combination with heparin and or warfarin. In administered in combination with the albuminfusion proteins another specific embodiment, compositions of the invention 55 and/or polynucleotides of the invention, include, but are not are administered in combination with warfarin. In another limited to, CRIXIVANR) (indinavir), NORVIR(R) (ritonavir), specific embodiment, compositions of the invention are INVIRASE(R) (saquinavir), and VIRACEPTR) (nelfinavir). In administered in combination with warfarin and aspirin. In a specific embodiment, antiretroviral agents, nucleoside another specific embodiment, compositions of the invention reverse transcriptase inhibitors, non-nucleoside reverse tran are administered in combination with heparin. In another 60 Scriptase inhibitors, and/or protease inhibitors may be used in specific embodiment, compositions of the invention are any combination with albumin fusion proteins and/or poly administered in combination with heparin and aspirin. nucleotides of the invention to treat AIDS and/or to prevent or In another embodiment, the albumin fusion proteins and or treat HIV infection. polynucleotides of the invention are administered in combi Additional NRTIs include LODENOSINE(R) (F-ddA; an nation with thrombolytic drugs. Thrombolytic drugs that may 65 acid-stable adenosine NRTI; Triangle/Abbott; COVI be administered with the compositions of the invention RACIL(R) (emitricitabine/FTC: structurally related to lamivu include, but are not limited to, plasminogen, lys-plasmino dine (3TC) but with 3- to 10-fold greater activity in vitro: US 8,946,156 B2 93 94 Triangle/Abbott); dOTC (BCH-10652, also structurally at cell Surface rather than being a true integrase inhibitor; related to lamivudine but retains activity against a substantial Arondex); and naphthols such as those disclosed in WO proportion of lamivudine-resistant isolates: Biochem 98/50347. Pharma); Adefovir (refused approval for anti-HIV therapy by Additional antiretroviral agents include hydroxyurea-like FDA: Gilead Sciences); PREVEONR) (Adefovir Dipivoxil, compounds such as BCX-34 (a purine nucleoside phospho the active prodrug of adefovir; its active form is PMEA-pp); rylase inhibitor: Biocryst); ribonucleotide reductase inhibi VIREADR) (tenofovir) (bis-POC PMPA, a PMPA prodrug: tors such as DIDOXTM (Molecules for Health); inosine mono Gilead); DAPD/DXG (active metabolite of DAPD; Triangle/ phosphate dehydrogenase (IMPDH) inhibitors such as Abbott); REVERSETR) (D-D4FC) (related to 3TC, with VX-497 (Vertex); and mycopholic acids such as CellCept activity against AZT/3TC-resistant virus); GW420867X 10 (mycophenolate mofetil: Roche). (Glaxo Wellcome); ZIAGENR) (abacavir/159U89; Glaxo Additional antiretroviral agents include inhibitors of viral Wellcome Inc.); CS-87 (3'azido-2',3'-dideoxyuridine: WO integrase, inhibitors of viral genome nuclear translocation 99/66936); and S-acyl-2-thioethyl (SATE)-bearing prodrug Such as arylene bis(methylketone) compounds; inhibitors of forms of B-L-FD4C and B-L-FddC (see, International Publi HIV entry such as AOP-RANTES, NNY-RANTES, cation No. WO981 17281). 15 RANTES-IgG fusion protein, soluble complexes of Additional NNRTIs include COACTINONTM (Emivirine RANTES and glycosaminoglycans (GAG), and AMD-3100: MKC-442, potent NNRTI of the HEPT class; Triangle/Ab nucleocapsid Zinc finger inhibitors such as dithiane com bott); CAPRAVIRINETM (AG-1549 s-1153, a next genera pounds, targets of HIV Tat and Rev. and pharmacoenhancers tion NNRTI with activity against viruses containing the Such as ABT-378. K103N mutation: Agouron); PNU-142721 (has 20- to 50-fold Other antiretroviral therapies and adjunct therapies include greater activity than its predecessor delaVirdine and is active cytokines and lymphokines such as MIP-1C, MIP-1B, SDF against K103N mutants; Pharmacia & Upjohn); DPC-961 1C, IL-2, PROLEUKINTM (aldesleukin/L2-7001: Chiron), and DPC-963 (second generation derivatives of efavirenz, IL-4, IL-10, IL-12, and IL-13; interferons such as IFN-C2C.; designed to be active against viruses with the K103N muta antagonists of TNFs, NFKB, GM-CSF, M-CSF, and IL-10; tion; DuPont); GW-420867X (has 25-fold greater activity 25 agents that modulate immune activation Such as cyclosporine than HBY097 and is active against K103N mutants; Glaxo and prednisone; vaccines such as REMUNER) (HIV Immu Wellcome), CALANOLIDE A (naturally occurring agent nogen), APL 400-003 (Apollon), recombinant gp120 and from the latex tree; active against viruses containing either or fragments, bivalent (B/E) recombinant envelope glycopro both the Y 181C and K103N mutations); and Propolis (see, tein, rgp120CM235, MN rgp120, SF-2 rgp120, gp120/ International Publication No. WO99/49830). 30 soluble CD4 complex, Delta JR-FL protein, branched syn Additional protease inhibitors include LOPINAVIRTM thetic peptide derived from discontinuous gp120 C3/C4 (ABT378/r; Abbott Laboratories): BMS-232632 (an azapep domain, fusion-competent immunogens, and Gag, Pol, Nef, tide; Bristol-Myres Squibb), TIPRANAVIRTM (PNU and Tat vaccines; gene-based therapies such as genetic Sup 140690, a non-peptic dihydropyrone; Pharmacia & Upjohn); pressor elements (GSEs: WO 98/54366), and intrakines (ge PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); 35 netically modified CC chemokines targeted to the ER to block BMS 232632 (an azapeptide: Bristol-Myers Squibb); L-756, surface expression of newly synthesized CCR5 (Yang et al., 423 (an indinavir analog; Merck); DMP-450 (a cyclic urea PNAS94:11567-72 (1997); Chen et al., Nat. Med. 3:1110-16 compound: Avid & DuPont): AG-1776 (a peptidomimetic (1997)); antibodies such as the anti-CXCR4 antibody 12G5, with in vitro activity against protease inhibitor-resistant the anti-CCR5 antibodies 2D7,5C7, PA8, PA9, PA10, PA 11, viruses; Agouron); VX-175/GW-433908 (phosphate prodrug 40 PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA of amprenavir; Vertex & Glaxo Welcome); CGP61755 T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies (Ciba): and AGENERASETM (amprenavir. GlaxoWellcome 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tatantibod Inc.). ies, anti-TNF-C. antibodies, and monoclonal antibody 33A. Additional antiretroviral agents include fusion inhibitors/ aryl hydrocarbon (AH) receptor agonists and antagonists gp41 binders. Fusion inhibitors/gp41 binders include T-20 (a 45 such as TCDD, 3.3'44'.5-pentachlorobiphenyl, 3.3'4,4'-tet peptide from residues 643-678 of the HIV gp41 transmem rachlorobiphenyl, and C-naphthoflavone (see, International brane protein ectodomain which binds to gp41 in its resting Publication No. WO 98/30213); and antioxidants such as state and prevents transformation to the fusogenic state; Tri Y-L-glutamyl-L-cysteine ethyl ester (y-GCE: WO99/56764). meris) and T-1249 (a second-generation fusion inhibitor; Tri In a further embodiment, the albumin fusion proteins and/ meris). 50 or polynucleotides of the invention are administered in com Additional antiretroviral agents include fusion inhibitors/ bination with an antiviral agent. Antiviral agents that may be chemokine receptor antagonists. Fusion inhibitors/chemok administered with the albumin fusion proteins and or poly ine receptor antagonists include CXCR4 antagonists Such as nucleotides of the invention include, but are not limited to, AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40 acyclovir, ribavirin, amantadine, and remantadine. 4C (a cationic peptide), T22 (an 18 amino acid peptide; Tri 55 In other embodiments, albumin fusion proteins and/or meris) and the T22 analogs T134 and T 140; CCR5 antago polynucleotides of the invention may be administered incom nists such as RANTES (9-68), AOP-RANTES, NNY bination with anti-opportunistic infection agents. Anti-oppor RANTES, and TAK-779; and CCR5/CXCR4 antagonists tunistic agents that may be administered in combination with such as NSC 651016 (a distamycin analog). Also included are the albumin fusion proteins and/or polynucleotides of the CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetor 60 invention, include, but are not limited to, TRIMETHOPRIM agonists such as RANTES, SDF-1, MIP-1C, MIP-1 B, etc., SULFAMETHOXAZOLETM (also known as BACTRIMR), may also inhibit fusion. COTRIMR, and SEPTRAR), DAPSONETM (4,4'-diamino Additional antiretroviral agents include integrase inhibi diphenylsulfone (DDS)), PENTAMIDINETM, ATOVA tors. Integrase inhibitors includedicaffeoylquinic (DFOA) QUONETM (trans-2-4-(4-chlorophenyl)cyclohexyl)-3-hy acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid): 65 droxy-1,4-naphthalenedione), ISONIAZIDTM (isonicotinic quinalizarin (QLC) and related anthraquinones; acid hydrazide INHI), RIFAMPINTM (3-(4-Methyl-1-pip ZINTEVIRTM (AR 177, an oligonucleotide that probably acts erazinyl)iminomethylrifamycin or 5.6.9.17, 19.21-hexahy US 8,946,156 B2 95 96 droxy-23-methoxy-2,4,12,16.20.22-heptamethyl-8-N-(4- tic cytomegalovirus infection. In another specific embodi methyl-1-piperazinyl)formimidoyl-2.7-(epoxypentadeca ment, albumin fusion proteins and/or polynucleotides of the 1,11,13trienimino)naphtho2.1-bfuran-1,11(2H)-dione invention are used in any combination with FLUCONA 21-acetate), PYRAZINAMIDETM (CHNO), ETHAMBU ZOLETM, ITRACONAZOLETM, and/or KETOCONA TOLTM (+)2.2' (Ethylenediimino)-di-1-butanol dihydrochlo ZOLETM to prophylactically treat or prevent an opportunistic ride. Also known as MYAMBUTOL(R), RIFABUTINTM (1", fungal infection. In another specific embodiment, albumin 4-didehydro-1-deoxy-1,4-dihydro-5'-(2-methylpropyl)-1- fusion proteins and/or polynucleotides of the invention are oxorifamycin XIV, also known as MYCOBUTINR), used in any combination with ACYCLOVIRTM and/or FAM CLARITHROMYCINTM (6-0-methylerythromycin, also CICOLVIRTM to prophylactically treat or prevent an oppor known as BIAXINR), AZITHROMYCINTM (2R,3S4R,5R, 10 8R,10R,11R,12S,13S, 14R)-13-(2,6-dideoxy-3-C-methyl tunistic herpes simplex virus type I and/or type II infection. In 3-O-methyl-a-L-ribo-hexopyranosyl)oxyl-2-ethyl-3,4,10 another specific embodiment, albumin fusion proteins and/or trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-3,4,6- polynucleotides of the invention are used in any combination trideoxy-3-(dimethylamino)-b-D-xylohexopyranosylloxy With PYRIMETHAMINETM and for LEUCOVORINTM to 1-Oxa-6-azacyclopentadecan-15-one, also known as 15 prophylactically treator prevent an opportunistic Toxoplasma ZITHROMAX(R), GANCICLOVIRTM (9-2-hydroxy-1- gondii infection. In another specific embodiment, albumin (hydroxymethyl) ethoxymethylguanine also known as fusion proteins and/or polynucleotides of the invention are CYTOVENE-1V(R) or CYTOVENER), FOSCARNETTM used in any combination with LEUCOVORINTM and/or also known as FOSCAVIRR (foscarnet sodium)), CIDO NEUPOGENTM to prophylactically treator prevent an oppor FOVIRTM (1-(S)-3-hydroxy-2 (phosphonomethoxy)propyl tunistic bacterial infection. cytosine dihydrate (HPMPC), also known as VISTIDE(R), In a further embodiment, the albumin fusion proteins and FLUCONAZOLETM (2,4-difluoro-aa1-bis(1H-1,2,4-tria or polynucleotides of the invention are administered in com Zol-1-ylmethyl) benzyl alcohol, also known as DIFLU bination with an antibiotic agent. Antibiotic agents that may CANR), ITRACONAZOLETM ((+)-1-(R*)-sec-butyl-4-p- be administered with the albumin fusion proteins and/or poly 4-p-(2R*.4S*)-2-(2,4-dichlorophenyl)-2-(1H-1,2,4- 25 nucleotides of the invention include, but are not limited to, triazol-1-ylmethyl)-1,3-dioxolan-4-yl)methoxyphenyl)-1- amoxicillin, beta-lactamases, aminoglycosides, beta-lactam piperazinylphenyl-delta2-1,2,4-triazolin-5-one mixture (glycopeptide), beta-lactamases, Clindamycin, chloram with (+)-1-((R)-sec-butyl-4-p-4-p-(2S*4R)-2-(2,4- phenicol, cephalosporins, ciprofloxacin, erythromycin, fluo dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl-1,3-diox roquinolones, macrollides, metronidazole, penicillins, quino olan-4-yl)methoxyphenyl-1-piperazinylphenyl-delta2-1, 30 lones, rapamycin, rifampin, streptomycin, Sulfonamide, 2,4-triazolin-5-one or (+)-1-(RS)-sec butyl-4p-4-p- tetracyclines, trimethoprim, trimethoprim-sulfamethox (2R,4S)-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1- azole, and Vancomycin. ylmethyl)-1,3-dioxolan-4-yl)methoxyphenyl)-1- In other embodiments, the albumin fusion proteins and/or piperazinylphenyl-delta2-1,2,4-triazolin-5-one, also polynucleotides of the invention are administered in combi known as SPORANOX(R), KETOCONAZOLETM (cis-1- 35 nation with immunestimulants. Immunostimulants that may acetyl-4-4-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl be administered in combination with the albumin fusion pro methyl)-1,3-dioxolan-4-yl)methoxylphenylpiperazine), teins and/or polynucleotides of the invention include, but are ACYCLOVIRTM (2-amino-1,9-dihydro-9-(2-hydroxy not limited to levamisole (e.g. ERG AMISOLTM), isopri ethoxy)methyl-6H-purin-6-one, also known as ZOVI nosine (e.g. INOSIPLEXTM), interferons (e.g. interferon RAX(R), FAMCICOLVIRTM (2-[2-(2-amino-9H-purin-9-yl) 40 alpha), and interleukins (e.g., IL-2). ethyl-1,3-propanediol diacetate, also known as FAMVIRR), In other embodiments, albumin fusion proteins and/or PYRIMETHAMINETM (5-(4-chlorophenyl)-6-ethyl-2,4-py polynucleotides of the invention are administered in combi rimidinediamine, also known as DARAPRIMR), LEUCOV nation with immunosuppressive agents. Immunosuppressive ORINTM (L-Glutamic acid, N-4-(2-amino-5-formyl 14.5, agents that may be administered in combination with the 6,7,8-hexahydro-4-oxo-6-pteridinyl)methylamino 45 albumin fusion proteins and/or polynucleotides of the inven benzoyl-, calcium salt (1:1)), NEUPOGENTM (filgrastim/G- tion include, but are not limited to, Steroids, cyclosporine, CSF), and LEUKINETM (sargramostim/GM-CSF). In a cyclosporine analogs, cyclophosphamide methylprednisone, specific embodiment, albumin fusion proteins and/or poly prednisone, azathioprine, FK-506, 15-deoxyspergualin, and nucleotides of the invention are used in any combination with other immunosuppressive agents that act by Suppressing the TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAP 50 function of responding T cells. Other immunosuppressive SONETM, PENTAMIDINETM, and/or ATOVAQUONETM to agents that may be administered in combination with the prophylactically treat or prevent an opportunistic Pneu albumin fusion proteins and/or polynucleotides of the inven mocystis carinii pneumonia infection. In another specific tion include, but are not limited to prednisolone, methotrex embodiment, albumin fusion proteins and/or polynucleotides ate, thalidomide, methoXSalen, rapamycin, leflunomide, of the invention are used in any combination with ISO 55 mizoribine (BREDININTM), brequinar, deoxyspergualin, and NIAZIDTM, RIFAMPINTM, PYRAZINAMIDETM, and/or azaspirane (SKF 105685), ORTHOCLONE OKTR 3 ETHAMBUTOLTM to prophylactically treat or prevent an (muromonab-CD3), SANDIIMUNETM, NEORALTM, opportunistic Mycobacterium avium complex infection. In SANGDYATM (cyclosporne), PROGRAF(R) (FK506, tacroli another specific embodiment, albumin fusion proteins and/or mus), CELLCEPTOR (mycophenolate motefil, of which the polynucleotides of the invention are used in any combination 60 active metabolite is mycophenolic acid), IMURANTM (aza with RIFABUTINTM, CLARITHROMYCINTM, and/or thioprine), glucocorticosteroids, adrenocortical steroids Such AZITHROMYCINTM to prophylactically treat or prevent an as DELTASONETM (prednisone) and HYDELTRASOLTM opportunistic Mycobacterium tuberculosis infection. In (predmnsolone), FOLEXTM and MEXATETM (methotrxate), another specific embodiment, albumin fusion proteins and/or OXSORALEN-ULTRATM (methosalen) and RAPAM polynucleotides of the invention are used in any combination 65 UNETM (sirolimus). In a specific embodiment, immunosup with GANCICLOVIRTM, FOSCARNETTM, and/or CIDO pressants may be used to prevent rejection of organ or bone FOVIRTM to prophylactically treat or prevent an opportunis marrow transplantation. US 8,946,156 B2 97 98 In an additional embodiment, albumin fusion proteins and tungstate, calcium tungstate, Sodium tungstate dihydrate, and or polynucleotides of the invention are administered alone or tungstic acid. Suitable tungsten oxides include tungsten (IV) in combination with one or more intravenous immune globu oxide and tungsten (VI) oxide. Suitable oxo molybdenum lin preparations. Intravenous immune globulin preparations complexes include molybdate, molybdenum oxide, and that may be administered with the albumin fusion proteins molybdenyl complexes. Suitable molybdate complexes and/or polynucleotides of the invention include, but not lim include ammonium molybdate and its hydrates, Sodium ited to, GAMMARTM, IVEEGAMTM, SANDOGLOBU molybdate and its hydrates, and potassium molybdate and its LINTM, GAMMAGARDS DTM, ATGAMTM (antithymocyte hydrates. Suitable molybdenum oxides include molybdenum glubulin), and GAMIMUNETM. In a specific embodiment, (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suit albumin fusion proteins and/or polynucleotides of the inven 10 able molybdenyl complexes include, for example, molybde tion are administered in combination with intravenous nyl acetylacetonate. Other Suitable tungsten and molybde immune globulin preparations in transplantation therapy (e.g. num complexes includehydroxo derivatives derived from, for bone marrow transplant). example, glycerol, tartaric acid, and Sugars. In certain embodiments, the albuminfusion proteins and/or A wide variety of other anti-angiogenic factors may also be polynucleotides of the invention are administered alone or in 15 utilized within the context of the present invention. Repre combination with an anti-inflammatory agent. Anti-inflam sentative examples include, but are not limited to, platelet matory agents that may be administered with the albumin factor 4, protamine Sulphate, Sulphated chitin derivatives fusion proteins and or polynucleotides of the invention (prepared from queen crab shells), (Murata et al., Cancer Res. include, but are not limited to corticosteroids (e.g. betametha 51:22-26, (1991)); Sulphated Polysaccharide Peptidoglycan Sone, budesonide, cortisone, dexamethasone, hydrocorti Complex (SP-PG) (the function of this compound may be Sone, methylprednisolone, prednisolone, prednisone, and tri enhanced by the presence of steroids such as estrogen, and amcinolone), nonsteroidal anti-inflammatory drugs (e.g. tamoxifen citrate); Staurosporine; modulators of matrix diclofenac, diflunisal, etodolac, fenoprofen, floctafenine, metabolism, including for example, proline analogs, cishy flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofe droxyproline, d.L-3,4-dehydroproline. Thiaproline, alpha,al namate, mefenamic acid, meloxicam, nabumetone, 25 pha-dipyridyl, aminopropionitrile fumarate, 4-propyl-5-(4- naproxen, oxaprozin, phenylbutaZone, piroXicam, Sulindac, pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone: tenoxicam, tiaprofenic acid, and tolmetin), as well as antihis Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 tamines, aminoarylcarboxylic acid derivatives, arylacetic (Pavloffetal. J. Bio. Chem. 267: 17321-17326, (1992)): Chy acid derivatives, arylbutyric acid derivatives, arylcarboxylic mostatin (Tomkinson et al., Biochem J. 286:475-480. acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, 30 (1992)); Cyclodextrin Tetradecasulfate: Eponemycin; Camp salicylic acid derivatives, thiazinecarboxamides, e-acetami tothecin; Fumagillin (Ingber et al. Nature 348:555-557, docaproic acid, S-adenosylmethionine, 3-amino-4-hydroxy (1990)); Gold Sodium Thiomalate (“GST': Matsubara and butyric acid, amiXetrine, bendazac, benzydamine, bucolome, Ziff. J. Clin. Invest. 79: 1440-1446, (1987)); anticollagenase difenpiramide, ditazol, emorfaZone, guaiaZulene, nabume serum; alpha2-antiplasmin (Holmes et al. J. Biol. Chem. 262 tone, nimeSulide, orgotein, oxaceprol, paranyline, perisoxal, 35 (4): 1659-1664, (1987)). Bisantrene (National Cancer Insti pifoXime, produaZone, proxazole, and tenidap. tute); Lobenzarit disodium (N-(2)-carboxyphenyl-4- In an additional embodiment, the compositions of the chloroanthronilic acid disodium or “CCA': (Takeuchi et al. invention are administered alone or in combination with an Agents Actions 36:312-316, (1992)); and metalloproteinase anti-angiogenic agent. Anti-angiogenic agents that may be inhibitors such as BB94. administered with the compositions of the invention include, 40 Additional anti-angiogenic factors that may also be uti but are not limited to, Angiostatin (Entremed, Rockville, lized within the context of the present invention include Tha Md.), Troponin-(Boston Life Sciences, Boston, Mass.), anti lidomide, (Celgene, Warren, N.J.); Angiostatic steroid: Invasive Factor, retinoic acid and derivatives thereof, pacli AGM-1470 (H. Brem and J. Folkman.JPediatr. Surg. 28:445 taxel (Taxol), Suramin, Tissue Inhibitor of Metalloprotein 51 (1993)); an integrin alpha v beta 3 antagonist (C. Storgard ase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI, 45 et al. J. Clin. Invest. 103:47-54 (1999)); carboxynaminolmi Plasminogen Activator Inhibitor-1, Plasminogen Activator dazole; Carboxyamidotriazole (CAI) (National Cancer Insti Inhibitor-2, and various forms of the lighter “d group' tran tute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXi sition metals. GENE, Boston, Mass.); Squalamine (Magainin Lighter “d group' transition metals include, for example, Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (Tap Vanadium, molybdenum, tungsten, titanium, niobium, and 50 Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca tantalum species. Such transition metal species may form (London, UK); APRA (CT2584); Benefin, Byrostatin-1 transition metal complexes. Suitable complexes of the above (SC339555); CGP-41251 (PKC 412); CM 101: Dexrazoxane mentioned transition metal species include oxo transition (ICRF187); DMXAA: Endostatin: Flavopridiol: Genestein: metal complexes. GTE: ImmTher: Iressa (ZD1839); Octreotide (Somatosta Representative examples of Vanadium complexes include 55 tin); Panretin: Penacillamine: Photopoint; P1-88: Prinomas oxovanadium complexes such as Vanadate and Vanadylcom tat (AG-3340) Purly tin; Suradista (FCE2664-4); Tamoxifen plexes. Suitable Vanadate complexes include metavanadate (Nolvadex); Tazarotene; Tetrathiomolybdate: Xeloda and orthovanadate complexes such as, for example, ammo (Capecitabine); and 5-Fluorouracil. nium metavanadate, sodium metavanadate, and sodium Anti-angiogenic agents that may be administrated in com orthovanadate. Suitable vanadyl complexes include, for 60 bination with the compounds of the invention may work example, Vanadyl acetylacetonate and Vanadyl Sulfate includ through a variety of mechanisms including, but not limited to, ing vanadyl Sulfate hydrates Such as Vanadyl Sulfate mono inhibiting proteolysis of the extracellular matrix, blocking the and trihydrates. function of endothelial cell-extracellular matrix adhesion Representative examples of tungsten and molybdenum molecules, by antagonizing the function of angiogenesis complexes also include oxo complexes. Suitable OXO tung 65 inducers such as growth factors, and inhibiting integrin recep Sten complexes include tungstate and tungsten oxide com tors expressed on proliferating endothelial cells. Examples of plexes. Suitable tungstate complexes include ammonium anti-angiogenic inhibitors that interfere with extracellular US 8,946,156 B2 99 100 matrix proteolysis and which may be administered in combi analogs (for example, Methotrexate (amethopterin)), pyrimi nation with the compositions of the invention include, but are dine analogs (for example, Fluorouacil (5-fluorouracil; not limited to, AG-3340 (Agouron, LaJolla, Calif.), BAY-12 5-FU). Floxuridine (fluorodeoxyuridine; FudR), and Cytara 9566 (Bayer, West Haven, Conn.), BMS-275291 (Bristol bine (cytosine arabinoside)), purine analogs and related Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, inhibitors (for example, Mercaptopurine (6-mercaptopurine; East Hanover, N.J.), Marimastat (British Biotech, Oxford, 6-NIP). Thioguanine (6-thioguanine: TG), and Pentostatin UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of (2'-deoxycoformycin)), Vinca alkaloids (for example, Vin anti-angiogenic inhibitors that act by blocking the function of blastine (VLB, vinblastine sulfate)) and Vincristine (vincris endothelial cell-extracellular matrix adhesion molecules and tine Sulfate)), epipodophyllotouins (for example, Etoposide which may be administered in combination with the compo 10 and Teniposide), antibiotics (for example, Dactinomycin (ac sitions of the invention include, but are not limited to, EMD tinomycin D), Daunorubicin (daunomycin; rubidomycin), 12 1974 (Merck KcgaA Darmstadt, Germany) and Vitaxin Doxorubicin, Bleomycin, Plicamycin (mithramycin), and (Ixsys, La Jolla, Calif/Medimmune, Gaithersburg, Md.). Mitomycin (mitomycin C), enzymes (for example, L-Aspara Examples of anti-angiogenic agents that act by directly ginase), biological response modifiers (for example, Inter antagonizing or inhibiting angiogenesis inducers and which 15 feron-alpha and interferon-alpha-2b), platinum coordination may be administered in combination with the compositions of compounds (for example, Cisplatin (cis-DDP) and Carbopl the invention include, but are not limited to, Angiozyme (Ri atin), anthracenedione (Mlitoxantrone), substituted ureas (for bozyme, Boulder, Colo.). Anti-VEGF antibody (Genentech, example, Hydroxyurea), methylhydrazine derivatives (for S, San Francisco, Calif.), PTK-787/ZK-225846 (Novartis, example, Procarbazine (N-methylhydrazine; MIH), adreno Basel, Switzerland), SU-101 (Sugen, S. San Francisco, corticosteroids (for example, Prednisone), progestins (for Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, example, Hydroxyprogesterone caproate, Medroxyprogest N.J.), and SU-6668 (Sugen). Otheranti-angiogenic agents act erone, Medroxyprogesterone acetate, and Megestrol acetate), to indirectly inhibit angiogenesis. Examples of indirect estrogens (for example, Diethylstilbestrol (DES), Diethylstil inhibitors of angiogenesis which may be administered in bestrol diphosphate, Estradiol, and Ethinyl estradiol), anties combination with the compositions of the invention include, 25 trogens (for example, Tamoxifen), androgens (Testosterone but are not limited to, IM-862 (Cytran, Kirkland, Wash.), proprionate, and Fluoxymesterone), antiandrogens (for Interferon-alpha, IL-12 (Roche, Nutley, N.J.), and Pentosan example, Flutamide), gonadotropin-releasing hormone ana polysulfate (Georgetown University, Washington, D.C.). logs (for example, Leuprolide), other hormones and hormone In particular embodiments, the use of compositions of the analogs (for example, methyltestosterone, estramustine, invention in combination with anti-angiogenic agents is con 30 estramustine phosphate Sodium, chlorotrianisene, and testo templated for the treatment, prevention, and/or amelioration lactone), and others (for example, dicarbazine, glutamic acid, of an autoimmune disease, such as for example, an autoim and mitotane). mune disease described herein. In one embodiment, the compositions of the invention are In a particular embodiment, the use of compositions of the administered in combination with one or more of the follow invention in combination with anti-angiogenic agents is con 35 ing drugs: infliximab (also known as REMICADETM Cento templated for the treatment, prevention, and, or amelioration cor, Inc.), TROCADETM (Roche, RO-32-3555), Leflunomide of arthritis. In a more particular embodiment, the use of (also known as ARAVATM from Hoechst Marion Roussel), compositions of the invention in combination with anti-an KINERETTM (an IL-1 Receptor antagonist also known as giogenic agents is contemplated for the treatment, prevention, Anakinra from Amgen, Inc.) and or amelioration of rheumatoid arthritis. 40 In a specific embodiment, compositions of the invention In another embodiment, the polynucleotides encoding a are administered in combination with CHOP (cyclophospha polypeptide of the present invention are administered in com mide, doxorubicin, Vincristine, and prednisone) or combina bination with an angiogenic protein, or polynucleotides tion of one or more of the components of CHOP. In one encoding an angiogenic protein. Examples of angiogenic pro embodiment, the compositions of the invention are adminis teins that may be administered with the compositions of the 45 tered in combination with anti-CD20 antibodies, human invention include, but are not limited to acidic and basic monoclonal anti-CD20 antibodies. In another embodiment, fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epi the compositions of the invention are administered in combi dermal growth factor alpha and beta, platelet-derived endot nation with anti-CD20 antibodies and CHOP, or anti-CD20 helial cell growth factor, platelet-derived growth factor, antibodies and any combination of one or more of the com tumor necrosis factor alpha, hepatocyte growth factor, insu 50 ponents of CHOP particularly cyclophosphamide and/or lin-like growth factor, colony stimulating factor, macrophage prednisone. In a specific embodiment, compositions of the colony Stimulating factor, granulocyte/macrophage colony invention are administered incombination with Rituximab. In stimulating factor, and nitric oxide synthase. a further embodiment, compositions of the invention are In additional embodiments, compositions of the invention administered with Rituximab and CHOP, or Rituximab and are administered in combination with a chemotherapeutic 55 any combination of one or more of the components of CHOP. agent. Chemotherapeutic agents that may be administered particularly cyclophosphamide and/or prednisone. In a spe with the albumin fusion proteins and/or polynucleotides of cific embodiment, compositions of the invention are admin the invention include, but are not limited to alkylating agents istered in combination with to situmomab. In a further Such as nitrogen mustards (for example, Mechlorethamine, embodiment, compositions of the invention are administered cyclophosphamide, Cyclophosphamide Ifosfamide, Mel 60 with to situmomab and CHOP or to situmomab and any com phalan (L-sarcolysin), and Chlorambucil), ethylenimines and bination of one or more of the components of CHOP particu methylmelamines (for example, Hexamethylmelamine and larly cyclophosphamide and/or prednisone. The anti-CD20 Thiotepa), alkyl sulfonates (for example, Busulfan), antibodies may optionally be associated with radioisotopes, nitrosoureas (for example, Carmustine (BCNU), Lomustine toxins or cytotoxic prodrugs. (CCNU), Semustine (methyl-CCNU), and Streptozocin 65 In another specific embodiment, the compositions of the (streptozotocin)), triaZenes (for example, Dacarbazine invention are administered in combination ZEVALINTM (DTIC: dimethyltriazenoimidazolecarboxamide)), folic acid (Ibritumomab Tiuxetan). In a further embodiment, composi US 8,946,156 B2 101 102 tions of the invention are administered with ZEVALINTM and Number WO 98.707832; and Vascular Endothelial Growth CHOP, or ZEVALINTM and any combination of one or more Factor-E (VEGF-E), as disclosed in German Patent Number of the components of CHOP particularly cyclophosphamide DE 19639601. The above mentioned references are herein and/or prednisone. ZEVALINTM may be associated with one incorporated by reference in their entireties. or more radioisotopes. Particularly preferred isotopes are 'Y In an additional embodiment, the albumin fusion proteins and '''In. and or polynucleotides of the invention are administered in In an additional embodiment, the albumin fusion proteins combination with Fibroblast Growth Factors. Fibroblast and/or polynucleotides of the invention are administered in Growth Factors that may be administered with the albumin combination with cytokines. Cytokines that may be adminis fusion proteins and or polynucleotides of the invention tered with the albumin fusion proteins and/or polynucleotides 10 include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, of the invention include, but are not limited to, IL2, IL3, IL4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-II, IL5, IL6, IL7, IL 10, IL12, IL 13, IL15, anti-CD-R4, CD40L, FGF-12, FGF-13, FGF-14, and FGF-15. IFN-gamma and TNF-alpha. In another embodiment, albu In an additional embodiment, the albumin fusion proteins min fusion proteins and/or polynucleotides of the invention and/or polynucleotides of the invention are administered in may be administered with any interleukin, including, but not 15 combination with hematopoietic growth factors. Hematopoi limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, etic growth factors that may be administered with the albumin IL-7, IL-S, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, fusion proteins and/or polynucleotides of the invention IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21. include, but are not limited to, granulocyte macrophage In one embodiment, the albumin fusion proteins and, or colony stimulating factor (GM-CSF) (sargramostim, LEUK polynucleotides of the invention are administered in combi INETM, PROKINETM), granulocyte colony stimulating factor nation with members of the TNF family, TNF, TNF-related or (G-CSF) (filgrastim, NEUPOGENTM), macrophage colony TNF-like molecules that may be administered with the albu stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin min fusion proteins and/or polynucleotides of the invention alfa, EPOGENTM, PROCRITTM), stem cell factor (SCF, c-kit include, but are not limited to soluble forms of TNF-alpha, ligand, Steel factor), megakaryocyte colony stimulating fac lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT 25 tor, PIXY321 (a GMCSF/IL-3 fusion protein), interleukins, beta (found in complex heterotrimer LT-alpha2-beta), OPGL, especially any one or more of IL-1 through IL-12, interferon FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, gamma, or thrombopoietin. TNF-gamma (International Publication No. WO96/14328), In certain embodiments, albumin fusion proteins and/or AIM-1 (International Publication No. WO97/33899), endok polynucleotides of the present invention are administered in ine-alpha (International Publication No. WO 98/07880), 30 combination with adrenergic blockers, such as, for example, OPG, and neutrokine-alpha (International Publication No. acebutolol, atenolol, betaxolol, bisoprolol, carteolol, labe WO 98/18921, OX40, and nerve growth factor (NGF), and talol, metoprolol, nadolol, Oxprenolol, penbutolol, pindolol, soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 propranolol, Sotalol, and timolol. (International Publication No. WO 96/34095), DR3 (Interna In another embodiment, the albumin fusion proteins and/or tional Publication No. WO 97/33904), DR4 (International 35 polynucleotides of the invention are administered in combi Publication No. WO 98/32856), TR5 (International Publica nation with an antiarrhythmic drug (e.g., adenosine, ami tion No. WO 98/30693), TRANK, TR9 (International Publi doarone, bretylium, digitalis, digoxin, digitoxin, diliazem, cation No. WO 98/56892), TR10 (International Publication disopyramide, esmolol, flecamide, lidocaine, mexiletine, No. WO 98/54202), 312C2 (International Publication No. moricizine, phenyloin, procainamide. N-acetyl procaina WO98/06842), and TR12, and soluble forms CD154, CD70, 40 mide, propafenone, propranolol, quinidine, Sotalol, and CD153. tocuinide, and Verapumil). In an additional embodiment, the albumin fusion proteins In another embodiment, the albumin fusion proteins and or and/or polynucleotides of the invention are administered in polynucleotides of the invention are administered in combi combination with angiogenic proteins. Angiogenic proteins nation with diuretic agents, such as carbonic anhydrase-in that may be administered with the albumin fusion proteins 45 hibiting agents (e.g., acetazolamide, dichlorphenamide, and and or polynucleotides of the invention include, but are not methazolamide), osmotic diuretics (e.g., glycerin, isosorbide, limited to, Glioma Derived Growth Factor (GDGF), as dis mannitol, and urea), diuretics that inhibit Na"—K"-2Cl closed in European Patent Number EP-3998 16: Platelet Symport (e.g. furosemide, bumetanide, azosemide, piret Derived Growth Factor-A (PDGF-A), as disclosed in Euro anide, tripamide, ethacrynic acid, muZolimine, and pean Patent Number EP-6821.10: Platelet Derived Growth 50 torsemide), thiazide and thiazide-like diuretics (e.g. bendrof Factor-B (PDGF-B), as disclosed in European Patent Number lumethiazide, benzthiazide, chlorothiazide, hydrochlorothi EP-282317: Placental Growth Factor (PIGF), as disclosed in azide, hydroflumethiazide, methyclothiazide, polythiazide, International Publication Number WO92/06194: Placental trichormethiazide, chlorthalidone, indapamide, metolaZone, Growth Factor-2 (PIGF-2), as disclosed in Hauser et al. and quinethaZone), potassium sparing diuretics (e.g. Growth Factors, 4:259-268 (1993): Vascular Endothelial 55 amiloride and triamterene), and mineralcorticoid receptor Growth Factor (VEGF), as disclosed in International Publi antagonists (e.g. spironolactone, canrenone, and potassium cation Number WO 90/13649; Vascular Endothelial Growth canrenoate). Factor-A (VEGF-A), as disclosed in European Patent Num In one embodiment, the albumin fusion proteins and/or ber EP-506477: Vascular Endothelial Growth Factor-2 polynucleotides of the invention are administered in combi (VEGF-2), as disclosed in International Publication Number 60 nation with treatments for endocrine and or hormone imbal WO 96/39515: Vascular Endothelial Growth Factor B ance disorders. Treatments for endocrine and/or hormone (VEGF-3): Vascular Endothelial Growth Factor B-186 imbalance disorders include, but are not limited to, '''I, (VEGF-B 186), as disclosed in International Publication radioactive isotopes of iodine such as ''I and 'I; recombi Number WO 96/26736: Vascular Endothelial Growth Fac nant growth hormone, such as HUMATROPETM (recombi tor-D (VEGF-D), as disclosed in International Publication 65 nant Somatropin): growth hormone analogs such as PRO Number WO 98/02543: Vascular Endothelial Growth Fac TROPINTM (somatrem); dopamine agonists such as tor-D (VEGF-D), as disclosed in International Publication PARLODELTM (bromocriptine): somatostatin analogs such US 8,946,156 B2 103 104 as SANDOSTATINTM (octreotide): gonadotropin prepara ILONTM (methyltestosterone), and OXANDRINTM (oxan tions such as PREGNYLTM, A.P.L.TM and PROFASITM drolone); testosterone transdermal systems such as (chorionic gonadotropin (CG)), PERGONALTM (menotro TESTODERMTM; androgen receptor antagonistand 5-alpha pins), and METRODINTM (urofollitropin (uFSH)): synthetic reductase inhibitors such as ANDROCURTM (cyproterone human gonadotropin releasing hormone preparations such as 5 acetate), EULEXINTM (flutamide), and PROSCARTM (finas FACrRELTM and LUTREPULSETM (gonadorelin hydrochlo teride); adrenocorticotropic hormone preparations such as ride); synthetic gonadotropin agonists such as LUPRONTM CORTROSYNTM (cosyntropin); adrenocortical steroids and (leuprolide acetate), SUPPRELINTM (histrelin acetate), their synthetic analogs such as ACLOVATETM (alclometa SYNARELTM (nafarelin acetate), and ZOLADEXTM (goser elin acetate); synthetic preparations of thyrotropin-releasing 10 sone dipropionate), CYCLOCORTTM (amcinonide), hormone Such as RELEFACT TRHTM and THYPINONETM BECLOVENTTM and VANCERILTM (beclomethasone dipro (protirelin); recombinant human TSH such as THYRO pionate), CELESTONETM (betamethasone), BENISONETM GENTM: synthetic preparations of the sodium salts of the and UTICORTTM (betamethasone benzoate), natural isomers of thyroid hormones such as L-TTM, SYN DIPROSONETM (betamethasone dipropionate), CELE THROIDTM and LEVOTHROIDTM (levothyroxine sodium), 15 STONE PHOSPHATETM (betamethasone sodium phos L-TTM, CYTOMELTM and TRIOSTATTM (liothyroine phate), CELESTONE SOLUSPANTM (betamethasone sodium), and THY ROLARTM (liotrix); antithyroid com sodium phosphate and acetate), BETA-VALTM and VAL pounds such as 6-n-propylthiouracil (propylthiouracil), ISONETM (betamethasone valerate), TEIMOVATETM (clobe 1-methyl-2-mercaptoimidazole and TAPAZOLETM (methi tasol propionate), CLODERMTM (clocortolone pivalate), mazole), NEO-MERCAZOLETM (carbimazole): beta-adren- 20 CORTEFTM and HYDROCORTONETM (cortisol (hydrocor ergic receptor antagonists Such as propranolol and esmolol: tisone)), HYDROCORTONE ACETATETM (cortisol (hydro Ca" channel blockers: dexamethasone and iodinated radio cortisone) acetate), LOCOIDTM (cortisol (hydrocortisone) logical contrast agents such as TELEPAQUETM (iopanoic butyrate), HYDROCORTONE PHOSPHATETM (cortisol acid) and ORAGRAFINTM (sodium ipodate). (hydrocortisone) sodium phosphate), A-HYDROCORTTM Additional treatments for endocrine and or hormone 25 and SOLU CORTEFTM (cortisol (hydrocortisone) sodium imbalance disorders include, but are not limited to estrogens succinate), WESTCORTTM (cortisol (hydrocortisone) valer or conjugated estrogens such as ESTRACETM (estradiol), ate), CORTISONE ACETATETM (cortisone acetate), DESO ESTINYLTM (ethinyl estradiol), PREMARINTM, WENTM and TRIDESILONTM (desonide), TOPICORTTM ESTRATABTM, ORTHO-ESTTM, OGENTM and estropipate (desoximetasone), DECADRONTM (dexamethasone), DEC (estrone), ESTROVISTM (quinestrol), ESTRADERMTM (es- 30 ADRON LATM (dexamethasone acetate), DECADRON tradiol), DELESTROGENTM and VALERGENTM (estradiol PHOSPHATETM and HEXADROL PHOSPHATETM (dex valerate, DEPO-ESTRADIOL CYPIONATETM and amethasone sodium phosphate), FLORONETM and MAXI ESTROJECT LATM (estradiol cypionate): antiestrogens such FLORTM (diflorasone diacetate), FLORINEF ACETATETM as NOLVADEXTM (tamoxifen), SEROPHENETM and CLO (fludrocortisone acetate), AEROBIDTM and NASALIDETM, MIDTM (clomiphene): progestins such as DURALUTINTM 35 (flunisolide), FLUONIDTM and SYNALARTM (fluocinolone (hydroxyprogesterone caproate), NMPATM and DEPO acetonide), LIDEXTM (fluocinonide), FLUOR-OPTM and PROVERATM (medroxyprogesterone acetate), PROVERATM FILTM (tfluorometholone), CORDRANTM (flurandrenolide), and CYCRINTM (NIPA), MEGACETM (megestrol acetate), HALOGTM (halcinonide), HMSLIZLIFILMTM (medrysone), NORLLTINTM (norethindrone), and NORLUTATETM and MEDROLTM (methylprednisolone), DEPO-MEDROLTM AYGESTINTM (norethindrone acetate); progesterone 40 and MIEDROL ACETATETM (methylprednisone acetate), implants such as NORPLANT MTM (subdermal implants of A-METHAPREDTM and SOLUMEDROLTM (methylpred norgestrel); antiprogestins such as RU486TM (mifepristone): nisolone sodium succinate), ELOCONTM (mometasone hormonal contraceptives such as ENOVIDTM (norethynodrel furoate), HALDRONETM (paramethasone acetate), DELTA plus mestranol), PROGESTASERTTM (intrauterine device CORTEFTM (prednisolone), ECONOPREDTM (prednisolone that releases progesterone), LOESTRINTM, BREVICONTM, 45 acetate), HYDELTRASOLTM (prednisolone sodium phos MODICONTM, GENORATM, NELONATM, NORINYLTM, phate), HYDELTRA-T.B.ATM (prednisolone tebutate), DEL OVACON-35TM and OVACON-50TM (ethinyl estradiol/nore TASONETM (prednisone), ARISTOCORTTM and KENA thindrone), LEVLENTM, NORDETTETM, TRI-LEV LENTM CORTTM (triamcinolone), KENALOGTM (triamcinolone and TRIPHASIL-21 TM (ethinyl estradiol/levonorgestrel) acetonide), ARISTOCORTTM and KENACORT DIAC LO/OVRALTM and OVRALTM (ethinyl estradiol/norgestrel), 50 ETATETM (triamcinolone diacetate), and ARISTOSPANTM DEMULENTM (ethinyl estradiol/ethynodiol diacetate), (triamcinolone hexacetonide); inhibitors of biosynthesis and NORINYLTM, ORTHO-NOVUMTM, NORETHINTM, action of adrenocortical Steroids such as CYTADRENTM GENORATM, and NELOVATM (norethindrone/mestranol), (aminoglutethimide), NIZORALTM (ketoconazole), DESOGENTM and ORTHO-CEPTTM (ethinyl estradiol/ MODRASTANETM (trilostane), and METOPIRONETM desogestrel), ORTHO-CYCLENTM and ORTHO-TRICY. 55 (metyrapone); bovine, porcine or human insulin or mixtures CLENTM (ethinyl estradiol/norgestimate), MICRONORTM thereof; insulin analogs; recombinant human insulin Such as and NOR-QDTM (norethindrone), and OVRETTETM (norg HUMULINTM and NOVOLINTM: oral hypoglycemic agents estrel). such as ORAMIDETM and ORINASETM (tolbutamide), Additional treatments for endocrine and/or hormone DIABINESETM (chlorpropamide), TOLAMIDETM and imbalance disorders include, but are not limited to testoster- 60 TOLINASETM (tolazamide), DYMELORTM (acetohexam one esters such as methenolone acetate and testosterone ide), glibenclamide, MICRONASETM, DIBETATM and GLY undecanoate; parenteral and oral androgens Such as TEST NASETM (glyburide), GLUCOTROLTM (glipizide), and OJECT-50TM (testosterone), TESTEXTM (testosterone propi DIAMICRONTM (gliclazide), GLUCOPHAGETM (met onate), DELATESTRYLTM (testosterone enanthate), DEPO formin), ciglitaZone, pioglitaZone, and alpha-glucosidase TESTOSTERONETM (testosterone cypionate), 65 inhibitors; bovine or porcine glucagon; Somatostatins such as DANOCRINETM (danazol), HALOTESTINTM (fluoxymes SANDOSTATINTM (octreotide); and diazoxides such as terone), ORETON METHYLTM, TESTREDTM and VIR PROGLYCEMTM (diazoxide). US 8,946,156 B2 105 106 In one embodiment, the albumin fusion proteins and/or the albumin fusion proteins and/or polynucleotides of the polynucleotides of the invention are administered in combi invention include, but are not limited to, Angiotensin Con nation with treatments for uterine motility disorders. Treat Verting Enzyme (ACE) inhibitors (e.g. papaverine, isoxSu ments for uterine motility disorders include, but are not lim prine, benazepril, captopril, cilaZapril, enalapril, enalaprilat, ited to, estrogen drugs such as conjugated estrogens (e.g., fosinopril, lisinopril, moexipril, perindopril, quinapril, rami PREMARINR) and ESTRATAB(R), estradiols (e.g., CLI pril, spirapril, trandolapril, and nylidrin), and nitrates (e.g. MARAR) and ALORAR), estropipate, and chlorotrianisene: isosorbide dinitrate, isosorbide mononitrate, and nitroglyc progestin drugs (e.g., AMENR) (medroxyprogesterone), erin). Examples of calcium channel blocking agents that may MICRONORR) (norethidrone acetate), PROMETRIUM(R) be administered in combination with the albumin fusion pro progesterone, and megestrol acetate); and estrogen progest 10 teins and or polynucleotides of the invention include, but are erone combination therapies Such as, for example, conjugated not limited to amlodipine, bepridil, diltiazem, felodipine, flu estrogens, medroxy progesterone (e.g. PREMPROTM and narizine, isradipine, nicardipine, nifedipine, nimodipine, and PREMPHASE(R) and norethindrone acetate ethinyl estsra Verapamil. diol (e.g. FEMHRTTM). In certain embodiments, the albumin fusion proteins and, In an additional embodiment, the albumin fusion proteins 15 or polynucleotides of the invention are administered in com and or polynucleotides of the invention are administered in bination with treatments for gastrointestinal disorders. Treat combination with drugs effective in treating iron deficiency ments for gastrointestinal disorders that may be administered and hypochromic anemias, including but not limited to fer with the albumin fusion protein and/or polynucleotide of the rous sulfate (iron sulfate, FEOSOLTM), ferrous fumarate (e.g. invention include, but are not limited to. He histamine recep FEOSTATTM), ferrous gluconate (e.g. FERGONTM), polysac tor antagonists (e.g., TAGAMETTM (cimetidine), ZAN charide-iron complex (e.g. NIFEREXTM), iron dextran injec TACTM (ranitidine), PEPCIDTM (famotidine), and AXIDTM tion (e.g. INFEDTM), cupric sulfate, pyroxidine, riboflavin, (nizatidine)); inhibitors of H, K ATPase (e.g. PRE Vitamin B, cyancobalamin injection (e.g., REDISOLTM, VACIDTM (lansoprazole) and PRILOSECTM (omeprazole)); RUBRAMIN PCTM), hydroxocobalamin. folic acid (e.g., Bismuth compounds (e.g. PEPTO-BISMOLTM (bismuth sub FOLVITETM), leucovorin (folinic acid, 5-CHOH4PteClu, 25 salicylate) and DE-NOLTM (bismuth subcitrate)); variousant citrovorum factor) or VELLCOVORIN (Calcium salt of leu acids; sucralfate; prostaglandin analogs (e.g. CYTOTECTM covorin), transferrin or ferritin. (misoprostol)); muscarinic cholinergic antagonists; laxatives In certain embodiments, the albuminfusion proteins and/or (e.g. Surfactant laxatives, stimulant laxatives, saline and polynucleotides of the invention are administered in combi osmotic laxatives); antidiarrheal agents (e.g., LOMOTILTM nation with agents used to treat psychiatric disorders. Psychi 30 (diphenoxylate), MOTOFENTM (diphenoxin), and IMO atric drugs that may be administered with the albumin fusion DIUMTM (loperamide hydrochloride)), synthetic analogs of proteins and or polynucleotides of the invention include, but somatostatin such as SANDOSTATINTM (octreotide), anti are not limited to antipsychotic agents (e.g. chlorpromazine, emetic agents (e.g. ZOFRANTM (ondansetron), KYTRILTM chlorprothixene, clozapine, fluiphenazine, haloperidol, loxap (granisetron hydrochloride), tropisetron, dolasetron, meto ine, mesoridazine, molindone, olanzapine, perphenazine, 35 clopramide, chlorpromazine, perphenazine, prochlorpera pimozide, quetiapine, risperidone, thioridazine, thiothixene, Zine, promethazine, thiethylperazine, triflupromazine, dom trifluoperazine, and triflupromazine), antimanic agents (e.g. peridone, haloperidol, droperidol, trimethobenzamide, carbamazepine, divalproex sodium, lithium carbonate, and dexamethasone, methylprednisolone, dronabinol, and lithium citrate), antidepressants (e.g., amitriptyline, amoxap nabilone); D2 antagonists (e.g., metoclopramide, tri ine, bupropion, citalopram, clomipramine, desipramine, dox 40 methobenzamide and chlorpromazine); bile salts; chenode epin, fluvoxamine, fluoxetine, imipramine, isocarboxazid, oxycholic acid; urSodeoxycholic acid; and pancreatic enzyme maprotiline, mirtazapine, nefazodone, nortriptyline, paroxet preparations such as pancreatin and pancrelipase. ine, phenelzine, protriptyline, Sertraline, tranylcypromine, In additional embodiments, the albumin fusion proteins traZodone, trimipramine, and Venlafaxine), antianxiety and/or polynucleotides of the invention are administered in agents (e.g., alprazolam, buspirone, chlordiazepoxide, clo 45 combination with other therapeutic or prophylactic regimens, razepate, diazepam, halazepam, lorazepam, oxazepam, and Such as, for example, radiation therapy. prazepam), and stimulants (e.g., d-amphetamine, meth The invention also provides a pharmaceutical pack or kit ylphenidate, and pemoline). comprising one or more containers filled with one or more of In other embodiments, the albumin fusion proteins and/or the ingredients of the pharmaceutical compositions compris polynucleotides of the invention are administered in combi 50 ing albumin fusion proteins of the invention. Optionally asso nation with agents used to treat neurological disorders. Neu ciated with Such container(s) can be a notice in the form rological agents that may be administered with the albumin prescribed by a governmental agency regulating the manu fusion proteins and/or polynucleotides of the invention facture, use or sale of pharmaceuticals or biological products, include, but are not limited to antiepileptic agents (e.g., car which notice reflects approval by the agency of manufacture, bamazepine, clonazepam, ethoSuximide, phenobarbital, phe 55 use or sale for human administration. nyloin, primidone, Valproic acid, divalproex sodium, felbam Gene Therapy ate, gabapentin, lamotrigine, levetiracetam, oXcarbazepine, Constructs encoding albumin fusion proteins of the inven tiagabine, topiramate, Zonisamide, diazepam, lorazepam, and tion can be used as a part of a gene therapy protocol to deliver clonazepam), antiparkinsonian agents (e.g., levodopa/carbi therapeutically effective doses of the albumin fusion protein. dopa, selegiline, amantidine, bromocriptine, pergolide, rop 60 A preferred approach for in vivo introduction of nucleic acid inirole, pramipexole, benztropine; biperiden; ethopropazine; into a cell is by use of a viral vector containing nucleic acid, procyclidine; trihexyphenidyl, tolcapone), and ALS thera encoding an albumin fusion protein of the invention. Infec peutics (e.g. riluzole). tion of cells with a viral vector has the advantage that a large In another embodiment, albumin fusion proteins and or proportion of the targeted cells can receive the nucleic acid. polynucleotides of the invention are administered in combi 65 Additionally, molecules encoded within the viral vector. e.g., nation with vasodilating agents and or calcium channel block by a cDNA contained in the viral vector, are expressed effi ing agents. Vasodilating agents that may be administered with ciently in cells which have taken up viral vector nucleic acid. US 8,946,156 B2 107 108 Retrovirus vectors and adeno-associated virus vectors can nantly from specificity of transfection provided by the gene be used as a recombinant gene delivery system for the transfer delivery vehicle, cell-type or tissue-type expression due to the of exogenous nucleic acid molecules encoding albumin transcriptional regulatory sequences controlling expression fusion proteins in vivo. These vectors provide efficient deliv of the receptor gene, or a combination thereof. In other ery of nucleic acids into cells, and the transferred nucleic embodiments, initial delivery of the recombinant gene is acids are stably integrated into the chromosomal DNA of the more limited with introduction into the animal being quite host. The development of specialized cell lines (termed localized. For example, the gene delivery vehicle can be "packaging cells') which produce only replication-defective introduced by catheter (see U.S. Pat. No. 5,328,470) or by retroviruses has increased the utility of retroviruses for gene Stereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3 therapy, and defective retroviruses are characterized for use in 10 gene transfer for gene therapy purposes (for a review see 054-3 05 7). The pharmaceutical preparation of the gene Miller, A. D. (1990) Blood 76:271). A replication defective therapy construct can consist essentially of the gene delivery retrovirus can be packaged into virions which can be used to system in an acceptable diluent, or can comprise a slow infect a target cell through the use of a helper virus by stan release matrix in which the gene delivery vehicle is imbed dard techniques. Protocols for producing recombinant retro 15 ded. Where the albumin fusion protein can be produced intact viruses and for infecting cells in vitro or in vivo with such from recombinant cells, e.g. retroviral vectors, the pharma viruses can be found in Current Protocols in Molecular Biol ceutical preparation can comprise one or more cells which ogy, Ausubel, F. M. et al., (eds.) Greene Publishing Associ produce the albumin fusion protein. ates, (1989), Sections 9.10-9.14 and other standard laboratory Additional Gene Therapy Methods manuals. Also encompassed by the invention are genetherapy meth Another viral gene delivery system useful in the present ods for treating or preventing disorders, diseases and condi invention uses adenovirus-derived vectors. The genome of an tions. The gene therapy methods relate to the introduction of adenovirus can be manipulated Such that it encodes and nucleic acid (DNA. RNA and antisense DNA or RNA) expresses a gene product of interest but is inactivated in terms sequences into an animal to achieve expression of an albumin of its ability to replicate in a normal lytic viral life cycle. See, 25 fusion protein of the invention. This method requires a poly for example, Berkner et al., BioTechniques 6:616 (1988); nucleotide which codes for an albumin fusion protein of the Rosenfeld et al. Science 252:431-434 (1991): and Rosenfeld present invention operatively linked to a promoter and any et al. Cell 68:143-155 (1992). Suitable adenoviral vectors other genetic elements necessary for the expression of the derived from the adenovirus strain Ad type 5 d1324 or other fusion protein by the target tissue. Such gene therapy and strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to 30 delivery techniques are known in the art, see, for example, those skilled in the art. Recombinant adenoviruses can be WO90/11092, which is herein incorporated by reference. advantageous in certain circumstances in that they are not Thus, for example, cells from a patient may be engineered capable of infecting nondividing cells and can be used to with a polynucleotide (DNA or RNA) comprising a promoter infect a wide variety of cell types, including epithelial cells operably linked to a polynucleotide encoding an albumin (Rosenfeld et al., (1992) cited supra). Furthermore, the virus 35 fusion protein of the present invention ex vivo, with the engi particle is relatively stable and amenable to purification and neered cells then being provided to a patient to be treated with concentration, and as above, can be modified so as to affect the fusion protein of the present invention. Such methods are the spectrum of infectivity. Additionally, introduced adenovi well-known in the art. For example, see Belldegrun, A. et al., ral DNA (and foreign DNA contained therein) is not inte J. Natl. Cancer Inst.85: 207-216 (1993); Ferrantini, M. et al., grated into the genome of a host cell but remains episomal, 40 Cancer Research 53: 1107-1112 (1993: Ferrantini, M. et al., J. thereby avoiding potential problems that can occuras a result Immunology 153: 4604-4615 (1994): Kaido, T. et al. Int. J. of insertional mutagenesis in situations where introduced Cancer 60: 221-229 (1995); Ogura, H. et al., Cancer Research DNA becomes integrated into the host genome (e.g., retrovi 50: 5102-5106 (1990): Santodonato, L. et al. Human Gene ral DNA). Moreover, the carrying capacity of the adenoviral Therapy 7:1-10 (1996): Santodonato, L. et al. Gene Therapy genome for foreign DNA is large (up to 8 kilobases) relative 45 4:1246-1255 (1997); and Zhang, J.-F. et al. Cancer Gene to other gene delivery vectors (Berkner et al. cited supra; Therapy 3: 31-38 (1996)), which are herein incorporated by Haj-Ahmand et al. J. Virol. 57:267 (1986)). reference. In one embodiment, the cells which are engineered In another embodiment, non-viral gene delivery systems of are arterial cells. The arterial cells may be reintroduced into the present invention rely on endocytic pathways for the the patient through direct injection to the artery, the tissues uptake of the subject nucleotide molecule by the targeted cell. 50 Surrounding the artery, or through catheter injection. Exemplary gene delivery systems of this type include liposo As discussed in more detail below, the polynucleotide con mal derived systems, poly-lysine conjugates, and artificial structs can be delivered by any method that delivers injectable viral envelopes. In a representative embodiment, a nucleic materials to the cells of an animal. Such as, injection into the acid molecule encoding an albumin fusion protein of the interstitial space of tissues (heart, muscle, skin, lung, liver, invention can be entrapped in liposomes bearing positive 55 and the like). The polynucleotide constructs may be delivered charges on their surface (e.g. lipofectins) and (optionally) in a pharmaceutically acceptable liquid or aqueous carrier. which are tagged with antibodies against cell Surface antigens In one embodiment, polynucleotides encoding the albumin of the target tissue (Mizuno et al. (1992) No Shinkei Geka fusion proteins of the present invention is delivered as a naked 20:547-551; PCT publication WO91/06309; Japanese patent polynucleotide. The term “naked’ polynucleotide, DNA or application 1047381; and European patent publication EP-A- 60 RNA refers to sequences that are free from any delivery 43075). vehicle that acts to assist, promote or facilitate entry into the Gene delivery systems for a gene encoding an albumin cell, including viral sequences, viral particles, liposome for fusion protein of the invention can be introduced into a patient mulations, lipofectin or precipitating agents and the like. by any of a number of methods. For instance, a pharmaceu However, polynucleotides encoding the albumin fusion pro tical preparation of the gene delivery system can be intro 65 teins of the present invention can also be delivered in lipo duced systemically, e.g. by intravenous injection, and specific Some formulations and lipofectin formulations and the like transduction of the protein in the target cells occurs predomi can be prepared by methods well known to those skilled in the US 8,946,156 B2 109 110 art. Such methods are described, for example, in U.S. Pat. The preferred route of administration is by the parenteral Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein route of injection into the interstitial space of tissues. How incorporated by reference. ever, other parenteral routes may also be used. Such as, inha The polynucleotide vector constructs used in the gene lation of an aerosol formulation particularly for delivery to therapy method are preferably constructs that will not inte lungs or bronchial tissues, throat or mucous membranes of the grate into the host genome nor will they contain sequences nose. In addition, naked DNA constructs can be delivered to that allow for replication. Appropriate vectors include pWL arteries during angioplasty by the catheter used in the proce NEO, pSV2CAT, pCG44, pXT1 and pSG available from dure. Stratagene: pSVK3, pBPV, pMISG and pSVL available from The naked polynucleotides are delivered by any method Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 avail 10 known in the art, including, but not limited to, direct needle able from Invitrogen. Other suitable vectors will be readily injection at the delivery site, intravenous injection, topical apparent to the skilled artisan. administration, catheter infusion, and so-called "gene guns'. Any strong promoter known to those skilled in the art can These delivery methods are known in the art. be used for driving the expression of the polynucleotide The constructs may also be delivered with delivery sequence. Suitable promoters include adenoviral promoters, 15 vehicles such as viral sequences, viral particles, liposome Such as the adenoviral major late promoter, or heterologous formulations, lipofectin, precipitating agents, etc. Such meth promoters, such as the cytomegalovirus (CMV) promoter, the ods of delivery are known in the art. respiratory syncytial virus (RSV) promoter; inducible pro In certain embodiments, the polynucleotide constructs are moters, such as the MMT promoter, the metallothionein pro complexed in a liposome preparation. Liposomal prepara moter: heat shock promoters; the albumin promoter, the tions for use in the instant invention include cationic (posi ApoA1 promoter; human globin promoters; viral thymidine tively charged), anionic (negatively charged) and neutral kinase promoters, such as the Herpes Simplex thymidine preparations. However, cationic liposomes are particularly kinase promoter; retroviral LTRs; the b-actin promoter; and preferred because a tight charge complex can be formed human growth hormone promoters. The promoter also may between the cationic liposome and the polyanionic nucleic be the native promoter for the gene corresponding to the 25 acid. Cationic liposomes have been shown to mediate intra Therapeutic protein portion of the albumin fusion proteins of cellular delivery of plasmid DNA (Felgner et al. Proc. Natl. the invention. Acad. Sci. USA (1987) 84:7413-7416, which is herein incor Unlike other gene therapy techniques, one major advan porated by reference); mRNA (Malone et al., Proc. Natl. tage of introducing naked nucleic acid sequences into target Acad. Sci. USA (1989) 86:6077-6081, which is herein incor cells is the transitory nature of the polynucleotide synthesis in 30 porated by reference); and purified transcription factors the cells. Studies have shown that non-replicating DNA (Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which sequences can be introduced into cells to provide production is herein incorporated by reference), in functional form. of the desired polypeptide for periods of up to six months. Cationic liposomes are readily available. For example, The polynucleotide construct can be delivered to the inter N1-2,3-dioleyloxy)propyl-N, N,N-triethylammonium Stitial space of tissues within the an animal, including of 35 (DOTMA) liposomes are particularly useful and are available muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, under the trademark LIPOFECTINR), from GIBCO BRL, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl Acad. bladder, Stomach, intestine, testis, ovary, uterus, rectum, ner Sci. USA (1987) 84:7413-7416, which is herein incorporated Vous system, eye, gland, and connective tissue, Interstitial by reference). Other commercially available liposomes space of the tissues comprises the intercellular, fluid, muco 40 include transfectace (DDAB/DOPE) and DOTAP/DOPE polysaccharide matrix among the reticular fibers of organ (Boehringer). tissues, elastic fibers in the walls of vessels or chambers, Other cationic liposomes can be prepared from readily collagen fibers of fibrous tissues, or that same matrix within available materials using techniques well known in the art. connective tissue ensheathing muscle cells or in the lacunae See, e.g. PCT Publication No. WO90/11092 (which is herein of bone. It is similarly the space occupied by the plasma of the 45 incorporated by reference) for a description of the synthesis circulation and the lymph fluid of the lymphatic channels. of DOTAP (1.2-bis(oleoyloxy)-3-(trimethylammonio)pro Delivery to the interstitial space of muscle tissue is preferred pane) liposomes. Preparation of DOTMA liposomes is for the reasons discussed below. They may be conveniently explained in the literature, see, e.g., P. Felgner et al. Proc. delivered by injection into the tissues comprising these cells. Natl. Acad. Sci. USA 84:7413-7417, which is herein incor They are preferably delivered to and expressed in persistent, 50 porated by reference. Similar methods can be used to prepare non-dividing cells which are differentiated, although delivery liposomes from other cationic lipid materials. and expression may be achieved in non-differentiated or less Similarly, anionic and neutral liposomes are readily avail completely differentiated cells, such as, for example, stem able, such as from Avanti Polar Lipids (Birmingham, Ala.), or cells of blood or skin fibroblasts. In vivo muscle cells are can be easily prepared using readily available materials. Such particularly competent in their ability to take up and express 55 materials include phosphatidyl, choline, cholesterol, phos polynucleotides. phatidyl ethanolamine, dioleoylphosphatidyl choline For the naked nucleic acid sequence injection, an effective (DOPC), dioleoylphosphatidyl glycerol (DOPG), dio dosage amount of DNA or RNA will be in the range of from leoylphosphatidyl ethanolamine (DOPE), among others. about 0.05 mg/kg body weight to about 50 mg/kg body These materials can also be mixed with the DOTMA and weight. Preferably the dosage will be from about 0.005 mg/kg 60 DOTAP starting materials in appropriate ratios. Methods for to about 20 mg/kg and more preferably from about 0.05 making liposomes using these materials are well known in the mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary art. skill will appreciate, this dosage will vary according to the For example, commercially dioleoylphosphatidylcholine tissue site of injection. The appropriate and effective dosage (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dio of nucleic acid sequence can readily be determined by those 65 leoylphosphatidyl ethanolamine (DOPE) can be used in vari of ordinary skill in the art and may depend on the condition ous combinations to make conventional liposomes, with or being treated and the route of administration. without the addition of cholesterol. Thus, for example, US 8,946,156 B2 111 112 DOPG/DOPC vesicles can be prepared by drying 50 mg each present invention. Retroviruses from which the retroviral of DOPG and DOPC under a stream of nitrogen gas into a plasmid vectors may be derived include, but are not limited to, Sonication vial. The sample is placed under a vacuum pump Moloney Murine Leukemia Virus, spleen necrosis virus. overnight and is hydrated the following day with deionized Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis water. The sample is then Sonicated for 2 hours in a capped 5 virus, gibbon ape leukemia virus, human immunodeficiency vial, using a Heat Systems model 350 Sonicator equipped virus. Myeloproliferative Sarcoma Virus, and mammary with an inverted cup (bath type) probeat the maximum setting tumor virus. while the bath is circulated at 15EC. Alternatively, negatively The retroviral plasmid vector is employed to transduce charged vesicles can be prepared without Sonication to pro packaging cell lines to form producer cell lines. Examples of duce multilamellar vesicles or by extrusion through nucle 10 opore membranes to produce unilamellar vesicles of discrete packaging cells which may be transfected include, but are not size. Other methods are known and available to those of skill limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14x, in the art. VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and The liposomes can comprise multilamellar vesicles DAN cell lines as described in Miller, Human Gene Therapy (MLVs), small unilamellar vesicles (SUVs), or large unila 15 1:5-14 (1990), which is incorporated herein by reference in its mellar vesicles (LUVs), with SUVs being preferred. The entirety. The vector may transduce the packaging cells various liposome-nucleic acid complexes are prepared using through any means known in the art. Such means include, but methods well known in the art. See, e.g., Straubinger et al., are not limited to, electroporation, the use of liposomes, and Methods of Immunology (1983), 101:512-527, which is CaPO precipitation. In one alternative, the retroviral plasmid herein incorporated by reference. For example, MLVs con vector may be encapsulated into a liposome, or coupled to a taining nucleic acid can be prepared by depositing a thin film lipid, and then administered to a host. of phospholipid on the walls of a glass tube and Subsequently The producer cell line generates infectious retroviral vec hydrating with a solution of the material to be encapsulated. tor particles which include polynucleotide encoding an albu SUVs are prepared by extended sonication of MLVs to pro min fusion protein of the present invention. Such retroviral duce a homogeneous population of unilamellar liposomes. 25 vector particles then may be employed, to transduce eukary The material to be entrapped is added to a Suspension of otic cells, either in vitro or in vivo. The transduced eukaryotic preformed MLVs and then sonicated. When using liposomes cells will express a fusion protein of the present invention. containing cationic lipids, the dried lipid film is resuspended In certain other embodiments, cells are engineered, ex vivo in an appropriate solution Such as sterile water or an isotonic or in Vivo, with polynucleotide contained in an adenovirus buffer solution such as 10 mM Tris/NaCl, sonicated, and then 30 vector. Adenovirus can be manipulated Such that it encodes the preformed liposomes are mixed directly with the DNA. and expresses fusion protein of the present invention, and at The liposome and DNA form a very stable complex due to the same time is inactivated in terms of its ability to replicate binding of the positively charged liposomes to the cationic in a normal lytic viral life cycle. Adenovirus expression is DNA. SUVs find use with small nucleic acid fragments, achieved without integration of the viral DNA into the host LUVs are prepared by a number of methods, well known in 35 cell chromosome, thereby alleviating concerns about inser the art. Commonly used methods include Ca"-EDTA chela tional mutagenesis. Furthermore, adenoviruses have been tion (Papahadjopoulos et al., Biochem. Biophys. Acta (1975) used as live enteric vaccines for many years with an excellent 394:483; Wilson et al., Cell 17:77 (1979)), ether injection safety profile (Schwartz et al. Am. Rev. Respir. Dis. 109:233 (Deamer, D. and Bangham, A. Biochem. Biophys. Acta 443: 238 (1974)). Finally, adenovirus mediated gene transfer has 629 (1976); Ostro et al. Biochem. Biophys. Res. Commun. 40 been demonstrated in a number of instances including trans 76:836 (1977): Fraley et al., Proc. Natl. Acad. Sci. USA ferofalpha-1-antitrypsin and CFTR to the lungs of cotton rats 76:3348 (1979)); detergent dialysis (Enoch. H, and Strittmat (Rosenfeld, M. A. et al. (1991) Science 252:431-434, Rosen ter, P., Proc. Natl. Acad. Sci. USA 76:145 (1979)); and feld et al. (1992) Cell 68:143-155). Furthermore, extensive reverse-phase evaporation (REV) (Fraley et al. J. Biol. Chem. studies to attempt to establish adenovirus as a causative agent 255:10431 (1980): Szoka, F, and Papahadjopoulos, D., Proc. 45 in human cancer were uniformly negative (Green, M. et al. Natl. Acad. Sci. USA 75:145 (1978): Schaefer-Ridder et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606). Science 215: 166 (1982)), which are herein incorporated by Suitable adenoviral vectors useful in the present invention reference. are described, for example, in Kozarsky and Wilson, Curr. Generally, the ratio of DNA to liposomes will be from Opin. Genet. Devel. 3:499-503 (1993); Rosenfeld et al. Cell about 10:1 to about 1:10. Preferably, the ration will be from 50 68:143-155 (1992): Engelhardt et al. Human Genet. Ther. about 5:1 to about 1:5. More preferably, the ration will be 4:759-769 (1993): Yang et al., Nature Genet. 7:362-369 about 3:1 to about 1:3. Still more preferably, the ratio will be (1994): Wilson et al. Nature 365:691-692 (1993): and U.S. about 1:1. Pat. No. 5,652.224, which are herein incorporated by refer U.S. Pat. No. 5,676,954 (which is herein incorporated by ence. For example, the adenovirus vector Ad2 is useful and reference) reports on the injection of genetic material, com 55 can be grown in human 293 cells. These cells contain the E1 plexed with cationic liposomes carriers, into mice. U.S. Pat. region of adenovirus and constitutively express Ela and E1b. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, which complement the defective adenoviruses by providing 5,693,622, 5,580,859, 5,703,055, and international publica the products of the genes deleted from the vector. In addition tion no. WO 94-9469 (which are herein incorporated by ref to Ad2, other varieties of adenovirus (e.g. Ad3, Ad5, and Ad7) erence) provide cationic lipids for use in transfecting DNA 60 are also useful in the present invention. into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, Preferably, the adenoviruses used in the present invention 5,580,859, 5,703,055, and international publication no. WO are replication deficient. Replication deficient adenoviruses 94/9469 provide methods for delivering DNA-cationic lipid require the aid of a helper virus and/or packaging cell line to complexes to mammals. form infectious particles. The resulting virus is capable of In certain embodiments, cells are engineered, ex vivo or in 65 infecting cells and can express a polynucleotide of interest Vivo, using a retroviral particle containing RNA which com which is operably linked to a promoter, but cannot replicate in prises a sequence encoding an albumin fusion protein of the most cells. Replication deficient adenoviruses may be deleted US 8,946,156 B2 113 114 in one or more of all or a portion of the following genes: E1a, The promoter-targeting sequence construct is delivered to E1b, E3, E4, E2a, or L1 through L5. the cells, either as naked polynucleotide, or in conjunction In certain other embodiments, the cells are engineered, ex with transfection-facilitating agents. Such as liposomes, viral Vivo or in Vivo, using an adeno-associated virus (AAV). AAVs sequences, viral particles, whole viruses, lipofection, precipi are naturally occurring defective viruses that require helper tating agents, etc. described in more detail above. The P viruses to produce infectious particles (Muzyczka, N., Curr. promoter-targeting sequence can be delivered by any method, Topics in Microbiol. Immunol. 158:97 (1992)). It is also one included direct needle injection, intravenous injection, topi of the few viruses that may integrate its DNA into non cal administration, catheter infusion, particle accelerators, dividing cells. Vectors containing as little as 300 base pairs of etc. The methods are described in more detail below. 10 The promoter-targeting sequence construct is taken up by AAV can be packaged and can integrate, but space for exog cells. Homologous recombination between the construct and enous DNA is limited to about 4.5 kb. Methods for producing the endogenous sequence takes place, Such that an endog and using Such AAVs are known in the art. See, for example, enous sequence is placed under the control of the promoter. U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, The promoter then drives the expression of the endogenous 5,474,935, 5,478,745, and 5,589,377. 15 Sequence. For example, an appropriate AAV vector for use in the The polynucleotide encoding an albumin fusion protein of present invention will include all the sequences necessary for the present invention may contain a secretory signal sequence DNA replication, encapsidation, and host-cell integration. that facilitates secretion of the protein. Typically, the signal The polynucleotide construct is inserted into the AAV vector sequence is positioned in the coding region of the polynucle using standard cloning methods, such as those found in Sam otide to be expressed towards or at the 5' end of the coding brook et al. Molecular Cloning: A Laboratory. Manual, Cold region. The signal sequence may be homologous or heterolo Spring Harbor Press (1989). The recombinant AAV vector is gous to the polynucleotide of interest and may be homolo then transfected into packaging cells which are infected with gous or heterologous to the cells to be transfected. Addition a helper virus, using any standard technique, including lipo ally, the signal sequence may be chemically synthesized fection, electroporation, calcium phosphate precipitation, 25 using methods known in the art. etc. Appropriate helper viruses include adenoviruses, Any mode of administration of any of the above-described cytomegaloviruses, vaccinia viruses, or herpesviruses. Once polynucleotides constructs can be used so long as the mode the packaging cells are transfected and infected, they will results in the expression of one or more molecules in an produce infectious AAV viral particles which contain the amount sufficient to provide a therapeutic effect. This polynucleotide construct. These viral particles are then used 30 includes direct needle injection, systemic injection, catheter to transduce eukaryotic cells, either ex vivo or in vivo. The infusion, biolistic injectors, particle accelerators (i.e. 'gene transduced cells will contain the polynucleotide construct guns), gelfoam sponge depots, other commercially available integrated into its genome, and will express a fusion protein of depot materials, osmotic pumps (e.g., Alza minipumps), oral the invention. or Suppositorial Solid (tablet or pill) pharmaceutical formula Another method of gene therapy involves operably associ 35 tions, and decanting or topical applications during Surgery. ating heterologous control regions and endogenous poly For example, direct injection of naked calcium phosphate nucleotide sequences (e.g. encoding a polypeptide of the precipitated plasmid into rat liver and rat spleen or a protein present invention) via homologous recombination (see, e.g. coated plasmid into the portal vein has resulted in gene U.S. Pat. No. 5,641,670, issued Jun. 24, 1997: International expression of the foreign gene in the rat livers (Kaneda et al., Publication No. WO 96/29411, published Sep. 26, 1996: 40 Science 243:375 (1989)). International Publication No. WO94/12650, published Aug. A preferred method of local administration is by direct 4, 1994: Koller et al. Proc. Natl. Acad. Sci. USA 86:8932 injection. Preferably, an albumin fusion protein of the present 8935 (1989); and Zijlstra et al. Nature 342:435-438 (1989), invention complexed with a delivery vehicle is administered which are herein incorporated by reference. This method by direct injection into or locally within the area of arteries. involves the activation of a gene which is present in the target 45 Administration of a composition locally within the area of cells, but which is not normally expressed in the cells, or is arteries refers to injecting the composition centimeters and expressed at a lower level than desired. preferably, millimeters within arteries. Polynucleotide constructs are made, using standard tech Another method of local administration is to contact a niques known in the art, which contain the promoter with polynucleotide construct of the present invention in or around targeting sequences flanking the promoter. Suitable promot 50 a Surgical wound. For example, a patient can undergo Surgery ers are described herein. The targeting sequence is Suffi and the polynucleotide construct can be coated on the Surface ciently complementary to an endogenous sequence to permit of tissue inside the wound or the construct can be injected into homologous recombination of the promoter-targeting areas of tissue inside the wound. sequence with the endogenous sequence. The targeting Therapeutic compositions useful in Systemic administra sequence will be sufficiently near the 5' end of the desired 55 tion, include fusion proteins of the present invention com endogenous polynucleotide sequence so the promoter will be plexed to a targeted delivery vehicle of the present invention. operably linked to the endogenous sequence upon homolo Suitable delivery vehicles for use with systemic administra gous recombination. tion comprise liposomes comprising ligands for targeting the The promoter and the targeting sequences can be amplified vehicle to a particular site. In specific embodiments, Suitable using PCR. Preferably, the amplified promoter contains dis 60 delivery vehicles for use with systemic administration com tinct restriction enzyme sites on the 5' and 3' ends. Preferably, prise liposomes comprising albumin fusion proteins of the the 3' end of the first targeting sequence contains the same invention for targeting the vehicle to a particular site. restriction enzyme site as the 5' end of the amplified promoter Preferred methods of systemic administration, include and the 5' end of the second targeting sequence contains the intravenous injection, aerosol, oral and percutaneous (topi same restriction site as the 3' end of the amplified promoter. 65 cal) delivery. Intravenous injections can be performed using The amplified promoter and targeting sequences are digested methods standard in the art. Aerosol delivery can also be and ligated together. performed using methods standard in the art (see, for US 8,946,156 B2 115 116 example, Stribling et al., Proc. Natl. Acad. Sci. USA 189: diseases, disorders or conditions as described under"Immune 11277-11281, 1992, which is incorporated herein by refer Activity”, “Infectious Disease', and/or “Hyperproliferative ence. Oral delivery can be performed by complexing a poly Disorders' infra. nucleotide construct of the present invention to a carrier In a preferred embodiment, albumin fusion proteins of the capable of withstanding degradation by digestive enzymes in 5 invention comprising a Therapeutic protein portion corre the gut of an animal. Examples of Such carriers, include sponding to human Growth hormone and/or fragments and/or plastic capsules or tablets, such as those known in the art. variants thereofmay be used to treat, prevent, diagnose, prog Topical delivery can be performed by mixing a polynucle nose, and/or detect disease, disorders and/or conditions otide construct of the present invention with a lipophilic related to growth hormone deficiency, including but not lim 10 ited to, Acromegaly, Growth failure, Growth failure and reagent (e.g. DMSO) that is capable of passing into the skin. endogenous growth hormone replacement, Growth hormone Determining an effective amount of substance to be deliv deficiency, Growth failure and growth retardation, Prader ered can depend upon a number of factors including, for Willi syndrome in children 2 years or older, Growth deficien example, the chemical structure and biological activity of the cies, Postmenopausal osteoporosis, burns, cachexia, cancer Substance, the age and weight of the animal, the precise 15 cachexia, dwarfism, metabolic disorders, obesity, renal fail condition requiring treatment and its severity, and the route of ure, Turner's Syndrome, fibromyalgia, fracture treatment, administration. The frequency of treatments depends upon a frailty, or as described under“Endocrine Disorders”, “Wound number of factors, such as the amount of polynucleotide Healing and Epithelial Cell Proliferation', and/or “Hyperpro constructs administered per dose, as well as the health and liferative Disorders' infra. history of the Subject. The precise amount, number of doses, In preferred embodiments, fusion proteins of the present and timing of doses will be determined by the attending invention may be used in the diagnosis, prognosis, prevention physician or veterinarian. and/or treatment of diseases and/or disorders relating to dis Albumin fusion proteins of the present invention can be eases and disorders of the endocrine system (see, for example, administered to any animal, preferably to mammals and "Endocrine Disorders' section below), the nervous system birds. Preferred mammals include humans, dogs, cats, mice, 25 (see, for example, “Neurological Disorders' section below), rats, rabbits sheep, cattle, horses and pigs, with humans being the immune system (see, for example, "Immune Activity” particularly preferred. section below), respiratory system (see, for example, "Res Biological Activities piratory Disorders' section below), cardiovascular system Albumin fusion proteins and/or polynucleotides encoding (see, for example, “Cardiovascular Disorders' section albumin fusion proteins of the present invention, can be used 30 below), reproductive system (see, for example, “Reproduc in assays to test for one or more biological activities. If an tive System Disorders' section below digestive system (see, albumin fusion protein and/or polynucleotide exhibits an for example, "Gastrointestinal Disorders' section below), activity in a particular assay, it is likely that the Therapeutic diseases and/or disorders relating to cell proliferation (see, for protein corresponding to the fusion protein may be involved example, “Hyperproliferative Disorder” section below), and/ in the diseases associated with the biological activity. Thus, 35 or diseases or disorders relating to the blood (see, for the fusion protein could be used to treat the associated dis example, “Blood Related Disorders' section below). CaSC. In preferred embodiments, the present invention encom Members of the secreted family of proteins are believed to passes a method of treating a disease or disorder listed in the be involved in biological activities associated with, for “Preferred Indication Y column of Table 1 comprising example, cellular signaling. Accordingly, albumin fusion pro 40 administering to a patient in which Such treatment, prevention teins of the invention and polynucleotides encoding these or amelioration is desired an albumin fusion protein of the proteins, may be used in diagnosis, prognosis, prevention invention that comprises a Therapeutic protein portion corre and/or treatment of diseases and/or disorders associated with sponding to a Therapeutic protein disclosed in the "Thera aberrant activity of secreted polypeptides. peutic Protein X column of Table 1 (in the same row as the In a preferred embodiment, albumin fusion proteins of the 45 disease or disorder to be treated is listed in the "Preferred invention comprising a Therapeutic protein portion corre Indication Y column of Table 1) in an amount effective to sponding to EPO, immunoglobulins, hirudin, applaggin, treat, prevent or ameliorate the disease or disorder. serum cholinesterase, alpha-1 antitrypsin, aprotinin, and In certain embodiments, an albumin fusion protein of the coagulation factors in both pre and active forms (e.g., includ present invention may be used to diagnose and/or prognose ing, but not limited to von Willebrand factor, fibrinogen, 50 diseases and or disorders associated with the tissue(s) in factor II, factor VII, factor VIIA activated factor, factor VIII, which the gene corresponding to the Therapeutic protein factor IX, factor X, factor XIII, c1 inactivator, antithrombin portion of the fusion protein of the invention is expressed. III, thrombin and prothrombin, apo-lipoprotein, c-reactive Thus, fusion proteins of the invention and polynucleotides protein, and protein C) and/or fragments and/or variants encoding albumin fusion proteins of the invention are useful thereof may be used to modulate hemostatic (the stopping of 55 in the diagnosis, detection and/or treatment of diseases and, bleeding) or thrombolytic (clot dissolving) activity and % or or disorders associated with activities that include, but are not treat, prevent, diagnose, prognose, and/or detect blood-re limited to prohormone activation, neurotransmitter activity, lated disorders or cardiovascular disorders and/or diseases, cellular signaling, cellular proliferation, cellular differentia disorders or conditions as described under “Blood Related tion, and cell migration. Disorders”, “Anti-Angiogenesis Activity”, and/or “Cardio 60 More generally, fusion proteins of the invention and poly vascular Disorders' infra. nucleotides encoding albumin fusion proteins of the inven In a preferred embodiment, albumin fusion proteins of the tion may be useful for the diagnosis, prognosis, prevention invention comprising a Therapeutic protein portion corre and/or treatment of diseases and/or disorders associated with sponding to Interferon alpha, G-CSF, GM-CSF, scatter factor, the following systems. MCP/MCAF, M-CSF and/or fragments and/or variants 65 Immune Activity thereof may be used to treat, prevent, diagnose, prognose, Albumin fusion proteins of the invention and polynucle and/or detect diseases or disorders of the immune system, or otides encoding albumin fusion proteins of the invention may US 8,946,156 B2 117 118 be useful in treating, preventing, diagnosing and/or prognos diagnosed, and/or prognosed using fusion proteins of the ing diseases, disorders, and/or conditions of the immune sys invention and/or polynucleotides encoding albumin fusion tem, by, for example, activating or inhibiting the prolifera proteins of the invention. tion, differentiation, or mobilization (chemotaxis) of immune Other immunodeficiencies that may be treated, prevented, cells. Immune cells develop through a process called hemato diagnosed, and/or prognosed using fusion proteins of the poiesis, producing myeloid (platelets, red blood cells, neu invention and, or polynucleotides encoding albumin fusion trophils, and macrophages) and lymphoid (B and T lympho proteins of the invention, include, but are not limited to cytes) cells from pluripotent stem cells. The etiology of these chronic granulomatous disease, Chediak-Higashi syndrome, immune diseases, disorders, and/or conditions may be myeloperoxidase deficiency, leukocyte glucose-6-phosphate genetic, Somatic, Such as cancer and some autoimmune dis 10 dehydrogenase deficiency, X-linked lymphoproliferative eases, acquired (e.g., by chemotherapy or toxins), or infec syndrome (XLP), leukocyte adhesion deficiency, comple tious. Moreover, fusion proteins of the invention and/or poly ment component deficiencies (including C1, C2, C3, C4, C5, nucleotides encoding albumin fusion proteins of the C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, invention can be used as a marker or detector of a particular thymic alymphoplasia-aplasia, immunodeficiency with thy immune system disease or disorder. 15 moma, severe congenital leukopenia, dysplasia with immu In another embodiment, a fusion protein of the invention nodeficiency, neonatal neutropenia, short limbed dwarfism, and/or polynucleotide encoding an albumin fusion protein of and Nezelof syndrome-combined immunodeficiency with the invention, may be used to treat diseases and disorders of Igs. the immune system and/or to inhibit or enhance an immune In a preferred embodiment, the immunodeficiencies and/or response generated by cells associated with the tissue(s) in conditions associated with the immunodeficiencies recited which the polypeptide of the invention is expressed. above are treated, prevented, diagnosed and or prognosed Albumin fusion proteins of the invention and or polynucle using fusion proteins of the invention and/or polynucleotides otides encoding albumin fusion proteins of the invention may encoding albumin fusion proteins of the invention. be useful in treating, preventing, diagnosing, and/or prognos In a preferred embodiment fusion proteins of the invention ing immunodeficiencies, including both congenital and 25 and/or polynucleotides encoding albumin fusion proteins of acquired immunodeficiencies. Examples of B cell immuno the invention could be used as an agent to boost immunore deficiencies in which immunoglobulin levels B cell function sponsiveness among immunodeficient individuals. In specific and/or B cell numbers are decreased include: X-linked agam embodiments, fusion proteins of the invention and/or poly maglobulinemia (Bruton's disease), X-linked infantile agam nucleotides encoding albumin fusion proteins of the inven maglobulinemia, X-linked immunodeficiency with hyper 30 tion could be used as an agent to boost immunoresponsive IgM, non X-linked immunodeficiency with hyper IgM, ness among B cell and/or T cell immunodeficient individuals. X-linked lymphoproliferative syndrome (XLP), agamma The albumin fusion proteins of the invention and/or poly globulinemia including congenital and acquired agamma nucleotides encoding albumin fusion proteins of the inven globulinemia, adult onset agammaglobulinemia, late-onset tion may be useful in treating, preventing, diagnosing and/or agammaglobulinemia, dysgammaglobulinemia, hypogam 35 prognosing autoimmune disorders. Many autoimmune disor maglobulinemia, unspecified hypogammaglobulinemia, ders result from inappropriate recognition of self as foreign recessive agammaglobulinemia (Swiss type). Selective IgM material by immune cells. This inappropriate recognition deficiency, selective IgA deficiency, selective IgG Subclass results in an immune response leading to the destruction of deficiencies, IgG subclass deficiency (with or without IgA the host tissue. Therefore, the administration of fusion pro deficiency), Ig deficiency with increased IgM, IgG and IgA 40 teins of the invention and/or polynucleotides encoding albu deficiency with increased IgM, antibody deficiency with nor min fusion proteins of the invention that can inhibit an mal or elevated Igs, Ig heavy chain deletions, kappa chain immune response, particularly the proliferation, differentia deficiency, B cell lymphoproliferative disorder (BLPD), tion, or chemotaxis of T-cells, may be an effective therapy in common variable immunodeficiency (CVID), common vari preventing autoimmune disorders. able immunodeficiency (CVI) (acquired), and transient 45 Autoimmune diseases or disorders that may be treated, hypogammaglobulinemia of infancy. prevented, diagnosed and/or prognosed by fusion proteins of In specific embodiments, ataxia-telangiectasia or condi the invention and/or polynucleotides encoding albumin tions associated with ataxia-telangiectasia are treated, pre fusion proteins of the invention include, but are not limited to vented, diagnosed, and/or prognosing using the fusion pro one or more of the following: systemic lupus erythematosus, teins of the invention and/or polynucleotides encoding 50 rheumatoid arthritis, ankylosing spondylitis, multiple Sclero albumin fusion proteins of the invention. sis, autoimmune thyroiditis, Hashimoto's thyroiditis, autoim Examples of congenital immunodeficiencies in which T mune hemolytic anemia, hemolytic anemia, thrombocytope cell and/or B cell function and/or number is decreased nia, autoimmune thrombocytopenia purpura, autoimmune include, but are not limited to: DiGeorge anomaly, severe neonatal thrombocytopenia, idiopathic thrombocytopenia combined immunodeficiencies (SCID) (including, but not 55 purpura, purpura (e.g. Henloch-Scoenlein purpura), autoim limited to, X-linked SCID, autosomal recessive SCID, munocytopenia, Goodpasture’s syndrome, Pemphigus Vul adenosine deaminase deficiency, purine nucleoside phospho garis, myasthenia gravis, Grave's disease (hyperthyroidism), rylase (PNP) deficiency, Class II MHC deficiency (Bare lym and insulin-resistant diabetes mellitus. phocyte syndrome), Wiskott-Aldrich syndrome, and ataxia Additional disorders that are likely to have an autoimmune telangiectasia), thymic hypoplasia, third and fourth pharyn 60 component that may be treated, prevented, and/or diagnosed geal pouch syndrome, 22d 11.2 deletion, chronic mucocuta with the albumin fusion proteins of the invention and/or poly neous candidiasis, natural killer cell deficiency (NK), idio nucleotides encoding albumin fusion proteins of the inven pathic CD4+ T-lymphocytopenia, immunodeficiency with tion include, but are not limited to type II collagen-induced predominant T cell defect (unspecified), and unspecified arthritis, antiphospholipid syndrome, dermatitis, allergic immunodeficiency of cell mediated immunity. 65 encephalomyelitis, myocarditis, relapsing polychondritis, In specific embodiments, DiGeorge anomaly or conditions rheumatic heart disease, neuritis, uveitis ophthalmia, polyen associated with DiGeorge anomaly are treated, prevented, docrinopathies, Reiter's Disease, Stiff-Man Syndrome, US 8,946,156 B2 119 120 autoimmune pulmonary inflammation, autism, Guillain In another specific preferred embodiment IgA nephropathy Barre Syndrome, insulin dependent diabetes mellitus, and is treated, prevented, and/or diagnosed using fusion proteins autoimmune inflammatory eye disorders. of the invention and/or polynucleotides encoding albumin Additional disorders that are likely to have an autoimmune fusion proteins of the invention. component that may be treated, prevented, diagnosed and/or In a preferred embodiment, the autoimmune diseases and prognosed with the albumin fusion proteins of the invention disorders and or conditions associated with the diseases and and/or polynucleotides encoding albumin fusion proteins of disorders recited above are treated, prevented, diagnosed and/ the invention include, but are not limited to scleroderma with or prognosed using fusion proteins of the invention and/or anti-collagen antibodies (often characterized, e.g. by nucle polynucleotides encoding albumin fusion proteins of the 10 invention. olar and other nuclear antibodies), mixed connective tissue In preferred embodiments, fusion proteins of the invention disease (often characterized, e.g., by antibodies to extractable and/or polynucleotides encoding albumin fusion proteins of nuclear antigens (e.g. ribonucleoprotein)), polymyositis (of the invention are used as a immunosuppressive agent(s). ten characterized, e.g. by nonhistone ANA), pernicious ane Albumin fusion proteins of the invention and/or polynucle mia (often characterized, e.g., by antiparietal cell, 15 otides encoding albumin fusion proteins of the invention may microsomes, and intrinsic factorantibodies), idiopathic Addi be useful in treating, preventing, prognosing, and/or diagnos son's disease (often characterized, e.g., by humoral and cell ing diseases, disorders, and/or conditions of hematopoietic mediated adrenal cytotoxicity, infertility (often character cells. Albumin fusion proteins of the invention and/or poly ized. C.2. by antispermatozoal antibodies), nucleotides encoding albumin fusion proteins of the inven glomerulonephritis (often characterized, e.g., by glomerular tion could be used to increase differentiation and proliferation basement membrane antibodies or immune complexes), of hematopoietic cells, including the pluripotent stem cells, in bullous pemphigoid (often characterized, e.g., by IgG and an effort to treat or prevent those diseases, disorders, and/or complement in basement membrane), Sjogren's syndrome conditions associated with a decrease in certain (or many) (often characterized, e.g., by multiple tissue antibodies, and/ types hematopoietic cells, including but not limited to, leu or a specific nonhistone ANA (SS-B)), diabetes mellitus (of 25 kopenia, neutropenia, anemia, and thrombocytopenia. Alter ten characterized, e.g., by cell-mediated and humoral islet natively, fusion proteins of the invention and/or polynucle cell antibodies), and adrenergic drug resistance (including otides encoding albumin fusion proteins of the invention adrenergic drug resistance with asthma or cystic fibrosis) could be used to increase differentiation and proliferation of (often characterized, e.g., by beta-adrenergic receptor anti hematopoietic cells, including the pluripotent stem cells, in bodies). 30 an effort to treat or prevent those diseases, disorders, and/or Additional disorders that may have an autoimmune com conditions associated with an increase in certain (or many) ponent that may be treated, prevented, diagnosed and/or prog types of hematopoietic cells, including but not limited to nosed with the albuminfusion proteins of the invention and/or histiocytosis. polynucleotides encoding albumin fusion proteins of the Allergic reactions and conditions, such as asthma (particu invention include, but are not limited to, chronic active hepa 35 larly allergic asthma) or other respiratory problems, may also titis (often characterized, e.g., by Smooth muscle antibodies), be treated, prevented, diagnosed and, or prognosed using primary biliary cirrhosis (often characterized, e.g., by mito fusion proteins of the invention and/or polynucleotides chondria antibodies), other endocrine gland failure (often encoding albumin fusion proteins of the invention. Moreover, characterized, e.g. by specific tissue antibodies in some these molecules can be used to treat, prevent, prognose, and/ cases), vitiligo (often characterized, e.g., by melanocyte anti 40 or diagnose anaphylaxis, hypersensitivity to an antigenic bodies), Vasculitis (often characterized, e.g., by Ig and molecule, or blood group incompatibility. complement in vessel walls and/or low serum complement), Additionally, fusion proteins of the invention and or poly post-MI (often characterized, e.g. by myocardial antibodies), nucleotides encoding albumin fusion proteins of the inven cardiotomy syndrome (often characterized, e.g., by myocar tion, may be used to treat, prevent, diagnose and/or prognose dial antibodies), urticaria (often characterized, e.g. by IgG 45 IgE-mediated allergic reactions. Such allergic reactions and IgM antibodies to IgE), atopic dermatitis (often charac include, but are not limited to asthma, rhinitis, and eczema. In terized, e.g. by IgG and IgM antibodies to IgE), asthma (often specific embodiments, fusion proteins of the invention and/or characterized, e.g. by IgG and IgM antibodies to IgE), and polynucleotides encoding albumin fusion proteins of the many other inflammatory, granulomatous, degenerative, and invention may be used to modulate IgE concentrations in atrophic disorders. 50 vitro or in vivo. In a preferred embodiment, the autoimmune diseases and Moreover, fusion proteins of the invention and/or poly disorders and/or conditions associated with the diseases and nucleotides encoding albumin fusion proteins of the inven disorders recited above are treated, prevented, diagnosed and/ tion have uses in the diagnosis, prognosis, prevention, and/or or prognosed using for example, fusion proteins of the inven treatment of inflammatory conditions. For example, since tion and/or polynucleotides encoding albuminfusion proteins 55 fusion proteins of the invention and/or polynucleotides of the invention. In a specific preferred embodiment, rheuma encoding albumin fusion proteins of the invention may inhibit toid arthritis is treated, prevented, and/or diagnosed using the activation, proliferation and or differentiation of cells fusion proteins of the invention and/or polynucleotides involved in an inflammatory response, these molecules can be encoding albumin fusion proteins of the invention. used to prevent and/or treat chronic and acute inflammatory In another specific preferred embodiment, systemic lupus 60 conditions. Such inflammatory conditions include, but are not erythematosus is treated, prevented, and/or diagnosed using limited to for example, inflammation associated with infec fusion proteins of the invention and or polynucleotides tion (e.g. septic shock, sepsis, or systemic inflammatory encoding albumin fusion proteins of the invention. In another response syndrome), ischemia-reperfusion injury, endotoxin specific preferred embodiment, idiopathic thrombocytopenia lethality, complement-mediated hyperacute rejection, purpura is treated, prevented, and/or diagnosed using fusion 65 nephritis, cytokine or chemokine induced lung injury, inflam proteins of the invention and/or polynucleotides encoding matory bowel disease, Crohn's disease, over production of albumin fusion proteins of the invention. cytokines (e.g. TNF or IL-1), respiratory disorders (e.g. US 8,946,156 B2 121 122 asthma and allergy); gastrointestinal disorders (e.g. inflam detected, and/or prevented. The immune response may be matory bowel disease); cancers (e.g., gastric, ovarian, lung, increased by either enhancing an existing immune response, bladder, liver, and breast); CNS disorders (e.g., multiple scle or by initiating a new immune response. Alternatively, fusion rosis; ischemic brain injury and/or stroke, traumatic brain proteins of the invention and/or polynucleotides encoding injury, neurodegenerative disorders (e.g., Parkinson's disease albumin fusion proteins of the invention may also directly and Alzheimer's disease); AIDS-related dementia; and prion inhibit the infectious agent (refer to section of application disease); cardiovascular disorders (e.g., atherosclerosis, listing infectious agents, etc), without necessarily eliciting an myocarditis, cardiovascular disease, and cardiopulmonary immune response. bypass complications); as well as many additional diseases, In another embodiment, albumin fusion proteins of the conditions, and disorders that are characterized by inflamma 10 tion (e.g., hepatitis, rheumatoid arthritis, gout, trauma, pan invention and/or polynucleotides encoding albumin fusion creatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion proteins of the invention are used as a vaccine adjuvant that injury, Grave's disease, systemic lupus erythematosus, dia enhances immune responsiveness to an antigen. In a specific betes mellitus, and allogenic transplant rejection). embodiment, albumin fusion proteins of the invention and or Because inflammation is a fundamental defense mecha 15 polynucleotides encoding albumin fusion proteins of the nism, inflammatory disorders can effect virtually any tissue invention are used as an adjuvant to enhance tumor-specific of the body. Accordingly, fusion proteins of the invention immune responses. and/or polynucleotides encoding albumin fusion proteins of In another specific embodiment, albumin fusion proteins of the invention, have uses in the treatment of tissue-specific the invention and/or polynucleotides encoding albumin inflammatory disorders, including, but not limited to adren fusion proteins of the invention are used as an adjuvant to alitis, alveolitis, angiocholecystitis, appendicitis, balanitis, enhance anti-viral immune responses. Anti-viral immune blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, responses that may be enhanced using the compositions of the cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cys invention as an adjuvant, include virus and virus associated titis, dermatitis, diverticulitis, encephalitis, endocarditis, diseases or symptoms described herein or otherwise known in esophagltis, eustachtis, fibrositis, folliculitis, gastritis, gastro 25 the art. In specific embodiments, the compositions of the enteritis, gingivitis, glossitis, hepatosplenitis, keratitis, laby invention are used as an adjuvant to enhance an immune rinthitis, laryngitis, lymphangitis, mastitis, media otitis, men response to a virus, disease, or symptom selected from the ingitis, metritis, mucitis, myocarditis, myosititis, myringitis, group consisting of AIDS, meningitis, Dengue, EBV, and nephritis, neuritis, orchitis, osteochondritis, otitis, pericardi hepatitis (e.g. hepatitis B). In another specific embodiment, tis, peritendonitis, peritonitis, pharyngitis, phlebitis, polio 30 the compositions of the invention are used as an adjuvant to myelitis, prostatitis, pulpitis, retinitis, rhinitis, Salpingitis, enhance an immune response to a virus, disease, or symptom scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, selected from the group consisting of HIV/AIDS, respiratory Steatitis, stomatitis, synovitis, Syringitis, tendonitis, tonsilli syncytial virus, Dengue, rotavirus, Japanese B encephalitis, tis, urethritis, and vaginitis. influenza A and B, parainfluenza, measles, cytomegalovirus, In specific embodiments, fusion proteins of the invention 35 rabies, Junin, Chikungunya, Rift Valley Fever, herpes sim and/or polynucleotides encoding albumin fusion proteins of plex, and yellow fever. the invention, are useful to diagnose, prognose, prevent, and/ In another specific embodiment, albumin fusion proteins of or treat organ transplant rejections and graft-versus-host dis the invention and/or polynucleotides encoding albumin ease. Organ rejection occurs by host immune cell destruction fusion proteins of the invention are used as an adjuvant to of the transplanted tissue through an immune response. Simi 40 enhance anti-bacterial or anti-fungal immune responses. larly, an immune response is also involved in GVHD (graft Anti-bacterial or anti-fungal immune responses that may be Versus host disease), but, in this case, the foreign transplanted enhanced using the compositions of the invention as an adju immune cells destroy the host tissues. Polypeptides, antibod vant, include bacteria or fungus and bacteria or fungus asso ies, or polynucleotides of the invention, and/or agonists or ciated diseases or symptoms described herein or otherwise antagonists thereof, that inhibit an immune response, particu 45 known in the art. In specific embodiments, the compositions larly the activation, proliferation, differentiation, or chemot of the invention are used as an adjuvant to enhance an immune axis of T-cells, may be an effective therapy in preventing response to a bacteria or fungus, disease, or symptom selected organ rejection or GVHD. In specific embodiments, fusion from the group consisting of tetanus, Diphtheria, botulism, proteins of the invention and/or polynucleotides encoding and meningitis type B. albumin fusion proteins of the invention, that inhibit an 50 In another specific embodiment, the compositions of the immune response, particularly the activation, proliferation, invention are used as an adjuvant to enhance an immune differentiation, or chemotaxis of T-cells, may be an effective response to a bacteria or fungus, disease, or symptom selected therapy in preventing experimental allergic and hyperacute from the group consisting of Vibrio cholerae, Mycobacte Xenograft rejection. riwn leprae, Salmonella ryphi, Salmonella pararyphi, Meis In other embodiments, fusion proteins of the invention 55 seria meningitidis, Streptococcus pneumoniae, Group B and/or polynucleotides encoding albumin fusion proteins of streptococcus, Shigella spp., Enterotoxigenic Escherichia the invention, are useful to diagnose, prognose, prevent, and/ coli, Enterohemorrhagic E. coli, and Borrelia burgdorferi. or treat immune complex diseases, including, but not limited In another specific embodiment, albumin fusion proteins of to, serum sickness, post streptococcal glomerulonephritis, the invention and/or polynucleotides encoding albumin polyarteritis nodosa, and immune complex-induced vasculi 60 fusion proteins of the invention are used as an adjuvant to tis. enhance anti-parasitic immune responses. Anti-parasitic Albumin fusion proteins of the invention and/or polynucle immune responses that may be enhanced using the composi otides encoding albumin fusion proteins of the invention can tions of the invention as an adjuvant, include parasite and be used to treat, detect, and/or prevent infectious agents. For parasite associated diseases or symptoms described herein or example, by increasing the immune response, particularly 65 otherwise known in the art. In specific embodiments, the increasing the proliferation activation and/or differentiation compositions of the invention are used as an adjuvant to of B and/or T cells, infectious diseases may be treated, enhance an immune response to a parasite. In another specific US 8,946,156 B2 123 124 embodiment, the compositions of the invention are used as an after the beginning of recovery of T cell populations, but prior adjuvant to enhance an immune response to Plasmodium to full recovery of B cell populations. (malaria) or Leishmania. In another specific embodiment, albumin fusion proteins of In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin the invention and/or polynucleotides encoding albumin 5 fusion proteins of the invention are used as an agent to boost fusion proteins of the invention may also be employed to treat immunoresponsiveness among individuals having an infectious diseases including silicosis, sarcoidosis, and idio acquired loss of B cell function. Conditions resulting in an pathic pulmonary fibrosis; for example, by preventing the acquired loss of B cell function that may be ameliorated or recruitment and activation of mononuclear phagocytes. treated by administering the albumin fusion proteins of the In another specific embodiment, albumin fusion proteins of 10 invention and/or polynucleotides encoding albumin fusion the invention and, or polynucleotides encoding albumin proteins of the invention, include, but are not limited to, HIV fusion proteins of the invention are used as an antigen for the Infection, AIDS, bone marrow transplant, and B cell chronic generation of antibodies to inhibit or enhance immune medi lymphocytic leukemia (CLL). ated responses against polypeptides of the invention. In another specific embodiment, albumin fusion proteins of In one embodiment, albumin fusion proteins of the inven 15 the invention and/or polynucleotides encoding albumin tion and or polynucleotides encoding albumin fusion proteins fusion proteins of the invention are used as an agent to boost of the invention are administered to an animal (e.g. mouse, immunoresponsiveness among individuals having a tempo rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, rary immune deficiency. Conditions resulting in a temporary camel, goat, horse, cow, sheep, dog, cat, non-human primate, immune deficiency that may be ameliorated or treated by and human, most preferably human) to boost the immune administering the albumin fusion proteins of the invention system to produce increased quantities of one or more anti and/or polynucleotides encoding albumin fusion proteins of bodies (e.g. IgG, IgA, IgM, and IgE), to induce higheraffinity the invention, include, but are not limited to, recovery from antibody production and immunoglobulin class Switching viral infections (e.g., influenza), conditions associated with (e.g. IgG, IgA, IgM, and IgE), and/or to increase an immune malnutrition, recovery from infectious mononucleosis, or response. 25 conditions associated with stress, recovery from measles, In another specific embodiment, albumin fusion proteins of recovery from blood transfusion, and recovery from Surgery. the invention and/or polynucleotides encoding albumin In another specific embodiment, albumin fusion proteins of fusion proteins of the invention are used as a stimulator of B the invention and/or polynucleotides encoding albumin cell responsiveness to pathogens. fusion proteins of the invention are used as a regulator of In another specific embodiment, albumin fusion proteins of 30 antigen presentation by monocytes, dendritic cells, and or the invention and/or polynucleotides encoding albumin B-cells. In one embodiment, albumin fusion proteins of the fusion proteins of the invention are used as an activator of T invention and/or polynucleotides encoding albumin fusion cells. proteins of the invention enhance antigen presentation or In another specific embodiment, albumin fusion proteins of antagonizes antigen presentation in vitro or in vivo. More the invention and/or polynucleotides encoding albumin 35 over, in related embodiments, this enhancement or antago fusion proteins of the invention are used as an agent that nism of antigen presentation may be useful as an anti-tumor elevates the immune status of an individual prior to their treatment or to modulate the immune system. receipt of immunosuppressive therapies. In another specific embodiment, albumin fusion proteins of In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin the invention and/or polynucleotides encoding albumin 40 fusion proteins of the invention are used as an agent to direct fusion proteins of the invention are used as an agent to induce an individual’s immune system towards development of a higher affinity antibodies. humoral response (i.e. TH2) as opposed to a TH1 cellular In another specific embodiment, albumin fusion proteins of response. the invention and/or polynucleotides encoding albumin In another specific embodiment, albumin fusion proteins of fusion proteins of the invention are used as an agent to 45 the invention and/or polynucleotides encoding albumin increase serum immunoglobulin concentrations. fusion proteins of the invention are used as a means to induce In another specific embodiment, albumin fusion proteins of tumor proliferation and thus make it more susceptible to the invention and/or polynucleotides encoding albumin anti-neoplastic agents. For example, multiple myeloma is a fusion proteins of the invention are used as an agent to accel slowly dividing disease and is thus refractory to virtually all erate recovery of immunocompromised individuals. 50 anti-neoplastic regimens. If these cells were forced to prolif In another specific embodiment, albumin fusion proteins of erate more rapidly their susceptibility profile would likely the invention and/or polynucleotides encoding albumin change. fusion proteins of the invention are used as an agent to boost In another specific embodiment, albumin fusion proteins of immunoresponsiveness among aged populations and or neo the invention and or polynucleotides encoding albumin nates. 55 fusion proteins of the invention are used as a stimulator of B In another specific embodiment, albumin fusion proteins of cell production in pathologies such as AIDS, chronic lym the invention and/or polynucleotides encoding albumin phocyte disorder and or Common Variable Immunodefi fusion proteins of the invention are used as an immune system ciency. enhancer prior to during, or after bone marrow transplant In another specific embodiment, albumin fusion proteins of and/or other transplants (e.g., allogeneic or Xenogeneic organ 60 the invention and or polynucleotides encoding albumin transplantation). With respect to transplantation, composi fusion proteins of the invention are used as a therapy for tions of the invention may be administered prior to concomi generation and/or regeneration of lymphoid tissues following tant with, and/or after transplantation. In a specific embodi Surgery, trauma or genetic defect. In another specific embodi ment, compositions of the invention are administered after ment, albumin fusion proteins of the invention and/or poly transplantation, prior to the beginning of recovery of T-cell 65 nucleotides encoding albumin fusion proteins of the inven populations. In another specific embodiment, compositions tion are used in the pretreatment of bone marrow samples of the invention are first administered after transplantation prior to transplant. US 8,946,156 B2 125 126 In another specific embodiment, albumin fusion proteins of tion may also be employed to treat idiopathic hyper-eosino the invention and/or polynucleotides encoding albumin philic syndrome by, for example, preventing eosinophil fusion proteins of the invention are used as a gene-based production and migration. therapy for genetically inherited disorders resulting in In another specific embodiment, albumin fusion proteins of immuno-incompetence/immunodeficiency Such as observed the invention and/or polynucleotides encoding albumin among SCID patients. fusion proteins of the invention are used to enhance or inhibit In another specific embodiment, albumin fusion proteins of complement mediated cell lysis. the invention and/or polynucleotides encoding albumin In another specific embodiment, albumin fusion proteins of fusion proteins of the invention are used as a means of acti the invention and/or polynucleotides encoding albumin 10 fusion proteins of the invention are used to enhance or inhibit Vating monocytes/macrophages to defend against parasitic antibody dependent cellular cytotoxicity. diseases that effect monocytes such as Leishmania. In another specific embodiment, albumin fusion proteins of In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed for fusion proteins of the invention are used as a means of regu 15 treating atherosclerosis, for example, by preventing mono lating secreted cytokines that are elicited by polypeptides of cyte infiltration in the artery wall. the invention. In another specific embodiment, albumin fusion proteins of In another embodiment, albumin fusion proteins of the the invention and/or polynucleotides encoding albumin invention and/or polynucleotides encoding albumin fusion fusion proteins of the invention may be employed to treat proteins of the invention are used in one or more of the adult respiratory distress syndrome (ARDS). applications described herein, as they may apply to Veterinary In another specific embodiment, albumin fusion proteins of medicine. the invention and/or polynucleotides encoding albumin In another specific embodiment, albumin fusion proteins of fusion proteins of the invention may be useful for stimulating the invention and/or polynucleotides encoding albumin wound and tissue repair, stimulating angiogenesis, and/or fusion proteins of the invention are used as a means of block 25 stimulating the repair of vascular or lymphatic diseases or ing various aspects of immune responses to foreign agents or disorders. Additionally, fusion proteins of the invention and/ self. Examples of diseases or conditions in which blocking of or polynucleotides encoding albumin fusion proteins of the certain aspects of immune responses may be desired include invention may be used to stimulate the regeneration of autoimmune disorders such as lupus, and arthritis, as well as mucosal Surfaces. immunoresponsiveness to skin allergies, inflammation, 30 In a specific embodiment, albumin fusion proteins of the bowel disease, injury and diseases/disorders associated with invention and/or polynucleotides encoding albumin fusion pathogens. proteins of the invention are used to diagnose, prognose, treat, and/or prevent a disorder characterized by primary or In another specific embodiment, albumin fusion proteins of acquired immunodeficiency, deficient serum immunoglobu the invention and/or polynucleotides encoding albumin 35 lin production, recurrent infections, and or immune system fusion proteins of the invention are used as a therapy for dysfunction. Moreover, fusion proteins of the invention and preventing the B cell proliferation and Ig secretion associated or polynucleotides encoding albumin fusion proteins of the with autoimmune diseases such as idiopathic thrombocy invention may be used to treat or prevent infections of the topenic purpura, systemic lupus erythematosus and multiple joints, bones, skin, and/or parotid glands, blood-borne infec Sclerosis. 40 tions (e.g., sepsis, meningitis, septic arthritis, and/or osteo In another specific embodiment, polypeptides, antibodies, myelitis), autoimmune diseases (e.g. those disclosed herein), polynucleotides and or agonists or antagonists of the present inflammatory disorders, and malignancies, and/or any dis fusion proteins of the invention and or polynucleotides ease or disorder or condition associated with these infections, encoding albumin fusion proteins of the invention are used as diseases, disorders and/or malignancies) including, but not a inhibitor of B and/or T cell migration in endothelial cells. 45 limited to, CVID, other primary immune deficiencies, HIV This activity disrupts tissue architecture or cognate responses disease. CLL, recurrent bronchitis, sinusitis, otitis media, and is useful, for example in disrupting immune responses, conjunctivitis, pneumonia, hepatitis, meningitis, herpes and blocking sepsis. Zoster (e.g., severe herpes Zoster), and/or pneumocystis car In another specific embodiment, albumin fusion proteins of nii. Other diseases and disorders that may be prevented, diag the invention and or polynucleotides encoding albumin 50 nosed, prognosed, and/or treated with fusion proteins of the fusion proteins of the invention are used as a therapy for invention and/or polynucleotides encoding albumin fusion chronic hypergammaglobulinemia evident in Such diseases as proteins of the invention include, but are not limited to, HIV monoclonal gammopathy of undetermined significance infection, HTLV-BLV infection, lymphopenia, phagocyte (MGUS), Waldenstrom's disease, related idiopathic mono bactericidal dysfunction anemia, thrombocytopenia, and clonal gammopathies, and plasmacytomas. 55 hemoglobinuria. In another specific embodiment, albumin fusion proteins of In another embodiment, albumin fusion proteins of the the invention and/or polynucleotides encoding albumin invention and/or polynucleotides encoding albumin fusion fusion proteins of the invention may be employed for instance proteins of the invention are used to treat, and/or diagnose an to inhibit polypeptide chemotaxis and activation of macroph individual having common variable immunodeficiency dis ages and their precursors, and of neutrophils, basophils, B 60 ease (“CVID'; also known as “acquired agammaglobuline lymphocytes and some T-cell Subsets, e.g., activated and CD8 mia' and “acquired hypogammaglobulinemia') or a Subset of cytotoxic T cells and natural killer cells, in certain autoim this disease. mune and chronic inflammatory and infective diseases. In a specific embodiment, albumin fusion proteins of the Examples of autoimmune diseases are described herein and invention and/or polynucleotides encoding albumin fusion include multiple Sclerosis, and insulin-dependent diabetes. 65 proteins of the invention may be used to diagnose, prognose, The albumin fusion proteins of the invention and/or poly prevent, and/or treat cancers or neoplasms including immune nucleotides encoding albumin fusion proteins of the inven cell or immune tissue-related cancers or neoplasms. US 8,946,156 B2 127 128 Examples of cancers or neoplasms that may be prevented, might accompany angioplasty procedures, for reducing the diagnosed, or treated by fusion proteins of the invention and risk of stroke in patients with atrial fibrillation including or polynucleotides encoding albumin fusion proteins of the nonrheumatic atrial fibrillation, for reducing the risk of embo invention include, but are not limited to acute myelogenous lism associated with mechanical heart valves and or mitral leukemia, chronic myelogenous leukemia. Hodgkin’s dis 5 valves disease. Other uses for the albumin fusion proteins of ease, non-Hodgkin’s lymphoma, acute lymphocytic anemia the invention and or polynucleotides encoding albumin (ALL) Chronic lymphocyte leukemia, plasmacytomas, mul fusion proteins of the invention, include, but are not limited to tiple myeloma, Burkitt's lymphoma, EBV-transformed dis the prevention of occlusions in extracorporeal devices (e.g. eases, and/or diseases and disorders described in the section intravascular canulas, vascular access shunts in hemodialysis entitled “Hyperproliferative Disorders' elsewhere herein. 10 patients, hemodialysis machines, and cardiopulmonary In another specific embodiment, albumin fusion proteins of bypass machines). the invention and/or polynucleotides encoding albumin In another embodiment, albumin fusion proteins of the fusion proteins of the invention are used as a therapy for invention and or polynucleotides encoding albumin fusion decreasing cellular proliferation of Large B-cell Lymphomas. proteins of the invention, may be used to prevent, diagnose, In another specific embodiment, albumin fusion proteins of 15 prognose, and/or treat diseases and disorders of the blood the invention and or polynucleotides encoding albumin and/or blood forming organs associated with the tissue(s) in fusion proteins of the invention are used as a means of which the polypeptide of the invention is expressed. decreasing the involvement of B cells and Ig associated with The fusion proteins of the invention and/or polynucleotides Chronic Myelogenous Leukemia. encoding albumin fusion proteins of the invention may be In specific embodiments, the compositions of the invention used to modulate hematopoietic activity (the formation of are used as an agent to boost immunoresponsiveness among B blood cells). For example, the albumin fusion proteins of the cell immunodeficient individuals, such as, for example, an invention and/or polynucleotides encoding albumin fusion individual who has undergone a partial or complete splenec proteins of the invention may be used to increase the quantity tomy. of all or subsets of blood cells, such as, for example, eryth Blood-Related Disorders 25 rocytes, lymphocytes (B or T cells), myeloid cells (e.g., baso In a preferred embodiment, albumin fusion proteins of the phils, eosinophils, neutrophils, mast cells, macrophages) and invention comprising a Therapeutic protein portion corre platelets. The ability to decrease the quantity of blood cells or sponding to immunoglobulins, serum cholinesterase, alpha-1 subsets of blood cells may be useful in the prevention, detec antitrypsin, aprotinin, and coagulation factors in both pre and tion, diagnosis and/or treatment of anemias and leukopenias active forms (e.g. including, but not limited to, von Will 30 described below. Alternatively, the albumin fusion proteins of ebrand factor, fibrinogen, factor II, factor VII, factor VIIA the invention and/or polynucleotides encoding albumin activated factor, factor VIII, factor IX, factor X, factor XIII, c.1 fusion proteins of the invention may be used to decrease the inactivator, antithrombin III, thrombinand prothrombin, apo quantity of all or Subsets of blood cells. Such as, for example, lipoprotein, c-reactive protein, and protein C) and fragments erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., and/or variants thereof may be used to modulate hemostatic 35 basophils, eosinophils, neutrophils, mast cells, macrophages) (the stopping of bleeding) or thrombolytic (clot dissolving) and platelets. The ability to decrease the quantity of blood activity and/or treat, prevent, diagnose, prognose, and/or cells or subsets of blood cells may be useful in the prevention, detect blood-related disorders. detection, diagnosis and/or treatment of leukocytoses, such The albumin fusion proteins of the invention and/or poly as, for example eosinophilia. nucleotides encoding albumin fusion proteins of the inven 40 The fusion proteins of the invention and/or polynucleotides tion may be used to modulate hemostatic (the stopping of encoding albumin fusion proteins of the invention may be bleeding) or thrombolytic (clot dissolving) activity. For used to prevent, treat, or diagnose blood dyscrasia. example, by increasing hemostatic or thrombolytic activity, Anemias are conditions in which the number of red blood fusion proteins of the invention and/or polynucleotides cells or amount of hemoglobin (the protein that carries oxy encoding albumin fusion proteins of the invention could be 45 gen) in them is below normal. Anemia may be caused by used to treat or prevent blood coagulation diseases, disorders, excessive bleeding, decreased red blood cell production, or and/or conditions (e.g. afibrinogenemia, factor deficiencies, increased red blood cell destruction (hemolysis). The albu hemophilia), blood platelet diseases, disorders, and/or condi min fusion proteins of the invention and or polynucleotides tions (e.g. thrombocytopenia), or wounds resulting from encoding albumin fusion proteins of the invention may be trauma, Surgery, or other causes. Alternatively, fusion pro 50 useful in treating, preventing, and/or diagnosing anemias. teins of the invention and/or polynucleotides encoding albu Anemias that may be treated prevented or diagnosed by the min fusion proteins of the invention that can decrease hemo albumin fusion proteins of the invention and/or polynucle static or thrombolytic activity could be used to inhibit or otides encoding albumin fusion proteins of the invention dissolve clotting. These molecules could be important in the include iron deficiency anemia, hypochromic anemia, micro treatment or prevention of heart attacks (infarction), strokes, 55 cytic anemia, chlorosis, hereditary sideroblastic anemia, idio or Scarring. pathic acquired sideroblastic anemia, red cell aplasia, mega In specific embodiments, the albuminfusion proteins of the loblastic anemia (e.g. pernicious anemia, (vitamin B12 invention and/or polynucleotides encoding albumin fusion deficiency) and folic acid deficiency anemia), aplastic ane proteins of the invention may be used to prevent, diagnose, mia, hemolytic anemias (e.g. autoimmune helolytic anemia, prognose, and/or treat thrombosis, arterial thrombosis, 60 microangiopathic hemolytic anemia, and paroxysmal noctur venous thrombosis, thromboembolism, pulmonary embo nal hemoglobinuria). The albumin fusion proteins of the lism, atherosclerosis, myocardial infarction, transient invention and/or polynucleotides encoding albumin fusion ischemic attack, unstable angina. In specific embodiments, proteins of the invention may be useful in treating, prevent the albumin fusion proteins of the invention and/or poly ing, and/or diagnosing anemias associated with diseases nucleotides encoding albumin fusion proteins of the inven 65 including but not limited to anemias associated with systemic tion may be used for the prevention of occlusion of saphenous lupus erythematosus, cancers, lymphomas, chronic renal dis grafts, for reducing the risk of periprocedural thrombosis as ease, and enlarged spleens. The albumin fusion proteins of the US 8,946,156 B2 129 130 invention and/or polynucleotides encoding albumin fusion compared to normal is known as leukocytosis. The body proteins of the invention may be useful in treating, prevent generates increased numbers of white blood cells during ing, and/or diagnosing anemias arising from drug treatments infection. Thus, leukocytosis may simply be a normal physi Such as anemias associated with methyldopa, dapsone, and/or ological parameter that reflects infection. Alternatively, leu Sulfadrugs. Additionally, fusion proteins of the invention and/ kocytosis may be an indicator of injury or other disease Such or polynucleotides encoding albumin fusion proteins of the as cancer. Leokocytoses, include but are not limited to, eosi invention may be useful in treating, preventing, and/or diag nophilia, and accumulations of macrophages. In specific nosing anemias associated with abnormal red blood cell embodiments, the albumin fusion proteins of the invention architecture including but not limited to, hereditary sphero and/or polynucleotides encoding albumin fusion proteins of cytosis, hereditary elliptocytosis, glucose-6-phosphate dehy 10 the invention, may be useful in diagnosing, prognosing, pre drogenase deficiency, and sickle cell anemia. venting, and/or treating leukopenia. In other specific embodi The albumin fusion proteins of the invention and/or poly ments, the albumin fusion proteins of the invention and/or nucleotides encoding albumin fusion proteins of the inven polynucleotides encoding albumin fusion proteins of the tion may be useful in treating, preventing, and/or diagnosing invention may be useful in diagnosing, prognosing, prevent hemoglobin abnormalities, (e.g., those associated with sickle 15 ing, and/or treating leukocytosis. cell anemia, hemoglobin C disease, hemoglobin S-C disease, Leukopenia may be a generalized decreased in all types of and hemoglobin Edisease). Additionally, the albumin fusion white blood cells, or may be a specific depletion of particular proteins of the invention and/or polynucleotides encoding types of white blood cells. Thus, in specific embodiments, the albumin fusion proteins of the invention may be useful in albumin fusion proteins of the invention and/or polynucle diagnosing, prognosing, preventing, and/or treating thalas otides encoding albumin fusion proteins of the invention may semia, including, but not limited to, major and minor forms of be useful in diagnosing, prognosing, preventing, and/or treat alpha-thalassemia and beta-thalassemia. ing decreases in neutrophil numbers, known as neutropenia. In another embodiment, the albumin fusion proteins of the Neutropenias that may be diagnosed, prognosed, prevented, invention and/or polynucleotides encoding albumin fusion and/or treated by the albumin fusion proteins of the invention proteins of the invention may be useful in diagnosing, prog 25 and/or polynucleotides encoding albumin fusion proteins of nosing, preventing, and/or treating bleeding disorders includ the invention include, but are not limited to infantile genetic ing, but not limited to, thrombocytopenia (e.g. idiopathic agranulocytosis, familial neutropenia, cyclic neutropenia, thrombocytopenic purpura, and thrombotic thrombocy neutropenias resulting from or associated with dietary defi topenic purpura), Von Willebrand’s disease, hereditary plate ciencies (e.g. vitamin B12 deficiency or folic acid defi let disorders (e.g., storage pool disease such as Chediak 30 ciency), neutropenias resulting from or associated with drug Higashi and Hermansky-Pudlak syndromes, thromboxane treatments (e.g. antibiotic regimens Such as penicillin treat A2 dysfunction, thromboasthenia, and Bernard-Soulier syn ment, sulfonamide treatment, anticoagulant treatment, anti drome), hemolytic-uremic syndrome, hemophelias Such as convulsant drugs, anti-thyroid drugs, and cancer chemo hemophelia A or Factor VII deficiency and Christmas disease therapy), and neutropenias resulting from increased or Factor IX deficiency. Hereditary Hemorhhagic Telangiect 35 neutrophil destruction that may occur in association with sia, also known as Rendu-Osler-Weber syndrome, allergic Some bacterial or viral infections, allergic disorders, autoim purpura (Henoch Schonlein purpura) and disseminated intra mune diseases, conditions in which an individual has an vascular coagulation. enlarged spleen (e.g. Felty syndrome, malaria and sarcoido The effect of the albumin fusion proteins of the invention sis), and some drug treatment regimens. and/or polynucleotides encoding albumin fusion proteins of 40 The albumin fusion proteins of the invention and/or poly the invention on the cloning time of blood may be monitored nucleotides encoding albumin fusion proteins of the inven using any of the clotting tests known in the art including, but tion may be useful in diagnosing, prognosing, preventing, not limited to whole blood partial thromboplastintime (PTT), and/or treating lymphocytopenias (decreased numbers of B the activated partial thromboplastin time (aPTT), the acti and/or T lymphocytes), including, but not limited to lympho vated clotting time (ACT), the recalcified activated clotting 45 cytopenias resulting from or associated with stress, drug time, or the Lee-White Clotting time. treatments (e.g., drug treatment with corticosteroids, cancer Several diseases and a variety of drugs can cause platelet chemotherapies, and/or radiation therapies), AIDS infection dysfunction. Thus, in a specific embodiment, the albumin and/or other diseases Such as, for example, cancer, rheuma fusion proteins of the invention and/or polynucleotides toid arthritis, Systemic lupus erythematosus, chronic infec encoding albumin fusion proteins of the invention may be 50 tions, Some viral infections and/or hereditary disorders (e.g., useful in diagnosing, prognosing, preventing, and/or treating DiGeorge syndrome, Wiskott-Aldrich Syndrome, severe acquired platelet dysfunction Such as platelet dysfunction combined immunodeficiency, ataxia telangiectsia). accompanying kidney failure, leukemia, multiple myeloma, The albumin fusion proteins of the invention and/or poly cirrhosis of the liver, and systemic lupus erythematosus as nucleotides encoding albumin fusion proteins of the inven well as platelet dysfunction associated with drug treatments, 55 tion may be useful in diagnosing, prognosing, preventing, including treatment with aspirin, ticlopidine, nonsteroidal and/or treating diseases and disorders associated with mac anti-inflammatory drugs (used for arthritis, pain, and rophage numbers and/or macrophage function including, but sprains), and penicillin in high doses. not limited to, Gaucher's disease, Niemann-Pick disease, In another embodiment, the albumin fusion proteins of the Letterer-Siwe disease and Hand-Schuller-Christian disease. invention and/or polynucleotides encoding albumin fusion 60 In another embodiment, the albumin fusion proteins of the proteins of the invention may be useful in diagnosing, prog invention and/or polynucleotides encoding albumin fusion nosing, preventing, and/or treating diseases and disorders proteins of the invention may be useful in diagnosing, prog characterized by or associated with increased or decreased nosing, preventing, and/or treating diseases and disorders numbers of white blood cells. Leukopenia occurs when the associated with eosinophil numbers and/or eosinophil func number of white blood cells decreases below normal. Leuko 65 tion including, but not limited to idiopathic hypereosinophilic penias include, but are not limited to, neutropenia and lym syndrome, eosinophilia-myalgia syndrome, and Hand phocytopenia. An increase in the number of white blood cells Schuller-Christian disease. US 8,946,156 B2 131 132 In yet another embodiment, the albumin fusion proteins of otides encoding albumin fusion proteins of the invention may the invention and/or polynucleotides encoding albumin proliferate other cells which can inhibit the hyperproliferative fusion proteins of the invention may be useful in diagnosing, disorder. prognosing, preventing, and/or treating leukemias and lym For example, by increasing an immune response, particu phomas including, but not limited to acute lymphocytic larly increasing antigenic qualities of the hyperproliferative (lymphpblastic) leukemia (ALL), acute myeloid (myelocytic, disorder or by proliferating, differentiating, or mobilizing myelogenous, myeloblastic, or myelomonocytic) leukemia, T-cells, hyperproliferative disorders can be treated. This chronic lymphocytic leukemia (e.g., B cell leukemias, T cell immune response may be increased by either enhancing an leukemias, Sezary syndrome, and Hairy cell leukemia), existing immune response, or by initiating a new immune chronic myelocytic (myeloid, myelogenous, or granulocytic) 10 response. Alternatively, decreasing an immune response may also be a method of treating hyperproliferative disorders, leukemia, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma. Such as a chemotherapeutic agent. Burkitt's lymphoma, and mycosis fungoides. Examples of hyperproliferative disorders that can be In other embodiments, the albumin fusion proteins of the treated or detected by fusion proteins of the invention and/or invention and/or polynucleotides encoding albumin fusion 15 polynucleotides encoding albumin fusion proteins of the proteins of the invention may be useful in diagnosing, prog invention include, but are not limited to neoplasms located in nosing, preventing, and or treating diseases and disorders of the: colon, abdomen, bone, breast, digestive system, liver, plasma cells including, but not limited to, plasma cell dyscra pancreas, peritoneum, endocrine glands (adrenal, parathy sias, monoclonal gammaopathies, monoclonal gammopa roid, pituitary, testicles, ovary, thymus, thyroid), eye, head thies of undetermined significance, multiple myeloma, mac and neck, nervous (central and peripheral), lymphatic system, roglobulinemia. Waldenstrom's macroglobulinemia, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract. cryoglobulinemic, and Raynaud's phenomenon. Similarly, other hyperproliferative disorders can also be In other embodiments, the albumin fusion proteins of the treated or detected by fusion proteins of the invention and/or invention and/or polynucleotides encoding albumin fusion polynucleotides encoding albumin fusion proteins of the proteins of the invention may be useful in treating, prevent 25 invention. Examples of such hyperproliferative disorders ing, and/or diagnosing myeloproliferative disorders, includ include, but are not limited to: Acute Childhood Lymphoblas ing but not limited to, polycythemia Vera, relative poly tic Leukemia, Acute Lymphoblastic Leukemia, Acute Lym cythemia, secondary polycythemia, myelofibrosis, acute phocytic Leukemia, Acute Myeloid Leukemia, Adrenocorti myelofibrosis, agnogenic myeloid metaplasia, thromb cal Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult ocythemia, (including both primary and secondary thromb 30 (Primary) Liver Cancer, Adult Acute Lymphocytic Leuke ocythemia) and chronic myelocytic leukemia. mia, Adult Acute Myeloid Leukemia, Adult Hodgkin’s Dis In other embodiments, the albumin fusion proteins of the ease. Adult Hodgkin’s Lymphoma, Adult Lymphocytic Leu invention and/or polynucleotides encoding albumin fusion kemia, Adult Non-Hodgkin’s Lymphoma, Adult Primary proteins of the invention may be useful as a treatment prior to Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Surgery, to increase blood cell production. 35 Lymphoma, AIDS-Related Malignancies, Anal Cancer, In other embodiments, the albumin fusion proteins of the Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Can invention and/or polynucleotides encoding albumin fusion cer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Can proteins of the invention may be useful as an agent to enhance cer of the Renal Pelvis and Ureter, Central Nervous System the migration, phagocytosis, Superoxide production, anti (Primary) Lymphoma, Central Nervous System Lymphoma, body dependent cellular cytotoxicity of neutrophils, eosiono 40 Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical phils and macrophages. Cancer, Childhood (Primary) Hepatocellular Cancer, Child In other embodiments, the albumin fusion proteins of the hood (Primary) Liver Cancer, Childhood Acute Lymphoblas invention and/or polynucleotides encoding albumin fusion tic Leukemia, Childhood Acute Myeloid Leukemia, Child proteins of the invention may be useful as an agent to increase hood Brain Stem Glioma, Childhood Cerebellar the number of stem cells in circulation prior to stem cells 45 Astrocytoma, Childhood Cerebral Astrocytoma, Childhood pheresis. In another specific embodiment, the albumin fusion Extracranial Germ Cell Tumors, Childhood Hodgkin’s Dis proteins of the invention and % or polynucleotides encoding ease, Childhood Hodgkin’s Lymphoma, Childhood Hypotha albumin fusion proteins of the invention may be useful as an lamic and Visual Pathway Glioma, Childhood Lymphoblastic agent to increase the number of stem cells in circulation prior Leukemia, Childhood Medulloblastoma, Childhood Non to platelet pheresis. 50 Hodgkin’s Lymphoma, Childhood Pineal and Supratentorial In other embodiments, the albumin fusion proteins of the Primitive Neuroectodermal Tumors, Childhood Primary invention and/or polynucleotides encoding albumin fusion Liver Cancer, Childhood Rhabdomyosarcoma, Childhood proteins of the invention may be useful as an agent to increase Soft Tissue Sarcoma, Childhood Visual Pathway and Hypo cytokine production. thalamic Glioma, Chronic Lymphocytic Leukemia, Chronic In other embodiments, the albumin fusion proteins of the 55 Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell invention and/or polynucleotides encoding albumin fusion Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, proteins of the invention may be useful in preventing, diag Endometrial Cancer, Ependymoma, Epithelial Cancer, nosing, and/or treating primary hematopoietic disorders. Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Hyperproliferative Disorders Exocrine Pancreatic Cancer, Extracranial Germ. Cell Tumor, In certain embodiments, fusion proteins of the invention 60 Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Can and/or polynucleotides encoding albumin fusion proteins of cer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, the invention can be used to treat or detect hyperproliferative Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carci disorders, including neoplasms. Albumin fusion proteins of noid Tumor, Gastrointestinal Tumors, Germ. Cell Tumors, the invention and or polynucleotides encoding albumin Gestational Trophoblastic Tumor, Hairy Cell Leukemia, fusion proteins of the invention may inhibit the proliferation 65 Head and Neck Cancer, Hepatocellular Cancer, Hodgkin’s of the disorder through direct or indirect interactions. Alter Disease, Hodgkin’s Lymphoma, Hypergammaglobulinemia, natively, fusion proteins of the invention and/or polynucle Hypopharyngeal Cancer, Intestinal Cancers, Intraocular US 8,946,156 B2 133 134 Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Can tory fibrous hyperplasia, inflammatory papillary hyperplasia, cer, Kaposi’s Sarcoma, Kidney Cancer, Laryngeal Cancer, intravascular papillary endothelial hyperplasia, nodular Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, hyperplasia of prostate, nodular regenerative hyperplasia, Lymphoproliferative Disorders, Macroglobulinemia, Male pseudoepitheliomatous hyperplasia, senile sebaceous hyper Breast Cancer, Malignant Mesothelioma, Malignant Thy plasia, and Verrucous hyperplasia. moma, Medulloblastoma, Melanoma, Mesothelioma, Meta Metaplasia is a form of controlled cell growth in which one static Occult Primary Squamous Neck Cancer, Metastatic type of adult or fully differentiated cell substitutes for another Primary Squamous Neck Cancer, Metastatic Squamous Neck type of adult cell. Metaplastic disorders which can be diag Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell nosed, prognosed, prevented, and/or treated with fusion pro Neoplasm, Myelodysplastic Syndrome, Myelogenous Leu 10 teins of the invention and/or polynucleotides encoding albu kemia, Myeloid Leukemia, Myeloproliferative Disorders, min fusion proteins of the invention include, but are not Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal limited to, agnogenic myeloid metaplasia, apocrine metapla Cancer, Neuroblastoma, Non-Hodgkin’s Lymphoma During sia, atypical metaplasia, autoparenchymatous metaplasia, Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell connective tissue metaplasia, epithelial metaplasia, intestinal Lung Cancer, Occult Primary Metastatic Squamous Neck 15 metaplasia, metaplastic anemia, metaplastic ossification, Cancer, Oropharyngeal Cancer, Osteo-?Malignant Fibrous metaplastic polyps, myeloid metaplasia, primary myeloid Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma, metaplasia, secondary myeloid metaplasia, squamous meta Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, plasia, squamous metaplasia of amnion, and symptomatic Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ova mycluid metaplasia. rian Low Malignant Potential Tumor, Pancreatic Cancer, Dysplasia is frequently a forerunner of cancer, and is found Paraproteinemias, Purpura, Parathyroid Cancer, Penile Can mainly in the epithelia; it is the most disorderly form of cer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neo non-neoplastic cell growth, involving a loss in individual cell plasm/Multiple Myeloma, Primary Central Nervous System uniformity and in the architectural orientation of cells. Dys Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal plastic cells often have abnormally large, deeply stained Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, 25 nuclei, and exhibit pleomorphism. Dysplasia characteristi Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Can cally occurs where there exists chronic irritation or inflam cer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, mation. Dysplastic disorders which can be diagnosed, prog Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue nosed, prevented, and/or treated with fusion proteins of the Sarcoma, Squamous Neck Cancer, Stomach Cancer, Suprat invention and/or polynucleotides encoding albumin fusion entorial Primitive Neuroectodermal and Pineal Tumors, 30 proteins of the invention include, but are not limited to, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid anhidrotic ectodermal dysplasia, anterofacial dysplasia, Cancer, Transitional Cell Cancer of the Renal Pelvis and asphyxiating thoracic dysplasia, atriodigital dysplasia, bron Ureter, Transitional Renal Pelvis and Ureter Cancer, Tropho chopulmonary dysplasia, cerebral dysplasia, cervical dyspla blastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral sia, chondroectodermal dysplasia, cleidocranial dysplasia, Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, 35 congenital ectodermal dysplasia, craniodiaphysial dysplasia, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, craniocarpotarsal dysplasia, craniometaphysial dysplasia, Waldenstrom's Macroglobulinemia, Wilms Tumor, and any dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, other hyperproliferative disease, besides neoplasia, located in enamel dysplasia, encephalo-ophthalmic dysplasia, dyspla an organ system listed above. sia epiphysialis hemimelia, dysplasia epiphysialis multiplex, In another preferred embodiment, albumin fusion proteins 40 dysplasia epiphysialis punctata, epithelial dysplasia, facio of the invention and/or polynucleotides encoding albumin digitogenital dysplasia, familial fibrous dysplasia of jaws, fusion proteins of the invention are used to diagnose, prog familial white folded dysplasia, fibromuscular dysplasia, nose, prevent, and/or treat premalignant conditions and to fibrous dysplasia of bone, florid osseous dysplasia, hereditary prevent progression to a neoplastic or malignant state, includ renal-retinal dysplasia, hidrotic ectodermal dysplasia, hypo ing but not limited to those disorders described above. Such 45 hidrotic ectodermal dysplasia, lymphopenic thymic dyspla uses are indicated in conditions known or Suspected of pre sia, mammary dysplasia, mandibulofacial dysplasia, meta ceding progression to neoplasia or cancer, in particular, where physical dysplasia, Mondini dysplasia, monostatic fibrous non-neoplastic cell growth consisting of hyperplasia, meta dysplasia, mucoepithelial dysplasia, multiple epiphysial dys plasia, or most particularly, dysplasia has occurred (for plasia, oculoauriculovertebral dysplasia, oculodentodigital review of such abnormal growth conditions, see Robbins and 50 dysplasia, oculovertebral dysplasia, odontogenic dysplasia, Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co. ophthalmomandibulomelic dysplasia, periapical, cemental Philadelphia, pp. 68-79.) dysplasia, polyostotic fibrous dysplasia, pseudoachondro Hyperplasia is a form of controlled cell proliferation, plastic spondyloepiphysial dysplasia, retinal dysplasia, involving an increase in cell number in a tissue or organ, septo-optic dysplasia, spondyloepiphysial dysplasia, and without significant alteration in structure or function. Hyper 55 Ventriculoradial dysplasia. plastic disorders which can be diagnosed, prognosed, pre Additional pre-neoplastic disorders which can be diag vented, and or treated with fusion proteins of the invention nosed, prognosed, prevented, and/or treated with fusion pro and/or polynucleotides encoding albumin fusion proteins of teins of the invention and/or polynucleotides encoding albu the invention include, but are not limited to angiofollicular min fusion proteins of the invention include, but are not mediastinal lymph node hyperplasia, angiolymphoid hyper 60 limited to, benign dysproliferative disorders (e.g., benign plasia with eosinophilia, atypical melanocytic hyperplasia, tumors, fibrocystic conditions, tissue hypertrophy, intestinal basal cell hyperplasia, benign giant lymph node hyperplasia, polyps, colon polyps, and esophageal dysplasia), leuko cementum hyperplasia, congenital adrenal hyperplasia, con plakia, keratoses, Bowen's disease, Farmer's Skin, Solar genital sebaceous hyperplasia, cystic hyperplasia, cystic cheilitis, and Solar keratosis. hyperplasia of the breast, denture hyperplasia, ductal hyper 65 In another embodiment, albumin fusion proteins of the plasia, endometrial hyperplasia, fibromuscular hyperplasia, invention and/or polynucleotides encoding albumin fusion focal epithelial hyperplasia, gingival hyperplasia, inflamma proteins of the invention, may be used to diagnose and/or US 8,946,156 B2 135 136 prognose disorders associated with the tissue(s) in which the noma, choriocarcinoma, seminoma, embryonal carcinoma, polypeptide of the invention is expressed. Wilm's tumor, cervical cancer, testicular tumor, lung carci In another embodiment, albumin fusion proteins of the noma, Small cell lung carcinoma, bladder carcinoma, epithe invention and/or polynucleotides encoding albumin fusion lial carcinoma, glioma, astrocytoma, medulloblastoma, cran proteins of the invention conjugated to a toxin or a radioactive iopharyngioma, ependymoma, pinealoma, isotope, as described herein, may be used to treat cancers and emangioblastoma, acoustic neuroma, oligodendroglioma, neoplasms, including, but not limited to those described menangioma, melanoma, neuroblastoma, and retinoblas herein. In a further preferred embodiment, albumin fusion tOma. proteins of the invention and/or polynucleotides encoding Diseases associated with increased apoptosis that could be albumin fusion proteins of the invention conjugated to a toxin 10 diagnosed, prognosed, prevented, and/or treated by fusion or a radioactive isotope, as described herein, may be used to proteins of the invention and/or polynucleotides encoding treat acute myelogenous leukemia. albumin fusion proteins of the invention, include AIDS: neu Additionally, fusion proteins of the invention and/or poly rodegenerative disorders (such as Alzheimer's disease, Par nucleotides encoding albumin fusion proteins of the inven kinson's disease, amyotrophic lateral Sclerosis, retinitis pig tion may affect apoptosis, and therefore, would be useful in 15 mentosa, cerebellar degeneration and brain tumor or prior treating a number of diseases associated with increased cell associated disease): autoimmune disorders (such as, multiple Survival or the inhibition of apoptosis. For example, diseases Sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, bil associated with increased cell survival or the inhibition of iary cirrhosis, Behcet’s disease, Crohn's disease, polymyosi apoptosis that could be diagnosed, prognosed, prevented, and tis, systemic lupus erythematosus and immune-related glom or treated by polynucleotides, polypeptides, and/or agonists erulonephritis and rheumatoid arthritis) myelodysplastic or antagonists of the invention, include cancers (such as fol syndromes (such as aplastic anemia), graft V, host disease, licular lymphomas, carcinomas with p53 mutations, and hor ischemic injury (such as that caused by myocardial infarction, mone-dependent tumors, including, but not limited to colon stroke and reperfusion injury), liver injury (e.g., hepatitis cancer, cardiac tumors, pancreatic cancer, melanoma, retino related liver injury, ischemia/reperfusion injury, cholestosis blastoma, glioblastoma, lung cancer, intestinal cancer, tes 25 (bile duct injury) and liver cancer); toxin-induced liver dis ticular cancer, stomach cancer, neuroblastoma, myxoma, ease (Such as that caused by alcohol), septic shock, cachexia myoma, lymphoma, endothelioma, osteoblastoma, osteo and anorexia. clastoma, osteosarcoma, chondrosarcoma, adenoma, breast Hyperproliferative diseases and/or disorders that could be cancer, prostate cancer, Kaposi's sarcoma and ovarian can diagnosed, prognosed, prevented, and/or treated by fusion cer); autoimmune disorders such as, multiple Sclerosis, 30 proteins of the invention and/or polynucleotides encoding Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrho albumin fusion proteins of the invention, include, but are not sis, Behcet’s disease, Crohn's disease, polymyositis, sys limited to, neoplasms located in the liver, abdomen, bone, temic lupus erythematosus and immune-related glomerulo breast, digestive system, pancreas, peritoneum, endocrine nephritis and rheumatoid arthritis) and viral infections (such glands (adrenal, parathyroid, pituitary, testicles, ovary, thy as herpes viruses, pox viruses and adenoviruses), inflamma 35 mus, thyroid), eye, head and neck, nervous system (central tion, graft V, host disease, acute graft rejection, and chronic and peripheral), lymphatic system, pelvis, skin, soft tissue, graft rejection. spleen, thorax, and urogenital tract. In preferred embodiments, fusion proteins of the invention Similarly, other hyperproliferative disorders can also be and/or polynucleotides encoding albumin fusion proteins of diagnosed, prognosed, prevented, and/or treated by fusion the invention are used to inhibit growth, progression, and/or 40 proteins of the invention and/or polynucleotides encoding metastasis of cancers, in particular those listed above. albumin fusion proteins of the invention. Examples of such Additional diseases or conditions associated with hyperproliferative disorders include, but are not limited to: increased cell Survival that could be diagnosed, prognosed, hypergammaglobulinemia, lymphoproliferative disorders, prevented, and/or treated by fusion proteins of the invention paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, and/or polynucleotides encoding albumin fusion proteins of 45 Waldenstrons macroglobulinemia, Gaucher's Disease, his the invention, include, but are not limited to, progression, tiocytosis, and any other hyperproliferative disease, besides and/or metastases of malignancies and related disorders such neoplasia, located in an organ system listed above. as leukemia (including acute leukemias (e.g., acute lympho Another preferred embodiment utilizes polynucleotides cytic leukemia, acute myelocytic leukemia (including myelo encoding albumin fusion proteins of the invention to inhibit blastic, promyelocytic, myelomonocytic, monocytic, and 50 aberrant cellular division, by gene therapy using the present erythroleukemia)) and chronic leukemias (e.g. chronic invention, and/or protein fusions or fragments thereof. myelocytic (granulocytic) leukemia and chronic lymphocytic Thus, the present invention provides a method for treating leukemia)), polycythemia Vera, lymphomas (e.g. Hodgkin’s cell proliferative disorders by inserting into an abnormally disease and non-Hodgkin’s disease), multiple myeloma, proliferating cell a polynucleotide encoding an albumin Waldenstrom's macroglobulinemia, heavy chain disease, and 55 fusion protein of the present invention, wherein said poly Solid tumors including, but not limited to sarcomas and car nucleotide represses said expression. cinomas Such as fibrosarcoma, myxosarcoma, liposarcoma, Another embodiment of the present invention provides a chondrosarcoma, osteogenic sarcoma, chordoma, angiosar method of treating cell-proliferative disorders in individuals coma, endotheliosarcoma, lymphangiosarcoma, lymphan comprising administration of one or more active gene copies gioendotheliosarcoma, synovioma, mesothelioma, Ewings 60 of the present invention to an abnormally proliferating cell or tumor, leiomyosarcoma, rhabdomyosarcoma, colon carci cells. In a preferred embodiment, polynucleotides of the noma, pancreatic cancer, breast cancer, ovarian cancer, pros present invention is a DNA construct comprising a recombi tate cancer, Squamous cell carcinoma, basal cell carcinoma, nant expression vector effective in expressing a DNA adenocarcinoma, Sweat gland carcinoma, sebaceous gland sequence encoding said polynucleotides. In another preferred carcinoma, papillary carcinoma, papillary adenocarcinomas, 65 embodiment of the present invention, the DNA construct cystadenocarcinoma, medullary carcinoma, bronchogenic encoding the fusion protein of the present invention is carcinoma, renal cell carcinoma, hepatoma, bile duct carci inserted into cells to be treated utilizing a retrovirus, or more US 8,946,156 B2 137 138 preferably an adenoviral vector (See GJ. Nabel, et. al. PNAS “biologically inhibiting is meant partial or total growth inhi 1999 96: 324-326, which is hereby incorporated by refer bition as well as decreases in the rate of proliferation or ence). In a most preferred embodiment, the viral vector is growth of the cells. The biologically inhibitory dose may be defective and will not transform non-proliferating cells, only determined by assessing the effects of the polynucleotides of proliferating cells. Moreover, in a preferred embodiment, the the present invention on target malignant or abnormally pro polynucleotides of the present invention inserted into prolif liferating cell growth in tissue culture, tumor growth in ani erating cells either alone, or in combination with or fused to mals and cell cultures, or any other method known to one of other polynucleotides, can then be modulated via an external ordinary skill in the art. stimulus (i.e. magnetic, specific Small molecule, chemical, or Moreover, fusion proteins of the invention and/or poly drug administration, etc.), which acts upon the promoter 10 nucleotides encoding albumin fusion proteins of the inven upstream of said polynucleotides to induce expression of the tion of the present invention are useful in inhibiting the angio encoded protein product. AS Such the beneficial therapeutic genesis of proliferative cells or tissues, either alone, as a affect of the present invention may be expressly modulated protein fusion, or in combination with other polypeptides (i.e. to increase, decrease, or inhibit expression of the present directly or indirectly, as described elsewhere herein. In a most invention) based upon said external stimulus. 15 preferred embodiment, said anti-angiogenesis effect may be Polynucleotides of the present invention may be useful in achieved indirectly, for example, through the inhibition of repressing expression of oncogenic genes or antigens. By hematopoietic, tumor-specific cells, such as tumor-associated “repressing expression of the oncogenic genes' is intended macrophages (See Joseph IB. etal.JNatl Cancer Inst. 90(21): the Suppression of the transcription of the gene, the degrada 1648-53 (1998), which is hereby incorporated by reference). tion of the gene transcript (pre-message RNA), the inhibition Albumin fusion proteins of the invention and or polynucle of splicing, the destruction of the messenger RNA, the pre otides encoding albumin fusion proteins of the invention may vention of the post-translational modifications of the protein, be useful in inhibiting proliferative cells or tissues through the the destruction of the protein, or the inhibition of the normal induction of apoptosis. These fusion proteins and or poly function of the protein. nucleotides may act either directly, or indirectly to induce For local administration to abnormally proliferating cells, 25 apoptosis of proliferative cells and tissues, for example in the polynucleotides of the present invention may be administered activation of a death-domain receptor, such as tumor necrosis by any method known to those of skill in the art including, but factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor not limited to transfection, electroporation, microinjection of related apoptosis-mediated protein (TRAMP) and TNF-re cells, or in vehicles Such as liposomes, lipofectin, or as naked lated apoptosis-inducing ligand (TRAIL) receptor-1 and -2 polynucleotides, or any other method described throughout 30 (See Schulze-OsthoffK. et al., EurJ Biochem 254(3):439-59 the specification. The polynucleotide of the present invention (1998), which is hereby incorporated by reference). More may be delivered by known gene delivery systems such as, over, in another preferred embodiment of the present inven but not limited to retroviral vectors (Gilboa, J. Virology tion, these fusion proteins and/or polynucleotides may induce 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., apoptosis through other mechanisms, such as in the activation Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system 35 of other proteins which will activate apoptosis, or through (Chakrabarty et al. Mol. Cell. Biol. 5:3403 (1985) or other stimulating the expression of these proteins, either alone or in efficient DNA delivery systems (Yates et al. Nature 313:812 combination with Small molecule drugs or adjuvants, such as (1985)) known to those skilled in the art. These references are apoptonin, galectins, thioredoxins, anti-inflammatory pro exemplary only and are hereby incorporated by reference. In teins (See for example, Mutat Res 400(1-2):447-55 (1998), order to specifically deliver or transfect cells which are abnor 40 Med. Hypotheses. 50(5):423-33 (1998), Chem Biol Interact. mally proliferating and spare non-dividing cells, it is prefer April 24; 111-112:23-34 (1998), J Mol. Med. 76(6):402-12 able to utilize a retrovirus, or adenoviral (as described in the (1998), Int J Tissue React; 2001):3-15 (1998), which are all art and elsewhere herein) delivery system known to those of hereby incorporated by reference). skill in the art. Since host DNA replication is required for Albumin fusion proteins of the invention and or polynucle retroviral DNA to integrate and the retrovirus will be unable 45 otides encoding albumin fusion proteins of the invention are to self replicate due to the lack of the retrovirus genes needed useful in inhibiting the metastasis of proliferative cells or for its life cycle. Utilizing such a retroviral delivery system for tissues. Inhibition may occur as a direct result of administer polynucleotides of the present invention will target said gene ing these albumin fusion proteins and/or polynucleotides, or and constructs to abnormally proliferating cells and will spare indirectly, such as activating the expression of proteins the non-dividing normal cells. 50 known to inhibit metastasis, for example alpha 4 integrins. The polynucleotides of the present invention may be deliv (See, e.g. Curr Top Microbiol Immunol 1998; 231:125-41, ered directly to cell proliferative disorder/disease sites in which is hereby incorporated by reference). Such therapeutic internal organs, body cavities and the like by use of imaging affects of the present invention may be achieved either alone, devices used to guide an injecting needle directly to the dis or in combination with Small molecule drugs or adjuvants. ease site. The polynucleotides of the present invention may 55 In another embodiment, the invention provides a method of also be administered to disease sites at the time of Surgical delivering compositions containing the albumin fusion pro intervention. teins of the invention and/or polynucleotides encoding albu By "cell proliferative disease' is meant any human orani min fusion proteins of the invention to targeted cells express mal disease or disorder, affecting any one or any combination ing the a polypeptide bound by, that binds to, or associates of organs, cavities, or body parts, which is characterized by 60 with an albumin fusion protein of the invention. Albumin single or multiple local abnormal proliferations of cells, fusion proteins of the invention may be associated with het groups of cells, or tissues, whether benign or malignant. erologous polypeptides, heterologous nucleic acids, toxins, Any amount of the polynucleotides of the present invention or prodrugs via hydrophobic, hydrophilic, ionic and/or cova may be administered as long as it has a biologically inhibiting lent interactions. effect on the proliferation of the treated cells. Moreover, it is 65 Albumin fusion proteins of the invention are useful in possible to administer more than one of the polynucleotide of enhancing the immunogenicity and/or antigenicity of prolif the present invention simultaneously to the same site. By erating cells or tissues, either directly, such as would occur if US 8,946,156 B2 139 140 the albumin fusion proteins of the invention vaccinated the natremia, hypokalemia, hyperkalemia, hypocalcemia, hyper immune response to respond to proliferative antigens and calcemia, hypophosphatemia, and hyperphosphatemia). immunogens, or indirectly, Such as in activating the expres Compositions of the invention may be administered using sion of proteins known to enhance the immune response (e.g. any method known in the art, including, but not limited to chemokines), to said antigens and immunogens. direct needle, injection at the delivery site, intravenous injec Renal Disorders tion, topical administration, catheter infusion, biolistic injec Albumin fusion proteins of the invention and/or polynucle tors, particle accelerators, gelfoam sponge depots, other com otides encoding albumin fusion proteins of the invention, may mercially available depot materials, osmotic pumps, oral or be used to treat, prevent, diagnose, and/or prognose disorders Suppositorial Solid pharmaceutical formulations, decanting 10 or topical applications during Surgery, aerosol delivery. Such of the renal system, Renal disorders which can be diagnosed, methods are known in the art. Compositions of the invention prognosed, prevented, and/or treated with compositions of may be administered as part of a Therapeutic, described in the invention include, but are not limited to, kidney failure, more detail below. Methods of delivering polynucleotides of nephritis, blood vessel disorders of kidney, metabolic and the invention are described in more detail herein. congenital kidney disorders, urinary disorders of the kidney, 15 Cardiovascular Disorders autoimmune disorders, Sclerosis and necrosis, electrolyte Albumin fusion proteins of the invention and/or polynucle imbalance, and kidney cancers. otides encoding albumin fusion proteins of the invention, may Kidney diseases which can be diagnosed, prognosed, pre be used to treat, prevent, diagnose, and/or prognose cardio vented, and or treated with compositions of the invention vascular disorders, including, but not limited to peripheral include, but are not limited to acute kidney failure, chronic artery disease. Such as limb ischemia. kidney failure, atheroembolic renal failure, end-stage renal Cardiovascular disorders include, but are not limited to disease, inflammatory diseases of the kidney (e.g. acute cardiovascular abnormalities, such as arterio-arterial fistula, glomerulonephritis, postinfectious glomerulonephritis, rap arteriovenous fistula, cerebral arteriovenous malformations, idly progressive glomerulonephritis, nephrotic syndrome, congenital heart defects, pulmonary atresia, and Scimitar membranous glomerulonephritis, familial nephrotic Syn 25 Syndrome. Congenital heart defects include, but are not lim drome, membranoproliferative glomerulonephritis I and II, ited to, aortic coarctation, cor triatriatum, coronary vessel mesangial proliferative glomerulonephritis, chronic glom anomalies, crisscross heart, dextrocardia, patent ductus arte erulonephritis, acute tubulointerstitial nephritis, chronic riosus, Ebstein's anomaly, Eisenmenger complex, hypoplas tubulointerstitial nephritis, acute post-streptococcal glomeru tic left heart syndrome, levocardia, tetralogy of fallot, trans lonephritis (PSGN), pyelonephritis, lupus nephritis, chronic 30 position of great vessels, double outlet right ventricle, nephritis, interstitial nephritis, and post-streptococcal glom tricuspidatresia, persistent truncus arteriosus, and heart sep erulonephritis), blood vessel disorders of the kidneys (e.g. tal defects, such as aortopulmonary septal defect, endocardial kidney infarction, atheroembolic kidney disease, cortical cushion defects, Lutembacher's Syndrome, trilogy of Fallot, necrosis, malignant nephrosclerosis, renal vein thrombosis, ventricular heart septal defects. renal underperfusion, renal retinopathy, renal ischemia-rep 35 Cardiovascular disorders also include, but are not limited erfusion, renal artery embolism, and renal artery Stenosis), to, heart disease, Such as arrhythmias, carcinoid heart disease, and kidney disorders resulting form urinary tract disease high cardiac output, low cardiac output, cardiac tamponade, (e.g., pyelonephritis, hydronephrosis, urolithiasis (renal lithi endocarditis (including bacterial), heart aneurysm, cardiac asis, nephrolithiasis), reflux nephropathy, urinary tract infec arrest, congestive heart failure, congestive cardiomyopathy, tions, urinary retention, and acute or chronic unilateral 40 paroxysmal dyspnea, cardiac edema, heart hypertrophy, con obstructive uropathy.) gestive cardiomyopathy, left ventricular hypertrophy, right In addition, compositions of the invention can be used to Ventricular hypertrophy, post-infarction heart rupture, ven diagnose, prognose, prevent, and/or treat metabolic and con tricular septal rupture, heart valve diseases, myocardial dis genital disorders of the kidney (e.g., uremia, renal amyloido eases, myocardial ischemin, pericardial effusion, pericarditis sis, renal osteodystrophy, renal tubular acidosis, renal glyco 45 (including constrictive and tuberculous), pneumopericar Suria, nephrogenic diabetes insipidus, cystinuria, Fanconi's dium, postpericardiotomy syndrome, pulmonary heart dis syndrome, renal fibrocystic osteosis (renal rickets), Hartnup ease, rheumatic heart disease, Ventricular dysfunction, hype disease, Bartter's syndrome, Liddle's syndrome, polycystic remia, cardiovascular pregnancy complications, Scimitar kidney disease, medullary cystic disease, medullary sponge Syndrome, cardiovascular syphilis, and cardiovascular tuber kidney, Alport's syndrome, nail-patella syndrome, congenital 50 culosis. nephrotic syndrome, CRUSH syndrome, horseshoe kidney, Arrhythmias include, but are not limited to sinus arrhyth diabetic nephropathy, nephrogenic diabetes insipidus, anal mia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, gesic nephropathy, kidney Stones, and membranous nephr Adams-Stokes Syndrome, bundle-branch block, sinoatrial opathy), and autoimmune disorders of the kidney (e.g. sys block, long QT syndrome, parasystole, Lown-Ganong-Le temic lupus erythematosus (SLE), Goodpasture syndrome, 55 vine Syndrome, Mahaim-type pre-excitation syndrome, IgA nephropathy, and IgM mesangial proliferative glomeru Wolff-Parkinson-White syndrome, sick sinus syndrome, lonephritis). tachycardias, and Ventricular fibrillation, Tachycardias Compositions of the invention can also be used to diag include paroxysmal tachycardia, Supraventricular tachycar nose, prognose, prevent, and/or treat Sclerotic or necrotic dia, accelerated idioventricular rhythm, atrioventricular disorders of the kidney (e.g. glomerulosclerosis, diabetic 60 nodal reentry tachycardia, ectopic atrial tachycardia, ectopic nephropathy, focal segmental glomerulosclerosis (FSGS), junctional tachycardia, Sinoatrial nodal reentry tachycardia, necrotizing glomerulonephritis, and renal papillary necrosis), sinus tachycardia, Torsades de Pointes, and Ventricular tachy cancers of the kidney (e.g. nephroma, hypernephroma, neph cardia. roblastoma, renal cell cancer, transitional cell cancer, renal Heart valve diseases include, but are not limited to aortic adenocarcinoma, squamous cell cancer, and Wilm's tumor), 65 valve insufficiency, aortic valve Stenosis, hear murmurs, aor and electrolyte imbalances (e.g. nephrocalcinosis, pyuria, tic valve prolapse, mitral valve prolapse, tricuspid valve pro edema, hydronephritis, proteinuria, hyponatremia, hyper lapse, mitral valve insufficiency, mitral valve Stenosis, pull US 8,946,156 B2 141 142 monary atresia, pulmonary valve insufficiency, pulmonary Albumin fusion proteins of the invention and/or polynucle valve Stenosis, tricuspidatresia, tricuspid valve insufficiency, otides encoding albumin fusion proteins of the invention may and tricuspid valve Stenosis. be administered using any method known in the art, includ Myocardial diseases include, but are not limited to alco ing, but not limited to, direct needle injection at the delivery holic cardiomyopathy, congestive cardiomyopathy, hyper site, intravenous injection, topical administration, catheter trophic cardiomyopathy, aortic Subvalvular Stenosis, pulmo infusion, biolistic injectors, particle accelerators, gelfoam nary Subvalvular Stenosis, restrictive cardiomyopathy, sponge depots, other commercially available depot materials, Chagas cardiomyopathy, endocardial fibroelastosis, osmotic pumps, oral or Suppositorial Solid pharmaceutical endomyocardial fibrosis, Kearns Syndrome, myocardial rep formulations, decanting or topical applications during Sur 10 gery, aerosol delivery. Such methods are known in the art. erfusion injury, and myocarditis. Methods of delivering polynucleotides are described in more Myocardial ischemias include, but are not limited to coro detail herein. nary disease, such as angina pectoris, coronary aneurysm, Respiratory Disorders coronary arteriosclerosis, coronary thrombosis, coronary Albumin fusion proteins of the invention and/or polynucle vasospasm, myocardial infarction and myocardial stunning. 15 otides encoding albumin fusion proteins of the invention may Cardiovascular diseases also include vascular diseases be used to treat, prevent, diagnose, and, or prognose diseases Such as aneurysms, angiodysplasia, angiomatosis, bacillary and/or disorders of the respiratory system. angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay Diseases and disorders of the respiratory system include, Weber Syndrome, Sturge-Weber Syndrome, angioneurotic but are not limited to, nasal vestibulitis, nonallergic rhinitis edema, aortic diseases, Takayasu's Arteritis, aortitis, Ler (e.g. acute rhinitis, chronic rhinitis, atrophic rhinitis, vasomo iche's Syndrome, arterial occlusive diseases, arteritis, enar torrhinitis), nasal polyps, and sinusitis, juvenile angiofibro teritis, polyarteritis nodosa, cerebrovascular disorders, dia mas, cancer of the nose and juvenile papillomas, Vocal cord betic angiopathies, diabetic retinopathy, embolisms, polyps, nodules (singer's nodules), contact ulcers, Vocal cord thrombosis, erythromelalgia, hemorrhoids, hepatic veno-Oc paralysis, laryngoceles, pharyngitis (e.g. viral and bacterial), clusive disease, hypertension, hypotension, ischemia, periph 25 tonsillitis, tonsillar cellulitis, parapharyngeal abscess, laryn eral vascular diseases, phlebitis, pulmonary veno-occlusive gitis, laryngoceles, and throat cancers (e.g., cancer of the disease, Raynaud's disease, CREST syndrome, retinal vein nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g. occlusion, Scimitar syndrome, Superior vena cava syndrome, squamous cell carcinoma, Small cell (oat cell) carcinoma, telangiectasia, atacia telangiectasia, hereditary hemorrhagic large cell carcinoma, and adenocarcinoma), allergic disorders telangiectasia, varicocele, varicose veins, varicose ulcer, Vas 30 (eosinophilic pneumonia, hypersensitivity pneumonitis (e.g. culitis, and venous insufficiency. extrinsic allergic alveolitis, allergic interstitial pneumonitis, Aneurysms include, but are not limited to dissecting aneu organic dust pneumoconiosis, allergic bronchopulmonary rysms, false aneurysms, infected aneurysms, ruptured aneu aspergillosis, asthma, Wegener's granulomatosis (granulo rysms, aortic aneurysms, cerebral aneurysms, coronary aneu matous vasculitis), Goodpasture's syndrome)), pneumonia rysms, heart aneurysms, and iliac aneurysms. 35 (e.g. bacterial pneumonia (e.g. Streptococcus pneumoniae Arterial occlusive diseases include, but are not limited to (pneumococcal pneumonia), Staphylococcus aureus (staphy arteriosclerosis, intermittent claudication, carotid Stenosis, lococcal pneumonia), Gram-negative bacterial pneumonia fibromuscular dysplasias, mesenteric vascular occlusion, (caused by, e.g. Klebsiella and Pseudomas spp.), Myco Moyamoya disease, renal artery obstruction, retinal artery plasma pneumoniae pneumonia, Hemophilus influenzae occlusion, and thromboangiitis obliterans. 40 pneumonia, Legionella pneumophila (Legionnaires dis Cerebrovascular disorders include, but are not limited to ease), and Chlamydia psittaci (Psittacosis)), and viral pneu carotidartery diseases, cerebral amyloid angiopathy, cerebral monia (e.g. influenza, chickenpox (varicella). aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral Additional diseases and disorders of the respiratory system arteriovenous malformation, cerebral artery diseases, cere include, but are not limited to bronchiolitis, polio (poliomy bral embolism and thrombosis, carotid artery thrombosis, 45 elitis), croup, respiratory syncytial viral infection, mumps, sinus thrombosis, Wallenberg's syndrome, cerebral hemor erythema infectiosum (fifth disease), roseola infantum, pro rhage, epidural hematoma, Subdural hematoma, Subaraxh gressive rubella panencephalitis, german measles, and Sub noid hemorrhage, cerebral infarction, cerebral ischemia (in acute Sclerosing panencephalitis), fungal pneumonia (e.g., cluding transient). Subclavian steal syndrome, periventricular Histoplasmosis, Coccidioidomycosis, Blastomycosis, fungal leukomalacia, Vascular headache, cluster headache, 50 infections in people with severely Suppressed immune sys migraine, and vertebrobasilar insufficiency. tems (e.g., cryptococcosis, caused by Cryptococcus neofor Embolisms include, but are not limited to air embolisms, mans; aspergillosis, caused by Aspergillus spp.; candidiasis, amniotic fluid embolisms, cholesterol embolisms, blue toe caused by Candida; and mucormycosis)). Pneumocystis cari syndrome, fat embolisms, pulmonary embolisms, and thro nii (pneumocystis pneumonia), atypical pneumonias (e.g., moboembolisms, Thrombosis include, but are not limited to 55 Mycoplasma and Chlamydia spp.), opportunistic infection coronary thrombosis, hepatic vein thrombosis, retinal vein pneumonia, nosocomial pneumonia, chemical pneumonitis, occlusion, carotid artery thrombosis, sinus thrombosis, Wal and aspiration pneumonia, pleural disorders (e.g., pleurisy, lenberg's syndrome, and thrombophlebitis. pleural effusion, and pneumothorax (e.g. simple spontaneous Ischemic disorders include, but are not limited to cerebral pneumothorax, complicated spontaneous pneumothorax, ischemia, ischemic colitis, compartment syndromes, anterior 60 tension pneumothorax)), obstructive airway diseases (e.g., compartment syndrome, myocardial ischemia, reperfusion asthma, chronic obstructive pulmonary disease (COPD), injuries, and peripheral limb ischemia, Vasculitis includes, emphysema, chronic or acute bronchitis), occupational lung but is not limited to, aortitis, arteritis, Behcet’s Syndrome, diseases (e.g. silicosis, black lung (coal workers’ pneumoco Churg-Strauss Syndrome, mucocutaneous lymph node syn niosis), asbestosis, berylliosis, occupational asthsma, byssi drome, thromboangiitis obliterans, hypersensitivity vasculi 65 nosis, and benign pneumoconiosis), Infiltrative Lung Disease tis, Schoenlein-Henoch purpura, allergic cutaneous vasculi (e.g. pulmonary fibrosis (e.g., fibrosing alveolitis, usual inter tis, and Wegener's granulomatosis. Stitial pneumonia), idiopathic pulmonary fibrosis, descquama US 8,946,156 B2 143 144 tive interstitial pneumonia, lymphoid interstitial pneumonia, kemias. For example, fusion proteins of the invention and/or histiocytosis X (e.g. Letterer-Siwe disease, Hand-Schüller polynucleotides encoding albumin fusion proteins of the Christian disease, eosinophilic granuloma), idiopathic pull invention may be delivered topically, in order to treat cancers monary hemosiderosis, sarcoidosis and pulmonary alveolar Such as skin cancer, head and neck tumors, breast tumors, and proteinosis), Acute respiratory distress syndrome (also 5 Kaposi's sarcoma. called, e.g. adult respiratory distress syndrome), edema, pull Within yet other aspects, fusion proteins of the invention monary embolism, bronchitis (e.g. viral, bacterial), bron and or polynucleotides encoding albumin fusion proteins of chiectasis, atelectasis, lung abscess (caused by, e.g., Staphy lococcus aureus or Legionella pneumophila), and cystic the invention may be utilized to treat superficial forms of fibrosis. 10 bladder cancer by, for example, intravesical administration. Anti-Angiogenesis Activity Albumin fusion proteins of the invention and/or polynucle The naturally occurring balance between endogenous otides encoding albumin fusion proteins of the invention may stimulators and inhibitors of angiogenesis is one in which be delivered directly into the tumor, or near the tumor site, via inhibitory influences predominate, Rastinejad et al. Cell injection or a catheter. Of course, as the artisan of ordinary 56:345-355 (1989). In those rare instances in which neovas- 15 skill will appreciate, the appropriate mode of administration cularization occurs under normal physiological conditions, will vary according to the cancerto be treated. Other modes of Such as wound healing, organ regeneration, embryonic devel delivery are discussed herein. opment, and female reproductive processes, angiogenesis is Albumin fusion proteins of the invention and/or polynucle stringently regulated and spatially and temporally delimited. otides encoding albumin fusion proteins of the invention may Under conditions of pathological angiogenesis Such as that 20 be useful in treating other disorders, besides cancers, which characterizing Solid tumor growth, these regulatory controls involve angiogenesis. These disorders include, but are not fail. Unregulated angiogenesis becomes pathologic and Sus limited to:benign tumors, for example hemangiomas, acous tains progression of many neoplastic and non-neoplastic dis tic neuromas, neurofibromas, trachomas, and pyogenic eases. A number of serious diseases are dominated by abnor granulomas; artheroscleric plaques, ocular angiogenic dis mal neovascularization including Solid tumor growth and 25 eases, for example, diabetic retinopathy, retinopathy of pre metastases, arthritis, Some types of eye disorders, and psoria maturity, macular degeneration, corneal graft rejection, sis. See, e.g. reviews by Moses et al., Biotech. 9:630-634 neovascular glaucoma, retrolental fibroplasia, rubeosis, ret (1991): Folkman eral. N. Engl. J. Med. 333:1757-1763 inoblastoma, uvietis and Pterygia (abnormal blood vessel (1995); Auerbachetal.J. Microvasc. Res. 29:401–411 (1985): growth) of the eye; rheumatoid arthritis; psoriasis; delayed Folkman, Advances in Cancer Research, eds. Klein and 30 wound healing; endometriosis; vasculogenesis; granulations; Weinhouse, Academic Press, New York, pp. 175-203 (1985); hypertrophic scars (keloids); nonunion fractures; Sclero Patz, Am. J. Opthalmol. 94:715–743 (1982): and Folkman et derma; trachoma; vascular adhesions; myocardial angiogen al. Science 221:719-725 (1983). In a number of pathological esis; coronary collaterals; cerebral collaterals; arteriovenous conditions, the process of angiogenesis contributes to the malformations; ischemic limb angiogenesis; Osler-Webber disease State. For example, significant data have accumulated 35 Syndrome; plaque neovascularization; telangiectasia; hemo which Suggest that the growth of solid tumors is dependent on philiac joints; angiofibroma; fibromuscular dysplasia; wound angiogenesis. Folkman and Klagsbrun, Science 235:442-447 granulation; Crohn's disease; and atherosclerosis. (1987). For example, within one aspect of the present invention The present invention provides for treatment of diseases or methods are provided for treating hypertrophic scars and disorders associated with neovascularization by administra- 40 keloids, comprising the step of administering albumin fusion tion offusion proteins of the invention and/or polynucleotides proteins of the invention and/or polynucleotides encoding encoding albumin fusion proteins of the invention. Malignant albumin fusion proteins of the invention to a hypertrophic and metastatic conditions which can be treated with the poly scar or keloid. nucleotides and polypeptides, or agonists or antagonists of Within one embodiment of the present invention fusion the invention include, but are not limited to, malignancies, 45 proteins of the invention and/or polynucleotides encoding Solid tumors, and cancers described herein and otherwise albumin fusion proteins of the invention are directly injected known in the art (for a review of such disorders, see Fishman into a hypertrophic scar or keloid, in order to prevent the et al., Medicine, 2d Ed., J. B. Lippincott Co. Philadelphia progression of these lesions. This therapy is of particular (1985)). Thus, the present invention provides a method of value in the prophylactic treatment of conditions which are treating an angiogenesis-related disease and/or disorder, 50 known to result in the development of hypertrophic Scars and comprising administering to an individual in need thereof a keloids (e.g. burns), and is preferably initiated after the pro therapeutically effective amount of an albumin fusion protein liferative phase has had time to progress (approximately 14 of the invention and/or polynucleotides encoding an albumin days after the initial injury), but before hypertrophic scar or fusion protein of the invention. For example, fusion proteins keloid development. As noted above, the present invention of the invention and/or polynucleotides encoding albumin 55 also provides methods for treating neovascular diseases of the fusion proteins of the invention may be utilized in a variety of eye, including for example, corneal neovascularization, additional methods in order to therapeutically treat a cancer neovascular glaucoma, proliferative diabetic retinopathy, ret or tumor. Cancers which may be treated with fusion proteins rolental fibroplasia and macular degeneration. of the invention and or polynucleotides encoding albumin Moreover, Ocular disorders associated with neovascular fusion proteins of the invention include, but are not limited to 60 ization which can be treated with the albumin fusion proteins Solid tumors, including prostate, lung, breast, ovarian, stom of the invention and/or polynucleotides encoding albumin ach, pancreas, larynx, esophagus, testes, liver, parotid, biliary fusion proteins of the invention include, but are not limited to: tract, colon, rectum, cervix, uterus, endometrium, kidney, neovascular glaucoma, diabetic retinopathy, retinoblastoma, bladder, thyroid cancer, primary tumors and metastases; retrolental fibroplasia, uveitis, retinopathy of prematurity melanomas; glioblastoma; Kaposi's sarcoma: leiomyosar- 65 macular degeneration, corneal graft neovascularization, as coma; non-small cell lung cancer; colorectal cancer, well as other eye inflammatory diseases, ocular tumors and advanced malignancies; and blood born tumors such as leu diseases associated with choroidal or iris neovascularization. US 8,946,156 B2 145 146 See, e.g. reviews by Waltman et al. Am. J. Ophthal. 85:704 amount of an albumin fusion protein of the invention and/or 710 (1978) and Gartner et al. Surv. Ophthal. 22:291-312 polynucleotides encoding an albumin fusion protein of the (1978). invention to the eye, such that the formation of blood vessels Thus, within one aspect of the present invention methods is inhibited. In one embodiment, the compound may be are provided for treating neovascular diseases of the eye Such 5 administered topically to the eye in order to treat early forms as corneal neovascularization (including corneal graft of neovascular glaucoma. Within other embodiments, the neovascularization), comprising the step of administering to a compound may be implanted by injection into the region of patient a therapeutically effective amount of a compound the anterior chamber angle. Within other embodiments, the (e.g., fusion proteins of the invention and/or polynucleotides compound may also be placed in any location Such that the encoding albumin fusion proteins of the invention) to the 10 compound is continuously released into the aqueous humor. cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue which normally lacks blood Within another aspect of the present invention, methods are vessels. In certain pathological conditions however, capillar provided for treating proliferative diabetic retinopathy, com ies may extend into the cornea from the pericorneal vascular prising the step of administering to a patient a therapeutically plexus of the limbus. When the cornea becomes vascularized, 15 effective amount of an albumin fusion protein of the invention it also becomes clouded, resulting in a decline in the patients and/or polynucleotides encoding an albumin fusion protein of visual acuity. Visual loss may become complete if the cornea the invention to the eyes, such that the formation of blood completely opacitates. A wide variety of disorders can result vessels is inhibited. in corneal neovascularization, including for example, corneal Within particularly preferred embodiments of the inven infections (e.g., trachoma, herpes simplex keratitis, leishma tion, proliferative diabetic retinopathy may be treated by niasis and onchocerciasis), immunological processes (e.g., injection into the aqueous humor or the vitreous, in order to graft rejection and Stevens-Johnson's syndrome), alkali increase the local concentration of the polynucleotide, burns, trauma, inflammation (of any cause), toxic and nutri polypeptide, antagonist and/or agonist in the retina. Prefer tional deficiency states, and as a complication of wearing ably, this treatment should be initiated prior to the acquisition contact lenses. 25 of severe disease requiring photocoangulation. Within particularly preferred embodiments of the inven Within another aspect of the present invention, methods are tion, may be prepared for topical administration in Saline provided for treating retrolental fibroplasia, comprising the (combined with any of the preservatives and antimicrobial step of administering to a patient a therapeutically effective agents commonly used in ocular preparations), and adminis amount of an albumin fusion protein of the invention and/or tered in eyedrop form. The Solution or Suspension may be 30 polynucleotides encoding an albumin fusion protein of the prepared in its pure form and administered several times invention to the eye, such that the formation of blood vessels daily. Alternatively, anti-angiogenic compositions, prepared is inhibited. The compound may be administered topically, as described above, may also be administered directly to the via intravitreous injection and/or via intraocular implants. cornea. Within preferred embodiments, the anti-angiogenic Additionally, disorders which can be treated with fusion composition is prepared with a muco-adhesive polymer 35 proteins of the invention and/or polynucleotides encoding which binds to cornea. Within further embodiments, the anti albumin fusion proteins of the invention include, but are not angiogenic factors or anti-angiogenic compositions may be limited to hemangioma, arthritis, psoriasis, angiofibroma, utilized as an adjunct to conventional steroid therapy. Topical atherosclerotic plaques, delayed wound healing, granula therapy may also be useful prophylactically in corneal lesions tions, hemophilic joints, hypertrophic scars, nonunion frac which are known to have a high probability of inducing an 40 tures, Osler-Weber syndrome, pyogenic granuloma, Sclero angiogenic response (such as chemical burns). In these derma, trachoma, and vascular adhesions. instances the treatment, likely in combination with steroids, Moreover, disorders and/or states, which can be treated, may be instituted immediately to help prevent Subsequent prevented, diagnosed, and/or prognosed with the albumin complications. fusion proteins of the invention and or polynucleotides Within other embodiments, the compounds described 45 encoding albumin fusion proteins of the invention of the above may be injected directly into the corneal stroma by an invention include, but are not limited to solid tumors, blood ophthalmologist under microscopic guidance. The preferred born tumors such as leukemias, tumor metastasis, Kaposi's site of injection may vary with the morphology of the indi sarcoma, benign tumors, for example hemangiomas, acoustic vidual lesion, but the goal of the administration would be to neuromas, neurofibromas, trachomas, and pyogenic granulo place the composition at the advancing front of the vascula 50 mas, rheumatoid arthritis, psoriasis, ocular angiogenic dis ture (i.e., interspersed between the blood vessels and the eases, for example, diabetic retinopathy, retinopathy of pre normal cornea). In most cases this would involve perilimbic maturity, macular degeneration, corneal graft rejection, corneal injection to “protect’ the cornea from the advancing neovascular glaucoma, retrolental fibroplasia, rubeosis, ret blood vessels. This method may also be utilized shortly after inoblastoma, and uvietis, delayed wound healing, a corneal insult in order to prophylactically prevent corneal 55 endometriosis, vasculogenesis, granulations, hypertrophic neovascularization. In this situation the material could be Scars (keloids), nonunion fractures, Scleroderma, trachoma, injected in the perilimbic cornea interspersed between the vascular adhesions, myocardial angiogenesis, coronary col corneal lesion and its undesired potential limbic blood Supply. laterals, cerebral collaterals, arteriovenous malformations, Such methods may also be utilized in a similar fashion to ischemic limb angiogenesis, Osler-Webber Syndrome, prevent capillary invasion of transplanted corneas. In a sus 60 plaque neovascularization, telangiectasia, hemophiliac tained-release form injections might only be required 2-3 joints, angiofibroma fibromuscular dysplasia, wound granu times per year. A steroid could also be added to the injection lation, Crohn's disease, atherosclerosis, birth control agent Solution to reduce inflammation resulting from the injection by preventing vascularization required for embryo implanta itself. tion controlling menstruation, diseases that have angiogen Within another aspect of the present invention, methods are 65 esis as a pathologic consequence Such as cat scratch disease provided for treating neovascular glaucoma, comprising the (Rochele minalia quintosa), ulcers (Helicobacter pylori), step of administering to a patient a therapeutically effective Bartonellosis and bacillary angiomatosis. US 8,946,156 B2 147 148 In one aspect of the birth control method, an amount of the Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metal compound Sufficient to block embryo implantation is admin loproteinase-2, Plasminogen Activator Inhibitor-1, Plasmi istered before or after intercourse and fertilization have nogen Activator Inhibitor-2, and various forms of the lighter occurred, thus providing an effective method of birth control, 'd group' transition metals. possibly a “morning after method. Albumin fusion proteins 5 Lighter "d group' transition metals include, for example, of the invention and/or polynucleotides encoding albumin Vanadium, molybdenum, tungsten, titanium, niobium, and fusion proteins of the invention may also be used in control tantalum species. Such transition metal species may form ling menstruation or administered as either a peritoneal lav transition metal complexes. Suitable complexes of the above age fluid or for peritoneal implantation in the treatment of mentioned transition metal species include OXO transition endometriosis. 10 metal complexes. Albumin fusion proteins of the invention and or polynucle Representative examples of Vanadium complexes include otides encoding albumin fusion proteins of the invention may OXO Vanadium complexes such as Vanadate and Vanadylcom be incorporated into Surgical Sutures in order to prevent Stitch plexes. Suitable Vanadate complexes include metavanadate granulomas. and orthovanadate complexes such as, for example, ammo Albumin fusion proteins of the invention and/or polynucle 15 nium metavanadate, sodium metavanadate, and sodium otides encoding albumin fusion proteins of the invention may orthovanadate. Suitable vanadyl complexes include, for be utilized in a wide variety of surgical procedures. For example, Vanadyl acetylacetonate and Vanadyl Sulfate includ example, within one aspect of the present invention a com ing vanadyl Sulfate hydrates Such as Vanadyl Sulfate mono positions (in the form of for example, a spray or film) may be and trihydrates. utilized to coat or spray an area prior to removal of a tumor, in Representative examples of tungsten and molybdenum order to isolate normal Surrounding tissues from malignant complexes also include oxo complexes. Suitable OXO tung tissue, and/or to prevent the spread of disease to Surrounding Sten complexes include tungstate and tungsten oxide com tissues. Within other aspects of the present invention, com plexes. Suitable tungstate complexes include ammonium positions (e.g. in the form of a spray) may be delivered via tungstate, calcium tungstate, Sodium tungstate dihydrate, and endoscopic procedures in order to coat tumors, or inhibit 25 tungstic acid. Suitable tungsten oxides include tungsten (IV) angiogenesis in a desired locale. Within yet other aspects of oxide and tungsten (VI) oxide. Suitable oxo molybdenum the present invention, Surgical meshes which have been complexes include molybdate, molybdenum oxide, and coated with anti-angiogenic compositions of the present molybdenyl complexes. Suitable molybdate complexes invention may be utilized in any procedure whereina Surgical include ammonium molybdate and its hydrates, Sodium mesh might be utilized. For example, within one embodiment 30 molybdate and its hydrates, and potassium molybdate and its of the invention a Surgical mesh laden with an anti-angiogenic hydrates. Suitable molybdenum oxides include molybdenum composition may be utilized during abdominal cancer resec (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suit tion Surgery (e.g. Subsequent to colon resection) in order to able molybdenyl complexes include, for example, molybde provide Support to the structure, and to release an amount of nyl acetylacetonate. Other Suitable tungsten and molybde the anti-angiogenic factor. 35 num complexes includehydroxo derivatives derived from, for Within further aspects of the present invention, methods example, glycerol, tartaric acid, and Sugars. are provided for treating tumor excision sites, comprising A wide variety of other anti-angiogenic factors may also be administering albumin fusion proteins of the invention and/or utilized within the context of the present invention. Repre polynucleotides encoding albumin fusion proteins of the sentative examples include platelet factor 4, protamine Sul invention to the resection margins of a tumor Subsequent to 40 phate: Sulphated chitin derivatives (prepared from queen crab excision, Such that the local recurrence of cancer and the shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sul formation of new blood vessels at the site is inhibited. Within phated Polysaccharide Peptidoglycan Complex (SP-PG) (the one embodiment of the invention, the anti-angiogenic com function of this compound may be enhanced by the presence pound is administered directly to the tumor excision site (e.g., of steroids such as estrogen, and tamoxifen citrate); Stauro applied by Swabbing, brushing or otherwise coating the resec 45 sporine; modulators of matrix metabolism, including for tion margins of the tumor with the anti-angiogenic com example, proline analogs, cishydroxyproline, d. L-3,4-dehy pound). Alternatively, the anti-angiogenic compounds may droproline. Thiaproline, alpha,alpha-dipyridyl, aminopropi be incorporated into known Surgical pastes prior to adminis onitrile fumarate, 4-propyl-5-(4-pyridinyl)-2(3H)-ox tration. Within particularly preferred embodiments of the azolone: Methotrexate; Mitoxantrone; Heparin; interferons: invention, the anti-angiogenic compounds are applied after 50 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. hepatic resections for malignancy, and after neuroSurgical Chem. 267:17321-17326, (1992)): Chymostatin (Tomkinson operations. et al. Biochem J. 286:475-480, (1992)): Cyclodextrin Tet Within one aspect of the present invention, fusion proteins radecasulfate; Eponemycin; Camptothecin, Fumagillin (Ing of the invention and/or polynucleotides encoding albumin ber et al. Nature 348:555-557, 1990): Gold Sodium Thioma fusion proteins of the invention may be administered to the 55 late (“GST': Matsubara and Ziff, J. Clin. Invest. 79:14-40 resection margin of a wide variety of tumors, including for 1446. (1987)): anticollagenase-serum; alpha2-antiplasmin example, breast, colon, brain and hepatic tumors. For (Holmes et al. J. Biol. Chem. 262(4): 1659-1664, (1987)); example, within one embodiment of the invention, anti-an Bisantrene (National Cancer Institute); Lobenzarit disodium giogenic compounds may be administered to the site of a (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or neurological tumor Subsequent to excision, such that the for 60 “CCA': Takeuchi et al. Agents Actions 36:312-316, (1992)); mation of new blood vessels at the site are inhibited. Thalidomide: Angostatic steroid; AGM-1470; carboxynami The albumin fusion proteins of the invention and/or poly nolmidazole; and metalloproteinase inhibitors such as BB94. nucleotides encoding albumin fusion proteins of the inven Diseases at the Cellular Level tion may also be administered along with other anti-angio Diseases associated with increased cell survival or the inhi genic factors. Representative examples of other anti 65 bition of apoptosis that could be treated, prevented, diag angiogenic factors include: Anti-Invasive Factor, retinoic nosed, and/or prognosed using fusion proteins of the inven acid and derivatives thereof, paclitaxel, Suramin, Tissue tion and/or polynucleotides encoding albuminfusion proteins US 8,946,156 B2 149 150 of the invention, include cancers (such as follicular lympho mia), graft V, host disease, ischemic injury (such as that mas, carcinomas with p53 mutations, and hormone-depen caused by myocardial infarction, stroke and reperfusion dent tumors, including, but not limited to colon cancer, car injury), liver injury (e.g., hepatitis related liver injury, diac tumors, pancreatic cancer, melanoma, retinoblastoma, ischemia/reperfusion injury, cholestosis (bile duct injury) and glioblastoma, lung cancer, intestinal cancer, testicular cancer, liver cancer); toxin-induced liver disease (such as that caused stomach cancer, neuroblastoma, myxoma, myoma, lym by alcohol), septic shock, cachexia and anorexia. phoma, endothelioma, osteoblastoma, osteoclastoma, Wound Healing and Epithelial Cell Proliferation osteosarcoma, chondrosarcoma, adenoma, breast cancer, In accordance with yet a further aspect of the present inven prostate cancer, Kaposi's sarcoma and ovarian cancer); tion, there is provided a process for utilizing fusion proteins autoimmune disorders (such as, multiple Sclerosis, Sjogren's 10 of the invention and/or polynucleotides encoding albumin syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Beh fusion proteins of the invention, for therapeutic purposes, for cet’s disease, Crohn's disease, polymyositis, systemic lupus example, to stimulate epithelial cell proliferation and basal erythematosus and immune-related glomerulonephritis and keratinocytes for the purpose of wound healing, and to stimu rheumatoid arthritis) and viral infections (such as herpes late hair follicle production and healing of dermal wounds. viruses, pox viruses and adenoviruses), inflammation, graft V, 15 Albumin fusion proteins of the invention and/or polynucle host disease, acute graft rejection, and chronic graft rejection. otides encoding albumin fusion proteins of the invention, may In preferred embodiments, fusion proteins of the invention be clinically useful in stimulating wound healing including and/or polynucleotides encoding albumin fusion proteins of Surgical wounds, excisional wounds, deep wounds involving the invention are used to inhibit growth, progression, and/or damage of the dermis and epidermis, eye tissue wounds, metasis of cancers, in particular those listed above. dental tissue wounds, oral cavity wounds, diabetic ulcers, Additional diseases or conditions associated with dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis increased cell survival that could be treated or detected by ulcers, burns resulting from heat exposure or chemicals, and fusion proteins of the invention and/or polynucleotides other abnormal wound healing conditions such as uremia, encoding albumin fusion proteins of the invention include, malnutrition, Vitamin deficiencies and complications associ but are not limited to, progression, and/or metastases of 25 ated with systemic treatment with steroids, radiation therapy malignancies and related disorders such as leukemia (includ and antineoplastic drugs and antimetabolites. Albumin fusion ing acute leukemias (e.g., acute lymphocytic leukemia, acute proteins of the invention and/or polynucleotides encoding myelocytic leukemia (including myeloblastic, promyelo albumin fusion proteins of the invention, could be used to cytic, myelomonocytic, monocytic, and erythroleukemia)) promote dermal reestablishment Subsequent to dermal loss and chronic leukemias (e.g., chronic myelocytic (granulo 30 Albumin fusion proteins of the invention and/or polynucle cytic) leukemia and chronic lymphocytic leukemia)), poly otides encoding albumin fusion proteins of the invention, cythemia vera, lymphomas (e.g., Hodgkin’s disease and non could be used to increase the adherence of skin grafts to a Hodgkin’s disease), multiple myeloma, Waldenstrom's wound bed and to stimulate re-epithelialization from the macroglobulinemia, heavy chain disease, and Solid tumors wound bed. The following are types of grafts that fusion including, but not limited to, sarcomas and carcinomas Such 35 proteins of the invention and or polynucleotides encoding as fibrosarcoma, myxosarcoma liposarcoma, chondrosar albumin fusion proteins of the invention, could be used to coma, osteogenic sarcoma, chordoma, angiosarcoma, endot increase adherence to a wound bed; autografts, artificial skin, heliosarcoma, lymphangiosarcoma, lymphangioendothe allografts, autodermic graft, autoepdermic grafts, avacular liosarcoma, synovioma, mesothelioma, Ewings tumor, grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, 40 cutis graft, delayed graft, dermic graft, epidermic graft, fascia pancreatic cancer, breast cancer, ovarian cancer, prostate can graft, full thickness graft, heterologous graft, Xenograft, cer, Squamous cell carcinoma, basal cell carcinoma, adeno homologous graft, hyperplastic graft, lamellar graft, mesh carcinoma, Sweat gland carcinoma, sebaceous gland carci graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, noma, papillary carcinoma, papillary adenocarcinomas, patch graft, pedicle graft, penetrating graft, split skin graft, cystadenocarcinoma, medullary carcinoma, bronchogenic 45 thick split graft. Albumin fusion proteins of the invention carcinoma, renal cell carcinoma, hepatoma, bile duct carci and/or polynucleotides encoding albumin fusion proteins of noma, choriocarcinoma, seminoma, embryonal carcinoma, the invention, can be used to promote skin strength and to Wilm's tumor, cervical cancer, testicular tumor, lung carci improve the appearance of aged skin. noma, Small cell lung carcinoma, bladder carcinoma, epithe It is believed that fusion proteins of the invention and/or lial carcinoma, glioma, astrocytoma, medulloblastoma, cran 50 polynucleotides encoding albumin fusion proteins of the iopharyngioma, ependymoma, pinealoma, invention, will also produce changes in hepatocyte prolifera hemangioblastoma, acoustic neuroma, oligodendroglioma, tion, and epithelial cell proliferation in the lung, breast, pan menangioma, melanoma, neuroblastoma, and retinoblas creas, stomach, Small intestine, and large intestine. Albumin tOma. fusion proteins of the invention and/or polynucleotides Diseases associated with increased apoptosis that could be 55 encoding albumin fusion proteins of the invention, could treated, prevented, diagnosed, and, or prognosed using fusion promote proliferation of epithelial cells such as Sebocytes, proteins of the invention and/or polynucleotides encoding hair follicles, hepatocytes, type II pneumocytes, mucin-pro albumin fusion proteins of the invention, include, but are not ducing goblet cells, and other epithelial cells and their pro limited to, AIDS, neurodegenerative disorders (such as genitors contained within the skin, lung, liver, and gas Alzheimer's disease, Parkinson's disease, Amyotrophic lat 60 trointestinal tract. Albumin fusion proteins of the invention eral Sclerosis, Retinitis pigmentosa, Cerebellar degeneration and/or polynucleotides encoding albumin fusion proteins of and brain tumor or prior associated disease); autoimmune the invention, may promote proliferation of endothelial cells, disorders (such as, multiple Sclerosis, Sjogren's syndrome, keratinocytes, and basal keratinocytes. Hashimoto's thyroiditis, biliary cirrhosis, Behcet’s disease, Albumin fusion proteins of the invention and/or polynucle Crohn's disease, polymyositis, systemic lupus erythematosus 65 otides encoding albumin fusion proteins of the invention, and immune-related glomerulonephritis and rheumatoid could also be used to reduce the side effects of guttoxicity that arthritis) myelodysplastic syndromes (such as aplastic ane result from radiation, chemotherapy treatments or viral infec US 8,946,156 B2 151 152 tions. Albumin fusion proteins of the invention and/or poly and toxic Substances (i.e., acetaminophen, carbon tetrachlo nucleotides encoding albumin fusion proteins of the inven ride and other hepatotoxins known in the art). tion, may have a cytoprotective effect on the Small intestine In addition, fusion proteins of the invention and/or poly mucosa. Albumin fusion proteins of the invention and/or nucleotides encoding albumin fusion proteins of the inven polynucleotides encoding albumin fusion proteins of the tion, could be used treat or prevent the onset of diabetes invention, may also stimulate healing of mucositis (mouth mellitus. In patients with newly diagnosed Types I and II ulcers) that result from chemotherapy and viral infections. diabetes, where some islet cell function remains, fusion pro Albumin fusion proteins of the invention and/or polynucle teins of the invention and/or polynucleotides encoding albu otides encoding albumin fusion proteins of the invention, min fusion proteins of the invention, could be used to main 10 tain the islet function so as to alleviate, delay or prevent could further be used in full regeneration of skin in full and permanent manifestation of the disease. Also, fusion proteins partial thickness skin defects, including burns, (i.e. repopu of the invention and/or polynucleotides encoding albumin lation of hair follicles, Sweat glands, and sebaceous glands), fusion proteins of the invention, could be used as an auxiliary treatment of other skin defects Such as psoriasis. Albumin in islet cell transplantation to improve or promote islet cell fusion proteins of the invention and or polynucleotides 15 function. encoding albumin fusion proteins of the invention, could be Neural Activity and Neurological Diseases used to treat epidermolysis bullosa, a defect in adherence of The albumin fusion proteins of the invention and/or poly the epidermis to the underlying dermis which results in fre nucleotides encoding albumin fusion proteins of the inven quent, open and painful blisters by accelerating reepithelial tion may be used for the diagnosis and or treatment of dis ization of these lesions. Albumin fusion proteins of the inven eases, disorders, damage or injury of the brain and/or nervous tion and/or polynucleotides encoding albuminfusion proteins system. Nervous system disorders that can be treated with the of the invention, could also be used to treat gastric and duode compositions of the invention (e.g. fusion proteins of the nal ulcers and help heal by Scar formation of the mucosal invention and/or polynucleotides encoding albumin fusion lining and regeneration of glandular mucosa and duodenal proteins of the invention), include, but are not limited to, mucosal lining more rapidly. Inflammatory bowel diseases, 25 nervous system injuries, and diseases or disorders which Such as Crohn's disease and ulcerative colitis, are diseases result in either a disconnection of axons, a diminution or which result in destruction of the mucosal surface of the small degeneration of neurons, or demyelination. Nervous system or large intestine, respectively. Thus, fusion proteins of the lesions which may be treated in a patient (including human invention and/or polynucleotides encoding albumin fusion and non-human mammalian patients) according to the meth proteins of the invention, could be used to promote the resur 30 ods of the invention, include but are not limited to, the fol facing of the mucosal Surface to aid more rapid healing and to lowing lesions of either the central (including spinal cord, prevent progression of inflammatory bowel disease. Treat brain) or peripheral nervous systems: (1) ischemic lesions, in ment with fusion proteins of the invention and/or polynucle which a lack of oxygen in a portion of the nervous system otides encoding albumin fusion proteins of the invention, is results in neuronal injury or death, including cerebral infarc expected to have a significant effect on the production of 35 tion or ischemia, or spinal cord infarction or ischemia; (2) mucus throughout the gastrointestinal tract and could be used traumatic lesions, including lesions caused by physical injury to protect the intestinal mucosa from injurious Substances that or associated with Surgery, for example, lesions which severa are ingested or following Surgery. Albumin fusion proteins of portion of the nervous system, or compression injuries; (3) the invention and/or polynucleotides encoding albumin malignantlesions, in which a portion of the nervous system is fusion proteins of the invention, could be used to treat dis 40 destroyed or injured by malignant tissue which is either a eases associate with the under expression. nervous system associated malignancy or a malignancy Moreover, fusion proteins of the invention and/or poly derived from non-nervous system tissue; (4) infectious nucleotides encoding albumin fusion proteins of the inven lesions, in which a portion of the nervous system is destroyed tion, could be used to prevent and heal damage to the lungs or injured as a result of infection, for example, by an abscess due to various pathological states. Albumin fusion proteins of 45 or associated with infection by human immunodeficiency the invention and/or polynucleotides encoding albumin virus, herpes Zoster, or herpes simplex virus or with Lyme fusion proteins of the invention, which could stimulate pro disease, tuberculosis, or syphilis; (5) degenerative lesions, in liferation and differentiation and promote the repair of alveoli which a portion of the nervous system is destroyed or injured and bronchiolar epithelium to preventor treat acute or chronic as a result of a degenerative process including but not limited lung damage. For example, emphysema, which results in the 50 to degeneration associated with Parkinson's disease, Alzhe progressive loss of alveoli, and inhalation injuries, i.e., result imer's disease, Huntington's chorea, or amyotrophic lateral ing from Smoke inhalation and burns, that cause necrosis of sclerosis (ALS); (6) lesions associated with nutritional dis the bronchiolar epithelium and alveoli could be effectively eases or disorders, in which a portion of the nervous system is treated using polynucleotides or polypeptides, agonists or destroyed or injured by a nutritional disorder or disorder of antagonists of the present invention. Also fusion proteins of 55 metabolism including, but not limited to vitamin B12 defi the invention and/or polynucleotides encoding albumin ciency, folic acid deficiency, Wernicke disease, tobacco-alco fusion proteins of the invention, could be used to stimulate the hol amblyopia, Marchiafava-Bignami disease (primary proliferation of and differentiation of type II pneumocytes, degeneration of the corpus callosum), and alcoholic cerebel which may help treat or prevent disease Such as hyaline mem lar degeneration; (7) neurological lesions associated with brane diseases, such as infant respiratory distress syndrome 60 systemic diseases including, but not limited to diabetes (dia and bronchopulmonary displasia, in premature infants. betic neuropathy, Bell's palsy). Systemic lupus erythemato Albumin fusion proteins of the invention and/or polynucle Sus, carcinoma, or sarcoidosis; (8) lesions caused by toxic otides encoding albumin fusion proteins of the invention, Substances including alcohol, lead, or particular neurotoxins: could stimulate the proliferation and differentiation of hepa and (9) demyelinated lesions in which a portion of the ner tocytes and, thus, could be used to alleviate or treat liver 65 Vous system is destroyed or injured by a demyelinating dis diseases and pathologies such as fulminant liver failure ease including, but not limited to multiple Sclerosis, human caused by cirrhosis, liver damage caused by viral hepatitis immunodeficiency virus-associated myelopathy, transverse US 8,946,156 B2 153 154 myelopathy or various etiologies, progressive multifocal leu In specific embodiments, motor neuron disorders that may koencephalopathy, and central pontine myelinolysis. be treated according to the invention include, but are not In one embodiment, the albumin fusion proteins of the limited to, disorders such as infarction, infection, exposure to invention and or polynucleotides encoding albumin fusion toxin, trauma, Surgical damage, degenerative disease or proteins of the invention are used to protect neural cells from malignancy that may affect motor neurons as well as other the damaging effects of hypoxia. In a further preferred components of the nervous system, as well as disorders that embodiment, the albumin fusion proteins of the invention selectively affect neurons such as amyotrophic lateral sclero and/or polynucleotides encoding albumin fusion proteins of sis, and including, but not limited to, progressive spinal mus the invention are used to protect neural cells from the dam cular atrophy, progressive bulbar palsy, primary lateral scle aging effects of cerebral hypoxia. According to this embodi 10 rosis, infantile and juvenile muscular atrophy, progressive ment, the compositions of the invention are used to treat or bulbar paralysis of childhood (Fazio-Londe syndrome), prevent neural cell injury associated with cerebral hypoxia. In poliomyelitis and the post polio syndrome, and Hereditary one non-exclusive aspect of this embodiment, the albumin Motorsensory Neuropathy (Charcot-Marie-Tooth Disease). fusion proteins of the invention and/or polynucleotides Further, fusion proteins of the invention and/or polynucle encoding albumin fusion proteins of the invention, are used to 15 otides encoding albumin fusion proteins of the invention may treat or prevent neural cell injury associated with cerebral play a role in neuronal Survival; synapse formation; conduc ischemia. In another non-exclusive aspect of this embodi tance; neural differentiation, etc. Thus, compositions of the ment, the albumin fusion proteins of the invention and/or invention (including fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neural cell injury asso invention) may be used to diagnose and/or treat or prevent ciated with cerebral infarction. diseases or disorders associated with these roles, including, In another preferred embodiment, albumin fusion proteins but not limited to, learning and/or cognition disorders. The of the invention and/or polynucleotides encoding albumin compositions of the invention may also be useful in the treat fusion proteins of the invention are used to treat or prevent ment or prevention of neurodegenerative disease states and/or neural cell injury associated with a stroke. In a specific 25 behavioural disorders. Such neurodegenerative disease states embodiment, albumin fusion proteins of the invention and/or and/or behavioral disorders include, but are not limited to, polynucleotides encoding albumin fusion proteins of the Alzheimer's Disease, Parkinson's Disease, Huntington's invention are used to treat or prevent cerebral neural cell Disease, Tourette Syndrome, Schizophrenia, mania, demen injury associated with a stroke. tia, paranoia, obsessive compulsive disorder, panic disorder, In another preferred embodiment, albumin fusion proteins 30 learning disabilities, ALS, psychoses, autism, and altered of the invention and/or polynucleotides encoding albumin behaviors, including disorders in feeding, sleep patterns, bal fusion proteins of the invention are used to treat or prevent ance, and perception. In addition, compositions of the inven neural cell injury associated with a heart attack. In a specific tion may also play a role in the treatment, prevention and/or embodiment, albumin fusion proteins of the invention and/or detection of developmental disorders associated with the polynucleotides encoding albumin fusion proteins of the 35 developing embryo, or sexually-linked disorders. invention are used to treat or prevent cerebral neural cell Additionally, fusion proteins of the invention and or poly injury associated with a heart attack. nucleotides encoding albumin fusion proteins of the inven The compositions of the invention which are useful for tion, may be useful in protecting neural cells from diseases, treating or preventing a nervous system disorder may be damage, disorders, or injury, associated with cerebrovascular selected by testing for biological activity in promoting the 40 disorders including, but not limited to carotid artery diseases survival or differentiation of neurons. For example, and not (e.g. carotid artery thrombosis, carotid Stenosis, or by way of limitation, compositions of the invention which Moyamoya Disease), cerebral amyloid angiopathy, cerebral elicit any of the following effects may be useful according to aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral the invention: (1) increased Survival time of neurons in cul arteriovenous malformations, cerebral artery diseases, cere ture either in the presence or absence of hypoxia or hypoxic 45 bral embolism and thrombosis (e.g. carotid artery thrombo conditions: (2) increased sprouting of neurons in culture or in sis, sinus thrombosis, or Wallenberg's Syndrome), cerebral Vivo: (3) increased production of a neuron-associated mol hemorrhage (e.g. epidural or Subdural hematoma, or Sub ecule in culture or in vivo, e.g. choline acetyltransferase or arachnoid hemorrhage), cerebral infarction, cerebral acetylcholinesterase with respect to motor neurons; or (4) ischemia (e.g. transient cerebral ischemia, Subclavian Steal decreased symptoms of neuron dysfunction in vivo. Such 50 Syndrome, or vertebrobasilar insufficiency), vascular demen effects may be measured by any method known in the art. In tia (e.g. multi-infarct), leukomalacia, periventricular, and preferred, non-limiting embodiments, increased Survival of vascular headache (e.g. cluster headache or migraines). neurons may routinely be measured using a method set forth In accordance with yet a further aspect of the present inven herein or otherwise known in the art, Such as, for example, in tion, there is provided a process for utilizing fusion proteins Zhanget al. Proc Natl AcadSci USA 97:3637-42 (2000) or in 55 of the invention and/or polynucleotides encoding albumin Arakawa et al. J. Neurosci. 10:3507-15 (1990): increased fusion proteins of the invention, for therapeutic purposes, for sprouting of neurons may be detected by methods known in example, to stimulate neurological cell proliferation and/or the art, such as, for example, the methods set forth in Pestronk differentiation. Therefore, fusion proteins of the invention et al., Exp. Neurol.., 70:65-82 (1980), or Brown etal. Ann. Rev. and/or polynucleotides encoding albumin fusion proteins of Neurosci. 4:17-42 (1981); increased production of neuron 60 the invention may be used to treat and/or detect neurologic associated molecules may be measured by bioassay, enzy diseases. Moreover, fusion proteins of the invention and/or matic assay, antibody binding, Northern blot assay, etc, using polynucleotides encoding albumin fusion proteins of the techniques known in the art and depending on the molecule to invention, can be used as a marker or detector of a particular be measured: and motor neuron dysfunction may be mea nervous system disease or disorder. Sured by assessing the physical manifestation of motor neu 65 Examples of neurologic diseases which can be treated or ron disorder, e.g. weakness, motor neuron conduction Veloc detected with fusion proteins of the invention and/or poly ity, or functional disability. nucleotides encoding albumin fusion proteins of the inven