CA2405525A1 - Albumin fusion proteins - Google Patents

Abstract

The present invention encompasses albumin fusion proteins. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of maki ng the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells. Additionally the present invention encompasses pharmaceutical compositions comprising albumin fusion proteins and methods o f treating, preventing, or ameliorating diseases, disorders or conditions usin g albumin fusion proteins of the invention.

Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

~~ TTENANT LES PAGES 1 A 223 NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
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NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
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ALBUMIN FUSION PROTEINS

BACKGROUND OF THE INVENTION
The invention relates generally to Therapeutic-proteins (including, but not limited to, a polypeptide, antibody, or peptide, or fragments and variants thereof) fused to albumin or fragments or variants of albumin. The invention further relates to Therapeutic proteins , (including, but not limited to, a,polypeptide, antibody, or peptide, or fragments and variants .thereof) fused to albumin or fragments or variants. of albumin, that exhibit extended shelf life and/or extended or therapeutic activity in solution. These fusion proteins are herein collecfively referred to as "albumin fusion .proteins of the invention." The 'invention ' encompasses therapeutic .albumin fusion proteins, compositions, pharmaceutical compositions, formulations _ and kits. Nucleic , acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these .nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention using these nucleic acids, vectors, andlor host.cells.
The invention is also directed to rriethods of in vitro stabilizing a Therapeutic protein via fusion or conjugation of the Therapeutic protein to albumin or fragments ~or variants of . albumin.
Human serum albumin (HSA, or HA), a protein of 585 amino acids in its mature.
form (as shown in Figure IS or in SEQ~ ID N0:18), is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. At present, HA for clinical use is produced by extraction from human blood. The production of recombinant HA (rHA) in microorganisms has been disclosed in EP

and EP 361 991. .
' The role of albumin as a carrier molecule and its inert nature are desirable properties for use as a earner and transporter of polypeptides in vivo. The use of albumin as a ' component of an albumin fusion protein as a carrier for various proteins. has been suggested .
in WO' 93115199, WO 93/,15200, and EP 413 622. The use of N-terminal fragments of HA

Therapeutic protein may be achieved by genetic manipulation, such that the DNA, coding-for HA, or a. fragment thereof, is joined to the DNA coding for the Therapeutic protein. A
.suitable host is then transformed or transfected with the fused nucleotide sequences, so arranged on a suitable plasmid as to. express a fusion ~polypeptide. The expression may be effected in vitro from, for example, prokaryotic or eukaryotic cells, or in vivo e.g. from a transgenic organism.
. Therapeutic proteins 'in their native state or when recombinantly produced, such as interferons and growth hormones, are typically labile molecules exhibiting short shelf lives, particularly when formulated in aqueous solutions. The instability in these molecules when formulated for administration dictates that many of the molecules must be lyophilized and refrigerated at all times during storage, thereby rendering the molecules difficult to transport.
and/or store. Storage problems are particularly acute when pharmaceutical formulations must be stored and~dispensed outside of the hospital environment., Many protein and peptide drugs also require the addition of high concentrations of other protein such as albumin to reduce or prevent loss of protein due to binding to the container. This is a major concern with respect to proteins such as IFN. For this reason, many Therapeutic proteins are formulated in combination with large proportion of albumin carrier molecule (100-1000 fold excess), though this is an undesirable and expensive feature of the formulation. .
Few practical solutions to the storage ,problems of 'labile protein molecules have been proposed., Accordingly, there is _a need for stabilized, long lasting formulations of proteinaceous therapeutic molecules that are easily dispensed, preferably with a simple formulation requiring minimal post-storage manipulation.
SUMMARY OF THE INVENTION
- The present invention is based, in part, on the discovery that Therapeutic proteins may be stabilized to extend the shelf life, and%or to retain the Therapeutic protein's activity for extended periods of time in solution, in vitro and/or in vivo, by genetically or chemically fusing or conjugating the Therapeutic protein to albumin or a fragment (portion) ~or variant of albumin, that is sufficient to stabilize the protein and/or its activity. In addition it has been determined that the use of albumin-fusion proteins or albumin conjugated proteins may reduce the need 'to formulate protein solutions with large excesses of carrier proteins , (such as 'albumin, unfused) to prevent loss of Therapeutic proteins due to factors such as binding to the container.
,.
The present invention encompasses albumin fusion proteins comprising a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof) fused to albumin of a fragment (portion) or variant of albumin.. The present invention also encompasses albumin fusion proteins comprising a Therapeutic protein~(e.g., 'a polypeptide, antibody, or peptide, or fragments and variants thereof) fused. to albumin ,or a fragment (portion) or variant of albumin, that is sufficient to prolong the shelf life of the Therapeutic protein, and/or stabilize the Therapeutic protein andlor its activity in solution (or in a pharmaceutical composition) in vitro and/or in vivo. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids, vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells. . ' .
The invention also encompasses pharmaceutical formulations comprising an albumin ~ fusion protein of the invention and a pharmaceutically acceptable diluent or carrier. Such formulations may be in a kit or container. , Such kit, or container may be packaged with instructions pertaining to the extended shelf life of the Therapeutic protein.
Such formulations may be used in methods of treating,-.preventing, ameliotationg ar diagnosing a disease or disease symptom in a patient, preferably a mammal, most preferably a human, comprising the ' step of administering the pharmaceutical formulation to the patient.
. In other embodiments, the present invention encompasses methods of preventing treating, or ameliorating a disease or disorder. In preferred embodiments; .
the present invention encompasses, a method of treating ~a disease or disorder listed in the "Preferred .
Indication Y"- column of Table ,1 comprising administering to a patient ~in which such treatment, prevention or amelioration is desired an albumin fusion protein of the invention that comprises a Therapeutic protein portion corresponding to a Therapeutic protein (or fragment , or variant thereof) disclosed in the "Therapeutic Protein X" column of Table 1 (in the same row as the disease or disorder to be treated is listed in the "Preferred Indication Y" column of , Table 1) -in an amount effective to treat prevent or ameliorate the disease or disorder.
- In another embodiment, the invention includes a method of extending the shelf life of a Therapeutic protein (e.g:, a polypeptide, antibody, or peptide, or fragments and variants thereof) comprising the step of fusing or conjugating the Therapeutic protein to albumin or a fragment (portion) or variant of albumin, that, is sufficient to extend the shelf-life of the . Therapeutic protein. In a preferred embodiment, the Therapeutic_ protein used according to .
this method is fused to the albumin, or the -fragment or .variant of albumin.
In a most preferred embodiment, the Therapeutic protein used according to this method is fused to albumin, or a fragment or variant of albumin, via recombinant DNA technology or genetic engineering.
In another embodiment, the invention includes a method of stabilizing a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof) in solution, comprising the step of.fusing or conjugating the Therapeutic protein to albumin or a fragment (portion) or variant of albumin, that is sufficient to stabilize the Therapeutic protein.

WO 01/79271 PCT/iJS01/12009 In a preferred embodiment,-the Therapeutic protein used according to this method is fused to .
the albumin, or the fragment or variant of .albumin. In, a most preferred embodiment,, the Therapeutic protein used according to this method is fused to albumin, or a fragment . or variant of albumin, via recombinant DNA technology or genetic engineering.
The present invention further includes transgenic organisms modified-to contain the nucleic acid molecules of the invention, preferably modified to express the albumin fusion proteins encoded by the nucleic acid molecules.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 depicts the extended shelf life of an HA fusion protein in terms of the biological activity (Nb2 cell proliferation) of HA-hGH remaining after incubation in cell culture media for up to 5 weeks at 37°C. Under these conditions, hGH
has no observed activity by week 2. ' . -Figure 2 depicts the extended shelf life of an HA fusion protein in terms of the stable biological activity (Nb2 cell proliferation) of ~ HA-hGH remaining after .
incubation in cell culture media for up to 3 weeks at ~4, 37, or 50°C. Data is normalized to the biological activity of hGH at time zero. , Figures 3A and 3B compare the biological activity of HA-hGH .with hGH in the Nb2 cell proliferation assay. Figure 3A shows proliferation after 24 hours of incubation with various concentrations. of hGH . or the albumin fusion protein, and Figure 3B
shows proliferation after 48 hours of incubation with various concentrations of hGH
or the albumin fusion protein.
Figure 4 shows a map of a plasmid (pPPC0005) that can be used as the base vector into which polynucleotides encoding the Therapeutic proteins (including polypeptides and fragments and variants thereof) may ,be cloned to form HA-~usions. Plasmid Map-key: , PRBIp: PRBI S. cerevisiae promoter; FL: Fusion leader sequence; rHA: cDNA
encoding HA: ADHlt: ADHI S. cerevisiae terminator; T3: T3 sequencing primer site; T7:

sequencing primer site; Amp R: (3-lactamase gene; ori: origin of replication.
Please note that in the provisional applications to which this application claims priority,-the plasmid in Figure 4 was labeled pPPC0006, instead of pPPC0005. In addition the drawing of this plasmid~did not show certain pertinent restriction sites in this vector. Thus in the present application, the drawing is labeled pPPC0005 and more,iestriction sites of the same vector: are shown.
Figure 5 compares the recovery of vial-stored HA-IFN solutions of various concentrations with a stock' solution. after 48. or 72 hours of storage.
~ Figure 6 compares the activity. of an HA-a.-IFN fusion protein after administration to monkeys via IV or SC:

Figure 7 describes the bioavailability and stability of an HA-cc-IFN fusion protein.
_ Figure 8 is a map of an expression vector for the production of HA-a.-1FN.
Figure 9 shows the location of loops in HA.
Figure 10 is ~an example of the modification of an HA loop.
Figure l I is a representation of the HA loops.
. ~ Figure 12 shows the HA loop IV.
Figure 13 show's the tertiary structure of HA.
Figure ~14 shows an example of a scFv-HA fusion Figure 15 shows the amino acid sequence of the mature form of human albumin (SEQ
ID N0:18) and a polynucleotide encoding it (SEQ ID N0:17). .
DETAILED DESCRIPTION
As described above, the present invention is based, in part, on the discovery that a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants . thereof) may be stabilized to extend the shelf-life and/or retain the Therapeutic protein's activity for extended periods of time in solution (or in a pharmaceutical composition) in vitro andlor in vivo, by genetically, fusing or chemically conjugating the Therapeutic protein, polypeptide or peptide to all or a portion of albumin sufFcient to stabilize the protein and its activity. ' ~ ~ ~ .
The present invention relates generally to albumin fusion proteins and methods of treating, preventing, or ameliorating diseases or disorders. ~ As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a Therapeutic protein (or fragment or variant thereof). An albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumi~i, , which are associated with one-another, preferably by genetic fusion (i.e., the albumin fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a ' portion of a Therapeutic protein. is joined in-frame with a polynucleotide encoding all or a .portion of albumin) or chemical conjugation to one . another. The Therapeutic protein and albumizi protein, once part of the albumin fusion protein, may be. referred to as a "portion", "region"'or "moiety" of the albumin fusion protein (e.g., a "Therapeutic protein portion" or an "albumin protein portion"). ' .
In one 'embodiment, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein (e.g:, as described in Table _1) and a serum albumin protein: In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment of a Therapeutic protein and a serum 'albumin protein. In other embodiments,..the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active 'variant of a Therapeutic protein and a serum albumin protein. In preferred embodiments, the serum albumin protein component of the albumin fusion protein is the mature portion of serum albumin. -In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein, and a ,biologically active and/or therapeutically active fragment of serum albumin. In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of;
a Therapeutic protein and a- biologically active and/or therapeutically active variant of serum albumin. In preferred embodiments, the Therapeutic protein portion of the albumin fusion.
protein is the mature portion of the Therapeutic protein. In a further preferred embodiment, the Therapeutic protein portion of the albumin fusion- protein is the extracellular soluble domain of the Therapeutic protein. In an alternative embodiment, the Therapeutic protein -portion of the albumin fusion protein is the active form of the Therapeutic protien. - .
In further embodiments, the invention provides an. albumin fusion,protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment or variant of a Therapeutic protein and a biologically active and/or therapeutically active fragment - or variant of serum albumin. In preferred embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, the mature portion of a Therapeutic protein and the mature portion of serum alburriin.
Therapeutic proteins , . - .
As stated above, an albumin fusion protein of the invention comprises at least a . fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with ,oiie another, preferably by genetic fusion or chemical conjugation.
' As used herein; "Therapeutic protein" refers to proteins, polypeptides, antibodies, peptides or fragments or variants thereof, having one or more therapeutic andJor biological activities. Therapeutic proteins encompassed by the invention include but are not limited to, proteins, polype~tides, peptides, antibodies, and biologics. (The terms peptides, proteins, and polypeptides are used interchangeably herein.) It is. specifically contemplated that the term "Therapeutic protein" encompasses antibodies and fragments and variants thereof. Thus an albumin fusion protein of the invention may contain at least a fragment or variant of a Therapeutic protein, and/or at least a fragment or variant of an antibody.
Additionally, the term "Therapeutic protein" may refer to the endogenous or naturally occurring correlate of a Therapeutic protein. ~ -By a polypeptide displaying a "therapeutic activity" or a protein that is~
"therapeutically WO 01/79271 PCT/iTS01/12009 active" is meant a polypeptide that possesses one or more known biological and/or therapeutic activities associated with a Therapeutic protein such as one or more of the Therapeutic proteins described herein or otherwise known in the art. As a non-limiting example, a "Therapeutic protein" ~is a protein that is , useful to' treat, prevent or ameliorate a . disease, condition or disorder. As a non-limiting example, a "Therapeutic protein" may be one that binds specifically to a particular cell type (normal (e.g., lymphocytes) or abnormal e.g., (cancer cells)) and therefore may be used to target a compound (drug, or cytotoxic agent) to.
' that cell type specifically. ~ ' ' In another non-limiting example, a "Therapeutic protein" is a protein that has a biological activity, and in particular, a biological activity that is useful for treating preventing or ameliorating a disease. A non-inclusive list of biological activities that~may be possessed by a Therapeutic protein includes, enhancing the immune response, promoting angiogenesis, inhibiting angiogenesis, regulating hematopoietic functions, stimulating -nerve growth, enhancing an immune response, inhibiting an immune response, or any one or more of the biological activities described in the "Biological Activities" section below.
. ~ As used herein, "therapeutic activity" or "activity" may refer to an activity whose effect is consistent with a desirable therapeutic outcome in huinaris, or to desired effects in non-human mammals or in other species or organisms. Therapeutic activity may be measured . in vivo or in vitro. For example, ~a desirable effect may be assayed in cell culture. As an . example, when hGH is the Therapeutic protein, the effects of hGH on cell proliferation as described in Example 1 may be used as the endpoint for which therapeutic activity is measured.. Such in vitro or cell culture assays are commonly available for many Therapeutic proteins as described in tl~e art. ' . ~ , . Examples of useful ~ assays for particular Therapeutic proteins include, but 'are not limited to, GMCSF'(Eaves, A.C. and Eaves C.J., Erythropoiesis in culture. In:
McCullock EA (edt) Cell culture techniques - Clinics in hematology. WB Saunders, Eastbourne, pp 371 91 (1984); Metcalf,. D., International Journal of Cell Cloning 10: 116-25 (1992); Testa, N.G., et al., Assays for hematopoietic growth factors. In: Balkwill FR (edt) Cytokines, A
practical Approach, pp 229-44; IRL Press Oxford 1991) EPO (bioassay: Kitamura et al., J.
CeII. Physiol.. 140 p323 (1989)); Hirudin (platelet aggregation assay: Blood Coagul Fibrinolysis 7(2):259-61 (1996)); IFNa (anti-viral ; assay: Rubinstein et.
al., J. Virol.
37(2):755-8 (1981); anti-proliferative assay: Gao Y, et al Mol .Cell Biol.
19(11):7305-13 (1999); and bioassay: Czarniecki et al., J. Virol. 49 p490 (1984)); GCSF
(bioassay: Shirafuji et al., EXp. Hematol. 17 p116 (1989); proliferation of murine NFS-60 cells (Weinstein et.al, . 35 Proc Natl Acad Sci 83:5010-4 (19$6)); insulin (3H-glucose uptake assay:
Steppan'et al., Nature 409(6818):307-12 (2001)); hGH (BalF3-hGHR proliferation assay: J Clin Endocrinol Metab 85(11):4274-9 (2000); International standard for growth hormone: ~Horm Res, .51 Suppl 1:7-I2 (1999)); factor X (factor X activity assay: Van Wijk et al.
.Thromb Res 22:681-686 (1981)); factor VII (coagulation assay using prothrombin clotting time:
Belaaouaj et al., J. Biol: Chem. 275:27123-8(2000); Diaz-Collier et al., Thromb Haemost 71:339-46 (1994)), . or as shown in Table 1 in the "Exemplary Activity Assay" column.
. Therapeutic proteins corresponding to, a Therapeutic protein portion of ari albumin fusion protein of the invention, such as cell surface and secretory proteins, are often modified by the attachment of one or more oligosaccharide groups. The modification, referred to as glycosylation, .can dramatically affect the physical properties of proteins and can be important in protein stability, secretion, and localization. Glycosyiadon occurs at specific locations along the polypeptide backbone. There are usually two major types of glycosylation:
glycosylation characterized by O-linked oligosaccharides, which are attached to serine or threonine residues; and glycosylation characterized by N-linked oligosaccharides; ,which are attached to asparagine residues in an Asn-X-Ser/Thr sequence, where X can be any amino acid except proline.~ N-acetylneuramic acid (also-known as sialic acid) is usually the terminal .
i5 residue of both N-linked and 0-linked oligosaccharides. Variables such as protein structure and cell type influence the number and nature of the carbohydrate units within the chains at different glycosylation sites. Glycosylation isomers are also comyon at the same site within a given cell type.
For example', several types of human interferon are glycosylated. Natural human interferon-a2 is O-glycosylated at threonine 106, and N-glycosylation occurs at asparagine 72 in interferon-a14 (Adolf et al., J. Biochem 276:511 (1991); Nyman TA et cal., J. Biochem 329:295 (1998)). The oligosaccharides at asparagine 80 in natural interferon-(31a may play an important factor in the solubility and stability of the protein, but may not be essential for its ' biological activity. This permits the production of an unglycosylated analog (interferon-~ilb) engineered. with sequence modifications to enhance stability (Hosoi et al., J.
Interferon Res.

8:375 (1988; Karpusas et al., Cell Mol Life Sci 54:1203 (1998); Knight, J.
Interferon Res.
2:42I~ (1982); Runkei ~et cal., Pharm Res 15:641 (1998); Lin, Dev. Biol..
Stand. 96:97 (1998))1. Interferon-'y contains two N-linked oligosaccharide chains at positions 25 and 97;
both important for the efficient formation of the bioactive recombinant protein, and having an .
.30 -influence on the pharmacokinetic properties of the protein (Sareneva et cal., Eur. J. Biochem 242:191 (1996); Sareneva et al,. Biochem J. 3.03:831 (1994); Sareneva et al., J. Interferon Res. 13:267 (1993)). Mixed O-linked and N-linked glycosylation also, occurs, for example in human erythropoietin, N-linked glycosylation occurs at ~ asparagine residues located at positions 24, 38 and 83 while O-linked glycosylation occurs at a~ serine residue located at position 126 (Lai et al., .J. Biol. Chem. 261:3116 (1986); Broudy et al., Arch. Biochem.
Biophys. 265:329 (1988)). ~ ' Therapeutic proteins corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, as well as analogs and variants thereof,, may be modified so that glycosylation at one or more sites is altered as a result of manipulations) of their nucleic acid sequence, by the host cell in which they are expressed, or due to other conditions of their expression. For example, glycosylation isomers may be produced by abolishing or introducing glycosylation sites, e.g., by substitution or deletion of amino acid residues, such as substitution of glutamine for asparagine, or~unglycosylated recombinant proteins may be ,produced by expressing the proteins in host cells that will not gIycosylate them, e.g. in E. coli or glycosylation-deficient yeast. These approaches are described in more detail below and' are known in the art. . , ' Therapeutic proteins corresponding to a Therapeutic protein portion of an albumin fusion protein of the- invention include, but are not limited to, . plasma proteins. More specifically, such Therapeutic proteins include, but are not limited to, immunoglobulins, serum cholinesterase, alpha-1 antJtrypsin, aprotiniri, coagulation factors in both pre and active forms including but not limited to, von Willebrand factor, fibrinogen, factor II, factor VII, factor VILA activated factor, factor VIII; -factor IX, factor X, factor XIII, c1 inactivator, antithrombin III, thrombin; prothrombin, apo-lipoprotein, c-reactive protein, and protein C.
Therapeutic proteins corresponding to a Therapeutic protein -portion bf an albumin fusion protein of the invention further include, but are not limited to, human growth hormone (hGH), a-interferon, erythropoietin (EPO), granulocyte-colony stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor .(GMCSF), insulin, single chain antibodies, autocrine motility factor, scatter factor, laminin, hirudin, applaggin, . monocyte chemotactic protein (MCP/MCAF), macrophage colony-stimulating _ factor (M-CSF), osteopontin, platelet factor 4, tenascin, vitronectin; in addition to those described in Table .1.
' These proteins and nucleic acid sequences encoding these proteins' are well known and available in public databases such as Chemical Abstracts Services Databases (e.g., the CAS
Registry), GeriBank, and GenSeq as shown in Table 1.. , .
Additional Therapeutic proteins corresponding to ~a Therapeutic protein portion of an albumin fusion protein of the invention include, but are not limited to, one or~more of the Therapeutic proteins or peptides disclosed in the "Therapeutic Protein X"
column of Table 1, or fragment or variable thereof.
. Table 1.~ provides a non-exhaustive list of Therapeutic proteins that correspond to a Therapeutic protein portion of an albumin fusion protein of the invention. The '°Therapeutic Protein X" column discloses Therapeutic protein ' molecules followed by parentheses containing scientific and brand names that comprise, ' or alternatively consist, of, that Therapeutic protein molecule or a fragment or variant thereof. '~'herapeutic protein X" as used herein may refer either to an individual Therapeutic protein molecule (as defined by the amino acid sequence obtainable from the CAS and Genbank accession numbers), or to the entire group of Therapeutic proteins associated with a given Therapeutic-protein molecule disclosed -in this column. The "Exemplary Identifier" column provides Chemical Abstracts Services (CAS) Registry Numbers (published by the American Chemical Society) andlor Genbank _Accession Numbers ((e.g., Locus ID, NP XXXXX (Reference Sequence Protein), - - and XP XXXXX (Model Protein) identifiers available through the national Center for Biotechnology Information (NCBI) webpage at www.ncbi.nlm.nih.gov)' that correspond to entries in the CAS Registry or Genbank database which contain an amino acid sequence of the Therapeutic Protein Molecule or of a fragment or variant of the Therapeutic Protein Molecule.
The summary pages associated with each of these CAS and Genbank Accession Numbers are each incorporated by reference in their entireties, particularly with respect to the amino acid - sequences described therein. The "PCTIPatent Reference" column provides U.S.
Patent numbers, or PCT International Publication Numbers corresponding to patents and/or published patent applications that describe the Therapeutic protein molecule.
Each Qf 'the patents and/or published -patent applications cited in the "PCT/Patent Reference" column are herein incorporated by reference in their entireties. In particular, the amino acid 'sequences of - the specified polypeptide set forth in the .sequence listing of each cited "PCT/Patent Reference", the variants of these amino- acid sequences (mutations, fragments, etc.). set forth, for example, in the detailed description of each cited "PCT/Patent.
Reference", the therapeutic indications set forth, for example, in the detailed description of each cited "PCT/Patent Reference", and the activity asssaysfor the _ specified polypeptide set forth in the detailed description, and more particularly, the examples of each cited "PCTIPatent Reference" are incorporated herein by reference. The "Biological activity" column describes Biological activities associated with the Therapeutic protein molecule. The "Exemplary Activity Assay"
column provides references that describe assays which may be used, to test the therapeutic and/or biological activity. of a Therapeutic protein or an albumin fusion protein of the invention comprising a Therapeutic protein X portion. Each of the references cited in the "Exemplary Activity Assay" column are herein incorporated by reference in their entireties, _ 'particularly~with respect to the description of the respective activity assay described in the reference {see Methods section, for example) -for assaying the corresponding biological activity set forth in the "Biological Activity" column of Table 1. The "Preferred Indication Y"
' column describes disease, disorders, -and/or conditions that may be treated, prevented, diagnosed, or ameliorated by Therapeutic protein X or an albumin fusion protein of the invention comprising a'Therapeutic protein X portion:

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In preferred embodiments, the albumin fusion proteins of the invention are capable of a therapeutic activity and/or biologic activity corresponding to the therapeutic activity and/or biologic activity of the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin fusion protein listed in the corresponding row of Table 1. (See, e.g., the "Biological Activity" and "Therapeutic Protein X"columns of Table l.) In further preferred embodiments, the therapeutically active protein portions of the albunun fusion proteins of the invention are fragments or variants of the reference sequence cited in the "Exemplary Identifier" column of Table 1, and are capable of the therapeutic activity and/or biologic activity of the corresponding Therapeutic protein disclosed in "Biological Activity" column of Table 1.
Polypeptcde and Polynucleohde Fragments and Variants Fragments . .
The present invention is further directed to fragments of the Therapeutic proteins described in Table 1, albumin proteins, and/or albumin fusion proteins of the invention.
Even if deletion of one or more amino acids from the N-terminus of a protein results in modification or_loss of one or more biological functions of the Therapeutic protein, albumin protein, and/or albumin fusion protein, other Therapeutic activities and/or functional activities (e.g., biological activities, ability to multimerize, ability to bind a~iigand) may still be retained.
For example, the ability of polypeptides with N-terminal deletions to induce and/or bind to antibodies. which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking ~ N-terminal residues ~of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
Accordingly, fragments of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, include the full length protein as well.as polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the reference polypeptide (e.g., a Therapeutic protein as disclosed in Table 1). In particular, N-terminal deletions may be described by the general formula m-q, where q is a whole integer representing the total number of amino acid residues in a reference polypeptide (e.g., a Therapeutic protein referred to in Table I),'and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In addition, fragments of serum albumin polypeptides corresponding to an albumin protein portion of an albumin fusion protein of the invention, include the full length protein as well as polypeptides having one or more residues deleted from the amino terminus :of the amino acid sequence of the reference polypeptide (i.e., serum albumin}. In particular, N-terminal deletions may be described by the general formula m-585, where 585 is a whole integer representing the total number of amino acid residues in serum albumin (SEQ ID
N0:18), and m~is defined as any integer ranging from 2 to 579.
Polynucieotides'encoding these polypeptides are also encompassed by the invention.
. Moreover, fragments of albumin fusion proteins of the invention, include the full length albumin fusion protein as well as polypeptides having one -or more residues deleted from the amino terminus of the albumin fusion protein. In particular, N-terminal deletions may be described by the general 'formula m-q, where q is a whole integer representing the total number of amino acid residues in the albumin fusion protein, and m is defined as any integer ranging from 2 to q-6. ~Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the N-terminus or C-terminus of a reference polypeptide (e.g., a Therapeutic protein andlor serum albumin protein) results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) andlor Therapeutic activities may still be retained. For example the ability of polypeptides with C-terminal deletions to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will ~be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking the N-temunal andlor C-terminal residues of a reference polypeptide retains Therapeutic activity can readily be detem~ined by routine methods described herein and/or otherwise known in the art.
The present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention (e.g., a Therapeutic protein referred to in Table 1). In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole,integer ranging from 6 to q-1, and where q is a whole integer representing the total number of amino acid residues in a reference polypeptide (e.g., a~Therapeutic protein referred to in Table 1):
Polynucleotides encoding these polypeptides are also encompassed by the invention.
In addition, the present invention provides polypeptides having one or more residues .deleted from the carboxy terminus' of the amino acid sequence of an albumin protein corresponding to an albumin protein portion of an albumin fusion protein of the invention _ (e.g., serum albumin). In particular, C-terminal deletions may be. described by' the general formula 1-n, where -n is any whole integer ranging from.6 to 584, where 584-is the whole integer representing the total number of amino acid residues in serum albumin (SEQ ll~
NO:18) minus 1. Polynucleotides encoding these polypeptides are also encompassed by the invention.
. Moreover, the present invention provides polypeptides having one or more residues - deleted from the carboxy terminus of an albumin fusion protein of the invention. In ' particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to q-1, and where q is a whole integer representing the total number of amino acid residues in an albumin fusion protein of the invention.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
- In addition, any of the above described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted reference polypepfide. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of a reference polypeptide :(e.g., a Therapeutic protein referred to in Table 1, or serum albumin (e.g., SEQ ID NO:IB), or an albumin fusion protein of the invention) where n and m are integers as described above.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical . to a reference polypeptide sequence (e.g., a Therapeutic protein, serum albumin protein or an albumin fusion protein of the invention) set -forth herein, or fragments ,thereof. In preferred embodiments, the application is directed to proteins comprising polypeptides at least 80%; 85%, 90%, 95%,.
96%, 97%, 98% or 99% identical to reference polypeptides having the amino acid sequence of N- and C-terminal deletions as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
- ~ Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of,.an amino acid sequence that displays a Therapeutic activity and/or functional activity (e.g. biological. activity) of the polypeptide sequence of the Therapeutic - , ~ protein or serum albumin protein of which the amino acid sequence.is a fragment.
Other preferred polypeptide fragments are biologically active fragments.
Biologically active fragments are those exhibiting activity similar; but not necessarily .identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include ari improved desired activity, or a decreased undesirable activity.
Variants WO 01/79271 PCT/iJS01/12009 "Variant" refers to a polynucleotide or nucleic acid differing from a reference nucleic -acid or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the reference nucleic acid or polypeptide.
As used herein, "variant", refers to a Therapeutic protein portion of an albumin fusion protein of the invention, albumin portion of an albumin fusion protein of the invention, or albumin fusion protein differing in sequence from a Therapeutic ~ protein.
(e.g. see "therapeutic" column of Table 1), albumin protein,y andlor albumin fusion protein of the invention, respectively, but retaining at least one functional and/or therapeutic property thereof (e.g., a therapeutic activity and/or biological activity as disclosed in the "Biological Activity" column of Table 1) as described elsewhere herein or otherwise known in the art.
Generally, variants are overall very similar, and, in many regions, identical to the amino acid sequence of the Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, albumin protein corresponding~to an albumin protein porEion of an albumin fusion 'protein of the invention, and/or albumin fusion protein of the invention. Nucleic acids encoding these variants are also encompassed by the invention.
The present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%o, 97%, . . 98%, 99% or 100%, identical to, for example, the amino acid sequence of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the ~20 invention (e.g., an amino acid sequence disclosed in the "Exemplary Identifier" column of Table 1, or fragments or variants thereof), albumin proteins (e.g., SEQ ID
N0:18 or fragments or variants thereof) corresponding to an albumin protein portion of an albumin fusion protein of the invention, and/or albumin fusion proteins of the invention: Fragments of these polypeptides are also provided (e.g., those .fragments described herein). Further 25, polypeptides ~ encompassed by the invention are polypeptides encoded by polynucleotides which hybridize to the complement of a nucleic acid molecule encoding an amino acid . sequence of the invention under stringent hybridization conditions (e.g., hybridization to filter bound DNA in 6X Sodium chloride/Sodium citrate {SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2X SSC, 0.1% SDS at about 50 - 65 degrees Celsius), 30 under highly stringent conditions (e.g., hybridization to filter bound DNA
in 6X sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in O.1X SSC, 0.2% SDS at about 68 degrees Celsius), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F.M. et al., eds:, 1989 Current protocol in Molecular Biology, Green publishing associates, Inc., and 35 John Wiley & Sons Inca, New York, at pages 6.3.1 - 6.3.6 and 2.10.3).
Polynucleotides encoding these polypeptides are also encompassed by the invention.
By a golypeptide having an amino acid sequence at least, for example, 95%
"identical"

to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence; up to 5%
of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino-or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence~or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%,, 97%, 98% or 99% identical to, for instance, the amino acid sequence, of an albumin fusion protein of the invention or a fragment thereof (such as the Therapeutic protein portion of the albumin fusion protein or the albumin portion of the albumin fusion protein), ~ .can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the; FASTDB computer program based on the algorithm of Bnitlag et al.
(Comp. App. Biosci.6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization ~ .Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results.
This is because the FASTDB program does not account for N-'and C-terminal truncations of the subject sequence when calculating global percent ,identity. For subject sequences truncated at the N- and C=termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject .
residue, as a percent of the total, bases of. the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent identity, calculated by the above FASTDB
program using the specified parameters, to arrive at a final percent identity score. This final ~percent.identity score is what is used for the purposes of the present invention. ' Only residues to the N- and C-termini of the subject sequence, which are not matchedlaIigned with the query sequence, are considered for the purposes of manually adjusting the percent identity score.
That is, only query residue positions outside the farthest N- and C- terminal residues of the subject sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to detemune percent identity. The delefion occurs at the N-teiminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the~first 10 residues -at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the .final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence.' This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matchedlaligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected.
Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence~are manually corrected for. No othermanual corrections are to made for the purposes of the present invention The variant will usually have at least 75 % (preferably at least about 80%, 90%, 95%
or 99%) sequence identity with a length of normal HA or Therapeutic protein which is the same length as the variant. Homology or identity at the nucleotide or amino acid sequence level is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, .I. Mol. Evol.
36: 290-300 (1993), fully incorporated by reference) which are tailored for sequence similarity searching.
The approach used by the BLAST program is to first consider similar segments between a~query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al., (Nature Genetics 6: 119-129 (1994)) which is fully incorporated by reference. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter aie at the default settings. The default scoring matrix used' by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., Pioc. Natl.
Acad. Sci. USA 89: 10915-10919 (1992), fully incorporated by reference). For blastn, the' scoring, matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively. Four blastn parameters may be adjusted as follows: . Q=IO (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every wink'h position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings were Q=9;
R=2; wink=1;
and gapw=32. A BestFt comparison Between sequences, available , in the GCG
package ' version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.
The polynucieotide variants of the invention may contain alterarions in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities .of the encoded polypeptide: Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred.
Moreover, polypeptide variants in which less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted,. or added in .any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host, such as, yeast or E. coli).
In a preferred embodiment, a polynucleotide encoding an albumin portion of an albumin fusion protein of the invention is optimized for expression in yeast or mammalian cells. In further preferred embodiment, a polynucleotide encoding a Therapeutic protein portion of an albumin fusion protein of the invention is~ optimized for expression in yeast or mammalian cells. In a still further preferred embodiment, a polynucleotide encoding an ' albumin fusion protein of the invention is optimized .for expression in yeast or mammalian ~ cells.
In an alternative embodiment, a codon optimized polynucleotide encoding a Therapeutic protein portion of an albumin fusion protein of the invention does not hybridize to the wild type polynucleotide encoding the Therapeutic protein under sfiringent hybridization conditions as described herein. In a further embodiment, a codon optimized polynucleotide encoding an albumin portion of an albumin fusion protein of the invention does not hybridize to the wild type polynucleotide encoding the albumin protein under stringent hybridization conditions as described herein. In another embodiment, a codon optimized polynucleotide encoding an albumin fusion protein of the invention does not hybridize ~to the wild type polynucleotide encoding the Therapeutic protein portin or the albumin protein portion under - .stringent hybridization conditions as described herein.
In an additional embodiment, polynucleotides encoding a Therapeutic protein portion of an albumin fusion protein of the.invention do not comprise, or alternatively 'consist of, the naturally occurring sequence of that Therapeutic protein. In a further embodiment, polynucleotides encoding an albumin protein portion of an albumin fusion protein of the invention , do not comprise, or alternatively consist of, the naturally occurring sequence of albumin protein. In an alternative embodiment, polynucleotides encoding an albumin fusion protein of the invention do not comprise, or alternatively consist of, the naturally occurring sequence of a Therapeutic protein portion or the albumin protein portion.
Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B:, ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention.
Alternatively, non-naturally occurring variants may be produced by rriutagenesis techniques or by direct synthesis.
Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988 (1993)) reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).) Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For. example, Gayle and coworkers (J. Biol.
Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-la. They used random mutagenesis to generate over 3,500 individual IL-la mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined.at every possible amino acid position. The investigafors found that "[m]ost of the molecule could ~be altered with Iittie effecf on either [binding or biological activity]." In fact, only 23 unique amino acid sequences, out of more than 3;500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
Furthermore; even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce, and/or to bind antibodies which recognize the secreted .form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
Thus, the invention further includes polypeptide variants which have a functional activity (e.g., biological activity andlor therapeutic activity). In highly preferred embodiments the invention provides variants of albumin fusion proteins that have a functional activity (e.g., biological activity andlor therapeutic activity, such as that disclosed in the "Biological Activity" column in Table 1) that corresponds to one or more biological and/or therapeutic activities of the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin fusion protein. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
In preferred embodiments, the variants of the invention have conservative substitutions. By "conservative substitutions" is intended swaps within groups such as replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and-Ile;
replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu;
replacement of the amide residues Asn -and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
Guidance concerning how to make phenotypically silent amino acid substitutions is provided, for exainpIe, in Bowie et al., "Deciphering the Message in Protein -Sequences:
Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identif ed. These conserved amino acids are likely important for protein, function. In contrast, the amino acid positions where substitutions have .been tolerated by natural selection indicates that these positions are not critical for. protein function.
Thus, positions tolerating amino acid substitution could be~mbdified while still maintaining biological activity of the protein.
.The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.
As the authors state, these two strategies have revealed that proteins are, surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes 4f are likely to be permissive at certain amino acid positions in the protein.
For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.
Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and IIe; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acid substitution, variants of :the present invention ~ include (i) polypeptides containing substitutions of one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded -by .
. the genetic code, or (ii) polypeptides containing substitutions of one or more of the amino acid - residues having a substituent group, or (iii) polypeptides which have been fused with or chemically conjugated to another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), (iv) polypeptide containing additional amino acids, such as, for example, an IgG Fc fusion region peptide, .
Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
For example, polypeptide variants containing amino acid substitutions ~ of charged amino acids with other charged, or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleiand et al., Crit. Rev. Therapeutic Drug Carrier Systems.10:307-377 (1993).
In specif c embodiments, the polypeptides of the invention comprise; or alternatively, consist of, fragments or variants of the amino acid sequence of a Therapeutic protein described herein and/or human serum albumin, and/or albumin fusion protein of the invention, wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions, substitutions, and/or,deletions when compared to the reference . ~ amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Nucleic acids encoding these polypeptides , are also encompassed by the invention.
- The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The pol~ypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art_ Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifcations can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be _ appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without-branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of ~a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization,~ disulfide bond formation, deW ethylation, formation of covalent cross-links, ~ formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methyiation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
(See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (I993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12~ (1983); Seifter et al., Meth. Enzymol.
182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)). . ' Functional activity "A polypeptide having functional activity" refers to a polypeptide capable of displaying one or more known functional activities- associated with the full-length, pro-protein, and/or mature form of a Therapeutic protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability . to bind (or compete with a polypeptide for binding) to an anti-polypeptide antibody], immunogenicity {ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.
"A polypeptide having biological activity" refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a Therapeutic protein of the present ~ invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the .case where dose dependency does exist, it need not be identical to that of~the poIypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably; not more than about three-fold less activity relative to the polypeptide of the present invention).
In preferred embodiments, an albumin fusion protein of the invention has at least one biological and/or therapeutic activity associated with the Therapeutic protein (or fragment or variant thereof) when it is not fused to.albumin.
The albumin fusion proteins of the invention can be assayed for functional activity (e.g., biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Specifically, albumin fusion proteins may be assayed for functional . activity (e.g., biological activity or therapeutic activity) using the assay referenced in the "Exemplary Activity Assay" column of Table 1. Additionally, one of skill in the art may routinely assay fragments of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, for activity using assays referenced in its corresponding row of Table 1. Further, one of skill in the art may routinely assay fragments of an albumin protein corresponding to an albumin protein portion of an albumin fusion protein of the invention,.for activity using assays known in the art and/or as described in the. Examples section below.
For example, in one embodiment where one is assaying for the ability of an albumin fusion protein of the invention to bind or compete with a Therapeutic protein for binding to an anti Therapeutic polypeptide antibody and/or anti-albumin antibody; various immunoassays known in the art can . be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA
(enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A
assays, and immunoelectrophoresis assays, etc: In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
In a preferred embodiment, where a binding partner (e.g., a receptor or a ligand) of a Therapeutic protein is identified, binding to that binding partner by an albumin fusion protein containing that Therapeutic protein as the Therapeutic protein portion of the fusion can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., lVlicrobiol. Rev. 59:94-123 (1995). In another embodiment, the ability of physiological correlates of an albumin fusion protein - of the present invention to bind to a substrates) of the Therapeutic.polypeptide corresponding to the Therapeutic portion of the albumin fusion protein of the invention can be routinely assayed using techniques known in the art.
In an alternative embodiment, where the ability.of an albumin fusion protein of the invention to multimerize is being evaluated, association with other components of the multimer can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and- afFnity blotting. See generally, Phizicky et al., supra.
In addition, assays described herein (see Examples and Table 1) and otherwise known in the art--mayroutinely be applied to -measure the ability of albumin fusion proteins of the present invention and fragments, variants and derivatives thereof to elicit biological-activity and/or Therapeutic activity (either in vitro or in vivo) related to either the Therapeutic protein portion and/or albumin portion of the albumin fusion protein of the present invention. Other methods will be known to the skilled artisan and are.within the scope of the invention.
Albumin - As described above, an albumin fusion. protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion or chemical conjugation. ~ -The terms, human serum albumin ~(HSA) and human albumin (HA) are used interchangeably herein. The terms, "albumin and "serum albumin" are broader, and encompass human serum albumin - (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).
As used herein, "albumin" refers collectively to albumin protein or amino acid - sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to -human albumin or fragments thereof (see EP 201239, EP 322 094 WO-97/24445, W095123857) especially the i mature form of human albumin as shown in Figure 15 and SEQ ID N0:18, or albumin from ' other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof.
In preferred embodiments, the human serum albumin protein used in the albumin fusion proteins of the invention contains one or both of the following sets of point mutations with reference to SEQ ID NO:IB: Leu-407 to AIa, Leu-408 to VaI, Val-409, to' Ala; and Arb 4I0 to AIa; or Arg-4I0 to A, Lys-4.13 to Gln, and Lys-4I4 to GIn (see, e.g., International Publication No. W095/23857, hereby incorporated in its entirety by reference herein). In even more preferred embodiments, albumin fusion proteins of the invention that contain one or both of above-described sets of point mutations nave improved stability/resistance to yeast Yap3p, proteolytic cleavage, allowing increased production of recombinant albumin fusion proteins expressed in yeast host cells.
As used herein, a portion of albumin sufFcient to prolong the therapeutic activity or shelf-life . of the Therapeutic protein refers to a .portion of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity of the protein so that the shelf life of the Therapeutic protein portion of the albumin fusion ~ protein is prolonged or extended compared~to the shelf life in the non-fusion state. The albumin portion of the albumin fusion proteins .may comprise the full length of the HA sequence as described above or as shown in Figure 15, or may include one or more fragments thereof that ale capable of stabilizing or prolonging the therapeutic activity. Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the HA
sequence or may include part or all of specific domains of HA. For instance, one or more fragments of HA spanning the first two immunoglobulin-like domains may be used.
The albumin portion of the albumin fusion proteins of the invention may be a variant of normal HA. The Therapeutic protein -portion of the albumin fusion proteins of the invention may also be variants of the Therapeutic proteins as described ~
herein. The term "variants" includes insertions, ~ deletions and substitutions, either conservative or non conservative, where such changes do not substantially alter orie or more of the oncotic, useful ligand-binding and non-immunogenic properties of albumin, or the active site, or active domain which confers the therapeutic activities of the Therapeutic proteins.
In particular, the albumin fusion proteins of the invention may include~.naturally .
occurring polymorphic variants of human albumin and fragments of human albumin, for example those fragments disclosed in EP 322 094 (namely HA (Pn), where n is 369 ~to 419).
The albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Non=mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein may be from a different animal than the Therapeutic protein portion. ~ .
Generally speaking, an HA fragment or variant will be at,least 100 amino acids long, preferably at least 150 amino acids long: The HA variant may consist of or alternatively comprise at least one whole domain of HA, for example domains 1 (amino acids 1-.194 of SEQ ID N0:18),.2 (amino acids 195-387 of SEQ ID N0:18), 3 (amino acids 388-585 of SEQ
ID N0:18), 1 + 2 (1-387 of SEQ ID,N0:18); 2 + 3 (195-585 of SEQ ID N0:18) or 1 + 3 (amino acids 1-194 of SEQ ID N0:18 + amino acids 388-585 of SEQ ID N0:18).
Each domain is itself made up of two homologous subdomains namely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, with flexible inter-subdomain linker regions comprising residues Lys106 to Glul l9, G1u292 to Va1315 and G1u492 to A1a511.
Preferably, the albumin portion of an albumin fusion protein of the invention comprises at least one subdomain or domain of HA or conservative modifications thereof. If the fusion is based on subdomains, some or a1I of the adjacent linker is preferably used to link to the Therapeutic piotein moiety.
Albumin Fusion Proteins The present invention relates generally to albumin fusion proteins and methods of treating, preventing, or ameliorating diseases~or disorders. As used herein, "albumin fusion protein"refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a Therapeutic protein (or fragment or variant thereof). An albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion (i.e., the albumin fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a Therapeutic protein is joined in-frame with a polynucleotide encoding all or a portion of albumin) or chemical conjugation to one another. The Therapeutic protein and albumin protein, once part of the albumin fusion protein, may be referred to as a "portion", "region" or "moiety" of the albumin fusion protein.
In one embodiment, the invention provides an albumin fusion protein comprising, or alternatively consisting ~of,,a Therapeutic protein (e.g., as described in Table 1) and a serum albumin protein: In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment of a Therapeutic protein and a serum albumin protein. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active variant of a Therapeutic protein and a serum albumin protein. In preferred embodiments, the serum albumin protein component of the albumin fusion protein is the mature portion of serum albumin.
In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein, and a biologically active and/or therapeutically active fragment of serum albumin. In further' embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein and a biologically active and/or therapeutically active variant of serum albumin. In . - preferred embodiments, the Therapeutic protein portion of the albumin fusion protein is the mature portion of the Therapeutic protein.
51' In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment or variant of a Therapeutic protein and a biologically active and/or therapeutically active fragment or variant of serum albumin. In preferred embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, the mature portion of a Therapeutic protein and the mature portion of serum albumin. ~ ~ ~ -, Preferably, the albumin fusion protein comprises HA as the N-terminal portion, and a Therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising HA as the C-terminal portion,and~a Therapeutic protein as the N-terminal portion may also be used.
In other embodiments, the albumin fusion protein has a Therapeutic protein fused to both the N-terminus and the C-terminus . of albumin. In a preferred embodiment, the Therapeutic proteins fused at the N- and C- termini are the same Therapeutic proteins. In a preferred embodiment, the Therapeutic proteins fused at the N- and C- termini are different Therapeutic proteins. In another preferred embodiment, the Therapeutic proteins fused at the N- and C- termini are different Therapeutic proteins which may be used to treat or prevent the same.disease, disorder, or condition (e.g. as,listed in the "Preferred Indication Y" column of Table 1). In~~another preferred embodiment, the Therapeutic proteins fused at the N- and C-temiini are different Therapeutic proteins which may be used to treat or prevent diseases or disorders (e.g. as listed in the "Preferred Indication Y" column of Table 1) which are known in the art to commonly occur in patients simultaneously.
In addition to albumin fusion protein in _which the albumin portion is fused N-terminal and/or C-terminal of the Therapeutic piotein portion, albumin fusion proteins of the invention may also be produced by inserting the Therapeutic protein or peptide of interest (e:g., Therapeutic protein X as diclosed in Table 1) into an internal region of HA. For instance, within the protein sequence of the HA molecule a'. number of loops or turns exist between the end and beginning of a-helices, which are stabilized by disulphide bonds (see Figures 9-11). The loops, as determined from the crystal structure of HA
.(Fig. 13) (PDB
identifiers lAO6, 1BJ5, fBKE, 1BM0, lE7E to lE7I and lUOR) for. the most part extend away from the body of the molecule. These loops are-useful for the insertion, or internal fusion, of therapeutically active peptides, particularly -those requiring a secondary structure to be functional, or Therapeutic proteins, to essentially generate an albumin molecule with specific biological activity.
. Loops in human albumin structure into which peptides or polypeptides may be inserted to generate albumin fusion proteins of the invention include: V'a154-Asn6l, Thr76-Asp89, A1a92-GIu100, G1n170-Alal76, His247-G1u252, G1u266-G1u277, G.1u280-His288, A1a362-G1u368, Lys439-Pro447,Va1462-Lys475, Thr478-Pro486, and Lys560-Thr566.
~ In more preferred embodiments, peptides or polypeptides are inserted into the VaI54-Asn6I, G1n170-AlaI76, andlor Lys560-Thr566 loops of mature human albumin (SEQ ID
N0:18).
Peptides to be inserted may be derived from either phage display or synthetic peptide libraries screened for specific biological activity or from the active portions of a molecule with the desired function. Additionally, random peptide libraries may be generated within particular loops or by insertions of randomized peptides into particular loops of the HA
molecule and in which all possible combinations of amino acids are represented.
Such library(s) could be generated on HA or domain fragments of HA by one of the following methods:
(a) randomized mutation of amino acids within one or more peptide loops of HA.
or HA domain fragments. Either one, more or all the residues within a loop could be mutated in this manner (for example see Fig. 10a);
(b) ~ replacement of, or insertion into one or more loops of HA or HA domain fragments (i.e., internal fusion) of a randomized peptides) of length X"
(where-X is an amino acid and n is the number of residues (for example see Fig. 10b);
(c) N-, C- or N- and C- terminal peptide/protein fusions in addition to (a) and/or (b) .
The HA or HA domain fragment may also be made multifunctional by grafting the peptides derived from different screens of different loops against different targets into the same HA or HA domain fragment.
In preferred embodiments, peptides inserted into a loop of human serum albumin are peptide fragments or peptide variants of the Therapeutic proteins disclosed in Table 1. . More particulary, the invention encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least-25, at least 30, at least 35, or at least 40 amino acids in length inserted into a loop of human serum albumin. The invention also encompasses albumin fusion proteins which comprise peptide fragments or.peptide variants at least 7 at least 8, at least 9, at least 10, at least 1 l, at least 12, at least 13, at least 14, at least 15, at least 20, at least-25, at least 30, at least 35, or at least 40 amino acids fused to the N-terminus of human serum albumin. The invention also encompasses albumin fusion proteins . which comprise peptide fragments or peptide variants at least 7 ~ at least 8, at least 9, at least, 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least ~30, at least 35, orrat least 40 amino acids fused to the C-terminus of human serum albumin.
. ~ Generally, the albumin fusion proteins of the invention may have one HA-derived region and one Therapeutic protein-derived region. Multiple regions of each protein, however, may be used to make an albumin fusion protein of the invention.
Similarly, more than one Therapeutic protein may be used to make an albumin fusion protein of the invention.

For instance, a Therapeutic protein may be fused to both the N- and C-terminal ends of the HA. In such a configuration, the Therapeutic protein portions may be the same or different Therapeutic protein molecules. The structure of bifunctional albumin fusion proteins may be represented as: X-HA-Y or Y-HA-X.
For example, an anti-BLyS T"' scFv-HA-IFNa-2b fusion may be prepared to modulate the immune response to IFNcc-Zb by anti-BLyST"' . scFv. An alternative is making a bi (or ' even mufti) functional dose of HA-fusions e.g. HA-IFNa-2b fusion mixed with HA-anti-BLyST"' scFv fusion or other HA-fusions in various ratio's depending on function, half-life etc.
Bi- or mufti-functional albumin fusion proteins may also be prepared to target the Therapeutic protein portion of a fusion to a target organ or cell type via protein or peptide at the opposite terminus of HA.
. As an alternative to the fusion of known therapeutic molecules, the peptides could be obtained by screening libraries constructed as fusions to the N-, C- or N- and C- termini of HA, or domain fragment of HA, of typically 6, 8, 12, 20 or 25 or Xn (where X
is an amino acid (aa) and n equals the number of residues) randomized amino acids, and in which all possible combinations of amino acids were represented. A particular advantage of this approach is that the peptides may be selected in situ on the HA molecule and the properties of the peptide would therefore be as selected for rather than, potentially, modified as might be the case for a peptide derived by any other method then being attached to HA.
Additionally, the albumin fusion proteins of the invention may include a linker peptide between the fused portions to provide greater physical separation between the moieties and thus maximize the accessibility of the Therapeutic protein portion, for instance, for binding to its cognate receptor. The linker peptide may consist of amino acids such that it is flexible or more rigid.
The linker sequence may be_cleavable by a protease or chemically to yield the growth hormone related moiety. Preferably, the protease is one which is produced naturally by the host, for example the S. cerevisic~e protease kex2 or equivalent proteases.
a Therefore, as~ described above, the albumin fusion proteins of the invention may have ~ the following formula Rl-L-R2; R2-L-Rl; or R1-L-R2-L-Rl, wherein Rl is at least one Therapeutic . protein, peptide or polypeptide sequence, and not necessarily the same Therapeutic protein, L is a linker and R2 is a serum albumin sequence.
In preferred embodiments, Albumin fusion proteins of the invention comprising a Therapeutic protein have extended shelf life compared to the shelf life the same Therapeutic protein when not fused to albumin. Shelf-life typically refers to the time period over which the therapeutic activity of a Therapeutic protein in solutiom or in some ~
other storage a formulation, is stable without undue loss of therapeutic activity. Many of the Therapeutic proteins are highly labile in their unfused state. As described below, the typical,shelf Life of these Therapeutic proteins is markedly prolonged upon incorporation into the albumin fusion protein of the invention.
Albumin fusion proteins of the invention with "prolonged" or "extended" shelf life exhibit greater therapeutic activity relative to a standard that has been subjected to the same storage and handling conditions. The standard may be. the unfused full-length Therapeutic protein. When the Therapeutic protein portion of the albumin fusion protein is an analog, a variant, or is otherwise altered or does not include the complete sequence for that protein, the prolongation of therapeutic activity may alternatively be compared to the unfused equivalent of that analog, variant, altered peptide or incomplete sequence. As an. example, an albumin fusion protein of the invention may retain greater than about 100% of the therapeutic activity, or greater than about 105%, 110%, 120%, 130%, 150% or 200% of the therapeutic activity of a standard when subjected to the same storage and handling conditions as the standard when compared at a given time point.
Shelf life may also be assessed in terms of therapeutic activity_remaining after storage, normalized to therapeutic activity when storage began. Albumin fusion proteins of the invention with prolonged or extended shelf life . as exhibited by prolonged or extended therapeutic activity may retain greater than about 50% of the therapeutic activity, about 60%, 70%, 80%, or 90% or more of the therapeutic. activity of the equivalent unfused Therapeutic protein when subjected to the same conditions. For example, as discussed in Example l, an albumin fusion protein of the invention comprising hGH fused to the full length HA sequence may retain about 80% or more of its original activity in solution for periods of up to 5 weeks or more under various temperature conditions.
Expression of Fusion Proteins .
'' The albumin fusion proteins of the invention may 'be produced as recombinant molecules by secretion from yeast, a microorganism such as a bacterium, or a human or - animal cell line. Preferably, the polypeptide is secreted from the host cells. We have found that, by fusing the hGH coding sequence to the HA coding sequence, either to the 5' end or 3' end, it is possible to secrete the albumin fusion protein from yeast without the requirement for a yeast-derived pro sequence. This was surprising, as other workers have found that a yeast derived pro sequence was needed for efficient secretion of hGH in yeast.
For example, Hiramatsu et al. (Appl Environ Microbiol 56:2125 (1990); App1 Envixon Microbiol 57:2052 (1991)) found that the N-terminal portion of the pro sequence in the Mucor pusilltw rennin pre-pro leader was important. Other authors, using the MFa-1 signal, have always included the MFa-1 pro sequence when secreting hGH. The pro sequences were believed to assist in the folding of the hGH by acting as an intramolecuIar chaperone. The present invention shows that HA or fragments of HA can perform a.similar function.
Hence, a particular embodiment of the invention comprises a DNA construct encoding a signal sequence effective for directing secretion in yeast, particularly a yeast-derived signal sequence (especially one which is homologous to the yeast host), and the fused molecule of the first aspect of the invention, there being no yeast-derived pro sequence between the signal and the mature polypeptide.
The Saccharomyces cerevisiae invertase signal is a preferred example of a yeast-derived signal sequence.
Conjugates of the kind prepared by Poznansky et al., (FEBS Lett. 239:18 (1988)), in which separately-prepared polypeptides are joined by chenucal cross-linking, are not contemplated.
-The present invention also includes a cell, preferably a - yeast cell transformed to express an albumin fusion protein of the invention. In addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. If the polypeptide is secreted, the medium will contain the polypeptide, with the cells, or without the cells if they have . been filtered _ or centrifuged away. Many expression systems are known and may be used, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharonzyces cerevisiae, Kluyveromyces lactis and Pichia pastoris, filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.
Preferred yeast strains to be used in the. production. of albumin fusion proteins are D88, DXY 1 and BXP10. D88 [leLC2-3, leac2-122, canl , ~pral , ubc4] is a derivative of parent strain AH22his+ (also known as DB l; see, e.g., Sleep et al. Biotechnology 8:42-46 (1990)).
The strain contains a leu2 mutation which allows for auxotropic selection of 2 micron-based plasmids that contain the LEU2 gene. D88 also exhibits a derepression of PRB1 in glucose excess. The PRB 1 promoter is normally controlled by two checkpoints that monitor glucose levels and growth stage. The promoter is activated in wild type yeast upon glucose depletion and entry into stationary phase. Strain D88 exhibits the repression by glucose but maintains the induction~upon entry into stationary phase. The PRA1 gene encodes a yeast ,vacuolar protease, YscA endoprotease A, that is localized in the ER. The UBC4 gene is in the ubiquitination pathway and is involved in targeting short lived and abnormal proteins for ubiquitin dependant degradation. Isolation of this ubc4 mutation was found to increase the copy number of an expression plasmid in the cell and .cause'an increased level of expression of a desired protein expressed from the plasmid (see, e.g., International Publication No.
W099/00504, hereby incorporated in its entirety by reference herein).

DXY1, a derivative of D88, has the following genotype: [leu2-3, leu2-122, carol, pral, ubc4, ura3: yap3]. In addition to the mutations isolated in D88, this strain also has .a knockout of the YAP3 protease. This protease causes cleavage of mostly di-basic residues (RR, RK, KR, KK) but can also promote cleavage at single basic residues in proteins.
Isolation of this yap3 mutation resulted in higher levels of full length HSA
production (see, e.g., U.S. Patent No. 5,965,386, and Kerry-Williams et al., Yeast 14:161-169 (1998), hereby incorporated in their entireties by reference herein).
BXP10 has .the following genotype: leu2-3, leu2-122, canl,, pral, ube4, ura3, . yap3: : URA3, lys2, hspl S0: : LYS2, pmtl : : UI~A3. In addition to the mutations isolated in DXY1, this strain also has a knockout of the PMTl gene and the HSP150 gene.
The PMT1 gene is a member of the evolutionarily conserved family of dolichyl-phosphate-D-mannose protein O-mannosyltransferases (Pmts). The transmembraiie topology of Pmtlp suggests that it is an integral membrane protein of the endoplasmic reticulum with a role in O-linked glycosyIation. This mutation serves to reduce/eliminate O-linked glycosylation of HSA
fusions (see; e.g., International Publication No. W000/44772, hereby incorporated in its entirety by reference herein). Studies revealed that the Hsp150 protein is inefficiently separated from rHA by ion exchange chromatography. The mutation in the HSPI50 gene removes a potential contaminant that has proven difficult to remove by standard purification techniques. See, e.g., U.S. Patent No. 5,783,423, hereby incorporated in its entirety by reference herein.
The desired protein is produced in conventional ways, for example from a coding sequence inserted in the host chromosome or on a free plasmid. The yeasts are transformed with a coding sequence for the desired protein in any of the usual ways, for example electroporation. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.
Successfully transformed cells, i.e., cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of .an expression construct can .be grown to produce the desired polypeptide.
Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern. (1975) J. Mol. Biol.
98, 503, or Berent et al. (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies. ' Useful yeast plasmid vectors include pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
Plasmids pRS403, pRS4.04, pRS405 and pRS406 are. Yeast Integrating ~ plasmids (YIps) and incorporate the. yeast selectable markers HIS3, 7RP1, LEU2 and URA3. Plasmids pRS413-4.16 are Yeast Centromere plasmids (Ycps). _ ~ _ ' Preferred vectors for making albumin fusion proteins for expression in yeast include pPPC0005, , pScCHSA, pScNHSA, and pC4:HSA which are described in detail in Example 2. Figure 4 shows a map of'the pPPC0005 plasmid that can be used as the base vector into which polynucleotides encoding Therapeutic~proteins may be cloned to form HA-fusions. It contains a PRBI S. cerevisiae promoter (PRBTp), a Fusion leader sequence (FL), DNA
encoding HA (rHA) and an A.DHI S. cerevisiae terminator sequence. The sequence of the fusion leader sequence consists of the first 19 amino acids of the signal peptide of human serum albumin (SEQ ID N0:29) and the last five amino acids of the mating factor alpha 1 promoter (SLDKR, see EP-A-387 319 which is hereby incorporated by reference in its entirety.
The plasmids, pPPC0005, pScCHSA, pScNHSA, and pC4:HSA were deposited on April 11, 2001 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209 and given accession numbers ATCC , , ' ~ , and , respectively. Another vector useful~for expressing an albumin fusion protein in yeast the pSAC35 vector which is described in Sleep et al., BioTechnology 8:42 (1990) which is hereby incorporated by reference in its entirety.
A variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA
segment are then joined by hydrogen bonding between the complementary homopolymeric tails .to form recombinant DNA molecules.
Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion, is treated with bacteriophage T4 DNA polymerase or E.
coli DNA
polymerase~ I, .enzymes that remove protruding, y-single-stranded termini -with their 3' 5'-exonucleolytic activities, and fill in recessed 3'-ends with their polymerizing activities.
The combination of these activities therefore generates blunt-ended DNA
segments.
The blunt-ended segments are then incubated with a large molar excess of linker molecules in " the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments - carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved .
with an enzyme that produces termini compatible with those of the DNA segment.
Synthetic linkers containing a variety of restriction endonuciease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CT, USA. ' A desirable way to modify the DNA in accordance with the invention, if, for example, HA variants are to be prepared, is to use the polymerase chain reaction as disclosed by Saiki et al. (1988) Science 239, 487-491. In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
Exemplary genera of yeast contemplated to be useful in the practice of the present invention as hosts for expressing the albumin fusion proteins are Pichia (formerly classified as Hansenula), Saccharonzyces, Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora, SchiZOSaccharomyces, Citeromyces, Pachysolen, Zygosaccharomyces, Debaromyces, Trichodenna, Cephalosporizcm, Humicola, Mucor, Neurospora, Yarrowia, Metschunikowia, Rhodosporidium, Leucosporidium, ~Botryoascus, Sporidiobolus, Endomycopsis, .and the like. Preferred genera are those selected from the group consisting of Sacclzaromyces, Schizosyccharomyces, Kluyveromyces, Pichia and Torulaspora.
Examples of Saccharomyces spp. are S. cerevisiae, S. italicus and S. i-ouxii.
Examples of Kluyveromyces spp. are K. fragilis, K. lactic and K. marxianus. A
suitable Torulaspora species is T. delbrueckii. Examples of Pichia (Hansenula) spp. are P.
angusta (formerly H. polymorpha), P. anomaly (formerly H. anomaly) and P.
pastoris.
Methods for the transformation of S. cerevisiae are taught generally in EP 251 744, EP 258 067 and WO 90/01063, all of which are incorporated herein by reference.
Preferred exemplary species of Saccharomyces include S. cerevisiae, S.
italicus, S.
diastaticus, and Zygosaccharomyces rouxii. Preferred exemplary species of Kluyveromyces include K. fragilis and K. lactic. Preferred exemplary species of Hansenula include H.
polymorpha (now Pichia angusta), H. anomaly (now Pichia anomaly), and Pichia capsulate.
Additional preferred exemplary species of Pichia include P. pectoris.
Preferred exemplary species of Aspergillus include A. niger and A. nidulans. ' Preferred .exemplary species of Yarrowia include Y. lipolytica. Many preferred yeast species are available from the ATCC.
For example, the ,following preferred yeast species are available from the ATCC and are useful in the expression of albumin fusion proteins: Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 yap3 mutant (ATCC Accession No. 4022731);
Saccharomy~es cerevisiae Hansen,. teleomorph strain BY4743 hsp150 mutant (ATCC Accession No.
4021266); ~ Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 pmtl mutant (ATCC Accession No. 4023792); Saccharomyces cerevisiye Hansen, teleomorph (ATCC
Accession Nos. 20626; 44773; 44774; and 62995); Saccharomyces diastaticus Andrews et Gilliland ex van der Walt, teleomorph (ATCC Accession No. 62987);
Kluyveromyces lactis (Dombrowski) van ~der Walt, teleomorph (ATCC Accession No. 76492); Pichia angusta (Teunisson et al.) Kurtzman, teleoinorph deposited as Hansenula polymorpha de Morais et Mafia, teleomorph (ATCC Accession No. 26012); Aspergillus niger van Tieghem, anamorph 59 ' (ATCC Accession No. 9029); Aspergillus niger van Tieghem, anamorph (ATCC
Accession No. 16404); ~Aspergillacs nidulans (Eidam) Winter, anamorph (ATCC Accession No. 48756);
and Yarrowia lipolytica (Wickerham et al.) van der Walt et von Arx, teleomorph (ATCC
Accession No. 201847). ' .
Suitable promoters for S. cerevisiae include those associated with. the PGKI
gene, GALh or GAL10 genes, CYCI, PHOS, TRPI, ADHI, ADH2, the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase, phosphoglucose isomerase, glucokinase, alpha-mating factor pheromone, [a mating factor pheromone, the PRBI promoter, the GUT2 promoter, the GPDI promoter, and hybrid promoters involving hybrids of parts of 5' regulatory regions with parts of 5' regulatory regions of other promoters or with upstream activation sites (e.g. the promoter of EP-A-258 067).
Convenient regulatable promoters for use in Schizosaccharomyces. pombe are the thiamine-repressible promoter from the nmt gene as described ~by Maundrell (1990).~J.~ Biol.
Chem: 265, 10857-10864 and the glucose repressible jbpl 'gene promoter as described by Hoffman & Winston (1990) Genetics 124, 807-816.
. Methods of transforming Pichia for expression of foreign genes are taught in, for example, Cregg et al. (1993); and various Phillips patents (e.g. US 4 857 467, incorporated herein by reference), and Pichia expression kits are commercially available from Invitrogen BV, Leek, Netherlands, and Invitrogen Corp., Saii Diego, California. Suitable promoters include AOXI and AOX2: Gleeson et al. (1986) J. Gen. Microbiol. 132, 3459-3465 include information on Hansenula vectors and transformation, suitable promoters being MOX1 and FMD1; whilst EP 361 991, Fleer et al. (1991) and other- publications from Rhone-Poulenc Rorer teach how to express foreign proteins in Kl uyveromyces spp., a suitable promoter being PGKI.
The transcription termination signal -is preferably the ,3' flanking sequence' of a eukaryotic gene which contains proper signals for transcription termination and polyadenylation. Suitable 3' flanking, sequences may, for example, be those of the gene naturally linked to the expression control sequence used, 1.e. may correspond to the promoter.
Alternatively, they may be different in which case the termination signal of.
the S. cerevisiae ADHI gene is preferred.
The desired albumin fusion protein may be initially expressed with a secretion leader sequence, ~ which may be any leader effective in the yeast chosen. Leaders useful in S .
cerevisiae include that from the mating factor a polypeptide (MF a-1) and the hybrid leaders bf EP-A-387 319. Such leaders (or signals) .are cleaved by the yeast before the mature albumin is released into the surrounding medium. Further such leaders include those of S .
cerevisiae invertase (SUC2) disclosed in JP 62-096086 (granted as 911036516), ' acid phosphatase (PHOS), the pre-sequence of MFa.-1, 0 glucanase (BGL2) and killer toxin; S.
diastaticus glucoarnylase II; S. carlsbergercsis a-galactosidase (MELD; K.
lactic killer toxin;
and Carcdida glucoarnylase.
Additional Methods of Recombinant and Synthetic Production of Albumin Fusion Proteins .
The present invention also relates to vectors containing a polynucleotide encoding an albumin fusion protein of the present invention, host cells, and the production of albumin fusion proteins by synthetic and recombinant techniques. The vector may, be, for example, a phage, plasmid, viral, or.retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells. . v The polynucleotides encoding albumin fusion proteins of the invention may be joined to a vector containing-a selectable marker for propagation in a host.
Generally,. a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged.lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter; the E. - coli lac, trp, phoA and tac promoters, the 20. SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other .suitable promoters will be known to the skilled artisan. The expression constructs will further cpntain' sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs. will preferably include a translation initiating codon at the beginning and ~ a termination codon (UAA, UGA or UAG) appropriately positioned at ~ the end of the polypeptide to be translated. _ As indicated, the expression vectors will preferably include at least one selectable marker. Such ' markers include dihydrofolate reductase, 6418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhiinurium cells; fungal cells, such as yeast cells (e.g.,' Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS,NSO, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria include pQE70, pQE60~ and pQE-9, ~61 available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNHBA, pNH 16a, pNHl8A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from.Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO; pSV2CAT, pOG44; pXTI and pSG
available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZaIph, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Carlbad, CA).
Other suitable vectors,will be readily apparent to the skilled artisan. .
In one embodiment, polynucleotides encoding an albumin fusion protein of the -invention may be fused to signal sequences which will direct the localization of a protein of the invention to particular compartments of a prokaryotic or euka.ryotic cell and/or direct the secretion of a protein of the invention from a prokaryotic or eukaryotic cell.
For example, in ,E. coli, one may wish to direct the expression of the protein to the periplasmic space.
Examples of signal sequences or proteins (or fragments thereof) to which the albumiin fusion proteins of the invention may be fused in order to direct the expression of the polypeptide to J
the periplasmic space of bacteria include, but are not limited to, the pelB
signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit, and the signal sequence of alkaline phosphatase. Several vectors are commercially .available for the construction of fusion proteins which will direct the localization of a protein, such as the pMAL series of vectors (particularly the pMAL-p series) available from New England Biolabs.
In a specific embodiment, polynucleotides albumin fusion proteins of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides .in Gram-negative bacteria. See, U.S. Patent Nos. 5,576,195 and 5,846,818, the contents of which are herein incorporated by reference in their entireties.
Examples of signal peptides that may be fused to an albumin fusion protein of the invention in order to direct its secretion in mammalian cells include, but are not limited to, the , MPIF-1 signal sequence (e.g., - amino acids 1-21 of ~ GenBank Accession number AAB51134), the stanniocalcin signal sequence (MLQNSAV.LLLLVISASA, SEQ ID
N0:34), and a consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID N0:35). A
suitable signal sequence that may be used .in conjunctionwith, baculoviral expressiow systems is the gp67 signal sequence (e.g., amino acids 1-19 of GenBank Accession Number AAA72759).
Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively.
An advantage of glutamine synthase based vectors are the availabilty of cell tines (e.g., the WO 01/79271 PCT/iTS01/12009 murine myeloma cell line, NSO) which are glutamine synthase negative.
Glutamine synthase expression systems can also function in. glutamine synthase expressing cells (e.g., Chinese Hamster, Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: W087/04462; W086/05807; W089/01036; W089I10404;
and W091/06657, which are hereby incorporated in their entireties by reference herein.
Additionally, glutamine synthase expression vectors can be obtained- from Lonza Biologics, Inc. (Portsmouth, NH). -Expression and production of monoclonal antibodies using a GS
expression system in murine myeloma cells is described in Bebbington et al.,.
Bioltechnology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are herein incorporated by reference.
The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control ' regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired.
Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled.
Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins.
Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
Introduction ~ of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAF-dextran mediated transfection,, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
In addition to encompassing host cells containing the' vector corsstructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence corresponding to a Therapeutic protein may be replaced with an albumin fusion protein corresponding to the Therapeutic 63'.

protein), and/or to include genetic material (e.g., heterologous polynucleotide sequences such as for example, an albumin fusion protein of the invention corresponding to the Therapeutic protein may be included). The genetic material operably associated with the endogenous polynucleotide may activate, alter, andlor amplify 'endogenous polynucleotides.
~ In addition, techniques known in the art may be .. used to operably associate heterologous polynucleotides (e.g., polynucleotides encoding an albumin protein, or a fragment or variant thereof) and/or heterologous control regions (e.g., promoter and/or enhancer) with endogenous polynucleotide sequences encoding a Therapeutic protein via homologous recombination (see, e.g., US Patent Number 5,641,670, issued June 24, 1997;
International Publication Number WO 96/29411; International Publication Number WO
94./12650; Koller et al., Proc. Nat': Acad. ~Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties). .
Albumin fusion proteins of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation; acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic' interaction chromatography, affinity chromatography, hydroxylapatite chromatography, hydrophobic charge interaction chroriiatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
In preferred embodiments the albumin fusion proteins of the invention are purified using Anion Exchange Chromatography including, but not limited to, chromatography on Q-sepharose, DEAF sepharose, poros HQ, poros DEAE, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/Source Q acid DEAE, Fractogel Q and DEAE columns.
In specific embodiments the albumin fusion proteins of the invention are purified using Cation Exchange Chromatography including, but not limited to, SP-sepharose, CM
sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource/Source S .
and CM, Fractogel S and CM columns and their equivalents and comparables.
In specific embodiments the albumin fusion proteins of the invention are purified using Hydrophobic Interaction Chromatography including, but not limited to, Phenyl, Butyl, Methyl, Octyl, Hexyl-sepharose, poros Phenyl; Butyl, Methyl, Octyl, Hexyl , Toyopearl Phenyl, Butyl, Methyl, Octyl, Hexyl ResourcelSource Phenyl, Butyl, Methyl, Octyl, Hexyl, - Fractogel Phenyl, Butyl, Methyl, Octyl, Hexyl columns and their equivalents and comparables. , ' In specific embodiments the albumin fusion proteins of the invention are purified using Size Exclusion Chromatography including, but not limited to, sepharose S100, 5200, 5300, superdex resin columns and their equivalents and comparables.

WO 01/79271 PCT/iTS01/12009 In specific embodiments the albunun fusion proteins of the invention are purified using Affinity Chromatography including, but not linuted to, Mimetic Dye affinity, peptide affinity and antibody affinity-columns that are selective for either the HSA
or the "fusion target" molecules. . _ In preferred embodiments albumin fusion proteins of the invention are purified using one or more Chromatography methods listed above. - In other preferred embodiments, albumin fusion proteins of the invention are purified using one or more of the following Chromatography columns, Q sepharose FF column, SP Sepharose FF column, Q
Sepharose High Performance Column, Blue Sepharose FF column , Blue Column, Phenyl Sepharose FF.column, DEAE Sepharose FF, or Methyl Column.
Additionally, albumin fusion proteins of the invention may be purified using the process described in International Publication No. W000/44772 which is herein incorporated by reference in its entirety. One of -skill in the art could easily modify the process described therein for use in the purification of albumin fusion proteins of the invention. -Albumin fusion proteins of the present invention may be recovered from:
products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production.
procedure, the polypeptides of the present invention may be glycosylated or may be non- .
glycosylated. In addition, albumin fusion proteins of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well 'known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
In one embodiment, the yeast Pichia pastoris is used to express albumin fusion proteins of the invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O2.
This reaction -is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O2. Consequently, in ~a growth medium depending on .methanol, as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOXl) is highly active. ~In the presence of methanol, alcohol oxidase produced from the ADXI gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P.J, et al., Yeast 5:167-77 (1989); Tschopp, J.F., -et al., Nucl. Acids Res.
15:3859-76 (1987). Thus, a heterologous coding sequeilce, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the ADXI regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
In one example, the plasmid vector pPIC9K is used to express DNA encoding an albumin fusion protein of the invention, as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology,"
D.R: Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOXI
promoter Linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located~upstream of a multiple cloning site.
Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDl, pTEFI/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9; pPIC3.5, pHIL-D2; pHIL
Sl, pPIC3.SK, and PA0815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG
as required.
In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide encoding an albumin fusion protein of the ,present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol. ~ ' In addition, albumin fusion proteins of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins:
Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 31~0:105 111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, ~ citruiline, homocitrulline, cysteic . acid, t-buiylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D
(dextrorotary)~ or L
(Ievorotary). .
The-invention encompasses albumin fusion proteins of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, .
phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide,. trypsin, Ichymotrypsin, papain, V8 .
protease, NaBH~; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-Linked carbohydrate chains, processing of N-terminal or C-terminal ends); attachment of chemical moieties to the amino acid backbone,' chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
The albumin fusion proteins may also be modified with a detectable label, such as ~an enzymatic, fluorescent, isotopic or affinity IabeI to allow for detection and isolation of the protein.
Examples of suitable enzymes include horseradish peroxidase, alkaline phosphata.se, beta-galactosidase, or acetylcholinesterase;. examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine. (~z'I~ lash izsh ~s~I)~ carbon (14C), sulfur (3sS), tritium (3H), indium (i~lln, , "ZIn, '13'"Ln, nsmln), technetium (~Tc,99"'Tc), thallium (z°'Ti), gallium (68Ga, 6'Ga), palladium ('°3fd), molybdenum (99Mo), xenon (issXe)~ fluorine (is~~ isssm~ m~Lu~ is9Gd~ iasPm~ ~4oLa~ msYb~ i6sHoy 9oY~
~~Sc~ issRe~ _ issRe~ mzPT~ ios~~ and 9'Ru. ' In specific embodiments, albumin fusion proteins of the present invention or fragments , or variants thereof are attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, 1"Lu, 9oY~ 166Ho, and 's3Sm, to polypeptides. ' In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators is jl'In. ~ In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is 9°Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10 tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are corrimonly known in the art - see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.
As mentioned, the albumin fusion proteins of the invention may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Polypeptides of the invention may be branched, for' example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation;
GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR
PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York '(1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.' Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol.
182:626-646 ( 1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 ( 1992)).
Albumin fusion proteins of the invention and antibodies that bind a Therapeutic protein or fragments or-variants thereof can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton .
Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein.
Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.. ~ . -Further, an albumin fusion protein of the invention may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A
cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, ,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, ~mithramycin, actinomycin D, 1-dehydrotestosterone, ..
glucocorticoids, , procaine, tetracaine, lidocaine, propranolol, and. puromycin and analogs or homologs thereof. Therapeutic agents - include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, ~ melphalan, carmustine . 10 (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, ~
dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). , , The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed ~as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, l3-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF alpha, TNF-beta, AIM I
(See, International Publication No. WO 97133899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., ~ Int. Immunol., 6:1567-1574 (1994)), VEGI {See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such. as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Techniques for conjugating such therapeutic moiety to proteins (e.g., albumin fusion proteins) are well known in the art.
Albumin fusion proteins may also be attached to solid supports,, which are particularly useful for immunoassays or purification of polypeptides that are bound by, that bind to, or . associate with albumin fusion proteins of the invention. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
Albumin fusion proteins, with or without a therapeutic moiety conjugated to it, administered alone or i~n combination with cytotoxic factors) and/or cytokine(s) can be used as-a therapeutic. ' .

WO 01/79271 PCT/iTS01/12009 Also provided by the invention are chemically modified derivatives of the .albumin fusion proteins of the. invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179;337): The chemical moieties for derivitization may be selected from water soluble polymers 'such as polyethylene glycol, ethylene glycollpropylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The albumin fusion proteins may ~be modified at random positions within the molecule, or at predetermined .,.
positions within the molecule and may include one, two, three or more attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or unbranched.
For polyethylene glycol, the preferred mo(ecular~weight is between about 1 kDa and about 100 kDa (the term. "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and, manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., .
the duration of sustained release ,desired, the effects, if any on biological activity, the ease in .
handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a Therapeutic protein or, analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, '1000, 1500, 2000, .2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500,' 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
As noted above, the polyethylene glycol may have a branched structure.
Branched polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575;
Morpurgo et al., Appl. Bioc_hem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999)'; and Caliceti et~ al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
The polyethylene glycol molecules (or other chemical moieties) should be attached to ' the protein with consideration of effects on -functional or antigenic domains of the protein.
There,are a number of attachment methods available to those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein incorporated by reference; see also. Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl chloride: , For example, polyethylene glycol may be covalently bound through amino acid residues via reactive group; such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol ~_ molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl. group may include aspartic acid residues glutamic acid residues and the C-temunal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino - group, such as attachment at the N-terminus or lysine group.
As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine; histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and~combinations thereof) of the protein.
One may specifically desire proteins chemically modified at the N-terminus.
Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion~of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a popularion of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modif cation may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups . (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
As indicated above, pegylation of the albumin fusion proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the albumin fusion protein either directly or by an intervening linker.
~.inkerless systems for ~ attaching polyethylene glycollto proteins are described in Delgado et al., Crit. Rev. Thera.
Diug Carrier Sys. 9:249-304 (1992); Francis et al., Intern..3. of Hematol.
68:1-18 0998);
U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO
98/32466, the disclosures of each of which are incorporated herein by reference.
One system for . attaching polyethylene glycol directly to amino ~ acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (CISOZCHZCF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is WO 01/79271 PCT/USOl/12009 directly attached to amine groups of the protein. ' Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane~sulphonyl group. .
Polyethylene glycol can also be attached to proteins ,using a number of different intervening linkers. For example, U.S. Patent No. 5,612,460, the entire disclosure of, which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be ~ produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol. derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by .reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.
The number of polyethylene glycol moieties attached to each albumin fusion protein of ~e invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to .1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-I6, 15-17, 16-18, 17-19, ~or 18-20 _polyethylene glycol moieties per protein molecule. _ Methods for determining the degree of substitution are discussed, for,example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992). .
The polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, -but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphoceilulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
72 .

The presence and quantity of albumin fusion proteins of the invention may be determined using ELISA,. a well known immunoassay known in the art. In one ELISA
protocol that would be useful for detecting/quantifying albumin fusion proteins of the invention, comprises the steps of coating an EI,ISA plate with an anti-human serum albumin antibody, blocking the plate to prevent non-specific binding, washing the ELISA plate, adding a solution containing the albumin fusion protein of the invention (at one or rrtore different concentrations), adding a secondary anti-Therapeutic. protein specific antibody coupled to a detectable label (as described herein or otherwise known in the art), and detecting the presence of the secondary antibody. In an alternate version of this protocol, the ELISA
plate might be coated with the anti-Therapeutic protein specific antibody and the labeled secondary reagent might be the anti-human albumin specific antibody.
Uses of the Polynncleotides Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following. description should be considered exemplary and utilizes known techniques.
The polynucleotides of the present invention are useful to produce the albumin fusion proteins of the invention. As described in more detail below, polynucleoddes of the invention (encoding albumin fusion proteins) may be ,used in recombinant DNA methods useful in . genetic engineering to make cells, cell lines, or tissues that express the albumin fusion protein encoded by the polynucleotides encoding albumin fusion proteins of the invention.
Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct -the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genorne, thereby producing a new trait in the host cell. Additional 'non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the sections labeled "Gene Therapy", and Examples 17 and 18).
Uses of the Poly~eptides Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.
Albumin fusion proteins of the invention are useful to provide immunological probes for differential identification of the tissues) (e.g., immunohistochemistry .assays- such as, for example, ABC immunoperoxidase (Hsu et al.,_J.~Histochem. Cytochem. 29:577-580 (1981)) or cell types) (e.g., immunocytochemistry assays).

Albumin fusion proteins can be used to assay levels of polypeptides in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J.
Cell. Biol.
105:3087-3096 (I987)). Other methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent , assay (ELISA) and the radioimmunoassay (RIA). Suitable assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (13'I, lzsh lz3h lz~i)~ carbon (14C), sulfur (3sS), tritium (3H), indium ('ls'-"In,, 113mIn, 112In, 11'In), and technetium (99Tc, 99m~LC)~ thallium (z°1Ti), gallium ('~Ga, 6'Ga),. palladium (1°3Pd), molybdenum {~9Mo), xenon (133Xe), fluorine (18~, 153sm' 177Lu' ls9Gd' 149Pm' 140La' 175Yb' 166H~' 90Y' 47SC' ls6Re, lssRe, 142.' lose 9~RLi; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
Albumin fusion proteins of the invention can also be detected in vivo by imaging.
Labels or markers for in vivo imaging of protein include those detectable by X-radiography, nuclear magnetic resonance (NMR) or election spin relaxtion (ESR). For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR
include those with a detectable characteristic spin, such as deuterium, which may ~be incorporated into the albumin fusion protein by labeling of nutrients given to a cell line expressing the albumin fusion protein of the invention.' An albumin fusion protein which has been labeled with an appropriate detectable imaging moiety, such.as ~a radioisotope (for example, 1311, llzln, 99~.c, (131h lzsl~ lzsl~ -lzll)~
carbon (14C), sulfur (3sS), tritium- (3H), indium (' lsmln, ' l3mln, llzln, 111In), and technetium (~fc, 99"'Tc), thallium (zolTi), gallium (68Ga, 6'Ga), palladium (1°3Pd), molybdenum (99Mo), xenon (133Xe), fluorine. (18F, 153Sm~ l~7Lu~ ls9Gd~ 149Pm~ laoLa~ 17s.~,b~
166,HQ, 9oY~ a~Sc~
la6Re~ lesRe, l4zPr~ lose 97Ru), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be~ examined for immune system disorder.
~ It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled albumin fusion protein will then preferentially accumulate at locations in the body (e.g., organs, cells, extracellular spaces or matrices) where one or more receptors, ligands or substrates (corresponding to that of the 35' Therapeutic protein used to make~the albumin fusion protein of the invention) are located.
Alternatively, in the case where the albumin fusion protein comprises at Least a fragment or variant of a Therapeutic antibody, the labeled albumin fusion protein will then preferentially accumulate ,at the locations in the body (e.g., organs; cells, extracelluiar spaces or matrices) where the polypeptides/epitopes corresponding to those bound by the Therapeutic antibody (used to make the albumin fusion protein of the invention) are located. In vivo tumor imaging is described in S.W. Burchiel et, al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radioclzemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds.; Masson Publishing Inc.
{1982)). The protocols described therein could easily be modified by one of skill in the art for use with the albumin fusion proteins of the invention.
- In one embodiment, the invention provides a method for. the specific delivery of . albumin fusion proteins of the invention to cells by administering albumin fusion proteins' of the invention (e.g., polypeptides encoded by polynucleotides encoding albumin fusion ' proteins of the invention and/or antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example; the invention provides a method for delivering a Therapeutic protein into the targeted cell. In another example, ' the invention provides a .
method for delivering a single. stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell. ' , In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering albumin. fusion proteins of the invention in association with toxins or cytotoxic prodrugs.
By "toxin" is meant one or more compounds that bind and activate endogenous cytotoxic ~effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits ~of x toxins, or any molecules or enzymes not normally. present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, ~saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. "Toxin" also inctudes a~ cytostatic~ or cytocidal agent, a therapeutic agent ar a radioactive metal ion, e.g., alpha-emitters such as, for example, Z'3Bi, or other radioisotopes Such as, for example, '°3Pd, 133xe,131I, 6gGe, 57Co' 65zn' 85Sr' 32P' 35S' 90Y' 153Sm' 153Gd, '69Yb, SICr; ~'Mn, 75Se, "3Sn, 9°Yttrium, "'Tin, '$6Rhenium, "Holmium, and 'Rhenium;
luminescent labels, such , as luminol; and fluorescent labels, such as.
fluorescein and rhodamine, and biotin. In a specific embodiment, the invention provides a method for the specific destruction -of cells (e.g., the destruction of tumor cells) by administering polypeptides of . the invention or .antibodies of the invention in association with the radioisotope 9°Y. In another specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the. destruction of tumor ,cells) byadministering polypeptides of the invention or antibodies of the invention in association with the radioisotope "'In. In a further specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope '3'I.
Techniques known in the art may be applied to label.polypeptides of the invention.
Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Patent Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931;
5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety).
The albumin fusion proteins of the .present invention are useful ' for diagnosis, treatment, prevention andlor prognosis of various disorders in mammals, preferably humans.
Such disorders include, but are not limited to,.- those described herein under the section heading "Biological Activities," below.
Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression level of a certain polypeptide in cells or body fluid of an individual using an albumin fusion protein of the invention; and (b) comparing the assayed polypeptide expression level, with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide. expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more defnitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Moreover, albumin fusion proteins of the present invention can be used to treat or prevent diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and . conditions. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., ' insulin), to supplement absent or decreased levels. of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor); to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF-receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
In particular, albumin fusion proteins comprising of at least a fragment or variant of a Therapeutic antibody can also be used -to treat disease (as described -supra, and elsewhere herein). For example,~administration of an albumin fusion protein comprising of at least a fragment or variant of a Therapeutic antibody can bind, and/or neutralize the polypeptide to which the Therapeutic antibody used to make the albumin fusion protein immunospecificaIly binds, and/or reduce overproduction of the polypeptide to which the Therapeutic antibody used to make the albumin fusion protein immunospecifically binds. Similarly, administration of an~albumin fusion protein comprising of at least a fragment or variant of a Therapeutic antibody can activate the polypeptide to which the Therapeutic antibody used to make the ' albumin fusion protein immunospecifically binds, by binding to the polypeptide bound to a .
membrane (receptor).
At the very least, the albumin fusion proteins of the invention of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
Albumin fusion proteins of the invention can also be used .to raise antibodies, which in turn may be used to measure protein expression of the Therapeutic protein, alburriin protein, and/or the. albumin fusion protein of the invention from a recombinant cell, as a way of assessing transformation of the host cell, or in a biological sample. Moreover, the albumin fusion proteins of the present invention can be used to test the biological activities described herein.
Diagnostic Assays The compounds of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans. Such .
disorders include, but are not limited to, those described for each Therapeutic protein in the corresponding row of Table l and herein under the section headings "Immune Activity,"
"Blood Related Disorders,". "Hyperproliferative , Disorders," "Renal Disorders,"
"Cardiovascular Disorders," "Respiratory Disorders," "Anti-Arigiogenesis Activity,"
"Diseases at the Cellular Level," "Wound Healing and Epithelial Cell Proliferation," "Neural Activity and Neurological Diseases," "Endocrine Disorders,". "Reproductive .System Disorders," "Infectious Disease," "Regeneration," and/or "Gastrointestinal Disorders," infra.
For a number of disorders, substantially altered (increased or decreased) levels of gene expression can be detected in tissues, cells or bodily fluids (e.g:, sera, plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual having.such a disorder, relative to a "standard" gene expression level, that is,. the expression level in tissues or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves measuring the expression Level of the gene encoding a polypeptide in tissues, cells or body fluid from an individual and comparing the measured gene expression level with a standard gene expression level, whereby an increase or decrease in the gene expression levels) compared to the standard is indicative of a disorder. These diagnostic assays may be performed in vivo or in vitf-o, such as, for example, on blood samples; biopsy tissue or autopsy tissue.
The present invention is also -useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome By "assaying the expression level of the gene encoding a polypeptide" is intended qualitatively or quantitatively measuring or estimating the level of a particular polypeptide (e.g. a'polypeptide corresponding to a Therapeutic protein disclosed in Table 1) or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or W RNA level in a second biological sample).
Preferably; the polypeptide expression Level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA
level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from, a population of individuals not having the disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By "biological sample" is intended any biological sample obtained from an individual, cell line,-tissue culture, or other source containing polypeptides of the invention (including portions thereof) or mRNA. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue, sources found to express the full length or fragments thereof of ~a polypeptide or mRNA. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological .
sample is to include mRNA, a tissue biopsy is the preferred source. ' Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the polypeptides of the invention are then assayed using any appropriate method. These include Northern blot analysis, S1 nuclease mapping, the polymera'se chain , reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR)..
The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of polypeptides that bind to, are bound iiy, or associate with albumin fusion proteins of the invention, in a biological sample (e.g., cells and tissues), including determination of normal and abnormal levels of polypeptides. Thus, for instance, a diagnostic assay in accordance with the invention for detecting abnormal expression of polypeptides that bind to, are bound by, or associate with albumin fusion proteins compared to normal control tissue samples may be used to .detect the,presence of~
tumors. Assay techniques that can be used to determine levels of a polypeptide that bind to, are bound by, oi~
associate with albumin fusion proteins of the present invention in a sample derived from a ' host are well-known to those of skill in the art. Such 'assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA
assays.
Assaying polypeptide levels in a biological sample can occur using any art-known method.
Assaying polypeptide levels in a biological sample can occur using a variety of techniques. For example, polypeptide expression in tissues can be .studied with classical immunohistological methods (Jalkanen et=al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096 (1987)). Other methods useful for detecting .polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable. antibody 'assay labels are known in the art and include enzyme labels, ~ such as, glucose oxidase, and radioisotopes, such as iodine (lzsh lzll), carbon (14C), sulfur (35S), tritium (3H), indium (llzln), and technetium (99mTC),'and fluorescent labels, such as fluorescein and rhodamine, and biotin.
The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the gene of interest (such as, for example, cancer). The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is incorporated herein by reference in its entirety. The isolated cells. can be derived from cell culture or from a patient.
The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the gene.
For example, albumin fusion proteins may be used to quantitatively or qualitatively detect the presence of polypeptides that bind to, are bound by, of associate with albumin fusion proteins of the present invention. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled albumin fusion protein coupled with light microscopic, flow cytoinetric, or fluorimetric detection.
In a preferred embodiment, albumin fusion proteins comprising at least a fragment or . variant of.an antibody that immunospecifically binds at least a Therapeutic protein disclosed herein (e.g., the Therapeutic proteins disclosed in Table 1) or otherwise known in the .art may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be _ accomplished, for example, by imrnunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection. ~ .
The albumin fusion proteins of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of polypeptides that bind to, are bound by, or associate with an albumin fusion protein of the present invention. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or polypeptide of the present invention. The albumin fusion proteins are preferably applied by overlaying the labeled albumin fusion proteins onto a biological sample.
Through the use of such. a procedure, it is possible to determine not only the presence of the polypeptides that bind to, are bound by, or associate with albumin fusion proteins, but also its distribution in ' the examined tissue. Using the present invention, those of ordinary skill will readily perceive 15. that any of a wide variety of histological methods (such as ~ staining procedures) can be modified in order to achieve such in situ detection.
- Immunoassays and non-immunoassays that detect polypeptides that bind to, are bound by, or associate with alburriin fusion proteins will typically comprise incubating a sample, such as a biological fluid, ~a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture,.in the presence bf a delectably labeled antibody capable of binding gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
The biological ~ sample may be brought in contact with and immobilized onto a solid phase support or carrier such . as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detestably labeled albumin fusion protein of the invention. The solid phase support may then be washed with the buffer a second time to remove unbound.' antibody or polypeptide. Optionally the antibody is subsequently labeled.
The amount of bound label on solid support may then be detected by conventional means.
By "solid phase support or carrier" is intended any support capable of binding a polypeptide (e.g., an albumin fusion protein, or polypeptide that binds, is bound by, or associates with an albumin fusion protein of the invention.) Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural ' and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. , The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a polypeptide. Thus, the support conf guration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, ~etc.
Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation. .
The binding activity of a given lot of albumin fusion protein may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
In addition to assaying polypeptide levels in a biological sample obtained from an IO . individual, polypeptide can also be detected in vivo by imaging. For example., in one embodiment of the invention, albumin fusion proteins of, the invention are used to image diseased or.neoplastic cells.
IJabels or markers for in vivo imaging of albumin fusion proteins of the invention include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For IS X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the albumin fusion protein by labeling of nutrients of a cell line (or bacterial or yeast strain) engineered.
20 . Additionally, albumin fusion proteins of the invention whose presence can be detected, can be administered. For example, albumin fusion proteins of the invention labeled with a radio-opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further, such polypeptides can be utilized for in vitro diagnostic procedures.
25 ~ A polypeptide-specific antibody or antibody fragment which has been labeled with an . appropriate detectable imaging moiety, such as a radioisotope (for example, ~31I,'~zln, 9s"~Tc), a radio=opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenteratly, subcutaneously or intraperitoneally) into the mammal to be examined for a disorder. It will be understood in the art that the size of the subject and the 30 imaging system used will determine the quantity . of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled albumin fusion protein will then preferentially accumulate at the locations in the body which contain a polypeptide or other substance that binds to, is bound by or associates with an 35 albumin fusion protein of the present invention. In vivo tumor imaging is described in S.W.
Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments"
(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W.
Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)). .
One of the ways in which an albumin fusion protein of the present invention can be detectably labeled is by linking the same to a reporter enzyme and using the linked product in an enzyme immunoassay (EIA) (Volley, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Micro~biological Associates_ Quarterly Publication, Walkersville, MD); Volley et al., J. Clin.. Pathol. 31:507-520 (1978); Butler, J.E., Meth. Erezymol. 73:482-523 (1981); Maggio, E. (ed.), I980, Enzyme Immunoassay, CRC Press, Boca Raton, FL,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The reporter enzyme which is bound fo the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Reporter enzymes which can be used to detectably label the antibody .include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose IS phosphate isomerase, horseradish peroxidase, all~aline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be .
accomplished by colorimetric methods which employ a chromogenic substrate for the reporter enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
. Albumin fusion proteins may also be. radiolabelled and used in any of a variety of other immunoassays. For example, by radioactively labeling the albumin fusion proteins, it is possible to the use the albumin fusionproteins in a radioimmunoassay (RiA) (see, for _ example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography. ~ .
It is also possible to label the albumin fusion proteins with a fluorescent compound.
When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate,: rhodamine, phycoerythrin, phycocyanin, allophycocyanin; ophthaldehyde and fluorescamine.
The albumin fusion protein can also be detectably labeled using fluorescence emitting metals such as 1'ZEu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The albumin fusion proteins can also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged albumin fusion protein is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester; imidazole, acridinium salt ~ and oxalate ester.
Likewise, a bioluminescent compound may be used to label albumin fusion proteins of the present invention. Bioluminescence is a type of chemiluminescence found in biological a systems in, which a catalytic protein increases the efFciency of the chemiluminescent reaction.
. The presence of a bioluminescent. protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are Iuciferin, luciferase and aequorin.
Transonic Or,~anisms ' -~Transgenic organisms that express the albumin fusion proteins of the invention are also included in the invention. Transgenic organisms are genetically modified organisms into which recombinant, exogenous or cloned genetic material has been transferred.
Such genetic material is often referred to as a transgene. The nucleic acid sequence of the transgene may ' include one or more transcripfional regulatory sequences and other nucleic acid sequences such ~as introns, that may be necessary for optimal expression and secretion of the encoded protein. The transgene may be designed to direct the expression of the encoded protein tin a manner that facilitates its recovery from the organism -or from a product produced by the-organism, e.g. from the milk, blood; urine, eggs, hair or seeds of the organism. The transgene may consist of nucleic acid sequences derived from the genome of the same species or 'of a different species than the.species of the target animal. The transgene maybe integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene.
The term "germ cell line transgenic organism" refers to a transgen~ic organism in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic organism to transfer the genetic information to offspring. If such offspring in fact possess some or ' all of that alteration or genetic information, then they too are transgenic organisms. The alteration or genetic information may be foreign to the species of organism to which the recipient belongs, foreign only to the particular- individual recipient, or may be genetic information already possessed by the recipient. Iii the last case, the altered or introduced gene may be expressed differently than the native gene.
A transgenic organism may be a transgenic animal -or a transgenic plant.
Transgenic animals can be produced by a variety . of . ' different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent No. 5,602,307;
Mullins et al. (1993) Hypertension 22(4):630-633; Brenin et al. (1997) Surg.
Oncol. 6(2)99-110; Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biology No. 62, Humana Press (1997)). The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any. method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Patent No. 5,489,743 and U.S. Patent No. 5,602,307.
A number of recombinant or transgenic mice have been produced, including those which express an activated oncogene sequence (U.S. Patent-No. 4,736,866);
express simian SV40 T-antigen (U.S. Patent No. 5,728,915); lack the expression of interferon regulatory -factor 1 (IRF-1) (U.S. Patent No: 5,73I,490); exhibit dopaminergic dysfunction (U.S.
Patent No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent No. 5,731,489); display greater similarity to the conditions existing~in naturally occurring Alzheimer's disease (U.S. Patent No. 5,720,936); have a reduced capacity to mediate cellular adhesion (LJ.S: Patent No. 5,602,307); possess a bovine growth hormone gene (Clutter et al. (1996) Genetics 143(4):1753-1760); or, are capable of generating a fully human antibody response (McCarthy (1997) The Lancet 349(9049):405).
While mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species.
Transgenic procedures have been successfully utilized in a variety of non-marine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits; cows and guinea pigs (see, e.g., Kim et al. (1997) Mol. Reprod. Dev.- 46(4}:515-526; Houdebine (1995) Reprod. Nutr. Dev. 35(6):609-617; Petters-(1994) Reprod. Fertil. Dev.
6(5):643-645;
Schnieke et al. (I997) Science 278(5346):2130-2133; and Amoah (I997) J. Animal Science 75(2):578-585). . -To direct the secretion of the transgene-encoded protein of the invention into the milk of transgenic mammals, it may be put under the control of a promoter that is preferentially .
activated ~ in mammary epithelial cells. Promoters that control the genes encoding milk proteins are preferred, for example .the promoter for casein, beta lactoglobulin, uvhey acid protein, or lactalbumin (see, e.g., DiTullio (1992) BioTechnology 10:74-77;
Clark et al.
01989) BioTechnology 7:487-492; Gorton et al. {1987) BioTechnology 5:1183-1187; and Soulier et al. '(1992) FEBS Lefts. 297:13). The transgenic mammals of choice would produce large volumes of milk and have long lactating periods, for example goats, cows, camels or sheep.-An albumin fusion protein of the invention can also be expressed~in a transgenic plant, e.g. a plant in which the DNA transgene is inserted into the nuclear or plastidic genome.
Plant transformation procedures used to introduce foreign nucleic acids into plant cells or protoplasts are known in the art (e.g., see Example .19). See, in general,' Methods in Enzymology Vol. 153 {"Recombinant DNA Part D") 1987, Wu , and Grossman Eds., Academic Press and European Patent Application EP 693554. Methods for generation of genetically engineered plants are further described in US Patent No.
5,283,184, US Patent No. 5, 482,852, and European Patent Application EP 693 554, all of which are hereby incorporated by reference.
Pharmaceutical or Therapeutic Compositions The albumin fusion proteins of the invention or formulations thereof may be administered by any conventional method including, parenteral (e.g.
subcutaneous or intramuscular) injection or intravenous infusion. The treatment may consist of a single dose or a plurality of doses over a period of time. , . .
While it is possible for an albumin fusion protein of the invention to,be administered alone, it is preferable to present it as a pharmaceutical~formulation, together with one or more acceptable carriers. The carriers) must be "acceptable" in the sense of being compatible with the albumin fusion protein and not deleterious to the recipients thereof.
Typically, the carriers . will be water or saline,which will be sterile and pyrogen free. Albumin fusion proteins of the invention are particularly well suited to formulation in aqueous carriers such as sterile pyrogen free water, saline or other isotonic solutions because of their extended shelf life in solution.
For instance, pharmaceutical compositions of the invention may ~be formulated well in .
advance in aqueous form; for instance, weeks or months or longer.time periods before being dispensed. _ For example, wherein the Therapeutic protein is hGH, EPO, alpha-IFN or beta-IFN, formulations containing the albumin fusion protein may be prepared taking into. account the . extended shelf life of the albumin fusion protein in aqueous formulations.
As exhibited in Table 2, most Therapeutic proteins are unstable with short shelf lives after formulation with an aqueous carrier. As discussed above, the shelf life of many of these Therapeutic proteins are markedly increased or prolonged after fusion to HA. - _ Table 2 Protein Tradename, RouteFormulation Storage Conditions of Manufacturer Non Fusion Protein Interferon,Roferon-A, sc sol_n 4-.8.C .

alpha-2a Hoffmann-l.aRochelm (vial or _ pre-filled~ ' .
.

syringe) WO 01!79271 PCT/USO1/12009 Protein Tradename, Route Formulation Storage Conditions ~ of Manufacturer Non-Fusion Protein Interferon,Intron-A, iv sc so! n; ~4-8C
lm alpha-2b Schering Plough powder + dl!. (all preps, before and after dilution) COMBO Rebetron (Intron-Apo Rebetol capsule_ ;

Interferon + Rebetol) + + Intron-A injection alpha-2b + Schering Ploughsc Ribavirin Interferon,Infergen . sc , sol_n ~L-8C

Alphacon-1 Amgen Interferon,Wellferon, sc sol_n ' 4-8C
~ .

alpha-nl, Wellcome lm (with albumin as Lympho- stablizer~

Mastoid -interferon,Avonex, lm powder + dl!. 4-8C
' beta-la Biogen (with' albumin)(before and after dilution) (Use within 3-6h of _ reconstitution) Rebif, sc soI_n, Ares=Serono in pre-filled .
syringe ~

(Europe only) Interferon,Betaseron, sc powder + dl!. 4-8C

beta-Ib Chiron (with albumin) (before and after ~ dilution) (Europe: Betaferon) ~ (Use within 3h of reconstitution) ~

Single use vials.
, Interferon,Actimmune, sc . 4-8C .

Gamma-lb InterMune (before and after dilution) Pharmaceuticals (Use' within 3h of reconstitution).

Growth ~ Genotropin, powder/dil cartridges4-8C

Hormone Pharmacia (single or multi-use);(before and after Upjohn dilution);

{somatropin) - single use MiniQuicksingle use lVIiniQuick injector . Delivery Device' should . be refrigerated until use.

- Humatrope, sc powder + dl!. 4-8C

Eli Lilly lm (Vial or pen (before and after . cartridge) dilution) (Use vials within 25h, cartridges within 28d, of reconstitutiori).

Norditropin, . -~

Novo ~ Nordisk Pharmaceuticals Nutropin, sc powder + dl!. 4.-8C

Genentech (stable for 14d ' , after dil_n) ' (all preps, before and after ' dilution) .

8'b Protein Tradename, Route Formulation Storage Conditions _~ of Manufacturer _ Non-Fusion Protein Nutropin AQ, sc sol_n ~L-8C

Genentech (Stable for 28 d after 1 st use) Nutropin Depot,sc microsphere ~l-8C -suspension ' Genentech as Single use pkges.
Dose powder + dil. 1-2x/month , (Protease micro-encapsulation technol.) Saizen, ~ sc. powder + dil. Powder should be stored ' (Serono) ~ im , at Rm Temp_.
After reconstitution store 4-8C for a to 14d.

Serostim, Powder should be stored Serono ' at Rm Temp_.
After reconstitution store in ~

8C for a to 14d.

hGH, with Protropin, sc powder + dil. 4-8C , .

N-term. Genentech im (all preps, before . Met and (somatrem) ' after dilution) ErythropoietinEpogen, iv sol_n - 4-8C ' (Epoetin Amgen sc _ (use within 21d alfa) ~ , of first . tee) .

(Single & multi-dose vials) Procrit, iv sol_n 4-8C

Amgen sc (use within 21d of first . _ ' use) .

(Single & multi-dose . vials) In instances where -aerosol administration is appropriate, the albumin fusion proteins of the invention can be formulated as aerosols using standard procedures. The term "aerosol"
~ includes any gas-borne suspended phase of an albumin fusion protein of the instant invention which is capable of being inhaled into the bronchioles or nasal passages.
Specifically, aerosol includes a gas-borne suspension of -droplets of an albumin -fusion protein of the instant invention, as may be produced in a metered dose inhaler or nebulizer, or in a mist'sprayer. .
Aerosol also includes a dry powder composition of a compound of the instant invention suspended in air or other carrier gas, which may be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones, Dn~g Delivery to the Respiratory Tract, Elks Honvood (19 87); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Sysfems 6:273-313; and Raeburn et al,. (1992) Pharmacol. Toxicol. Methods 27:143-1'59.

. The formulations of the invention are also' typically non-immunogenic, in part, because of the use of the components of the albumin fusion protein being derived from the proper species. For instance,- for human use, both the Therapeutic protein and albumin portions of the albumin fusion protein will typically be human. In some cases, wherein either component is non human-derived, that component may be humanized by substitution of key amino acids so that specific .epitopes appear to the human immune system to be human in nature rather than foreign.
The formulations may conveniently be presented in unit dosage form and' may be prepared by any, of the methods well known in the art of .pharmacy. Such methods include the step of bringing into association the albumin fusion protein with the carrier that constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carnets or finely divided solid carriers or both, and then, if necessary, shaping the product.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation appropriate for the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
The formulations may be presented in unit-dose or mufti-dose containers, for example sealed ampules, vials or syringes, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders. Dosage formulations may contain the Therapeutic protein portion ~at a lower molar concentration or lower dosage compared to the non-fused standard formulation for the Therapeutic protein given the extended serum half life exhibited by many of the albumin fusion proteins of the invention.
As an example, when an albumin fusion protein of the invention. comprises growth hormone as one or more of the Therapeutic protein regions, the dosage form can be calculated on the basis of the potency of the albumin fusion protein relative to the potency of hGH, while taking into account the prolonged serum half life and shelf life of the albumin fusion proteins compared to that of native hGH. Growth hormone is typically administered at 0.3 to 30.0 IU/kg/week, for example 0.9 to 12.0 IU/kg/week, given in three or seven divided doses for a year or more. In an albumin fusion protein consisting of full length HA
fused to full .Iength.GH, an equivalent dose in terms of units would represent a greater weight of agent but the dosage frequency can be reduced, for example to twice a week, once a week or less.
Formulations or compositions of the invention may be packaged together with, or included in a kit with, instructions or a package insert referring to the extended shelf life of the albumin fusion protein component. For instance, such instructions or package inserts may address recommended storage conditions, such as time, temperature and light, taking into account the extended or prolonged shelf-life of the albumin fusion proteins of the invention. Such instructions or package inserts may also address the particular advantages of the albumin fusion proteins of the inventions, such as the ease of storage for formulations that may require use in the field, outside of controlled hospital, clinic or office conditions. As described above, formulations of the invention may be in aqueous form and may be stored under less than ideal circumstances without significant loss of therapeutic activity.
.Albumin fusion proteins of the invention can also be included in nutraceuticals. For instance, certain albumin fusion proteins of the invention may be administered in natural products, including milk or milk product obtained from a transgenic mammal which expresses albumin fusion protein. Such compositions can also include plant or plant products obtained from a transgenic plant which e~cpresses the albumin fusion protein. The albumiri fusion protein can also be provided in powder or tablet form, with or without other known additives, carriers, fillers and diluents. Nutraceuticals are described in Scott Hegenhart, Food Product Design, Dee: 1993.
The invention also provides methods of treatment and/or prevention of diseases or disorders (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount. of an albumin fusion protein of the invention or a polynucleotide encoding an albumin fusion proteiw of the invention ("albumin fusion polynucleotide") in a pharmaceutically acceptable carrier.
The albumin fusion protein andlor polynucleotide will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the albumin fusion protein and/or polynucleotide alone), the site of, ~ delivery, the method of administration, the ~ scheduling of administration, and other factors known to . practitioners.
The "effective amount" for purposes herein is thus determined.by such considerations.
. As a general proposition, the total pharmaceutically effective, amount of the albumin fusion protein administered parenterally per dose will be in the range .of about lug/kglday to 10 mg/kg/day of -patient body weight, although, . as noted above, this 'will be subject to ~ therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably -for humans between about 0.01 and 1 mg/kg/day for the hormone. ~
If given continuously, the albumin fusion protein is typically administered at a dose rate of about 1 ug/kglhour to about 50 ~ug/kg/hour, either by 1-4. injections per day or by continuous subcutaneous- infusions, for example, using , a mini-pump. An intravenous bag solution may 35. also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
Albumin fusion proteins andfor polynucleotides can be ~ are administered orally, rectally, parenterally; intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray.
"Pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material 'or formulation auxiliary of any. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal°, subcutaneous and intraarticular injection and infusion.
Albumin fusion proteins and/or polynucleotides of the invention are also suitably administered by sustained-release systems. Examples of sustained=release albumin fusion proteins and/or polynucleotides are administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. "Pharmaceutically acceptable carrier"
refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of . administration which include intravenous, intramuscular, intraperitoneal, intrasternal,.
subcutaneous and intraarticular injection and infusion. Additional examples of sustained release albumin fusion proteins and/or polynucleotides include suitable-polymeric materials (such as, for~example, semi-permeable polymer matrices in the form of shaped articles, e. g., films, or mirocapsules), suitable hydrophobic materials (foi example as an emulsion in an acceptable oil) or ion ~ exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt). , . , Sustained-release matrices include polylactides (U.S. . Pat. No. '3,773,919, EP
58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2- hydroxyethyl methacrylate) (Langer et al., J.
Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), . ethylene vinyl acetate (Langer et al., Id.) or poly-D- (-)-3-hydroxybutyric acid (EP 133,988).
Sustained-release albumin fusion proteins aridlor polynucIeotides also include liposomally entrapped albumin fusion proteins and/or polynucleotides of the invention (see generally, Langer; Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease ai~cd Cancer, Lopez-Berestein and Fidler (eds.), Liss, -New York, pp.
. 317 -327 and 353-365 (1989)): Liposomes containing the albumin fusion.
protein and/or ' polynucleotide are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc.
Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad.
Sci.(USA) 77:403'0-4034 (i980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;
Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4;544,545; and EP
102,324.
Ordinarily, the Iiposomes are of the small (about 200-800 Angstroms) unilamellar type in which the. lipid- content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal. Therapeutic- ' ' In yet an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.
Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et aL, N.
Engl. J. Med. 321:574 ( 1989)).
Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 {1990)).
For parenteral administration, in one embodiment, the albumin fusion protein and/or polynucleotide is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form ~ (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.
Generally, -the formulations are prepared by contacting the albumin fusion protein and/or polynucleotide uniformly and intimately with liquid carriers .or finely divided solid carriers or both., Then, if necessary, the product is shaped into the desired formulation.
Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
The carrier suitably contains minor amounts of additives such' as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers , such as phosphate, ~ citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid;
. low molecular weight (less than about ten residues) polypeptides, ~ e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,. aspartic acid, or arginine; monosaccharides; disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA;
sugar alcohols such as niannitol or sorbitol; ~counterions such as sodium;
and/or nonionic surfactants such as polysorbates, poloxamers, or PEG. . ' ' The albumin fusion protein is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result " 35 in the formation of polypeptide salts.
Any pharmaceutical used for therapeutic administration can be sterile.
Sterility is readily accomplished by filtration through sterile fltration membranes (e.g:, 0.2 micron membranes). Albumin fusion proteins and/or polynucleotides generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial, having a stopper pierceable by a hypodermic injection needle.
Albumin fusion proteins and/or polynucleotides ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous albumin fusion protein and/or polynucleotide solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized albumin fusion protein and/or polynucleotide using bacteriostatic Water-for-Injection.
In a specific and preferred embodiment, the Albumin fusion protein formulations comprises 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromole sodium octanoatelmilligram of fusion protein, 15 micrograms/milliliter polysorbate 80, pH 7.2. In another specific and preferred embodiment, the Albumin fusion protein formulations consists 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 microinole sodium octanoate/milligram of fusion protein, IS micrograms/milliliter polysorbate 80, pH 7.2. The pH and buffer are chosen to match physiological conditions and the salt is added as a tonicifier. Sodium octanoate has been chosen due to its reported ability to increase the thermal stability of the protein in solution. Finally, polysorbate has been added as a generic surfactant, ~ which lowers the surface tension of the solution and lowers note-specific adsorption of the albumin fusion protein to the container closure system.
The invention also provides a pharmaceutical pack or kit comprising one or more containets'filled with one or more of the ingredients of the albumin fusion proteins and/or polynucleotides of the invention. Associated with such containers} can be a notice in the form prescribed by a governmental agency regulating 'the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. in addition, the albumin fusion proteins and/or polynucleotides may be employed in conjunction with other therapeutic compounds.
The albumin fusion proteins and/or polynucleotides of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not Limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), (Genentech, ~Inc.), BCG (e.g., THERACYS~), MPL 'and nonviable preparations of CorynelZacteriacm _ parvum. In 'a specific embodiment, albumin fusion ~
proteins . and/or polynucleotides of the invention are administered in combination . with alum.
In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with QS-21. Further adjuvants ,that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but. are not limited to, Monophosphoryl Lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the ~ albumin fusion proteins and/or polynucleotides of the invention . 5 include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, Haemophilus influenzc~e B, whooping cough, pneumonia, influenza, Lyme's Disease;
rotavirus, cholera, yellow fever, 3apanese encephalitis, ~ poliomyelitis, rabies, typhoid fever, and pertussis:
Combinations may be, administered either concomitantly, e.g., as an admixture, separately but 14 simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration "in combination" further includes the separate administration of one of the compounds or agents given first, followed 15 by the second.
The albumin fusion proteins and/or polynucleotides of the invention may be administered alone or in combination with other therapeutic agents. Albumin fusion protein and/or poiynucleotide agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include but not limited to, chemotherapeutic 20 agents, antibiotics, steroidal and non-steroidal anti-inflammatories, coilventional immunotherapeutic agents, and/or therapeutic treatments described below:
Combinations may .
be administered either concomitantly, e.g., as an admixture, separately. but simultaneously or ' concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined 25 agents are administered separately but simultaneously, e.g., as through separate- intravenous lines into the same individual. Administration "in combination" further includes the separate administration of one of the compounds or agents given first, followed by the second.
In one embodiment, the albumin fusion proteins and%or polynucleotides of the invention are administered in combination with an anticoagulant.
Anticoagulants that may be ' 30 administered with~the compositions of the invention include, but are not~limited to,~ heparin, low molecular weight heparin, warfarin sodium (e.g., COUMADIN~), dicumarol, 4-hydroxycoumarin, anisindione (e.g., MIRADONT'"), acenocoumarol (e:g., nicoumalone, SINTHROMETM), indan-1,3-dione, ~ phenprocoumon (e:g~., MARCUMARTM), ethyl biscoumacetate~ (e:g., TROMEXANTM ), and aspirin. In a specific embodiment, compositions 35 of the invention are administered in combination with heparin and/or warfarin. In another specific embodiment, compositions of the invention are administered iri combination with warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin and aspirin. In another specific embodiment, compositions of the invention are administered in combination with heparin. ~ In another specific embodiment, compositions of the invention are adrriinistered inlcombination with heparin and aspirin.
In another embodiment, the albumin fusion 'proteins and/or polynucleotides of the invention are administered in combination with thrombolytic drugs.
Thrombolytic drugs that may be administered with the compositions of the invention include, but are not limited to, . -plasminogen, lys-plasminogen, 'alpha2-antiplasmin, streptokinae (e.g., KABIKINASET'"), antiresplace (e.g., EIVIINASET'"), tissue plasminogen activator (t-PA, altevase, ACTIVASET'"), urokinase (e.g., ABBOKINASETM), sauruplase, (Prourokinase, single chain urok~nase), and aminocapro'ic acid (e.g., AMICART"°). In a specific embodiment, compositions of the invention are administered in combination with tissue plasminogen activator and aspirin.
In another embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with antiplatelet drugs.
Antiplatelet drugs that may be administered with the compositions of the invention include, but are not limited to, aspirin, dipyridamole (e.g., PERSANTINET'" ), and ~ticlopidine (e.g., TICLIDT"' ).
In specific embodiments, the use of anti-coagulants, thrombolytic and/or antiplatelet drugs in combination with albumin fusion proteins and/or. polynucleotides of the invention is contemplated for the prevention, diagnosis, and/or treatment of thrombosis, arterial thrombosis, venous thrombosis, thromboemboli~m, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the use of anticoagulants, thrombolytic drugs and/or antiplatelet drugs in combination with albumin fusion proteins and/or polynucleotides of the invention is contemplated for the prevention of occulsion of saphenous grafts, for reducing the risk of ~
periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atiial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses . for the therapeutics of the invention, alone or in combination with antiplatelet, anticoagulant, and/or thrombolytic drugs, include, but are not limited to, the prevention of occlusions in extracorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
In certain embodiments, albumin fusion proteins andlor polynucleotides of the invention are administered in combination with antiretroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors .
. (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, RETROVIRT"" (zidovudine/AZT), VIDEXT"' (didanosine/ddI), HIVIDT""

(zalcitabinelddC), ZERITT'" (stavudine/d4T), EPIVIRT"' (lamivudine/3TC), and COMBIVIRT"' (zidovudinellamivudine). NNRTIs that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, VIRAMUNET"' (nevirapine), RESCRIPTORT"' (delavirdine), and SUSTIVAT"' (efavirenz).
Protease inhibitors that may be administered in combination with the albumin fusion proteins ' and/or polynucleotides of the invention, include; but are not limited to, CRIXIVANT"' (indinavir), NORVIRT"' (ritonavir), INVIRASET"" (saquinavir), and VIRACEPTT""
(nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with albumin fusion proteins and/or polynucleotides of the invention .
to treat AIDS and/or to prevent or treat HIV infection.
Additional NRTIs include LODENOSINET"" (F-ddA; an acid-stable adenosine NRTI;
Triangle/Abbott; COVIRACILT"" (emtricitabine/FTC; structurally related to lainivudine (3TC) but with 3- to IO-fold greater activity in vitro; Triangle/Abbott); dOTC (BCH-10652, also structurally related to lamivudine but retains activity against a substantial proportion of lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused approval for anti-HIV
therapy by FDA; Gilead Sciences); PREVEON~ (Adefovir Dipivoxil, the active prodrug of .
adefovir; its active 'form. is PMEA-pp); TENOFOVIR'"' (bis-POC PMPA, a PMPA
prodrug;
Gilead); DAPD/DXG (active metabolite of DAPD; Triangle/Abbott); D-D4FC
(related to 3TC, with activity against AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome);
ZIAGENT""
(abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3'azido-2',3'-dideoxyuridine;
WO
99/66936); and S-acyl-2-thioethyl (SATE)-bearing prodrug forms of [3-L-FD4C
and (3-L-FddC (see, International Publication No. WO 98/17281).
Additional NNRTIs include COACTINONT"' (Emivirine/MKC-442, potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINET"" (AG-1549/S-1153, a next generation NNRTI with activity against viruses containing the K103N mutation; Agouron);
PNU-142721 (has 20- to 50-fold greater activity than its predecessor delavirdine and is active against K103N mutants; Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivatives of efavirenz, designed to be active against viruses with the K103N
mutation;
DuPont); GW-420867X (has 25-fold greater activity than HBY097 and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A (naturally occurring agent, from the latex tree; active against viruses containing either or both the Y181C and K103N mutations);
and Propolis (see, International Publication No. WO 99/49830).
Additional protease inhibitors . include LOPINAVIRr"' (ABT378/r; Abbott Laboratories); BMS-232632 (an azapeptide; Bristol-Myres. Squibb);
TIPRANAVIRT"" (PNU-140690, a .non-peptic dihydropyrone; Pharmacia ~& Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-Myers Squibb);
L-756,423 (an indinavir analog; Merck); DMP-4-50 (a cyclic urea compound; Avid &
DuPont);
AG-1776 (a peptidomimetic with in vitro activity against protease inhibitor-resistant viruses;
Agouron); VX-175/GW-4.33908 (phosphate prodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); and AGENERASET"' (amprenavir; Giaxo Wellcome Inc.).
Additional antiretroviral agents include fusion inhibitors/gp41 binders.
Fusion inhibitors/gp4l binders include T-20. (a peptide from residues 643-678 of the HIV gp41 transmembrane protein ectodomain which binds to gp41 in its resting state and prevents transformation to the fusogenic state; Trimeris) and T-1249 (a second-generation fusion.
inhibitor; Trimeris). . . ' , Additional antiretroviral agents include fusion inhibitors/chemokine receptor antagonists. Fusion inhibitors/chemokine receptor ~ antagonists include CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and its analogs; and ALX40~1.C (a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and the T22 analogs T 134 and T 140;
CCRS
antagonists such-as RANTES (9-68), AOP RANTES, NNY-RANTES, and TAK-779; and CCRS/CXCR4 antagonists such as NSC 651016 (a distamycin. analog). Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetor agonisfs such as RANTES, SDF-1, MIP-la, MIP-1(3, etc., may also inhibit fusion.
Additional antiretroviral agents include integrase inhibitors. Integrase inhibitors include dicaffeoyiquinic (DFQA) acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid);
quinalizarin (QLC) and related anthraquinones; ZINTEVIRT"" (AR 177, an oligonucleotide that probably acts at cell surface rather than being a true integrase inhibitor;
Arondex); and naphthols such as those disclosed in WO 98/50347.
Additional antiretroviral agents include hydroxyurea-like compunds such as BCX-(a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide~
reductase inhibitors such as DIDOXT"" (Molecules for Health); inosine ~ monophosphate, dehydrogenase (IMPDH) inhibitors sucha as VX-497 (Vertex); and mycopholic acids such as CelICept (mycophenolate mofetil; Roche).
Additional antiretroviral agents include inhibitors of viral integrase, inhibitors of viral 35. genome nuclear translocation such as arylene bis(methylketone) compounds;
inhibitors of HIV entry such ~as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES and glycosaminogIycans (GAG), and AMD-3100; nucleocapsid zinc finger inhibitors such as dithiane compounds; targets . of HIV Tat and Rev;
and pharmacoenhancers such as ABT-378.
Other antiretroviral therapies and adjunct therapies include cytokines and lymphokines such as MIP-la, MIP-1[3, SDF-la, IL-2,. PROLEUKIN~" (aldesleukin/L2-7001;
Chiron), IL-4-, IL-10, IL-12, and IL-13; interferons such as IFN-a2a; antagonists of TNFs, NFKB, GM-CSF, M-CSF, and IL-10; agents that modulate immune activation such as cyclosporin and prednisone; vaccines such as RemuneT"" (HIV Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinant envelope glycoprotein,-rgp120CM235, MN rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL
protein, branched synthetic peptide derived from discontinuous gp120 C3/C4 domain, fusion-competent immunogens, and Gag; Pol, Nef, and Tat vaccines; gene-based therapies such as genetic suppressor elements (GSEs; WO 98/54366), and intrakines (genetically modified CC
chemokines targetted to the ER to block surface expression of newly synthesized CCRS
(Yang et al., PNAS 94:11567-72 (1997); Chen et al., Nat. Med. 3:1110-16 (1997));
. antibodies such as the anti-CXCR4 antibody 1265, the anti-CCRS antibodies 2D7, SC7, PAB, PA9, PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the . anti-CCR3 antibody 7B 11, the anti-gp 120 antibodies 17b, 48d, 447-52D, 257-D, 268-D and .
50:1, anti-Tat antibodies, anti-TNF-a antibodies, and monoclonal antibody 33A;
aryl hydrocarbon (AH) receptor agonists and antagonists such as TCDD, 3,3',4,4',5-pentachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and a-naphthoflavone (see, International Publication No. WO 98130213); and antioxidants such as y-L-glutamyl-L-cysteine ethyl ester ('y-GCE; WO 99/56764).
, _ ' In a further embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.
In other, embodiments, albumin fusion proteins . and/or . polynucleotides of the invention may be administered in combination with anti-opportunistic infection' agents. Anti opportunistic agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, TRIMETHOPRIM
SULFAMETHOXAZOLET"", ,DAPSONET'", PENTAMIDINET"', ATOVAQUONET"', ISONIAZID'"', RIFAMPINt"', PYRAZINAMIDET"', ETHAMBUTOLT"", RIFABUTINT"', CLARITHROMYCIN'"', AZITHROMYCINT"', GANCICLOVIRT"", FOSCARNET~', CIDOFOVIRT"", FLUCONAZOLET"', ITRACONAZOLET"", KETOCONAZOLET"', ACYCLOVIRTM, FAMCICOLVIRT"', PYRiMETHAMINET"', LEUCOVORINT"", NEUPOGENT'" (filgrastim/G-CSF), and LEUKINET"" .(sargramostim/GM-CSF). Iri a specific embodiment, albumin fusion , proteins and/or polynucleotides of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLET"', DAPSONET'", PENTAMIDINET"", and/or ATOVAQUONET"' to prophylactically treat or prevent an opportunistic Pneccmocystis carinii . pneumonia infection. In another specific embodiment, albumin fusion proteins and/or polynucieotides of the invention are used in any combination with ISONIAZIDT"', RIFAMPINT"", PYRAZINAMIDET"', and/or ETHAMBUTOLT"' to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection.
In another specific embodiment, albumin fusion proteins andlor polynucIeotides of the invention are used in any combination with RIFABUTINT"", CLARITHROMYCINT"', and/or AZ1THROMYCINT"' to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific- embodiment, ' albumin fusion proteins and/or polynucleo6des of the invention ~ are used in any combination with GANCICLOVIR'"", FOSCARNETT'", and/or CIDOFOVIRT"" to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with FLUCONAZOLET"", ITRACONAZOLET"', and/or KETOCONAZOLET"' to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with ACYCLOVIRT'"
and/or FAMCICOLVIRT"" to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with PYRIMETHAMINET"' and/or LEUCOVORINT"' to prophylactically treat or .prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with LEUCOVORINT"" and/or NEUPOGENT"' to prophyiactically treat or prevent an opportunistic bacterial infection. - -In a further embodiment, the albumin fusion proteins andlor polynucleotides of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to; amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), . beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.
In other embodiments, the albumin fusion. proteins and/or polynucleotides of the invention are administered in combination with immunestimulants.
Imrimnostimulants that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, levamisole (e.g., ERGAMISOLTM), '15 isoprinosine (e.g. INOSIPLEXTM), interferons (e.g. interferon' alpha), and interleukins (e.g., IL-2).
In other embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with immunosuppressive agents.
immunosuppressive agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, steroids, cyclosporine; cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, -15-deoxysperguaiin, and ~ other immunosuppressive agents that act by suppressing the function of responding T cells. Other immunosuppressive agents that. may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide,, . methoxsaien, rapamycin, leflunomide, mizoribine (BREDININ''M ), brequinar, deoxyspergualin, and azaspirane (SKF 105685), ORTHOCLONE OKT~ 3 (muromonab CD3), SANDIMMUNET"', NEORALT".", SANGDYAT"' (cyclosporine), PROGRAF~
- (FK506,.tacrolimus), CELLCEPT~ (mycophenolate motefil, of which the active metabolite is mycophenolic acid), IMURANT"' (azathioprine), glucocorticosteroids, adrenocortical steroids such as DELTASONET"' (prednisone) and HYDELTRASOLT'" (prednisolone), FOLEXTM .
and MEXATET"' (methotrxate), OXSORALEN-ULTRAT'" (methoxsalen) and RAPAMUNET"' (sirolimus). In a~ specific embodiment, immunosuppressants may be used to.
prevent rejection of organ or bone marrow transplantation.
In an additional embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered alone or in combination with one or more intravenous immune WO 01!79271 PCT/USO1/12009 globulin preparations. Intravenous immune globulin preparations that may be administered with the albumin fusion proteins andlor polynucleotides of the invention include, but not limited to, GAMMART"", IVEEGAMT"", SANDOGLOBULINT'", GAMMAGARD S/DT"', ATGAMT'~ (antithymocyte glubulin), and GAMIMUNE'"'. In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with intravenous immune globulin' preparations in transplantation therapy (e.g., bone marrow transplant).
In certain embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-.10 inflammatory agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, corticosteroids (e.g.
betarriethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, ,prednisone, and triamcinolone), nonsteroidal anti-inflanunatory , drugs (e. g., ' - diclofenac, diflunisal, etodolac, fenoprofen, floctafenine, flur.biprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, and tolmetin.), as well as antihistamines, amiiZOarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thia.zinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.
In an additional embodiment, the compositions of the invention are administered alone or in combination with an anti-angiogenic agent. Anti-angiogenic agents that may be administered with the compositions of the invention include, but are not limited to, Angiostatin (Entremed, Rockville, MD), Troponin-1 (Boston Life Sciences, Boston, MA), .
anti-Invasive Factor, retinoic acid aiid derivatives thereof, paclitaxel (Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1; Plasminogen Activator Inhibitor-2, and various forms of the lighter "d group" transition metals.- ~ _ Lighter "d group"' transition metals- include, for example, . vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. ' Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate. complexes include rrietavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
. Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten ,oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dehydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyi complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes IS include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
A wide variety of other anti=angiogenic factors may also be utilized within the context of the present invention. RepresentativeJexamples include, but are not limited to,. platelet factor 4;.protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, (i991)); Sulphated Polysaccharide Peptidoglycan Complex (SP- PG) (the function of this compound may be enhanced by the presence of . steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proIine analogs, cishydroxyproline, d,L-3,4 dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5 (4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin;
Interferons; : 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Biv. Chem. 267:17321-17326, (1992));
Chymostatin (Tomkinson et al., Biochem J. 286:475-480, (1992)); Cyciodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, (1990)); Gold Sodium Thiomalate ("GST"; Matsubara and Ziff, J. Clin. Invest.
79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J.
Biol. Chem.
262(4):1659-1664, (1987)); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4- chloroanthronilic acid disodium or "CCA"; (Takeuchi et al., ,Agents Actions 36:312-316, (1992)); and metalloproteinase inhibitors such as BB94.
Additional anti-angiogenic factors that may also be utilized within the context of the present invention include Thalidomide, {Celgene, Warren, ~NJ); Angiostatic steroid; AGM
1470 (H. Brem and J. Folkman J Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C. Storgard.et al., J Clin. Invest. 103:47-54 (1999));
carboxynaminolmidazole;
Carboxyamidotriazole (CAI) (National Cancer Institute, Bethesda,. M~);
Conbretastatin A~1-10i (CA4P) (OXiGENE, Boston, MA); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, PA); TNP-470, (Tap Pharmaceuticals, Dee~eld, IL); ZD-OI01 AstraZeneca (London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251 (PKC
412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol;
Genestein; GTE;
ImmTher; Iressa (ZD1839); Octreotide (Somatostatin); Panretin; Penacillamine;
Photopoint;
PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex);
Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and S-F'luorouracil.
Anti-angiogenic agents that may be administed in combination with the compounds of the invention may work through a variety of mechanisms including, but not limited to, inhibiting proteolysis of the extraceilular matrix, blocking the function of endothelial cell-extracellular matrix adhesion molecules, by antagonizing the function of angiogenesis inducers such as growth factors, and inhibiting integrin receptors expressed on proliferating endothelial cells. Examples of anti-angiogenic inhibitors that interfere with extracellular matrix proteolysis and which may be administered in combination with the compositons ~of the I S invention include, but are not lmited to, AG-3340 (Agouron, La Jolla, CA), (Bayer, West Haven, CT), BMS-275291 (Bristol Myers Squibb, Princeton, NJ), CGS-27032A (Novartis, East Hanover, NJ), Marimastat (British Biotech, Oxford, UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and which may be administered in combination with the compositons of the invention include, but are not lmited to,- EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla; CAlMedimmune, Gaithersburg, MD). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting angiogenesis inducers and which may be administered in combination with the compositons. of the invention include, but are not lmited to, Angiozyme (Ribozyme, Boulder, CO), Anti-VEGF antibody (Genentech, S. San Francisco, CA), PTK-787-IZK-225846 (Novartis, -Basel, Switzerland), SU-101 (Sugen, S. San Francisco, CA), SU-5416 (Sugenl Pharmacia Upjohn, Bridgewater, NJ), and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis which may be administered in combination with the compositoiis of the invention include, but are not limited to, IM-862 (Cytran, Kirkland, WA), Interferon-alpha, IL-12 (Roche, Nutley, NJ), and Pentosan polysulfate (Georgetown University, Washington, DC).
In particular embodiments, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration 35. tif,an autoimmune disease, such as for example, an autoimmune disease described.herein.
In a particular embodiment, the use ~of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, andlor amelioration of arthritis. In a more particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of rheumatoid arthritis.
In another embodiment, the polynucleotides encoding a polypeptide of the present , invention are administered in combination' with an angiogenic protein, or polynucleotides encoding an angiogenic protein. Examples of angiogenic proteins that may be administered with the compositions of the invention include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alpha and beta, platelet-derived endothelial cell 'growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
In additional embodiments, compositions of the invention are administered in combination with a chemotherapeutic . -agent. Chemotherapeutic agents that may. be I S administered with the albumiil fusion proteins and/or poIynucleotides of the invention include, but are not limited to ~ alkylating agents such as nitrogen mustards (for example, Mechlorethamine, cyclophosphamide, Cyclophosphamide Ifosfamide, Melphalan (L-sarcolysin), and Chlorambucil), ethylenimines and methylmelamines (for example, Hexamethylmelamine and Thiotepa), alkyl sulfonates (for example, Busulfan), nitrosoureas (for example, Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), and . Streptozocin (streptozotocin)), triazenes . (for example, Dacarbazine (DTIC;
dimethyltriazenoimidazolecarboxamide)), folic acid analogs (for example, -Methotrexate (amethopterin)), pyrimidine analogs (for example, Fluorouacil (5-fluorouracil;
5-FU), Floxuridine (fluorodeoxyuridine; FudR), and Cytarabine (cytosine arabinoside)), purine , analogs and related inhibitors (for example, Mercaptopurine~ (6-mercaptopurine; 6-MP), Thioguanine (6-thioguanine; TG), and Pentostatin (2'-deoxycoformycin)), vinca alkaloids (for example, Vinblastine (VL,B, vinblastine sulfate)) and Vincristine (vincristine sulfate)), epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics (for example, Dactinomycin (actinornycin D); Daunorubicin (daunomycin; rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), and Mitomycin (mitomycin C), enzymes ' (for example, L-Asparaginase), biological response modifiers (for example, Interferon-alpha and interferon-alpha-2b), platinum coordination compounds (for example, Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone), substituted, ureas (for example, Hydroxyurea), methylhydrazine derivatives (for example, Procarbazine (N-methylhydrazine;
MIH), adrenocorticostero'ids (for example, Prednisone); ' progestins (for example, Hydroxyprogesterone - caproate, Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol acetate), estrogens (for example, Diethylstilbestrol (DES), ~Dlethylstilbestrol 103.

WO 01/79271 PCT/USOl/12009 diphosphate, Estradiol, and Ethinyl estradiol), antiestrogens (for example, Tamoxifen), androgens (Testosterone proprionate, and Fluoxymesterone), antiandrogens (for example, Flutamide), gonadotropin-releasing horomone analogs, (for example, Leuprolide), other hormones and hormone analogs (for example, methyltestosterone; estramustine, estramustine phosphate sodium, chlorotrianisene, and testolactone), and others (for example, dicarbazine, .
glutamic acid, and mitotane).
In one embodiment, the compositions of the invention are administered in combination with one or more of the following drugs: infliximab (also known as RemicadeT"' Centocor, Inc.); Trocade (Roche, RO-32-3555), Leflunomide (also known as AravaTM from Hoechst Marion Roussel), KineretTM (an IL-1 Receptor antagonist aIso._known as Anakinra from Amgen, Inc.) . ~ ~ -- In a specific embodiment, compositions of the invention are administered in - combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or combination of one or more of the components of CHOP. In one embodiment, the compositions of the invention are administered ~in combination with anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. In another embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies and CHOP, or anti-CD20 antibodies and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In-a specific embodiment, compositions of the invention are administered in combination with Rituximab. In a further embodiment, compositions of the invention are administered with Rituximab and CHOP, or Rituximab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered . in combination with tositumomab. In a further embodiment, compositions of the invention are administered with tositurnomab and CHOP, or. tositumomab and any combination of .one or more of the components of CHOP, particularly cyclophosphamide andlor prednisone. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs. .
In another specific embodiment, the~compositions of the invention are administered in ~ combination ZevalinT"'. In a further embodiment, compositions of the invention are administered with ZevalinT"' and CHOP, or ZevaIinT'", and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone.
ZevalinT"' may 'be associated with one or more radisotopes. Particularly preferred isotopes are 9°Y and "lln.
In an additional embodiment, the albumin fusion proteins and/or polynucleotides of - ~ the invention are administered in combination . with cytokines. Cytokines that may be administered with the albumin fusion proteins and/or polynucieotides of the invention include, but are not limited to, IL2, IL3, IL4, ILS, IL6, IL7, ILiO, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF~alpha. In another embodiment, albumin fusion proteins and/or polynucleotides of the invention may be administered with any interleukin, including, but not limited to, IL-/alpha, IL-!beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-I1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-2i.
In one embodiment, the albumin fusion proteins andlor polynucleotides of the invention are administered in combination with members of the TNF~family. TNF, TNF-related or TNF-like molecules that may be administered with ~ the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, Fast, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I
(International Publication No. - -WO 97/33899), endokine-alpha (International Publication No.
WO
98!07880), OPG, and neutrokine-alpha (International Publication No. WO
98/18921, OX40~, .
and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TRZ (International Publication No. WO 96/34095), DR3 (International Publication No. WO
.- 97!33904), DR4 (International Publication No. WO- 98/32856), TR5 (International Publication No. WO 98/30693), TRANK, TR9 (International Publication No.~ WO
98/56892),TR10 (International Publication No. WO 98/54202), , 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.
In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with angiogenic proteins.
Angiogenic proteins that may be administered' with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth-Factor-A (PDGF-.-.-A), as disclosed ~in European Patent Number EP-682110; Platelet Derived Growth Factor-B
(PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PIGF), as disclosed in International Publication Numbe-r WO 92/06194;
Placental Growth Factor-2 (P1GF-2); .as disclosed in Hauser et al., Growth Factors, 4:259-268 (1993);
Vascular Endothelial Growth Factor {VEGF), as disclosed in Internatior<al Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO. 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); V ascular Endothelial Growth Factor B-186 (VEGF-B 186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D
(VEGF-D), as disclosed in International Publication Number WO 98/02543;
Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in 'International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are herein incorporated by reference iwtheir entireties.
.In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with Fibroblast Growth Factors.
Fibroblast Growth Factors that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, FGF-l, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11., FGF-12, -FGF-13, - v FGF-14, and FGF-15.
In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with hematopoietic growth factors.
Hematopoietic growth factors that may be administered with the albumin fusion proteins andlor polynucleotides of the invention include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostim, LEUKINETM , . PROKINETM), granuiocyte colony stimulating factor (G-CSF) (filgrastim, NEUPOGENT"'), macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin aifa, EPOGENTM, PROCRITT"'), stem cell factor (SCF, c-kit Iigand, steel factor), megakaryocyte colony stimulating factor, PIXY321 (a GMCSF/IL-3 fusion, protein), interleukins, especially any one or more of IL-1 through-IL-12, interferon-gamma, or thrombopoietin.
In certain embodiments, albumin fusion proteins andlor polynucleotides of the present invention are administered in combination with adrenergic blockers, such as, for example, acebutolol, atenolol, .betaxolol, bisoprolol, carteolol, labetalol, metoprolol, ' nadolol, oxprenolol, penbutolol, pindoiol, propranolol, sotalol, and timolol.
In another embodiment, the albumin fusion proteins and/or poIynucleotides of the invention are administered in combination with an antiarrhythmic drug (e.g., adenosine, amidoarone, bretylium, digitalis, digoxiri, digitoxin, diliazem, disopyramide, esmolol, flecainide, lidocaine, mexiletine, moricizine, phenytoin, procainamide, N-acetyl procainamide, propafenone, propranolol, quinidine, sotalol, tocainide,~and verapamil). .
In another embodiment, the albumin fusion proteins andlor polynucleotides of the , invention are administered in combination with diuretic agents, such as carbonic anhydrase inhibiting agents (e.g., acetazolamide, dichlorphenamide, and methazolamide), osmotic diuretics (e.g., -glycerin, isosorbide, mannitol, and urea), diuretics 'that inhibit Na+-K+-2C1~
symport (e.g., furosemide, bumetanide, azosemide, piretanide, tripamide, ethacrynic acid, muzolimine, and' torsemide), thiazide and thiazide-like diuretics (e.g., bendroflumethiazide, 35~ benzthiazide,, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, polythiazide., trichormethiazide, chlorthalidone, indapamide, metolazone, and quinethazone), potassium sparing diuretics (e.g., amiloride and triamterene), and mineralcorticoid receptor antagonists (e.g., spironolactone, canrenone, and potassium canrenoate).
In one embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with treatments for endocrine and/or hormone imbalance disorders. Treatments for endocrine and/or hormone imbalance disorders include, ~ but are not limited to, '2'I, radioactive isotopes of iodine such as 13'1 and '~I; recombinant growth hormone, such as HUMATROPET"' (recombinant somatropin); growth hormone analogs such as PROTROPINT"' (somatrem); dopamine agonists such as PARLODELT"' (bromocriptine); somatostatin analogs such as SANDOSTATINT"' (octreotide);
gonadotropin preparations such as PREGNYLT"", A.P.L.T'" and PROFASIT"' (chorionic gonadotropin (CG)), PERGONAL'"" (menotropins), and METRODINT"' (urofollitropin (uFSH));
synthetic human go~adotropin releasing hormone preparations such as FACTRELT"" and LUTREPULSET"' (gonadorelin hydrochloride); synthetic gonadotropin agonists such as LUPRONT"" (leuprolide acetate), SUPPRELINT"' (histrelin acetate), SYNARELT"' (nafarelin acetate), and ZOLADEXT"" (goserelin acetate); synthetic preparations of thyrotropin-releasing hornione such as RELEFACT TRHT"' and THYPINONET"' (protirelin); recombinant human TSH such as THYROGENT'"; synthetic preparations of the sodium salts of the natural isomers of thyroid hormones such as L-T4T"', SYNTHROIDT"" and LEVOTHROIDT""
(levothyroxine sodium), L-T3T"", CYTOMELr"' and TRIOSTATT"' (liothyroine sodium), and THYROLAR'""
(liotrix); antithyroid compounds such as 6-n-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazole and TAPAZOLET"' (methimazole), NEO-MERCAZOLE'"' (carbimazole);
beta-adrenergic receptor antagonists such as propranolol and esmolol; Ca2+
channel blockers;
dexamethasone and iodinated radiological contrast agents such as TELEPAQUET'"
(iopanoic acid) and ORAGRAFINT"' (sodium ipodate).
Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, estrogens or congugated estrogens such as ESTRACET""
(estradiol), ESTINYLT'" (ethinyl estradiol), PREMARINT"", ESTRATABT"", ORTHO-ESTT"', OGENTM
and estropipate (estrone), ESTROVISr"' (quinestrol), ESTRADERMT"" (estradiol), DELESTROGENTM and VALERGENT'" (estradiol valerate), DEPO-ESTRADIOL
_ <

CYPIONATETM and ESTROJECT LAT"' (estradiol cypionate); antiestrogens such as.
NOLVADEXT"' (tamoxifen), SEROPHENET"' and CLOMIDT"' (clomiphene); progestins such as DURALUTINT"' (hydroxyprogesterone caproate), MPAT"" and DEPO-PROVERAT"' (medroxyprogesterone acetate), PROVERAT"" and CYCRINT"" (MPA), MEGACET""
(megestrol acetate), NORLUTINT'" (norethindrone), and NORLUTATET"" and AYGESTINT"' (norethindrone acetatej; progesterone implants such as NORPLANT SYSTEMT'"
(subdermal implants of norgestrel); antiprogestins such as RU 486T"' (mifepristone);
hormonal contraceptives such as ENOVIDT"' (norethynodrel plus mestranol), PROGESTASERTT"' (intrauterine device that releases progesterone), LOESTRINT"', BREVICONT"', MODICONT"", GENORAT"', NELONAT"", NORINYLT"", OVACON-35T"' and OVACON-50T"' (ethinyl estradiol/norethindrone), LEVLENT"", NORDET'fE'"", TRI-LEVLENT"'~and TRIPHASIL-21T""
(ethinyl estradiol/levonorgestrel) LO/OVRALT"' and OVRALTM (ethinyl estradiol/norgestrel), DEMULENT"" (ethinyl estradiol/ethynodiol diacetate), NORINYLT"", ORTHO-NOVUMT"", NORETHIN'"", GENORAT"", and NELOVAT"' (norethindronelmestranol), DESOGENT""
and ORTHO-CEPTT"" (ethinyl estradiol/desogestiel), ORTHO-CYCLENT"' and ORTHO-TRICYCLENT'" (ethinyl estradiol/norgestimate), MICRONORT"" and NOR-QDT'"
(norethindrone), and OVRETTET"" (norgestrel).
Additional treatments~for endocrine and/or hormone imbalance disorders include, but are not limited to, testosterone esters such as methenolone acetate and testosterone undecanoate; parenteral and oral androgens such as TESTOJECT-50T'"
(testosterone), TESTEXT"" (testosterone propionate), DELATESTRYLT"' (testosterone enanthate), DEPO-TESTOSTERONET"' (testosterone cypionate), DANOCRINET"" (danazol), HALOTESTINT"' (fluoxymesterone), ORETON METHYLT"', TESTREDT"' and VIRILON'"' (methyltestosterone), and OXANDRINT"" (oxandrolone); testosterone transdermal systems such as TESTODERMT""; androgen receptor antagonist and 5-alpha-reductase inhibitors such as ANDROCURT'" (cyproterone acetate), EULEXINT"' (flutamide), and . PROSCART"' (finasteride); adrenocorticotropic hormone preparations such as CORTROSYNT"' (cosyntropin); adrenocortical steroids and their synthetic analogs such as ACLOVATE'""
(alclometasone dipropionate), CYCLOCORTT"' (amcinonide), BECLOVENTT"' and VANCERILT'" (beclomethasone dipropionate), CELESTONET"" (betamethasone), BENISONET"' and UTICORTT"' (betamethasone benzoate), DIPROSONET"' (betamethasone dipropionate), ~CELESTONE. PHOSPHATE?"" (betamethasone sodium phosphate), CELESTONE SOLUSPANT"' (betamethasone sodium phosphate and acetate), BETA-VALT"' and VALISONET'" (betamethasone valerate), TEMOVATE1"' (clobetasol propionate), CLODERMT"" (clocortolone pivalate), CORTEFT"' and HYDROCORTONET."' . (cortisol (hydrocortisone)), HYDROCORTONE AC>JTATET"' (cortisol (hydrocortisone) acetate), LOCOIDT"" (cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATE?"' (cortisol (hydrocortisone) sodium phosphate), A-HYDROCORTT"" and SOLU
CORTEFT"' (cortisol (hydrocortisone) sodium succinate), WESTCORTT"' (cortisol (hydrocortisone) valerate), CORTISONE ACETATE?"' (cortisone acetate), DESOWENT"" and TRIDESILONT"", (desonide), TOPICORTT'" (desoximetasone), DECADRONT"' (dexamethasone), DECADRON
~TM (dexamethasone acetate), DECADRON PHOSPHATET""~ and HFX_A_DROL
PHOSPHATE'"" (dexametliasone sodium phosphate), FLORONET"" and MAXIFLORT"' (diflorasone diacetate), FLORINEF ACETATE?"' (fludrocortisone acetate), AEROBIDr"' and NASALIDET"' (flunisolide), FLUONIDT"' and SYNALART"" (fluocinolone acetonide), LIDEX'"" (fluocinonide), FLUOR-OPT"° and FMLT"°
(fluoiometholone), CORDRAN'"' (flurandrenolidej, HALOGT"" (halcinonide), HMS LIZUIFILMT"" (medrysone), MEDROLT""
(methylprednisolone), DEPO-MEDROLT"" and MEDROL ACETATE?"' (methylprednisone acetate), A-METHAPREDT"" and SOLUMEDROLT'" (methylprednisolone sodium succinate), ELOCON'"" (mometasone furoate), HALDRONE'"' (paramethasone acetate), DELTA-CORTEFT"' (prednisolone), ECONOPREDT"" (prednisolone acetate), HYDELTRASOLT'"
(prednisolone sodium phosphate), HYDELTRA-T.S.AT"" (prednisolone tebutate), DELTASONE~"' (prednisone), ARISTOCORTT"' and KENACORTT"' (triamcinolone), KENALOGT"' (triamcinolone acetonide), ARISTOCORTT"' and KENACORT DIACETATET"~
(triamcinolone diacetate), and ARISTOSPANT"' (triamcinolone hexacetonide);
inhibitors of biosynthesis and ~ action . of adrenocortical steroids such as CYTADREN'""
(aminoglutethimide), NIZORALT"" (ketoconazole), MODRASTANET"" (trilostane), and METOPIRONET"" (metyrapone); bovine, porcine or human insulin or mixtures thereof; insulin analogs; recombinant human insulin such as HUMULINT"" and NOVOLINT""; oral hypoglycemic agents such as ORAMIDET"" and ORINASET"' (tolbutamide), DiABINESET""
(chlorpropamide), TOLAMmET'" and TOLINASET"' (tolazamide), . DYMELORT""
(acefohexamide), glibenclamide, MICRONASET"', DIBETA"" and GLYNASET"' (glyburide), GLUCOTROL'"" (glipizide), and DIAMICRONT"' . (gliclazide), GLUCOPHAGET"' (metformin), ciglitazone, pioglitazone, and alpha-glucosidase inhibitors;
bovine or porcine gIucagon; somatostatins such as SANDOSTATIN7"° (octreotide); and diazoxides such as PROGLYCEMT"' (diazoxide).
In one embodiment, the albumin fusion ~ proteins and/or polynucleotides of the . invention are administered in combination with treatments for uterine motility disorders.
Treatments~for uterine motility disorders include, but are not limited to, estrogen drugs such as conjugated estrogens (e.g., PREMARIN~ and ESTRATAB~), estradiols (e.g., CLIMARA° and ALORA~), estropipate, and chlorotrianisene; progestin drugs (e.b., AMEN~
(medroxyprogesterone), MICRONOR~ (norethidrone acetate), PROMETRIUM~
progesterone, and megestrol acetate); and- estrogen/progesterone combination therapies such as, for example, conjugated estrogenslmedroxyprogesterone (e.g.;. PREMPROT"' and PREMPHASE~) and norethindrone acetate/ethinyl estsradiol (e.g., FEMHRTT'").
L
In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with drugs effective in treating iron deficiency and hypochromic anemias, including but not limited to, ferrous ~ sulfate (iron sulfate, FEOSOLTM), ferrous fuinarate (e.g., FEOSTATT~'), ferrous gluconate (e.g., FERGONTM), polysaccharide-iron complex (e.g., NIF'EREXT"'), iron dextran injection (e.g., INFEDT'"), cupric sulfate, pyroxidine, riboflavin, Vitamin B,2, cyancobalamin injection (e.g., REDISOLT."' , RUBRAMIN PCT'" ), hydroxocobalamin, folic acid (e.g., FOLVITE''M
), leucovorin (folinic acid, 5-CHOH4PteGlu, citrovorum factor) or WELLCOV012IN
(Calcium salt of leucovorin), transferrin-or ferritiri.
Iw certain embodiments, the albumin fusion proteins andlor polynucleotides of the invention are administered in combination with agents used to treat psychiatric disorders.
Psychiatric drugs that may be ' administered with the albumin fusion proteins and/or polynucieotides of the invention include, but are not limited to, antipsychotic agents (e.g., chlorpromazine, chlorprothixene, clozapine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, quetiapine, risperidone, thioridazine, thiothixene, trifluoperazine, and triflupromazine), antimanic agents (e.g., carbamazepine, divalproex sodium, lithium carbonate, and lithium 'citrate), antidepressants (e.g., amitriptyline, amoxapine, bupropion, citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, ~isocarboxazid, maprotiIine, mirtazapine, nefazodone, nortriptyline, paroxetine,_,phenelzine, protriptyline, ~sertraline, tranylcypromine, trazodone, trimipramine, and venlafaxine), antianxiety agents (e.g., - alprazolam, buspirone, chlordiazepoxide, clorazepate, diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulants.(e.g., d-amphetamine, methylphenidate, and pemoline).
- In other embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with agents used to treat neurological disorders. ' .
Neurological agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, antiepileptic agents (e.g., carbamazepine; clonazepam, ethosuximide, phenobarbital, phenytoin, primidone, valproic acid, divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazeparn, and clonazepam), antiparkinsonian-agents (e.g., levodopa/carbidopa, selegiline, -amantidine, bromocriptine, pergolide, ropinirole, pramipexole, benztropine; biperiden; ethopropazine; procyclidine;
trihexyphenidyl, tolcapone), and ALS therapeutics (e.g. riluzole).
In another embodiment, albumin fusion proteins andlor polynucleotides of the invention are administered in' combinatioil with vasodilating agents and/or calcium. channel blocking agents. Vasodilating agents that may be administered with the albumin fusion . proteins andlor polynucleotides of the invention include, but are not limited to, Angiotensin Converting Enzyme (ACE) inhibitors (e.g., papaverine, isoxsuprine, benazepril, captopril, cilazapril, enatapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril, and nylidrin), and nitrates (e.g., isosorbide dinitrate,, isosorbide mononitrate, and nitroglycerin). Examples of calcium channel blocking agents that may be administered in combination with the albumin fusion proteins andlor polynucleotides of the invention include, but are not Limited to amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, nimodipirle, and verapamil.
In certain embodiments, the albumin fusion proteins andlor polynucleotides of the invention are administered in combination with treatments for gastrointestinal disorders.
Treatments for gastrointestinal disorders that. may be administered with the albumin,fusion protein and/or polynucleotide of the invention include, but are not limited to, Hz histamine receptor antagonists (e.g., TAGAMETTM (cimetidine), ZANTACTM (ranitidine), PEPCIDTM
(famotidine), and AXID~'~'' (nizatidine)); inhibitors of H+, K~ ATPase (e.g., PREVACIDT'~' (lansoprazole) and - PRILOSECTM (omeprazole}); Bismuth compounds (e. g., PEP'TO-BISMOLT"' (bismuth subsalicylate) and DE-NOL~'~'~' (bismuth subcitrate});
various antacids;
sucralfate; prostaglandin analogs (e.g. CYTOTEC'~"'' (misoprostol));
muscarinic cholinergic antagonists; laxatives (e.g., surfactant laxatives, stimulant laxatives, saline and osmotic laxatives); antidiarrheal agents (e.g., LOMOTIL~ (diphenoxyiate), MOTOFENT"' (diphenoxin); and IMODIUM~ (loperamide hydrochloride)), synthetic analogs of somatostatin such as SANDOSTATINTM (octreotide), antiemetic agents (e.g., ZOFRAN~
(ondansetron), KYTRILTM (granisetron hydrochloride), _ tropisetron, dolasetron, rrietoclopramide, chlorpromazine, perphenazine, prochlorperazine, .
promethazine, thiethylperazine; triflupromazine, domperidone, haloperidol, droperidol, trimethobenzamide, dexamethasone, methylprednisolone, dronabinol, and nabilone); D2 antagonists (e.g:, metoclopramide, trimethobenzamide and chlorpromazine); bile salts;
chenodeoxycholic acid;
~ ursodeoxycholic acid; and pancreatic enzyme preparations such as pancreatin and pancrelipase.
In additional, embodiments, the, albumin fusion proteins andlor polynucleotides of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions comprising albumin fusion proteins of the invention. Optionally associated with such containers) can be'a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human.administration. , Gene Theraw Constructs encoding albumin fusion proteins of the invention can be used as a part of a gene therapy protocol to deliver therapeutically effective doses of the albumin fusion protein. A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing, nucleic acid, encoding an albumin. fusion protein of the invention.

Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA~contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
Retrovirus vectors and adeno-associated- virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous nucleic acid molecules encoding albumin fusion proteins i~ vivo. These vectors provide efficient delivery of nucleic acids into cells, and the transferred riucleic acids are stably integrated into the chromosomal DNA of the host.
The development of specialized cell lines , (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:27 1). A replication defective retrovirus can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al., (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.
Another viral gene delivery system useful in the present invention uses adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of , interest but is inactivated in terms of its ability' to replicate in a normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al.,' Cell 68:143-155 (1992). Suitable adenoviral vectors derived from the adenoyirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., ,(1992) cited supra).
Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modif ed so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign~DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional-mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g.; retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., cited supra; Haj-Ahmand et al., J. Virol. 57:267 (1986)).
In another.embodiment, non-viral gene delivery systems of the present'invention rely on endocytic pathways for the uptake of the subject nucleotide molecule by the targeted cell.

Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and -artificial viral envelopes. In a representative embodiment, a nucleic acid molecule encoding an albumin fusion protein of the invention can be entrapped in liposomes bearing positive charges on their surface ~(e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-5 5 1; PCT publication W091106309; Japanese patent application 1047381; and European patent publication EP-A-43075).
Gene delivery systems for a gene encoding an albumin fusion protein of 'the invention can be introduced into a patient by any of a number of methods. For instance, a ~ pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g.
by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to -the . transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more Limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S.
Patent 5,328,470) or by Stereotactic injection (e.g. Chen et al. (1994) PNAS
91: 3 054-3 05 7). The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Where the albumin fusion protein can be produced intact' from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can comprise one or more cell's which produce the albumin fusion protein.
Additional Gene Therapy Methods - Also encompassed by the invention are gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of an albumin fusion protein of the invention.
This method requires a polynticleotide which codes for an albumin fusion protein of the present invention , operatively linked to a promoter and any other genetic elements necessary for the expression of the fusion protein by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, W090111092, which is herein incorporated by reference.
Thus, for example, cells from a, patient may be engineered with.a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide encoding an albumin fusion protein of the present invention ex vivo; with the engineered cells then being provided to a patient to be treated with the fusion protein of the present invention. Such methods are well-known in the art. For example, see Belldegrun, A., et al., .1~. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini,~ M. et al., Cancer Research 53: iI07-1112 (1993);
Ferrantini, M. et al., J. Immunology 153: 4604-4615 (I994); Kaido, T., et al.,, Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990);
Santodonato, L., et al., Human Gene. Therapy 7:1=10 (1996); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injectioin to the artery, the tissues surrounding the artery, or through catheter injection.
As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
In 'one embodiment, poIynucleotides encoding the albumin fusion proteins of the.
present invention is delivered as a naked polynucleotide. The term "naked"
polynucleotide, .
DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the Like. However, polynucleotides encoding the albumin fusion proteins of the present invention can also be delivered in liposome formulations and lipofectin formulations and the -like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S.
. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein ~
incorporated by reference.
The polynucleotide vector constructs used in the gene therapy method are.
preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia;
and pEFl/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.
Any strong promoter known to those skilled in the art can be used for driving the . , expression of the polynucleotide sequence. Suitable promoters include adenoviral promoters, such as the adenoviral major late° promoter; , or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the 'metallothionein promoter; heat shock promoters;
the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes SirnpIex thymidine kinase promoter; retroviral LTRs; the b-acon promoter; and human 'growth hornione promoters. The promoter also may be the native promoter for the gene corresponding to the Therapeutic protein portion of the albumin fusion proteins of the invention.
Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to .six months.
The polynucleotide construct can be delivered, to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial . space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such .20 ~ as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express-polynucleotides.
.For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to aiiout 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mglkg and more 25~ preferably from about 0.05 mglkg to about 5 mg/kg. Of course, as the artisan of ordinary skill will 'appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.
30 The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may .also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous ~membranes~ of the nose. In ~ addition, naked DNA constructs can be delivered to arteries during angioplasty by,the catheter used in the procedure.
35 The naked polynucleotides are delivered by any method known in the 'art, including, but not limited to, , direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called ".gene guns". These delivery .methods are known in the art.
The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc.
Such methods of .delivery are known in the art.
In certain embodiments, the polynucleotide constructs are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively chaiged) and neutral preparations.
However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl.
Acad. Sci. USA
(1987) 84:7413-7416, which .is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci.. USA (1989) 86:6077-6081, which is herein incorporated by reference); ~ and purified transcription factors (Debs et al., '. J. ' Biol.
Chem. (1990) 265:10189-10192, which is herein incorporated by reference), in functional form.
- 15 ~ Cationic liposomes . are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammoniuin (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, - N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA {1987) 84:7413-7416, which is herein incorporated by reference). Other commercially available liposomes .
include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
Other cationic liposomes can be prepared from readily available materials using .
techniques well known in the art. See, e.g. PCT Publication No. WO '90/11092 '(which is herein' incorporated by reference) for a description ~ of the synthesis of DOTAP ( 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOrTMA
liposomes is explained in the literature, see, e.g., P. Felgner et al., Proc. Natl.
Acad.-~Sci. USA
84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials. ~ . y .
Similarly, anionic and neutral Iiposomes are readily available, such as from Avanti Polar Lipids (Birmingham,. Ala.), or can be easily prepared using readily available materials.
Such materials include phosphatidyl, choline,. cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making . liposomes using these materials are well known in the art.
3 S For example, ~ commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol' (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial.
The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized w water. The sample~is then sonicated for 2 hours in a capped-vial, using a Heat Systems model 5__ 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles. or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art. .
The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et aL, Methods of Immunology (1983), .101:512-527, which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by IS depositing a thin. film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar Iiposomes. The material to be entrapped is added to a suspension of. preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water br an isotonic buffer solution such as 10 mM
Tris/NaCI, sonicated, and then the preformed liposomes are mixed directly with the DNA.
The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the~cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca2+-EDTA
chelation (Papahadjopoulos et al., Biochim.- Biophys. 'Acts (1975) 394:483;
Wilson et al., Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A.,_ Biochim.
Biophys. Acta 443:629 (1976);~Ostro et al., Biochem.. Biophys. Res. Commun. 76:836 (1977);
Fraley et al., Proc. Natl. Acad. Sci. USA 76:3348 (1979)); detergent dialysis (Enoch, H.
and Strittmatter, P., Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc. ~Natl. Acad. Sci. USA 75:1.45 (1978); Schaefei-Ridder et al., Science 215:166 (-I982)), which are herein incorporated by reference.
Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10.
Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:l.
. U.S. Patent No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S.

Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication 'no. WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for .use in transfecting DNA into cells and mammals. U.S. Patent Nos. x,589,466, 5,693,622, 5,580,859,. 5,703,055, and international publication no. WO 94/9469 provide methods for delivering DNA-cationic lipid complexes to mammals.
In certain embodiments, cells are engineered, ex- vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding an albumin fusion protein of the present invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. _ The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86; GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in its entirety.. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP04 precipitation. Iri one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid; and then administered to a host.
The producer cell line generates infectious retroviral vector particles which include _ polynucleotide encoding an albumin fusion protein of the present invention.
Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
The transduced eukaryotic cells will express a fusion protin of the present invention.
In certain ~ other embodiments, cells are engineered, ' ex viyo or in vivo, with polynucleotide contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses fusion protein of the present invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been-used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al. Am. Rev. Respir. Dis.109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M. A. ~ et al. (1991) Science 252:431-434;
Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensive studies' to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M: et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).
Suitable adenoviral vectors~useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet: Devel. 3:499-503 ~( 1993);
Rosenfeld et al., Cell 68:143-155 (i992); Engelhardt et al., Human Genet: Ther. 4:759-769 (1993);
Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692 (1993); and U.S.
Patent No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the El region.-of adenovirus and constitutively express Ela and Elb, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, AdS, and ~Ad7) are also useful in the present invention. .
Preferably, the adenoviruses used in the present invention are replication deficient.
Replication deficient adenoviruses .require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: Ela; Elb, E3, E4, E2a, or L1 through L5. ~ ~ ' In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr. Topics in Microbiol.
Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. .
Methods for ~ producing and using such AAVs are known in the art. See, for example, U.S.
Patent Nos.
5,139,941, 5,173,414, 5,354,678,.5,436,146, 5,474,935, 5,478,745, and 5,589,377.
For example, an appropriate AAV vector foi- use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleot'ide construct-is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., . Molecular Cloning: A Laboratory Manual, .Cold Spring Harbor Press ( 1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, 'including lipofection, electroporation, calcium phosphate precipitation, etc.
Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses., Once the packaging cells are transfected and infected, they will produce infectious AAV
viral particles which contain the polynucleotide construct. These viral particles are then used, to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will , contain the polynucleotide construct integrated into its genome, and will express a fsuion protein of the invention.
Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding a polypeptide of the present invention) via homologous recombination (see, e.g., U.S. Patent No. 5,641,670, issued June 24, 1997; International Publication No. WO 96/29411, published September 26, '1996;
International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc.
Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), which are herein encorporated by reference. This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter.
Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the S' end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. ' .
_ The promoter and the targeting sequences can be amplified using PCR.
Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5' and 3' ends.
Preferably, the 3' end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5' end of the second targeting sequence contains the same restriction site as the 3' end of the amplified promoter..
The amplified promoter and targeting sequences are digested and ligated together.
The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating,agents, such as liposomes, viral sequences, viral -particles, whole viruses, lipofection, precipitating agents, etc.;
described in more detail above. The P promoter-targeting sequence can be delivered by arty method, included direct needle injection, intravenous injection, topical administration; catheter infusion, particle accelerators, etc. The methods are,described in more detail below. , The promoter-targeting sequence .construct is taken up by cells: ' Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous.sequence is placed under the control of the promoter. The promoter.then drives the expression of the endogenous sequence.
. The polynucleotide encoding an albumin fusion protein of the present invention may contain a secretory signal sequence that facilitates secretion of the protein.
Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5' end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.
. Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., "gene guns"), gelfoam sponge depots, other commercially available depot materials, osmotic-pumps ~ (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasinid into the portal vein has resulted in gene expression of the foreign gene in the rat livers (Kaneda et al., Science 243:375 (1989)). , ~ A preferred method of local administration is by direct injection.
Preferably, an albumin fusion protein of the present invention complexed with a delivery ~
vehicle is administered by direct injectiowinto or locally within the area of arteries.
Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries. . ~ ~, Another method of local adrriinistration is to contact a polynucleotide coristruct of the present invention in or around a surgical wound. For example, a patient can undergo surgery . and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the iwound.
Therapeutic compositions useful in systemic administration, include fusion proteins of the present invention complexed to a targeted delivery vehicle ~of the present invention.
. Suitable delivery' vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site. In specific embodiments, suitable delivery vehicles for use with systemic administration comprise liposomes comprising albumin fusion proteins of the invention for targeting the vehicle to a particular site.
Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) ' delivery. 'Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for. example, Stribling et al., Proc. Natl. Acad. Sci. USA
189:11277-11281, 1992,. which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct. of the present invention to a carrier capable of withstaniiing degradation by digestive enzymes in the gut .of an animal_ Examples of such carriers, include plastic capsules or tablets, such as these known in the art.
Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing.into the skin.
Determining an effective amount of substance to be delivered can depend upon a ' number of factors including, for example, the chemical structure and biological activity of the substance, the 'age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatrnents depends upon a number of factors,. such-as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian.
Albumin fusion proteins of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.
Biological Activities Albumin fusion proteins and/or polynucleotides encoding albumin fusion proteins of the present invention, can,be used in assays to test for one or more biological activities. If an ' albumin fusion protein and/or polynucleotide exhibits an activity in a particular assay, it is likely that the Therapeutic protein corresponding to the fusion portein may be involved in the diseases associated with the biological activity. Thus, the fusion protein could be used to treat the associated disease.
Members of the secreted family of proteins are believed to be involved in biological activities associated with, for example, cellular signaling. Accordingly, albumin fusion _ proteins of the invention and polynucleotides encoding these protiens, may be used in ' . diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders associated . with aberrant activity of secreted polypeptides.
In a preferred embodiment, albumin fusion proteins of the invention comprising a Therapeutic protein portion corresponding to EPO, iminunoglobulins, hirudin, applaggiri, serum cholinesterase, alpha-1 antitrypsin, aprotinin, and coagulation factors in both pre and active forms (e.g., including, but not limited to, eon Willebrand factor, fibrinogen, factor Ii, factor VII, factor VIIA activated factor, factor VIII, factor IX, factor X, factor XIII, c1 inactivator, antithrombin III, thrombin and prothrombin, apo-lipoprotein, c-reactive protein, and protein C) and/or fragments and/or variants thereof may be used to modulate hemostatic (the stopping of bleeding) .or thrombolytic (clot dissolving) activity .
and/or treat, prevent, diagnose, prognose, and/or detect blood-related disorders or. cardiovascular disorders andlor diseases, disorders or conditions as described under "Blood Related Disorders", "Anti-WO 01/79271 PCT/iTS01/12009 Angiogenesis Activity", and/or "Cardiovascular Disorders" infra.
In a preferred embodiment, albumin fusion proteins of the invention.
comprising a Therapeutic protein portion corresponding to Interferon alpha, G-CSF, GM-CSF, scatter factor, MCP/MCAF, M-CSF andlor fragments and/or variants thereof may be used to treat, prevent, diagnose,.prognose, and/or detect diseases or disorders of the immune system, or diseases, disorders or conditions as described under "Immune Activity", "Infectious Disease", and/or "Hyperproliferative Disorders" infra.
In . a preferred embodiment, albumin fusion proteins of the invention comprising a Therapeutic protein portion 'corresponding to human Growth hormone and/or fragments andlor variants thereof may be used to treat, prevent, diagnose, prognose, and/or detect disease, disorders and/or conditions related to growth hormone deficiency, including but not, limited to, Acromegaly, Growth failure, Growth failure and endogenous growth hormone replacement, Growth hormone ~ deficiency, ' Growth failure and growth retardation, Prader-Willi syndrome in children 2 years or older, Growth deficiencies, Postmenopausal . osteoporosis, burns, cachexia,~cancer cachexia, dwa~sm, metabolic disorders, obesity, renal failure, Turner's Syndrome, fibrorriyalgia, fracture treatment, frailty, or as described under "Endocrine Disorders", "Wound Healing and Epithelial Cell Proliferation", and/or "Hyperproliferative Disorders" infra. , In preferred embodiments, fusion proteins of the present invention may be used in the diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders relating to diseases .and disorders of the endocrine system (see, for example, "Endocrine Disorders"
section below), the nervous system {see, for example, "Neurological Disorders"
section below), .the immune system (see, for example, "Immune Activity" section below), respiratory system (see, for example, "Respiratory .Disorders" section below), ' cardiovascular system ~ (see, for,example, "Cardiovascular Disorders" section below), reproductive system (see, for . example, "Reproductive System Disorders" section- . below) digestive system (see, for example, "Gastrointestinal Disorders" section below), diseases and/or disorders relating to cell proliferation (see, for example, "Hyperproliferative Disorders" section below), and/or diseases or' disorders relating to the blood (see, for example, '.'Blood-Related Disorders"
section below).
In preferred embodiments, the present invention encompasses.a method of treating a disease or disorder listed in the "Preferred Indication'~Y" column of Table 1 comprising administering to a patient in which such treatment, prevention or amelioration is desired an albumin fusion protein of the invention that comprises a - Therapeutic protein portion corresponding to a Therapeutic protein disclosed in the "Therapeutic Protein X" column ~ of Table 1 (in the same row as the disease or disorder to be treated is listed in the "Preferred Indication Y" column~bf Table 1) in an amount effective to treat, prevent or'ameliorate the disease or disorder.
In certain embodiments, an albumin fusion protein of the present invention may be used to diagnose and/or prognose diseases and/or disorders associated with the tissues) in which the gene corresponding to the Therapeutic protein portion of the fusion portien of the 5- invention is expressed. - .
Thus, fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the- invention are useful~,in the diagnosis, detection andlor, treatment of. diseases andlor disorders associated with activities that include, but are 'not limited to, prohormone activation, neurotransmitter activity, cellular signaling, cellular proliferation, cellular ~10 differentiation, and cell migration.
More generally, fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention may be useful for the diagnosis, prognosis, prevention and/or treatment of diseases ~ and/or disorders 'associated with the. following systems.
Immune Activity Albumin fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, diagnosing and/or prognosing diseases, disorders, and/or conditions of the immune system, by, for example, activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T
lymphocytes) cells from. pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy or toxins), or infectious.
Moreover,. fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as a marker or detector of a particular immune system disease or disorder.
In another embodiment, a fusion protein of the .,invention and/or polynucleotide encoding an albumin fusion protein of the invention, may be used to treat diseases and disorders of the immune system and/or to inhibit or enhance an immune response generated by cells associated with the tissues) in which the polypeptide of the invention is, expressed.
Albumin fusion proteins of the invention- and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, diagnosing, and/or . 35 prognosing immunodeficiencies, including both congenital and acquired immunodeficiencies.
Examples of B cell ,immunodeficiencies in which immunoglobulin levels B cell function and%or B cell numbers are decreased include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with' hyper IgM, X-linked lymphoproliferative~
syndrome (XLP), agammaglobulinemia , including congenital and acquired . agammaglobulinemia, adult onset agammagiobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type),' Selective IgM deficiency, selective IgA
deficiency, selective IbG subclass deficiencies, IgG subclass deficiency (with or without. IgA
deficiency), Ig deficiency with increased IgM, IgG and IgA defciency with increased IgM, antibody. deficiency with normal or elevated Igs, Ig heavy chain deletions, kappa chain deficiency, B cell Iymphoproliferative disorder (BLPD), common variable immunodeficiency (CVID), common ~ variable immunodeficiency (CVI) {acquired), and transient hypogamrriaglobulinemia of infancy.
In specific embodiments, ataxia=telangiectasia or conditions associated with ataxiar ~-telangiectasia are treated, prevented, diagnosed, and/or prognosing using the, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
Examples of congenital immunodeficiencies in which T cell and/or B cell function and/or number is decreased include, but are not limited to: DiGeorge anomaly, severe combined immunodeficiencies (SCID) {including, but not limited to, X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome, and. ataxia telangiectasia), thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous candidiasis, natural killer cell defciency (NK), idiopathic CD4+ T-lymphocytopenia, immunodeficiency with predominant T
cell defect (unspecified), and unspecified immunodeficiency of cell mediated immunity.
~ In specific embodiments, DiGeorge anomaly or conditions associated with DiGeorge anomaly are treated, prevented, diagnosed, and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
Other immunodeficiencies that may be' treated, prevented, diagnosed, and/or prognosed using fusion proteins of the invention and/or polynucleotides .
encoding albumin fusion proteins of the invention, include, but are not limited to, chronic granulomatous disease, Chediak-Higashi. syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency, - X-linked Iymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, rcomplement component deficiencies (including Cl, C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphoplasia-aplasia; immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short Iimbed dwarfism, .and N,ezelof syndrome-combined immunodeficiency with Igs:

In a preferred embodiment, the immunodeficiencies and/or conditions associated with the imrnunodeficiencies recited above are treated, prevented, diagnosed and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
In a preferred embodiment fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals. ~ In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used as an agent to boost immunoresponsiveness among B
cell and/or T cell immunodeficient individuals. .
The albumin fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins .of the invention may be useful in treating, preventing, diagnosing , and/or prognosing autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue.
Therefore, the administration of fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention that. can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective , therapy in preventing autoimmune disorders.
Autoimmune diseases or disorders that may be treated, prevented, diagnosed and/or prognosed by fusion proteins of the invention and/or polynucIeotides encoding albumin fusion proteins of the invention include, but are not limited to, one or more of the following:
systemic lupus erythematosus, rheumatoid arthritis; ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, purpura (e.g., Henloch-Scoenle~n purpura), autoimmunocytopenia, 'Goodpasture's syndrome., Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and insulin-resistant diabetes mellitus.
_ Additional disorders that are likely to have an autoimmune component that may be treated, prevented, and/or diagnosed with the albumin fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention include, but are not limited .to, type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff Man Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disorders.
Additional disorders that are likely to have art autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not~limited to, scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease , (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis {often characterized, 10. e.g., by glomerular basement membrane' antibodies or immune complexes), bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus (often~characterized, e.g., by cell-mediated and humoral islet cell - antibodies), and adrenergic drug resistance (including adrenergic drug ~15 resistance with asthma or cystic fibrosis) (often characterized, e.g:, by beta-adrenergic .
receptor antibodies).
Additional disorders that may have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not 20 Limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitochondria antibodies), other endocrine gland failure (often characterized,.e.g., by specific tissue antibodies in some cases), vitiligo Often characterized, e.g., by. melanQCyte antibodies), vascuIitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often 25 characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., .by IgG
and IgM
antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to - IgE), asthma (often characterized, e.g., by IgG and IgM antibodies to IgE), and many other inflammatory; granulomatous, degenerative, and atrophic disorders. .
30 In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using for example, fusion proteins of the invention and/or polynucleotides .encoding albumin fusion proteins of the invention. In a specific preferred embodiment, rheumatoid arthritis is treated; prevented, and/or diagnosed using fusion proteins of the 35 invention and/or polynucleotides encoding albumin fusion proteins of the invention:
In another specific preferred embodiment, systemic lupus erythematosus is treated, prevented, and/or diagnosed using fusion proteins of the. invention and/or polynucleotides encoding albumin fusion proteins of the invention. In another specific preferred embodiment, idiopathic thrombocytopenia purpura is treated, prevented, and/or diagnosed using fusion proteins of the invention and/or poIynucleotides encoding albumin fusion proteins of the invention.
In another specific preferred embodiment IgA nephropathy is treated, prevented, and/or diagnosed using- fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
- In preferred embodiments, fusion proteins of . the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a immunosuppressive agent(s). .
Albumin fusion- proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, ~ preventing, prognosing, andlor diagnosing diseases, disorders, and/or .conditions of hematopoietic cells.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells, including but not limited to, leukopenia, . neutropenia, anemia, and thrombocytopenia.
Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of~ the invention could be used to increase differentiation and proliferation of . hematopoietic cells, including the pluripotent stem cells, in an effort to treat oi- prevent those diseases, disorders, and/or conditions associated with an increase in certain (or many) types of ~hematopoietic cells, including but not limited to, histiocytosis.
Allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, diagnosed and/or prognosed using fusion proteins of the invention .and/or polynuoleotides encoding albumin fusion ' proteins of the invention. Moreover, these molecules can be used to treat, prevent, prognose, and/or. diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
Additionally, fusion proteins of the invention and/or polynucleotides .
encoding.
albumin fusion proteins of the invention, may be used to treat, prevent, diagnose and/or . prognose IgE-mediated allergic reactions. Such allergic reactions include, but are not. limited to, asthma, rhinitis, and eczema. In specific embodiments, fusion proteins of the invention and/or polynucleotides encodinb albumin fusion proteins of the invention may be used to modulate IgE concentrations in vitro or in vivo.
Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention have uses in the diagnosis, prognosis, prevention, and/or treatment of inflammatory conditions. For example, since fusion proteins of the invention f and/or polynucleotides encoding albumin fusion proteins of the invention may inhibit the activation, proliferation and/or differentiation of cells involved in an inflammatory response, these molecules can be used to prevent and/or treat chronic and acute inflammatory . conditions. Such inflammatory conditions include, ~ but are not limited to, for example, inflammation associated with. infection (e.g., septic shock, sepsis, or systemic inflammatory ,response syndrome), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, , nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, over production ~of cytokines (e.g., TNF or IL-1.), -respiratory disorders (e.g., asthma and allergy); gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS .
disorders (e.g., multiple sclerosis; ischemic brain injury and/or stroke, traumatic brain injury, neurodegenerative disorders (e.g., Parkinson's ~ disease and Alzheimer's disease); AIDS-related dementia; and prion disease); cardiovascular disorders (e.g., ~atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); as well as many additional .diseases, conditions, and disorders that are characterized by inflammation (e.g., hepatitis, rheumatoid arthritis, gout, trauma, pancreatitis;
sarcoidosis, dermatitis, renal ischemia-reperfusion ~ injury, Grave's disease, systemic lupus erythematosus, diabetes mellitus, and allogenic transplant rejection), Because inflammation is a fundamental defense mechanism, inflammatory disorders can effect virtually any tissue of ,the body. Accordingly, fusion proteins of the. invention and/or polynucleotides encoding albumin fusion proteins of the invention, have uses in the.

treatment of tissue=specific inflammatory disorders, including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachitis, fibrositis, foliiculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis, mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis, orchitis, osteochondritis, otitis, pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis, ' scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and vaginitis_ ~ . -In specific embodiments, fusion _proteins of the invention and/or polynucleotides WO 01/79271 PCT/USO1l12009 encoding albumin fusion proteins of the inventiori, are useful to diagnose, prognose, prevent, ~. and/or treat organ transplant rejections and graft-versus-host disease.
Organ rejection occurs by host immune cell. destruction of the transplanted tissue through an immune response..
Similarly, an immune, response is also involved in GVHD, but, in this case, the .foreign transplanted ' immune cells destroy the host tissues. ' Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy,in preventing organ rejection or GVHD. In specific - embodiments; fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, that inhibit an immune response, particularly the activation, proliferation, ~ differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing experimental allergic and hyperacute xenograft rejection.
In other embodiments, fusion proteins of , the invention and/or polynucleotides encoding albumin fusion proteins of the invention, are useful to diagnose, prognose, prevent, and/or treat immune complex diseases, including, but not~limited to, serum sickness, post streptococcal. glomerulonephritis, polyarteritis nodosa, and immune complex-induced vasculitis.
Albumin fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention can be used to treat, detect, andlor prevent infectious agents.
For example, by increasing the immune response, particularly increasing the proliferation activation and/or differentiation -of B and/or T cells, infectious diseases may be treated, detected, and/or prevented. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins. of the invention may also directly inhibit the infectious agent (refer to section of application listing infectious agents, etc), without necessarily eliciting an immune response. . ' in another embodiment, albumin fusion . proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used ~
as a vaccine adjuvant that enhances immune responsiveness. to an antigen. In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance tumor-specific immune responses.
In..another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvailt to , enhance anti-viral immune responses.. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include virus and virus associated diseases or symptoms described herein or otherwise known in the art: In specific embodiments, the compositions of, the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of:
AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIVIAIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B , parainfluenza, measles, cytomegalovirus, rabies, 3unin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, Botulism, and meningitis type B.
In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom ~ selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B
streptococcus, Shigella spp.; Enterotoxigenic Escherichia coli,.
Enterohemorrhagic E. coli, and Borrelia burgdorferi.
In.. another. specif c embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art., In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an 30~. immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria) or Leishmania. . ' In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin. fusion proteins of the invention may also-be employed to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis;
for example, by preventing the recruitment and activation of mononuclear phagocytes:
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an antigen for the generation of antibodies to inhibit or enhance immune mediated responses against polypeptides of the invention.
In one embodiment; albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are administered to an animal (e. g., mouse, . rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, LgA, IgM, and IgE), to induce higher amity antibody production and immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), andlor to increase an immune response.
In another specific embodiment, albumin fusion proteins of the invention and/or _ polynuc(eotides encoding albumin fusion proteins of the invention are used as a stimulator of B cell responsiveness to.pathogens. ' ' _ In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an activator of T cells.
In another specific embodiment, albumin fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention are used as an agent that elevates the immune status of an individual ~ prior to their receipt of immunosuppressive therapies.
In another specific embodiment, albumin fusiom proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to induce higher afFnity antibodies.
In another specific embodiment, albumin fusion proteins of the invention and/or 25- polynucleotides encoding albumin fusion proteins of the invention are used as an agent to increase serum imrimnoglobulin concentrations.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to accelerate recovery of immunocompromised individuals.
In another specif c embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding alburriin fusion proteins of the invention are used as- an agent to boost immunoresponsiveness among aged populations and/or neonates.
In another specific embodiment, albumin fusion proteins of the invention and/or ' polynucieotides encoding albumin fusion proteins of the invention are used as an immune system enhancer prior to, during, .or _after bone marrow transplant andlor other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, andlor after 133 ~ ' WO 01/79271 PCTlIJSOI/12009 transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, prior to the beginning of recovery of T-cell populations. In another specific embodiment, compositions of the invention are f rst administered after transplantation after the beginning of recovery of T cell populations; but prior to full recovery of B cell populations.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention .are used as an agent to boost immunoresponsiveness among individuals having an acquired loss bf B cell function.
Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the albumin fusion proteins.of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to., HN
Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
In another specific embodiment, albumin fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency.
Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the albumin fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, and recovery from surgery.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albunun fusion proteins of the invention are used as a regulator of antigen presentation by monocytes, dendritic cells, and/or B-cells. In one embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention enhance antigen presentation or antagonizes antigen presentation in vitro or in vivo. Moreover, in related embodiments, this enhancement or antagonism of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to direct an individual's immune system towards development of a humoral response (i.e. ~TH2) as opposed to a TH1 cellular response.
~In another specific- embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins ~of the invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and i~s thus refractory' to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a stimulator of B cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable Immunodificiency.
. In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect. In another specific embodiment, albumin fusion proteins of the' invention and/or polynucleotides encoding albumin fusion proteins of the invention ar-a used in the pretreatment of bone marrow samples prior to transplant.
.In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used. as a gene-based therapy for genetically inherited disorders resulting in , immuno incompetence/immunodeficiency such as observed among SCID patients.
In another specif c embodiment, albumin fusion proteins of the invention and/or - polynucleotides encoding albumin fusion proteins of the invention are used ~as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes ' such as Leishmania.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of regulating secreted cytokines that are elicited by polypeptides of the invention.
In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used in one or more of the applications decribed herein, as they may apply to veterinary medicine.
In another specific embodiment, albumin fusion proteins of the invention and/or -polynucleotides encoding albumin fusion proteins of the invention are used as a means of blocking various aspects of immune - responses to foreign agents or self.
Examples of diseases or conditions in which blocking of certain aspects of immune responses may be desired include autoimmune disorders such _ as lupus, and arthritis, as well as immunoresponsiveness to skin allergies, inflammation, bowel disease, injury _ and diseases/disorders associated with pathogens. .
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for preventing the B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis. , WO 01179271 PCT/i1S01/12009 In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention invention are used as a inhibitor of B
and/or T cell migration in endothelial cells. This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.
In another specific embodiment, albumin fusion proteins of the indention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for chronic hyperga.mmaglobulinemia evident in such diseases as monoclonal gammopathy of 10- undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monoclonal gammopathies, and plasmacytomas.
In another specific embodiment, albumin fusion proteins . of the invention and/or polynucleotides encoding albumin fusion proteins of the 'invention may be employed for instance to inhibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CDS cytotoxic T cells and natural killer cells, in certain autoimmune and chronic inflammatory and infective diseases. Examples of autoimmune diseases are described herein and include multiple sclerosis, and insulin-dependent diabetes.
The albumin fusion proteins of the invention and/or polynucleotides encoding albumin ~ fusion proteins of the invention may also be employed to treat idiopathic hyper-eosinophilic-syndrome by, for example, preventing eosinophil production and migration.
In another specific embodiment, albumin fusion proteins of the .invention and/or polynucleotides. encoding albumin fusion proteins of the invention are used to enhance or inhibit complement mediated cell lysis.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to enhance or inhibit antibody dependent cellular cytotoxicity.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed for treating atheiosclerosis, for example, by preventing monocyte infiltration in the artery wall.
~In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be employed to treat adult respiratory distress syndrome CARDS). -In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful for stimulating wound and tissue repair, stimulating angiogenesis, and/or stimulating the repair of vascular or lymphatic diseases or disorders: Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to stimulate the regeneration of mucosal surfaces.
~In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used ,to diagnose, prognose, treat, and/or prevent ~ a disorder characterized by primary or acquired . immunodeficiency, deficient serum immunoglobulin production, recurrent infections, andlor immune system dysfunction. Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis,' septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, ' and malignancies, and/or any disease or disorder or condition associated with these, infections, diseases, disorders and/or malignancies) including, but not limited,to, CVID, other primary immune deficiencies, HN
disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carnii.
Other diseases and disorders that may be prevented, diagnosed, prognosed, and/or treated with fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, HN infection, HTLV-BLV
infection, lymphopenia, phagocyte, bactericidal dysfunction anemia, thrombocytopenia, and hemoglobinuria. , In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are , used to treat, andlor diagnose an individual ~ having common variable immunodeficiency disease ('~CVID"; also ' known as "acquired agammaglobulinemia" and "acquired hypogammaglobulinemia") or a subset of this disease. ,.
In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to diagnose, prognose, prevent, and/or treat cancers or neoplasms including immune cell. or immune tissue-related cancers or neoplasms. Examples- of cancers or neoplasms that may be prevented, diagnosed, or treated by fusion proteins of the invention andlor polynucleotides - encoding albumin fusion proteins of the invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases, and/or ,diseases and disorders described in the section entitled "Hyperproliferative Disorders"'elsewhere herein.
. ~ In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for decreasing cellular proliferation of Large B-cell Lymphomas.
In another ~ specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of decreasing the involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.
In specific embodiments, the compositions of the invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy.
Blood-Related Disorders In a preferred embodiment, albumin fusion proteins of the invention comprising a Therapeutic.~protein portion corresponding to immunoglobulins, serum cholinesterase, alpha-1 antitrypsin, aprotinin, and coagulation factors y both pre and active forms (e.g., including, but not limited to, von Willebrand factor, fibrinogen, factor II, factor VII, factor VILA
activated factor, factor VIII, factor IX, factor X, factor XIII, c1 inactivatox., ~antithrombin III, thrombin and prothrombin, apo-lipoprotein, c-reactive protein, and protein C) and fragments and/or variants thereof may be used to modulate hemostatic (the stopping of bleeding) or thrombolytic (clot dissolving) activity and/or treat, prevent, diagnose, prognose, and/or detect blood-related disorders. .
The albumin fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention may be used to- modulate. hemostatic (the stopping of bleeding) or thrombolytic (clot dissolving) activity. For example, by increasing hemostatic or thrombolytic activity, fusion proteins of the invention and/or polynucleotides encoding albumin fusiom proteins of the invention could be-used to treat or prevent blood coagulation diseases, disorders, and/or conditions . (e.g., afibrinogenemia, factor deficiencies, hemophilia),~blood platelet diseases, disorders, and/or conditions (e.g.;~
thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. -Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting.
These molecules could be important in the treatment or prevention of heart attacks (infarction), strokes, ~or scarring.
In specific embodiments, the albumin fusion , proteins of the ~ invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to prevent, diagnose, prognose, and/or treat thrombosis arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, ~ atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the albumin fusion proteins of the inventiow and/or polynucleotides encoding albumin fusion proteins of the invention may be used for , the prevention of occulsion of saphenous grafts, for reducing the risk of .periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atria/ fibrillation including nonrheumatic atria/ f brillation, for reducing the risk of embolism associated with=mechanical heart valves and or nutral valves disease. Other uses for the albumin fusion proteins of the invention and/or polynucleotides encoding alburriiri fusion proteins of the invention, include, but are not limited' to, the prevention of occlusions in extrcorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis ~ machines, and cardiopulmonary bypass machines). ~ .
In another embodiment, albumin fusion ~ proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to prevent, diagnose, prognose, and/or treat diseases and disorders of the blood and/or blood forming organs associated with the tissues) in which the polypeptide of the invention is expressed.
The fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to modulate hematopoietic activity (the formation of blood cells). For example, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to increase the quantity of all or subsets of blood cells, such as, for, example,-erythrocytes, lymphocytes (B
or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of anemias and leukopenias , described below. Alternatively, the albumin fusion proteins of the invention and/or polynucleotides encoding~albumin fusion proteins of the invention may be used to decrease the quantity of all or subsets of blood cells, such ~as, for example, erythrocytes, lymphocytes (B~ or T cells), .myeloid cells (e.g., basophils, -eosinophils, neutrophils, mast cells, macrophages) and platelets.. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of leukocytoses, such as, for example eosinophilia.
'The fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to prevent, treat, or diagnose blood dyscrasia.
Anemias are conditions in which the number of red blood cells or amount of hemoglobin (the protein that carries oxygen) in them is below normal. Anemia may be caused by excessive bleeding,, decreased red blood cell production, or increased red blood cell destruction (hemolysis). The albumin_fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the, invention may be ' useful in treating, preventing, and/or diagnosing anemias. Anemias that may be treated prevented or diagnosed liy the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion 13'9 ' proteins of the invention include iron deficiency anemia, hypochromic anemia, microcytic anemia, chlorosis, hereditary siderob;astic anemia, idiopathic acquired sideroblastic anemia, red cell aplasia, megaloblastic anemia (e.g., pernicious anemia, (vitamin BI2 deficiency) arid folic acid deficiency anemia), aplastic anemia, hemolytic anemias-(e.g., autoimmune helolytic anemia, ~microangiopathic hemolytic anemia, and paroxysmal nocturnal hemoglobinuria). The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing,, and/or diagnosing anemias associated with diseases including but not limited to, anemias associated with systemic lupus erythematosus, cancers, lymphomas, chronic renal disease, and enlarged spleens. The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing anemias arising from drug treatments such as anemias associated with methyldopa, dapsone, andlor sulfadrugs. Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or I5 diagnosing anemias associated with abnormal red blood cell architecture including but not limited to; hereditary spherocytosis, hereditary elliptocytosis, glucose-6-phosphate dehydrogenase deficiency, and sickle cell anemia..
'The albumin fusion proteins of the invention and/or polynucleotides ~
encoding albumin fusion proteins of the invention may be useful in treating, preventing, , and/or diagnosing hemoglobin abnormalities, (e.g., those associated with sickle cell anemia, hemoglobin C
disease, hemoglobin S-C disease, and hemoglobin E disease). Additionally, the albumin.
fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of . ' the invention may be useful in diagnosing, prognosing, preventing, andlor treating thalassemias, including, but not limited to, major and minor forms of alpha-thalassemia and beta-thalassemia. ' . In another embodiment; the ~ albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may. be useful in . diagnosing, prognosing, preventing, and/or treating bleeding disorders including, but not limited to, thromliocytopenia (e.g., idiopathic thrombocytopenic~purpura, and ,thrombotic thrombocytopenic purpura), Von Willebrand's disease, hereditary platelet disorders (e.g., storage pool disease such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome), hemolytic uremic syndrome, hemophelias such as hemophelia A or Factor VII deficiency and Christmas disease or Factor IX deficiency, Hereditary . Hemorhhagic Telangiectsia, also known as Rendu-Osler-Weber syndrome, allergic ' purpura . (Henoch Schonlein ' purpura) and disseminated intravascular coagulation. . .
The effect of the albumin fusion proteins' of the invention andlor polynucleotides encoding albumin fusion proteins of the invention on the clotting time of blood may be monitored using any of the clotting tests known in the art including, but not limited to, whole blood partial thromboplastin time (PTT), the activated partial thromboplastin time (aPTT), the activated clotting time (ACT), the recalcified activated clotting time, or the Lee-White Clotting time.
Several diseases and a variety of drugs can cause platelet dysfunction. Thus, in a specific embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating acquired platelet dysfunction such as platelet dysfunction accompanying kidney failure,leukemia, multiple myeloma, cirrhosis of the liver, and systemic lupus erythematosus as well as platelet dysfunction associated with drug treatments, including treatment with aspirin, ticlopidine, nonsteroidal anti-inflammatory drugs (used for arthritis, pain, and sprains), and penicillin in high doses. . .
~In another embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders characterized by or associated with increased or decreased numbers of white blood cells.
Leukopenia occurs when the number of white blood cells decreases below normal. L_eukopenias include, but are not limited to, neutropenia and lymphocytopenia. An increase in the number of white blood cells compared to normal is known as leukocytosis. The body generates increased numbers of white blood cells during infection. Thus, Ieukocytosis may simply be a normal physiological parameter that reflects infection. Alternatively, leukocytosis may be an indicator of injury or other disease such as cancer. Leokocytoses, include but are. not limited to, ~eosinophilia, and accumulations of macrophages.. In specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin.fusion proteins of the invention may be useful in diagnosing, , prognosing, preventing, andlor treating leukopenia. In other specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating leukocytosis.
Leukopenia may be a generalized decreased in all types of white blood cells, or may be a specific depletion of particular types of white blood cells. Thus, in specific embodiments, the albumin fusion proteins of the_invention andlor polynucIeotides encoding albumin fusion 'proteins of , the invention may be useful in diagnosing, ~prognosing, preventing, and/or treating decreases in neutrophil numbers, known as neutropenia. , Neutropenias that may be diagnosed, prognosed, prevented, and/or treated by the albumin fusion proteins -of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, infantile genetic agranulocytosis, familial neutropenia, cyclic neutropenia, neutropenias resulting from or associated with dietary deficiencies (e.g., vitamin B 12 deficiency or folic acid deficiency), .neutropenias resulting from' or associated with drug treatments (e.g., antibiotic regimens such as penicillin treatment, sulfonamide treatment, anticoagulant treatment, anticonvulsant drugs, anti-thyroid drugs, and - cancer chemotherapy), and neutropenias resulting from increased neutrophil destruction that ' may occur in association with - some bacterial - or viral infections, allergic disorders, autoimmune diseases, conditions imwhich an individual has an enlarged spleen (e.g., Felty syndrome, malaria and sarcoidosis), and some drug treatment regimens. ' The albumin fusion proteins of the invention and/or polynucieotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating lymphocytopenias (decreased numbers of B and/or T lymphocytes), including, but not limited to, lymphocytopenias resulting from or associated with stress, drug treatments (e.g., drug treatment with corticosteroids, cancer chemotherapies, and/or radiation therapies), AIDS infection and/or other diseases such as, for example, cancer, rheumatoid arthritis, systemic lupus erythematosus, chronic infections, some viral infections and/or hereditary disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich Syndome, severe combined iminunodeficiency, ataxia telangiectsia). ~ ' ' The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders associated with macrophage numbers and/or macrophage function including, but not limited to, Gaucher's disease, Niemann-Pick disease, Letterer-Siwe disease and Hand-Schuller-Christian disease.
In another '-embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, andlor treating diseases and disorders associated with eosinophil numbers and/or eosinophil function including, but not limited to, idiopathic hypereosinophilic syndrome, ebsinophilia-myalgia syndrome, and Hand-Schuller-Christian disease. - .
In yet another embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin . fusion -proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating leukemias and lymphomas including, but not.liinited to, acute lymphocytic (lymphpblastic) leukemia (ALL), acute myeloid (myelocytic, , myelogenous, myeloblastic, or myelomonocytic)- leukemia, chronic lymphocytic leukemia (e.g., B cell Ieukemias, T cell leukemias, Sezary syndrome, and- Hairy cell leukenia), chronic myelocytic (myeloid, myelogenous, or granulocytic) leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, and mycosis fungoides. -In other embodiments, the albumin fusion proteins of . the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders of plasma cells including, but not limited to, plasma cell dyscrasias, monoclonal gammaopathies, monoclonal gammopathies of ~ undetermined significance, multiple myeloma, macroglobulinemia, Waldenstrom's macroglobulinemia, cryoglobulinemia, and Raynaud's phenomenon.
In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the.invention may be useful in treating, preventing, and/or. diagnosing myeloproliferative disorders, including but not limited to, polycythemia vera, relative polycythemia, secondary polycythemia, myelofibrosis, acute myelofibrosis, agnogenic myelod metaplasia, thrombocythemia, (including both primary and seconday thrombocythemia) and chronic myelocytic leukemia. , In other 'embodiments, the albumin fusion proteins of the invention andlor . polynucleotides encoding albumin fusion proteins of the invention may be useful, as a treatment prior to surgery, to increase blood cell production.
In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to enhance'the migration,. phagocytosis, superoxide production, antibody dependent cellular cytotoxicity of neutrophils, eosionophils arid macrophages.
In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to increase the number .of stem cells in circulation prior to stem cells pheresis. In another specific- embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to increase the number of stem cells in circulation prior to platelet pheresis.
~ In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to increase cytokine production.
In other embodiments, the ~ albumin fusion proteins of the invention and/or . polynucleotides encoding albumin fusion proteins of the 'invention may be useful in preventing, diagnosing, and/or treating primary hematopoietic disorders.
H~neruroliferative Disorders .
In certain embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be ~ used to treat or detect . hyperproliferative disorders, including neoplasms. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may inhibit the proliferation of the disorder through direct or indirect interactions.
Alternatively, fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention may proliferate other cells which can inhibit the hyperproliferative disorder.
For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative disorders can be treated. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
Alternatively, decreasing an immune response ~ may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.
Examples of hyperproliferative disorders that can be treated or detected by fusion proteins of . the invention and/or polynucleotides -encoding albumin fusion proteins of the invention include, bat are not' limited to neoplasms located in the: colon, abdomen, bone, breast, digestive 'system; liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and IS peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.
Similarly, other hyperproliferative disorders can also be treated or detected by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Examples of such hyperproliferative disorders include, but are not limited to:
Acute Childhood Lymphoblastic Leukemia, Acute .Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer,~Adult (Primary) .Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Li.'ver Cancer, Adult Soft Tissue. Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Boiie Cancer, Brain Stem Glioma, Brain Tumors, Breast. Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytbma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastlc Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma,' Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell, Tumors, Childhood. Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentoriai Primitive Neuroectodernial Tumors, ~ Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic ' Myelogenous' Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Ext~'agonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy CeII Leukemia, Head and Neck Cancer, HepatoceIlular Cancer, Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancex, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, . Lymphoproliferative Disorder's, Macroglobulinemia, ~
Male Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, -Melanoma, Mesothelioma, Metastatic Occult Primary 'Squamous Neck Cancer, Metastatic Primary Squamious Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Mvtiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, , Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic - Squamous Neck Cancer, Oropharyr<geal Cancer, Osteo-/Malignant Fibrous Sarcoma, OsteosarcomalMalignant Fibrous . Histiocytoma, OsteosarcomalMalignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell 'tumor, Ovarian Low Malignant Potential Tumoi, Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary Liver, Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma; Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer ~ of the Renal Pelvis and. Ureter; Transitional Renal Pelvis and Ureter Cancer, Trophoblastic .
Tumors, Ureter and Renal Pelvis Ceil Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and any other.hyperproliferative disease,, besides neoplasia, located in an organ system listed above.
~ ~ In another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to diagnose, prognose, prevent, and/or treat premalignant conditions and to prevent progression to a .
' 145 neoplastic or malignant state, including but not limited to those disorders described above.
Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell,~ 1976, Basic Pathology, 2d Ed., W. -.B.
Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a form of controlled cell proliferation, involving an increase in cell number in a tissue or organ, without significant alteration in structure or function.
Hyperplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, angiofollicular . mediastinal lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, cementum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, .cystic hyperplasia, cystic hyperplasia of the breast, denture hyperplasia, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular papillary endothelial hyperplasia, nodular hyperplasia , of prostate, nodular regenerative liyperplasia, pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia, and verrucous hyperplasia.
Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, agnogenic ' myeloid metaplasia, apocrine metaplasia, atypical metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic ossification, metaplastic polyps, myeloid , metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.
Dysplasia is frequently a forerunner of cancer, and is found mainly in the -epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation. Dysplastic disorders which can be _ diagnosed, prognosed, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, anhidrotic- ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata; epithelial dysplasia, faciodigitogenital dysplasia, familial fibrous dysplasia of jaws, familial white_folded dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous dysplasia, . hereditary renal-retinal dysplasia, 'hidrotic ectodermal . dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammary dysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia, oculoauriculovertebral dysplasia, oculodentodigital dysplasia, oculovertebral dysplasia,.
odontogenic dysplasia, ophthalmomandibulomelic dysplasia, periapical, cemental dysplasia, polyostotic fibrous' dysplasia, ~pseudoachondroplastic spondyloepiphysial ~dysplasia, retinal IS dysplasia, .septo-optic dysplasia, spondyloepiphysial dysplasia, and ventriculoradial dysplasia. .
. ~ ~ Additional pre-neoplastic disorders which can be diagnosed, prognbsed, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, benign ~dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
In another embodiment, ~ albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to diagnose and/or prognose disorders associated with the tissues) in which the polypeptide of the invention is expressed.
In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention conjugated to a toxin or a' radioactive isotope, as described herein, may be used to treat cancers and neoplasms, including, but not limited to, those described herein. In a further , preferred embodiment, -albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat acute myelogenous leukemia.
' Additionally, fusion proteins of the invention. and/or polynucleotides encoding albumin fusion proteins of the invention may affect apoptosis, and therefore, would be useful in treating a number of -diseases associated with increased 'cell survival or the inhibition of apoptosis. For example, diseases associated~with increased cell survival or the inhibition of apoptosis that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders such as, multiple sclerosis;
Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related - glomeiulonephritis and' rheumatoid arthritis) arid viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.
In preferred embodiments, fusion proteins. of the invention andJor polynucleotides encoding albumin fusion proteins of the invention are used to inhibit growth, progression, ~ andlor metastasis of cancers, in particular those listed above.
Additional diseases or conditions associated with increased cell survival that could be diagnosed, prognosed, prevented, andlor treated by fusion proteins of the invention and/or polynucleotides encoding. albumin fusion proteins of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myeIomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g.., chronic myelocytic (granulocytic) leukemia and chronic.lymphocytic leukemia)), poiycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, .liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, ' rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, .sebaceous _ gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic' carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonaI carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neurobIastoma, and retiiloblastoma. ' 148 .

Diseases associated with increased apoptosis that could be diagnosed, prognosed, prevented, and/or treated. by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or prior associated disease);
autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease;_polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial 1~0 infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis {bile duct injury) and liver cancer);
toxin-induced ' liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
' Hyperproliferative diseases andlor disorders that could-be diagnosed, prognosed, prevented, and/or treated by fusion proteins of the invention and/or .polynucleotides encoding albumin fusion proteins of the invention, include, but are not iimited~to, neoplasms located in the liver, abdomen, bone, breast, digestive system, pancreas, peritoneum, endocrine glands (adrenal, parath~!roid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract. .
Similarly, other hyperproliferative disorders can also be diagnosed, prognosed, prevented; and/or treated by fusion proteins of the invention and/or polynucleotides encoding . albumin fusion proteins of the invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other -hyperproliferative ' disease, besides neoplasia, located in an organ system listed above. - ' .
Another preferred embodiment utilizes polynucleotides encoding albumin fusion proteins of the invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof. . ' ' Thus, the present invention provides. a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide encoding an albumin fusion protein of the present invention, wherein said polynucleotide represses said expression.
Another embodiment of the present invention provides a method of treating cell proliferative disorders in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell -or cells.
In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a . recombinant expression vector effective i'n expressing a DNA sequence 'encoding said polynucleotides. In another preferred embodiment of the ~ present invention, the DNA
construct encoding the fusion protein of the present invention is inserted into cells to be treated utilizing a retrovirus, or more preferably an adenoviral vector (See G
J. Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells.either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e.
magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect .of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus. ' Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By "repressing expression of ttie oncogenic genes " is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.
For local administration to abnormally proliferating cells, polynucleotides of the -. present invention may. be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of ,cells, or in ' vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et aL, Proc.
~Natl. Acid. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol.
5:3403 (1985) or other e~cient DNA delivery systems (Pates et al., Nature 313:812 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or f adenoviral (as described,in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the' retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a -retroviral delivery system for polynucleotides of. the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.

The polynucleotides of the present. invention may be delivered directly to cell proliferative disorder/disease sites in -internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at- the time of surgical intervention.
By "cell proliferative disease" is meant any human or animal. disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.
- 'Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells.
Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By "biologically inhibiting" is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells.
The biologically inhibitory dose may .be determined by assessing the effects of the polynucleotides- of -the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art. -Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin ~ fusion proteins -of the invention of the , present invention are ,useful in inhibiting the - angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. I~ a - most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for I
example, through the inhibition of hematopoietic, tumor-specifc cells, such as tumor-associated macrophages (See Joseph IB, et al. J Nati Cancer Inst, 90(21):148-53 (1998), which is hereby incorporated by reference).
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful ~in inhibitinig proliferative cells or tissues through the induction of apoptosis. These fusion protieins andlor polynucleotides may act-either directly, or indirectly to induce -apoptosis of proliferative cells and-tissues, for example in the activation of a death-domain receptor, such as tumor. necrosis factor (TNF) receptor-1, CD95 (Fas/AP0-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et.al., Eur J -Biochem -254(3):439-59 (1998), which is hereby incorporated by reference).
Moreover, in another preferred embodiment of the present invention, these fusion- proteins - and/or polynucleotides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the ' - - 151 expression of these proteins, either alone or in combination with small molecule drugs or adjuviants, such as apoptonin, galectins, thioredoxins, anti-inflammatory proteins (See for example, Mutat Res 400(1-2):44.7-55 (1998), Med Hypotheses.50(5):423-33 (1998), 'C>~em Biol Interact. Apr 24;111-112:23-34 (1998),.J Mol Med.76(6):402-12 (1998), Int J Tissue React;20(1):3-15 (1998), which are all hereby incorporated by reference). .
Albumin fusion proteins of the invention and/or polynucleotides encoding 'albumin fusion proteins of the invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result ~of administering these albumin fusion -proteins and/or polynucleotides, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, '(See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is hereby incorporated by reference). Such thereapeutic affects of . the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.
In another embodiment, the invention provides a method of delivering compositions containing the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention to targeted cells expressing the a polypeptide bound by, that binds to, or associates with an albumin fuison protein of the invention. Albumin fusion proteins of the invention may be associated with with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. - ' Albumin fusion proteins of the invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as' would occur if the albumin fusion proteins of the invention 'vaccinated' the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known ~to enhance the immune response (e.g. chemokines), to said antigens and immunogens.
Renal Disorders Albumin fusion-proteins of the invention and/or polynucleotides encoding albumin fusion proteins ~vf the invention, may be used to treat, prevent, diagnose, and/or prognose disorders of the renal system. Renal ~ disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention include, but are not limited to, kidney failure, nephritis, blood vessel disorders of kidney, metabolic and congenital kidney disorders, urinary disorders of the kidney,-autoimmune disorders; sclerosis and necrosis, electrolyte imbalance, and kidney cancers.
Kidney diseases which can 'be diagnosed, prognosed, prevented,' and/br treated with compositions.of the invention include, but are not limited to, acute kidney failure, chronic kidney failure, atheroembolic renal failure, end-stage renal disease, inflammatory diseases of the kidney (e.g., acute glomerulonephritis, postinfectious glomerulonephritis, rapidly progressive glomerulonephritis, riephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoproliferative glomerulonephritis I and II, mesangial p~roliferative glomerulonephritis, chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis; lupus nephritis, chronic nephritis, interstitial nephritis;
and post-streptococcal glomerulonephritis), blood vessel disorders ~ of ~ the kidneys (e.g., kidney infarction, atheroembolic kidney disease, cortical necrosis, malignant nephrosclerosis, renal vein thrombosis, renal underperfusion, -renal retinopathy, renal ischemia-reperfusion, renal artery embolism, and renal artery stenosis), and kidney disorders resulting form urinary tract disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renal lithiasis, nephrolithiasis), reflux nephropathy, urinary tract infections, urinary ~ retention, and acute' or chronic unilateral obstructive uropathy-.) ~ ' .
In addition; compositions of the invention can be used to diagnose, .prognose, prevent, and/or treat metabolic and congenital disorders of the kidney (e.g., uremia, renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal gIycosuria, nephxogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocystic osteosis (renal rickets), Hartnup ydisease, Bartter's syndrome, Liddle's syndrome, polycystic kidney disease, medullary cystic disease, medullary sponge kidney; Alport's syndrome, nail-patella syndrome, congenital nephrotic syndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes insipidus, , analgesic nephropathy, kidney stones, and membranous nephropathy), and autoimrriune disorders of the kidney (e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis). . ' .
Compositions of the invention can also be used to diagnose, prognose, prevent, and/or treat sclerotic or necrotic disorders of the kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), necrotizing glomeruloilephritis, and renal papillary necrosis), cancers of the kidney (e.g., nephroma, hypernephroma, nephroblastoma, renal cell cancer, ~ transitional cell cancel, renal adenocarcinoma, squamous cell cancer., and Wilm's tumor), and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria, hyponatremia, hypernatremia, hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).
._ ._ Compositions of the invention may. be administered using any method known ' in the art, including, but not limited to, direct needle- injection at the delivery site, 'intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam. sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Compositions of the invention may be administered as part of a Therapeutic, described in more detail below.
Methods of delivering polynucleotides.of the invention are described in more detail herein.
Cardiovascular Disorders Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose, and/or prognose cardiovascular disorders, including, but not limited to, peripheral artery disease, such as limb ischemia.
Cardiovascular disorders include, but are not limited to, cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, i5 congenital.heart defects, pulmonary atresia, and Scimitar Syndrome.
Congenital heart~defects include, but are not limited to, aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of . great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart ~septal defects, , such as aortopulmonary septal defect, ~ endocardial cushion defects, Lutembacher's Syndrome, trilogy of FaIIot, ventricular heart septal defects.
Cardiovascular disorders also include, but are not limited to, heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac .edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy; post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular ~
pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
Arrhythmias include, but are not limited to, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block; sinoatrial block; long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre excitation syndrome, Wolff Parkinson-White 'syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic functional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
Heart valve diseases include, but are not limited to, aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve ins~~ciency, mitral valve stenosis, pulmonary atresia, pulmonary valve~insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis. .
Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy, congestive cardiomyopathy, hyperbrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular- stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, ~ endomyocardiai fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
Myocardial ischemias include, but are not limited to, coronary disease; such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning. _ Cardiovascular ~ diseases also include vascular diseases such as aneurysms,.
angiodysplasia,. angiornatosis, bacillary angiomatosis, HippeI-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, .Sturge-Weber Syndrome, angioneurotic edema, ~
aortic diseases, , Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic . angiopathies, diabetic ~retinopathy; embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST
syndrome, retinal vein occlusion, Scimitar 'syndrome, superior vena cava syndrome, telangiectasia, atacia 25. telangiectasia, hereditary hemorrhagic telangiectasia,~ varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.
Aneurysms include, but are not Limited to, dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
. Arterial occlusive diseases include, but are not limited to, arteriosclerosis, intermittent claudication, carotid : stenosis; fibromuscular dysplasias, mesenteric, vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and throw boangiitis . obliterans.
Cerebrovascular disorders include, but are not limited to., carotid artery diseases, cerebral amyloid angiopathy; cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral WO 01/79271 PCT/iJS01/12009 hemorrhage, epidural hematpma, subdural hematoma, subaraxhnoid hemorrhage, cerebral 1 infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, , migraine, and vertebrobasilar .
insufficiency. , ~ Embolisms include, but are not limited to, air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include, but are not limited to, coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, ' Wallenberg's syndrome, and thrombophIebitis.
Ischemic disorders include, but are not limited to, cerebral ischemia, ischemic~ colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion . injuries, and peripheral limb ischemia. Vasculitis includes, but is not limited to, aortitis, arteritis, Behcet's Syndrome, Churl-Strauss Syndrome. mucocutaneous lvmnh nnc~e syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch - .
purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.~ -Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be administered 'using any method known 'in the art, including,.but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other ~ commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Methods of delivering polynucleotides are described in more detail herein. - ~ ' Res~iratorv Disorders.
Albumin fusion proteins of the invention andJor polynucleotides encoding albumin fusion proteins of the invention may be used to treat, prevent, diagnose, andlor prognose diseases and/or disorders of the respiratory system.
, ~ Diseases and disorders of the respiratory system include, but are not limited to, nasal vestibulitis, nonallergic rhinitis (e:g., acute rhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis), nasal polyps, and -sinusitis, juvenile angiofibromas, cancer of the nose and juvenile papillomas, vocal cord polyps, nodules (singer's nodules), contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis (e.g., viral and bacterial), tonsillitis, tonsillar cellulitis, parapharyngeal abscess, laryngitis, laryngoceles, and throat cancers (e.g., cancer of the nasopharynx, tonsil cancer, larynx cancer), 1'ung cancer (e.g., squamous cell carcinoma, small cell (oat cell) carcinoma, large cell carcinoma, and adenocarcinoma), allergic disorders (eosinophilic pneumonia, hypersensitivity pneumonitis (e.g., extrinsic allergic alveolitis, allergic interstitial pneumonitis, organic dust pneumoconiosis, allergic bronchopulmonary aspergillosis, asthma, Wegener'_s granulomatosis (granulomatous vasculitis), Goodpasture's syndrome)), pneumonia (e:g., bacterial pneumonia (e.g., Streptococcus pneumoniae (pneumoncoccal pneumonia), Staphylococcus aureus (staphylococcal pneumonia);
Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and Pseudomas spp.), Mycoplasma pneumoniae pneumonia; Hemophilus . influenzae pneumonia, Legionella pneumophila . (Legionnaires'rdisease), and Chlamydia psittaci (Psittacosis)), and viral pneumonia (e.g., influenza; chickenpox (varicella).
Additional diseases and disorders of the respiratory system include, but are not limited to bronchiolitis, polio (poliomyelitis), croup, respiratory syncytial viral infections mumps, erythema infectiosum (fifth disease), roseola infantum, progressive rubella panencephalitis, german measles, and subacute sclerosing panencephalitis), fungal pneumonia (e.g., ' Histoplasmosis, Coccidioidomycosis, Blastomycosis, fungal infections in people' with severely suppressed immune systems (e.g., .cryptococcosis, caused by Cryptococez~r ueoformans; aspergillosis, caused by Aspergillus spp.; candidiasis, caused by Candida; and mucorinyeosis)), Pneumocystis carinii {pneumocystis pneumonia), atypical pneumonias (e.g., lt~ycoplasma and Chlamydia spp.), opportunistic infection pneumonia, nosocomial pneumonia, chemical pneumonitis, and aspiration pneumonia, pleural disorders (e.g., pleurisy, pleural effusion, and pneumothorax (e.g., simple spontaneous pneumothorax, complicated spontaneous pneurnothorax, tension pneumothorax)), . obstructive, airway diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis, black lung (coal workers' pneumoconiosis), asbestosis, berylliosis, occupational asthsma, byssinosis, -and benign pneumoconioses), Infiltrative Lung Disease (e.g., pulmonary. fibrosis (e.g., fibrosing alveolitis, usual interstitial pneumonia), idiopathic pulmonary fibrosis, desquamative interstitial' pneumonia, lymphoid interstitial ~ pneumonia, histiocytosis X
(e. g., Letterer-Siwe . disease, Hand-Schiiller-Christian disease, eosinophilic granuloma), idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary alveolar proteinosis), Acute respiratory distress syndrome (also called, e.g., adult .respiratory distress syndrome), edema, pulmonary embolism, bronchitis (e.g., viral, bacterial), bronchiectasis, atelectasis, lung abscess (caused by, e.g., Staphylococcus aureus or Legionella pneumophila), and cystic fibrosis.
Anti-An,~,io;~nesis Activity The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences. predominate. Rastinejad et al:, Cell 56:345-355 (1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that ' characterizing solid tumor growth, these regulatory controls fail.
Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases.
A number of serious diseases .are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman et al., N. Engl.
J. Med., 333:1757-1763 (1995); Auerbach et al., ' J. Microvasc. Res. 29:401-411 (1985);
Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp.
175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., ' Science 221:719-725 (1983). In a number of pathological conditions, the process of arigiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).
The present invention provides for treatment of diseases or disorders associated with neovascularization by administration of fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman. et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia ,(1985)).Thus, the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention. For example, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be utilized in a variety of additional methods in order to therapeutically~treat a cancer or tumor.
Cancers which may .30 be treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary~~tract, colon, rectum, cervix, uterus, endometrium, kidney, .bladder, thyroid cancer;
primary. tumors and metastases; melanomas; glioblastoma; ~ Kaposi's sarcoma;
. Ieiomyosarcoma; non- small cell Lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias. For example, fusiow proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be delivered topically, in order to treat cancels such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma. ' Within yet other aspects, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be utilized to treat supe~cial forms of S bladder cancer by, for example, intravesical administration. Albumin fusion proteins of .the invention and/or polynucIeotides encoding albumin fusion proteins of the invention may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.
~ Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating other disorders, besides cancers, which involve angiogeilesis. These disorders include, but are not limited to:
benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular .angiogenic ~ diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular . degeneration, corneal graft ~ rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel- growth) of the eye; rheumatoid arthritis; psoriasis;
delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis;
coronary collaterals; cerebral - collate_rals; arteriovenous malformations; ischemic Limb angiogenesis;
Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints;
angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's - disease;
and atherosclerosis. .
For .example, within one aspect of the present invention methods are provided for treating hypertrophic scars and keloids, comprising the step of administering albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins. of the invention to a hypertrophic scar or keloid. . . .
Within one embodiment of the present invention fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the-invention. are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions. This .
therapy is of particular value ~in the prophylactic treatment of conditions which are known to result in the development of~hypertrophic scars and keloids~(e.g., burns), and is preferably' initiated after the proliferative.phase has had time to progress.(approximately 14 days after the . . initial injury), but before hypertrophic scar or keloid development. As noted above, the ' present invention also provides ,methods for treating neovascular diseases of .the eye, including for example; corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental f broplasia and macular degeneration. - ~ .

Moreover, Ocular disorders associated with neovascularization which can be treated with the albumin fusion proteins of the invention and/or polynucleotides encoding albumin - fusion proteins of the invention include, but are not limited to:
neovascular glaucoma, diabetic retinopathy,.retirioblastoma, retrolerital fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv.
Ophthal. 22:291-312 ( 1978).
Thus, within one aspect of the present invention methods are provided for treating neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (e.g., fusion proteins of the invention andlor polynucIeotides encoding. albumin fusion proteins of the invention) 'to the cornea, such that the formation of blood vessels is inhibited.. Briefly, the cornea is a tissue which normally lacks blood vessels.
' In certain pathological conditions however, capillaries may extend into the cornea from the pericorneal vascular plexus of the Iimbus. When the cornea becomes vascularized, it also becomes clouded,~resulting in a decline in the patient's visual acuity. Visual loss may become complete-if the cornea completely opacitates. A wide variety of disorders can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, herpes simplex keratitis,_ leishmaniasis and onchocerciasis), immunological processes ~(e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.
. Within particularly preferred embodiments of the. invention, may be prepared for topical administration in saline (combined with any of the preservatives and antiriiicrobial , agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily.
Alternatively, anti-angiogenic compositions, prepared as described. above, may also be administered directly to the cornea. Within preferred embodiments, the anti=angiogenic composition is. prepared with a muco-adhesive polymer -which binds to.
cornea.. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical bums). In these instances the treatment, likely in -combination ' with steroids, may be instituted immediately to help preveilt subsequent 3S complications.
Within other embodiments, the compounds described above may be injected directly into the,corneal strorina by an ophthalmologist under microscopic guidance.
The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the ~ advancing front of the vasculature (i.e., interspersed between ,the blood vessels and the normal cornea). In most cases this .would involve perilimbic corileal injection to."protect" the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected. in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary .invasion of transplanted corneas. In a sustained-release form injections might only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.
Within another aspect of the present invention, methods are provided for treating neovascular glaucoma, comprising the step of administering to a patient a -therapeutically effective amount of an .albumin fusiori protein of 'the invention .and/or polynucleotides encoding an albumin fusion protein of the invention to the eye, such that the formation of blood vessels is inhibited. , In one embodiment, the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle.
Within other embodiments,. the compound may also be placed in any location such that the ' compound is continuously released into the aqueous humor. Within another aspect of the present invention, methods are provided for treating proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of an _ albumin fusion protein of the invention andlor polynucleotides encoding an albumin fusion .
protein of the invention to the eyes, such that the formation of blood vessels is inhibited.
. ~ Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into.the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist~andlor agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.
Within another aspect of the present invention, methods are provided for treating retrolental fibroplasia, comprising the step of administering ~ to a patient a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention to the eye; such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants. .
Additionally, disorders which can be treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, hemangioma, arthritis; psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic grariuloma, scleroderma, trachoma, and vascular adhesions.
Moreover, disorders andlor states, which can, be treated, prevented, diagnosed, and/or prognosed with the the albumin fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention of the invention include, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas,. rheumatoid arthritis, psoriasis, ~ ocular angiogenic diseases, for example,,diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, , endometriosis, vascluogenesis, grailulations, hypertrophic scars (keloids), . nonunion fractures, scleroderma, trachoma, ~ vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic - consequence such as cat scratch disease (Rocliele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.
In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a "morning after"
method. Albumin fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention may also be used in controlling.ri~enstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the' treatment .of endometriosis. .
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be incorpbrated into surgical sutures in order to prevent stitch granulomas.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be utilized in a wide variety of surgical procedures. For example, within, one ~ aspect of the present invention . a compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, ' 162 compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical . mesh might be utilized. For example, within one embodiment of the invention a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.
Within further aspects of the present invention, methods are provided for treating tumor excision sites, comprising administering albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the -formatiori o~ new blood vessels at the site is inhibited. Within ~ one .
embodiment of the invention, the_anti-angiogenic compound is administered directly to the tumor excision site . (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly~preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.
Within one aspect of -the present invention, fusion proteins of the invention and/or ~ , polynucleotides encoding albumin fusion proteins of the invention may be administered to the , resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to~the site of a neurological tumor subsequent to excision, ~ such that the formation of new blood vessels at the site are inhibited.
The albumin fusion proteins of the invention and/or polynucleotides.encoding albumin fusion proteins of the invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include:
Anti-Invasive Factor, retinoic acid and derivatives thereof, , paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, ~ Tissue Inhibitor of Metalloproteinase-2, ~ Plasminogen Activator Inhibitor-1, Plasminogen.Activator Inhibitor-2, and various forms of the lighter "d group"
-. transition metals. . .~ , Lighter "d group" transition metals include, for example, vanadium, molybdenum, .
tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal' complexes.
Representative examples of vanadium complexes include oxo vanadium complexes 163 ~ -such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable,vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide . complexes. Suitable tungstate .complexes include ammonium tungstate, calcium tungstate, sodium tungstate dehydrate, and tungstic acid.- Suitable tungsten oxides include tungsten (IV) ~ oxide and tungsten (VI) , oxide. Suitable ~ oxo molybdenum complexes include moIybdate, molybdenum -oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate , and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) 'oxide, molybdenum (VI) oxide, and, molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. . Representative examples include platelet factor 4;
protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP- PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including, for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; ~ 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum;
ChIMP-3 (Pavloff et ~al., J. Bio. Chem. 267:17321-17326, ,(1992)); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, ( 1992)); Cyclodextrin Tetradecasulfate;
Eponemycin; Camptothecin; Fumagillin {Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST"; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987));
anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem.
262(4):1659-1664, (1987)); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl ' 4- chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions 36:312-316, . (1992)); Thalidomide; ~ Angostatic steroid; AGM-1470;
carboxynaminolmidazole; and metalloproteinase inhibitors such as BB94. , Diseases at the Cellular Level -Diseases associated with increased cell survival or the inhibition of 'apoptosis that could be treated, prevented, diagnosed, and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include cancers (such as follicular lymphomas, carcinomas with p~3 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma. osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.
In preferred embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.
Additional diseases or conditions associated with increased cell survival that could be treated or detected by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases. associated with increased apoptosis that could be treated, prevented, diagnosed, and/or prognesed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion -,proteins of the ~ invention, include, but are not limited to, AIDS;
neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease;
Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and . immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, choiestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia. ' ' Wound Healing and Epithelial Cell Proliferation . Iri accordance with,yet~ a further aspect of the present invention, there is provided a process for utilizing fusion proteins of the invention and/or polynucleotides .encoding albumin fusion proteins of the invention, for therapeutic purposes, for example, to stimulate epithelial -cell proliferation and basal .keratinocytes for the purpose of wound healing, and to stimulate hair: follicle production and healing of dermal wounds. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, -, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy and antineoplastic drugs and .antimetabolites. Albumin fusion _ proteins of the invention ,and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to promote .
dermal reestablishment subsequent to derinal loss Albumin fusion proteins ~of the invention andlor polynucleotides encoding albumin fusion proteins of the invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are types of grafts that fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, 'could be used to inciease adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cubs graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, Iamellar graft, mesh graft; mucosal graft, Oilier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. Albumin fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention, can be used to promote skin strength and to improve the appearance of aged skin.
It. is believed that fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, will also produce changes in hepatocyte 10. proliferation, and epithelial cell proliferation in the Lung, breast, pancreas, stomach, small intestine, and large intestine. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and~other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes. , Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may have a cytoprotective effect on the small intestine mucosa.
Albumin fusion proteins. of the invention-and/or polynucleotides encoding albumin fusion proteins of the invention, may. also, stimulate healing of mucositis (mouth ulcers) that result from ~ chemotherapy and viral infections. .
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which, results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly.
Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus; fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to promote the resurfacing of the inucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with ' fusion proteins of the invention and/or polynucleotides encoding ~albunun fusion proteins of the invention, is expected to have a significant effect on the production of mucus throughout the - gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to ' ,10 treat diseases associate with the under expression. .
l~Ioreover, fusion. proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to prevent and heal damage to the lungs due to various pathological. states. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, which could, stimulate proliferation and ~ differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For. example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that ' cause necrosis of the. bronchiolar epithelium and alveoli could be effectively treated ~ using polynucleotides or polypeptides, agonists or antagonists of the present invention. Also fusion . ~ 20 proteins of the invention and/or ~polynucleotides encoding albumin fusion proteins of the invention, could be used to stimulate the proliferation of and differentiation of type II
pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.
- Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by.cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).
In addition, fusion proteins of the invention and/or polynucIeotides encoding albumin fusion proteins of the invention, could be used treat or prevent the onset of diabetes mellitus.
In patients with newly diagnosed Types I and II diabetes, where some islet ~
cell function remains, fusion proteins of the invention and/or polynucleotides , encoding albumin fusion -proteins of the'invention, could be used to maintain the islet function so as, to alleviate, delay or prevent permanent manifestation of the disease. Also, fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention, could be used as an r auxiliary in islet cell transplantation to improve or promote islet cell function. . -Neural Activity and Neurological Diseases The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used for the diagnosis and/or treatment of diseases, disorders, damage or injury of the brain and/or nervous system. Nervous system disorders that can be 'treated with ahe compositions of the invention (e.g., fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention), include, but are not limited to, nervous system injuries; and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.
Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the methods of the invention, include but ara not limited to, -the following lesions of either the central (including spi~ial cord, brain) or peripheral nervous systems: (1) ischemic lesions, in-which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction .or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is -' destroyed or injured by malignant tissue which is either a nervous system associated malignancy'or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for -example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5) degenerative lesions, in- which a portion of the nervous system is destroyed 'or injured as a - 25 result of a degenerative process including but not limited to, degeneration associated with Parkinson's ~ disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases or -disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of , metabolism including, but not limited to, vitamin B 12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol -amblyopia; Marchiafava-Bignami disease (primary - degeneration of the corpus callosum), and alcoholic cerebellar degeneration;
(7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic - neuropathy, Bell's palsy), systemic lupus erythematosus,-'carcinoma, or, sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in'which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human - . - immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, f69 progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
In one embodiment, the albumin fusion proteins of , the . invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to protect neural cells from the damaging effects of hypoxia. In a further preferred embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia.
According to~this embodiment, the compositions of the invention are used to treat or prevent neural cell injury associated with cerebral hypoxia. In one non-exclusive aspect of ' this embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the !invention, are used to treat or prevent neural cell injury associated with cerebral ischemia. In another-non-exclusive aspect of this embodiment, the albumin fusion proteins of. the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat~or~prevent neural cell injury associated with cerebral infarction.
In another preferred embodiment, albumin fusion proteins ~ of the invention and/oi polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neural cell injury associated with a stroke. In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent cerebral neural cell injury associated with a stroke.
In another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent , neural cell injury associated with a heart attack. In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent cerebral neural cell injury associated with a heart attack.
The compositions, of the invention which are useful- for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) 'increased survival time ,of neurons in culture either in the presence or absence of hypoxia or hypoxic conditions; (2) increased sprouting of neurons in culture or in vivo; (3) iilcreased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect~to motor neurons; or (4) decreased symptoms of neuron dysfunction in .vivo. Such effects may be measured by ' any method known in the art. In preferred, non-limiting embodiments, increased survival of .
neurons may routinely >je measured using a method set forth herein or otherwise known in the art, such as, for example, in Zhang et al., Proc Natl Acad Sci USA '97:3637-42 (2000) or in Arakawa et al., J. Neurosci., 10:3507-15 (i990); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk . et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann. Rev. Neurosci., 4:17-4.2 {1981); increased production of neuron-associated molecules~may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the ,molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
In specific embodiments, motor neuron disorders that may be treated according to the.
invention include, but are not limited to, disorders' such as infarction, infection, exposure to . toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively .affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
Further, fusion proteins ~of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may play a role in neuronal survival; synapse formation;
conductance; neural differentiation, etc. Thus, compositions of the invention (including fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention) may be used to diagnose and/or treat or prevent diseases or disorders associated with these roles, including, but not limited to, learning. and/or cognition disorders. The' compositions of . the invention may also be useful - in the treatment or prevention of neurodegenerative disease states and/or behavioural disorders. Such neurodegenerative , disease states and/or behavioral disorders include, but are not limited to, Alzheimer's Disease, . . Parkinson's Disease, Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities;
ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, compositions of the invention may also play a role in~the treatment, prevention and/or detection of developmental disorders associated, with -the developing embryo, or sexually-linked disorders. -Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins 'of the invention, may -be useful in protecting neural cells from diseases, damage, disorders, or injury, associated with cerebrovascular disorders including, but not Limited to, carotid artery diseases (e.g., carotid artery thrombosis, carotid stenosis, or Moyamoya Disease), . cerebral amyloid angiopathy, . cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis (e.g., carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or subdural hematoma, or subarachnoid hemorrhage), cerebral infarction, cerebral ischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome, or vertebrobasilar insufficiency), vascular dementia (e.g., mufti-infarct), leukomalacia; periventricular, and vascular headache (e.g., cluster headache or migraines). , In accordance with yet a further aspect of the present invention, there is provided a process for utilizing fusion proteins of the invention and/or polynucleotides.encoding albumin fusion proteins of the invention, for therapeutic purposes, for example, to .stimulate neurological cell . proliferation and/or differentiation. Therefore, fusion proteins of the invention and/or pblynucleotides encoding albumin fusion proteins of the invention may be used to treat and/or detect neurologic diseases. Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, can be used as a marker or detector of a particular nervous system disease or disorder.
.Examples of neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, brain diseases, such as metabolic brain diseases which includes phenylketonuria such as maternal phenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke's Encephalopathy, brain edema, brain neoplasms such as cerebellar neoplasms which include infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavan disease, cerebellar diseases such as cerebellar ataxia which include spinocerebellar degeneration such as. ataxia telangiectasia, cerebellar dyssynergia, Friederich's Ataxia, Machado-Joseph Disease, olivopontocerebellar atrophy, ~cerebellar neoplasms such as infratentorial neoplasms, diffuse cerebral sclerosis such as. encephalitis periaxialis, globoid cell leukodystrophy, metachromatic leukodystrophy and subacute sclerosing panencephalitis.
Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include cerebrovascular disorders (such as carotid artery diseases which include carotid artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery' diseases, cerebral embolism and thrombosis such as carotid artery thrombosis, sinus thrombosis and Wallenberg's Syndrome, cerebral hemorrhage such as epidural herriatoma; subdural hematoma and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as transient cerebral ischemia, .Subclavian Steal Syndrome ar<d vertebrobasilar insufficiency, vascular dementia such as mufti-infarct dementia, periventricular leukomalacia, vascular headache such as cluster headache and migraine. ' 1.72 Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention .
include dementia such as AIDS Dementia Complex, presenile dementia such' as Alzheimer's Disease and Creutzfeldt-Jakoli Syndrome, senile dementia such as Alzheimer's Disease and progressive supranuclear palsy, vascular dementia such as mufti-infarct dementia, encephalitis which include encephalitis periaxialis, viral encephalitis such as epidemic encephalitis, . Japanese Encephalitis, St. Louis Encephalitis, tick-borne encephalitis and West Nile Fever, ' acute disseminated encephalomyelitis, meningoencephalitis such as uveomeningoencephalitic syndrome, Postencephalitic Parkinson Disease and subacute sclerosing panencephalitis, encephalomalacia such as periventricular leukomalacia, epilepsy such as generalized epilepsy which includes infantile spasms, absence epilepsy, myoclonic, epilepsy which includes MERRF Syndrome, tonic-clonic' epilepsy, partial epilepsy such as complex partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic epilepsy;
status epilepticus _ such as Epilepsia Partialis Continua, and Hallervorden-Spatz Syndrome.
Additional neurologic diseases which can be treated or detected with fusipn proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include hydrocephalus such as Dandy-Walker~Syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neopl'asms, cerebral malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranial tuberculoma and Zellweger ' Syndrome, central nervous system infections such as AIDS Dementia Complex, Brain Abscess, ' subdural empyema, encephalomyelitis such as Equine Encephalomyelitis, ' Venezuelan Equine Encephalomyelitis, Necrotizing Hemorrhagic Encephalomyelitis, Visna, and cerebral malaria.
' Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include meningitis such as arachnoiditis, aseptic meningtitis such as viral meningtitis which includes lymphocytic choriomeiiingitis, Bacterial meningtitis which 'includes Haemophilus Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such as Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal tuberculosis, fungal meningitis such as Cryptococcal Meningtitis, subdural effusion, meningoencephalitis such as uvemeningoencephalitic syndrome, myelitis such as transverse myelitis, neurosyphilis such as tabes dorsalis, ~ poliomyelitis which includes bulbar poliomyelitis and postpoLiomyelitis syndrome, , prion diseases (such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform 35. Encephalopathy, Gerstmann-Straussler ' Syndrome, Kuru, Scrapie), and cerebral toxoplasmosis. .
Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include central nervous system neoplasms such as brain neoplasms that include cerebellar neoplasms such as infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms and supratentorial , neoplasms, meningeal neoplasms, spinal cord neoplasms which include epidural neoplasms, demyelinating diseases such as Canavan Diseases, diffuse cerebral sceloris which includes adrenoleukodystrophy, encephalitis periaxialis, globoid cell leukodystrophy, diffuse cerebral sclerosis such as metachromatic . leukodystrophy, ~ allergic encephalomyelitis, ~ necrotizing hemorrhagic encephalomyelitis, progressive multifocal leukoencephalopathy, multiple -sclerosis, . central pontine myelinolysis, transverse myelitis, neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal cord diseases such as'amyotonia congenita, amyotrophic lateral, sclerosis, spinal muscular atrophy such as Werdnig-H'offmann.Disease, spinal cord cbmpression, spinal cord neoplasms such as epidural neoplasms, syringomyelia, Tabes Doi~salis, Stiff-Man Syndrome, mental retardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome, Down Syndrome; Gangliosidoses such as gangliosidoses G(M1), Sandhoff Disease, Tay-Sachs Disease, Hartnup Disease,, homocystinuria, Lawrence-Moon- Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis such as fucosidosis, neuronal ceroid-,lipofuscinosis, oculocerebrorenal. syndrome, phenylketonuria ~ such as maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome, Rubinstein-Taybi Syndrome, . Tuberous Sclerosis; WAGR Syndrome, nervous system abnormalities such . as holoproseneephaiy, neuial tube . defects such- as anencephaly which includes hydrangencephaly, ' Arnold-Chairi Deformity, encephalocele, meningocele, meningomyelocele, spinal dysraphism such as spina bifida cystica and spina bifida occulta.
Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides~ encoding albumin fusion proteins of the invention include hereditary motor and sensory neuropathies which include Charcot-Marie Disease, Hereditary optic atrophy, Refsum's. Disease, hereditary spastic - paraplegia, Werdnig-- Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies such as Congenital .Analgesia and Familial Dysautonomia, Neurologic manifestations, (such as agnosia that .
include Gerstmann's Syndrome, Amnesia such-as retrograde amnesia, apraxia, neurogenic bladder, cataplexy, communicative disorders such as hearing disorders that includes deafness, partial hearing loss, loudness recruitment 'and tinnitus, language disorders such as aphasia which include agraphia, anomia, broca aphasia, and Wernicke Aphasia, Dyslexia such as Acquired Dyslexia, language development disorders, speech disorders such as aphasia which includes anomia, broca aphasia and Wernicke Aphasia, articulation disorders, communicative disorders such as speech disorders-which include dysarthria, echolalia, mutism~and stuttering, voice disorders such as aphonia and hoarseness, decerebrate state, 'delirium, fasciculation, hallucinations, meningism, movement disorders such as angelman syndrome, ataxia, athetosis,, chorea, dystonia, hypokinesia, muscle hypotonia; myoclonus, tic, torticollis and tremor, muscle hypertonia such as muscle rigidity such as stiff man syndrome, muscle spasticity, paralysis such as facial paralysis which includes Herpes Zoster Oticus, Gastroparesis, Hemiplegia, ophthalmoplegia such as diplopia, Duane's Syndrome, Homer's - Syndrome, Chronic progressive.external.ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis, Tropical Spastic Paraparesis, Paraplegia such as Brown-Sequard Syndrome, quadriplegia, respiratory paralysis and vocal ~ cord paralysis, paresis, phantom Limb, taste disorders such as ageusia and dysgeusia, vision disorders such as amblyopia, blindness, color vision defects, diplopia, hemianopsia, scotoma and subnormal vision, sleep disorders such as hypersomnia which includes Kleine-Levin Syndrome, insomnia, and somnambulism, spasm such as ~ trismus, unconsciousness such as coma, persistent vegetative state and syncope and vertigo,'neuromuscular diseases such as amyotonia congenita, amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motor neuron disease, muscular atrophy such as spinal muscular atrophy, Charcot-Marie Disease and Werdnig-Hoffmann . Disease, Postpoliomyelitis Syndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia Atrophica, lVIyotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome, peripheral nervous ' system diseases such as aciodynia, amyloid neuropathies, autonomic nervous , system diseases such as Adie's Syndrome, . Barre-Lieou Syndrome, Familial Dysautonomia, Horner's Syndrome, Reflex Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial V Nerve Diseases such as Acoustic Nerve Diseases such as .Acoustic Neuroma which includes Neurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia,Melkersson-Rosenthal Syndrome, ocular motility disorders which includes _ amblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia such as Duane's Syndrome, Homer's Syndrome, Chronic Progressive External Ophthalmoplegia which includes Kearns Syndrome, Strabismus such as Esotropia and Exotropia, Oculomotor Nerve Paralysis, Optic Nerve Diseases such as Optic . Atrophy which includes Hereditary Optic. Atrophy, Optic-Disk Drus~n, Optic Neuritis such as Netiromyelitis Optica, Papilledema, Trigeminal , Neuralgia, Vocal Cord Paralysis, Demyelinating Diseases such as Neuromyelitis Optica and Swayback, and Diabefic neuropathies such as diabetic foot:
Additional neurologic diseases vvhi'ch can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include nerve compression syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome such ~as cervical rib syndrome, ulnar nerve compression syndrome, neuralgia such as causalgia, cervico-brachial neuralgia, .facial 'neuralgia and trigeminal neuralgia, neuritis such as experimental allergic neuritis, optic neuritis, polyneuritis, poiyradicuIoneuritis and radiculities such as polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-Marie Disease, Hereditary. Optic Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and Werdnib Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies which include Congenital Analgesia and- Familial Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating and Tetany).
Endocrine Disorders Albumin fusion proteins of the invention' and/or polynucleotides encoding albumin fusion proteins of the 'invention, may be used to treat, prevent, diagnose, and/or prognose disorders and/or diseases related to hormone imbalance, and/or disorders or diseases of the endocrine system.
Hormones secreted by the glands of the endocrine system control physical growth, sexual function, metabolism, and other functions. Disorders may be classified -in two ways:
disturbances in the production of hormones, and the inability of tissues to respond to hormones. The etiology of these hormone imbalance or endocririe system diseases, disorders or conditions may be genetic; somatic, such as cancer and some autoimmune diseases, acquired.(e.g., by chemotherapy, injury or toxins), or infectious. Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can - be used as a marker or detector of a particular disease or disorder related to the endocrine system and/or hormone imbalance..
Endocrine system and/or hormone imbalance and/or diseases encompass disorders of - uterine motility including, but not limited to: complications with pregnancy and labor (e.g.., pre-term labor, post-term pregnancy, spontaneous abortion, and .stow or stopped labor); and disorders and/or diseases of xhe menstrual cycle (e.g., dysmenorrhea and endometriosis).
Endocrine system and/or hormone imbalance disorders and/or diseases include disorders and/or diseases of the pancreas, such as, for example, diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis, pheochromocytoma--islet cell tumor syndrome;
disorders and/or diseases of the adrenal glands such as, for example, Addison's Disease, corticosteroid deficiency, virilizing disease, _ hirsutism, Cushing's Syndrome, hyperaldosteronism, pheochromocytoma; disorders and/or diseases of the pituitary gland, such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism, pituitary adenoma, ~.panhypopituitarism, acromegaly, ' gigantism; disorders and/or diseases of the thyroid, including but not limited to, hyperthyroidism; hypothyroidism, Plummer's disease, Graves' disease (toxic diffuse goiter), toxic nodular ~ goiter, thyroiditis (Hashimoto's ~_ thyroiditis, subacute granulomatous thyroiditis, and silent lymphocytic thyroiditis), Pendred's syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormone coupling defect, thymic aplasia, Hurthle cell tumours of the thyroid, thyroid cancer, thyroid carcinoma, Medullary thyroid carcinoma; disorders and/or diseases of the parathyroid, such as, for example, hyperparathyroidism, hypoparathyroidism; disorders and/or diseases of the hypothalamus.
In addition, endocrine system andlor hormone imbalance disorders and/or diseases may also include disorders and/or diseases of the testes' or ovaries, including cancer. Other disorders and/or diseases of the testes or ovaries further include, for example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome, vanishing testes syndrome (bilateral anorchia), congenital absence of Leydig's cells, cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the testis (benign), neoplasias of the testis and neo-testis.
Moreover, endbcrine system andlor hormone imbalance disorders andlor diseases may also include disorders and/or diseases such as, for example, polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.
In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to diagnose, - prognose, prevent; and/or treat endocrine diseases and/or disorders associated with the tissues) in which the Therapeutic protein corresponding to the Therapeutic protein portion of 20~ the albumin protein of the~invention is expressed, -. ' ' Reproductive System Disorders .
The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used for the diagnosis, treatment, or prevention of . diseases and/or disorders of the reproductive system. Reproductive system disorders that can be treated by the compositions of the inventioil, include, but are not limited to, reproductive system injuries, infections, neoplastic disorders, congenital defects, and diseases or disorders which result in infertility, complications with pregnancy, labor, or parturition, and postpartum difficulties.
Reproductive system disorders andlor diseases include diseases andor disorders of the testes, including testicular atrophy, testicular feminization, cryptorchism (unilateral. and bilateral), anorchia, ectopic testis, epididymitis and orchitis (typically resulting from infections . ~ .
such as; for example, gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion, vasitis ~nodosa, germ cell ~ tumors (e.g., seminomas, embryonal cell carcinomas, teratocarcinomas, choriocarcinomas,. yolk sac tumors, and teratomas); stromal tumors (e.g., Leydig cell tumors), hydrocele, hematocele; varicocele, spermatocele, inguinal hernia, and .
disorders of sperm production (e.g., immotile cilia syndrome, aspermia, asthenozoospermia, azoospermia, oligospermia, and teratozoospermia).
. Reproductive system disorders also include disorders. of the prostate gland, such as acute non-bacterial prostatitis, chronic non-bacterial prostatitis; acute bacterial prostatitis, chronic bacterial prostatitis, prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia, benign prostatic hypertrophy or hyperplasia, and prostate neoplastic disorders, including adenocarcinomas, transitional cell carcinomas, ductal carcinomas, and squamous cell carcinomas.
Additionally, the compositions of the invention may be useful iri the diagnosis, treatment, and/or prevention of disorders or diseases of the penis and urethra, including inflammatory disorders, such as balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal .
urethritis, chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and pearly penile papules; urethra( abnormalities, such as hypospadias, epispadias, and phimosis; premalignant lesions, including Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile cancers, including squamous cell carcinomas, carcinoma in ~ situ, verrucous carcinoma, and disseminated penile carcinoma; urethral neoplastic disorders, including penile urethra( carcinoma, bulbomembranous urethra( carcinoma, ,and prostatic urethra( carcinoma; and erectile disorders; such as priapism, Peyronie's disease, erectile dysfunction, and impotence. . .
Moreover, diseases- and/or disorders of the vas deferens include vasculititis and CBAVD (congenital bilateral absence of the vas deferens); additionally, the -albumin fusion proteins of the invention and/or polynucleotides~ encoding albumin fusion proteins of the . . invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders of the seminal vesicles, including hydatid disease, congenital chloride diarrhea, and polycystic kidney disease:
Other disorders and/or diseases of the male reproductive system include, for example, Klinefelter's syndrome, Young's syndrome; premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's,syndrome, high fever, multiple sclerosis, and gynecomastia.
Further, the polynucleotides, fusion proteins of the invention and/or polynucleotides ' encoding albumin fusion proteins of the invention may be used in the diagnosis, treatment, and/or prevention. of. diseases and/or disorders of the vagina and vulva, .including bacterial vaginosis, .candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma, vulvar trauma, adenosis, 'chlamydia vaginitis, gonorrhea, trichomorias vaginitis, condyloma acuminatum, syphilis, molluscum contagiosum, atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis, vulvar vestibulitis, 178 ' . .

and neoplastic disorders, such as squamous cell hyperplasia, clear cell carcinoma, basal cell carcinoma, melanomas, cancer of Bartholin's gland, and vulvar intraepithelial neoplasia.
Disorders andlor diseases of the uterus include dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals), and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, and sarcomas. Additionally, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as a marker or detector of, as well as in the diagnosis, treatment, and/or prevention of congenital uterine abnormalities, such as bicornuate uterus, septate uterus, simple unicornuate uterus, unicomuate uterus with a noncavitary rudimentary horn, unicornuate uterus with a non-communicating cavitary rudimentary horn, unicornuate uterus with a communicating cavitary horn, arcuate uterus, uterine didelfus, and T-shaped uterus. ' Ovarian diseases andlor disorders include anovulation, polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction, ovarian insensitivity to gonadotropins, ovarian overproduction of androgens, right ovarian vein syndrome, amenorrhea~ hirutism, and ovarian cancer (including, but not limited to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endometriod~ carcinoma of the ovary,' ovarian papillary serous adenocarcinoina, ovarian mucinous adenocarcinoma, and Ovarian Krukenberg tumors).
Cervical diseases and/or disorders include cervicitis, chronic cervicitis, 'mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, and cervical neoplasms (including, for example, cervical carcinoma, squarrious .
. metaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, and columnar cell . neoplasia). ' Additionally, diseases and/or disorders of the reproductive system include disorders .
and/or diseases of pregnancy, including miscarriage and stillbirth, such as early abortion, late abortion, spontaneous abortion, induced abortion, therapeutic abortion, threatened abortion, .
missed abortion, incomplete abortion, complete abortion, habitual abortion, missed abortion, and septic abortion; ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational diabetes, intrauterine growth retardation, polyhydramnios, HELLP
syndrome, abruptio placentae, placenta previa, hyperemesis, preeclampsia, eclampsia, herpes gestationis, and -urticaria of pregnancy. Additionally, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used in the diagnosis, treatment, and/or prevention of diseases that can complicate pregnancy,.
. including heart disease, heart failure, ~ rheumatic heart disease, congenital heart 'disease, mural ' valve prolapse, high blood pressure, anemia, kidney disease, infectious disease (e.g.., rube-IIa, cytomegalovirus, toxoplasmosis; infectious hepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus, Graves' disease, thyroiditis, hypothyroidisril, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis of the liver, primary biliary cirrhosis, asthma, systemic lupus eryematosis, . rheumatoid arthritis, myasthenia' gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian cysts, gallbladder disorders,and obstruction of the intestine.
. Complications associated with labor and parturition include premature rupture of the membranes, pre-term labor, post-term pregnancy, postinaturity, labor that progresses too' slowly, fetal distress {e.g., abnormal heart rate (fetal or maternal), breathing problems, and abnormal fetal position), shoulder dystocia, prolapsed umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding. ~ . -Further, diseases and/or disorders of the postdelivery period, including endometritis, myometritis, parametritis, peritonitis, pelvic thrombophlebitis, pulmonary embolism, ~ endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage, and inverted uterus.
Other disorders and/or diseases of the female reproductive system that may be diagnosed, treated, and/or prevented by the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins -of the invention include, for example, Turner's syndrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion {vascular engorgement), frigidity, anorgasmia, dyspareunia, ruptured fallopian tube, and Mittelschmerz.
Infectious Disease .
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat or detect infectious.
agents. For example, by increasing the immune response, particularly increasing the proliferation and difFerentiation . of B and/or T cells, infectious diseases may be treated. The immune response may be 30~ ~ ~. increased by either enhancing an existing immune response, or by initiating a new immune.
response. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also directly inhibit the infectious agent; without necessarily eliciting an immune response.' .
Viruses are one example of an -infectious agent that can cause disease or symptoms that can be treated or detected by albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins ~of the invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not 'limited to the .

following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, 8unyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex; Herpes Zostei), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to:
arthritis; bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome,, hepatitis (A, B, C, E, Chronic Active, Delta),, Japanese B
encephalitis,, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections.
(e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, . Polio, leukemia, Rubella, ' sexually ' transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia.
Albumin fusion proteins of the invention and/or polynucleotides . encoding albumin fusion proteins of the invention, can be used to treat or detect any of these - symptoms or diseases.
In specific embodiments, . fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the. invention are used to treat: meningitis, Dengue, EBV, andlor hepatitis (e.g., hepatitis B). In an additional specific embodiment fusion proteins of the invention . and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. I,n a . further specific embodiment fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat AIDS. . ._ Similarly, bacterial and fungal agents that can cause disease or symptoms and that can be treated or detected by albumin fusion proteins of the invention and/or polynueleotides encoding albumin fusion proteins of the invention include, but not limited to, the following .
Gram-Negative and Gram-positive bacteria, bacterial families, and fungi:
Actinomyces (e.g., Norcardia), Acinetobacter, Cryptococcus neoformans, Aspergillus, Bacillaceae (e.g., Bacillus .
~ anthrasis), Bacteroides (e.g:, Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g:, Borrelia burgdorferi), Brucella, Candidia; Campylobacter, Chlamydia, Clostridium (e.g., Clostridiaim botulinum, Clostridium dificile, Clostridium perfrangens, Clostridium tetarti), Coccidioides, Corynebacterium (e.g.,. Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. ,coli and Enterohemorrhagic E. coli), . Enterobactei (e.g. Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia, Shigella), Erysipelothrix, Haemophilus (e.g., Haemophilus influenza type B), Helicobacter, Legionella 181 ' (e.g., Legionella pneumophila), Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio ' cholerae); Neisseriaceae (e.g., ~' Neisseria gonorrhea, Neisseria rneningitidis), Pasteurellacea, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponema spp.,' Leptospira spp., Borrelia spp.), Shigella spp., Staphylococcus (e.g., Staphylococcus aureus), Meningiococcus, Pneumococ.cus_ and Streptococcus (e.g., Streptococcus pneaimoniae and Groups A, B, and C
Streptococci), and Ureaplasmas. These bacterial, parasitic, and fungal faiiiilies can cause diseases or symptoms, including, but not. limited to: ~ antibiotic-resistant infections, bacteremia, endocarditis, septicemia, eye infections (e.g., conjunctivitis), uveitis, tuberculosis, gingivitis, bacterial diarrhea; opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, dental caries, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery, paratyphoid fever, food poisoning, Legionella disease,, chronic and acute inflammation, erythema, yeast infections, typhoid, pneumonia, gonorrhea, meningitis (e.g., mengitis types A and B), chlamydia, syphillis, diphtheria, leprosy, brucellosis, peptic ulcers, anthrax, spontaneous abortions, birth defects,~pneumonia, lung infections, ear infections, deafness, blindness, lethargy, malaise, vomiting, chronic diarrhea, Crohn's disease, colitis, vaginosis, sterility, pelvic inflammatory diseases, candidiasis, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses); toxemia, urinary tract infections, wound infections, noscomial infections. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, fusion proteins of the , invention and/or polynucleotides encoding albumin fusion proteins, of the invention are used to treat: tetanus, diptheria, botulism, and/or meningitis type B.
Moreover,. parasitic agents causing disease ~ or symptoms , that can be treated, . prevented, and/or diagnosed by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but not limited to, the following families ar class: Amebiasis; Babesio'sis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardias, Helminthiasis, Leishmaniasis, Schistisoma, Theileriasis, Toxoplasmosis,- Trypanosomiasis, and Trichomonas and Sporozoaiis. (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). .
These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., .dysentery, giardiasis), liver disease, ' lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. Albunun fusion.proteins of. the invention and/or polynucleotides encoding 1$2' albumin fusion proteins of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat, .prevent, and/or diagnose malaria.
- ~ Albumin fusion proteins of the invention andlor polynucleotides encoding albumin fusion proteins of the invention could either be by administering an effective amount of an albumin fusion protein of the invnetion to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to'the patient (ex vivo therapy). Moreover, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
Regeneration Albumin fusion proteins. of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997)). The regeneration of tissues could be used ' to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease ~(e.g.
osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
Tissues that could be regenerated using the present invention include organs (~e. g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilabe, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.
Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may increase regeneration of tissues difficult to heal. For example, increased tendon/ligariient regeneration would quicken recovery time after damage.
Albumin fusion proteins of the. invention and/or polynucleotides encoding albumin fusion _proteins of the invention could, also be used proghylactically in an' effort to avoid damage.
Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds . includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by,using fusion proteins of . the invention and/or polynucleotides encoding albumin fusion proteins of the invention, to proliferate and differentiate. nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, , or mechanical and traumatic disorders~(e.g., spinal .cord disorders, head trauma, cerebrovascular disease, and .stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other.medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the albumin fusion proteins of the invention and/or.polynucleotides encoding albumin fusion proteins of the invention.
Gastrointestinal Disorders Albumin fusion proteins of the invention andlor polynucleotides encoding albunun .
fusion proteins of the invention, may be used to treat, prevent, diagnose, and/or prognose gastrointestinal disorders, including inflarrimatory diseases and/or conditions, infections, ~ cancers (e.g., intestinal neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of the small intestine, small bowl lymphoma)), and ulcers, such as peptic ulcers.
Gastrointestinal disorders include .dysphagia, odynophagia,~ inflammation of the esophagus,. peptic esophagitis, gastric reflux, submucosal f brosis and stricturing, Mallory Weiss lesions, leiomyomas, lipomas, epidermal cancers, adeoncarcinomas, gastric retention ~ disorders, gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps of the stomach, autoimmune disorders such as pernicious 'anemia, pyloric stenosis, gastritis (bacterial, viral, eosinophilic, stress-induced, chronic erosive, . atrophic, plasma cell, and Menetrier's), and peritoneal diseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst, -mesenteric Iymphadenitis, mesenteric vascular occlusion, ' panniculitis, . neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess,).
Gastrointestinal disorders also include disorders associated with the small intestine, such as malabsorption syndromes, distension, irritable bowel syndrome, sugar intolerance, .
celiac disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's disease, intestinal lymphangiectasia, Crohn's disease; appendicitis, obstructions of the ileum, Meckel's diverticulum, multiple diverticula, failure of complete rotation of the small and large intestine, lymphoma, and bacterial and parasitic diseases (such as Traveler's diarrhea, typhoid and paratyphoid, cholera, infection by Roundworms (Ascariasis lumbricoides), Hookworms .
(Ancylostama dacodenale), Threadworms (Enterobius vermicularis), Tapeworms (Taenia sagirzata, Echinococcus granulosus, Diphyllobothriurri spp., and T. solium)..
. Liver diseases and/or disorders include intrahepatic cholestasis (alagille' syndrome,_ ..
biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye syndrome), fiepatic vein thrombosis, ~ hepatolentricular ~ degeneration, hepatomegaly, hepatopulmonary syndrome, 184 ' ' hepatorenal syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary and experimental), alcoholic liver diseases (fatty liver, hepatitis, cirrhosis), parasitic {hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocellular, and cholestatic), cholestasis, portal hypertension, liver enlargement, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune, hepatitis B, hepatitis C, hepatitis D, drug ~
induced), toxic hepatitis, viral, human hepatitis (hepatitis A, hepatitis 'B, hepatitis C, hepatitis D, hepatitis E), Wilson's _ disease, granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, portal hypertension, varices, hepatic encephalopathy, primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangiomas, bile stones, liver . failure (hepatic encephalopathy, acute liver failure), . and liver neoplasms (angiomyolipoma, calcified liver metastases, cystic liver metastases, epithelial tumors, filirolamellar hepatocarcinoma, focal nodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma, hepatoblastoma; hepatocellular carcinoma, hepatoma, liver cancer, liver ~ hemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors of liver, nodular regenerative hyperplasia, benign liver tumors (Hepatic cysts [Simple cysts, Polycystic liver disease, Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymal hamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis, Lipoma5, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors [Bile duct epithelium (Bile duct hamartoma, Bile duct adenoma), Hepatocyte (Adenoma, Focal nodular hyperplasia, Nodular regenerative hyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma, hepatocellular- carcinoma, cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors bf blood vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma; other tumors, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma,- carcinoid, squamous carcinoma, primary lymphoma]), peliosis hepatis, erythrohepatic .porphyria, .hepatic porphyria (acute. intermittent porphyria, porphyria cutanea tarda), Zellweger syndrome).
Pancreatic diseases and/or disorders include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas, cystadenocarcinoma, insulinoma; gastrinoma, and glucagonoma, cystic neoplasms, islet cell tumors, pancreoblastoma), and other pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency)).
- Gallbladder diseases include gallstones (cholelithiasis and choledocholithiasis), postcholecystectomy syndrome, diverticulosis of the gallbladder, acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.
Diseases and/or disorders .of the large intestine include =antibiotic-associated colitis, diverticulitis, , ulcerative colitis, acquired megacolon, ~ abscesses, fungal ~ and bacterial 1$5 WO 01/79271 PCT/iJS01/12009 infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases (colitis, colonic neoplasms [colon cancer, adenomatous colon polyps (e.g., vinous adenoma), colon carcinoma, colorectal cancerJ, colonic diverticulitis, colonic diverticulosis, megacolon [Hirschsprung disease, toxic megacolon]; ' sigmoid diseases . [proctocolitis, sigmoin neoplasms]),constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery), duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenal ulcer,.
duodenitis), enteritis (enterocolitis), HN enteropathy, ileal diseases (ileal neopiasms, ileitis), immunoproliferative small intestinal disease, inflammatory bowel disease (ulcerative colitis, Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis, .balantidiasis, blastocystis~ infections, cryptosporidiosis, dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula (rectal fistula), intestinal neoplasms (cecal neoplasms, colonic neoplasms, duodenal neoplasms, deal neoplasms, intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferent loop syndrome, duodenal obstruction, impacted feces, intestinal pseudo-obstruction [cecal volvulus], intussusception), intestinal perforation, intestinal polyps (colonic polyps, :gardner syndrome, peutz jeghers syndrome), jejunal diseases (jejunal neoplasms), malabsorption syndromes (blind, loop syndrome, celiac disease, lactose intolerance, short bowl syndrome, tropical sprue, whipple's disease), , mesenteric vascular occlusion, pneumatosis cystoides intestinalis, protein-losing enteropathies {intestinal lymphagiectasis), rectal diseases (anus diseases, fecal incontinence, hemorrhoids, proctitis, rectal fistula, rectal - - prolapse, rectocelej; peptic ulcer (duodenal wlcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger-Ellison syndrome), postgastrectomy syndromes (dumping syndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux (bile reftux), gastric antral vascular ectasia, gastric fistula, gastric outlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis, stomach dilatation, stomach diverticulum, stomach neoplasms . (gastric ~carlcer, gastric polyps, gastric adenocarcinoma, hyperplastic gastric polyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis, visceroptosis;
vomiting (e.g., hematemesis, hypererriesis gravidaruin, postoperative nausea and vomiting) and hemorrhagic colitis. ' . .
Further diseases and/or disorders of the gastrointestinal system include biliary tract _ diseases, such as, gastr_oschisis,~ fistula ~(e.g., , biliary fistula, esophageal fistula, gastric fistula,, intestinal fistula, pancreatic fistula), neopiasms (e.g., biliary tract neoplasms, esophageal neoplasms, such as adenocarcinoma of the esophagus, . esophageal squamous cell carcinoma, gastrointestinal neoplasms, pancreatic' neoplasms, such as adenocarcinoma of the pancreas, mucinous cystic neoplasm of the pancreas, pancreatic cystic neoplasms, .
pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g., bullous diseases, candidiasis, glycogenic acanthosis, ulceration, barrett esophagus va.riees, atresia, cyst, diverticuIurii (e.g., Zenker's diverticulum), fistula (e.g., tracheoesophageal fistula), motility WO 01/79271 PCT/iTS01/12009 disorders (e.g., CREST syndrome, deglutition disorders, achalasia, spasm, gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaave syndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal hernia);
gastrointestinal diseases, such as, gastroenteritis (e.g., cholera morbus, norwalk virus infection}, hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, stomach cancer)}, hernia (e.g., congenital diaphragmatic hernia, femoral hernia; inguinal hernia, obturator hernia, umbilical hernia, ventral hernia}, and intestinal diseases (e.g., cecal diseases (appendicitis, cecal neoplasms)).
Chemotaxis Albumin fusion proteins ,of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, .neutrophils, T-cells, mast cells, eosiriophils, epithelial and/or endothelial cells) to a particular site in the bob, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the ' particular trauma or abnormality.
Albumin fusion proteins of the invention andlor polynucleotides encoding.
albumin fusion proteins of the invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to, tissues by attracting irrimune cells to the injured location.
Chemotactic molecules of he present invention can also attract fibroblasts, which can be used to treat wounds_ ' ' ' It is also contemplated that fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used as an ' inhibitor of chemotaxis.
Binding Activity.
Albumin fusion proteins of the- invention may be used to screen for molecules that bind to the Therapeutic protein portion of the fusion protein or for molecules to which the Therapeutic protein portion of the fusion protein binds. The binding of the fusion protein and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the fusion protein or the molecule bound. ~ Examples of such molecules include antibodies, WO 01/79271 PCT/iTS01/12009 oligonucleotides, proteins (e.g., receptors), or small molecules.
Preferably, the molecule is closely related to the natural ligand of the Therapeutic protein portion of the fusion protein of the invention, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current ~ Protocols in Immunology I(2):Chapter 5 (1991)). Similarly,~the molecule can be closely related to the natural receptor to which the Therapeutic protein portion of an albumin fusion protein of the invention binds, or at least, a fragment of the receptor capable of being bound by the Therapeutic protein portion of an albumin fusion protein of'the invention (e.g., -active site). In either case, the molecule can be rationally designed using known techniques.
Preferably, the screening for these molecules involves producing appropriate cells which express the albumin fusion proteins of the invention. Preferred cells include cells from mammals, yeast; Drosophila, or E. coli.
The assay may simply test binding of a candidate compound to an albumin fusion protein bf the invention, wherein bindiilg is detected by a label, or in an assay involving competition with a labeled competitor. Further,' the assay may test whether the candidate compound results in a signal generated by binding to the fusion protein.
Alternatively, the. assay can be carried out using cell-free preparations, fusion protein/molecule affixed to a solid support, ~ chemical libraries, or natural product mixtures. -The assay may also simply comprise the steps of mixing a ,candidate compound with a solution containing an albumin fusion protein, measuring fusion protein/molecule activity or binding, and comparing the.fusion,protein/molecule activity or binding to a standard.
Preferably, an ELISA assay can measure fusion protein level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can . measure fusion protein level or activity by either binding, directly or indirectly, to. the albumin fusion.protein or by competing with the albumin fusion protein for a substrate. .
Additionally, the receptor to which a Therapeutic protein portion of an albumin fusion protein of the invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan,-et al., Current Protocols in Immun., 1(2), Chapter 5, (199I)). For example, in cases wherein the Therapeutic protein portion, of the fusion protein corresponds to FGF; expression cloning may be employed wherein polyadenylated RNA is prepared from a cell responsive to the albumin fusion .
protein, for example, NIH3T3. cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA.library created from this RNA
is divided into pools and used to transfect COS cells or other cells that are not responsive to the albumin fusion protein. Transfected cells which are grown on.glass~ slides are exposed to the albumin fusion protein of the present invention, after they have been labeled. ~ The albumin fusion proteins can be labeled by_a variety of means including iodination or inclusion of a recognition ' site for a site-specific protein kinase.
Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identif ed and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.
As an alternative approach for receptor identification, a labeled albumin fusion protein can be photoafFnity linked with cell membrane or extract preparations that express the receptor rriolecule for the Therapeutoc . protein component of an albumin fusion protein of the invention, the linked material may be resolved by PAGE analysis and exposed to, X-ray film.
~ The labeled complex containing the receptors of the fusion protein can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained. from microsequencing would be used to design,a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.' Moreover, the techniques of gene-shuffling, motif shuffling, exon-shuffling;
and/or colon-shuffling (collectively referred to as "DNA shuffling") may be employed to modulate the activities of the fusion protein, and/or Therapeutic protein portion or albumin component of an albumin fusion protein of. the present invention, thereby effectively generating agonists and antagonists of an albumin fusion protein of the present invention. See generally, U.S.
Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P.
A., et al., Curr. Opinion Biotechreol. 8:724-33 (I997); Harayama, S. Trends Biotechnol.
16(2):76-82 (1998); Hansson, L: O., et al., J. ~Mol. Biol. 287:265-76 (1999);
and Lorenzo, M. M. and Blasco,.R. Biotechniques 24(2):308-13 (1998); each' of these patents and publications are hereby incorporated by reference). In one' embodiment, alteration of polynucleotides encoding albumin fusion proteins of the invention and thus, the albumin.
fusion proteins encoded thereby, may be achieved by DNA shuffling. DNA
shuffling involves the assembly of tvvo or more DNA segments into a desired molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides encoding albumin fusion proteins of the invention and thus, the albumin fusion proteins encoded thereby, may be altered by being subjected to random mutagenesis by error-prone . PCR, random nucleotide insertion or other methods prior to recombination. _ In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of an albumin fusion protein of the present invention may be recombined with one or more components, motifs, sections, parts; domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In . further preferred embodiments, the heterologous. molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-S, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-..alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF-betas, and glial-derived neurotrophic factor (GDNF).
~ Other preferred fragments are biologically active fragments of the Therapeutic protein portion and/or albumin component of the albumin fusion proteins of the present invention.
Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of a Therapeutic protein portion andlor albumin component of .the albumin fusion proteins of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
Additionally, this invention provides a method of screening compounds to identify those which modulate the action of an albumin fusion protein of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, an albumin fusion protein of the present invention, and the compound to be screened and 3 [H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate.. A control .
assay may be performed in the absence of the compound to be screened and compared to the ,amount of fibroblast proliferation in the presence of the compound to determine if the compound.stimulates proliferation by determining the uptake of 3[H] thymidine in each case.
The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3[H] thymidine. Both agonist ,and antagonist compounds may be identified by this procedure.
In another method, a mammalian cell or membrane preparation expressing a receptor for the Therapeutic protien component of a fusion protine of the invention is incubated with a labeled fusion protein -of the present invention in the presence of the compound. The ability .
of the compound to enhance or block this interaction could then be measured.
Alternatively, the response of a known second messenger system following interaction of a compound to be .
screened and the receptor is measured and the' ability of the compound~to bind to the receptor . and elicit a second messenger response is measured to determine if the compound is a potential fusion protein. - Such second messenger systems include but are not limited to, -cAIVIP guanylate cyclase, ion channels or phosphoinositide hydrolysis. ~ ~ .
All of these above assays can be used as diagnostic or prognostic markers. The molecules ,discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the fusion protein/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the albumin fusion proteins of the invention from suitably manipulated cells or tissues. ~ ' ' Therefore, the invention includes a method of identifying compounds which bind. to an albumin fusion protein of the invention comprising the steps of: (a) incubating a candidate binding compound with an albumin fusion protein of the present invention; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound ' with ~an albumin fusion protein of the present invention; (b) assaying a biological activity, and (b) determining if a biological activity of the fusion protein has been altered.
Targeted Delivery In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a component of an albumin fusion protein of the invention. .
As discussed herein, fusion proteins of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, .
hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a , method for the specific delivery of compositions of the invention to cells by administering ., fusion proteins of the invention (including antibodies) that are associated with heterologous polypeptides oi- nucleic acids. In one example, the invention provides a method for delivering ' a Therapeutic protein into the targeted cell. In another example, the invention -provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g.,, DNA that can integrate into - the cell's genome or replicate , episomally and that can be transcribed)'into the targeted cell. .
In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering an albumin fusion protein of the invention (e.g., polypeptiøes of the invention. or antibodies of the invention) in association With toxins or cytotoxic prodrugs. .
By "toxin" is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the 'methods of the invention include, but are not~limited to, radioisotopes known in the art, compounds such as, .for example, antibodies (or complement fixing containing portions 'thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, ~RNAse, alpha oxin, ricin, abrin, Pseudomorcas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin.
By "cytotoxic prodrug" is meant a non-toxic compound that is converted by ari enzyme, normally present in the cell, - into a ~ cytotoxic compound. ~ Cytotoxic prodrugs that may be used according.to the methods of the invention include,_but are not limited to, glutainyl derivatives WO 01/79271 PCT/iJS01/12009 of benzoic acid-mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.
Drug, Screening Further contemplated is the use of the albumin fusion proteins of the present invention, or .the polynucleotides. encoding these fusion proteins, to screen for molecules which modify the activities of the albumin fusion protein of the present invention or proteins corresponding to the Therapeutic protein portion of the albumin fusion protein. Such a method would include contacting the fusion protein with a selected compounds) suspected of having antagonist or agonist activity, and assaying the activity of the fusion protein following binding.
This invention is particularly useful for screening therapeutic compounds by using the albumin fusion proteins of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The albumin fusion protein employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with~recombinant nucleic acids expressing the albumin fusion protein. Drugs are screened against such transformed cells or supernatants obtained from culturing such cells, in. competitive binding . assays: One may measure, for example, the formulation of complexes between the agent being tested and an albumin fusion protein of the present invention.
Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the albumin fusion proteins of the present invention. These methods comprise contacting such an agent with an albumin fusion protein of the present invention or a fragment thereof and assaying for, the .presence of a _ complex between the agent and the albumin fusion protein or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a_ measure of the ability of a particular agent to bind to the alburiiin fusion protein of the present invention.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to an albumin fusion protein of the present invention, and is described in great detail in European Patent Application 84/03564, published on September 13, 1984,.which is-incorporated herein by reference herein.
Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds 'are reacted with ,an albumin fusion protein of the present invention and washed. Bound peptides are then detected by methods well known in the art. Purified albumin fusion protein may be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.
'. This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding an albumin fusion protein of the present invention specifically compete with a test compound for binding to the albumin fusion protein or fragments thereof. In this manner, the antibodies are used to detect the presence of any IO peptide which shares one or more antigenic epitopes with, an albumin fusion protein of the invention. .
Binding Peptides and Other Molecules IS - The invention also encompasses screening methods for identifying polypeptides and nonpolypeptides that bind albumin fusion proteins of the invention, and the binding molecules identified thereby. These binding molecules are useful, for example, as agonists and antagonists -of the albumin fusion proteins of the invention. Such agonists and antagonists can be used, in accordance with the invention, i-n the therapeutic embodiments described in 20 detail, below This method comprises the steps of:
contacting an albumin fusion protein of the invention with a pluralifiy of molecules;
and identifying a molecule that binds the albumin fusion protein.
25 - The step of contacting the albumin fusion protein of the invention with the plurality~of . molecules may be effected in a number of ways. For example, one may contemplate immobilizing the albumin fusion protein on, a solid support and bringing a solution of the plurality of molecules in contact with the immobilized polypeptides. Such a procedure would be akin to arl affinity chromatographic process, with the affinity matrix being comprised of the 30 immobilized albumin fusion protein of the invention. The molecules having a selective affinity for the albumin fusion protein can then be purified by affinity selection. The nature of the solid support, process for attachment of the albumin fusion protein to the solid support, solvent; and conditions of the affinity isolation or selection are largely conventional and well known to those of ordinary skill in the art. ' _ 35 , Alternatively, one may also separate a plurality of polypeptides into substantially separate fractions comprising a subset of or individual. polypeptides. For instance, ~ one can separate the plurality of polypeptides by gel electrophoresis, column chromatography, or like method known to those of ordinary skill for the separation of polypeptides.
The individual polypeptides can also be produced by a transformed host cell in such a way as to be expressed on or about its outer surface (e.g., a recombinant phage). Individual isolates can then be "probed" by an albumin fusion protein of the invention, optionally in the presence of an inducer should one be required for expression, to determine if any selective a~niiy interaction takes place between the albumin fusion protein and the individual clone. Prior to contacting the albumin fusion protein with each fraction comprising individual polypeptides, the polypeptides could first be transferred to a solid support for additional convenience. Such a solid support may simply be a piece of filter membrane, such as one made of nitrocellulose or ~ nylon. In this manner, .positive clones could be identified from a collection of transformed host cells of an expression library, which harbor a DNA construct encoding a polypeptide having a selective affinity for an albumin fusion protein of the invention.
Furthermore, the amino acid sequence of the polypeptide having a selective affinity. for an albumin fusion protein of the invention can be determined directly by conventional means or the coding sequence of the DNA encoding the polypeptide can frequently be determined more coriveniently.~ The primary sequence can then be deduced from the corresponding DNA
sequence. If the amino acid sequence is to be determined from the polypeptide itself, one may use ~microsequencing techniques. The sequencing technique may include mass spectroscopy.
In certain situations, it may be desirable to wash away any unbound polypeptides from a mixture of an albumin fusion protein of the invention and the plurality of polypeptides prior to attempting to~ determine or to detect the presence of a selective affinity interaction.
Such a wash step may be particularly desirable when the albumin fusion protein of the invention or the plurality of polypeptides are bound to a solid support.
The plurality of molecules provided according to this method may be provided by way of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind an albumin fusion protein of the invention: Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries.
Examples of chemically synthesized libraries are described in Fodor et aL, Science 251:767 ~ 773 (1991); Houghten et al., Nature 354:84=86 (1.991); Lam et al.', Nature 354:82-84(1991);
Medynski, Bio/TechnoIogy 12:709-710_ (I994); Gallop et al., J.. Medicinal Chemistry 37(9):1233-1251 (1994); Ohlmeyer et al., Proc. Natl.' Acad. Sci. USA 90:10922-(1993); Erb et al., Proc. Natl. Acad. Sci. USA 91:11422-11426 (1994); Houghten et al., Biotechniques 13:412 (1992); Jayawickreme et al., Proc. Natl. Acad. Sci. USA
91:1614-1618 (1994); Salmon et al., Proc. Natl. Acad. Sci. USA 90:11708-11?12 (1993);
PCT
Publication No. WO 93/20242; and Brenner and Lerner, Proc. Natl. Acad. Sci.
USA
89:5381-5383 (1992). ' - Examples of phage display libraries are described in Scott et al., Science 249:386-390 (1990); Devlin et al., Science, 249:404-406 (1990); Christian et al., 1992, J.
Mol. Biol.
227:711-718.1992); Lenstra, J. Immunol. Meth. 152:149-157 (1992}; Kay et al., Gene 128:59-65 (1993); and PCT Publication No. WO 94%I8318 dated Aug. 18, 1994.
~ ~ In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al., Proc. Natl.
Acad. Sci. USA 91:9022-9026 (1994).
By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., Bunin et al., Proc. Natl. Acad. Sci. USA 91:4708-4712 (1994)) can be adapted for use. Peptoid libraries (Simon et al., Proc. Natl. Acad. Sci. USA 89:9367-9371 (1992)) can also be used.
Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by.Ostresh et al. (Proc. Natl. Acad. Sci. USA 91:11138-11142 (1994)).
The variety of non-peptide libraries that are useful in the present invention is great.
For example, Ecker and Crooke (Bio/Technology 13:351-360 (1995) -list benzodiazepines, hydantoins, piperaainediones, biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, and oxazolones as among the chemical species that form the basis of various libraries.
Non-peptide libraries can be classified broadly into two types: decorated monomers and oligomers. Decorated monomer libraries employ a relatively simple scaffold structure upon which a variety functional groups is added. Often the scaffold will be a molecule-with a known useful pharmacological activity. For example, the scaffold might be the benzodiazepine structure.
Non-peptide oligomer libraries utilize a large number of monomers that are assembled together in ways that create new shapes that depend on the order of the monomers. Among the monomer units that have been used are carbamates, pyrrolinones, and morpholinos.
Peptoids, peptide-like oligomers in~which the side chain is attached to the alpha amino group rather than the alpha carbon, form the basis of another version of non-peptide oligomer libraries. The first non-peptide oligomer libraries utilized a single.type of monomer and thus contained a repeating backbone. Recent libraries have utilized more than one monomer, giving the libraries added flexibility. .
Screening~the libraries can be accomplished by any of a variety of commonly known methods.' See, e.g., the following references, which disclose screening of peptide libraries:
Parmley et al., Adv. Exp. Med. Biol. 251:215-218 (1989); Scott et al,. Science 249:386-390 (1990); Fowlkes et al., BioTechniques 13:422-4.27 (1992); Oldenburg et al., Proc. Natl.
.Acad. Sci. USA 89:5393-5397. (1992); Yu et al., Cell 76:933-945 (1994};
Staudt .et al., Science 241:577-580 (1988); Bock et al.,- Nature 355:564-566 (1992); Tuerk et al., Proc.

Natl. Acad. Sci. USA 89:6988-6992 (1992); Ellington et al., Nature 355:850-852 (1992);
U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar et al., Science 263:671-673 (1993); and PCT Publication No. WO
94/18318.
In a specific embodiment, screening to identify a molecule that, binds an, albumin fusion protein of the invention can be carried out by contacting the library members with an albumin fusion protein of the invention immobilized on a solid phase and harvesting those .
library members that bind to the albumin fusion protein. Examples of such screening .
methods, termed "panning" techniques~are described by way of example in Parmley et al., Gene 73:305-318 (1988); Fowlkes et al., BioTechniques 13:422-427 (1992); PCT
Publication No. WO 94/18318; and in references cited herein.
In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields et al., Nature 340:245-246 (1989); Chien et al., Proc.- Natl.
Acad. Sci. US.A
88:9578-9582 (1991) can be used to identify molecules that specifically bind to polypeptides of the invention. ~ ~ .
Where the binding molecule is a polypeptide, the polypeptide can be conveniently selected from any peptide library, including random peptide libraries, combinatorial peptide libraries, or biased peptide libraries. The term "biased" is used herein to mean that the method of generating the library is manipulated so as to restrict one or more parameters that.govern the diversity of the resulting collection of molecules, in this case peptides.
Thus, a truly random peptide library would generate a collection of peptides in which the probability of finding a particular amino acid at a given position of the peptide is the same for all 20 amino acids. A bias can be introduced into the library, however, by specifying, for example, that a lysine occur every fifth amino acid or that positions 4, 8, and 9 of a decapeptide library be fixed to include only arginine. Clearly, many types of biases can be contemplated, and the present invention is not restricted to any particular .bias. Furthermore, the present invention contemplates specific types of peptide libraries, such as phage displayed peptide libraries and.those that utilize a DNA constmct comprising a lambda phage vector with a DNA insert. .
As mentioned above, in the case of a binding molecule that is a polypeptide, the polypeptide may have about 6 to less than about 60 amino acid residues, preferably about 6 to about 10 amino acid residues, and most preferably, about 6 to about 22 amino acids. In another embodiment, a bidding polypeptide has in the range of 15-.100 amino acids, or 20-50 amino acids.
' The selected binding polypepfiide ~ can be obtained by chemical synthesis or recombinant expression.

Other Activities An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions ~ such as thrombosis, ' arteriosclerosis, and other cardiovascular conditions. The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the ' ~ repair or replacement of damaged or diseased tissue. ' - , An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed stimulate neuronal growth and to. treat and prevent neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may have the ability to stimulate chondrocyte growth, -therefore, they may be employed to enhance bone and periodontal regeneration and aid in -tissue transplants, or bone grafts.
An albumin fusion protein of the invention andlor polynucleotide encoding an albumin fusion protein of the invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed for- preventing hair loss, since FGF
family members activate hair-forming cells and promotes melanocyte growth.
Along the same lines, an albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues. An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also increase or decrease the differentiation ~or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.

An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue;
pigmentation, size, and shape (e.g., cosmetic surgery). ' Similarly, an albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing; utilization, and storage of energy.
An albumin fusion protein of the invention andlor polynucleotide encoding an albumin fusion protein of the invention may be used to change a mammal's mental state or physical ' IO state by influencing biorhythms, caricadic rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other. cognitive qualities.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.
The above-recited applications have uses in a wide variety of hosts. Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, moose, , rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
30, Without further description, it is. believed that one of ordinary skill 'in the art can, using the preceding description and the following illustrative examples, make and utilize the alterations detected in the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
EXAMPLES
- ~ 198 WO 01/79271 PCT/i1S01/12009 Example 1: Preparation of HA-hGH Fusion Proteins An HA-hGH fusion protein was prepared as follows: , Cloning of hGH cDNA
The hGH cDNA was obtained from a human pituitary gland cDNA library (catalogue number HL1097v, Clontech Laboratories, Inc) by PCR amplification. Two oligonucleotides suitable for PCR amplification of the hGH cDNA, HGH1 and HGH2, were synthesized using an Applied~Biosystems 380B Oligonucleotide Synthesizer.
HGH1: 5' - CCCAAGAATTCCCTTATCCAGGC - 3' (SEQ ID NO: 1) HGH2: 5' - GGGAAGCTTAGAAGCCACAGGATCCCTCCACAG - 3' (SEQ m NO: 2) HGH l and HGH2 differed from the equivalent portion of the hGH cDNA sequence (Martial et. al., 1979)- by two ~ and three nucleotides, respectively, such-that after PCR
amplification an EcoRI site would be introduced to the 5' end of the cDNA and a BamH 1 site would be introduced into the 3' end of the cDNA. In addition, HGH2 contained a HindIII
site immediately downstream of the hGH sequence. .
PCR amplification using ~a Perkin-Elmer-Cetus Thermal Cycler 9600 and a Perkin-Elmer-Cetus PCR kit, was performed using single-stranded DNA template isolated from the phage particles of the cDNA.library as follows: 10 p,L phage particles were lysed by the addition of 10 ~uL phage lysis buffer (280 p,g/mL proteinase K in TE
buffer) and incubation at 55°C for 15 min followed by 85°C for 15 min. After a 1 min. incubation on ice, phage debris was pelleted by centrifugation at 14,000 rpm for 3 min. The PCR ' mixture contained 6 ~L of this DNA template, 0.1 lZM of each primer and 200 p,M of each.
deoxyribonucleotide. PCR was carried out for 30 cycles, denaturing at 94°C for .30 s, annealing at 65°C for 30 s and extending at 72°C for 30 s, increasing the extension time by 1 s per cycle. , .
Analysis of the reaction by gel electrophoresis showed a single product of the expected size (589 base pairs). ' . . , The PCR product was purified . using Wizard PCR Preps DNA Purification System (Promega Corp) and then digested with EcoRI and HincIIII. After further purification of the EcoRl-HindIII fragment by gel electrophoresis, the product was cloned into pUCl9 (GIBCO
BRL) digested with EcoRI and HindIII, to give pHGHl. DNA sequencing of the EcoRl HindIII region showed that the PCR product was identical in sequence to the hGH sequence (Martial et a1:,.1979), except at the 5'. and 3' ends, where the EcoRl and BamHI sites had been introduced, respectively. .
Expression of the hGH cD ,NA.

The polylinker sequence of,the phagemid pBluescribe (+) (Stratagene) was replaced by inserting an oligonucleotide linker, formed by annealing two 75-mer oligonucleotides, between the EcoRI and HindIII sites to form pBST(+). The new polyIinker included a unique NotI site.
The Notl HA expression cassette of pAYE309 (EP 431 880) comprising the PRBI
promoter, DNA encoding the HA/MFa-1 hybrid leader sequence, DNA encoding HA
and the ADHl terminator, was transferred to pBST(+) to form pHAl. The HA. coding sequence was removed from this plasmid by digestion with HindIII followed by religation to form pHA2.
Cloning of the hGH cDNA, as described in Example ,I, provided the hGH coding region lacking the pro-hGH sequence and the first 8 base pairs (bp) of the mature- hGH
sequence. In order to construct an expression plasmid for secretion of hGH
from yeast., a ~~ yeast promoter, signal peptide and the first 8 by of the hGH sequence were attached to the 5' end of the cloned hGH sequence as follows: The HindIII-SfaNI fragment from pHA
1 was attached to the 5' 1 end of the EcoRIlHindIII fragment from pHGHI via two synthetic oligonucleotides, HGH3 and HGH4 (which can anneal to one another in such a way as to generate a double stranded fragment of DNA with sticky ends that can anneal with SfaNIand EcoRI sticky ends): ' HGH3: 5' - GATAAAGATTCCCAAC = 3'-(SEQ ID NO: 3) HGH4: 5' - AATTGTTGGGAATC"FTT- 3' (SEQ ID NO: 4) The. HindIII fragment so formed was cloned' into HindI~I-digested pHA2 to make pHGH2, such that the hGH cDNA was positioned downstream of the PRBI promoter and HAIMFa-1 fusion leader sequence (see, International Publication No. WO
90/01063). The NotI expression cassette contained in pHGH2, which included the ADHI
terminator downstream of the hGH cDNA, was cloned into Noti-digested pSAC35 (Sleep et al., BioTechnology 8:42 (1990)) to make pHGHl2. This plasmid comprised the entire 2 ~,m plasmid to provide replication functions and the LEU2 gene for selection of transformants.
pHGHl2 was introduced into S. cerevisiae D88 by transformation and individual transformants were grown for 3 days at 30°C in 10 mL YEPD (1% ww yeast extract, 2 w/v, peptone, 2 % w/v; dextrose).
. .After centrifugation of the cells, . the ' supernatants were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were found to contain protein which was of the expected size and which was recognized by anti-hGH antiserum (Sigma, Poole, UK) on Western blots.
- Cloning and expression of an HA-hGH fusion protein.
In order to fuse the HA cDNA to the , 5' end of the hGH cDNA, the pHAl HindIlI-Bsu361. fragment (containing most of the HA cDNA) vvas joined. to the pHGHl EcoRI-HinclIll fragment (containing most of the hGH cDNA) via two oligonucleotides, HGH7 and HGH8 HGH?: 5' - TTAGGCTTATTCCCAAC 3' (SEQ ID NO: 5) HGHB: 5' - AATTGTTGGGAATAAGCC 3' (SEQ ID NO: 6) The HincIIII fragment so formed was cloned into pHA2 digested with HindIII to make pHGHlO, and the Notl expression cassette of this plasmid was cloned into. Notl-digested pSAC35 to make pHGHl6.
pHGHl6 was used to transform S. cerevisiae D88 and supernatants of cultures were analyzed as described above. A predominant band was observed that had a molecular weight of approximately 88 kD; corresponding to the combined masses of HA and hGH.
Western blotting using anti-HA and anti-hGH antisera (Sigma) confirmed the presence of the two constituent parts of the albumin fusion protein.
The albumin fusion protein was purified from culture supernatant by cation exchange chromatography, followed by anion exchange and gel permeation chromatography.
Analysis of the N-terminus of the protein by amino acid sequencing confirmed the presence of the expected albumin sequence.
An in vitro growth hormone activity assay (Ealey et al., Growth Regulation 5:36 (1995)) indicated that the albumin fusion protein possessed full hGH activity.
In a hypophysectomised rat weight gain mode(, performed essentially as described in the European Pharmacopoeia (1987, monograph 556), the fusion molecule was more potent than hGH when the same number of units of activity (based on the above in vitro assay) were administered daily. Further experiments in which the albumin fusion protein was administered once every four days showed a similar overall growth ~ response to a daily administration of . ' hGH. .Pharmacokinetic experiments in which '25I- labeled protein was administered to rats indicated an approximately ten-fold increase in: circulatory half life for the albumin fusion protein compared to hGH.
A similar plasmid was constricted in which DNA encoding the S. cerevisiae invertase (SUC2) leader sequence replaced the sequence for the hybrid leader, such that the encoded leader and the junction (~, ) with the HA sequence were as follows:
... MLLQAFLFLLAGFAAKISA ,~ DAHKS ..... (SEQ ID NO: 7) Invertase leader HA sequence ...
On introduction into S. cerevisiae DBI, this plasmid~ directed the expression and secretion of the albumin fusion protein at a level similar to that obtained with pHGHl6.
Analysis of the N-terminus of the albumin fusion protein indicated precise and efficient cleavage of the leader sequence from the mature protein.

Cloning arid expression of ati hGH-HA fusion protein. . -In order to fuse the hGH cDNA to the 5' end of the HA cDNA, the HA cDNA was first altered by site-directed mutagenesis to introduce ~an EcoNl site near the 5' end of the coding region. This was dotle by the method of Kunkel et al. (Methods in Enzymol. 154:367 (1987)) using single-stranded DNA template prepared from pHAI .and a synthetic oligonucieotide, LEU4:
LEU4: 5' - GAGATGCACACCTGAGTGAGG - 3' {SEQ ID NO: 8) Site-directed mutagenesis using this oligonucleotide changed the coding sequence of the HA cDNA from Lys4 to Leu4 (K4L). However, this change was repaired when the hGH
cDNA was subsequently joined at the 5' end by linking the pHGH2 Notl-BamHI
fragment to the EcoNI-Notl fragment of the mutated pHA.I, via the two oligonucleotides HGHS and HGH6:
HGHS: S' - GATCCTGTGGCTTCGATGCACACAAGA - 3' (SEQ ID NO: 9) HGH6: 5' - C'TCTTGTGTGCATCGAAGCCACAG - 3' (SEQ ID NO: 10) , The Notl fragment so formed was cloned into Notl-digested pSAC35 to make pHGHl4. pHGHl4 was used to transform S. cerevisiae D88 and supernatants of culture were analyzed as above. A predominant band was observed that had a molecular weight of approximately 88 kD, ~ corresponding to .the combined masses of hGH and HA.
Western blotting using anti-HA and anti-hGH antisera confirmed the presence of the two constituent parts of the albumin fusion protein.
The albumin fusion protein was purified from culture supernatant by ration exchange chromatography, followed by anion exchange and gel permeation chromatography.
Analysis of .the N-terminus ~of the protein by amino acid sequencing confirmed the presence of the expected hGH sequence.
1h vitro studies showed that the albumin fusion protein retained hGH activity, but was significantly less potent than an albumin fusion protein comprising full length HA (1-585) as the N-terminal portion and hGH as the C-terminal portion, as described above.
Constrtaction of plasmids for the expression of hGH fusiorcs to domains of HA.
Fusion poIypeptides were made in which the hGH molecule was fused to the first two domains of HA (residues 1 to 387). Fusion to the N terminus of hGH was achieved by joining the pHAl HindIll-Sapl fragment, which contained most of the coding sequence for domains 1 and 2 of HA, to the pHGHI EcoRl-HindIIl fragment, .via the oligonucleotides HGH 11 and HGH 12:
HGH11: 5' - TGTGGAAGAGCCTCAGAATTTATTCCCAAC - 3' (SEQ ID NO:
11) HGH 12: 5' - AATTGTTGGGAATAAATTCTGAGGCTC'fTCC - 3' (SEQ ID NO:

12) The Hi~zdIII fragment so formed was cloned into HindIII-digested pHA2 to make pHGH37 and the Notl expression cassette of this plasmid was cloned into Notl-digested pSAC35.
The resulting plasmid, pHGH38, contained an expression cassette that was found to direct secretion of the fusion polypeptide into the supernatant when transformed into S .
cerevisiae DB 1. Western blotting using anti-HA and anti-hGH antisera confirmed the presence of the two constituent parts of the albumin fusion protein.
The albumin fusion protein was purified from culture supernatant by cation exchange chromatography followed by gel permeation chromatography.
In vivo studies with purified protein indicated that the circulatory half life was longer than that of hGH, and similar to that of an albumin fusion protein comprising full-length HA
(1-585) as the N-terminal portion and hGH as the C-terminal portion, as described above..ln vitro studies showed that the albumin fusion protein retained hGH activity.
Using a similar strategy as detailed above, an albumin fusion protein comprising the first domain of HA (residues 1-194) as the N-terminal portion and hGH as the C-terminal.
- portion, was cloned and expressed in S. cerevisiae DBL. Western blotting of culture supernatant using anti-HA and anti-hGH antisera confirmed the presence of the two constituent parts of the albumin fusion protein. -Fusion of HA to hGH using a flexible linker sequence - Flexible.linkers, comprising'repeating units of [Gly-Gly-Gly-Gly-Ser]~, where n was either 2 or 3, were introduced between the HA and hGH albumin fusion protein by cloning of the oligonucleotides HGH16, HGH17, HGHl8~and HGH19:
HGH16:5'-TTAGGCTTAGGTGGCGGTGGATCCGGCGGTGGTGGATCTTTCCCA AC-3' (SEQ
ID NO: 13) ' HGH17:S'-AATTGTTGGG.AAAGATCCACCACCGCCGGATCCACCGCCACCTAAGCC-3' (SEQ ID ~NO: 14) . - .
HGH18:5'-TTAGGCI'TAGGCGGTGGTGGATCTGGTGGCGGCGGA~'CTGGTGGCGGTGGATCC
TTCCCAAC-3' (SEQ ID NO: 15) HGH 19: 5'-AATTGTTGGGAAGGATCCACCGCCACCAGATCCGCCGCCACCA
GATCCACCACCGCCTAAGCC-3' (SEQ ID NO: 16) Annealing of HGH16 with HGH17 resulted in n=2, while HGH18 annealed to HGH19 resulted in n=3. After annealing, the- double- stranded oligonucleotides were cloned with the EcoRI-Bsu361 frabQment isolated from pHGHl into Bszs361-digested pHGHlO to makepHGH56 (where n=2) and pHGH57 (where n=3). The Notl expression~cassettes from 203 .

these plasmids were cloned into NotI-digested pSAC35 to .make pHGH58 and .pHGH59, respectively. , , Cloning of the oligonucleotides to make pHGH56 and pHGH57 introduced a BamHI
site in the linker sequences. It was therefore possible toconstruct linker sequences in which n=I and n = 4, by joining either the HindIII-BamHl fragment from pHGH56 to the BamHI-Hindlll fragment frorra pHGH57 (making n = I ), or the HindIII-BamHI
fragment from pHGH57 to the BamHI-HindIII fragment from pHGH56 (making n=2). Cloning of these fragments into the HindIII site ~of pHA2, , resulted in pHGH60 (n= I) and pHGH61 (n=4). The . Notl expression cassettes from pHGH60 and pHGH61 were cloned into Notl-digested pSAC35 to make pHGH62 and pHGH63, respectively.
Transformation of S. cerevisiae with pHGH58, pHGH59, pHGH62 and pHGH63 . resulted in transformants that secreted the fusion polypeptides into the supernatant. Western blotting using arid-HA and anti-hGH antisera confirmed the presence of the two constituent parts of the albumin fusion proteins. ~ ' . The albumin fusion proteins were purified from culture supernatant by cation exchange chromatography, followed by anion exchange and gel permeation chromatography.
Analysis of the N-termini ~of the proteins by amino acid sequencing confirmed the presence of the expected albumin sequence. Analysis of the purified proteins by electrospray mass spectrometry confirmed an increase in mass of 315 D (n=1), 630 D (n=2), 945 D
(n=3) and 1260 D (n=4.) compared to the HA-hGH fusion protein described above. The purified protein was found to be active in vitro.
Increased Shelf=Life o Hf A-hGH,~'usion proteins: Methods HA-hGH and hGH were separately diluted in cell culture media containing 5%
horse .
serum to final concentrations of 100-200 p,glml and incubated at 4; 37 or 50°C, At time zero and at ,weekly intervals thereafter, aliquots of the samples were tested for their biological activity in the Nb2 cell proliferation assay, and the data normalized to the biological activity of the control (hGH solution at time zero). In other assays hGH and HA-hGH were incubated . in phosphate buffer saline in at 4, 37 and 50 degree C.
Nb2 cell proliferation assay: The growth of these cells is dependent on hGH or other , lactogenic hormones. In a typical experiment 104 cells lwell are plated in 96-well plate in the presence of different concentration of hGH or HA-hGH in media such as DMEM
containing 5-10% horse serum for 24-4.8 hrs in the incubator. After the incubation period, 1:10 volume . of MTT (5mg/riil in H20) is added to each well and the plate is incubated for a further 6-I6 hrs.. The growing cells convert MTf to insoluble formazan. The formazan is solublized by acidic isopropanol, and the color produced is measured at 570 nm on microtiter plate reader.
The extent of formazamformation reflects the level of cellular proliferation.
.

Increased shelf life of HA-hGH fission proteins: Restclts' The fusion of Therapeutic proteins to albumin confers stability in aqueous or other solution. Figure 1 depicts the extended shelf-life of an HA fusion protein in terms of the biological activity of HA-hGH remaining after storage in cell culture media for up to 5 weeks at 37°C. A solution of 200 ~g/ml HA-hGH was prepared in tissue culture media containing 5% horse serum, and the solution incubated at 37°C starting at time zero. At the indicated times, a sample was removed and tested for its biological activity in the Nb2 cell assay, at 2 ng/ml final concentration. As shown in Figure 1, the biological activity of.HA-hGH remains essentially intact (within experimental variation) after 5 weeks of incubation at 37°C. The recombinant hGH used as control for this experiment lost its biological activity in the first week of the experiment.
Figure 2 shows the stability of HA-hGH after storage in cell culture media for up to 3 weeks at 4, 37, or 50°C. At time zero, a solution of HA-hGH was prepared in tissue culture media containing 5% horse serum, and incubated at 4, 37, and 50°C. At the indicated periods a sample was removed and assayed for its biological activity in the Nb2 cell proliferation assay, at.60 ng/ml final concentration. HA-hGH retains over 90% of its initial activity at all temperatures tested for at least 3 weeks after incubation while hGH loses its biological activity within the first week. This level of activity is further retained for at least 7 weeks at 37° C and 5 weeks at SO° C. 'These results indicate that HA-hGH is highly stable in aqueous solution even under temperature stress.
Figures 3A and 3B show the stable biological activity of HA-hGH compared to hGH
in the Nb2 cell proliferation assay. Nb2 cells were grown in the presence of 'increasing concentrations of recombinant hGH or HA-hGH, added at time zero. The cells were incubated for 24 or 48 hours before measuring the extent of proliferation by the MTT method.-The increased stability of HA-hGH in the assay results in essentially the same proliferative~
activity at 24 hours (Figure 3A) as at 48 hours (Figure 3B) while hGH shows a significant reduction in its proliferative activity after 48 hours of incubation (Figures 3A and 3B).
Compared to hGH, the HA-hGH has lower biological potency after 1 day; the albumin fusion protein is about 5 fold less potent than hGH. However, after 2 days the HA-hGH
shows essentially the same potency as hGH due to the short life of hGH in the assay.
This increase in the stability of the hGH as an albumin fusion protein has a major unexpected impact on the biological activity of the protein. Although the potency of the albumin fusion proteins is slightly lower than the unfused counterparts in rapid bioassays, their biological stability results in much higher biological activity in the longer term in vitro assayor in vivo assays.

Example 2: ~ Preparationw of HA fusion proteins.
Figure 4 shows a map of a plasmid (pPPC0005) that can be used as the base vector for cloning the cDNAs of therapeutic partners to form HA-fusions. For example, digestion of this vector with the restriction enzymes Bsu36IIPartial HindIII will allow for the insertion of a cDNA modified at the 5' end to encode the last S amino acids of HA including the~Bsu361 site and at the 3' end to include a double stop codon and HindIII site. As another example, digestion of this vector with the restriction enzymes Bsu361 /, Sphl allows for the insertion of a cDNA modified at the 5' end to encode the last 5 amino acids of HA including the Bsu361 site.and at the 3' end to include a double stop codon,. HindIII site and the ADHI terminator sequence up to and including the Sphl site.
This plasmid may easily be modified by one of skill in the art, for example, to modify, add or delete restriction sites so that one may more easily clone a Therapeutic protein, or fragment or variant of into the vector for the purpose of making an albumin fusion protein.of the invention.
For example, for the purpose of making an albumin fusion protein where the Therapeutic moiety is placed N-terminal to the (mature) albumin protein, restriction sites were added at the 5' end of the DNA encoding HA in pPPC0005 shown in Figure 4).
Because it was desired to add unique XhoI and CIaI sites at the 5' end of the DNA
encoding the HA protein in pPPC0005, it was first necessary to remove those same sites from the plasmid (located 3' of the ADHl terminator sequence). This was accomplished by cutting pPPC0005 ~ with XIaoI and CIaI, filling in the sticky ends with T4 DNA
polymerase, and religating the blunt ends to create pPPC0006 Engineering the Xho and Cla I restriction sites into the Fusion leader sequence just 5' 25' of the DNA encoding the HA protein in pPPC0006 was accomplished using two rounds of PCR. The first pair of oligonucleotides are those of SEQ ID N0:19 and SEQ ID
N0:20.
SEQ ID- I9 contains four point mutations relative to the DNA sequence encoding the Fusion leaadr sequence and the beginning of the.HA protein. These mutations are necessary to create the Xhol site in the fusion leader sequence and the Cla I site just at the beginning of the DNA
encoding.the HA protein. These four mutations are underlined in the sequence shown below.
In pPPC0006 the nucleotides at these four positions from 5' to 3' are T, G, T, and G.
5'-GCCTC_GAGAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCGATTTAAAGAT
TTGGG-3' (SEQ ID N0:19) 5'-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCIqJ'TTCTCGAGGCTCCTGGAA
TAAGC-3' (SEQ ID N0:20). A second round of PCR is then performed with an upstream flanking primer, 5'-TACAAACTTAAGAGTCCAATTAGC-3' (SEQ ID N0:21) and a downstream flanking primer 5'-CACTTCTCTAGAGTGGTTTCATATGTCTT-3' (SEQ ID

N0:22). The resulting PCR product is then purif ed and then digested with AflI
and XbaI
and ligated into the same sites in pPPC0006 creating pScCHSA. The resulting plasmid will have an XhoI sites engineered into the fusion leader sequence. The presence of the XhoI site creates a single amino acid change in the end of fusion leader sequence from LDKR to LEKR.
The D to E change will not be present in the final albumin fusion protein expression plasmid if one ligates into the XhoI and CIa.I sites a fragment comprising the Therapeutic moiety which has a 5'. SaII sticky end (which is compatible with the XhoI end) and a 3' CIaI end.
Ligation of the XhoI to the SaII restores the original amino acid sequence of the Fusion leader 'sequence. The Therapeutic protein moiety may be inserted after the Kex2 site (Kex2 claeves after the dibasic amino acid sequence KR at the end of the Fusion leader sequence) and before the CIaI site.
In addition, for the purpose of making an albumin fusion protein _ where the Therapeutic moiety is placed C-terminal to the (mature) albumin protein, four, eight-base-pair restriction sites were added at the 3' end of the DNA encoding HA in pScCHSA.
As an - example, it was felt to be desirable to incorporate AscI, FseI, and PmeI
restriction sites in between the Bsu36I and HindIII sites at the end ,of the DNA encoding the HA
protein in pScCHSA. This was accomplished through the use of two complementary synthetic oligonucleotides (SEQ ID N0:19 and SEQ ID N0:20) which contain the desired restriction sites.
5'-AAGCTGCCTTAGGCTTATAATAAGGCGCGCCGGCCGGCCGTTTAAACTAAGCT
TAATTCT-3' (SEQ ID N0:23) and CAGCTT-3' (SEQ.ID ~N0:24). These oligonucleotides may be annealed and digested with Bsu36I and HindIII and ligated into the same sites located at the end of the DNA, encoding the HA protein in pScCHSA creating pScNHSA, using techniques known in the art.
Making vectors comprising albumin fusion proteins where the albumin moiety is N terminal to the Therapeutic moiety.
The DNA encoding the Therapeutic moiety may be PCR amplif ed using primers that will add DNA encoding the last five amino acids of the HA~(and containing the Bsu36I site) onto the 5' end of the DNA encoding a Therapeutic protein and a STOP codon and appropriate cloning sites onto the 3' end of the coding sequence. For instance, the forward primer used to amplify the DNA encoding a Therapeutic protein might have the sequence, 5'-aagctGCCITAGGCTTA(N)15 3' (SEQ ID N0:25) where the underlined sequence is a Bsu36I site, the upper case nucleotides.encode the last four amino acids of.the mature HA
protein (ALGL), and .(N)15 is, identical to the first 15~ nucleotides encoding 'the Therapetic .
-1 protein of interest. Similarly; the reverse primer used to amplify the DNA
encoding a Therapeutic protein might -have, the . sequence, S'-GCGCGCGTTTAAACGGCCGGCCGGCGCGCC ATTA(N)15 3' (SEQ ID N0:26) , where the italicized nucleotides is a PmeI site, the double underlined nucleotides are a FseI
site, the singly underlined text is a PmeI site, the boxed nucleotides are the reverse complement of two. tandem stop codons, and (N)15 is identical to the reverse complement of the last 15 nucleotides encoding the Therapeutic protein of interest. Once the PCR product is amplified it may be cut with Bsu36I and one of (AscI, FseI, or PmeI) and ligated into -pScNHSA: - ' -Making vectors comprising albumin fusion proteins where the albumin moiety is N-terminal to the Therapeutic moiety.
The DNA encoding the Therapeutic moiety may be PCR amplified using primers that will add DNA encoding the last three amino acids of the Fusion leader sequence (and containing a SaII site) onto the S' end of the DNA encoding a Therapeutic protein and the first few amino acids of the HA (and containing a CIaI site. For instance, the forward primer used to amplify the DNA encoding a Therapeutic protein might - have the sequence, - 5'-aggagc cgt GACAAAAGA(N)1; 3' (SEQ ID N0:27) vc~here the underlined sequence is a Sal I site, the upper case nucleotides encode the last three amino acids of the Fusion leader sequence (DKR), and (N)~5 is identical to the first 15 nucleotides encoding the Therapetic protein of interest. Similarly,- the reverse primer used to amplify the DNA
encoding a Therapeutic protein ' - might have the , sequence, . , 5'-CTTTAAATCGATGAGCAACCTCACTCTTGTGTGCATC(N)is-3' (SEQ ID N0:28) where the italicized nucleotides are -a CIaI site, the underlined nucleotides are the reverse complement of the DNA encoding the first 9 amino acids of HA (DAHKSEVAH), and (N)ls is identical to the reverse complement of the last 15 nucleotides encoding the Therapeutic protein-of interest. Once the PCR product is amplified it may be-cut with,SalI
and CIaI and ligated into pScCHSA digested with XhoI and Cla I.
- Expression of an- Albumin Fusion Protein in yeast.
The Not I fragment containing the DNA encoding either an N-terminal or C-terminal albumin fusion protein generated from pScCHSA or pScNHSA may then be cloned in to the NotI site of pSAC35. ~ - -Expression of an Albumin Facsion Protein from Mammalian cell lines _ The HSA gene has also been cloned iilto a the pC4 vector which is more suitable for mammalian culture systems creating plasmid pC4:HSA. More specifically, pC4HSA
wa$
generated by PCR amplifying the mature HSA gene with a 5' primer-(SEQ ID
N0:30) that anneals to the 5' end of DNA encoding the mature form of the HSA protein (e.g, DNA in plasmid pScCHSA),incorporates BamHI (Shown in italics below) and HindIII
(shown singly underlined below) cloning sites, attaches a kozak sequence (shown double underlined below) and DNA encoding the natural HSA signal peptide (MKWVSFISLLFLFSSAYSRSLDKR;
SEQ ID N0:29) (shown~in bold below), and a 3' primer (SEQ m N0:31) that anneals to the 3' end of DNA encoding the mature form of the HSA protein arid incorporates an Asp718 restriction site (shown in bold below). The DNA encoding the natural human serum albumin leader sequence in SEQ ID N0:30 also contains a modification that introduces a XhoI site that is boxed below.
5'-TCAGGGATCCAAGCTTCCGCCACCATGAAGTGGGTAACCITTATTTCCCTTCTTTTTCTCITTAG
CTCGGCTTA CTCGAG GGGTGTGTTTCGTCGAGATGCACAC'AAGAGTGAG-3'-(SEQ ID N0:30) 5"-GCAGCGGTACCGAATT GGCGCGCCTTATAAGCCTAAGGCAGC-3' (SEQ ID N0:31) This PCR product (1.85kb) is then purified and digested with BamHI and Asp718 and cloned ii<to the same sites in pC4 (ATCC Accession No. 209646) to produce pC4:HSA .
Making vectors comprising~albumih fission proteins where the albumin rnoiety is C-termiruzl to the Therapeutic moiety casing the pC4:HSA vector Using pC4:HSA, albumin fusion proteins in which the Therapeutic protein moiety .is N terminal to the albumin sequence, one can clone DNA encoding a Therapeutic protein that has its own signal sequence between the Bam HI (or HindIII) and CIaI sites.
When cloning into either the BamHI or Hind III site . remember to include Kozak sequence - 25 (CCGCCACCATG) prior to translational start ~ codon of DNA encoding the Therapeutic Protein.to be subcloned. If the Therapeutic does. not have a signal sequence, the DNA
encoding that Therapeutic protein may be cloned in between the XhoI~ and CIaI
sites. When using the XhoI site, the following 5' (SEQ ID N0:32) and 3' (SEQ IDN0:33) PCR
primers may be used:
5'-CCGCCGCTCGAGGGGTGTGTTTCGTCGA(N)1$ 3' (SEQ ID NO: 32) . 5'-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATC(N)1$ 3' (SEQ ID N0:33) In SEQ ID N0:32, the underlined sequence is an .XhoI, site; and the XhoI site and the DNA following the XhoI site encode for the last seven amino acids of the leader sequence of natural human serum albumin. In SEQ ID- N0:33, the underlined sequence is a CIaI site; and the CIaI site and the DNA following it encode are the reverse complement of the DNA
encoding the first 10 amino acids of themature HSA protein (SEQ ID N0:~18).
'In SEQ ID
N0:32 "(N)18" is DNA identical to the first 18 nucleotides encoding the Therapeutic proteim of interest.). In SEQ ID N0:33 "(N),$" is the reverse complement of DNA encoding the last 18 nucleotides encoding the Therapeutic protein of interest. Using these two primers, one may PCR amplify the Therapeutic protein,of interest, purify the PCR product, digest it with XhoI
and CIaI restriction enzymes and then and clone it into the with XhoI and CIaI
sites in the pC4:HSA vector.
Making vectors comprising albumin fusion proteins where the albumin moiety is N terminal , to the Therapeutic moiety using the pC4:HSA vector Using pC4:HSA, albumin fusion proteins in which the Therapeutic protein moiety is N terminal to the albumin sequence, one can clone DNA encoding a Therapeutic protein between the Bsu36I and AscI restriction sites. _ When cloning into the- Bsu36I
and AscI, the -same primer design used to clone in the yeast vector system (SEQ ff~ N0:25 and 26) may be employed.
The pC4 vector is especially suitable for expression of albumin fusion proteins from CHO cells. For expression, in other mammalian cell types, e.g., NSO cells, it may be useful to subclone the HindIII - EcoRI fragment containing the DNA encoding an albumin fusion protein (from a pC4 vector in which the DNA encoding the Therapeutic protein has already - been cloned in frame with, the DNA encoding (the mature form of) hu-man serum albumin) into another expression vector (such as any of the marnmalian~ expression vectors described herein).
Example 3: Preparation of HA-cytokine or- HA-growth factor fusion proteins (such as EPO, GMCSF,- GCSF) -The cDNA for the cytokine or growth factor of -interest, such as-EPO, can ~be isolated - by a variety of means including from cDNA libraries, by RT-PCR and by PCR
using a series of overlapping synthetic oligonucleotide primers, all using standard methods.
The nucleotide sequences for all of these proteins are known and available, for instance, in U.S. Patents 4,703,008, 4,810,643 and 5,908,763. The cDNA can be tailored at the 5' and 3' ends to . generate restriction ~ sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the- N or C-terminus with or - without the use of a spacer sequence. EPO (or other cytokine) cDNA is cloned into a vector such as pPPC0005 (Figure 4), pScCHSA, pScNHSA , or pC4:HSA from which the complete expression cassette is then -excised and inserted into the plasmid pSAC35 to allow 35- the expression of the albumin fusion protein in yeast. The albumin fusion protein- secreted from the yeast can then 6e collected and purified from the media and tested for its biological activity. - For expression in mammalian cell lines, a similar procedure is adopted - except that the expression cassette used employs a mammalian promoter; leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 4: Preparation of HA-IFN fusion proteins (such as IFNa) The cDNA for the interferon of interest such as IFNa can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR and by PCR
using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences for interferons, such as IFNa are known and available, for instance, in U.S.~Patents 5.,326,859 and 4,588,585; in EP 32 134, as well as in public databases such as GenBank. The cDNA can be tailored at the 5' and 3' ends to generate restriction sites, such that oligonucleotide linkers can be used to clone the cDNA into a vector containing the cDNA
for HA. This can be at the N or C-terminus of the HA sequence, with or without the use of a spacer sequence. The IFNa (or other interferon) cDNA is cloned into a vector such as pPPC0005 (Figure 4), pScCHSA, pScNHSA , or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast (see Figure 8). The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Maximacm protein recovery from vials The albumin fusion proteins of the invention have a high degree of stability even when they . are packaged at low concentrations. In addition, in spite of the low' protein concentration, good fusion-protein recovery is observed even when ' the aqueous solution includes no other protein added to minimize binding to the vial walls. Figure 5 compares the , recovery of vial-stored HA-IFN solutions with a stock solution. 6 or 30 pg/ml HA-IFN
solutions were placed in vials and stored at 4°C. After 48 or 72 hrs a volume originally equivalent to 10 ng of sample was removed and measured in an IFN sandwich ELISA. The estimated values were compared to that of a high concentration stock solution.
As shown, there is essentially no loss of the sample in these vials, indicating that addition of exogenous material such as albumin is not ilecessary to prevent sample loss to the wall of the vials WO 01/79271 PCT/iJS01/12009 In vivo stability and bioavailability of HA-a-IFN fcisions To determine the in vivo stability and. bioavailabiIity of a HA-a-IFN fusion molecule, the purified fusion molecule (from yeast) was administered to monkeys at the dosages and time points described in Figures 6 and 7. Pharmaceutical compositions formulated from HA-a-IFN fusions may account for the extended serum half life and bioavailability exemplified in Figures 6 and 7. Accordingly, pharmaceutical compositions may be formulated to contain lower dosages of alpha-interferon activity compared to the native alpha-interferon molecule.
Pharmaceutical compositions containing HA-a-IFN fusions may be used to treat or prevent disease in patients with any disease or disease state that can be modulated by the administration of a-IFN. Such diseases include, but are not limited to, hairy cell leukemia, Kaposi's sarcoma, genital , and anal warts, chronic hepatitis ~B, chronic non-A, non-B
hepatitis, in particular hepatitis C, hepatitis D, chronic myelogenous leukemia, renal cell carcinoma, bladder carcinoma, ovarian and cervical carcinoma, skin cancers, recurrent respirator papillomatosis, non-Hodgkin's and cutaneous T-cell lymphomas, melanoma, multiple myeloma , AIDS, multiple sclerosis, gliobastoma, etc. (see Interferon Alpha, In:
AHFS Drug Information, 1997.
Accordingly, the invention includes pharmaceutical compositions containing a HA-a-IFN fusion protein, polypeptide or peptide formulated with the proper dosage for human administration. The invention also includes methods of treating patients in need of such treatment comprising at least ~ the step of administering a pharr a tical composition containing at least one HA-(-IFN fusion protein, polypeptide or peptide, Bifunctional HA-= IFNfusions The HA-a-IFN expression vector of Figure 8 is modified to include an insertion for the expression of bifunctional HA-a-IFN fusion proteins. -For instance, the, cDNA for a second protein of interest may be inserted in frame downstream of the "rHA-IFN" sequence after the double stop codon has been removed or shifted downstream of the coding sequence.
In one version of a bifunctional HA-a-IFN fusion protein, an antibody or fragment against B-lymphocyte stimulator protein (GenBaiik Acc 4455139) or polypeptide may be fused to one end of the HA component of the fusion molecule. This bifunctional protein is useful for modulating any immune response generated' by the a-IFN component of the fusion.
Example 5: Preparation of HA-hormone fusion protein (such as insulin, ,LH, FSH) The cDNA for the hormone of interest such as insulin can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR and by PCR
using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences for all of these proteins are known and available, for instance, in public databases such as GenBank. The cDNA can ~ be' tailored at the 5' and 3' ends to generate' restriction sites; such that oligonucleotide linkers can be used, for cloning of the cDNA into .a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The hormone cDNA ~is cloned into a vector such ~as pPPC0005 (Figure 4), , pScCHSA, pScNHSA , or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein .secreted from the yeast can then be collected and purified from the media and tested for its biological activity.' For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2).
This '. expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 6: , ,Preparation of HA-soluble receptor or HA-binding protein fusion protein 'such as HA-TNF receptor The cDNA for the soluble receptor or binding protein of interest such as TNF
receptor can be isolated by a variety of means including but not exclusively, from cDNA
libraries, by RT-PCR and by PCR using a series. of overlapping synthetic oligonucleotide primers, all , using standard methods. The nucleotide sequences for all of these proteins are known and available, for instance, in GenBank. The cDNA can be tailored at the S' and 3' ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning 'of the cDNA into a vector containing the cDNA for HA. This can be at the.N or C-terminus with or without the use of a spacer sequence. The receptor cDNA is cloned 'into a vector such as pPPC0005 (Figure 4), pScCHSA, pScNHSA_ , or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid. pSAC35 to allow the . expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from .
the yeast can then be collected and ,purified from the media and tested for its biological activity. For expression-in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Exafnple 7: Preparation of HA-growth factors such as HA-IGF-1 , fusion protein . _ The cDNA for the growth factor of interest such as IGF-1 can be isolated by a variety of means~including but not exclusively, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping _synthetic oligonucleotide primers, all using standard methods (see GenBank Acc. No.NP_000609). The cDNA can be tailored at the 5' and 3'' ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The growth factor cDNA is cloned into a vector such as pPPC0005 (Figure 4), pScCHSA, pScNHSA , or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in~ mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example. 2).
This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
. Example S: Preparation of HA-single chain antibody fusion proteins Single chain antibodies are produced by several methods including but not limited to:
selection from phage libraries, cloning of the variable region of a specific antibody by cloning the cDNA of the antibody and using the flanking constant regions as the primer to clone the variable region, or by synthesizing an oligonucleotide corresponding to the variable. region of any specific antibody. The cDNA can be tailored at, the 5' and 3' ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA
into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the-use of a spacer"sequence. The cell cDNA is cloned into a vector such as~ pPPC0005 (Figure 4), pScCHSA, pScNHSA , or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast.
In fusion molecules of the invention, the VH and VL Can be linked by one of the following means or a combination thereof: a peptide linker between the C-terminus of the VH
and the N-terminus of the V L: ; a Kex2p protease cleavage site between the V
H and V L such that the two are cleaved- apart upon secretion and 'then self associate; and cystine residues positioned such that the VH and VL can form a disulphide bond between them to link them together (see Figure 14). An alternative option would be to place the VH at the N-terminus of HA or an HA domain fragment and the VL at the C-terminus of the HA or HA
domain fragment.
The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its activity. For expression in_mammaiian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of miammalian cell Lines. The. antibody IO produced in this manner can be purified from media and tested for its binding to its antigen using standard immunochemical methods. .
., ~ Example 9: Preparation of HA-cell adhesion molecule fusion proteins The cDNA for the cell adhesion molecule of interest can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR and by PCR
using a series of overlapping synthetic oligonucleotide primers, all using standard methods. . The nucleotide sequences for the known cell adhesion molecules are known and available, for instance, in GenBank. ~ The cDNA can be tailored at the -5' and 3' ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA
into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The cell adhesion molecule cDNA is cloned into a vector such as pPPC0005 (Figure 4), pScCHSA, pScNHSA , or pC4-..HSA from which the complete expression cassette is then excised and inserted into the. plasmid pSAC35 .to allow the - expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the, media and tested for ~
its biological activity. For expression in mammalian cell lines a similar~procedure is adopted except that the ~- expression cassette used employs a mammalian promoter, leader sequence and terminator . (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 10: Preparation of inhibitory factors and peptides as HA
fusion proteins (such as ~ HA-antiviral, HA-antibiotic, HA-enzyme inhibitor and HA-anti-allergic proteins) The cDNA for the peptide of interest such as an antibiotic peptide can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR
and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard . methods. - The cDNA can be tailored at the 5' and 3' ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. Theypeptide cDNA is cloned into a vector such as pPPC0005 (Figure 4), pScCHSA, pScNHSA , or pC4:HSA. from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in. mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression IO cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 11: Preparation of targeted HA ,fusion proteins The cDNA for the protein of interest can be isolated from cDNA library or can be IS made synthetically using several overlapping oligonucleotides using standard molecular biology methods. The appropriate nucleotides can be engineered in the cDNA to form convenient restriction sites and also allow the attachment of the protein cDNA
to albumin cDNA similar to the method described for hGH. Also a targeting protein or peptide cDNA
such as single chain antibody or peptides, such as nuclear localization signals, that can direct 20 proteins inside the cells can be fused to the other end of albumin. The protein of interest and the targeting peptide is cloned into a vector such as pPPC0005 (Figure ~4), pScCHSA, pScNHSA , or pC4:HSA. which allows the fusion with albumin cDNA. In this manner both N- and C-terminal end of albumin are fused to other proteins. The fused cDNA
is then excised from pPPC0005 and is inserted into a plasmid~ such as pSAC35 to allow the 25 expression of the albumin fusion protein in yeast. All the above procedures can be performed using standard methods in molecular biology. The albumin fusion protein secreted from' yeast can- be collected and purified from the media and tested for its biological activity and its targeting activity using appropriate biochemical and biological tests.
30 . Example 12: Preparation of HA-enzymes fusions The cDNA for the enzyme of interest can be isolated by a.variety of means including but not exclusively; from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The cDNA can be tailored at the 5' and 3'_ends to generate restriction sites, such that oligonucleotide linkers can 35 be used, for cloning of the~cDNA into a vector containing the cDNA for HA.
This can be at the N or C-terminus with or without the use of .a spacer sequence. The enzyme cDNA is cloned into a vector such as pPPC0005 (Figure 4), pScCHSA, pScNHSA ; or ~pC4:HSA
. ., from which the complete expression cassette is then excised and inserted into the plasmid - pSAC35 ,to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and .purified from the media and tested for its biological activity: For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 13: Bacterial Expression of an Albumin Fusion Protein -A polynucleotide encoding an albumin fusion protein of the present invention - comprising a bacterial signal sequence is amplified using PCR
oligonucleotide primers corresponding to the 5' and 3' ends of the DNA sequence, to synthesize insertion fragments.
The primers used to amplify .the polynucleotide encoding insert should preferably contain I5 restriction sites, such as BamHI and XbaI, at the 5' end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI
correspond to the restriction enzyme sites on the bacterial expression vector pQE-9.
(Qiagen, Inc., Chatsworth, CA). This plasmid vector encodes antibiotic resistance (Amps, a bacterial origin of replication (ori),- an IPTG-regulatable promoter/operator (P/0), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4,~ which expresses the lacI
repressor and also confers kanamycin resistance (Kan~. Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis. -- Clones containing the desired constructs are grown overnight (0/N) in liquid culture in LB media supplemented.~with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate ~a large culture at a ratio of 1:100 to 1:250. - The cells are 'grown to an optical density 600 (O.D.boo) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the IacI repressor, clearing the P/O leading to increased gene expression. -- Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000Xg). The cell pellet is solubilized in the chaotropic~ agent 6 Molar. Guanidine HCl or preferably in 8 M urea and concentrations greater than 0.14 .M 2-mercaptoethanol by stirring for 3-4 hours at 4°C (see, e.g., Burton et al., Eur.. J.
Biochem.'179:379-387 (1989)).
' 217 The cell debris is removed by centrifugation, and_the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6 x His tag bind to the Ni-NTA resin with high affinity and can be purified in a . simple one-step procedure (for details see: The QIAexpressionist ( 1995) QIAGEN, Inc., supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCI, pH 8.
The column is first washed with 10 volumes of 6 M guanidine-HCI, pH 8, then washed with 10 volumes of 6 M guanidine-HCI pH 6, and finally the polypeptide is eluted with guanidine-HCl, pH 5.
The~purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCI. Alternatively, the protein can 'be successfully refolded.while immobilized on the Ni-NTA column. Exemplary conditions are as follows: renature using a linear 6M 1M urea gradient in 500 mM NaCI, 20%
glycerol, 20 mM Tris/HCi pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of i.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium~acetate pH 6 buffer plus 200 mM NaCI. The purified protein is stored at 4° C or frozen at -80° C.
In addition to the above .expression vector, the present invention further includes an expression vector, called pHE4a (ATCC Accession Number 209645, deposited on February 2S, 1998) which contains phage operator and promoter elements operatively linked to a polynucleotide encoding an. albumin fusion protein of the present invention, called pHE4a.
(ATCC Accession Number 209645, deposited on February 25, 1998.) This vector contains:
1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of ' replication, 3) a TS phage promoter sequence, 4) two lac operator sequences, '5) a Shine-Delgarno sequence, and 6) the lactose'operon repressor gene (laclq). The origin of replication .
(oriC) is derived from pLTCl9 (LTI, Gaithersburg, MD). The promoter and -operator.
sequences are made synthetically. .
DNA .can be inserted into 'the pHE4a by restricting the vector with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted -product on a gel; and isolating the larger fragment (the stuffer fragment should be about 310 base pairs): The DNA insert is generated according to PCR protocols described herein or otherwise known in. the art, using PCR
primers having restriction sites for NdeI (5' primer) 'and XbaI, BamHI, XhoI, or Asp718 (3' primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are iigated according to standard protocols.
The engineered vector may be substituted in the above protocol to express protein in a bacterial system.

WO 01/79271 PCT/iTS01/12009 Example 14: Expression of an Albumin Fusion Protein in Mammalian Cells . The albumin fusion proteins of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and- late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI
and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
Suitable expression vectors for use in practicing the present invention include, for example, vectors such as, pSVL and pMSG (Pharmacia, Uppsala~ Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBCI2MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3Ø Mammalian host cells that could be used include, but are not limited to, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV l, quail QCl-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells:
Alternatively, the albumin fusion protein can be expressed in stable cell lines containing the polynucleotide encoding the albumiw fusion protein integrated into a chromosome. The co-transfection with -a selectable marker such as DHFR, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
- The transfected polynucleotide encoding the fusion protein can also be amplified to express large amounts of the encoded fusion protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt et al., 'J. Biol. Chem.
253:1357-1370 (1978);
Hamlin et al., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page et al., Biotechnology I 9:64-68 (1991)). - Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al.;
Bio/Technology 10:169- -175 (1992). Using these markers, the mammalian cells are~grown in selective medium and 30- - the cells with the highest resistance are selected. These. cell lines contain the amplified genes) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are -often used for the production of proteins.
Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus. a fragment of the CMV.-enhancer (Boshart et al:, Cell 41:521-530 (1985)). Multiple cloning sites, e.g.; with the restriction enzyme' cleavage sites 219 _ BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.
Specifically, the plasinid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1 % agarose gel.
A polynucleotide . encoding an albumin fusion protein of the present invention is generated using techniques known in the art and this polynucleotide is amplified using PCR
technology known in the art. If a naturally occurring signal sequence is used to produce the fusion protein of the present invention, the vector does not need a second signal peptide.
Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified . to include a heterologous signal sequence. (See, e.g., International Publication No. WO
96/3489'1.) The amplified fragment encoding the fusion protein of the invention is isolated from a 1 % agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1 % agarose gel.
The amplified fragment encoding the albumin fusion protein of the invention is then digested with the same restriction enzyme and purified on a 1 % agarose gel.
The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
E. , toll HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene is used for transfection.
Five pg of the expression plasmid pC6 or pC4 is cotransfected with 0.5 y~g of the plasmid ' pSVileo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including 6418. The cells are seeded in alpha minus MEM
supplemented with 1 mglml 6418. After 2,days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of v methotrexate plus 1 mg/ml 6418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations - of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 ycM, 2 ~eM, 5 ~sM, 10 mM, 20 mM). ~
The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 ycM. Expression of the desired fusion protein is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis. , , Example 15: Multifusion Fusions The albumin fusion proteins (e.g,. containing a Therapeutic protein (or fragment or variant thereof) fused to albumin (or a fragment or variant thereof)) may additionally be fused to other proteins to generate "multifusion proteins". These multifusion proteins can be used for a variety of applications. For example, fusion of the albumin fusion proteins of the invention to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See e.g,. EP A 394,827; Traunecker et al.; Nature 331:84-86 (1988)). Nuclear localization signals fused to the polypeptides of the present invention can target the protein to.
I0 a specific subcellular localization, while covalent heterodimer of homodimers can increase or decrease the activity of an albumin fusion protein. Furthermore, the fusion of additional protein sequences to the albumin fusion proteins of the invention mad further increase the solubility and/or stability of the fusion protein. The fusion proteins described above can be made using or routinely modifting techniques known in the art and/or by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG
molecule.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified; using primers that span the 5' and 3' ends of the sequence described below. - These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian or yeast expression vector. .
For example, if pC4 (ATCC Accession No. . 209646). is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3' BamHI site should be destroyed.
Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and a polynucleotide encoding an albumin fusion protein of the present invention (generateed and isolated using techniques known in the art), is ligated into this BamHI site.
Note that the polynucleotide encoding the fusion pioteim of the invention is cloned without a stop ~codon, otherwise a Fc containing fusion protein will not be produced. .
If the naturally occurring signal sequence is used to produce the albumin fusion protein of the present invention, pC4 does not need a second signal peptide.
Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a ~ heterologous signal sequence. (See, e.g., International Publication No. WO
96134891.) Human IgG Fc region: ~ .
GGGATCCGGAGCCCAAATCITCTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACG
TAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGG
TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTG

TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACA
AGTGCAAGGTCfCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAA
AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
TGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCA
AGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA
GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC'TCTACAGCAAGCTC
ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG
CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTA
AATGAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ ID NO: 36) _ Example 16: Production of an Antibody from an Albumin Fusion Protein , a) Hybridoma Technology . .
Antib~dies.that bind:.the albumin fusion proteins of the present invention and portions of the albumin fusion proteins. of the present invention (e.g., the Therapeutic protein portion or albumin portion ~of the fusion protein) can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, a preparation of an albumin fusion protein of the invention or a portion.of an albumin fusion protein of the invention is prepared and purified to render it substantially free of natural contaminants.
Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
Monoclonal antibodies specific for an albumin fusion protein of the invention, or a portion of an albumin fusion protein ~of the invention, are prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J.
Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Haniimerling et al., in:
Monoclonal ~ Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with an albumin fusion protein of the invention, or a portion of an~ albumin fusion. protein of the invention. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)).
The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding an albumin fusion protein of Lhe invention, or a portion bf an .albumin fusion protein of the invention.
Alternatively, additional antibodies capable of binding to an albumin fusion protein of . the invention, or a portion of an albumin fusion protein of the invention can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an andbody~ which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody 'whose ability to bind to the an albumin fusion protein of the invention (or portion of an albumin fusion protein of the invention),-specific antibody can- be blocked by the fusion protein of the invention, or a portion of an albumin fusion protein of the invention. Such antibodies comprise anti-idiotypic antibodies to the fusion protein of the invention (or portion of an albumin fusion, protein of the invention) -specific antibody and are used to immunize an animal to induce formation of further fusion protein of the invention (or portion of an.albumin fusion protein of the invention) -specific antibodies.
For irc vivo use of antibodies in humans, an antibody is "humanized". Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S.
Patent No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO
8601533; Robinson et al., International Publication No. WO 8702671; Boulianne et al., _ Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985)) b) Isolation Of~Antibody Fragments Directed Against an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention From A
Library Of scFvs Naturally occurring V-genes isolated from .human PBLs 'are constructed into a library of antibody fragments which contain reactivities against an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention, to which the donor may or may not have been exposed (see e.g., U.S. .Patent 5,885,793 incorporated herein by reference in its entirety).
Rescue of the Library. A library of scFvs is constructed from the RNA of human PBLs as described in International Publication No. WO 92/01047. To rescue phage _, displaying antibody fragments, approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2xTY containing 1% glucose and 100 y~glml of ampicillin (2xTY AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this. culture is used to inoculate 50 ml of 2xTY-AMP-GLU, 2 x 108 TU of delta gene 3 helper (M13 delta gene III, see International Publication No. WO 92/01047) are added and the culture incubated at 37°C for minutes without shaking and then at 37°C~for. 45 minutes with shaking.
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Claims (42)

What is claimed:

1. An albumin fusion protein comprising a Therapeutic protein:X and albumin comprising the amino acid sequence of SEQ ID NO:18.

2. An albumin fusion protein comprising a Therapeutic protein:X -and a fragment or a variant of the amino acid sequence of SEQ ID NO:18, wherein said fragment or variant has albumin activity.

3. The albumin fusion protein of claim 2, wherein said albumin activity is the ability to prolong the shelf life of the Therapeutic protein:X compared to the shelf life of the Therapeutic protein:X in an unfused state.

4. The albumin fusion protein of claim 2, wherein the fragment or variant comprises the amino acid sequence of amino acids 1-387 of SEQ ID NO:18.

5. The albumin fusion protein of any one of claims 1-4, wherein said .
Therapeutic protein:X comprises interferon-alpha.

6. An albumin fusion protein comprising a fragment or variant of a Therapeutic protein:X, and albumin comprising the amino acid sequence of SEQ ID NO:18, wherein said fragment or variant has a biological activity of the Therapeutic protein:X.

7. The albumin fusion protein of claim 6, wherein said Therapeutic protein:X
comprises interferon-alpha, and wherein said fragment or variant has antiviral activity or inhibits cell proliferation.

8. The albumin fusion protein of any one of claims 1-4 or 6, wherein said Therapeutic protein:X, or fragment or variant thereof, comprises a protein selected from the group consisting of:

(a) serum cholinesterase;

(b) alpha-1 antitrypsin;

(c) aprotinin;

(d) coagulation complex;

(e) von Willebrand factor;

(f) fibrinogen;

(g) factor VII;

(h) factor VIIA activated factor;

(i) factor VIII;

(j) factor IX;

(k) factor X;

(l) factor XIII;

(m) cl inactivator;

(n) antithrombin III;

(o) thrombin;

(p) prothrombin;

(q) apo-lipoprotein;

(r) c-reactive protein;

(s) protein C; and (t) immunoglobulin.

9. The albumin fusion protein of any one of claims 1-8, wherein the Therapeutic protein:X, or fragment or variant thereof, is fused to the N-terminus of albumin, or the N-terminus of the fragment or variant of albumin.

10. The albumin fusion protein of any one of claims 1-8, wherein the Therapeutic protein:X, or fragment or variant thereof, is fused to the C-terminus of albumin, or the C-terminus of the fragment or variant of albumin.

11. The albumin fusion protein of any one of claims 1-8, wherein the Therapeutic protein:X, or fragment or variant thereof, is fused to the N-, terminus and C-terminus of albumin, or the N-terminus and the C-terminus of the fragment or variant of albumin.

12. The albumin fusion protein of any one of claims 1-8, which comprises a first Therapeutic protein:X, or fragment or variant thereof, and a second Therapeutic protein:X, or fragment or variant thereof, wherein said first Therapeutic protein:X, or fragment or variant thereof, is different from said second Therapeutic protein:X, or fragment or variant thereof.

13. The albumin fusion protein of any one of claims 1-11, wherein the Therapeutic protein:X, or fragment or variant thereof, is separated from the albumin or the fragment or variant of albumin by a linker.

14. The albumin fusion protein of any one of claims 1-11, wherein the albumin fusion protein has the following formula:

R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, wherein R1 is Therapeutic protein:X, or fragment or variant thereof, L is a peptide linker, and R2 is albumin comprising the amino acid sequence of SEQ ID NO:18 or fragment or variant of albumin.

15. The albumin fusion protein of any one of claims 1-14, wherein the shelf-life of the albumin fusion protein is greater than the shelf-life of the Therapeutic protein:X in an unfused state.

16. The albumin fusion protein of any one of claims 1-14, wherein the in vitro biological activity of the Therapeutic protein:X; or fragment or variant thereof, fused to albumin, or fragment or variant thereof, is greater than the in vitro biological activity of the Therapeutic protein:X, or fragment or variant thereof, in an unfused state:

17. The albumin fusion protein of any one of claims 1-14, wherein the in vivo biological activity of the Therapeutic protein:X, or fragment or variant thereof, fused to .
albumin, or fragment or variant thereof, is greater than the in vivo biological activity of the Therapeutic protein:X , or fragment or variant thereof, in an unfused state.

18. An albumin fusion protein comprising a peptide inserted into an albumin comprising the amino acid sequence of SEQ ID NO:18 or fragment or variant thereof.

19. An albumin fusion protein comprising a peptide inserted into an albumin comprising an amino acid sequence selected from the group consisting of:
(a) amino. acids 54 to 61 of SEQ ID NO:18;
(b) amino acids 76 to-89 of SEQ ID NO:18;
(c) amino acids 92 to 100 of SEQ ID NO:18;
(d) amino acids 170 to 176 of SEQ ID NO:18;
(e) amino acids 247 to 252 of SEQ ID NO:18;
(f) amino acids 266 to 277 of SEQ ID NO:18;
(g) amino acids 280 to 288 of SEQ ID NO:18;
(h) amino acids 362 to 368 of SEQ ID NO:18;
(i) amino acids 439 to 447 of SEQ ID NO:18;
(j) amino acids 462 to 475 of SEQ ID NO:18;
(k) amino acids 478 to 486 of SEQ ID NO:18; and (l) amino acids 560 to 566 of SEQ ID NO:18.

20. The albumin fusion protein of claims 18 or 19, wherein said albumin fusion protein comprises a portion of albumin sufficient to prolong the shelf-life of the peptide as compared to the shelf-life of the peptide in an unfused state.

21. The albumin fusion protein of claims 18 or 19, wherein said albumin fusion protein comprises a portion of albumin sufficient to prolong the in vitro biological activity of the peptide fused to albumin as compared to the in vitro biological activity of the peptide in an unfused state.

22. The albumin fusion protein of claims 18 or 19 wherein said albumin fusion protein comprises a portion of albumin sufficient to prolong the in vivo biological activity of the peptide fused to albumin compared to the in vivo biological activity of the peptide in an unfused state.

23. An albumin fusion protein comprising a single chain antibody or portion thereof and albumin comprising the amino acid sequence of SEQ ID NO:18 or fragment or variant thereof.

24. The albumin fusion protein of any one of claims 1-23, which is non-glycosylated.

25. The albumin fusion protein of any one of claims 1-23, which is expressed in yeast.

26. The albumin fusion protein of claim 25, wherein the yeast is glycosylation deficient.

27. The albumin fusion protein of claim 25 wherein the yeast is glycosylation and protease deficient.

28. The albumin fusion protein of any one of claims 1-23, which is expressed by a mammalian cell.

29. The albumin fusion protein of any one of claims 1-23, wherein the albumin fusion protein is expressed by a mammalian cell in culture.

30. The albumin fusion protein of any one of claims 1-23, wherein the albumin fusion protein further comprises a secretion leader sequence.

31. A composition comprising the albumin fusion protein of any one of claims 30 and a pharmaceutically acceptable carrier.

32. A kit comprising the composition of claim 31.

33. A method of treating a disease or disorder in a patient, comprising the step of administering the albumin fusion protein of any one of claims 1-30.

34. The method of claim 33, wherein the disease or disorder comprises indication:Y.

35. The method of claim 34, wherein the Therapeutic protein:X comprises interferon-alpha, or fragment or variant thereof, and the disease or disorder is selected from the group consisting of: Hairy cell leukemia; Kaposi's sarcoma; genital warts;
anal warts;
chronic hepatitis B; chronic non-A, non-B hepatitis; hepatitis C; hepatitis D;
chronic myelogenous leukemia; renal cell carcinoma; bladder carcinoma; ovarian carcinoma; cervical carcinoma;skin cancer; recurrent respirator papillomatosis; non-Hodgkin's lymphoma;
cutaneous T-cell lymphoma; melanoma; multiple myeloma; AIDS; multiple sclerosis; and glioblastoma.

36. A method of treating a patient with a disease or disorder that is modulated by Therapeutic protein:X, comprising the step of administering an effective amount of the albumin fusion protein of any one of claims 1-30.

37. The method of claim 36, wherein the disease or disorder is indication:Y.

38. The method of claim 37, wherein the Therapeutic protein:X is interferon-alpha, or fragment or variant thereof, and the disease or disorder is selected from the group consisting of: Hairy cell leukemia; Kaposi's sarcoma; genital warts; anal warts; chronic hepatitis B; chronic non-A, non-B hepatitis; hepatitis C; hepatitis D; chronic myelogenous leukemia; renal cell carcinoma; bladder carcinoma; ovarian carcinoma; cervical carcinoma; skin cancer; recurrent respirator papillomatosis; non-Hodgkin's lymphoma; cutaneous T-cell lymphoma; melanoma; multiple myeloma; AIDS; multiple sclerosis; and glioblastoma.

39. A method of extending the shelf-life of Therapeutic protein:X-comprising the step of fusing the Therapeutic protein:X, or fragment or variant thereof, to albumin or a fragment or variant of albumin sufficient to extend the shelf-life of the Therapeutic protein:X, or fragment or variant thereof, compared to the shelf-life of the Therapeutic protein:X , or fragment or variant thereof, in an unfused state.

40. A nucleic acid molecule comprising a polynucleotide sequence encoding the albumin fusion protein of any one of claims 1-30.

41. A vector comprising the nucleic acid molecule of claim 40.

42. A host cell comprising the nucleic acid molecule of claim 40.