Albumin fusion proteins

BACKGROUND OF THE INVENTION

0001The invention relates generally to an albumin fusion protein comprising two or more tandemly oriented GLP-1 polypeptides, wherein <ul id="ul0001" list-style="none" compact="compact"><li>(i) said GLP-1 polypeptides are selected from <ul id="ul0002" list-style="none" compact="compact"><li>(a) wild-type GLP-1</li><li>(b) GLP-1 (9-36);</li><li>(c) GLP-1 (7-36);</li><li>(d) GLP-1 (7-36(A8G));</li><li>(e) GLP-1 (7-36(A8S)); and</li><li>(f) other GLP-1 fragments and/or GLP-1, variants having GLP-1 activity;</li></ul> fused to albumin comprising the amino acid sequence of SEQ ID NO:1038, an albumin fragment, or albumin variant thereof.</li><li>(ii) said albumin fragment or albumin variant increases the serum plasma half life of the GLP-1 polypeptides, and</li><li>(iii) said fusion protein has GLP-1 activity.</li></ul> The invention encompasses polynucleotides encoding therapeutic albumin fusion proteins and therapeutic albumin fusion proteins.

0002Human serum albumin (HSA, or HA), a protein of 585 amino acids in its mature form (as shown in <figref idref="f0001 f0002 f0003 f0004">Figure 1</figref> ((SEQ ID NO: 1038)), 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 <patcit id="pcit0001" dnum="EP330451A"><text>EP 330 451</text></patcit> and <patcit id="pcit0002" dnum="EP361991A"><text>EP 361 991</text></patcit>.

0003Therapeutic 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. <patcit id="pcit0003" dnum="WO0069911A"><text>WO 00/69911</text></patcit> discloses the insulinotropic peptide hormone glucagon-like peptide (GLP-1) which has been covalently bound to albumin, in order to increase the half-life of GLP-1. <patcit id="pcit0004" dnum="WO0179258A"><text>WO 01/79258</text></patcit> discloses multiple copies of a polynucleotide encoding a fusion protein comprising albumin and a therapeutic protein in order to stabilize and extend shelf-life of the therapeutic protein. These polynucleotides have been integrated into the genome.

0004Few 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

0005<ul id="ul0003" list-style="none"><li>1. An albumin fusion protein comprising two or more tandemly oriented GLP-1 polypeptides, wherein <ol id="ol0001" compact="compact"><li>(i) said GLP-1 polypeptides are selected from <ol id="ol0002" compact="compact"><li>(a) wild-type GLP-1;</li><li>(b) Gap-1 (9-36);</li><li>(c) GLP-1 (7-36);</li><li>(d) GLP-1 (7-36(A8G));</li><li>(e) OLP-1 (7-36(A8S)); and</li><li>(f) other GLP-1 fragments and/or GLP-1 variants, having GLP-1 activity;</li></ol> fused to albumin comprising the amino acid sequence of SEQ ID NO:1038, an albumin fragment, or albumin variant thereof,</li><li>(ii) said albumin fragment or albumin variant increases the serum plasma half life of the GLP-1 polypeptides, and</li><li>(iii) said fusion protein has GLP-1 activity.</li></ol></li><li>2. A polynucleotides encoding the albumin fusion protein of the invention.</li><li>3. A vector comprising the polynucleotide encoding the albumin fusion protein of the invention.</li><li>4. A method of producing an albumin fusion protein comprising <ol id="ol0003" compact="compact"><li>(a) transforming a host cell comprising at least one vector comprising the polynucleotide encoding the albumin fusion protein of the invention;</li><li>(b) culturing the host cell in suitable conditions for expression of the albumin fusion protein; and</li><li>(c) isolating the albumin fusion protein.</li></ol></li><li>5. A pharmaceutical composition comprising the albumin fusion protein of the invention.</li><li>6. The albumin protein of the invention for use as a medicament</li><li>7. The albumin fusion protein of the invention for use in treating hyperglycemia; diabetes, diabetes insipidus; diabetes mellitus; type 1 diabetes; type 2 diabetes; insulin resistance; insulin deficiency; byperlipidemia; hyperketonemia; non-insulin dependent diabetes mellitus (NIDDM); insulin-dependent diabetes mellitus (IDDM); obesity; retinopathy; ulcers; suppression of body weight; suppression of appetite; syndrome X, metabolic, immune and vascular disorders associated with diabetes and a cardiovascular disorders.</li><li>8. Use of the albumin fusion protein of the invention for the manufacture of a medicament for treating hyperglycemia; diabetes; diabetes insipidus; diabetes mellitus; type 1 diabetes; type 2 diabetes; insulin resistance; insulin deficiency; hyperlipidemia; hyperketonemia; non-insulin dependent diabetes mellitus (NIDDM); insulin-dependent diabetes mellitus (IDDM); obesity; retinopathy; ulcers; suppression of body weight; suppression of appetite; syndrome X, metabolic, immune and vascular disorders associated with diabetes and Cardiovascular disorders.</li></ul>

0006In a preferred aspect of the invention, albumin fusion proteins include, but are not limited to, those GLP-1 constructs encoded by the polynucleotides described in Table 2.

0007The disclosure 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 treating, preventing, ameliorating or diagnosing a disease or diseases symptom in a patient, preferably a mammal, most preferably a human, comprising the step of administering the pharmaceutical formulation to the patient.

0008In other embodiments, the present disclosure encompasses methods of preventing, creating, or ameliorating a disease or disorder. 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 GLP-1 or portion corresponding to a GLP-1 fragment or variant thereof disclosed in the "Therapeutic Protein: X" (in the present case GLP-1) 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.

0009In one embodiment, an albumin fusion protein according to the invention has extended shelf life.

0010In a second embodiment, an albumin fusion protein according to the invention is more stable than the corresponding unfused GLP-1 molecule described in Table 1.

BRIEF DESCRIPTION OF THE FIGURES

0011<figref idref="f0001 f0002 f0003 f0004">Figure 1A-D</figref> shows the amino acid sequence of the mature form of human albumin (SEQ ID NO:1038) and a polynucleotide encoding it (SEQ ID NO:1037).

0012<figref idref="f0005">Figure 2</figref> shows the restriction map of the pPPC0005 cloning vector ATCC deposit PTA-3278.

0013<figref idref="f0006">Figure 3</figref> shows the restriction map of the pSAC35 yeast <i>S. cerevisiae</i> expression vector (<nplcit id="ncit0001" npl-type="s"><text>Sleep et al., BioTechnology 8:42 (1990</text></nplcit>)).

0014<figref idref="f0007">Figure 4</figref> shows the effect of various dilutions of EPO albumin fusion proteins encoded by DNA comprised in Construct ID NOS. (hereinafter CID) 1966 and 1981 and recombinant human EPO on the proliferation of TF-1 cells (see Examples 8 and 9). Cells were washed 3X to remove GM-CSF and plated at 10,000 cells/well for 72 hours in the presence of 3-fold dilutions of CID 1966 protein or CID 1981 protein. Concentrations used were calculated based on the weight of Epo alone, not HSA plus Epo. Recombinant human Epo (rhEpo) was used as the positive control and serially diluted 3 fold from 100 ng/ml to 0.01 ng/ml. Cells were exposed to 0.5 mCi/well of ³H-thymidine for an additional 18 hours. (□) rhEpo; (▼) HSA-Epo 1981; (●) Epo-HSA 1966.

0015<figref idref="f0008">Figure 5</figref> is a dose response analysis and shows the effect of various doses of recombinant human EPO and EPO albumin fusion proteins encoded by DNA comprised in CID 1966 and 1981 on the percent change in hematocrit from day 0 to day 7 (see Examples 8 and 9). 48 eight-week old female DBA/2NHsd mice were divided into 12 groups of 4 animals each. Recombinant human Epo (rhEpo) was administered subcutaneously at 0.5, 1.5, 4.5 and 12 µg/kg on days 0, 2, 4, and 6. Epo albumin fusion proteins made from constructs CID 1966 and CID 1981 were administered subcutaneously at 2, 6, 18, and 54 µg/kg on days 0, 2, 4, and 6. The higher doses of the Epo albumin fusion proteins allows a rough equimolar comparison with recombinant human Epo (note that the weight of the fusions is about 4.35 times the weight of non-glycosylated Epo). On days 0 and 7 of the experiment, the animals were bled via a tail vein and the hematocrit was determined by centrifugation. (■) rhEpo; (o) CID 1981; (▲) CID 1966.

0016<figref idref="f0009">Figure 6A</figref> shows the effect of various subcutaneous administrations of Epo albumin fusion proteins encoded by DNA comprised in CID 1966 and 1997, respectively, on the percent change in hematocrit from day 0 to day 8 (see Examples 8 and 10). *, p&lt;0.005 compared to rhEpo as determined by Mann-Whitney nonparametric analysis (n=6).

0017<figref idref="f0010">Figure 6B</figref> shows the effect of subcutaneous administrations of Epo albumin fusion proteins encoded by DNA comprised in CID 1997 and 1966 on the percent change in hematocrit from day 0 to day 14 (see Examples 8 and 10). *, p&lt;0.005 compared to rhEpo as determined by Mann-Whitney nonparametric analysis (n=6); **, p&lt;0.05 compared to rhEpo as determined by Mann-Whitney nonparametric analysis (n=6).

0018<figref idref="f0011">Figure 7</figref> shows the effect of various dilutions albumin fusion proteins encoded by DNA comprised in CID 1981 and 1997, respectively, on the proliferation of TF-1 cells (see Examples 9 and 10). Cells were washed 3X to remove GM-CSF and plated at 10,000 cells/well for 72 hours in the presence of 3-fold dilutions of Epo albumin fusion proteins encoded by CID 1981 or 1997. Equimolar amounts of rhEpo were used as a positive control (4.35 times less protein added since weight of non-glycosylated Epo is 20 kd, while Epo albumin fusion proteins are 87 kd). Cells were exposed to 0.5 µCi/well of ³H-thymidine for an additional 24 hours. (■) rhEpo Standard; (A) CID 1981 (CHO); (o) CID 1997 (NSO).

0019<figref idref="f0012">Figure 8</figref> shows the effect of various doses of recombinant human EPO (rhEpo) and EPO albumin fusion protein encoded by DNA comprised in construct 1997 (CID 1997) on the percent change in hematocrit from day 0 to day 8 (see Example 10). (▲) = rhEpo, (□) = CID 1997.

0020<figref idref="f0013">Figure 9</figref> shows the effect of various dilutions of IL2 albumin fusion proteins encoded by DNA comprised in CID 1812 (see Example 15) on CTLL-2 proliferation. 1x10⁴ cells/well were seeded in a 96-well plate in a final volume of 200 ul of complete medium containing the indicated amount of IL2 albumin fusion protein (CID 1812). All samples were run in triplicate. The cells were incubated for 40 hours at 37°C, then 20 ul of Alamar Blue was added and cells incubated for 8 hours. Absorbance at 530/590 was used as a measure of proliferation. EC50 = 0.386 ± 0.021. (Δ) = CID 1812.

0021<figref idref="f0014">Figure 10</figref> shows the effect of IL2 albumin fusion protein encoded by DNA comprised in CID 1812 on RENCA tumor growth at day 21 (see Example 15). BALB/c mice (n=10) were injected SC (midflank) with 10⁵ RENCA cells. 10 days later mice received 2 cycles (Day 10 to Day 14 and Days 17-21) of daily (QD) injections of rIL2 (0.9 mg/kg), IL2 albumin fusion protein (CID 1812 protein; 0.6 mg/kg), or PBS (Placebo) or injections every other day (QOD) of CID 1812 protein (0.6 mg/kg). The tumor volume was determined on Day 21 after RENCA inoculation. The data are presented in scatter analysis (each dot representing single animal). Mean value of each group is depicted by horizontal line. *, p=0.0035 between placebo control and CID 1812 protein. The number in parentheses indicates number of mice alive over the total number of mice per group. (o) = Placebo; (●) = IL2; (Δ) = CID 1812 protein (QD); (□) = CID 1812 protein (QOD).

0022<figref idref="f0015">Figure 11</figref> shows the effect of various dilutions of GCSF albumin fusion proteins encoded by DNA comprised in CID 1642 and 1643 on NFS-60 cell proliferation (see Examples 19 and 20). (■) = CID 1642; (A) = CID 1643; (○) = HSA.

0023<figref idref="f0016">Figure 12</figref> shows the effect of recombinant human GCSF (Neupogen) and GCSF albumin fusion protein on total white blood cell count (see Example 19). Total WBC (10³ cells/ul) on each day are presented as the group mean ± SEM. GCSF albumin fusion protein was administered sc at either 25 or 100 ug/kg every 4 days x 4 (Q4D), or at 100 ug/kg every 7 days x 2 (Q7D). Data from Days 8 and 9 for GCSF albumin fusion protein 100 ug/kg Q7 are presented as Days 9 and 10, respectively, to facilitate comparison with other groups. Controls were saline vehicle administered SC every 4 days x 4 (Vehicle Q4D), or Neupogen administered SC daily x 14 (Neupogen 5 ug/kg QD). The treatment period is considered Days 1-14, and the recovery period, Days 15-28.

0024<figref idref="f0017">Figure 13</figref> shows the effect of various dilutions of IFNb albumin fusion proteins encoded by DNA comprised in CID 2011 and 2053 on SEAP activity in the ISRE-SEAP/293F reporter cells (see Example 25). Proteins were serially diluted from 5e-7 to le-14 g/ml in DMEM/10% FBS and used to treat ISRE-SEAP/293F reporter cells. After 24 hours supernatants were removed from reporter cells and assayed for SEAP activity. IFNb albumin fusion protein was purified from three stable clones: 293F/#2011, CHO/#2011 and NSO/#2053. Mammalian derived IFNb, Avonex, came from Biogen and was reported to have a specific activity of 2.0e5 IU/ug.

0025<figref idref="f0018">Figure 14</figref> illustrates the steady-state levels of insulin mRNA in INS-1 (832/13) cells after treatment with GLP-1 or GLP-1 albumin fusion protein encoded by construct m 3070 (CID 3070 protein). Both GLP-1 and the CID 3070 protein stimulate transcription of the insulin gene in INS-1 cells. The first bar (black) represents the untreated cells. Bars 2-4 (white) represent cells treated with the indicated concentrations of GLP-1. Bars 5-7 (gray) represent cells treated with the indicated concentrations of CID 3070 protein.

0026<figref idref="f0019">Figure 15</figref> compares the anti-proliferative activity of IFN albumin fusion protein encoded by CID 3165 (Cm 3165 protein) and recombinant IFNa (rIFNa) on Hs294T melanoma cells. The cells were cultured with varying concentrations of either CID 3165 protein or rIFNa and proliferation was measured by BrdU incorporation after 3 days of culture. CID 3165 protein caused measurable inhibition of cell proliferation at concentrations above 10 ng/ml with 50% inhibition achieved at approximately 200 ng/ml. (■) = CID 3165 protein, (◆) = rIFNa.

0027<figref idref="f0020">Figure 16</figref> shows the effect of various dilutions of IFNa albumin fusion proteins on SEAP activity in the ISRE-SEAP/293F reporter cells. One preparation of IFNa fused upstream of albumin (◆) was tested, as well as two different preparations of IFNa fused downstream of albumin (▲) and (■).

0028<figref idref="f0021">Figure 17</figref> shows the effect of time and dose of IFNa albumin fusion protein encoded by DNA comprised in construct 2249 (CID 2249 protein) on the mRNA level of OAS (p41) in treated monkeys (see Example 31). Per time point: first bar = Vehicle control, 2^nd bar = 30 ug/kg CID 2249 protein day 1 iv, third bar = 30 ug/kg CID 2249 protein day 1 sc, 4^th bar = 300 ug/kg CID 2249 protein day 1 sc, 5^th bar = 40 ug/kg recombinant IFNa day 1, 3 and 5 sc.

0029<figref idref="f0022">Figure 18</figref> shows the effect of various dilutions of insulin albumin fusion proteins encoded by DNA comprised in constructs 2250 and 2276 on glucose uptake in 3T3-L1 adipocytes (see Examples 33 and 35). .

0030<figref idref="f0023">Figure 19</figref> shows the effect of various GCSF albumin fusion proteins, including those encoded by CID #1643 and #2702 (L-171, see Example 114), on NFS cell proliferation. The horizontal dashed line indicates the minimum level of detection.

DETAILED DESCRIPTION

<u>Definitions</u>

0031The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

0032As used herein, "polynucleotide" refers to a nucleic acid molecule having a nucleotide sequence encoding a fusion protein according to the invention as defined in the claims, said fusion protein comprises or alternatively consists of, at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least two tandemly oriented Therapeutic proteins X (or fragment or variant thereof) ("Therapeutic protein X" meaning herein GLP-1), a nucleic acid molecule having a nucleotide sequence encoding a fusion protein comprising, or alternatively consisting of, the amino acid sequence of SEQ ID NO:Y (i.e. the GLP-1 constructs, as described in column 6 of Table 2) or a fragment or variant thereof; a nucleic acid molecule having a nucleotide sequence comprising or alternatively consisting of one of those sequences shown in SEQ ID NO:X which encode a fusion protein of the invention; a nucleic acid molecule having a nucleotide sequence encoding a fusion protein comprising, or alternatively consisting of one of those amino acid sequences of SEQ ID NO:Z which encode a fusion protein of the invention; a nucleic acid molecule having a nucleotide sequence encoding an albumin fusion protein of the invention generated as described in Table 2 or in the Examples; a nucleic acid molecule having a nucleotide sequence contained in an albumin fusion construct of the invention described in Table 2, or a nucleic acid molecule having a nucleotide sequence contained in an albumin fusion construct of the invention deposited with the ATCC (as described in Table 3).

0033As used herein, "albumin fusion construct" refers to a nucleic acid molecule as defined above, optionally further comprising, for example, one or more of the following elements: (1) a functional self-replicating vector (including but not limited to, a shuttle vector, an expression vector, an integration vector, and/or a replication system), (2) a region for initiation of transcription (e.g., a promoter region, such as for example, a regulatable or inducible promoter, a constitutive promoter), (3) a region for termination of transcription, (4) a leader sequence, and (5) a selectable marker. The polynucleotide encoding the Therapeutic protein and albumin protein, once part of the albumin fusion construct, may each be referred to as a "portion", "region" or "moiety" of the albumin fusion construct.

0034The present invention relates generally to polynucleotides as defined above; and albumin fusion proteins encoded by such polynucleotides and as defined in the claims. 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 two tandemly oriented molecules of a Therapeutic protein (or fragment or variant thereof) ("Therapeutic protein" meaning herein "GLP-1"). An albumin fusion protein of the invention comprises the at least two tandemly oriented molecules of the Therapeutic protein (or fragment or variant thereof) and at least a fragment or variant of human serum albumin, which are associated with one another 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). The Therapeutic protein and albumin protein, once part of the albumin fusion protein, may each 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 a highly preferred embodiment, an albumin fusion protein of the invention comprises the at least two tandemly oriented molecules of the Therapeutic protein X or fragment or variant thereof (including, but not limited to a mature form of the Therapeutic protein X) and at least one molecule of albumin or fragment or variant thereof (including but not limited to a mature form of albumin).

0035In a further preferred embodiment, an albumin fusion protein of the invention is processed by a host cell and secreted into the surrounding culture medium. Processing of the nascent albumin fusion protein that occurs in the secretory pathways of the host used for expression may include, but is not limited to signal peptide cleavage; formation of disulfide bonds; proper folding; addition and processing of carbohydrates (such as for example, N- and O- linked glycosylation); specific proteolytic cleavages; and assembly into multimeric proteins. An albumin fusion protein of the invention is preferably in the processed form. In a most preferred embodiment, the "processed form of an albumin fusion protein" refers to an albumin fusion protein product which has undergone N- terminal signal peptide cleavage, herein also referred to as a "mature albumin fusion protein".

0036In several instances, a representative clone containing an albumin fusion construct of the invention was deposited with the American Type Culture Collection (herein referred to as "ATCC®"). Furthermore, it is possible to retrieve a given albumin fusion construct from the deposit by techniques known in the art and described elsewhere herein. The ATCC® is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC® deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

0037In one embodiment, the albumin fusion protein of the invention comprises two or more tandemly oriented biologically active and/or therapeutically active fragments of the Therapeutic protein as defined above and a serum albumin protein. In other embodiments, the albumin fusion protein of invention comprises, or alternatively consists of, two or more tandemly oriented molecules of a biologically active and/or therapeutically active variant of the 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. The invention further encompasses polynucleotides encoding these albumin fusion proteins.

0038In a further preferred embodiment, the Therapeutic protein portion of the albumin fusion protein of the invention 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 protein. The invention further encompasses polynucleotides encoding these albumin fusion proteins.

<u>Therapeutic proteins</u>

0039As stated above, a polynucleotide of the invention encodes a protein comprising or alternatively consisting of, at least two tandemly oriented fragments or variants of the Therapeutic protein as defined above and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion.

0040An additional embodiments includes a polynucleotide encoding a protein comprising or alternatively consisting of at least two tandemly oriented fragments or variants of the Therapeutic protein and at least a fragment or variant of human serum albumin, which are linked with one another by chemical conjugation.

0041As used herein, "Therapeutic protein" refers to GLP-1 polypeptides selected from wild-type GLP-1, GLP-1 fragments and GLP-1 variants having GLP-1 activity when present in the albumin fusion protein of the invention.

0042By a polypeptide displaying a "therapeutic activity" or a protein that is "therapeutically active" is meant a polypeptide that possesses one or more known biological and/or therapeutic activities associated with the Therapeutic protein described herein of otherwise known in the art. As a non-limituig 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.

0043The "Therapeutic protein" portions comprised by the albumin fusion protein of the invention is GLP-1 or GLP-1 fragments or GLP-1 variants.

0044In another non-limiting example, the "Therapeutic protein" is a GLP-1 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 the Therapeutic protein includes, one or more of the biological activities described in the "Biological Activities" section below and/or as disclosed for the given Therapeutic protein in Table 1 (column 2).

0045As used herein, "therapeutic activity" or "activity" may refer to an activity whose effect is consistent with a desirable therapeutic outcome in humans, or to desired effects in non-human mammals or in other species or organisms. Therapeutic activity may be measured <i>in</i> vivo or <i>in vitro.</i> For example, a desirable effect may be assayed in cell culture. Such <i>in vitro</i> or cell culture assays are commonly available for many Therapeutic proteins as described in the art. Examples of assays include, but are not limited to those described herein in the Examples section or in the "Exemplary Activity Assay" column (column 3) of Table 1.

0046Therapeutic proteins corresponding to the Therapeutic protein portion of an 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. Glycosylation 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 on threonine residues; and glycosylation characterized by N-linked oligosaccharides, which are attached to asparagine residues in an Asn-X-Ser or Asn-X-Thr sequence, where X can be any amino acid except proline. N-acetylneuramic acid (also known as sialic acid) is usually the terminal 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 common at the same site within a given cell type.

0047Therapeutic proteins corresponding to the 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 manipulation(s) 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, <i>e.g</i>., 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 glycosylate them, <i>e.g.</i> in <i>E. coli</i> or glycosylation-deficient yeast. These approaches are described in more detail below and are known in the art.

0048The therapeutic protein, disclosed in Table 1, and its nucleic acid and amino acid sequences are well known in the art and available in public databases such as Chemical Abstracts Services Databases (e.g., the CAS Registry), GenBank, and subscription provided databases such as GenSeq (e.g., Derwent). Exemplary nucleotide sequences of the Therapeutic protein which may be used to derive a polynucleotide of the invention are shown in column 7, "SEQ ID NO:X," of Table 2. Sequences shown as SEQ ID NO:X may be a wild type polynucleotide sequence encoding a given Therapeutic protein (e.g., either full length or mature), or in some instances the sequence may be a variant of said wild type polynucleotide sequence (<i>e.g</i>., a polynucleotide which encodes the wild type Therapeutic protein, wherein the DNA sequence of said polynucleotide has been optimized, for example, for expression in a particular species; or a polynucleotide encoding a variant of the wild type Therapeutic protein (i.e., a site directed mutant; an allelic variant)). It is well within the ability of the skilled artisan to use the sequence shown as SEQ ID NO:X to derive the construct described in the same row. For example, if SEQ ID NO:X corresponds to a full length protein, but only a portion of that protein is used to generate the specific CID, it is within the skill of the art to rely on molecular biology techniques, such as PCR, to amplify the specific fragment and clone it into the appropriate vector.

0049Table 1 provides the Therapeutic protein portion of an albumin fusion protein of the invention, or an albumin fusion protein encoded by a polynucleotide of the invention. The first column, "Therapeutic Protein X," discloses the Therapeutic protein molecule that may be followed by parentheses containing scientific and brand names of proteins that comprise, or alternatively consist of, that Therapeutic protein molecule or a fragment or variant thereof. "Therapeutic protein X" as used herein may refer either to an individual Therapeutic protein molecule, or to the entire group of Therapeutic proteins associated with a given Therapeutic protein molecule disclosed in this column. The "Biological activity" column (column 2) describes Biological activities associated with the Therapeutic protein molecule. Column 3, "Exemplary Activity Assay," provides references that describe assays which may be used to test the therapeutic and/or biological activity of the Therapeutic protein:X or an albumin fusion protein comprising the Therapeutic protein X (or fragment thereof) portion. The fourth column, "Preferred Indication: Y," describes disease, disorders, and/or conditions that may be treated, prevented, diagnosed, and/or ameliorated by the Therapeutic protein X or an albumin fusion protein comprising the Therapeutic protein X (or fragment thereof) portion. The "Construct ID" column (column 5) provides a link to an exemplary albumin fusion construct disclosed in Table 2 which encodes an albumin fusion protein comprising, or alternatively consisting of the referenced Therapeutic Protein X (or fragment thereof) portion. <tables id="tabl0001" num="0001"><table frame="all"><title><u>Table 1</u></title><tgroup cols="6"><colspec colnum="1" colname="col1" colwidth="30mm" /><colspec colnum="2" colname="col2" colwidth="25mm" /><colspec colnum="3" colname="col3" colwidth="29mm" /><colspec colnum="4" colname="col4" colwidth="32mm" /><colspec colnum="5" colname="col5" colwidth="25mm" /><colspec colnum="6" colname="col6" colwidth="27mm" /><thead><row><entry valign="top">Therapeutic Protein:X</entry><entry valign="top">Biological Activity</entry><entry valign="top">Exemplary Activity Assay</entry><entry valign="top">Preferred Indication:Y</entry><entry valign="top">Construct ID</entry><entry valign="top">Therapeutic Protein:Z</entry></row></thead><tbody><row><entry>Glucagon-Like-</entry><entry>Stimulates the synthesis</entry><entry>GLP1 activity may be</entry><entry>Hyperglycemia; Diabetes; Diabetes Insipidus;</entry><entry>2448, 2455, 2456, 2457,</entry><entry>See Table 2,</entry></row><row><entry>Peptide 1 (GLP1; Insulinotropin)</entry><entry>and release of insulin; enhances the sensitivity of adipose, muscle, and liver tissues towards insulin; stimulates glucose uptake; slows the digestive process; suppresses appetite; blocks the secretion of glucagon.</entry><entry>assayed in vitro using a [3-H]-glucose uptake assay. (<nplcit id="ncit0002" npl-type="s"><text>J Biol Chem 1999 Oct 22; 274(43):30864-30873</text></nplcit>).</entry><entry>Diabetes mellitus; Type 1 diabetes; Type 2 diabetes; Insulin resistance; Insulin deficiency; Hyperlipidemia; Hyperketonemia; Non-insulin dependent Diabetes Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A Condition Associated With Diabetes Including, But Not Limited To Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders; Obesity; Vascular Disorders; Suppression of Body Weight; Suppression of Appetite; Syndrome X.</entry><entry>2803, 2804, 2900, 2904, 2945, 2964, 2982, 3070, 2802, 3027, 3028, 3045, 3046, 3069, 3071, 3072, 3085, 3086, 3087, 3140, 3309</entry><entry>SEQ ID NO:Z for particular construct</entry></row></tbody></tgroup></table></tables><tables id="tabl0002" num="0002"><table frame="all"><title><u>Table 2</u></title><tgroup cols="11"><colspec colnum="1" colname="col1" colwidth="18mm" /><colspec colnum="2" colname="col2" colwidth="22mm" /><colspec colnum="3" colname="col3" colwidth="44mm" /><colspec colnum="4" colname="col4" colwidth="34mm" /><colspec colnum="5" colname="col5" colwidth="24mm" /><colspec colnum="6" colname="col6" colwidth="16mm" /><colspec colnum="7" colname="col7" colwidth="15mm" /><colspec colnum="8" colname="col8" colwidth="16mm" /><colspec colnum="9" colname="col9" colwidth="15mm" /><colspec colnum="10" colname="col10" colwidth="16mm" /><colspec colnum="11" colname="col11" colwidth="26mm" /><thead><row><entry align="center" valign="top">Fusion No.</entry><entry align="center" valign="top">Construct ID</entry><entry valign="top">Construct Name</entry><entry valign="top">Description</entry><entry valign="top">Expression Vector</entry><entry align="center" valign="top">SEQ ID NO:Y</entry><entry align="center" valign="top">SEQ ID NO: X</entry><entry align="center" valign="top">SEQ ID NO:Z</entry><entry align="center" valign="top">SEQ ID NO: A</entry><entry align="center" valign="top">SEQ ID NO:B</entry><entry align="center" valign="top">Leader Sequence</entry></row></thead><tbody><row><entry align="center">197</entry><entry align="center">2448</entry><entry>pSAC35:GLP-1(7-36).HSA</entry><entry>Amino acids H98 to R127 of preproglucagon (SEQ ID NO:630) (hereinafter this specific domain will be referred to as "GLP-1(7-36)") is fused upstream of mature HSA and downstream of HSA/kex2 leader sequence.</entry><entry>pSAC35</entry><entry align="center">414</entry><entry align="center">198</entry><entry align="center">630</entry><entry align="center">988</entry><entry align="center">989</entry><entry align="center">HSA/kex2</entry></row><row><entry align="center">199</entry><entry align="center">2455</entry><entry>pSAC35:HSA.GLP-1(7-36)</entry><entry>GLP-1(7-36) is fused downstream of mature HSA and HSA/kex2 leader sequence.</entry><entry>pSAC35</entry><entry align="center">416</entry><entry align="center">200</entry><entry align="center">632</entry><entry align="center">992</entry><entry align="center">993</entry><entry align="center">HSA/kex2</entry></row><row><entry align="center">200</entry><entry align="center">2456</entry><entry>pSAC35:GLP-1(7-36(A8G)).HSA</entry><entry>Amino acids H98 to R127 of Preproglucagon (SEQ ID NO:633)(also referred to as "GLP-1(7-36)") is mutated at amino acid 99 of SEQ ID NO:633 to replace the alanine with a glycine. This particular GLP-1 mutant will be hereinafter referred to as "GLP-1(7-36(A8G))" and corresponds to the sequence shown in SEQ ID NO:1808. GLP-1(7-36(A8G)) is fused upstream of mature HSA and downstream of HSA/kex2 leader sequence.</entry><entry>pSAC35</entry><entry align="center">417</entry><entry align="center">201</entry><entry align="center">633</entry><entry align="center">994</entry><entry align="center">995</entry><entry align="center">HSA/kex2</entry></row><row><entry align="center">201</entry><entry align="center">2457</entry><entry>pSAC35:HSA.GLP-1(7-36(A8G))</entry><entry>GLP-1(7-36(A8G)) (SEQ ID NO:1808) is fused downstream of mature HSA and HSA/kex2 leader sequence.</entry><entry>pSAC35</entry><entry align="center">418</entry><entry align="center">202</entry><entry align="center">634</entry><entry align="center">996</entry><entry align="center">997</entry><entry align="center">HSA/kex2</entry></row><row><entry align="center">276</entry><entry align="center">2802</entry><entry>pSAC35:GLP-1(7-36(A8G)).IP2.HSA</entry><entry>GLP-1(7-36(A8G)) (SEQ ID NO:1808) is fused downstream from the HSA/kex2 leader sequence and upstream from the intervening peptide-2 of proglucagon peptide and upstream from mature HSA.</entry><entry>pScNHSA</entry><entry align="center">1559</entry><entry align="center">1391</entry><entry align="center">1727</entry><entry align="center" /><entry align="center" /><entry align="center">HSA/kex2</entry></row><row><entry align="center">277</entry><entry align="center">2803</entry><entry>pSAC35:GLP-1(7-36(A8G))x2.HSA</entry><entry>GLP-1(7-36(A8G)) (SEQ ID NO:1808) is tandemly repeated and fused downstream of the HSA/kex2 signal sequence, and upstream of mature HSA.</entry><entry>pScCHSA</entry><entry align="center">1231</entry><entry align="center">1216</entry><entry align="center">1246</entry><entry align="center">1261</entry><entry align="center">1262</entry><entry align="center">HSA/kex2</entry></row><row><entry align="center">278</entry><entry align="center">2804</entry><entry>pSAC35:coGLP-1(7-36(A8G))x2.HSA</entry><entry>GLP-1(7-36(A8G)) (SEQ ID NO:1808) is tandemly repeated and fused downstream of the HSA/kex2 signal sequence, and upstream of mature HSA.</entry><entry>pScCHSA</entry><entry align="center">1232</entry><entry align="center">1217</entry><entry align="center">1247</entry><entry align="center">1263</entry><entry align="center">1264</entry><entry align="center">HSA/kex2</entry></row><row><entry align="center">312</entry><entry align="center">2900</entry><entry>pSAC:GLP-1(7-36)x2.HSA</entry><entry>GLP-1(7-36) is tandemly repeated and then fused downstream of the HSA/kex2 signal sequence and upstream of mature HSA.</entry><entry>pScCHSA</entry><entry align="center">1233</entry><entry align="center">1218</entry><entry align="center">1248</entry><entry align="center">1265</entry><entry align="center">1266</entry><entry align="center">HSA/kex2</entry></row><row><entry align="center">316</entry><entry align="center">2904</entry><entry>pSAC35:GLP-1(9-36).GLP-1(7-36).HSA</entry><entry>Amino acids E100 to R127 of preproglucagon (SEQ ID NO:1249) (hereinafter, this particular mutant is referred to as GLP-1(9-36)) is fused downstream from the HSA/kex2 signal sequence and upstream from GLP-1(7-36), and mature HSA.</entry><entry>pScCHSA</entry><entry align="center">1234</entry><entry align="center">1219</entry><entry align="center">1249</entry><entry align="center">1267</entry><entry align="center">1268</entry><entry align="center">HSA/kex2</entry></row><row><entry align="center">326</entry><entry align="center">2945</entry><entry>pSAC35:GLP-1(7-36(A8S)).GLP-1(7-36).HSA</entry><entry>Amino acids H98 to R127 of preproglucagon (SEQ ID NO:1250) is mutated at position 99 from alanine to serine (hereinafter, this particular mulant is referred to as GLP-1(7-36(A8S)), which is fused downstream from the HSA/kex2 signal sequence and upstream from GLP-1(7-36), and mature HSA.</entry><entry>pScCHSA</entry><entry align="center">1235</entry><entry align="center">1220</entry><entry align="center">1250</entry><entry align="center">1269</entry><entry align="center">1270</entry><entry align="center">HSA/kex2</entry></row><row><entry align="center">329</entry><entry align="center">2964</entry><entry>pSAC35:GLP-1(7-36)x2.HSA</entry><entry>GLP-1(7-36) is tandemly repeated as a dimer and fused downstream from the HSA/kex2 leader sequence and upstream from mature HSA.</entry><entry>pSAC35</entry><entry align="center">1236</entry><entry align="center">1221</entry><entry align="center">1251</entry><entry align="center">1271</entry><entry align="center">1272</entry><entry align="center">HSA/kex2</entry></row><row><entry align="center">332</entry><entry align="center">2982</entry><entry>pSAC35:GLP-1(7-36(A8G).GLP-1(7-36).HSA</entry><entry>GLP-1(7-36(A8G)) (SEQ ID NO:1808) is fused downstream from the HSA/kex2 signal sequence and upstream from GLP-1(7-36) and mature HSA.</entry><entry>pScCHSA</entry><entry align="center">1237</entry><entry align="center">1222</entry><entry align="center">1252</entry><entry align="center">1273</entry><entry align="center">1274</entry><entry align="center">HSA/kex2</entry></row><row><entry align="center">336</entry><entry align="center">3027</entry><entry>pSAC35:INV.GLP-1(7-36A8G)x2.HSA</entry><entry>Invertase signal peptide followed by GLP-1(7-36(A8G)) (SEQ ID NO:1808) tandemly repeated as a dimer, followed by mature HSA.</entry><entry>pSAC35</entry><entry align="center">1607</entry><entry align="center">1439</entry><entry align="center">1775</entry><entry align="center">2004</entry><entry align="center">2005</entry><entry align="center">invertase</entry></row><row><entry align="center">337</entry><entry align="center">3028</entry><entry>pSAC35:INV.GLP-1(7-36(A8G)).GLP-1(7-36).HSA</entry><entry>Invertase signal peptide followed by GLP-1(7-36(A8G)) (SEQ ID NO:1808), then GLP-1(7-36(A8G)), and then mature HSA.</entry><entry>pSAC35</entry><entry align="center">1608</entry><entry align="center">1440</entry><entry align="center">1776</entry><entry align="center">2006</entry><entry align="center">2007</entry><entry align="center">invertase</entry></row><row><entry align="center">338</entry><entry align="center">3045</entry><entry>pSAC35:DeltaKex.GLP-1(7-36A8G)x2.HSA</entry><entry>HSA/kex2 signal sequence, minus the last six amino acids of the leader, is fused to GLP-1(7-36(A8G)) (SEQ ID NO:1808) which is tandemly repeated as a dimer, followed by mature HSA.</entry><entry>pSAC35</entry><entry align="center">1609</entry><entry align="center">1440</entry><entry align="center">1776</entry><entry align="center">2008</entry><entry align="center">2009</entry><entry align="center">HSA/kex2 last six amino acids</entry></row><row><entry align="center">339</entry><entry align="center">3046</entry><entry>pSAC35:Delta Kex.GLP.1(7-36A8G).GLP-1(7-36).HSA</entry><entry>HSA/kex2 signal sequence, minus the last six amino acids of the leader, is fused to GLP-1(7-36(A8G)) (SEQ ID NO:1808), GLP-1(7-36), and mature HSA.</entry><entry>pSAC35</entry><entry align="center">1610</entry><entry align="center">1440</entry><entry align="center">1776</entry><entry align="center">2010</entry><entry align="center">2011</entry><entry align="center">HSA/kex2 last six amino acids</entry></row><row><entry align="center">349</entry><entry align="center">3066</entry><entry>pSAC35:CKB-1d8.GLP-1(7-36).HSA</entry><entry>Invertase signal peptide followed by amino acids G28-N93 of full length CKβ1 (SEQ ID NO:1788), followed by GLP-1(7-36), followed by mature HSA.</entry><entry>pScCHSA</entry><entry align="center">1620</entry><entry align="center">1452</entry><entry align="center">1788</entry><entry align="center">2030</entry><entry align="center">2031</entry><entry align="center">invertase</entry></row><row><entry align="center">350</entry><entry align="center">3069</entry><entry>pSAC35:INU.GLP-1(7-36(A8G)x2.HSA</entry><entry>The inulinase signal sequence is fused to GLP-1(7-36(A8G)) (SEQ ID NO:1808), which is tandemly repeated as a dimer and fused to mature HSA.</entry><entry>pSAC35</entry><entry align="center">1621</entry><entry align="center">1453</entry><entry align="center">1789</entry><entry align="center">2032</entry><entry align="center">2033</entry><entry align="center">inulinase</entry></row><row><entry align="center">351</entry><entry align="center">3070</entry><entry>pSAC35:KT.GLP-1(7-36(A8G))x2.HSA</entry><entry>GLP-1(7-36(A8G)) (SEQ ID NO:1808) is tandemly repeated as a dimer and fused upstream from mature HSA and downstream from the killer toxin signal sequence.</entry><entry>pSAC35</entry><entry align="center">1280</entry><entry align="center">1281</entry><entry align="center">1282</entry><entry align="center">1283</entry><entry align="center">1284</entry><entry align="center">Killer toxin</entry></row><row><entry align="center">352</entry><entry align="center">3071</entry><entry>pSAC35:MAF.GLP-1(7-36(A8G))x2.HSA</entry><entry>The yeast mating factor α-1 (hereinafter MFα-1) signal sequence is fused to tandemly repeated copies of GLP-1(7-36(A8G)) (SEQ ID NO:1808), which are fused to mature HSA.</entry><entry>pSAC35</entry><entry align="center">1622</entry><entry align="center">1454</entry><entry align="center">1790</entry><entry align="center">2034</entry><entry align="center">2035</entry><entry align="center">MFα-1</entry></row><row><entry align="center">353</entry><entry align="center">3072</entry><entry>pSAC35:AP.GLP-1(7-36(A8G))x2.HSA</entry><entry>The acid phosphatase signal sequence is fused to tandemly repeated copies of GLP-1(7-36(A8G)) (SEQ ID NO:1808), which are fused to mature HSA.</entry><entry>pSAC35</entry><entry align="center">1623</entry><entry align="center">1455</entry><entry align="center">1791</entry><entry align="center">2036</entry><entry align="center">2037</entry><entry align="center">Acid phosphatas e</entry></row><row><entry align="center">354</entry><entry align="center">3085</entry><entry>pSAC35:MAF.GLP-1(7-36(A8G)).GLP-1(7-36).HSA</entry><entry>The yeast mating factor α-1 (hereinafter MFα-1) signal sequence is fused to GLP-1(7-36(A8G)) (SEQ ID NO:1808), GLP-1(7-36), and mature HSA.</entry><entry>pSAC35</entry><entry align="center">1624</entry><entry align="center">1456</entry><entry align="center">1792</entry><entry align="center">2038</entry><entry align="center">2039</entry><entry align="center">MFα-1</entry></row><row><entry align="center">355</entry><entry align="center">3086</entry><entry>pSAC35:INU.GLP-1(7-36(A8G)).GLP.1(7-36).HSA</entry><entry>The inulinase signal sequence is fused to GLP-1(7-36(A8G)) (SEQ ID NO:1808), GLP-1(7-36), and mature HSA.</entry><entry>pSAC35</entry><entry align="center">1625</entry><entry align="center">1457</entry><entry align="center">1793</entry><entry align="center">2040</entry><entry align="center">2041</entry><entry align="center">inulinase</entry></row><row><entry align="center">356</entry><entry align="center">3087</entry><entry>pSAC35:AP.GLP-1(7-36(A8G)).GLP-1(7-36).HSA</entry><entry>The acid phosphatase signal sequence is fused to GLP-1(7-36(A8G)) (SEQ ID NO:1808), GLP-1(7-36), and mature HSA.</entry><entry>pSAC35</entry><entry align="center">1626</entry><entry align="center">1458</entry><entry align="center">1794</entry><entry align="center">2042</entry><entry align="center">2043</entry><entry align="center">Acid phosphatase</entry></row><row><entry align="center">370</entry><entry align="center">3140</entry><entry>pSAC35:GLP1(mut)DA HK.HSA</entry><entry>GLP-1(7-36(A8G)) (SEQ ID NO:1808) is linked to mature HSA by a 16 amino acid linker derived from the N-terminus of HSA. The HSA/kex2 signal sequence is used.</entry><entry>pSAC35</entry><entry align="center">1640</entry><entry align="center">1472</entry><entry align="center">1808</entry><entry align="center">2064</entry><entry align="center">2065</entry><entry align="center">HSA/kex2</entry></row><row><entry align="center">416</entry><entry align="center">3309</entry><entry>pSAC:KT.GLP-1(7-36(A8G))x2.MSA.E25-A608</entry><entry>Killer toxin leader sequence followed by GLP-1(7-36(A8G) followed by mature mouse serum albumin.</entry><entry>pSAC35</entry><entry align="center">2170</entry><entry align="center">2160</entry><entry align="center">2180</entry><entry align="center">2194</entry><entry align="center">2195</entry><entry align="center">Killer toxin</entry></row></tbody></tgroup></table></tables>

0050Table 2 provides a non-exhaustive list of polynucleotides of the invention comprising, or alternatively consisting of, nucleic acid molecules encoding an albumin fusion protein. The first column, "Fusion No." gives a fusion number to each polynucleotide. Column 2, "Construct ID" provides a unique numerical identifier for each polynucleotide of the invention. The Construct IDs may be used to identify polynucleotides which encode albumin fusion proteins comprising, or alternatively consisting of, a Therapeutic protein portion corresponding to the given Therapeutic Protein:X listed in the corresponding row of Table 1 wherein that Construct ID is listed in column 5. The "Construct Name" column (column 3) provides the name of a given albumin fusion construct or polynucleotide.

0051The fourth column in Table 2, "Description" provides a general description of a given albumin fusion construct, and the fifth column, "Expression Vector" lists the vector into which a polynucleotide comprising, or alternatively consisting of, a nucleic acid molecule encoding a given albumin fusion protein was cloned. Vectors are known in the art, and are available commercially or described elsewhere. For example, as described in the Examples, an "expression cassette" comprising, or alternatively consisting of, one or more of (1) a polynucleotide encoding a given albumin fusion protein, (2) a leader sequence, (3) a promoter region, and (4) a transcriptional terminator, may be assembled in a convenient cloning vector and subsequently be moved into an alternative vector, such as, for example, an expression vector including, for example, a yeast expression vector or a mammalian expression vector. In one embodiment, for expression in <i>S. cervisiae,</i> an expression cassette comprising, or alternatively consisting of, a nucleic acid molecule encoding an albumin fusion protein is cloned into pSAC35. In another embodiment, for expression in CHO cells, an expression cassette comprising, or alternatively consisting of, a nucleic acid molecule encoding an albumin fusion protein is cloned into pC4. In a further embodiment, a polynucleotide comprising or alternatively consisting of a nucleic acid molecule encoding the Therapeutic protein portion of an albumin fusion protein is cloned into pC4:HSA. In a still further embodiment, for expression in NS0 cells, an expression cassette comprising, or alternatively consisting of, a nucleic acid molecule encoding an albumin fusion protein is cloned into pEE12. Other useful cloning and/or expression vectors will be known to the skilled artisan.

0052Column 6, "SEQ ID NO:Y," provides the full length amino acid sequence of the albumin fusion protein of the invention. In most instances, SEQ ID NO:Y shows the unprocessed form of the albumin fusion protein encoded - in other words, SEQ ID NO:Y shows the signal sequence, a HSA portion, and a therapeutic portion all encoded by the particular construct. Specifically contemplated by the present invention are all polynucleotides that encode SEQ ID NO:Y. When these polynucleotides are used to express the encoded protein from a cell, the cell's natural secretion and processing steps produces a protein that lacks the signal sequence listed in columns 4 and/or 11 of Table 2. The specific amino acid sequence of the listed signal sequence is shown later in the specification or is well known in the art. Thus, most preferred embodiments of the present invention include the albumin fusion protein produced by a cell (which would lack the leader sequence shown in columns 4 and/or 11 of Table 2). Also most preferred are polypeptides comprising SEQ m NO:Y without the specific leader sequence listed in columns 4 and/or 11 of Table 2. Compositions comprising these two preferred embodiments, including pharmaceutical compositions, are also preferred. Moreover, it is well within the ability of the skilled artisan to replace the signal sequence listed in columns 4 and/or 11 of Table 2 with a different signal sequence, such as those described later in the specification to facilitate secretion of the processed albumin fusion protein.

0053The seventh column, "SEQ ID NO:X." provides the parent nucleic acid sequence from which a polynucleotide encoding the Therapeutic protein portion of a given albumin fusion protein may be derived. In one embodiment, the parent nucleic acid sequence from which a polynucleotide encoding the Therapeutic protein portion of an albumin fusion protein may be derived comprises the wild type gene sequence encoding the Therapeutic protein shown in Table 1. In an alternative embodiment, the parent nucleic acid sequence from which a polynucleotide encoding the Therapeutic protein portion of an albumin fusion protein may be derived comprises a variant or derivative of a wild type gene sequence encoding the Therapeutic protein shown in Table 1, such as, for example, a synthetic codon optimized variant of a wild type gene sequence encoding the Therapeutic: protein.

0054The eighth column, "SEQ ID NO:Z," provides a predicted translation of the parent nucleic acid sequence (SEQ ID NO:X). This parent sequence can be a full length parent protein used to derive the particular construct, the mature portion of a parent protein, a variant or fragment of a wildtype protein, or an artificial sequence that can be used to create the described construct. One of skill in the art can use this amino acid sequence shown in SEQ ID NO:Z to determine which amino acid residues of an albumin fusion protein encoded by a given construct are provided by the therapeutic protein. Moreover, it is well within the ability of the skilled artisan to use the sequence shown as SEQ ID NO:Z to derive the construct described in the same row. For example, if SEQ ID NO:Z corresponds to a full length protein, but only a portion of that protein is used to generate the specific CID, it is within the skill of the art to rely on molecular biology techniques, such as PCR, to amplify the specific fragment and clone it into the appropriate vector.

0055Amplification primers provided in columns 9 and 10, "SEQ ID NO:A" and "SEQ ID NO:B" respectively, are exemplary primers used to generate a polynucleotide comprising or alternatively consisting of a nucleic acid molecule encoding the Therapeutic protein portion of a given albumin fusion protein. In one embodiment of the disclosure, oligonucleotide primers having the sequences shown in columns 9 and/or 10 (SEQ ID NOS:A and/or B) are used to PCR amplify a polynucleotide encoding the Therapeutic protein portion of an albumin fusion protein using a nucleic acid molecule comprising or alternatively consisting of the nucleotide sequence provided in column 7 (SEQ m NO:X) of the corresponding row as the template DNA. PCR methods are well-established in the art. Additional useful primer sequences could readily be envisioned and utilized by those of ordinary skill in the art.

0056In an alternative embodiment, oligonucleotide primers may be used in overlapping PCR reactions to generate mutations within a template DNA sequence. PCR methods are known in the art.

0057As shown in Table 3, certain albumin fusion constructs disclosed in this application have been deposited with the ATCC®. <tables id="tabl0003" num="0003"><table frame="all"><title><b><u>Table 3</u></b></title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="25mm" /><colspec colnum="2" colname="col2" colwidth="60mm" /><colspec colnum="3" colname="col3" colwidth="40mm" /><thead><row><entry valign="top"><b>Construct ID</b></entry><entry valign="top"><b>Construct Name</b></entry><entry valign="top"><b>ATCC Deposit No</b>./ <b>Date</b></entry></row></thead><tbody><row><entry>3070</entry><entry>pSAC35:KT.GLP-1(7-36(A8G))x2.HSA</entry><entry>PTA-4671 Sept 16, 2002</entry></row></tbody></tgroup></table></tables>

0058It is possible to retrieve a given albumin fusion construct from the deposit by techniques known in the art and described elsewhere herein (see, Example 40). The ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

0059Described is an "expression cassette" comprising, or alternatively consisting of one or more of (1) a polynucleotide encoding a given albumin fusion protein, (2) a leader sequence, (3) a promoter region, and (4) a transcriptional terminator can be moved or "subcloned" from one vector into another. Fragments to be subcloned may be generated by methods well known in the art, such as, for example, PCR amplification (e.g., using oligonucleotide primers having the sequence shown in SEQ ID NO:A or B), and/or restriction enzyme digestion.

0060In 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. In further preferred embodiments, the therapeutically active protein portions of the albumin fusion proteins of the invention are fragments or variants of the protein encoded by the sequence shown in SEQ ID NO:X column of Table 2, and are capable of the therapeutic activity and/or biologic activity of the corresponding Therapeutic protein.

Non-human albumin fusion proteins of growth hormone.

0061In one embodiment, the albumin fusion proteins of the invention comprise one or more Serum Albumin proteins of a non-human animal species, fused in tandem and in-frame either at the N-terminus or the C-terminus to one or more Growth Hormone proteins of the same non-human animal species. Non-human Serum Albumin and Growth Hormone proteins are well known in the art and available in public databases. For example, Table 4 presents accession numbers corresponding to non-human Serum Albumin sequences (column 2) and non-human Growth Hormone sequences (column 3) found in GenBank. In a preferred embodiment, a Serum Albumin protein from a non-human animal species listed in Table 4 is fused to a Growth Hormone protein from the same non-human animal species.

0062In a specific embodiment, the albumin fusion protein of the invention comprises one or more Bos taurus Serum Albumin proteins listed in Table 4, column 2, fused in tandem and in-frame either at the N-terminus or the C-terminus to one or more Bos taurus Growth Hormone proteins listed in Table 4, column 3.

0063Fusion proteins comprising fragments or variants of non-human Serum Albumin, such as, for example, the mature form of Serum Albumin, are also encompassed by the invention. Fusion proteins comprising fragments or variants of non-human Growth Hormone proteins, such as, for example, the mature form of Growth Hormone, are also encompassed by the invention. Preferably the non-human Growth Hormone fragments and variants retain growth hormone activity.

0064Polynucleotides of the invention comprise, or alternatively consist of, one or more nucleic acid molecules encoding a non-human albumin fusion protein described above. For example, the polynucleotides can comprise, or alternatively consist of, one or more nucleic acid molecules that encode a Serum Albumin protein from a non-human animal species listed in Table 4, column 1 (such as, for example, the non-human Serum Albumin reference sequences listed in Table 4, column 2) fused in tandem and in-frame either 5' or 3' to a polynucleotide that comprises, or alternatively consists of, one or more nucleic acid molecules encoding the non-human Growth Hormone protein of the corresponding non-human animal species (for example, the Growth Hormone reference sequences listed in Table 4, column 3).

0065The above-described non-human albumin fusion proteins are encompassed by the invention, as are host cells and vectors containing these polynucleotides. In one embodiment, a non-human albumin fusion protein encoded by a polynucleotide as described above has extended shelf life. In an additional embodiment, a non-human albumin fusion protein encoded by a polynucleotide described above has a longer serum half-life and/or more stabilized activity in solution (or in a pharmaceutical composition) <i>in vitro</i> and/or in <i>vivo</i> than the corresponding unfused Growth Hormone molecule.

0066The present invention also encompasses methods of preventing, treating, or ameliorating a disease or disorder in a non-human animal species. In certain embodiments, the present invention encompasses a method of treating a veterinary disease or disorder comprising administering to a non-human animal species in which such treatment, prevention or amelioration is desired an albumin fusion protein of the invention that comprises a Growth Hormone portion corresponding to a Growth Hormone protein (or fragment or variant thereof) in an amount effective to treat, prevent or ameliorate the disease or disorder. Veterinary diseases and/or disorders which may be treated, prevented, or ameliorated include growth disorders (such as, for example, pituitary dwarfism), shin soreness, obesity, growth hormone-responsive dermatosis, dilated cardiomyopathy, eating disorders, reproductive disorders, and endocrine disorders.

0067Non-human albumin fusion proteins of the invention may also be used to promote healing of skin wounds, corneal injuries, bone fractures, and injuries of joints, tendons, or ligaments.

0068Non-human albumin fusion proteins of the invention may also be used to increase milk production in lactating animals. In a preferred embodiment, the lactating animal is a dairy cow.

0069Non-human albumin fusion proteins of the invention may also be used to improve body condition in aged animals.

0070Non-human albumin fusion proteins of the invention may also be used to increase fertility, pregnancy rates, and reproductive success in domesticated animals.

0071Non-human albumin fusion proteins of the invention may also be used to improve the lean-to-fat ratio in animals raised for consumption, as well as to improve appetite, and increase body size and growth rate. <tables id="tabl0004" num="0004"><table frame="all"><title><b><u>Table 4</u></b></title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="37mm" /><colspec colnum="2" colname="col2" colwidth="64mm" /><colspec colnum="3" colname="col3" colwidth="65mm" /><thead><row><entry align="center" valign="top"><b>Non-Human Species</b></entry><entry align="center" valign="top"><b>Non-Human Serum Albumin Reference Sequence(s): GenBank Protein Accession Nos.</b></entry><entry align="center" valign="top"><b>Non-Human Growth Hormone Reference Sequence(s): GenBank Protein Accession Nos.</b></entry></row></thead><tbody><row><entry>Bos taurus</entry><entry>ABBOS, CAA76847, P02769, CAA41735, 229552, AAA51411</entry><entry>STBO, BAA06379, A29864, AAF28806, AAF28805, AAF28804, P01246, AAF03132, AAC63901, AAB92549, A36506, I45901, JC1316, CAA23445, CAA00787, CAA00598, AAA30547, AAA30546, AAA30545, AAA30544, AAA30543, AAA30542</entry></row><row><entry>Sus scrofa</entry><entry>P08835, CAA30970, AAA30988</entry><entry>STPG, PC1017, AAB29947, AAB84359, I46585, I46584, PC1063, A01516, AAB17619, 226829, 225740, CAA37411, CAA00592, AAA73478, AAA73477, CAA00356, AAA31046, AAA31045, AAA31044, AA30543</entry></row><row><entry>Equus caballus</entry><entry>ABHOS, AAG40944, P35747, CAA52194</entry><entry>STHO, P01245, AAD25992, 227704, AAA21027</entry></row><row><entry>Ovis aries</entry><entry>ABSHS, P14639, CAA34903</entry><entry>STSH, AAB24467, AAC48679, 228487, 223932, CAA34098, CAA31063, CAA00828, AAA31527</entry></row><row><entry>Salmo salar</entry><entry>ABONS2, ABONS2, CAA36643, CAA43187</entry><entry>STONC, P07064, Q07221, P48096, P10814, P10607, I51186, S03709, JS0179, A23154, S06489, CAA42431, AAB29165, AAB24612, Q91221, Q91222, CAA43942, CAA32481, 738042, 224555, CAA00427, AAA50757, AAA49558, AAA49555, AAA49553, AAA49401, AAA49406, AAA49403, AAA49402</entry></row><row><entry>Gallus gallus</entry><entry>ABCHS, P19121, CAA43098</entry><entry>BAB62262, BAB69037, AAK95643, A60509, AAG01029, BAA01365, P08998, 226895, CAA31127, CAA35619, AAA48780</entry></row><row><entry>Felis catus</entry><entry>P49064, S57632, CAA59279, JC4660</entry><entry>JC4632, P46404, AAC00073, AAA96142, AAA67294</entry></row><row><entry>Canis familiaris</entry><entry>P49822, S29749, CAB64867, CAA76841, AAB30434</entry><entry>P33711, I46145, AAF89582, AAF21502, AAD43366, S35790, AAB34229, CAA80601</entry></row></tbody></tgroup></table></tables>

<b>Polypeptide</b> and <b>Polynucleotide Fragments and Variants</b>

<i>Fragments</i>

0072Even 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 of the invention, other Therapeutic activities and/or functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) 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.

0073Accordingly, fragments of the Therapeutic protein corresponding to the 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 (i.e., the Therapeutic protein referred to in Table 1, or a Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2). In particular, N-terminal deletions may be described by the general formula m to q, where q is a whole integer representing the total number of amino acid residues in a reference polypeptide (e.g., the Therapeutic protein referred to in Table 1, or a Therapeutic protein portion of an albumin fusion protein of the invention, or a Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2), and m is defined as any integer ranging from 2 to q minus 6. Polynucleotides encoding these polypeptides are also encompassed by the invention.

0074In 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, or a serum albumin portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2). In preferred embodiments, N-terminal deletions may be described by the general formula m to 585, where 585 is a whole integer representing the total number of amino acid residues in mature human serum albumin (SEQ ID NO:1038), and m is defined as any integer ranging from 2 to 579. Polynucleotides encoding these polypeptides are also encompassed by the invention. In additional embodiments, N-terminal deletions may be described by the general formula m to 609, where 609 is a whole integer representing the total number of amino acid residues in full length human serum albumin (SEQ ID NO:1094), and m is defined as any integer ranging from 2 to 603. Polynucleotides encoding these polypeptides are also encompassed by the invention.

0075Moreover, 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 (e.g., an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2; or an albumin fusion protein having the amino acid sequence disclosed in column 6 of Table 2). In particular, N-terminal deletions may be described by the general formula m to 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 minus 6. Polynucleotides encoding these polypeptides are also encompassed by the invention.

0076Also 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., the Therapeutic protein; serum albumin protein; or albumin fusion protein of the invention) 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) and/or 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-terminal and/or C-terminal residues of a reference polypeptide retains Therapeutic activity can readily be determined by routine methods described herein and/or otherwise known in the art

0077The present disclosure further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the Therapeutic protein corresponding to the Therapeutic protein portion of an albumin fusion protein of the invention (e.g., the Therapeutic protein referred to in Table 1, or a Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2). In particular, C-terminal deletions may be described by the general formula 1 to n, where n is any whole integer ranging from 6 to q minus 1, and where q is a whole integer representing the total number of amino acid residues in a reference polypeptide (e.g., the Therapeutic protein referred to in Table 1, or a Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2). Polynucleotides encoding these polypeptides are also encompassed by the invention.

0078In addition, the present disclosure 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 or an albumin protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2). In particular, C-terminal deletions may be described by the general formula I to 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 mature human serum albumin (SEQ ID NO:1038) minus 1. Polynucleotides encoding these polypeptides are also encompassed by the invention. In particular, C-terminal deletions may be described by the general formula 1 to n, where n is any whole integer ranging from 6 to 608, where 608 is the whole integer representing the total number of amino acid residues in serum albumin (SEQ ID NO:1094) minus 1. Polynucleotides encoding these polypeptides are also encompassed by the invention.

0079Moreover, the present disclosure 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 to n, where n is any whole integer ranging from 6 to q minus 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.

0080In addition, any of the above described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted reference polypeptide. The disclosure 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 to n of a reference polypeptide (e.g., the Therapeutic protein referred to in Table 1, or a Therapeutic protein portion of an albumin fusion protein of the invention, or a Therapeutic protein portion encoded by a polynucleotide or albumin fusion construct described in Table 2, or serum albumin (e.g., SEQ ID NO:1038), or an albumin protein portion of an albumin fusion protein of the invention, or an albumin protein portion encoded by a polynucleotide or albumin fusion construct described in Table 2, or an albumin fusion protein, or an albumin fusion protein encoded by a polynucleotide or albumin fusion construct of the invention) where n and m are integers as described above. Polynucleotides encoding these polypeptides are also disclosed.

0081The present disclosure 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., the Therapeutic protein referred to in Table 1, or a Therapeutic protein portion of an albumin fusion protein of the invention, or a Therapeutic protein portion encoded by a polynucleotide or albumin fusion construct described in Table 2, or serum albumin (e.g., SEQ ID NO: 1038), or an albumin protein portion of an albumin fusion protein of the invention, or an albumin protein portion encoded by a polynucleotide or albumin fusion construct described in Table 2, or an albumin fusion protein, or an albumin fusion protein encoded by a polynucleotide or albumin fusion construct of the invention) set forth herein, or fragments thereof. The disclosure 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 disclosed.

0082Preferred polypeptide fragments 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.

0083Other 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 an improved desired activity, or a decreased undesirable activity.

<i>Variants</i>

0084"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.

0085As used herein, "variant", refers to the 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 of the invention differing in sequence from the Therapeutic protein (e.g. see "therapeutic" column of Table 1), albumin protein, and/or albumin fusion protein, respectively, but retaining at least one functional and/or therapeutic property thereof 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 the Therapeutic protein portion of an albumin fusion protein, albumin protein corresponding to an albumin protein portion of an albumin fusion protein, and/or albumin fusion protein Nucleic acids encoding these variants are also encompassed by the invention.

0086The present disclosure is also directed to proteins' which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, the amino acid sequence of the Therapeutic protein corresponding to the Therapeutic protein portion of an albumin fusion protein of the invention (e.g., the amino acid sequence of the Therapeutic protein:X disclosed in Table 1 which is GLP-1; or the amino acid sequence of the Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 1 and 2, or fragments or variants thereof), albumin proteins corresponding to an albumin protein portion of an albumin fusion protein of the invention (e.g., the amino acid sequence of an albumin protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 1 and 2; the amino acid sequence shown in SEQ ID NO: 1038; or fragments or variants thereof), and/or albumin fusion proteins. Fragments of these polypeptides are also provided (e.g., those fragments described herein). Further described are polypeptides encoded by polynucleotide which hybridize to the complement of a nucleic acid molecule encoding an albumin fusion protein 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), 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 0.1X 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, <nplcit id="ncit0003" npl-type="b"><text>Ausubel, F.M. et al., eds., 1989 Current protocol in Molecular Biology, Green publishing associates, Inc., and John Wiley &amp; Sons Inc., New York, at pages 6.3.1 - 6.3.6 and 2 10.3</text></nplcit>). Polynucleotides encoding these polypeptides are also encompassed by the invention.

0087By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence, 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.

0088As 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 a Therapeutic protein portion of the albumin fusion protein or an 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 <nplcit id="ncit0004" npl-type="s"><text>Brutlag et al. (Comp. App. Biosci.6:237-245 (1990</text></nplcit>)). 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.

0089If 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 disclosure. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned 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.

0090For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus 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 matched/aligned 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 other manual corrections are to made for the purposes of the present invention.

0091The 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 (<nplcit id="ncit0005" npl-type="s"><text>Karlin et al., Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990</text></nplcit>) and <nplcit id="ncit0006" npl-type="s"><text>Altschul, J. Mol. Evol. 36: 290-300 (1993</text></nplcit>)) which are tailored for sequence similarity searching.

0092The 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 <nplcit id="ncit0007" npl-type="s"><text>Altschul et al., (Nature Genetics 6: 119-129 (1994</text></nplcit>)). The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (<nplcit id="ncit0008" npl-type="s"><text>Henikoff et al., Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992</text></nplcit>))_. 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=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every wink^th 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 Bestfit 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.

0093The polynucleotide variants of the disclosure may contain alterations 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 <i>E. coli</i>).

0094A polynucleotide of the disclosure which encodes the albumin portion of an albumin fusion protein is optimized for expression in yeast or mammalian cells. A polynucleotide which encodes the Therapeutic protein portion of an albumin fusion protein is optimized for expression in yeast or mammalian cells. A polynucleotide encoding an albumin fusion protein of the invention is optimized for expression in yeast or mammalian cells.

0095A codon optimized polynucleotide which encodes the Therapeutic protein portion of an albumin fusion protein does not hybridize to the wild type polynucleotide encoding the Therapeutic protein under stringent hybridization conditions as described herein. A codon optimized polynucleotide which encodes an albumin portion of an albumin fusion protein does not hybridize to the wild type polynucleotide encoding the albumin protein under stringent hybridization conditions as described herein. A cordon optimized polynucleotide which encodes an albumin fusion protein does not hybridize to the wild type polynucleotide encoding the Therapeutic protein portion or the albumin protein portion under stringent hybridization conditions as described herein.

0096In an additional embodiment, a polynucleotide which encodes the Therapeutic protein portion of an albumin fusion protein does not comprise, or alternatively consist of, the naturally occurring sequence of that Therapeutic protein. In a further embodiment, a polynucleotide which encodes an albumin protein portion of an albumin fusion protein does not comprise, or alternatively consist of, the naturally occurring sequence of albumin protein. In an alternative embodiment, a polynucleotide which encodes an albumin fusion protein does not comprise, or alternatively consist of, the naturally occurring sequence of a Therapeutic protein portion or the albumin protein portion.

0097Naturally 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. (<nplcit id="ncit0009" npl-type="b"><text>Genes II, Lewin, B., ed., John Wiley &amp; Sons, New York (1985</text></nplcit>)). 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 mutagenesis techniques or by direct synthesis.

0098Using 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, <nplcit id="ncit0010" npl-type="s"><text>Ron et al. (J. BioL Chem. 268: 2984-2988 (1993</text></nplcit>)) 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. (<nplcit id="ncit0011" npl-type="s"><text>Dobeli et al., J. Biotechnology 7:199-216 (1988</text></nplcit>).)

0099Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, <nplcit id="ncit0012" npl-type="s"><text>Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993</text></nplcit>)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a 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 investigators found that "[m]ost of the molecule could be altered with little effect 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.

0100Furthermore, 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.

0101Thus, the invention further includes polypeptide variants which have a functional activity (e.g., biological activity and/or therapeutic activity). In one embodiment, the invention provides variants of albumin fusion proteins that have a functional activity (e.g., biological activity and/or therapeutic activity) 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. In another embodiment, the invention provides variants of albumin fusion proteins that have a functional activity (e.g., biological activity and/or therapeutic activity) 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. Polynucleotides encoding such variants are also encompassed by the invention.

0102In 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.

0103Guidance concerning how to make phenotypically silent amino acid substitutions is provided, for example, in <nplcit id="ncit0013" npl-type="s"><text>Bowie et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990</text></nplcit>), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

0104The 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 identified. 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 modified while still maintaining biological activity of the protein.

0105The 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 <nplcit id="ncit0014" npl-type="s"><text>Cunningham and Wells, Science 244:1081-1085 (1989</text></nplcit>). The resulting mutant molecules can then be tested for biological activity.

0106As 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 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 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. 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.

0107For 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 <nplcit id="ncit0015" npl-type="s"><text>Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967</text></nplcit>); <nplcit id="ncit0016" npl-type="s"><text>Robbins et aL, Diabetes 36: 838-845 (1987</text></nplcit>); <nplcit id="ncit0017" npl-type="s"><text>Cleland et aL, Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993</text></nplcit>).

0108In specific embodiments, the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of the amino acid sequence of an albumin fusion protein, wherein the fragments or variants have the activities specified in the claims and 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.

0109The 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 polypeptides 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. Modifications 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, 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 ubiqnitination. (See, for instance, <nplcit id="ncit0018" npl-type="b"><text>PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993</text></nplcit>); <nplcit id="ncit0019" npl-type="b"><text>POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983</text></nplcit>); <nplcit id="ncit0020" npl-type="s"><text>Seifter et aL, Meth. Enzymol. 182:626-646 (1990</text></nplcit>); <nplcit id="ncit0021" npl-type="s"><text>Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992</text></nplcit>)).

Functional activity

0110"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 the 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.

0111"A polypeptide having biological activity" refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of the 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 polypeptide, 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).

0112The albumin fusion protein of the invention has GLP-1 activity.

0113The 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. Additionally, one of skill in the art may routinely assay fragments of the Therapeutic protein corresponding to the Therapeutic protein portion of an albumin fusion protein, for activity using assays referenced in its corresponding row of Table 1 (e.g., in column 3 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, for activity using assays known in the art and/or as described in the Examples section below.

0114For example, where one is assaying for the ability of an albumin fusion protein to bind or compete with the 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. Antibody binding is detected by detecting a label on the primary antibody. The primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. The secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay.

0115Where a binding partner (e.g., a receptor or a ligand) of the Therapeutic protein is identified, binding to that binding partner by an albumin fusion protein which comprises 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, <nplcit id="ncit0022" npl-type="s"><text>Phizicky et al., Microbiol. Rev. 59:94-123 (1995</text></nplcit>). The ability of physiological correlates of an albumin fusion protein to bind to a substrate(s) of the Therapeutic polypeptide corresponding to the Therapeutic protein portion of the fusion can be routinely assayed using techniques known in the art.

0116Where the ability of an albumin fusion protein 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 affinity blotting. See generally, Phizicky et al., <i>supra.</i>

0117Assays for the ability of the albumin fusion proteins to (Specifically) bind a specific protein or epitope may be performed in solution (<i>e.g.</i>, <nplcit id="ncit0023" npl-type="s"><text>Houghten, Bio/Techniques 13:412-421(1992</text></nplcit>)), on beads (<i>e.g.</i>, <nplcit id="ncit0024" npl-type="s"><text>Lam, Nature 354:82-84 (1991</text></nplcit>)), on chips (<i>e.g.</i>, <nplcit id="ncit0025" npl-type="s"><text>Fodor, Nature 364:555-556 (1993</text></nplcit>)), on bacteria (<i>e.g.</i>, <patcit id="pcit0005" dnum="US5223409A"><text>U.S. Patent No. 5,223,409</text></patcit>), on spores (<i>e.g.</i>, Patent Nos. <patcit id="pcit0006" dnum="US5571698A"><text>5,571,698</text></patcit>; <patcit id="pcit0007" dnum="US5403484A"><text>5,403,484</text></patcit>; and <patcit id="pcit0008" dnum="US5223409A"><text>5,223,409</text></patcit>), on plasmids (<i>e.g.</i>, <nplcit id="ncit0026" npl-type="s"><text>Cull et al., Proc. NatL Acad. Sci. USA 89:1865-1869 (1992</text></nplcit>)) or on phage (<i>e.g.</i>, <nplcit id="ncit0027" npl-type="s"><text>Scott and Smith, Science 249:386-390 (1990</text></nplcit>); <nplcit id="ncit0028" npl-type="s"><text>Devlin, Science 249:404-406 (1990</text></nplcit>); <nplcit id="ncit0029" npl-type="s"><text>Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990</text></nplcit>); and <nplcit id="ncit0030" npl-type="s"><text>Felici, J. Mol. Biol. 222:301-310 (1991</text></nplcit>)).

0118The binding affinity of an albumin fusion protein to a protein, antigen, or epitope and the off-rate of an albumin fusion protein-protein/antigen/epitope interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (<i>e.g.</i>, ³H or ¹²⁵I) with the albumin fusion protein of the invention in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the albumin fusion protein for a specific protein, antigen, or epitope and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second protein that binds the same protein, antigen or epitope as the albumin fusion proteins, can also be determined using radioimmunoassays. In this case, the protein, antigen or epitope is incubated with an albumin fusion protein conjugated to a labeled compound (<i>e.g.</i>, ³H or ¹²⁵I) in the presence of increasing amounts of an unlabeled second protein that binds the same protein, antigen, or epitope as the albumin fusion protein of the invention.

0119In a preferred embodiment, BLAcore kinetic analysis is used to determine the binding on and off rates of albumin fusion proteins of the invention to a protein, antigen or epitope. BIAcore kinetic analysis comprises analyzing the binding and dissociation of albumin fusion proteins, or specific polypeptides, antigens or epitopes from chips with immobilized specific polypeptides, antigens or epitopes or albumin fusion proteins, respectively, on their surface.

0120Antibodies that bind the Therapeutic protein corresponding to the Therapeutic protein portion of an albumin fusion protein may also be described or specified in terms of their binding affinity for a given protein or antigen, preferably the antigen which they specifically bind. Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10^-2 M, 10^-2 M, 5 X 10^-3 M, 10^-3 M, 5 X 10^-4 M, 10^-4 M. More preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10^-5 M, 10^-5 M, 5 X 10^-6 M, 10^-6M, 5 X 10^-7 M, 10⁷ M, 5 X 10^-8 M or 10^-8 M. Even more preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10^-9 M, 10^-9 M, 5 X 10^-10 M, 10^-10 M, 5 X 10^-11 M, 10^-11 M, 5 X 10^-12 M, ^10-11 M, 5 X 10^-13 M, 10^-13 M, 5 X 10^-14 M, 10^-14 M, 5 X 10^-15 M, or 10^-15 M. In addition, assays described herein (see Examples and Table 1) and otherwise known in the art may routinely be applied to measure the ability of albumin fusion proteins and fragments, variants and derivatives thereof to elicit biological activity and/or Therapeutic activity (either <i>in vitro</i> or <i>in vivo</i>) related to either the Therapeutic protein portion and/or albumin portion of the albumin fusion protein. Other methods will be known to the skilled artisan and are within the scope of the invention.

Albumin

0121As described above, an albumin fusion protein of the invention comprises at least two tandemly oriented fragments or variants of the Therapeutic protein and at fragments variants the Therapeutic protein and least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusions.

0122An additional embodiment comprises at least two tandemly orianted fragments or variants of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are linked to one another by chemical conjugation.

0123The 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).

0124As 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 for example, <patcit id="pcit0009" dnum="EP201239A"><text>EP 201 239</text></patcit>, <patcit id="pcit0010" dnum="EP322094A"><text>EP 322 094</text></patcit><patcit id="pcit0011" dnum="WO9714445A"><text>WO 97/14445</text></patcit>, <patcit id="pcit0012" dnum="WO9523857A"><text>WO95/23857</text></patcit>) especially the mature form of human albumin as shown in <figref idref="f0001 f0002 f0003 f0004">Figure 1</figref> and SEQ ID NO: 1038, or albumin from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof.

0125In 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: 1038: Leu-407 to Ala, Leu-408 to Val, Val-409 to Ala, and Arg-410 to Ala; or Arg-410 to A, Lys-413 to Gln, and Lys-414 to Gln (see, e.g., International Publication No. <patcit id="pcit0013" dnum="WO9523857A"><text>WO95/23857</text></patcit>). In even more preferred embodiments, albumin fusion proteins of the invention that contain one or both of above-described sets of point mutations have improved stability/resistance to yeast Yap3p proteolytic cleavage, allowing increased production of recombinant albumin fusion proteins expressed in yeast host cells.

0126As used herein, a portion of albumin sufficient 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 may include one or more fragments thereof that are 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. In a preferred embodiment, the HA fragment is the mature form of HA.

0127The 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 one 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.

0128In 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 <patcit id="pcit0014" dnum="EP322094A"><text>EP 322 094</text></patcit> (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.

0129Generally 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 NO: 1038), domain 2 (amino acids 195-387 of SEQ ID NO: 1038), domain 3 (amino acids 388-585 of SEQ ID NO: 1038), domains 1 and 2 (1-387 of SEQ ID NO: 1038), domains 2 and 3 (195-585 of SEQ ID NO: 1038) or domains 1 and 3 (amino acids 1-194 of SEQ ID NO: 1038 and amino acids 388-585 of SEQ ID NO: 1038). 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 Glu119, Glu292 to Val315 and Glu492 to Ala511.

0130Preferably, 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 all of the adjacent linker is preferably used to link to the Therapeutic protein moiety.

<i>Demonstration of Therapeutic or Prophylactic Activity</i>

0131The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then <i>in vivo</i> for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

<i>Therapeutic</i>/<i>Prophylactic Administration and Composition</i>

0132The compound or pharmaceutical composition of the invention can be used for treatment, inhibition and prophylaxis by administration to a subject of an effective amount. In a preferred embodiment, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.

0133Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.

0134Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., <nplcit id="ncit0031" npl-type="s"><text>Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987</text></nplcit>)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

0135It may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. When administering a protein, including an antibody, care must be taken to use materials to which the protein does not absorb.

0136The compound or composition can be delivered in a vesicle, in particular a liposome (see <nplcit id="ncit0032" npl-type="s"><text>Langer, Science 249:1527-1533 (1990</text></nplcit>); <nplcit id="ncit0033" npl-type="b"><text>Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989</text></nplcit>); <nplcit id="ncit0034" npl-type="b"><text>Lopez-Berestein, ibid., pp. 317-327</text></nplcit>; see generally ibid.)

0137The compound or composition can be delivered in a controlled release system. A pump may be used (see Langer, <i>supra</i>; <nplcit id="ncit0035" npl-type="s"><text>Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987</text></nplcit>); <nplcit id="ncit0036" npl-type="s"><text>Buchwald et al., Surgery 88:507 (1980</text></nplcit>); <nplcit id="ncit0037" npl-type="s"><text>Saudek et al., N. Engl. J. Med. 321:574 (1989</text></nplcit>)). Polymeric materials can be used (see <nplcit id="ncit0038" npl-type="b"><text>Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974</text></nplcit>); <nplcit id="ncit0039" npl-type="b"><text>Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984</text></nplcit>); <nplcit id="ncit0040" npl-type="s"><text>Ranger and Peppas, J., MacromoL Sci. Rev. Macromol. Chem. 23:61 (1983</text></nplcit>); see also <nplcit id="ncit0041" npl-type="s"><text>Levy et al., Science 228:190 (1985</text></nplcit>); <nplcit id="ncit0042" npl-type="s"><text>During et al., Ann. Neurol. 25:351 (1989</text></nplcit>); <nplcit id="ncit0043" npl-type="s"><text>Howard et al., J.Neurosurg. 71:105 (1989</text></nplcit>)). A controlled release system can be placed in proximity of the therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., <nplcit id="ncit0044" npl-type="s"><text>Goodson, in Medical Applications of Controlled Release, supra, voL 2, pp. 115-138 (1984</text></nplcit>)).

0138Other controlled release systems are discussed in the review by <nplcit id="ncit0045" npl-type="s"><text>Langer (Science 249:1527-1533 (1990</text></nplcit>)).

0139Where the compounds of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered <i>in vivo</i> to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see <patcit id="pcit0015" dnum="US4980286A"><text>U.S. Patent No. 4,980,286</text></patcit>), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox- like peptide which is known to enter the nucleus (see e.g., <nplcit id="ncit0046" npl-type="s"><text>Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991</text></nplcit>)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

0140The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound of the invention, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

0141The composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

0142The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

0143The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a Therapeutic protein can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

<u>Albumin fusion proteins</u>

0144The present invention relates to albumin fusion proteins as defined above and in the claims and to the use of said albumin fusion proteins treating, preventing, or ameliorating diseases or disorders. As used herein, "albumin fusion protein" refers to a protein formed the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least two tandemly oriented molecules of the Therapeutic protein (herein GLP-1)(or fragment or variant thereof), wherein said protein has the activity specified in the claims. An albumin fusion protein of the invention comprises at least two tandemly oriented fragments or variants of the 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 the Therapeutic protein is joined in-frame with a polynucleotide encoding all or a portion of albumin) or to one another. The Therapeutic protein and albumin protein, once part of the albumin fusion protein, may each be referred to as a "portion", "region" or "moiety" of the albumin fusion protein.

0145In a preferred embodiment, the invention provides an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 1 or Table 2. Polynucleotides encoding these albumin fusion proteins are also encompassed by the invention.

0146Preferred albumin fusion proteins of the invention, include, albumin fusion proteins encoded by a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least two tandemly oriented molecules of the Therapeutic protein (or fragment or variant thereof); a nucleic acid molecule comprising, or alternatively consisting of a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least two tandemly oriented molecules of the Therapeutic protein (or fragment or variant thereof) generated as described in Table 1, Table 2 or in the Examples; or a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least two tandemly oriented molecules of the Therapeutic protein (or fragment or variant thereof), further comprising, for example, one or more of the following elements: (1) a functional self-replicating vector (including but not limited to, a shuttle vector, an expression vector, an integration vector, and/or a replication system), (2) a region for initiation of transcription (e.g., a promoter region, such as for example, a regulatable or inducible promoter, a constitutive promoter), (3) a region for termination of transcription, (4) a leader sequence, and (5) a selectable marker.

0147In one embodiment, the invention provides an albumin fusion protein comprising, or alternatively consisting of, at least two tandemly oriented Therapeutic proteins (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, at least two tandemly oriented biologically active and/or therapeutically active fragments of the Therapeutic protein and a serum albumin protein. In other embodiments, the invention provides an album fusion protein comprising, or alternatively consisting of, at least two tandemly oriented biologically active and/or therapeutically active variants of the 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.

0148In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, at least two tandemly oriented Therapeutic proteins, 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, at least two tandemly oriented Therapeutic proteins 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.

0149In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, at least two tandemly oriented biologically active and/or therapeutically active fragments or variants 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 the Therapeutic protein and the mature portion of serum albumin.

0150Preferably, the albumin fusion protein comprises HA as the N-terminal portion, and the Therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising HA as the C-terminal portion, and the Therapeutic protein as the N-terminal portion may also be used.

0151In other embodiments, the albumin fusion protein has the 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 an alternative 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 or a related 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- termini are different Therapeutic proteins which may be used to treat, ameliorate, 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, concurrently, or consecutively, or which commonly occur in patients in association with one another.

0152Albumin fusion proteins of the invention encompass proteins containing one, two, three, four, or more molecules of the Therapeutic protein X or variant thereof fused to the N- or C- terminus of an albumin fusion protein of the invention, and/or to the N- and/or C- terminus of albumin or variant thereof. Molecules of the Therapeutic protein X, or variants thereof are present in the albumin fusion protein in tandem orientation, including a 'head to tail' orientation (e.g., wherein the C-terminus of one molecule of the Therapeutic protein X is fused to the N-terminus of another molecule of Therapeutic protein X).

0153In one embodiment, two, three, or more tandemly oriented Therapeutic protein X polypeptides (or fragments or variants thereof) are fused to the N- or C- terminus of an albumin fusion protein of the invention, and/or to the N- and/or C- terminus of albumin or variant thereof.

0154In another specific embodiment, two, three, four, five, or more tandemly oriented molecules of GLP-1 are fused to the N- or C-terminus of albumin or variant thereof. For example, two, three, four, five, or more tandemly oriented molecules of GLP-1 (including, but not limited to, molecules of GLP-1 comprising, or alternatively consisting of, amino acids 7 to 36, with residue 8 being mutated from an Alanine to a Glycine) (See for example the mutants disclosed in <patcit id="pcit0016" dnum="US5545618A"><text>U.S. Patent No. 5,545,618</text></patcit> are fused to the N- or C-terminus of albumin or variant thereof. Exemplary fusion proteins of the invention containing multiple protein portions of GLP-1, include, but are not limited to, GL1-GLP1-HSA, HSA-GLP1-GLP1, GLP1mutant-GLP1mutant-HSA, HSA-GLP1mutant-GLP1mutant, GLP1mutant-GLP1-HSA, HSA-GLP1mutant-GLP1, GLP1-GLP1mutant-HSA, or HSA-GLP1-GLP1mutant. Particularly preferred embodiments are GLP-1 tandem fusions such as construct ID #3070 and the protein encoded by such construct.

0155Albumin fusion proteins of the invention further encompass proteins containing two, three, four, or more molecules of the given Therapeutic proteins X or variant thereof fused to the N- or C- terminus of an albumin fusion protein of the invention, and/or to the N- and/or C- terminus of albumin or variant thereof, wherein the molecules are joined through peptide linkers. Examples include those peptide linkers described in <patcit id="pcit0017" dnum="US5073627A"><text>U.S. Pat. No. 5,073,627</text></patcit>. Albumin fusion proteins comprising multiple Therapeutic protein X polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology. Linkers are particularly important when fusing a small peptide to the large HSA molecule. The peptide itself can be a linker by fusing tandem copies of the peptide (see for example GLP-1) or other known linkers can be used. Constructs that incorporate linkers are described in Table 2 or are apparent when examining SEQ ID NO:Y.

0156Further, albumin fusion proteins of the invention may also be produced by fusing the Therapeutic protein X or variants thereof to the N-terminal and/or C-terminal of albumin or variants thereof in such a way as to allow the formation of intramolecular and/or intermolecular multimeric forms. In one embodiment of the invention, albumin fusion proteins may be in monomeric or multimeric forms (i.e., dimers, trimers, tetramers and higher multimers). In a further embodiment of the invention, the Therapeutic protein portion of an albumin fusion protein may be in monomeric form or multimeric form (i.e., dimers, trimers, tetramers and higher multimers). In a specific embodiment, the Therapeutic protein portion of an albumin fusion protein is in multimeric form (i.e., dimers, trimers, tetramers and higher multimers), and the albumin protein portion is in monomeric form.

0157In addition to albumin fusion proteins in which the albumin portion is fused N-terminal and/or C-terminal of the Therapeutic protein portion, albumin fusion proteins of the invention may also be produced by inserting the Therapeutic protein or peptide of interest (e.g., a Therapeutic protein X as disclosed 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 α-helices, which are stabilized by disulphide bonds. The loops, as determined from the crystal structure of HA (PDB identifiers 1A06, 1BJ5, 1BKE, 1BM0, 1E7E to 1E7I and 1UOR) 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.

0158Loops in human albumin structure into which peptides or polypeptides may be inserted to generate albumin fusion proteins of the invention include: Val54-Asn61, Thr76-Asp89, Ala92-Glu100, Gln170-Ala176, His 247 - Glu252, Glu 266 - Glu277, Glu 280-His288, Ala362-Glu368, Lys439-Pro447, Val462-Lys475, Thr478-Pro486, and Lys560-Thr566. In more preferred embodiments, peptides or polypeptides are inserted into the Val54-Asn61, Gln170-Ala176, and/or Lys560-Thr566 loops of mature human albumin (SEQ ID NO: 1 038).

0159Peptides 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.

0160Such library(s) could be generated on HA or domain fragments of HA by one of the following methods:

0161randomized 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;

0162replacement of, or insertion into one or more loops of HA or HA domain fragments (i.e., internal fusion) of a randomized peptide(s) of length X_n (where X is an amino acid and n is the number of residues;

0163N-, C- or N- and C- terminal peptide/protein fusions in addition to (a) and/or (b).

0164The 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.

0165In 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 particularly, 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 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 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, or at least 40 amino acids fused to the C-terminus of human serum albumin. For example, short peptides described in Table 1 and 2 (e.g., Therapeutic Y) can be inserted into the albumin loops.

0166Generally, 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, the 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.

0167Bi- or multi-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.

0168As 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 X_n (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 <i>in situ</i> 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.

0169Additionally, 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.

0170The 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 <i>S</i>. <i>cerevisiae</i> protease <i>kex</i>2 or equivalent proteases.

0171Therefore, as described above, the albumin fusion proteins may have the following formula R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, wherein R1 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.

0172In preferred embodiments, albumin fusion proteins of the invention comprising the 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 the Therapeutic protein in solution or in some other storage 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.

0173Albumin 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.

0174Shelf-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 38, 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.

<i>Expression of Fusion Proteins</i>

0175The 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.

0176A 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.

0177The <i>Saccharomyces cerevisiae</i> invertase signal is a preferred example of a yeast-derived signal sequence.

0178Conjugates of the kind prepared by <nplcit id="ncit0047" npl-type="s"><text>Poznansky et al., (FEBS Lett. 239:18 (1988</text></nplcit>)), in which separately-prepared polypeptides are joined by chemical cross-linking, are not contemplated.

0179The present disclosure 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, also disclosed is 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 <i>E. coli</i> and <i>Bacillus subtilis</i>), yeasts (for example <i>Saccharomyces cerevisiae. Kluyveromyces lactis</i> and <i>Pichia pastoris.</i> filamentous fungi (for example <i>Aspergillus</i>), plant cells, animal cells and insect cells.

0180Preferred yeast strains to be used in the production of albumin fusion proteins are D88, DXY1 and BXP10. D88 [<i>leu2-3, leu2-122, can1, pra1, ubc4</i>] is a derivative of parent strain AH22<i>his</i>⁺ (also known as DB1; see, e.g., <nplcit id="ncit0048" npl-type="s"><text>Sleep et al. Biotechnology 8:42-46 (1990</text></nplcit>)). The strain contains a <i>leu2</i> 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 PRB1 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 ABC4 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. <patcit id="pcit0018" dnum="WO9900504A"><text>WO99/00504</text></patcit>).

0181DXY1, a derivative of D88, has the following genotype: [<i>leu2-3</i>, <i>leu2-122, can1, pra1, ubc4, ura3::yap3</i>]<i>.</i> 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., <patcit id="pcit0019" dnum="US5965386A"><text>U.S. Patent No. 5,965,386</text></patcit> and <nplcit id="ncit0049" npl-type="s"><text>Kerry-Williams et al., Yeast 14:161-169 (1998</text></nplcit>)).

0182BXP10 has the following genotype: <i>leu2-3, leu2-122, can1, pra1, ubc4, ura3, yap3::URA3, lys2, hsp150::LYS2, pmt1::URA3.</i> In addition to the mutations isolated in DXY1, this strain also has a knockout of the PMT1 gene and the HSP150 gene. The PMT1 gene is a member of the evolutionarily conserved family of dolichyl-phosphate-mannose protein O-mannosyltransferases (Pmts). The transmembrane topology of Pmtlp suggests that it is an integral membrane protein of the endoplasmic reticulum with a role in O-linked glycosylation. This mutation serves to reduce/eliminate O-linked glycosylation of HSA fusions (see, e.g., International Publication No. <patcit id="pcit0020" dnum="WO0044772A"><text>WO00/44772</text></patcit>). Studies revealed that the Hsp150 protein is inefficiently separated from rHA by ion exchange chromatography. The mutation in the HSP150 gene removes a potential contaminant that has proven difficult to remove by standard purification techniques. See, e.g., <patcit id="pcit0021" dnum="US5783423A"><text>U.S. Patent No. 5,783,423</text></patcit>.

0183The 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 <nplcit id="ncit0050" npl-type="s"><text>Becker &amp; Guarente (1990) Methods Enzymol. 194, 182</text></nplcit>.

0184Successfully transformed cells, <i>i.e.</i>, 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 <nplcit id="ncit0051" npl-type="s"><text>Southern (1975) J. Mol. Biol. 98, 503</text></nplcit> or <nplcit id="ncit0052" npl-type="s"><text>Berent et al. (1985) Biotech. 3, 208</text></nplcit>. Alternatively, the presence of the protein in the supernatant can be detected using antibodies.

0185Useful yeast plasmid vectors include pRS403-406 and pro413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, 7RP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).

0186Preferred vectors for making albumin fusion proteins for expression in yeast include pPPC0005, pScCHSA, pScNHSA, and pC4:HSA which are described in detail in Example 1. <figref idref="f0005">Figure 2</figref> 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 <i>PRB1 S. cerevisiae</i> promoter (PRB1p), a Fusion leader sequence (FL), DNA encoding HA (rHA) and an <i>ADH1 S. cerevisiae</i> 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 NO: 1094) and the last five amino acids of the mating factor alpha 1 promoter (SLDKR, see <patcit id="pcit0022" dnum="EP397319A"><text>EP-A-397 319</text></patcit>).

0187The 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 PTA-3278, PTA-3276, PTA-3279, and PTA-3277, respectively. Another vector useful for expressing an albumin fusion protein in yeast the pSAC35 vector which is described in <nplcit id="ncit0053" npl-type="s"><text>Sleep et al., BioTechnology 8:42 (1990</text></nplcit>).

0188Another yeast promoter that can be used to express the albumin fusion protein is the MET25 promoter. See, for example, <nplcit id="ncit0054" npl-type="s"><text>Dominik Mumburg, Rolf Muller and Martin Funk. Nucleic Acids Research, 1994, Vol. 22, No. 25, pp. 5767-5768</text></nplcit>. The Met25 promoter is 383 bases long (bases -382 to -1) and the genes expressed by this promoter are also known as Met15, Met17, and YLR303W. A preferred embodiment uses the sequence below, where, at the 5' end of the sequence below, the Not 1 site used in the cloning is underlined and at the 3' end, the ATG start codon is underlined: <img file="EP1463751B1_D0001.tif" />

0189A 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.

0190Synthetic 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, gamma-single-stranded termini with their 3' 5'-exonucleolytic activities, and fill in recessed 3'-ends with their polymerizing activities.

0191The 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.

0192Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CT, USA.

0193A 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 <nplcit id="ncit0055" npl-type="s"><text>Saiki et al. (1988) Science 239, 487-491</text></nplcit>. 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.

0194Exemplary genera of yeast contemplated to be useful in the practice of the present invention as hosts for expressing the albumin fusion proteins are <i>Pichia</i> (Hansenula), <i>Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis,</i> and the like. Preferred genera are those selected from the group consisting of <i>Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia</i> and <i>Torulaspora.</i> Examples of <i>Saccharomyces spp.</i> are <i>S. cerevisiae, S. italicus and S. rouxii.</i>

0195Examples of <i>Kluyveromyces</i> spp. are <i>K</i>. <i>fragilis, K. lactis and K. marxianus. A</i> suitable <i>Torulaspora</i> species is <i>T. delbrueckii.</i> Examples of <i>Pichia</i> (Hansenula) spp. are <i>P</i>. <i>angusta</i> (formerly <i>H. polymorpha</i>), <i>P. anomala</i> (formerly <i>H. anomala)</i> and <i>P. pastoris.</i> Methods for the transformation of <i>S. cerevisiae</i> are taught generally in <patcit id="pcit0023" dnum="EP251744A"><text>EP 251 744</text></patcit>, <patcit id="pcit0024" dnum="EP258067A"><text>EP 258 067</text></patcit> and <patcit id="pcit0025" dnum="WO9001063A"><text>WO 90/01063</text></patcit>, all of which are incorporated herein by reference.

0196Preferred exemplary species of <i>Saccharomyces</i> include <i>S. cerevisiae, S. italicus, S. diastaticus,</i> and <i>Zygosaccharomyces rouxii.</i> Preferred exemplary species of <i>Kluyveromyces</i> include <i>K. fragilis</i> and <i>K. lactis.</i> Preferred exemplary species of <i>Hansenula</i> include <i>H. polymorpha</i> (now <i>Pichia angusta), H. anomala</i> (now <i>Pichia anomala),</i> and <i>Pichia capsulata.</i> Additional preferred exemplary species of <i>Pichia</i> include <i>P. pastoris.</i> Preferred exemplary species of <i>Aspergillus</i> include <i>A</i>. <i>niger</i> and <i>A. nidulans.</i> Preferred exemplary species of <i>Yarrowia</i> include <i>Y. lipolytica.</i> 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: <i>Saccharomyces cerevisiae</i> Hansen, teleomorph strain BY4743 <i>yap3</i> mutant (ATCC Accession No. 4022731); <i>Saccharomyces cerevisiae</i> Hansen, teleomorph strain BY4743 <i>hsp150</i> mutant (ATCC Accession No. 4021266); <i>Saccharomyces cerevisiae</i> Hansen, teleomorph strain BY4743 <i>pmt1</i> mutant (ATCC Accession No. 4023792); <i>Saccharomyces cerevisiae</i> Hansen, teleomorph (ATCC Accession Nos. 20626; 44773; 44774; and 62995); <i>Saccharomyces diastaticus</i> Andrews et Gilliland ex van der Walt, teleomorph (ATCC Accession No. 62987); <i>Kluyveromyces lactis</i> (Dombrowski) van der Walt, teleomorph (ATCC Accession No. 76492); <i>Pichia angusta</i> (Teunisson et al.) Kurtzman, teleomorph deposited as <i>Hansenula polymorpha</i> de Morais et Maia, teleomorph (ATCC Accession No. 26012); <i>Aspergillus niger</i> van Tieghem, anamorph (ATCC Accession No. 9029); <i>Aspergillus niger</i> van Tieghem, anamorph (ATCC Accession No. 16404); <i>Aspergillus nidulans</i> (Eidam) Winter, anamorph (ATCC Accession No. 48756); and <i>Yarrowia lipolytica</i> (Wickerham et al.) van der Walt et von Arx, teleomorph (ATCC Accession No. 201847).

0197Suitable promoters for <i>S. cerevisiae</i> include those associated with the PGKI gene, GAL1 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 (<i>e.g.</i> the promoter of <patcit id="pcit0026" dnum="EP258067A"><text>EP-A-258 067</text></patcit>).

0198Convenient regulatable promoters for use in <i>Schizosaccharomyces pombe</i> are the thiamine-repressible promoter from the nmt gene as described by <nplcit id="ncit0056" npl-type="s"><text>Maundrell (1990) J. Biol. Chem. 265, 10857-10864</text></nplcit> and the glucose repressible jbpl gene promoter as described by <nplcit id="ncit0057" npl-type="s"><text>Hoffman &amp; Winston (1990) Genetics 124, 807-816</text></nplcit>.

0199Methods of transforming <i>Pichia</i> for expression of foreign genes are taught in, for example, Cregg <i>et al</i>. (1993), and various Phillips patents (<i>e.g.</i><patcit id="pcit0027" dnum="US4857467A"><text>US 4 857 467</text></patcit>), and <i>Pichia</i> expression kits are commercially available from Invitrogen BV, Leek, Netherlands, and Invitrogen Corp, San Diego, California. Suitable promoters include AOXI and AOX2. <nplcit id="ncit0058" npl-type="s"><text>Gleeson et al. (1986) J. Gen. Microbiol. 132, 3459-3465</text></nplcit> include information on <i>Hansenula</i> vectors and trensformation, suitable promoters being MOX1 and FMD1; whilst <patcit id="pcit0028" dnum="EP361991A"><text>EP 361 991, Fleer et al. (1991</text></patcit>) and other- publications from Rhone-Poulenc Rorer teach how to express foreign proteins in <i>Kluyveromyces</i> spp., a suitable promoter being PGKI.

0200The transcription termination signal is preferably the 3' flanking sequence of a eukaryotic gene which contains proper signals for transcription termination and polyadenylation. Suitable 3' flaking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, <i>i.e.</i> may correspond to the promoter. Alternatively, they may be different in which case the termination signal of the <i>S</i>. <i>cerevisiae</i> ADHI gene is preferred.

0201The 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 yeast include any of the following: <ul id="ul0004" list-style="none" compact="compact"><li>a) the MPIF-1 signal sequence (e.g., amino acids 1-21 of GenBank Accession number AAB51134) MKVSVAALSCLMLVTALGSQA (SEQ ID NO:2132)</li><li>b) the stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:1054)</li><li>c) the pre-pro region of the HSA signal sequence (e.g., MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO:1176)</li><li>d) the pre region of the HSA signal sequence (e.g., MKWVTFISLLFLFSSAYS, SEQ ID NO:1171) or variants thereof, such as, for example, MKWVSFISLLFLFSSAYS, (SEQ ID NO:1168)</li><li>e) the invertase signal sequence (e.g., MLLQAFLFLLAGFAAKISA, SEQ ID NO:1108)</li><li>f) the yeast mating factor alpha signal sequence (e.g., MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV AVLPFSNSTNNGLLFINTTTIASIAAKEEGVSLEKR, SEQ ID NO:1109 or MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKR, SEQ ID NO:1109)</li><li>g) <i>K. lactis</i> killer toxin leader sequence</li><li>h) a hybrid signal sequence (e.g., MKWVSFISLLFLFSSAYSRSLEKR, SEQ ID NO:1110)</li><li>i) an HSA/MFα-1 hybrid signal sequence (also known as HSA/kex2) (e.g., MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:1111)</li><li>j) a <i>K. lactis</i> killer/ MFα-1 fusion leader sequence (e.g., MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO:1169)</li><li>k) the Immunoglobulin Ig signal sequence (e.g., MGWSCIILFLVATATGVHS, SEQ ID NO:1095)</li><li>l) the Fibulin B precursor signal sequence (e.g., MERAAPSRRVPLPLLLLGGLALLAAGVDA, SEQ ID NO:1096)</li><li>m) the clusterin precursor signal sequence (e.g., MMKTLLLFVGLLLTWESGQVLG, SEQ ID NO: 1097)</li><li>n) the insulin-like growth factor-binding protein 4 signal sequence (e.g., MLPLCLVAALLLAAGPGPSLG, SEQ ID NO:1098)</li><li>o) variants of the pre-pro-region of the HSA signal sequence such as, for example, MKWVSFISLLFLFSSAYSRGVFRR (SEQ ID NO:1167), MKWVTFISLLFLFAGVLG (SEQ ID NO:1099), MKWVTFISLLFLFSGVLG (SEQ ID NO:1100), MKWVTFISLLFLFGGVLG (SEQ ID NO:1101), Modified HSA leader HSA #64 MKWVTFISLLFLFAGVSG (SEQ ID NO:2133); Modified HSA leader HSA #66 MKWVTFISLLFLFGGVSG (SEQ ID NO:2134); Modified HSA (A14) leader- MKWVTFISLLFLFAGVSG (SEQ ID NO: 1102); Modified HSA (S14) leader (also known as modified HSA #65) - MKWVTFISLLFLFSGVSG (SEQ ID NO:1103), Modified HSA (G14) leader- MKWVTFISLLFLFGGVSG (SEQ ID NO:1104), or MKWVTFISLLFLFGGVLGDLHKS (SEQ ID NO:1105)</li><li>p) a consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:1055)</li><li>q) acid phosphatase (PH05) leader (e.g., MFKSVVYSILAASLANA SEQ ID NO:2135)</li><li>r) the pre-sequence of MFoz-1</li><li>s) the pre-sequence of 0 glucanase (BGL2)</li><li>t) killer toxin leader</li><li>u) the presequence of killer toxin</li><li>v) k. lactis killer toxin prepro (29 amino acids; 16 amino acids of pre and 13 amino acids of pro) MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQ ID NO:2136)</li><li>w) <i>S. diastaticus</i> glucoarrylase D secretion leader sequence</li><li>x) <i>S. carlsbergensis</i> α-galactosidase (MEL1) secretion leader sequence</li><li>y) <i>Candida glucoamylase</i> leader sequence</li><li>z) The hybrid leaders disclosed in EP-A-387 319 (herin incorporated by reference)</li><li>aa) the gp67 signal sequence (in conjunction with baculoviral expression systems) (e.g., amino acids 1-19 of GenBank Accession Number AAA72759) or</li><li>bb) the natural leader of the therapeutic protein X;</li><li>cc) S. cerevisiae invertase (SUC2) leader, as disclosed in JP 62-096086 (granted as 911036516, herein incorporate by reference); or</li><li>dd) Inulinase - MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO:2137).</li><li>ee) A modified TA57 propeptide leader variant #1 - MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTN SGGLDVVGLISMAKR (SEQ ID NO:2128)</li><li>ff) A modified TA57 propeptide leader variant #2 - MKLKTVRSAVLSSLFASQVLGQPIDDTESQTISVNLMADDTESAFATQTN SGGLDVVGLISMAEEGEPKR (SEQ ID NO:2129)</li></ul>

Additional Methods of Recombinant and Synthetic Production of Albumin Fusion Proteins

0202The present disclose 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.

0203The 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.

0204The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the <i>E</i>. <i>coli lac, trp, phoA</i> and <i>tac</i> promoters, the 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 contain 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.

0205As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in <i>E. coli</i> and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as <i>E</i>. <i>coli,</i> Streptomyces and <i>Salmonella typhimurium</i> cells; fungal cells, such as yeast cells (e.g., <i>Saccharomyces cerevisiae</i> or <i>Pichia pastoris</i> (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.

0206Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 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, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Carlbad, CA). Other suitable vectors will be readily apparent to the skilled artisan.

0207In 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 eukaryotic cell and/or direct the secretion of a protein of the invention from a prokaryotic or eukaryotic cell. For example, in <i>E. coli,</i> 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 albumin fusion proteins of the invention may be fused in order to direct the expression of the polypeptide to the periplasmic space of bacteria include, but are not limited to, the <i>pelB</i> signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the <i>ompA</i> signal sequence, the signal sequence of the periplasmic <i>E. coli</i> 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 <i>pelB</i> pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria. See, <patcit id="pcit0029" dnum="US5576195A"><text>U.S. Patent Nos. 5,576,195</text></patcit> and <patcit id="pcit0030" dnum="US5846818A"><text>5,846,818</text></patcit>.

0208Examples of signal peptides that may be fused to an albumin fusion protein of the invention in order to direct its secretion in mammalian cells includes <ul id="ul0005" list-style="none" compact="compact"><li>a) the MPIF-1 signal sequence (e.g., amino acids 1-21 of GenBank Accession number AAB51134) MKVSVAALSCLMLVTALGSQA (SEQ ID NO:2132)</li><li>b) the stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:1054)</li><li>c) the pre-pro region of the HSA signal sequence (e.g., MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO:1176)</li><li>d) the pre region of the HSA signal sequence (e.g., MKWVTFISLLFLFSSAYS, SEQ ID NO:1177) or variants thereof, such as, for example, MKWVSFISLLFLFSSAYS, (SEQ ID NO:1168)</li><li>e) the invertase signal sequence (e.g., MLLQAFLFLLAGFAAKISA, SEQ ID NO:1108)</li><li>f) the yeast mating factor alpha signal sequence (e.g., MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVL PFSNSTNNGLLFINTTIASIAAKEEGVSLEKR, SEQ ID NO:1109 or MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVL PFSNSTNNGLLFINTTIASIAAKEEGVSLDKR, SEQ ID NO:1109)</li><li>g) <i>K. lactis</i> killer toxin leader sequence</li><li>h) a hybrid signal sequence (e.g., MKWVSFISLLFLFSSAYSRSLEKR, SEQ ID NO:1110)</li><li>i) an HSA/MFα-1 hybrid signal sequence (also known as HSA/kex2) (e.g., MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:1111)</li><li>j) a <i>K. lactis</i> killer/ MFα-1 fusion leader sequence (e.g., MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO:1169)</li><li>k) the Immunoglobulin Ig signal sequence (e.g., MGWSCIILFLVATATGVHS, SEQ ID NO:1095)</li><li>1) the Fibulin B precursor signal sequence (e.g., MERAAPSRRVPLPLLLLGGLALLAAGVDA, SEQ ID NO:1096)</li><li>m) the clusterin precursor signal sequence (e.g., MMKTLLLFVGLLLTWESGQVLG, SEQ ID NO:1097)</li><li>n) the insulin-like growth factor-binding protein 4 signal sequence (e.g., MLPLCLVAALLLAAGPGPSLG, SEQ ID NO:1098)</li><li>o) variants of the pre-pro-region of the HSA signal sequence such as, for example, MKWVSFISLLFLFSSAYSRGVFRR (SEQ ID NO:1167), MKWVTFISLLFLFAGVLG (SEQ ID NO:1099), MKWVTFISLLFLFSGVLG (SEQ ID NO:1100), MKWVTFISLLFLFGGVLG (SEQ ID NO:1101), Modified HSA leader HSA #64 MKWVTFISLLFLFAGVSG (SEQ ID NO:2133); Modified HSA leader HSA #66 MKWVTFISLLFLFGGVSG (SEQ ID NO:2134); Modified HSA (A14) leader- MKWVTFISLLFLFAGVSG (SEQ ID NO: 1102); Modified HSA (S14) leader (also known as modified HSA #65) - MKWVTFISLLFLFSGVSG (SEQ ID NO:1103), Modified HSA (G14) leader - MKWVTFISLLFLFGGVSG (SEQ ID NO:1104), or MKWVTFISLLFLFGGVLGDLHKS (SEQ ID NO:1105)</li><li>p) a consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:1055)</li><li>q) acid phosphatase (PH05) leader (e.g., MFKSVVYSILAASLANA SEQ ID NO:2135)</li><li>r) the pre-sequence of MFoz-1</li><li>s) the pre-sequence of 0 glucanase (BGL2)</li><li>t) killer toxin leader</li><li>u) the presequence of killer toxin</li><li>v) k. lactis killer toxin prepro (29 amino acids; 16 amino acids of pre and 13 amino acids of pro) MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQ ID NO:2136)</li><li>w) <i>S</i>. <i>diastaticus</i> glucoamylase Il secretion leader sequence</li><li>x) <i>S. carlsbergensis</i> α-galactosidase (MEL1) secretion leader sequence</li><li><i>y) Candida glucoarnylase</i> leader sequence</li><li>z) The hybrid leaders disclosed in <patcit id="pcit0031" dnum="EP387319A"><text>EP-A-387 319</text></patcit> (herin incorporated by reference)</li><li>aa) the gp67 signal sequence (in conjunction with baculoviral expression systems) (e.g., amino acids 1-19 of GenBank Accession Number AAA72759) or</li><li>bb) the natural leader of the therapeutic protein X;</li><li>cc) S. cerevisiae invertase (SUC2) leader, as disclosed in <patcit id="pcit0032" dnum="JP62096086A"><text>JP 62-096086</text></patcit> (granted as 911036516, herein incorporate by reference); or</li><li>dd) Inulinase - MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO:2137).</li><li>ee) A modified TA57 propeptide leader variant #1-MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGG LDVVGLISMAKR (SEQ ID NO:2128)</li><li>ff) A modified TA57 propeptide leader variant #2 - MKLKTVRSAVLSSLFASQVLGQPIDTESQTTSVNLMADDTESAFATQTNSG GLDVVGLISMAEEGEPKR (SEQ ID NO:2129)</li></ul>

0209Vectors 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 availability of cell lines (e.g., the 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 <patcit id="pcit0033" dnum="WO8704462A"><text>PCT publications: WO87/04462</text></patcit>; <patcit id="pcit0034" dnum="WO8605807A"><text>WO86/05807</text></patcit>; <patcit id="pcit0035" dnum="WO8901036A"><text>WO89/01036</text></patcit>; <patcit id="pcit0036" dnum="WO8910404A"><text>WO89/10404</text></patcit>; and <patcit id="pcit0037" dnum="WO9106657A"><text>WO91/06657</text></patcit>. 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 <nplcit id="ncit0059" npl-type="s"><text>Bebbington et al., Bio/technology 10:169(1992</text></nplcit>) and in <nplcit id="ncit0060" npl-type="s"><text>Biblia and Robinson Biotechnol. Prog. 11:1 (1995</text></nplcit>).

0210The present disclosure 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 prokatyotic 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 curtain 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.

0211Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as <nplcit id="ncit0061" npl-type="s"><text>Davis et al., Basic Methods In Molecular Biology (1986</text></nplcit>). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.

0212In addition to describing host cells containing the vector constructs discussed herein, the disclosure also describes 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 the Therapeutic protein may be replaced with an albumin fusion protein corresponding to the Therapeutic 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, and/or amplify endogenous polynucleotides.

0213In 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 the Therapeutic protein via homologous recombination (see, e.g., <patcit id="pcit0038" dnum="US5641670A"><text>US Patent Number 5,641,670, issued June 24, 1997</text></patcit>; International Publication Number <patcit id="pcit0039" dnum="WO9629411A"><text>WO 96/29411</text></patcit>; International Publication Number <patcit id="pcit0040" dnum="WO9412650A"><text>WO 94/12650</text></patcit>; <nplcit id="ncit0062" npl-type="s"><text>Koller et al., Proc. Natl. Acad Sci. USA 86:8932-8935 (1989</text></nplcit>); and <nplcit id="ncit0063" npl-type="s"><text>Zijlstra et al., Nature 342:435-438 (1989</text></nplcit>)).

0214Albumin 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 chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.

0215In preferred embodiments the albumin fusion proteins of the invention are purified using Anion Exchange Chromatography including, but not limited to, chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAE, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/Source Q and DEAE, Fractogel Q and DEAE columns.

0216In specific embodiments the albumin fusion proteins of the invention are purified using Cation Exchange Chromatography including, SP-sepharose, CM sepharose, poros HS, poros CM, Toyopearl SP, Toynpearl CM, Resource/Source S and CM, Fractogel S and CM columns and their equivalents and comparables.

0217In 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, Toyopead Phenyl, Butyl, Methyl, Octyl, Hexyl Resource/Source Phenyl, Butyl, Methyl, Octyl, Hexyl, Frectogel Phenyl, Butyl, Methyl, Octyl, Hexyl columns and their equivalents and comparables.

0218In specific embodiments the albumin fusion proteins of the invention are purified using Size Exclusion Chromatography including, but not limited to, sepharose S100, S200, S300, superdex resin columns and their equivalents and comparables.

0219In specific embodiments the albumin fusion proteins of the invention are purified using Affinity Chromatography including, but not limited to, Mimetic Dye affinity, peptide affinity and antibody affinity columns that are-selective for either the HSA or the "fusion target" molecules.

0220In 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.

0221Additionally, albumin fusion proteins of the invention may be purified using the process described in <patcit id="pcit0041" dnum="WO0044772A"><text>PCT International Publication WO 00/44772</text></patcit>. 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.

0222Albumin 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-tenninal 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.

0223In one embodiment, the yeast <i>Pichia pastoris</i> is used to express albumin fusion proteins of the invention in a eukaryotic system. <i>Pichia pastoris</i> 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, <i>Pichia pastoris</i> 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 <i>(AOX1)</i> is highly active. In the presence of methanol, alcohol oxidase produced from the <i>AOX1</i> gene comprises up to approximately 30% of the total soluble protein in <i>Pichia pastoris.</i> See <nplcit id="ncit0064" npl-type="s"><text>Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985</text></nplcit>); <nplcit id="ncit0065" npl-type="s"><text>Koutz, P.J, et al., Yeast 5:167-77 (1989</text></nplcit>); <nplcit id="ncit0066" npl-type="s"><text>Tschopp, J.F., et al., Nuc/. Acids Res. 15:3859-76 (1987</text></nplcit>). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the <i>AOX1</i> regulatory sequence is expressed at exceptionally high levels in <i>Pichia</i> yeast grown in the presence of methanol.

0224In 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 <i>Pichea</i> yeast system essentially as described in <i>"</i><nplcit id="ncit0067" npl-type="b"><text>Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998</text></nplcit>. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong <i>AOX1</i> promoter linked to the <i>Pichia pastoris</i> alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.

0225Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, 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.

0226In 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.

0227In addition, albumin fusion proteins of the invention can be chemically synthesized using techniques known in the art (e.g., see <nplcit id="ncit0068" npl-type="b"><text>Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman &amp; Co., N.Y</text></nplcit>., and <nplcit id="ncit0069" npl-type="s"><text>Hunkapiller et al., Nature, 310:105-111 (1984</text></nplcit>)). 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, citrulline, homocitrulline, cysteic acid, t-butylglycine, 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 (levorotary).

0228The 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, chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

0229Additional 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 label to allow for detection and isolation of the protein.

0230Examples of suitable enzymes includes horseradish peroxidase, alkaline phosphatase, 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 phycocrythrin; 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 (¹¹¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹¹In, ¹¹²In, ^113mIn, ^115mIn), technetium (⁹⁹Tc, ^99mTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr,¹⁰⁵Rh, and ⁹⁷Ru.

0231In 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, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators is ¹¹¹In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is ⁹⁰Y. 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 commonly known in the art - see, for example, <nplcit id="ncit0070" npl-type="s"><text>DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998</text></nplcit>); <nplcit id="ncit0071" npl-type="s"><text>Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999</text></nplcit>); and <nplcit id="ncit0072" npl-type="s"><text>Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999</text></nplcit>).

0232As mentioned, the albumin fusion proteins of the invention may be modified by either natural processes, such as post-transtational 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, phosphotylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, <nplcit id="ncit0073" npl-type="b"><text>PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993</text></nplcit>); <nplcit id="ncit0074" npl-type="b"><text>POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983</text></nplcit>); <nplcit id="ncit0075" npl-type="s"><text>Seifter et al, Meth Enzymol. 182:626-646 (1990</text></nplcit>); <nplcit id="ncit0076" npl-type="s"><text>Rattan et al., Arm. N.Y. Acad. Sci. 663:48-62 (1992</text></nplcit>)).

0233Albumin fusion proteins of the invention and antibodies that bind the Therapeutic protein or fragments or variants thereof can be fused to marker sequences, such as a peptide to facilitate purification. The marker amino acid sequence may be 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 <nplcit id="ncit0077" npl-type="s"><text>Gentz et aL, Proc. Natl. Acad. Sci. USA 86:821-824 (1989</text></nplcit>), 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 (<nplcit id="ncit0078" npl-type="s"><text>Wilson et al., Cell 37:767 (1984</text></nplcit>)) and the "flag" tag.

0234Further, 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 (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).

0235The 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, ß-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. <patcit id="pcit0042" dnum="WO9733899A"><text>WO 97/33899</text></patcit>), AIM II (See, International Publication No. <patcit id="pcit0043" dnum="WO9734911A"><text>WO 97/34911</text></patcit>), Fas Ligand (<nplcit id="ncit0079" npl-type="s"><text>Takahashi et al., Int. Immunol., 6:1567-1574 (1994</text></nplcit>)), VEGI (See, International Publication No. <patcit id="pcit0044" dnum="WO9923105A"><text>WO 99/23105</text></patcit>), 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.

0236Albumin 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.

0237Albumin fusion proteins, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

0238Also 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 <patcit id="pcit0045" dnum="US4179337A"><text>U.S. Patent No. 4,179,337</text></patcit>). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene 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.

0239The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular 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.

0240As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in <patcit id="pcit0046" dnum="US5643575A"><text>U.S. Patent No. 5,643,575</text></patcit>; <nplcit id="ncit0080" npl-type="s"><text>Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996</text></nplcit>); <nplcit id="ncit0081" npl-type="s"><text>Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999</text></nplcit>); and <nplcit id="ncit0082" npl-type="s"><text>Caliceti et al., Bioconjug. Chem. 10:638-646 (1999</text></nplcit>).

0241The 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 <patcit id="pcit0047" dnum="EP0401384A"><text>EP 0 401 384</text></patcit> (coupling PEG to G-CSF); see also <nplcit id="ncit0083" npl-type="s"><text>Malik et al., Exp. Hematol. 20:1028-1035 (1992</text></nplcit>), 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-terminal 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.

0242As 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.

0243One 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 population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification 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.

0244As 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. Linkerless systems for attaching polyethylene glycol to proteins are described in <nplcit id="ncit0084" npl-type="s"><text>Delgado et aL, Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992</text></nplcit>); <nplcit id="ncit0085" npl-type="s"><text>Francis et al., Intern. J. of Hematol. 68:1-18 (1998</text></nplcit>); <patcit id="pcit0048" dnum="US4002531A"><text>U.S. Patent No. 4,002,531</text></patcit>; <patcit id="pcit0049" dnum="US5349052A"><text>U.S. Patent No. 5,349,052</text></patcit>; <patcit id="pcit0050" dnum="WO9506058A"><text>WO 95/06058</text></patcit>; and <patcit id="pcit0051" dnum="WO9832466A"><text>WO 98/32466</text></patcit>.

0245One 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 (ClSO₂CH₂CF₃). Upon reaction of protein with tresylated MPEG, polyethylene glycol is 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.

0246Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, <patcit id="pcit0052" dnum="US5612460A"><text>U.S. Patent No. 5,612,460</text></patcit> 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. <patcit id="pcit0053" dnum="WO9832466A"><text>WO 98/32466</text></patcit>. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.

0247The number of polyethylene glycol moieties attached to each albumin fusion protein of the 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, 14-16, 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 <nplcit id="ncit0086" npl-type="s"><text>Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992</text></nplcit>).

0248The 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, phosphocellulose 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.

0249The 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 ELISA 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 more 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.

<i>Uses of the Polynucleotides</i>

0250Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

0251The polynucleotides of the present invention are useful to produce the albumin fusion proteins of the invention. As described in more detail below, polynucleotides 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.

0252Polynucleotides 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 genome, 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 63 and 64).

<i>Uses of the Polypeptides</i>

0253Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.

0254Moreover, 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, reproductive disorders, gastrointestinal disorders, cardiovascular disorders and/or renal disorders. 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, catalyse, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor suppressor), 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).

0255In 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 <i>supra,</i> 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 specifically binds, and/or reduce overproduction of the polypeptide to which the Therapeutic antibody used to make the albumin fusion protein specifically 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 specifically binds, by binding to the polypeptide bound to a membrane (receptor).

0256At 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, albumin 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.

<i>Diagnostic Assays</i>

0257The 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 the Therapeutic protein in the corresponding row of Table 1 and herein under the section headings "Immune Activity," "Renal Disorders," "Cardiovascular Disorders," "Anti-Angiogenesis 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," <i>infra</i>.

0258For 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, described is 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 level(s) compared to the standard is indicative of a disorder. These diagnostic assays may be performed in <i>vivo</i> or in <i>vitro,</i> such as, for example, on blood samples, biopsy tissue or autopsy tissue.

0259The present disclosure is also useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome

0260By "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 the 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 mRNA 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.

0261By "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.

<i>Pharmaceutical or Therapeutic Compositions</i>

0262The albumin fusion proteins of the invention or formulations thereof may be administered by any conventional method including parenteral (<i>e.g.</i> 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.

0263While 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 carrier(s) 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.

0264For example, 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 discussed above, the shelf-life of the Therapeutic protein is markedly increased or prolonged after fusion to HA.

0265In 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 <nplcit id="ncit0087" npl-type="b"><text>Ganderton &amp; Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987</text></nplcit>); <nplcit id="ncit0088" npl-type="s"><text>Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313</text></nplcit>; and <nplcit id="ncit0089" npl-type="s"><text>Raeburn et al,. (1992) Pharmacol. Toxicol. Methods 27:143-159</text></nplcit>.

0266The 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.

0267The 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 carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

0268Formulations 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 suspension which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-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.

0269Formulations 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.

0270Albumin 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 expresses the albumin fusion protein. The albumin 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 <nplcit id="ncit0090" npl-type="s"><text>Scott Hegenhart, Food Product Design, Dec. 1993</text></nplcit>.

0271The albumin fusion protein and/or 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.

0272As 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/kg/day 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 I mg/kg/day for the hormone. If given continuously, the albumin fusion protein is typically administered at a dose rate of about 1 ug/kg/hour 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 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.

0273Albumin fusion proteins and/or polynucleotides can be 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.

0274Albumin 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 (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).

0275Sustained-release matrices include polylactides (<patcit id="pcit0054" dnum="US3773919A"><text>U.S. Pat. No. 3,773,919</text></patcit>, <patcit id="pcit0055" dnum="EP58481A"><text>EP 58,481</text></patcit>), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (<nplcit id="ncit0091" npl-type="s"><text>Sidman et al., Biopolymers 22:547-556 (1983</text></nplcit>)), poly (2- hydroxyethyl methacrylate) (<nplcit id="ncit0092" npl-type="s"><text>Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981</text></nplcit>), and <nplcit id="ncit0093" npl-type="s"><text>Langer, Chem. Tech. 12:98-105 (1982</text></nplcit>)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (<patcit id="pcit0056" dnum="EP133988A"><text>EP 133,988</text></patcit>).

0276Sustained-release albumin fusion proteins and/or polynucleotides also include liposomally entrapped albumin fusion proteins and/or polynucleotides of the invention (<i>see</i> generally, <nplcit id="ncit0094" npl-type="s"><text>Langer, Science 249:1527-1533 (1990</text></nplcit>); <nplcit id="ncit0095" npl-type="b"><text>Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317 - 327 and 353-365 (1989</text></nplcit>)). Liposomes containing the albumin fusion protein and/or polynucleotide are prepared by methods known per se: <patcit id="pcit0057" dnum="DE3218121"><text>DE 3,218,121</text></patcit>; <nplcit id="ncit0096" npl-type="s"><text>Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985</text></nplcit>); <nplcit id="ncit0097" npl-type="s"><text>Hwang et al., Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980</text></nplcit>); <patcit id="pcit0058" dnum="EP52322A"><text>EP 52,322</text></patcit>; <patcit id="pcit0059" dnum="EP36676A"><text>EP 36,676</text></patcit>; <patcit id="pcit0060" dnum="EP88046A"><text>EP 88,046</text></patcit>; <patcit id="pcit0061" dnum="EP143949A"><text>EP 143,949</text></patcit>; <patcit id="pcit0062" dnum="EP142641A"><text>EP 142,641</text></patcit>; Japanese Pat. AppL <patcit id="pcit0063" dnum="JP58118008A"><text>83-118008</text></patcit>; <patcit id="pcit0064" dnum="US4485045A"><text>U.S. Pat. Nos. 4,485,045</text></patcit> and <patcit id="pcit0065" dnum="US4544545A"><text>4,544,545</text></patcit>; and <patcit id="pcit0066" dnum="EP102324A"><text>EP 102,324</text></patcit>. Ordinarily, the liposomes 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.

0277The albumin fusion proteins and/or polynucleotides of the invention may be delivered by way of a pump (see Langer, <i>supra</i>; <nplcit id="ncit0098" npl-type="s"><text>Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987</text></nplcit>); <nplcit id="ncit0099" npl-type="s"><text>Buchwald et al., Surgery 88:507 (1980</text></nplcit>); <nplcit id="ncit0100" npl-type="s"><text>Saudek et al., N. Engl. J. Med. 321:574 (1989</text></nplcit>)).

0278Other controlled release systems are discussed in the review by <nplcit id="ncit0101" npl-type="s"><text>Langer (Science 249:1527-1533 (1990</text></nplcit>)).

0279For parenteral administration, the albumin fusion protein and/or polynucleotide may be 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.

0280Generally, 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.

0281The 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 mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

0282The 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 in the formation of polypeptide salts.

0283Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration 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.

0284Albumin 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.

0285In 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 octanoate/milligram 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 micromole sodium octanoate/milligram of fusion protein, 15 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 non-specific adsorption of the albumin fusion protein to the container closure system.

0286Also disclosed is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the albumin fusion proteins and/or polynucleotides of the invention. Associated with such container(s) 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.

0287The 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, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG (e.g., THERACYS®), MPL and nonviable preparations of <i>Corynebacterium parvum.</i> Albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with alum. Albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with QS-21. Further adjuvants that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Viresomal adjuvant technology. Vaccines that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, <i>Haemophilus influenzae</i> B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. 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 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.

0288The 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 polynucleotide agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include but not limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional 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 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.

0289The albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with an anticoagulant. Anticoagulants that may be 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., MIRADON™), acenocoumarol (e.g., nicoumalone, SINTHROME™), indan-1,3-dione, phenprocoumon (e.g., MARCUMAR™), ethyl biscoumacetate (e.g., TROMEXAN™), and aspirin. Compositions of the invention may be administered in combination with heparin and/or warfarin. Compositions of the invention may be administered in combination with warfarin. Compositions of the invention may be administered in combination with warfarin and aspirin. Compositions of the invention may be administered in combination with heparin. Compositions of the invention may be administered in combination with heparin and aspirin.

0290The albumin fusion proteins and/or polynucleotides of the invention may be 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., KABIKINASE™), antiresplace (e.g., EMINASE™), tissue plasminogen activator (t-PA, altevase, ACTIVASE™), urokinase (e.g., ABBOKINASE™), sauruplase, (Prourokinase, single chain urokinase), and aminocaproic acid (e.g., AMICAR™). Compositions of the invention may be administered in combination with tissue plasminogen activator and aspirin.

0291The albumin fusion proteins and/or polynucleotides of the invention may be 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., PERSANTINE™), and ticlopidine (e.g., TICLID™).

0292The use of anti-coagulants, thrombolytic and/or antiplatelet drugs in combination with albumin fusion proteins and/or polynucleotides of the invention may be contemplated for the prevention, diagnosis, and/or treatment of thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. The use of anticoagulants, thrombolytic drugs and/or antiplatelet drugs in combination with albumin fusion proteins and/or polynucleotides of the invention may be 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 atrial 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).

0293Albumin fusion proteins and/or polynucleotides of the invention may be 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, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERTT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). NNRTIs that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, VIRAMUNE™ (nevirapine), RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitors that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir). 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.

0294Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stable adenosine NRTI; Triangle/Abbott; COVIRACIL™ (emtricitabine/FTC; structurally related to lamivudine (3TC) but with 3- to 10-fold greater activity in <i>vitro</i>; 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); ZIAGEN™ (abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3'azido-2',3'-dideoxyuridine; <patcit id="pcit0067" dnum="WO9966936A"><text>WO 99/66936</text></patcit>); and S-acyl-2-thioethyl (SATE)-bearing prodrug forms of β-L-FD4C and β-L-FddC (<patcit id="pcit0068" dnum="WO9817281A"><text>WO 98/17281</text></patcit>).

0295Additional NNRTIs include COACTINON™ (Emivirine/MKC-442, potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINE™ (AG-15491/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 &amp; 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 (<patcit id="pcit0069" dnum="WO9949830A"><text>WO 99/49830</text></patcit>).

0296Additional protease inhibitors include LOPINAVIR™ (ABT378/r; Abbott Laboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb); TIPRANAVIR™ (PNU-140690, a non-peptic dihydropyrone; Pharmacia &amp; Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinavir analog; Merck); DMP-450 (a cyclic urea compound; Avid &amp; DuPont); AG-1776 (a peptidomimetic with <i>in vitro</i> activity against protease inhibitor-resistant viruses; Agouron); VX-175/GW-433908 (phosphate prodrug of amprenavir; Vertex &amp; Glaxo Welcome); CGP61755 (Ciba); and AGENERASE™ (amprenavir; Glaxo Wellcome Inc.).

0297Additional antiretroviral agents include fusion inhibitors/gp41 binders. Fusion inhibitors/gp41 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).

0298Additional 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 ALX40-4C (a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonists such as RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetor agonists such as RANTES, SDF-1, MIP-1α, MIP-1β, etc., may also inhibit fusion.

0299Additional antiretroviral agents include integrase inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and related anthraquinones; ZINTEVIR™ (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 <patcit id="pcit0070" dnum="WO9850347A"><text>WO 98/50347</text></patcit>.

0300Additional antiretroviral agents include hydroxyurea-like compunds such as BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide reductase inhibitors such as DIDOX™ (Molecules for Health); inosine monophosphate dehydrogenase (IMPDH) inhibitors sucha as VX-497 (Vertex); and mycopholic acids such as CellCept (mycophenolate mofetil; Roche).

0301Additional antiretroviral agents include inhibitors of viral integrase, inhibitors of viral 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 glycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc finger inhibitors such as dithiane compounds; targets of HIV Tat and Rev; and pharmacoenhancers such as ABT-378.

0302Other antiretroviral therapies and adjunct therapies include cytokines and lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKIN™ (aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12, and IL-13; interferons such as IFN-alpha2a, IFN-alpha2b, or IFN-beta; antagonists of TNFs, NFκB, GM-CSF, M-CSF, and IL-10; agents that modulate immune activation such as cyclosporin and prednisone; vaccines such as Remune™ (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; <patcit id="pcit0071" dnum="WO9854366A"><text>WO 98/54366</text></patcit>), and intrakines (genetically modified CC chemokines targetted to the ER to block surface expression of newly synthesized CCR5 (<nplcit id="ncit0102" npl-type="s"><text>Yang et al., PNAS 94:11567-72 (1997</text></nplcit>); <nplcit id="ncit0103" npl-type="s"><text>Chen et al., Nat. Med. 3:1110-16 (1997</text></nplcit>)); antibodies such as the anti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-α 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 α-naphthoflavone (<patcit id="pcit0072" dnum="WO9830213A"><text>WO 98/30213</text></patcit>); and antioxidants such as γ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; <patcit id="pcit0073" dnum="WO9956764A"><text>WO 99/56764</text></patcit>).

0303The albumin fusion proteins and/or polynucleotides of the invention may be 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, acyclovir, ribavirin, amantadine, remantidine, maxamine, or thymalfasin.

0304Albumin 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, TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™, ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN, CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™, FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™, PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastin/G-CSF), and LEUKINE™ (sargramostim/GM-CSF). Albumin fusion proteins and/or polynucleotides of the invention may be used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/or ATOVAQUONE™ to prophylactically treat or prevent an opportunistic <i>Pneumocystis carinii</i> pneumonia infection. Albumin fusion proteins and/or polynucleotides of the invention may be used in any combination with ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat or prevent an opportunistic <i>Mycobacterium avium</i> complex infection. Albumin fusion proteins and/or polynucleotides of the invention may be used in any combination with RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat or prevent an opportunistic <i>Mycobacterium tuberculosis</i> infection. Albumin fusion proteins and/or polynucleotides of the invention may be used in any combination with GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat or prevent an opportunistic cytomegalovirus inflection. Albumin fusion proteins and/or polynucleotides of the invention may be used in any combination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylactically treat or prevent an opportunistic fungal infection. Albumin fusion proteins and/or polynucleotides of the invention may be used in any combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. Albumin fusion proteins and/or polynucleotides of the invention may be used in any combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylactically treat or prevent an opportunistic <i>Toxoplasma gandii</i> infection. Albumin fusion proteins and/or polynucleotides of the invention may be used in any combination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent an opportunistic bacterial infection.

0305The albumin fusion proteins and/or polynucleotides of the invention may be 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, amoxicillin, beta-lactamases, aminoglycosides, betalactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

0306The albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with immunestimulants. Immunostimulants that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, levamisole (e.g., ERGAMISOL™), isoprinosine (e.g. INOSIPLEX™), interferons (e.g. interferon alpha), and interleukins (e.g., IL-2).

0307Albumin fusion proteins and/or polynucleotides of the invention may be 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, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, 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, prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine (BREDININ™), broquinar, deoxyspergualin, and azaspirane (SKF 105685), ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNE™, NEORAL™, SANGDYA™ (cyclosporine), PROGRAF® (FK506, tacrolimus), CELLCEPT® (mycophenolate motefil, of which the active metabolite is mycophenolic acid), IMURAN™ (azathioprine), glucocorticosteroids, adrenocortical steroids such as DELTASONE™ (prednisone) and HYDELTRASOL™ (prednisolone), FOLEX™ and MEXATE™ (methotrxate), OXSORALEN-ULTRA™ (methoxsalen) and RAPAMUNE™ (sirolimus). Immunosuppressants maybe used to prevent rejection of organ or bone marrow transplantation.

0308Albumin fusion proteins and/or polynucleotides of the invention may be administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but not limited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, ATGAM™ (antithymocyte glubulin), and GAMIMUNE™. Albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).

0309The albumin fusion proteins and/or polynucleotides of the invention may be administered alone or as part of a combination therapy, either in vivo to patients or in vitro to cells, for the treament of cancer. The albumin fusion proteins may be administered repeatedly during passive immunotherapy for cancer, such as adoptive cell transfer therapy for metastatic melanoma as described in <nplcit id="ncit0104" npl-type="s" url="www.scienceexpress.org"><text>Dudley et al. (Science Express, 19 September 2002, at www.scienceexpress.org</text></nplcit>).

0310The albumin fusion proteins and/or polynucleotides of the invention may be administered alone or in combination with an anti-inflammatory agent Anti-inflammatory agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, corticosteroids (e.g. betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs (e.g., diclofenac, diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, and tolmetin.), as well as antihistamines, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, 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.

0311The compositions of the invention may be 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 and 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.

0312Lighter "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.

0313Representative examples of vanadium complexes include oxo vanadium complexes 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.

0314Representative 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 dihydrate, 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 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.

0315A wide variety of other anti-angiogenic factors may also be utilized. Representative examples include, platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (<nplcit id="ncit0105" npl-type="s"><text>Murata et al., Cancer Res. 51:22-26, (1991</text></nplcit>)); 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,apha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (<nplcit id="ncit0106" npl-type="s"><text>Pavloff et al., J. Bio. Chem. 267:17321-17326, (1992</text></nplcit>)); Chymostatin (<nplcit id="ncit0107" npl-type="s"><text>Tomkinson et al., Biochem J. 286:475-480, (1992</text></nplcit>)); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (<nplcit id="ncit0108" npl-type="s"><text>Ingber et aL, Nature 348:555-557, (1990</text></nplcit>)); Gold Sodium Thiomalate ("GST"; <nplcit id="ncit0109" npl-type="s"><text>Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987</text></nplcit>)); anticollagenase-serum; alpha2-antiplasmin (<nplcit id="ncit0110" npl-type="s"><text>Holmes et al., J. Biol. Chem. 262(4):1659-1664, (1987</text></nplcit>)); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4- chloroanthronilic acid disodium or "CCA"; (<nplcit id="ncit0111" npl-type="s"><text>Takeuchi et al., Agents Actions 36:312-316, (1992</text></nplcit>)); and metalloproteinase inhibitors such as BB94.

0316Additional anti-angiogenic factors that may also be utilized include Thalidomide, (Celgene, Warren, NJ); Angiostatic steroid; AGM-1470 (<nplcit id="ncit0112" npl-type="s"><text>H. Brem and J. Folkman J Pediatr. Surg. 28:445-51 (1993</text></nplcit>)); an integrin alpha v beta 3 antagonist (<nplcit id="ncit0113" npl-type="s"><text>C. Storgard et al., J Clin. Invest. 103:47-54 (1999</text></nplcit>)); carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National Cancer Institute, Bethesda, MD); Conbretastatin A-4 (CA4P) (OXiGENE, Boston, MA); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, PA); TNP-470, (Tap Pharmaceuticals, Deerfield, IL); ZD-0101 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 5-Fluorouracil.

0317Anti-angiogenic agents that may be administed in combination with the compounds of the invention may work through a variety of mechanisms including, inhibiting proteolysis of the extracellular 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 invention include, AG-3340 (Agouron, La Jolla, CA), BAY-12-9566 (Bayer, West Haven, CT), BMS-275291 (Bristol Myers Squibb, Princeton, NJ), CGS-27032A (Novartis, East Hanover, NJ), Marimastat (British Biotech, Oxford, UK), and Metastat (Aetema, 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, EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, CA/Medimmune, 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, Angiozyme (Ribozyme, Boulder, CO), Anti-VEGF antibody (Genentech, S. San Francisco, CA), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. San Francisco, CA), SU-5416 (Sugen/ 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 compositons of the invention include, IM-862 (Cytran, Kirkland, WA), Interferon-alpha, IL-12 (Roche, Nutley, NJ), and Pentosan polysulfate (Georgetown University, Washington, DC).

0318The use of compositions of the invention in combination with anti-angiogenic agents may be contemplated for treating, preventing, and/or ameliorating an autoimmune disease, such as for example, an autoimmune disease described herein.

0319The use of compositions of the invention in combination with anti-angiogenic agents may be contemplated for treating, preventing, and/or ameliorating arthritis. The use of compositions of the invention in combination with anti-angiogenic agents may be contemplated for treating, preventing, and/or ameliorating rheumatoid arthritis.

0320The polynucleotides encoding a polypeptide of the present invention may be 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, 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.

0321Compositions of the invention may be administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, 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 (VLB, vinblastine sulfate)) and Vincristine (vincristine sulfate)), epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics (for example, Dactinomycin (actinomycin 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), adrenocorticosteroids (for example, Prednisone), progestins (for example, Hydroxyprogesterone caproate, Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol acetate), estrogens (for example, Diethylstilbestrol (DES), Diethylstilbestrol 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).

0322The compositions of the invention may be administered in combination with one or more of the following drugs: infliximab (also known as Remicade™ Centocor, Inc.), Trocade (Roche, RO-32-3555), Leflunomide (also known as Arava™ from Hoechst Marion Roussel), Kineret™ (an IL-1 Receptor antagonist also known as Anakinra from, Amgen, Inc.)

0323Compositions of the invention may be administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or combination of one or more of the components of CHOP. The compositions of the invention may be administered in combination with anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. The compositions of the invention may be 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. Compositions of the invention may be administered in combination with Rituximab. Compositions of the invention may be administered with Rituximab and CHOP, or Rituximab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. Compositions of the invention may be administered in combination with tositumomab. Compositions of the invention may be administered with tositumomab and CHOP, or tosittunomab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic produgs.

0324The compositions of the invention may be administered in combination Zevalin™. Compositions of the invention may be administered with Zevalin™ and CHOP, or Zevalin™ and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. Zevalin™ may be associated with one or more radisotopes. Isotopes may be ⁹⁰Y and ¹¹¹In_.

0325The albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with cytokines. Cytokines that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. Albumin fusion proteins and/or polynucleotides of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2; IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16; 1L-17, IL-18, IL-19, IL-20, and IL-21.

0326The albumin fusion proteins and/or polynucleotides of the invention may be 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, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. <patcit id="pcit0074" dnum="WO9614328A"><text>WO 96/14328</text></patcit>), AIM-I (International Publication No. <patcit id="pcit0075" dnum="WO9733899A"><text>WO 97/33899</text></patcit>), endokine-alpha (International Publication No. <patcit id="pcit0076" dnum="WO9807880A"><text>WO 98/07880</text></patcit>), OPG, and neutroline-alpha (International Publication No. <patcit id="pcit0077" dnum="WO9818921A"><text>WO 98/18921</text></patcit>, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. <patcit id="pcit0078" dnum="WO9634095A"><text>WO 96/34095</text></patcit>), DR3 (International Publication No. <patcit id="pcit0079" dnum="WO9733904A"><text>WO 97/33904</text></patcit>), DR4 (International Publication No. <patcit id="pcit0080" dnum="WO9832856A"><text>WO 98/32856</text></patcit>), TR5 (International Publication No. <patcit id="pcit0081" dnum="WO9830693A"><text>WO 98/30693</text></patcit>), TRANK, TR9 (International Publication No. <patcit id="pcit0082" dnum="WO9856892A"><text>WO 98/56892</text></patcit>),TR10 (International Publication No. <patcit id="pcit0083" dnum="WO9854202A"><text>WO 98/54202</text></patcit>), 312C2 (International Publication No. <patcit id="pcit0084" dnum="WO9806842A"><text>WO 98/06842</text></patcit>), and TR12_, and soluble forms CD154, CD70, and CD153.

0327The albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number <patcit id="pcit0085" dnum="EP399816A"><text>EP-399816</text></patcit>; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number <patcit id="pcit0086" dnum="EP682110A"><text>EP-682110</text></patcit>; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number <patcit id="pcit0087" dnum="EP282317A"><text>EP-282317</text></patcit>; Placental Growth Factor (PICF), as disclosed in International Publication Number <patcit id="pcit0088" dnum="WO9206194A"><text>WO 92/06194</text></patcit>; Placental Growth Factor-2 (PIGF-2), as disclosed in <nplcit id="ncit0114" npl-type="s"><text>Hauser et aL, Growth Factors, 4:259-268 (1993</text></nplcit>); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number <patcit id="pcit0089" dnum="WO9013649A"><text>WO 90/13649</text></patcit>; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number <patcit id="pcit0090" dnum="EP506477A"><text>EP-506477</text></patcit>; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number <patcit id="pcit0091" dnum="WO9639515A"><text>WO 96/39515</text></patcit>; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International Publication Number <patcit id="pcit0092" dnum="WO9626736A"><text>WO 96/26736</text></patcit>; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number <patcit id="pcit0093" dnum="WO9802543A"><text>WO 98/02543</text></patcit>; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number <patcit id="pcit0094" dnum="WO9807832A"><text>WO 98/07832</text></patcit>; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number <patcit id="pcit0095" dnum="DE19639601"><text>DE19639601</text></patcit>.

0328The albumin fusion proteins and/or polynucleotides of the invention may be 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, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8. FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

0329The albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostim, LEUKINE™, PROKINE™), granulocyte colony stimulating factor (G-CSF) (filgrastim, NEUPOGEN™), macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin alfa, EPOGEN™, PROCRIT™), stem cell factor (SCF, c-kit ligand, 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.

0330Albumin fusion proteins and/or polynucleotides of the present invention may be administered in combination with adrenergic blockers, such as, for example, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol<b>,</b> metoprolol, nadolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, and timolol.

0331The albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with an antiarrhythmic drug (e.g., adenosine, amidoarone, bretyrlium, digitalis, digoxin, digitoxin, diliazem, disopyramide, esmolol flecainide, lidocaine, mexiletine, moricizine, phenytoin, procainamide, N-acetyl procainamide, propafenone, propranolol, quinidine, sotalol, tocainide, and verapamil).

0332The albumin fusion proteins and/or polynucleotides of the invention may be 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⁺-2Cl^- symport (e.g., furosemide, bumetanide, azosemide, piretanide, tripamide, ethacrynic acid, muzolimine, and torsemide), thiazide and thiazide-like diuretics (e.g., bendroflumethiazide, 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).

0333The albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with treatments for endocrine and/or hormone imbalance disorders. Treatments for endocrine and/or hormone imbalance disorders include, ¹²⁷I, radioactive isotopes of iodine such as ¹³¹I and ¹²³I; recombinant growth hormone, such as HUMATROPE™ (recombinant somatropin); growth hormone analogs such as PROTROPIN™ (somatrem); dopamine agonists such as PARLODEL™ (bromocriptine); somatostatin analogs such as SANDOSTATIN™ (octreotide); gonadotropin preparations such as PREGNYL™, A.P.L.™ and PROFASI™(chorionic gonadotropin (CG)), PERGONAL™ (menotropins), and METRODIN™ (urofollitropin (uFSH)); synthetic human gonadotropin releasing hormone preparations such as FACTREL™ and LUTREPULSE™ (gonadorelin hydrochloride); synthetic gonadotropin agonists such as LUPRON™ (leuprolide acetate), SUPPRELIN™ (histrelin acetate), SYNAREL™ (nafarelin acetate), and ZOLADEX™ (goserelin acetate); synthetic preparations of thyrotropin-releasing hormone such as RELEFACT TRH™ and THYPINONE™ (protirelin); recombinant human TSH such as THYROGEN™; synthetic preparations of the sodium salts of the natural isomers of thyroid hormones such as L-T₄™, SYNTHROID™ and LEVOTHROID™ (levothyroxine sodium), L-T₃™, CYTOMEL™ and TRIOSTAT™ (liothyroine sodium), and THYROLAR™ (liotrix); antithyroid compounds such as 6-<i>n</i>-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazole and TAPAZOLE™ (methimazole), NEO-MERCAZOLE™ (carbimazole); beta-adrenergic receptor antagonists such as propranolol and esmolol; Ca²⁺ channel blockers; dexamethasone and iodinated radiological contrast agents such as TELEPAQUE™ (iopanoic acid) and ORAGRAFIN™ (sodium ipodate).

0334Additional treatments for endocrine and/or hormone imbalance disorders include, estrogens or congugated estrogens such as ESTRACE™ (estradiol), ESTINYL™ (ethinyl estradiol), PREMARIN™, ESTRATAB™, ORTHO-EST™, OGEN™ and estropipate (estrone), ESTROVIS™ (quinestrol), ESTRADERM™ (estradiol), DELESTROGEN™ and VALERGEN™ (estradiol valerate), DEPO-ESTRADIOL CYPIONATE™ and ESTROJECT LA™ (estradiol cypionate); antiestrogens such as NOLVADEX™ (tamoxifen), SEROPHENE™ and CLOMID™ (clomiphene); progestins such as DURALUTIN™ (hydroxyprogesterone caproate), MPA™ and DEPO-PROVERA™ (medroxyprogesterone acetate), PROVERA™ and CYCRIN™ (MPA), MEGACE™ (megestrol acetate), NORLUTIN™ (norethindrone), and NORLUTATE™ and AYGESTIN™ (norethindrone acetate); progesterone implants such as NORPLANT SYSTEM™ (subdermal implants of norgestrel); antiprogestins such as RU 486™ (mifepristone); hormonal contraceptives such as ENOVID™ (norethynodrel plus mestranol), PROGESTASERT™ (intrauterine device that releases progesterone), LOESTRIN™, BREVICON™, MODICON™, GENORA™, NELONA™, NORINYL™, OVACON-35™ and OVACON-50™ (ethinyl estradio/norethindrone), LEVLEN™, NORDETTE™, TRI-LEVLEN™ and TRIPHASIL-21™ (ethinyl estradiol/levonorgestrel) LO/OVRAL™ and OVRAL™ (ethinyl estradiol/norgestrel), DEMULEN™ (ethinyl estradiol/ethynodiol diacetate), NORINYL™, ORTHO-NOVUM™, NORETHIN™, GENORA™, and NELOVA™ (norethindrone/mestranol), DESOGEN™ and ORTHO-CEPT™ (ethinyl estradiol/desogestrel), ORTHO-CYCLEN™ and ORTHO-TRICYCLEN™ (ethinyl estradiol/norgestimate), MICRONOR™ and NOR-QD™ (norethindrone), and OVRETTE™ (norgestrel).

0335Additional treatments for endocrine and/or hormone imbalance disorders include, testosterone esters such as methenolone acetate and testosterone undecanoate; parenteral and oral androgens such as TESTOJECT-50™ (testosterone), TESTEX™ (testosterone propionate), DELATESTRYL™ (testosterone enanthate), DEPO-TESTOSTERONE™ (testosterone cypionate), DANOCRINE™ (danazol), HALOTESTIN™ (fluoxymesterone), ORETON METHYL™, TESTRED™ and VIRILON™ (methyltestosterone), and OXANDRIN™ (oxandrolone); testosterone transdermal systems such as TESTODERM™; androgen receptor antagonist and 5-alpha-reductase inhibitors such as ANDROCUR™ (cyproterone acetate), EULEXIN™ (flutamide), and PROSCAR™ (finasteride); adrenocorticotropic hormone preparations such as CORTROSYN™ (cosyntropin); adrenocortical steroids and their synthetic analogs such as ACLOVATE™ (alclometasone dipropionate), CYCLOCORT™ (anicinonide), BECLOVENT™ and VANCERIL™ (beclomethasone dipropionate), CELESTONE™ (betamethasone), BENISONE™ and UTICORT™ (betamethasone benzoate), DIPROSONE™ (betamethasone dipropionate), CELESTONE PHOSPHATE™ (betamethasone sodium phosphate), CELESTONE SOLUSPAN™ (betamethasone sodium phosphate and acetate), BETA-VAL™ and VALISONE™ (betamethasone valerate), TEMOVATE™ (clobetasol propionate), CLODERM™ (clocortolone pivalate), CORTEF™ and HYDROCORTONE™ (cortisol (hydrocortisone)), HYDROCORTONE ACETATE™ (cortisol (hydrocortisone) acetate), LOCOID™ (cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATE™ (cortisol (hydrocortisone) sodium phosphate), A-HYDROCORT™ and SOLU CORTEF™ (cortisol (hydrocortisone) sodium succinate), WESTCORT™ (cortisol (hydrocortisone) valerate), CORTISONE ACETATE™ (cortisone acetate), DESOWEN™ and TRIDESILON™ (desonide), TOPICORT™ (desoximetasone), DECADRON™ (dexamethasone), DECADRON LA™ (dexamethasone acetate), DECADRON PHOSPHATE™ and HEXADROL PHOSPHATE™ (dexamethasone sodium phosphate), FLORONE™ and MAXIFLOR™ (diflorasone diaectate), FLORINEF ACETATE™ (fludrocortisone acetate), AEROBID™ and NASALlDE™ (flunisolide), FLUONID™ and SYNALAR™ (fluocinolone acetonide), LIDEX™ (fluocinonide), FLUOR-OP™ and FML™ (fluommetholone), CORDRAN™ (flurandrenolide), HALOG™ (halcinonide), HMS LIZUIFILM™ (medrysone), MEDROL™ (methylprednisolone), DEPO-MEDROL™ and MEDROL ACETATE™ (methylprednisone acetate), A-METHAPRED™ and SOLUMEDROL™ (methylprednisolone sodium succinate), ELOCON™ (mometasone furoate), HALDRONE™ (paramethasone acetate), DELTA-CORTEF™ (prednisolone), ECONOPRED™ (prednisolone acetate), HYDELTRASOL™ (prednisolone sodium phosphate), HYDELTRA-T.B.A™ (prednisolone tebutate), DELTASONE™ (prednisone), ARISTOCORT™ and KENACORT™ (triamcinolone), KENALOG™ (triamcinolone acetomide), ARISTOCORT™ and KENACORT DIACETATE™ (triamcinolone diacetate), and ARISTOSPAN™ (triamcinolone hexacetonide); inhibitors of biosynthesis and action of adrenocortical steroids such as CYTADREN™ (aminoglutethimide), NIZORAL™ (ketoconazole), MODRASTANE™ (trilostane), and METOPIRONE™ (metyrapone); bovine, porcine or human insulin or mixtures thereof; insulin analogs; recombinant human insulin such as HUMULIN™ and NOVOLIN™; oral hypoglycemic agents such as ORAMIDE™ and ORINASE™ (tolbutamide), DIABINESE™ (chlorpropamide), TOLAMIDE™ and TOLINASE™ (tolazamide), DYMELOR™ (acetohexamide), glibenclamide, MICRONASE™, DIBETA™ and GLYNASE™ (glyburide), GLUCOTROL™ (glipizide), and DIAMICRON™ (gliclazide), GLUCOPHAGE™ (metformin), ciglitazone, pioglitazone, and alpha-glucosidase inhibitors; bovine or porcine glucagon; somatostatins such as SANDOSTATIN™ (octreotide); and diazoxides such as PROGLYCEM™ (diazoxide).

0336The albumin fusion proteins and/or polynucleotides of the invention may be administered in combination- with treatments for uterine motility disorders. Treatments for uterine motility disorders include, estrogen drugs such as conjugated estrogens (e.g., PREMARIN^® and ESTRATAB^®), estradiols (e.g., CLIMARA^® and ALORA^®), estropipate, and chlorotrianisene; progestin drugs (e.g., AMEN^® (medroxyprogesterone), MICRONOR^® (norethidrone acetate), PROMETRIUM^® progesterone, and megestrol acetate); and estrogen/progesterone combination therapies such as, for example, conjugated estrogens/medroxyprogesterone (e.g., PREMPRO^™ and PREMPHASE^®) and norethindrone acetate/ethinyl estsradiol (e.g., FEMHRT™).

0337The albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with drugs effective in treating iron deficiency and hypochromic anemias, including but not limited to, ferrous sulfate (iron sulfate, FEOSOL™)<b>,</b> ferrous fumarate (e.g., FEOSTAT^™), ferrous gluconate (e.g., FERGON™), polysaccharide-iron complex (e.g.. NIFEREX™), iron dextran injection (e.g., INFED™), cupric sulfate, pyroxidine, riboflavin, Vitamin B₁₂, cyancobalamin injection (e.g., REDISOL™, RUBRAMIN PC™), hydroxocobalamin, folic acid (e.g., FOLVITE™), leucovorin (folinic acid, 5-CHOH4PteGlu, citrovorum factor) or WELLCOVORIN (Calcium salt of leucovorin), transferrin or ferritin.

0338The albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with agents used to treat psychiatric disorders. Psychiatric drugs that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, 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, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, settraline, 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).

0339The albumin fusion proteins and/or polynucleotides of the invention may be 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, antiepileptic agents (e.g., carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, primidone, valproic acid, divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, 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).

0340Albumin fusion proteins and/or polynucleotides of the invention may: be administered in combination with vasodilating agents and/or calcium channel blocking agents. Vasodilating agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, Angiotensin Converting Enzyme (ACE) inhibitors (e.g., papaverine, isoxsuprine, benazepril, captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril quinapril, ramipril. spirapril, trandolapril, and nylidrin), and nitrates (e.g., isosorbide dinitrate, isosorbide mononitrate, and nitruglycerin). Examples of calcium channel blocking agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to amlodipine, bepridil, diltiazem, felodipine, flumarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil.

0341The albumin fusion proteins and/or polynucleotides of the invention may be 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, H₂ histamine receptor antagonists (e.g., TAGAMET™ (cimetidine), ZANTAC™ (ranitidine), PEPCID™ (famotidine), and AXID™ (nizatidine)); inhibitors of H⁺, K⁺ ATPase (e.g., PREVACID™ (Iansoprazole) and PRILOSEC™ (omeprazole)); Bismuth compounds (e.g., PEPTO-BISMOL™ (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™ (diphenoxylate), MOTOFEN™ (diphenoxin), and IMODIUM™ (loperamide hydrochloride)), synthetic analogs of somatostatin such as SANDOSTATIN™ (octreotide), antiemetic agents (e.g., ZOFRAN™ (ondansetron), KYTRIL™ (granisetron hydrochloride), tropisetron, dolasetron, metoclopramide, 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.

0342The albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

0343Also disclosed is 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 container(s) 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.

0344A method of local administration is by direct infection. An albumin fusion protein of the present invention may be complexed with a delivery vehicle is administered by direct injection into 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.

0345Therapeutic 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. 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.

0346Albumin fusion proteins of the present invention can be administered to any animal, preferably to mammals and birds. Mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs.

Biological Activities

0347Albumin 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.

0348In preferred embodiments, the present invention encompasses the use of the albumin fusion proteins defined in the claims for 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 the Therapeutic protein portion corresponding to the Therapeutic protein disclosed in the "Therapeutic Protein X" column of Table I (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.

0349In a further preferred embodiment the present invention encompasses the use of the albumin fusion proteins defined in the claims for treating a disease or disorder listed for a particular Therapeutic protein 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 the Therapeutic protein portion corresponding to the Therapeutic protein for which the indications in the Examples are related in an amount effective to treat, prevent or ameliorate the disease or disorder.

0350Specifically contemplated are albumin fusion proteins produced by a cell when encoded by the polynucleotides that encode SEQ ID NO:Y. When these polynucleotides are used to express the encoded proteins from a cell, the cell's natural secretion and processing steps produces a protein that lacks the signal sequence explicitly listed in columns 4 and/or 11 of Table 2. The specific amino acid sequence of the listed signal sequence is shown in the specification or is well known in the art. Thus, most preferred embodiments of the present invention include the albumin fusion protein produced by a cell (which would lack the leader sequence shown in columns 4 and/or 11 of Table 2). Also most preferred are polypeptides comprising SEQ ID NO:Y without the specific leader sequence listed in columns 4 and/or 11 of Table 2. Compositions comprising these two preferred embodiments, including pharmaceutical compositions, are also preferred. These albumin fusion proteins are specifically contemplated to treat, prevent, or ameliorate a disease or disorder listed for a particular Therapeutic protein in the "Preferred Indication:Y" column of Table 1.

0351In 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), cardiovascular system (see, for example, "Cardiovascular Disorders" section below), reproductive system (see, fpr example, "Reproductive System Disorders" section below) and/or digestive system (see, for example, "Gastrointestinal Disorders" section below)).

0352In 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 tissue(s) in which the gene corresponding to the Therapeutic protein portion of the fusion protein of the invention is expressed.

0353Thus, fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention are useful in the diagnosis, detection and/or treatment of diseases and/or disorders associated with activities that include, but are not limited to, prohormone activation, neurotransmitter activity, cellular signaling, cellular proliferation, cellular differentiation, and cell migration.

0354More 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.

<i>Immune Aclivity</i>

0355Albumin 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.

0356In 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 tissue(s) in which the polypeptide of the invention is expressed.

0357Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, diagnosing, and/or prognosing immunodeficiencies, including both congenital and acquired immunodeficiencies.

0358Autoimmune diseases or disorders that may be created, prevented, diagnosed and/or prognosed by fusion proteins of the invention and/or polynucleotides 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-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and insulin-resistant diabetes mellitus.

0359Additional disorders that are likely to have an autoimmune component that may be treated, presented, and/or diagnosed with the albumin fusion proteins of the invention and/or 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.

0360Additional disorders that are likely to have an autoimmune component that may be treated, presented, 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, 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 resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).

0361Moreover, 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 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 complication); 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).

0362In 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.

0363In 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 CD8 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.

<i>Renal Disorders</i>

0364Albumin 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 of the renal system. Renal disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention include, 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.

0365Kidney diseases which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention include, 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, nephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoproliferative glomerulonephritis I and II, mesangial proliferative 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.)

0366In 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 glycosuria, nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocystic osteosis (renal rickets), Hartnup disease, Bartter's syndrome, Liddie'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 autoimmune disorders of the kidney (e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis).

<i>Cardiovascular Disorders</i>

0367Albumin 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.

0368Cardiovascular disorders include, but are not limited to, cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, 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 Fallot, ventricular heart septal defects.

0369Cardiovascular 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.

0370Arrhythmias 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 junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia

0371Heart 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 insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.

0372Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.

0373Myocardial 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.

0374Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-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 telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

0375Aneurysms 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.

0376Arterial 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 thromboangiitis obliterans.

0377Cerebrovascular 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 hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.

0378Embolisms 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 thrombophlebitis.

0379Ischemic 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, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.

0380Albumin 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.

<i>Anti-Angiogenesis Activity</i>

0381The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. <nplcit id="ncit0115" npl-type="s"><text>Rastinejad et al., Cell 56:345-355 (1989</text></nplcit>). 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.

0382Albumin 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 angiogenesis. These disorders include, 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 collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis. 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 fibroplasia and macular degeneration.

0383Moreover, 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, retinoblastoma, retrolental 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 <nplcit id="ncit0116" npl-type="s"><text>Waltman et al., Am. J. Ophthal. 85:704-710 (1978</text></nplcit>) and <nplcit id="ncit0117" npl-type="s"><text>Gartner et al., Surv. Ophthal 22:291-312 (1978</text></nplcit>). Within another aspect of the present invention, the compounds of the invention may be used for treating proliferative diabetic retinopathy, by 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 eyes, such that the formation of blood vessels is inhibited.

0384Within 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 and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.

0385Moreover, disorders and/or states, which can be treated, prevented, diagnosed, and/or prognosed with the the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention of the invention include, 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, granulations, 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 (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

<i>Wound Healing and Epithelial Cell Proliferation</i>

0386In addition, fusion proteins of the invention and/or polynucleotides 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 and/or polynucleotides encoding albumin fusion proteins of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.

<i>Neural Activity and Neurological Diseases</i>

0387The 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 the 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 are not limited to, the following lesions of either the central (including spinal 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 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 B12 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, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

0388The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be 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. The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat or prevent neural cell injury associated with cerebral ischemia. The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat or prevent neural cell injury asssociated with cerebral infarction.

0389Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat or prevent neural cell injury associated with a stroke. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat or prevent cerebral neural cell injury associated with a stroke.

0390Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat or prevent neural cell injury associated with a heart attack. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat or prevent cerebral neural cell injury associated with a heart attack.

0391The 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 <i>in vivo</i>; (3) increased production of a neuron-associated molecule in culture or <i>in vivo, e.g.,</i> choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction <i>in vivo</i>. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, in <nplcit id="ncit0118" npl-type="s"><text>Zhang et al., Proc Natl Acad Sci USA 97:3637-42 (2000</text></nplcit>) or in <nplcit id="ncit0119" npl-type="s"><text>Arakawa et. al., J. Neurosci., 10:3507-15 (1990</text></nplcit>); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in <nplcit id="ncit0120" npl-type="s"><text>Pestronk et al., Exp. Neurol., 70:65-82 (1980</text></nplcit>), or <nplcit id="ncit0121" npl-type="s"><text>Brown et al., Ann. Rev. Neurosci., 4:17-42 (1981</text></nplcit>); 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.

0392Motor 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).

0393Further, 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.

0394Additionally, 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., multi-infarct), leukomalacia, periventricular, and vascular headache (e.g., cluster headache or migraines).

0395There 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 polynucleotides 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.

0396Examples 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.

0397Additional 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 hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as transient cerebral ischemia, Subclavian Steal Syndrome and vertebrobasilar insufficiency, vascular dementia such as multi-infarct dementia, periventricular leukomalacia, vascular headache such as cluster headache and migraine.

0398Additional 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-Jakob Syndrome, senile dementia such as Alzheimer's Disease and progressive supranuclear palsy, vascular dementia such as multi-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.

0399Additional 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 hydrocephalus such as Dandy-Walker Syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, 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.

0400Additional 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 choriomeningitis, 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 Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie), and cerebral toxoplasmosis.

0401Additional 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-Hoffmann Disease, spinal cord compression, spinal cord neoplasms such as epidural neoplasms, syringomyelia, Tabes Dorsalis, 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, Laurence-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 holoprosencephaly, neural 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.

0402Additional 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, Horner'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, Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome, peripheral nervous system diseases such as acrodynia, 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 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, Horner'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 Drusen, Optic Neuritis such as Neuromyelitis Optica, Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, Demyelinating Diseases such as Neuromyelitis Optica and Swayback, and Diabetic neuropathies such as diabetic foot.

0403Additional 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 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, polyradiculoneuritis and radiculities such as polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy, Refsam's Disease, Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies which include Congenital Analgesia and Familial Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating and Tetany).

<i>Endocrine Disorders</i>

0404Albumin 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.

0405Hormones 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 endocrine 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.

0406Endocrine system and/or hormone imbalance and/or diseases encompass disorders of uterine motility including: complications with pregnancy and labor (e.g., pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor); and disorders and/or diseases of the menstrual cycle (e.g., dysmenorrhea and endometriosis).

0407Endocrine 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, hyperpatathyroidism, hypoparathyroidism; disorders and/or diseases of the hypothalamus.

0408In addition, endocrine system and/or 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.

0409Moreover, endocrine system and/or hormone imbalance disorders and/or 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.

0410In 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 tissue(s) in which the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin protein of the invention is expressed,

<i>Reproductive System Disorders</i>

0411The 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.

0412Other 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.

0413Additionally, diseases and/or disorders of the reproductive system include disorders and/or diseases of pregnancy, including miscarriage and stillbirth, gestational diabetes, diabetes mellitus,

<i>Gastrointestinal Disorders</i>

0414Albumin 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 gastrointestinal disorders, including inflammatory 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.

0415Gastrointestinal disorders include dysphagia, odynophagia, inflammation of the esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis 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 Ménétrier's), and peritoneal diseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess,).

0416Gastrointestinal 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 (<i>Ascariasis lumbricoides</i>), Hookworms (<i>Ancylostoma duodenale</i>), Threadworms <i>(Enterobius vermicularis</i>), Tapeworms (<i>Taenia saginata, Echinococcus granulosus, Diphyllobothrium spp.,</i> and <i>T. solium</i>).

0417Liver diseases and/or disorders include intrahepatic cholestasis (alagille syndrome, biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye syndrome), hepatic vein thrombosis, hepatolentricular degeneration, hepatomegaly, hepatopulmonary syndrome, 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, fibrolamellar 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, Lipomas, 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 of 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).

0418Pancreatic 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)).

0419Gallbladder diseases include gallstones (cholelithiasis and choledocholithiasis), postcholecystectomy syndrome, diverticulosis of the gallbladder, acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.

0420Diseases and/or disorders of the large intestine include antibiotic-associated colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses, fungal and bacterial infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases (colitis, colonic neoplasms [colon cancer, adenomatous colon polyps (e.g., villous adenoma), colon carcinoma, colorectal cancer], 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), HIV enteropathy, ileal diseases (ileal neoplasms, 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, ileal 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, rectocele), peptic ulcer (duodenal ulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger-Ellison syndrome), postgastrectomy syndromes (dumping syndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux (bile reflux), gastric antral vascular ectasia, gastric fistula, gastric outlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis, stomach dilatation, stomach diverticulum, stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastric polyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis, visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum, postoperative nausea and vomiting) and hemorrhagic colitis.

0421Further diseases and/or disorders of the gastrointestinal system include biliary tract diseases, such as, gastroschisis, fistula (e.g., biliary fistula, esophageal fistula, gastric fistula, intestinal fistula, pancreatic fistula), neoplasms (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 varices, atresia, cyst, diverticulum (e.g., Zenker's diverticulum), fistula (e.g., tracheoesophageal fistula), motility 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)).

<i>Binding Activity</i>

0422Albumin 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, oligonucleotides, proteins (e.g., receptors), or small molecules.

0423Preferably, 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, <nplcit id="ncit0122" npl-type="b"><text>Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991</text></nplcit>)), 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.

0424Preferably, 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 <i>E. coli.</i>

0425The assay may simply test binding of a candidate compound to an albumin fusion protein of the invention, wherein binding 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.

0426Alternatively, 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.

0427Preferably, 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.

0428Additionally, 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 (<nplcit id="ncit0123" npl-type="b"><text>Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991</text></nplcit>)). 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.

0429Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified 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.

0430As an alternative approach for receptor identification, a labeled albumin fusion protein can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule 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.

0431Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-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. <i>See generally,</i><patcit id="pcit0096" dnum="US5605793A"><text>U.S. Patent Nos. 5,605,793</text></patcit>, <patcit id="pcit0097" dnum="US5811238A"><text>5,811,238</text></patcit>, <patcit id="pcit0098" dnum="US5830721A"><text>5,830,721</text></patcit>, <patcit id="pcit0099" dnum="US5834252A"><text>5,834,252</text></patcit>, and <patcit id="pcit0100" dnum="US5837458A"><text>5,837,458</text></patcit>, and <nplcit id="ncit0124" npl-type="s"><text>Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997</text></nplcit>); <nplcit id="ncit0125" npl-type="s"><text>Harayama, S. Trends Beotechnol. 16(2):76-82 (1998</text></nplcit>); <nplcit id="ncit0126" npl-type="s"><text>Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999</text></nplcit>); and <nplcit id="ncit0127" npl-type="s"><text>Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998</text></nplcit>); 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 two 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-5, 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-beta5, and glial-derived neurotrophic factor (GDNF).

0432Other 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 and/or 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.

0433Additionally, 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.

0434In 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, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

0435All 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.

0436Therefore, 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.

<i>Targeted Delivery</i>

0437In 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.

0438As 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 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.

0439In 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., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.

0440By "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 toxin, ricin, abrin, <i>Pseudomonas</i> 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 an 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, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

<i>Drug Screening</i>

0441Further 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 compound(s) suspected of having antagonist or agonist activity, and assaying the activity of the fusion protein following binding.

0442This 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.

0443Thus, 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 albumin fusion protein of the present invention.

0444Another 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 <patcit id="pcit0101" dnum="EP8403564A"><text>84/03564, published on September 13, 1984</text></patcit>, 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.

0445This 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 peptide which shares one or more antigenic epitopes with an albumin fusion protein of the invention.

Binding <b>Peptides</b> and Other Molecules

0446The 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, in the therapeutic embodiments described in detail, below.

0447This method comprises the steps of: <ul id="ul0006" list-style="none" compact="compact"><li>contacting an albumin fusion protein of the invention with a plurality of molecules; and</li><li>identifying a molecule that binds the albumin fusion protein.</li></ul>

0448The 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 an affinity chromatographic process, with the affinity matrix being comprised of the 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.

0449Alternatively, 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 affinity 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 conveniently. 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.

0450In 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.

0451The 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 <i>vitro</i> translation-based libraries. Examples of chemically synthesized libraries are described in <nplcit id="ncit0128" npl-type="s"><text>Fodor et al., Science 251:767-773 (1991</text></nplcit>); <nplcit id="ncit0129" npl-type="s"><text>Houghten et al., Nature 354:84-86 (1991</text></nplcit>); <nplcit id="ncit0130" npl-type="s"><text>Lam et aL, Nature 354:82-84 (1991</text></nplcit>); <nplcit id="ncit0131" npl-type="s"><text>Medynski, Bio/Technology 12:709-710 (1994</text></nplcit>); <nplcit id="ncit0132" npl-type="s"><text>Gallop et aL, J. Medicinal Chemistry 37(9):1233-1251 (1994</text></nplcit>); <nplcit id="ncit0133" npl-type="s"><text>Ohlmeyer et al., Proc. NatL Acad. Sci. USA 90:10922-10926 (1993</text></nplcit>); <nplcit id="ncit0134" npl-type="s"><text>Erb et al., Proc. Natl. Acad. Sci. USA 91:11422-11426 (1994</text></nplcit>); <nplcit id="ncit0135" npl-type="s"><text>Houghten et al., Biotechniques 13:412 (1992</text></nplcit>); <nplcit id="ncit0136" npl-type="s"><text>Jayawickreme et al., Proc. Natl. Acad. Sci. USA 91:1614-1618 (1994</text></nplcit>); <nplcit id="ncit0137" npl-type="s"><text>Salmon et aL, Proc. Natl. Acad. Sci. USA 90:11708-11712 (1993</text></nplcit>); <patcit id="pcit0102" dnum="WO9320242A"><text>PCT Publication No. WO-93/20242</text></patcit>; and <nplcit id="ncit0138" npl-type="s"><text>Brenner and Lerner, Proc. Natl. Acad. Sci. USA 89:5381-5383 (1992</text></nplcit>).

0452Examples of phage display libraries are described in <nplcit id="ncit0139" npl-type="s"><text>Scott et al., Science 249:386-390 (1990</text></nplcit>); <nplcit id="ncit0140" npl-type="s"><text>Devlin et al., Science, 249:404-406 (1990</text></nplcit>); <nplcit id="ncit0141" npl-type="s"><text>Christian et al., 1992, J. Mol. BioL 227:711-718 1992</text></nplcit>); <nplcit id="ncit0142" npl-type="s"><text>Lenstra, J. Immunol. Meth. 152:149-157 (1992</text></nplcit>); <nplcit id="ncit0143" npl-type="s"><text>Kay et al., Gene 128:59-65 (1993</text></nplcit>); and <patcit id="pcit0103" dnum="WO9418318A"><text>PCT Publication No. WO 94/18318 dated Aug. 18, 1994</text></patcit>.

0453In vitro translation-based libraries include but are not limited to those described in <patcit id="pcit0104" dnum="WO9105058A"><text>PCT Publication No. WO 91/05058 dated Apr. 18, 1991</text></patcit>; and <nplcit id="ncit0144" npl-type="s"><text>Mattheakis et aL, Proc. Natl. Acad. Sci. USA 91:9022-9026 (1994</text></nplcit>).

0454By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., <nplcit id="ncit0145" npl-type="s"><text>Bunin et al., Proc. Natl. Acad. Sci. USA 91:4708-4712 (1994</text></nplcit>)) can be adapted for use. Peptoid libraries (<nplcit id="ncit0146" npl-type="s"><text>Simon et aL, Proc. Natl. Acad. Sci. USA 89:9367-9371 (1992</text></nplcit>)) 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 <nplcit id="ncit0147" npl-type="s"><text>Ostresh et al. (Proc. NatL Acad. Sci. USA 91:11138-11142 (1994</text></nplcit>)).

0455The variety of non-peptide libraries that are useful in the present invention is great. For example, <nplcit id="ncit0148" npl-type="s"><text>Ecker and Crooke (Bio/Technology 13:351-360 (1995</text></nplcit>) list benzodiazepines, hydantoins, piperazinediones, 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.

0456Non-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.

0457Non-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.

0458Screening 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: <nplcit id="ncit0149" npl-type="s"><text>Parmley et aL, Adv. Exp. Med. BioL 251:215-218 (1989</text></nplcit>); <nplcit id="ncit0150" npl-type="s"><text>Scott et al,. Science 249:386-390 (1990</text></nplcit>); <nplcit id="ncit0151" npl-type="s"><text>Fowlkes et al., BioTechniques 13:422-427 (1992</text></nplcit>); <nplcit id="ncit0152" npl-type="s"><text>Oldenburg et al., Proc. Natl. Acad. Sci. USA 89:5393-5397 (1992</text></nplcit>); <nplcit id="ncit0153" npl-type="s"><text>Yu et aL, Cell 76:933-945 (1994</text></nplcit>); <nplcit id="ncit0154" npl-type="s"><text>Staudt et al., Science 241:577-580 (1988</text></nplcit>); <nplcit id="ncit0155" npl-type="s"><text>Bock et al., Nature 355:564-566 (1992</text></nplcit>); <nplcit id="ncit0156" npl-type="s"><text>Tuerk et al., Proc. Natl. Acad. Sci. USA 89:6988-6992 (1992</text></nplcit>); <nplcit id="ncit0157" npl-type="s"><text>Ellington et al., Nature 355:850-852 (1992</text></nplcit>); <patcit id="pcit0105" dnum="US5096815A"><text>U.S. Pat. No. 5,096,815</text></patcit>, <patcit id="pcit0106" dnum="US5223409A"><text>U.S. Pat. No. 5,223,409</text></patcit>, and <patcit id="pcit0107" dnum="US5198346A"><text>U.S. Pat. No. 5,198,346, all to Ladner et al.</text></patcit>; <nplcit id="ncit0158" npl-type="s"><text>Rebar et al., Science 263:671-673 (1993</text></nplcit>); and <patcit id="pcit0108" dnum="WO9418318A"><text>PCT Publication No. WO 94/18318</text></patcit>.

0459In 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 <nplcit id="ncit0159" npl-type="s"><text>Parmley et al., Gene 73:305-318 (1988</text></nplcit>); <nplcit id="ncit0160" npl-type="s"><text>Fowlkes et al., BioTechniques 13:422-427 (1992</text></nplcit>); <patcit id="pcit0109" dnum="WO9418318A"><text>PCT Publication No. WO 94/18318</text></patcit>; and in references cited herein.

0460In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (<nplcit id="ncit0161" npl-type="s"><text>Fields et aL, Nature 340:245-246 (1989</text></nplcit>); <nplcit id="ncit0162" npl-type="s"><text>Chien et al., Proc. Natl. Acad. Sci. USA 88:9578-9582 (1991</text></nplcit>) can be used to identify molecules that specifically bind to polypeptides of the invention.

0461where 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.

0462Thus, 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 construct comprising a lambda phage vector with a DNA insert.

0463As 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 binding polypeptide has in the range of 15-100 amino acids, or 20-50 amino acids.

0464The selected binding polypeptide can be obtained by chemical synthesis or recombinant expression.

<i>Other Activities</i>

0465An 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.

0466An 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, bums, 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.

0467An 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 neurodegenerative 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.

0468An albumin fusion protein of the invention and/or 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.

0469An 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.

0470An 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.

0471An 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.

0472An 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.

0473An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may be used to change a mammal's mental state or physical 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.

0474An 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.

0475The 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, mouse, 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.

0476Having generally described the invention, the same will be more readily understood by reference to the following examples.

EXAMPLES

<u>EXAMPLE 1: Generation of pScNHSA and pScCHSA.</u>

0477The vectors pScNHSA (ATCC Deposit No. PTA-3279) and pScCHSA (ATCC Deposit No. PTA-3276) are derivatives of pPPC0005 (ATCC Deposit No. PTA-3278) and are used as cloning vectors into which polynucleotides encoding a therapeutic protein or fragment or variant thereof is inserted adjacent to and in translation frame with polynucleotides encoding human serum albumin "HSA". pScCHSA may be used for generating Therapeutic protein-HSA fusions, while pScNHSA may be used to generate HSA-Therapeutic protein fusions.

<u>Generation of pScCHSA: albumin fusion with the albumin moiety C-terminal to the therapeutic portion.</u>

0478A vector to facilitate cloning DNA encoding a Therapeutic protein N-terminal to DNA encoding the mature albumin protein was made by altering the nucleic acid sequence that encodes the chimeric HSA signal peptide in pPPC0005 to include the <i>Xho</i> I and <i>Cla</i> I restriction sites.

0479First, the <i>Xho</i> I and <i>Cla</i> I sites inherent to pPPC0005 (located 3' of the ADH1 terminator sequence) were eliminated by digesting pPPC0005 with <i>Xho</i> I and <i>Cla</i> I, filling in the sticky ends with T4 DNA polymerase, and religating the blunt ends to create pPPC0006.

0480Second, the <i>Xho</i> I and <i>Cla</i> I restriction sites were engineered into the nucleic acid sequence that encodes the signal peptide of HSA (a chimera of the HSA leader and a kex2 site from mating factor alpha, "MAF") in pPPC0006 using two rounds of PCR. In the first round of PCR, amplification with primers shown as SEQ ID NO:1039 and SEQ ID NO:1040 was performed. The primer whose sequence is shown as SEQ ID NO:1039 comprises a nucleic acid sequence that encodes part of the signal peptide sequence of HSA, a kex2 site from the mating factor alpha leader sequence, and part of the amino-terminus of the mature form of HSA. Four point mutations were introduced in the sequence, creating the <i>Xho</i> I and <i>Cla</i> I sites found at the junction of the chimeric signal peptide and the mature form of HSA. These four mutations are underlined in the sequence shown below. In pPPC0005 the nucleotides at these four positions from 5' to 3' are T, G, T, and G. 5'-GC<u>C</u>T<u>C</u>GA<u>G</u>AAAAGAGATGCACACAAGAGTGAGGTTGCTCATCG<u>A</u>TTTAAAGAT TTGGG-3' (SEQ ID NO:1039) and 5'-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCTTTTCTCGAGGCTCCTGGAA TAAGC-3' (SEQ ID NO:1040). A second round of PCR was then performed with an upstream flanking primer, 5'-TACAAACTTAAGAGTCCAATTAGC-3' (SEQ ID NO:1041) and a downstream flanking primer 5'-CACTTCTCTAGAGTGGTTTCATATGTCTT-3' (SEQ ID NO:1042). The resulting PCR product was then purified and digested with <i>Afl</i> II and <i>Xba</i> I and ligated into the same sites in pPPC0006 creating pScCHSA. The resulting plasmid has <i>Xho</i> I and <i>Cla</i> I sites engineered into the signal sequence. The presence of the <i>Xho</i> I site creates a single amino acid change in the end of the signal sequence from LDKR to LEKR. The D to E change will not be present in the final albumin fusion protein expression plasmid when a nucleic acid sequence comprising a polynucleotide encoding the Therapeutic portion of the albumin fusion protein with a 5' <i>Sal</i> I site (which is compatible with the <i>Xho</i> I site) and a 3' <i>Cla</i> I site is ligated into the <i>Xho</i> I and <i>Cla</i> I sites of pScCHSA. Ligation of <i>Sal</i> I to <i>Xho</i> I restores the original amino acid sequence of the signal peptide sequence. DNA encoding the Therapeutic portion of the albumin fusion protein may be inserted after the Kex2 site (Kex2 cleaves after the dibasic amino acid sequence KR at the end of the signal peptide) and prior to the <i>Cla</i> I site.

<u>Generation of pScNHSA: albumin fusion with the albumin moiety N-terminal to the therapeutic portion.</u>

0481A vector to facilitate cloning DNA encoding a Therapeutic protein portion C-terminal to DNA encoding the mature albumin protein, was made by adding three, eight-base-pair restriction sites to pScCHSA. The <i>Asc</i> I, <i>Fse</i> I, and <i>Pme</i> I restriction sites were added in between the <i>Bsu36</i> I and <i>Hind</i> III sites at the end of the nucleic acid sequence encoding the mature HSA protein. This was accomplished through the use of two complementary synthetic primers containing the <i>Asc</i> I<b>,</b><i>Fse</i> I, and <i>Pme</i> I restriction sites underlined (SEQ ID NO:1043 and SEQ ID NO:1044). 5'-AAGCTGCCTTAGGCTTATAATAA<u>GGCGCGCCGGCCGGCCGTTTAAAC</u>TAAGCT TAATTCT-3' (SEQ ID NO:1043) and 5-AGAAWAAGCTTA<u>GTTTAAACGGCCGGCCGGCGCC</u>TATTATAAGCCTAAG GCAGCTT-3' (SEQ ID NO:1044). These primers were annealed and digested with <i>Bsu36</i> I and <i>Hind</i> III and ligated into the same sites in pScCHSA creating pScNHSA.

<u>EXAMPLE 2: General Construct Generation for Yeast Transformation.</u>

0482The vectors pScNHSA and pScCHSA may be used as cloning vectors into which polynucleotides encoding a therapeutic protein or fragment or variant thereof is inserted adjacent to polynucleotides encoding mature human serum albumin "HSA". pScCHSA is used for generating Therapeutic protein-HSA fusions, while pScNHSA may be used to generate HSA-Therapeutic protein fusions.

<u>Generation of albumin fusion constructs comprising HSA-Therapeutic protein fusion products,</u>

0483DNA encoding a Therapeutic protein (e.g., sequences shown in SEQ ID NO:X or known in the art) may be PCR amplified using the primers which facilitate the generation of a fusion construct (e.g., by adding restriction sites, encoding seamless fusions, encoding linker sequences, etc.) For example, one skilled in the art could design a 5' primer that adds polynucleotides encoding the last four amino acids of the mature form of HSA (and containing the <i>Bsu</i>36I site) onto the 5' end of DNA encoding a Therapeutic protein; and a 3' primer that adds a STOP codon and appropriate cloning sites onto the 3' end of the Therapeutic protein coding sequence. For instance, the forward primer used to amplify DNA encoding a Therapeutic protein might have the sequence, 5'-aagctG<u>CCTTAGG</u>CTTA(N)₁₅-3' (SEQ ID NO:1045) where the underlined sequence is a <i>Bsu</i>36I site, the upper case nucleotides encode the last four amino acids of the mature HSA protein (ALGL), and (N)₁₅ is identical to the first 15 nucleotides encoding the Therapetic protein of interest. Similarly, the reverse primer used to amplify DNA encoding a Therapeutic protein might have the sequence, 5'-GCGCGC<i>GTTTAAAC</i><u>GGCCGGCCGGCGCGCCTTATTA</u>(N)₁₅-3' (SEQ ID NO:1046) where the italicized sequence is a <i>Pme</i> I site, the double underlined sequence is an <i>Fse</i> I site, the singly underlined sequence is an <i>Asc</i> I 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 <i>Bsu</i>36I and one of <i>(Asc I, Fse</i> I, or <i>Pme</i> I) and ligated into pScNHSA.

0484The presence of the <i>Xho</i> I site in the HSA chimeric leader sequence creates a single amino acid change in the end of the chimeric signal sequence, i.e. the HSA-kex2 signal sequence, from LDKR (SEQ ID NO:2139) to LEKR (SEQ ID NO:2140).

<u>Generation of albumin fusion constructs comprising pene-HSA fusion products.</u>

0485Similar to the method described above, DNA encoding a Therapeutic protein may be PCR amplified using the following primers: A 5' primer that adds polynucleotides containing a <i>Sal</i>I site and encoding the last three amino acids of the HSA leader sequence, DKR, onto the 5' end of DNA encoding a Therapeutic protein; and a 3' primer that adds Polynucleotides encoding the first few amino acids of the mature HSA containing a <i>Cla</i> I site onto the 3' end of DNA encoding a Therapeutic protein. For instance, the forward primer used to amplify the DNA encoding a Therapeutic protein might have the sequence, 5'-aggagc<u>gtcGAC</u>AAAAGA(N)₁₅-3' (SEQ ID NO:1047) where the underlined sequence is a <i>Sal</i> I site, the upper case nucleotides encode the last three amino acids of the HSA leader sequence (DKR), and (N)₁₅ 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'-CTTTAA<i>ATCG</i><u><i>AT</i>GAGCAACCTCACTCTTGTGTGCATC</u>(N)₁₅-3'(SEQ ID NO: 1048) where the italicized sequence is a <i>Cla</i> I site, the underlined nucleotides are the reverse complement of the DNA encoding the first 9 amino acids of the mature form of HSA (DAHKSEVAH, SEQ ID NO:1106), 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 <i>Sal</i> I and <i>Cla</i> I and ligated into pScCHSA digested with <i>Xho I</i> and <i>Cla</i> I. A different signal or leader sequence may be desired, for example, invertase "INV" (Swiss-Prot Accession P00724), mating factor alpha "MAF" (Genbank Accession AAA18405), MPIF (Geneseq AAF82936), Fibulin B (Swiss-Prot Accession P23142), Clusterin (Swiss-Prot Accession P10909), Insulin-Like Growth Factor- Binding Protein 4 (Swiss-Prot Accession P22692), and permutations of the HSA leader sequence can be subcloned into the appropriate vector by means of standard methods known in the art.

<u>Generation of albumin fusion construct compatible for expression in veast S. cerevisiae.</u>

0486The <i>Not</i> I fragment containing the DNA encoding either an N-terminal or C-terminal albumin fusion protein generated from pScNHSA or pScCHSA may then be cloned into the <i>Not</i> I site of pSAC35 which has a LEU2 selectable marker. The resulting vector is then used in transformation of a yeast <i>S. cerevisiae</i> expression system.

<u>EXAMPLE 3: General Expression in Yeast <i>S. cerevisiae.</i></u>

0487An expression vector compatible with yeast expression can be transformed into yeast <i>S. cerevisiae</i> by lithium acetate transformation, electroporation, or other methods known in the art and or as described in part in <nplcit id="ncit0163" npl-type="b"><text>Sambrook, Fritsch, and Maniatis. 1989. "Molecular Cloning: A Laboratory Manual, 2nd edition", volumes 1-3</text></nplcit>, and in <nplcit id="ncit0164" npl-type="s"><text>Ausubel et al. 2000. Massachusetts General Hospital and Harvard Medical School "Current Protocols in Molecular Biology", volumes 1-4</text></nplcit>. The expression vectors are introduced into <i>S. cerevisiae</i> strains DXY1, D88, or BXP10 by transformation, individual transformants can be grown, for example, for 3 days at 30°C in 10 mL YEPD (1% w/v yeast extract, 2 % w/v, peptone, 2 % w/v, dextrose), and cells can be collected at stationary phase after 60 hours of growth. Supernatants are collected by clarifying cells at 3000g for 10 minutes.

0488pSAC35 (<nplcit id="ncit0165" npl-type="s"><text>Sleep et al., 1990, Biotechnology 8:42</text></nplcit> and see <figref idref="f0006">Figure 3</figref>) comprises, in addition to the LEU2 selectable marker, the entire yeast 2 µm plasmid to provide replication functions, the PRB1 promoter, and the ADH1 termination signal.

<u>EXAMPLE 4: General Purification of an Albumin Fusion Protein Expressed from an Albumin Fusion in Yeast <i>S. cerevisiae.</i></u>

0489In preferred embodiments, albumin fusion proteins of the invention comprise the mature form of HSA fused to either the N- or C- terminus of the mature form of a therapeutic protein or portions thereof (e.g., the mature form of a therapeutic protein listed in Table 1, or the mature form of a therapeutic protein shown in Table 2 as SEQ ID NO:Z). In one embodiment of the invention, albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature albumin fusion protein is secreted directly into the culture medium. Albumin fusion proteins of the invention preferably comprise heterologous signal sequences (e.g., the non-native signal sequence of a particular therapeutic protein) including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. Especially preferred as those signal sequence listed in Table 2 and/or the signal sequence listed in the "Expression of Fusion Proteins" and/or "Additional Methods of Recombinant and Synthetic Production of Albumin Fusion Proteins" section of the specification, above. In preferred embodiments, the fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.

0490Albumin fusion proteins expressed in yeast as described above can be purified on a small-scale over a Dyax peptide affinity column as follows. Supernatants from yeast expressing an albumin fusion protein is diafiltrated against 3 mM phosphate buffer pH 6.2, 20 mM NaCl and 0.01% Tween 20 to reduce the volume and to remove the pigments. The solution is then filtered through a 0.22 µm device. The filtrate is loaded onto a Dyax peptide affinity column. The column is eluted with 100 mM Tris/HCl, pH 8.2 buffer. The peak fractions containing protein are collected and analyzed on SDS-PAGE after concentrating 5-fold.

0491For large scale purification, the following method can be utilized. The supernatant in excess of 2 L is diafiltered and concentrated to 500 mL in 20 mM Tris/HCl pH 8.0. The concentrated protein solution is loaded onto a pre-equilibrated 50 mL DEAE-Sepharose Fast Flow column, the column is washed, and the protein is eluted with a linear gradient of NaCl from 0 to 0.4 M NaCl in 20 mM Tris/HCl, pH 8.0. Those fractions containing the protein are pooled, adjusted to pH 6.8 with 0.5 M sodium phosphate (NaH₂PO₄). A final concentration of 0.9 M (NH₄)₂SO₄ is added to the protein solution and the whole solution is loaded onto a pre-equilibrated 50 mL Buty1650S column. The protein is eluted with a linear gradient of ammonium sulfate (0.9 to 0 M (NH₄)₂SO₄). Those fractions with the albumin fusion are again pooled, diafiltered against 10 mM Na₂HPO₄/citric acid buffer pH 5.75, and loaded onto a 50 mL pre-equilibrated SP-Sepbarose Fast Flow column. The protein is eluted with a NaCl linear gradient from 0 to 0.5 M. The fractions containing the protein of interest are combined, the buffer is changed to 10 mM Na₂BPO₄/citric acid pH 6.25 with an Amicon concentrator, the conductivity is &lt; 2.5 mS/cm. This protein solution is loaded onto a 15 mL pre-equilibrated Q-Sepharose high performance column, the column is washed, and the protein is eluted with a NaCl linear gradient from 0 to 0.15 M NaCl. The purified protein can then be formulated into a specific buffer composition by buffer exchange.

<u>EXAMPLE 5: General Construct Generation for Mammalian Cell Transfection.</u>

<i><u>Generation plalbumin fusion construct compatible for expression in mammalian cell-lines.</u></i>

0492Albumin fusion constructs can be generated in expression vectors for use in mammalian cell culture systems. DNA encoding a therapeutic protein can be cloned N-terminus or C-terminus to HSA in a mammalian expression vector by standard methods known in the art (e.g., PCR amplification, restriction digestion, and ligation). Once the expression vector has been constructed, transfection into a mammalian expression system can proceed. Suitable vectors are known in the art including, but not limited to, for example, the pC4 vector, and/or vectors available from Lonza Biologics, Inc. (Portsmouth, NH).

0493The DNA encoding human serum albumin has been cloned into the pC4 vector which is suitable for mammalian culture systems, creating plasmid pC4:HSA (ATCC Deposit # PTA-3277). This vector has a DiHydroFolate Reductase, "DHFR", gene that will allow for selection in the presence of methotrexate.

0494The pC4:HSA vector is suitable for expression of albumin fusion proteins in CHO cells. For expression, in other mammalian cell culture systems, it may be desirable to subclone a fragment comprising, or alternatively consisting of, DNA which encodes for an albumin fusion protein into an alternative expression vector. For example, a fragment comprising, or alternatively consisting, of DNA which encodes for a mature albumin fusion protein may be subcloned into another expression vector including, but not limited to, any of the mammalian expression vectors described herein.

0495In a preferred embodiment, DNA encoding an albumin fusion construct is subcloned into vectors provided by Lonza Biologics, Inc. (Portsmouth, NH) by procedures known in the art for expression in NS0 cells.

<i><u>Generation of albumin fusion constructs comprising HSA-Therapeutic Protein fusion products.</u></i>

0496Using pC4:HSA (ATCC Deposit # PTA-3277), albumin fusion constructs can be generated in which the Therapeutic protein portion is C terminal to the mature albumin sequence. For example, one can clone DNA encoding a Therapeutic protein of fragment or variant thereof between the <i>Bsu</i> 36I and <i>Asc</i> I restriction sites of the vector. When cloning into the <i>Bsu</i> 36I and <i>Asc</i> I, the same primer design used to clone into the yeast vector system (SEQ ID NO:1045 and 1046) may be employed (see Example 2).

<i><u>Generation of albumin fusion constructs comprising gene-HSA fusion products.</u></i>

0497Using pC4:HSA (ATCC Deposit # PTA-3277), albumin fusion constructs can be generated in which a Therapeutic protein portion is cloned N terminal to the mature albumin sequence. For example, one can clone DNA encoding a Therapeutic protein that has its own signal sequence between the <i>Bam</i> HI (or <i>Hind</i> III) and <i>Cla</i> I sites of pC4:HSA. When cloning into either the <i>Bam</i> HI or <i>Hind</i> III site, it is preferrable to include a Kozak sequence (CCGCCACC<u>ATG</u>, SEQ ID NO:1107) prior to the translational start codon of the DNA encoding the Therapeutic protein. If a Therapeutic protein does not have a signal sequence, DNA encoding that Therapeutic protein may be cloned in between the <i>Xho</i> I and <i>Cla</i> I sites of pC4:HSA. When using the <i>Xho</i> I site, the following 5' (SEQ ID NO:1052) and 3' (SEQ ID NO:1053) exemplary PCR primers may be used: <ul id="ul0007" list-style="none" compact="compact"><li>5'-CCGCCG<u>CTCGAG</u>GGGTGTGTTTCGTCGA(N)₁₈-3' (SEQ ID NO: 1052)</li><li>5'-AGTCCC<u>ATCGAT</u>GAGCAACCTCACTCTTGTGTGCATC(N)₁₈-3' (SEQ ID NO:1053)</li></ul>

0498In the 5' primer (SEQ ID NO:1052), the underlined sequence is a <i>Xho</i> I site; and the <i>Xho</i> I site and the DNA following the <i>Xho</i> I site code for the last seven amino acids of the leader sequence of natural human serum albumin. In SEQ ID NO:1052, "(N)₁₈" is DNA identical to the first 18 nucleotides encoding the Therapeutic protein of interest. In the 3' primer (SEQ ID NO:1053), the underlined sequence is a <i>Cla</i> I site; and the <i>Cla</i> I site and the DNA following it are the reverse complement of the DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO:1038). In SEQ ID NO:1053 "(N)₁₈" 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 <i>Xho</i> I and <i>Cla</i> I restriction enzymes and clone it into the <i>Xho</i> I and <i>Cla</i> I sites in the pC4:HSA vector.

0499If an alternative leader sequence is desired, the native albumin leader sequence can be replaced with the chimeric albumin leader, i.e., the HSA-kex2 signal peptide, or an alternative leader by standard methods known in the art. (For example, one skilled in the art could routinely PCR amplify an alternate leader and subclone the PCR product into an albumin fusion construct in place of the albumin leader while maintaining the reading frame)

<u>EXAMPLE 6: General Expression in Mammalian Cell-Lines.</u>

0500An albumin fusion construct generated in an expression vector compatible with expression in mammalian cell-lines can be transfected into appropriate cell-lines by calcium phosphate precipitation, lipofectamine, electroporation, or other transfection methods known in the art and/or as described in <nplcit id="ncit0166" npl-type="b"><text>Sambrook, Fritsch, and Maniatis. 1989. "Molecular Cloning: A Laboratory Manual, 2nd editi</text></nplcit>on" and in <nplcit id="ncit0167" npl-type="s"><text>Ausubel et aL 2000. Massachusetts General Hospital and Harvard Medical School "Current Protocols in Molecular Biology", volumes 1-4</text></nplcit>. The transfected cells are then selected for by the presence of a selecting agent determined by the selectable marker in the expression vector.

0501The pC4 expression vector (ATCC Accession No. 209646) is a derivative of the plasmid pSV2-DHFR (ATCC Accession No. 37146). pC4 contains the strong promoter Long Terminal Repeats "LTR" of the Rous Sarcoma Viris (<nplcit id="ncit0168" npl-type="s"><text>Cullen et al., March 1985, Molecular and Cellular Biology, 438-447</text></nplcit>) and a fragment of the CytoMegaloVirus "CMV"-enhancer (<nplcit id="ncit0169" npl-type="s"><text>Bosbart et aL, 1985, Cell 41: 521-530</text></nplcit>). The vector also contains 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. Chinese hamster ovary "CHO" cells or other cell-lines lacking an active DHFR gene are used for transfection. Transfection of an albumin fusion construct in pC4 into CHO cells by methods known in the art will allow for the expression of the albumin fusion protein in CHO cells, followed by leader sequence cleavage, and secretion into the supernatant. The albumin fusion protein is then further purified from the supernatant.

0502The pEE12.1 expression vector is provided by Lonza Biologics, Inc. (Portsmouth, NH) and is a derivative of pEE6 (<nplcit id="ncit0170" npl-type="s"><text>Stephens and Cockett, 1989, Nucl. Acids Res. 17: 7110</text></nplcit>). This vector comprises a promoter, enhancer and complete 5'-untranslated region of the Major Immediate Early gene of the human CytoMegaloVirus, "hCMV-MIE" (International Publication # <patcit id="pcit0110" dnum="WO8901036A"><text>WO89/01036</text></patcit>), upstream of a sequence of interest, and a Glutamine Synthetase gene (<nplcit id="ncit0171" npl-type="s"><text>Murphy et al., 1991, Biochem J. 227: 277-279</text></nplcit>; <nplcit id="ncit0172" npl-type="s"><text>Bebbington et al., 1992, Bio/Technology 10:169-175</text></nplcit>; US patent <patcit id="pcit0111" dnum="US5122464A"><text>US 5,122,464</text></patcit>) for purposes of selection of transfected cells in selective methionine sulphoximine containing medium. Transfection of albumin fusion constructs made in pEE12.1 into NS0 cells (International Publication # <patcit id="pcit0112" dnum="WO8605807A"><text>WO86/05807</text></patcit>) by methods known in the art will allow for the expression of the albumin fusion protein in NS0 cells, followed by leader sequence cleavage, and secretion into the supernatant. The albumin fusion protein is then further purified from the supernatant using techniques described herein or otherwise known in the art.

0503Expression of an albumin fusion protein may be analyzed, for example, by SDS-PAGE and Western blot, reversed phase HPLC analysis, or other methods known in the art.

0504Stable CHO and NS0 cell-lines transfected with albumin fusion constructs are generated by methods known in the art (e.g., lipofectamine transfection) and selected, for example, with 100 nM methotrexate for vectors having the DiHydroFolate Reductase 'DHFR' gene as a selectable marker or through growth in the absence of glutamine. Expression levels can be examined for example, by immunoblotting, primarily, with an anti-HSA serum as the primary antibody, or, secondarily, with serum containing antibodies directed to the Therapeutic protein portion of a given albumin fusion protein as the primary antibody.

0505Expression levels are examined by immunoblot detection with anti-HSA serum as the primary antibody. The specific productivity rates are determined via ELISA in which the capture antibody can be a monoclonal antibody towards the therapeutic protein portion of the albumin fusion and the detecting antibody can be the monoclonal anti-HSA-biotinylated antibody <i>(or vice versa),</i> followed by horseradish peroxidase/streptavidin binding and analysis according to the manufacturer's protocol.

<u>EXAMPLE 7: General Purification of an Albumin Fusion Protein Expressed from an Albumin Fusion Construct in Mammalian Cell-lines.</u>

0506In preferred embodiments, albumin fusion proteins of the invention comprise the mature form of HSA fused to either the N- or C- terminus of the mature form of a therapeutic protein or portions thereof (e.g., the mature form of a therapeutic protein listed in Table 1, or the mature form of a therapeutic protein shown in Table 2 as SEQ ID NO:Z). In one embodiment of the invention, albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature albumin fusion protein is secreted directly into the culture medium. Albumin fusion proteins of the invention preferably comprise heterologous signal sequences (e.g., the non-native signal sequence of a particular therapeutic protein) including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. Especially preferred as those signal sequence listed in Table 2 and/or the signal sequence listed in the "Expression of Fusion Proteins" and/or "Additional Methods of Recombinant and Synthetic Production of Albumin Fusion Proteins" section of the specification, above. In preferred embodiments, the fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.

0507Albumin fusion proteins from mammalian cell-line supernatants are purified according to different protocols depending on the expression system used.

<i><u>Purification from CHO and 293T cell-lines.</u></i>

0508Purification of an albumin fusion protein from CHO cell supernatant or from transiently transfected 293T cell supernatant may involve initial capture with an anionic HQ resin using a sodium phosphate buffer and a phosphate gradient elution, followed by affinity chromatography on a Blue Sepharose FF column using a salt gradient elution. Blue Sepharose FF removes the main BSA/fetuin contaminants. Further purification over the Poros PI 50 resin with a phosphate gradient may remove and lower endotoxin contamination as well as concentrate the albumin fusion protein.

<i><u>Purification from NS0 cell-line.</u></i>

0509Purification of an albumin-fusion protein from NS0 cell supernatant may involve Q-Sepharose anion exchange chromatography, followed by SP-sepharose purification with a step elution, followed by Phenyl-650M purification with a step elution, and, ultimately, diafiltration.

0510The purified protein may then be formulated by buffer exchange.

<u>Example 40: Isolation of a Selected cDNA Clone From the Deposited Sample.</u>

0511Many of the albumin fusion constructs of the invention have been deposited with the ATCC as shown in Table 3. The albumin fusion constructs may comprise any one of the following expression vectors: the yeast <i>S. cerevisiae</i> expression vector pSAC35, the mammalian expression vector pC4, or the mammalian expression vector pEE12.1.

0512pSAC35 (<nplcit id="ncit0173" npl-type="s"><text>Sleep et al., 1990, Biotechnology 8:42</text></nplcit>), pC4 (ATCC Accession No. 209646; <nplcit id="ncit0174" npl-type="s"><text>Cullen et al., Molecular and Cellular Biology, 438-447 (1985</text></nplcit>); <nplcit id="ncit0175" npl-type="s"><text>Boshart et al., Cell 41: 521-530 (1985</text></nplcit>)), and pEE12.1 (Lonza Biologics, Inc.; <nplcit id="ncit0176" npl-type="s"><text>Stephens and Cockett, Nucl. Acids Res. 17: 7110 (1989</text></nplcit>); International Publication #<patcit id="pcit0113" dnum="WO8901036A"><text>WO89/01036</text></patcit>; <nplcit id="ncit0177" npl-type="s"><text>Murphy et al., Biochem J. 227: 277-279 (1991</text></nplcit>); <nplcit id="ncit0178" npl-type="s"><text>Bebbington et al., Bio/Technology 10:169-175 (1992</text></nplcit>); US patent <patcit id="pcit0114" dnum="US5122464A"><text>US 5,122,464</text></patcit>; International Publication #<patcit id="pcit0115" dnum="WO8605807A"><text>WO86/05807</text></patcit>) vectors comprise an ampicillin resistance gene for growth in bacterial cells. These vectors and/or an albumin fusion construct comprising them can be transformed into an <i>E. coli</i> strain such as Stratagene XL-1 Blue (Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, CA, 92037) using techniques described in the art such as Hanahan, spread onto Luria-Broth agar plates containing 100 µg/mL ampicillin, and grown overnight at 37 °C<b>.</b>

0513The deposited material in the sample assigned the ATCC Deposit Number cited in Table 3 for any given albumin fusion construct also may contain one or more additional albumin fusion constructs, each encoding different albumin fusion proteins. Thus, deposits sharing the same ATCC Deposit Number contain at least an albumin fusion construct identified in the corresponding row of Table 3.

0514Two approaches can be used to isolate a particular albumin fusion construct from the deposited sample of plasmid DNAs cited for that albumin fusion construct in Table 3.

<u>Method 1: Screening</u>

0515First, an albumin fusion construct may be directly isolated by screening the sample of deposited plasmid DNAs using a polynucleotide probe corresponding to SEQ ID NO:X for an individual construct ID number in Table 1, using methods known in the art. For example, a specific polynucleotide with 30-40 nucleotides may be synthesized using an Applied Biosysteins DNA synthesizer according to the sequence reported. The oligonucleotide can be labeled, for instance, with ³²P-γ-ATP using T4 polynucleotide kinase and purified according to routine methods. (E.g., <nplcit id="ncit0179" npl-type="b"><text>Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY (1982</text></nplcit>)). The albumin fusion construct from a given ATCC deposit is transformed into a suitable host, as indicated above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g., <nplcit id="ncit0180" npl-type="b"><text>Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104</text></nplcit>), or other techniques known to those of skill in the art.

Method 2: PCR

0516Alternatively, DNA encoding a given albumin fusion protein may be amplified from a sample of a deposited albumin fusion construct with SEQ ID NO:X, for example, by using two primers of 17-20 nucleotides that hybridize to the deposited albumin fusion construct 5' and 3' to the DNA encoding a given albumin fusion protein. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 µl of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 µM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94°C for 1 min; annealing at 55°C for 1 min; elongation at 72°C for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.

0517Several methods are available for the identification of the 5' or 3' non-coding portions of a gene which may not be present in the deposited clone. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5' and 3' "RACE" protocols which are known in the art. For instance, a method similar to 5' RACE is available for generating the missing 5' end of a desired full-length transcript. (<nplcit id="ncit0181" npl-type="s"><text>Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684 (1993</text></nplcit>)).

0518Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5' portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full length gene.

0519This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5' phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5' ends of messenger RNAs. This reaction leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.

0520This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5' end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the desired gene.

<u>Example 41: [</u>^<u>3</u><u>H]-2-Deoxyglucose Uptake Assay.</u>

0521Adipose, skeletal muscle, and liver are insulin-sensitive tissues. Insulin can stimulate glucose uptake/transport into these tissues. In the case of adipose and skeletal muscle, insulin initiates the signal transduction that eventually leads to the translocation of the glucose transporter 4 molecule, GLUT4, from a specialized intracellular compartment to the cell surface. Once on the cell surface, GLUT4 allows for glucose uptake/transport.

[³H]-2-Deoxyglucose Uptake

0522A number of adipose and muscle related cell-lines can be used to test for glucose uptake/transport activity in the absence or presence of a combination of any one or more of the therapeutic drugs listed for the treatment of diabetes mellitus. In particular, the 3T3-L1 murine fibroblast cells and the L6 murine skeletal muscle cells can be differentiated into 3T3-L1 adipocytes and into myotubes, respectively, to serve as appropriate in <i>vitro</i> models for the [³H]-2-deoxyglucose uptake assay (<nplcit id="ncit0182" npl-type="s"><text>Urso et al., J Biol Chem, 274(43): 30864-73 (1999</text></nplcit>); <nplcit id="ncit0183" npl-type="s"><text>Wang et al., J Mol Endocrinol, 19(3): 241-8 (1997</text></nplcit>); <nplcit id="ncit0184" npl-type="s"><text>Haspel et al., J Membr Biol, 169 (1): 45-53 (1999</text></nplcit>); <nplcit id="ncit0185" npl-type="s"><text>Tsakiridis et al., Endocrinology, 136(10): 4315-22 (1995</text></nplcit>)). Briefly, 2 x 10⁵ cells/100 µL of adipocytes or differentiated L6 cells are transferred to 96-well Tissue-Culture, "TC", treated, i.e., coated with 50 µg/mL of poly-L-lysine, plates in post-differentiation medium and are incubated overnight at 37 °C in 5% CO₂. The cells are first washed once with serum free low glucose DMEM medium and are then starved with 100 µL/well of the same medium and with 100 µL/well of either buffer or of a combination of any one or more of the therapeutic drugs listed for the treatment of diabetes mellitus, for example, increasing concentrations of 1 nM, 10 nM, and 100 nM of the therapeutics of the subject invention (e.g., specific fusions disclosed as SEQ ID NO:Y and fragments and variants thereof) for 16 hours at 37 °C in the absence or presence of 1 nM insulin. The plates are washed three times with 100 µL/well of HEPES buffered saline. Insulin is added at 1 nM in HEPES buffered saline for 30 min at 37 °C in the presence of 10 µM labeled [³H]-2-deoxyglucose (Amersham, #TRK672) and 10 µM unlabeled 2-deoxyglucose (SIGMA, D-3179). As control, the same conditions are carried out except in the absence of insulin. A final. concentration of 10 µM cytochalasin B (SIGMA, C6762) is added at 100 µL/well in a separate well to measure the non-specific uptake. The cells are washed three times with HEPES buffered saline. Labeled, i.e., 10 µM of [³H]-2-deoxyglucose, and unlabeled, i.e., 10 µM of 2-deoxyglucose, are added for 10 minutes at room temperature. The cells are washed three times with cold Phosphate Buffered <i>Sal</i> ine, "PBS". The cells are lysed upon then addition of 150 µL/well of 0.2 N NaOH and subsequent incubation with shaking for 20 minutes at room temperature. Samples are then transferred to a scintillation vial to which is added 5 mL of scintillation fluid. The vials are counted in a Beta-Scintillation counter. Uptake in duplicate conditions, the difference being the absence or presence of insulin, is determined with the following equation: [(Insulin counts per minute "cpm" - Non-Specific cpm)/(No Insulin cpm - Non-Specific cpm)]. Average responses fall within the limits of about 5-fold and 3-fold that of controls for adipocytes and myotubes, respectively.

<u>Differentiation of Cells</u>

0523The cells are allowed to become fully confluent in a T-75 cm² flask. The medium is removed and replaced with 25 mL of pre-differentiation medium for 48 hours. The cells are incubated at 37 °C, in 5% CO₂, 85% humidity. After 48 hours, the pre-differentiation medium is removed and replaced with 25 mL differentiation medium for 48 hours. The cells are again incubated at 37 °C, in 5% CO₂, 85% humidity. After 48 hours, the medium is removed and replaced with 30 mL post-differentiation medium. Post-differentiation medium is maintained for 14-20 days or until complete differentiation is achieved. The medium is changed every 2-3 days. Human adipocytes can be purchased from Zen-Bio, INC (# SA-1096).

<u>Example 42: In <i>vitro</i> Assay of [</u>^<u>3</u><u>H]-Thymidine Incorporation into Pancreatic Cell-lines.</u>

0524It has recently been shown that GLP-1 induces differentiation of the rat pancreatic ductal epithelial cell-line ARIP in a time- and dose-dependent manner which is associated with an increase in Islet Duodenal Homeobox-1 (IDX-1) and insulin mRNA levels (<nplcit id="ncit0186" npl-type="s"><text>Hui et at., 2001, Diabetes, 50(4): 785-96</text></nplcit>). The IDX-1 in turn increases mRNA levels of the GLP-1 receptor.

Cells Types Tested

0525<u>RIN-M cells:</u> These cells are available from the American Type Tissue Culture Collection (ATCC Cell Line Number CRL-20577). The RIN-M cell line was derived from a radiation induced transplantable rat islet cell tumor. The line was established from a nude mouse xenograft of the tumor. The cells produce and secrete islet polypeptide hormones, and produce L-dopa decarboxylase (a marker for cells having amine precursor uptake and decarboxylation, or APUD, activity).

0526<u>ARIP cells:</u> These are pancreatic exocrine cells of epithelial morphology available from the American Type Tissue Culture Collection (ATCC Cell Line Number CRL-1674). See also, references: <nplcit id="ncit0187" npl-type="s"><text>Jessop, N.W. and Hay, R.J., "Characteristics of two rat pancreatic exocrine cell lines derived from transplantable tumors," In Vitro 16: 212, (1980</text></nplcit>); <nplcit id="ncit0188" npl-type="s"><text>Cockell, M. et al., "Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas," Mol. Cell. Biol. 9: 2464-2476, (1989</text></nplcit>); <nplcit id="ncit0189" npl-type="s"><text>Roux, E., et al. "The cell-specific transcription factor PTF1 contains two different subunits that interact with the DNA" Genes Dev. 3: 1613-1624, (1989</text></nplcit>); and, <nplcit id="ncit0190" npl-type="s"><text>Hui, H., et al., "Glucagon-like peptide 1 induces differentiation of islet duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting cells," Diabetes 50: 785-796 (2001</text></nplcit>).

<u>Preparation of Cells</u>

0527The RIN-M cell-line is grown in RPMI 1640 medium (Hyclone, #SH300027.01) with 10% fetal bovine serum (HyClone, #SH30088.03) and is subcultured every 6 to 8 days at a ratio of 1:3 to 1:6. The medium is changed every 3 to 4 days.

0528The ARIP (ATCC #CRL-1674) cell-line is grown in Ham's F12K medium (ATCC, #30-2004) with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 10% fetal bovine serum. The ARIP cell-line is subcultured at a ratio of 1:3 to 1:6 twice per week. The medium is changed every 3 to 4 days.

<u>Assay Protocol</u>

0529The cells are seeded at 4000 cells/well in 96-well plates and cultured for 48 to 72 hours to 50% confluence. The cells are switched to serum-free media at 100 µL/well. After incubation for 48-72 hours, serum and/or the therapeutics of the subject invention (e.g., albumin fusion proteins of the invention and fragments and variants thereof) are added to the well. Incubation persists for an additional 36 hours. [³H]-Thymidine (5-20 Ci/mmol) (Amersham Pharmacia, #TRK120) is diluted to 1 microCuries/5 microliters. After the 36 hour incubation, 5 microliters is added per well for a further 24 hours. The reaction is terminated by washing the cells gently with cold Phosphate-Buffered <i>Sal</i> ine, "PBS", once. The cells are then fixed with 100 microliters of 10% ice cold TCA for 15 min at 4 °C. The PBS is removed and 200 microliters of 0.2 N NaOH is added. The plates are incubated for 1 hour at room temperature with shaking. The solution is transferred to a scintillation vial and 5 mL of scintillation fluid compatible with aqueous solutions is added and mixed vigorously. The vials are counted in a beta scintillation counter. As negative control, only buffer is used. As a positive control fetal calf serum is used.

<u>Example 43: Assaying for Glycosuria.</u>

0530Glycosuria (i.e., excess sugar in the urine), can be readily assayed to provide an index of the disease state of diabetes mellitus. Excess urine in a patient sample as compared with a normal patient sample is symptomatic of IDDM and NIDDM. Efficacy of treatment of such a patient having IDDM and NIDDM is indicated by a resulting decrease in the amount of excess glucose in the urine. In a preferred embodiment for IDDM and NIDDM monitoring, urine samples from patients are assayed for the presence of glucose using techniques known in the art. Glycosuria in humans is defined by a urinary glucose concentration exceeding 100 mg per 100 ml. Excess sugar levels in those patients exhibiting glycosuria can be measured even more precisely by obtaining blood samples and assaying serum glucose.

<u>Example 44: Occurrence of Diabetes in NOD Mice.</u>

0531Female NOD (non-obese diabetic) mice are characterized by displaying IDDM with a course which is similar to that found in humans, although the disease is more pronounced in female than male NOD mice. Hereinafter, unless otherwise stated, the term "NOD mouse" refers to a female NOD mouse. NOD mice have a progressive destruction of beta cells which is caused by a chronic autoimmune disease. Thus, NOD mice begin life with euglycemia, or normal blood glucose levels. By about 15 to 16 weeks of age, however, NOD mice start becoming hyperglycemic, indicating the destruction of the majority of their pancreatic beta cells and the corresponding inability of the pancreas to produce sufficient insulin. Thus, both the cause and the progression of the disease are similar to human IDDM patients.

0532<i>In vivo</i> assays of efficacy of the immunization regimens can be assessed in female NOD/LtJ mice (commercially available from The Jackson Laboratory, Bar Harbor, Me.). In the literature, it's reported that 80% of female mice develop diabetes by 24 weeks of age and onset of insulitis begins between 6-8 weeks age. NOD mice are inbred and highly responsive to a variety of immunoregulatory strategies. Adult NOD mice (6-8 weeks of age) have an average mass of 20-25 g.

0533These mice can be either untreated (control), treated with the therapeutics of the subject invention (e.g., albumin fusion proteins of the invention and fragments and variants thereof), alone or in combination with other therapeutic compounds stated above. The effect of these various treatments on the progression of diabetes can be measured as follows:

0534At 14 weeks of age, the female NOD mice can be phenotyped according to glucose tolerance. Glucose tolerance can be measured with the intraperitoneal glucose tolerance test (IPGTT). Briefly, blood is drawn from the paraorbital plexus at 0 minutes and 60 minutes after the intraperitoneal injection of glucose (1 g/kg body weight). Normal tolerance is defined as plasma glucose at 0 minutes of less than 144 mg %, or at 60 minutes of less than 160 mg %. Blood glucose levels are determined with a Glucometer Elite apparatus.

0535Based upon this phenotypic analysis, animals can be allocated to the different experimental groups. In particular, animals with more elevated blood glucose levels can be assigned to the impaired glucose tolerance group. The mice can be fed ad libitum and can be supplied with acidified water (pH 2.3).

0536The glucose tolerant and intolerant mice can be further subdivided into control, albumin fusion proteins of the subject invention, and albumin fusion proteins/therapeutic compounds combination groups. Mice in the control group can receive an interperitoneal injection of vehicle daily, six times per week. Mice in the albumin fusion group can receive an interperitoneal injection of the therapeutics of the subject invention (e.g., albumin fusion proteins of the invention and fragments and variants thereof) in vehicle daily, six times per week. Mice in the albumin fusion proteins/therapeutic compounds combination group can receive both albumin fusion proteins and combinations of therapeutic compounds as described above.

0537The level of urine glucose in the NOD mice can be determined on a bi-weekly basis using Labstix (Bayer Diagnostics, Hampshire, England). Weight and fluid intake can also be determined on a bi-weekly basis. The onset of diabetes is defined after the appearance of glucosuria on two consecutive determinations. After 10 weeks of treatment, an additional IPGTT can be performed and animals can be sacrificed the following day.

0538Over the 10 week course of treatment, control animals in both the glucose tolerant and glucose intolerant groups develop diabetes at a rate of 60% and 86%, respectively (see <patcit id="pcit0116" dnum="US5866546A"><text>US patent No. 5,866,546, Gross et al.</text></patcit>). Thus, high rates of diabetes occur even in NOD mice which are initially glucose tolerant if no intervention is made.

0539Results can be confirmed by the measurement of blood glucose levels in NOD mice, before and after treatment. Blood glucose levels are measured as described above in both glucose tolerant and intolerant mice in all groups described.

0540In an alternative embodiment, the therapeutics of the subject invention (e.g., specific fusions disclosed as SEQ ID NO:Y and fragments and variants thereof) can be quantified using spectrometric analysis and appropriate protein quantities can be resuspended prior to injection in 50 .mu.l phosphate buffered saline (PBS) per dose. Two injections, one week apart, can be administered subcutaneously under the dorsal skin of each mouse. Monitoring can be performed on two separate occasions prior to immunization and can be performed weekly throughout the treatment and continued thereafter. Urine can be tested for glucose every week (Keto-Diastix.RTM.; Miles Inc., Kankakee, III.) and glycosuric mice can be checked for serum glucose (ExacTech.RTM., MediSense, Inc., Waltham, Mass.). Diabetes is diagnosed when fasting glycemia is greater than 2.5g/L.

<u>Example 45: Histological Examination of NOD Mice.</u>

0541Histological examination of tissue samples from NOD mice can demonstrate the ability of the compositions of the present invention, and/or a combination of the compositions of the present invention with other therapeutic agents for diabetes, to increase the relative concentration of beta cells in the pancreas. The experimental method is as follows:

0542The mice from Example 44 can be sacrificed at the end of the treatment period and tissue samples can be taken from the pancreas. The samples can be fixed in 10% formalin in 0.9% saline and embedded in wax. Two sets of 5 serial 5 .mu.m sections can be cut for immunolabelling at a cutting interval of 150 .mu.m. Sections can be immunolabelled for insulin (guinea pig anti-insulin antisera dilution 1:1000, ICN Thames U.K.) and glucagon (rabbit anti-pancreatic glucagon antisera dilution 1:2000) and detected with peroxidase conjugated anti-guinea pig (Dako, High Wycombe, U.K.) or peroxidase conjugated anti-rabbit antisera (dilution 1:50, Dako).

0543The composition of the present invention may or may not have as strong an effect on the visible mass of beta cells as it does on the clinical manifestations of diabetes in glucose tolerant and glucose intolerant animals.

<u>Example 46: Pancreatic Beta-Cell Transplantation Combination Therapy.</u>

0544Transplantation is a common form of treatment of autoimmune disease, especially when the target self tissue has been severely damaged. For example, and not by way of limitation, pancreas transplantation and islet cell transplantation are common treatment options for IDDM (See, e.g., <nplcit id="ncit0191" npl-type="s"><text>Stewart et al., Journal of Clinical Endocrinology &amp; Metabolism 86 (3): 984-988 (2001</text></nplcit>); <nplcit id="ncit0192" npl-type="s"><text>Brunicardi, Transplant. Proc. 28: 2138-40 (1996</text></nplcit>); <nplcit id="ncit0193" npl-type="s"><text>Kendall &amp; Robertson, Diabetes Metab. 22: 157-163 (1996</text></nplcit>); <nplcit id="ncit0194" npl-type="s"><text>Hamano et al., Kobe J. Med. Sci. 42: 93-104 (1996</text></nplcit>); <nplcit id="ncit0195" npl-type="s"><text>Larsen &amp; Stratta, Diabetes Metab. 22: 139-146 (1996</text></nplcit>); and <nplcit id="ncit0196" npl-type="s"><text>Kinkhabwala, et al., Am. J. Surg. 171: 516-520 (1996</text></nplcit>)). As with any transplantation method, transplantation therapies for autoimmune disease patients include treatments to minimize the risk of host rejection of the transplanted tissue. However, autoimmune disease involves the additional, independent risk that the pre-existing host autoimmune response which damaged the original self tissue will exert the same damaging effect on the transplanted tissue. Accordingly, the present invention encompasses methods and compositions for the treatment of autoimmune pancreatic disease using the albumin fusion proteins of the subject invetion in combination with immunomodulators/immunosuppressants in individuals undergoing transplantation therapy of the autoimmune disease.

0545In accordance with the invention, the albumin fusion-based compositions and formulations described above, are administered to prevent and treat damage to the transplanted organ, tissue, or cells resulting from the host individual's autoimmune response initially directed against the original self tissue. Administration may be carried out both prior and subsequent to transplantation in 2 to 4 doses each one week apart.

0546The following immunomodulators/immunosuppressants including, but not limited to, AI-401, CDP-571 (anti-TNF monoclonal antibody), CG-1088, Diamyd (diabetes vaccine), ICM3 (anti-ICAM-3 monoclonal antibody), linomide (Roquinimex), NBI-6024 (altered peptide ligand), TM-27, VX-740 (HMR-3480), caspase 8 protease inhibitors, thalidomide, hOKT3gammal (Ala-ala) (anti-CD3 monoclonal antibody), Oral Interferon-Alpha, oral lactobacillus, and LymphoStat-B™ can be used together with the albumin fusion therapeutics of the subject invention in islet cell or pancreas transplantation.

<u>Example 47: <i>In vivo</i> Mouse Model of NIDDM.</u>

0547Male C57BL/6J mice from Jackson Laboratory (Bar Harbor, ME) can be obtained at 3 weeks of age and fed on conventional chow or diets enriched in either fat (35.5% wt/wt; Bioserv.Frenchtown, NJ) or fructose (60% wt/wt; Harlan Teklad, Madison, WI). The regular chow is composed of 4.5% wt/wt fat, 23% wt/wt protein, 31.9% wt/wt starch, 3.7% wt/wt fructose, and 5.3% wt/wt fiber. The high-fat (lard) diet is composed of 35.5% wt/wt fat, 20% wt/wt protein, 36.4% wt/wt starch, 0.0% wt/wt fructose, and 0.1% wt/wt fiber. The high-fructose diet is composed of 5% wt/wt fat, 20% wt/wt protein, 0.0% wt/wt starch, 60% wt/wt fructose, and 9.4% wt/wt fiber. The mice may be housed no more than five per cage at 22° +/- 3°C temperature- and 50% +/- 20% humidity-controlled room with a 12-hour light (6 am to 6 pm)/dark cycle (<nplcit id="ncit0197" npl-type="s"><text>Luo et al., 1998, Metabolism 47(6): 663-8, "Nongenetic mouse models of non-insulin-dependent diabetes mellitus</text></nplcit>"; <nplcit id="ncit0198" npl-type="s"><text>Larsen et al., Diabetes 50(11): 2530-9 (2001), "Systemic administration of the long-acting GLP-1 derivative NN2211 induces lasting and reversible weight loss in both normal and obese rats</text></nplcit>"). After exposure to the respective diets for 3 weeks, mice can be injected intraperitoneally with either streptozotocin, "STZ" (Sigma, St. Louis, MO), at 100 mg/kg body weight or vehicle (0.05 mol/L citric acid, pH 4.5) and kept on the same diet for the next 4 weeks. Under nonfasting conditions, blood is obtained 1, 2, and 4 weeks post-STZ by nipping the distal part of the tail. Samples are used to measure nonfasting plasma glucose and insulin concentrations. Body weight and food intake are recorded weekly.

0548To directly determine the effect of the high-fat diet on the ability of insulin to stimulate glucose disposal, the experiments can be initiated on three groups of mice, fat-fed, chow-fed injected with vehicle, and fat-fed injected with STZ at the end of the 7-week period described above. Mice can be fasted for 4 hours before the experiments. In the first series of experiments, mice can be anesthetized with methoxyflurane (Pitman-Moor, Mundelein, IL) inhalation. Regular insulin (Sigma) can be injected intravenously ([IV] 0.1 U/kg body weight) through a tail vein, and blood can be collected 3, 6, 9, 12, and 15 minutes after the injection from a different tail vein. Plasma glucose concentrations can be determined on these samples, and the half-life (t½) of glucose disappearance from plasma can be calculated using WinNonlin (Scientific Consulting, Apex, NC), a pharmacokinetics/pharmacodynamics software program.

0549In the second series of experiments, mice can be anesthetized with intraperitoneal sodium pentobarbital (Sigma). The abdominal cavity is opened, and the main abdominal vein is exposed and catheterized with a 24-gauge IV catheter (Johnson-Johnson Medical, Arlington, TX). The catheter is secured to muscle tissue adjacent to the abdominal vein, cut on the bottom of the syringe connection, and hooked to a prefilled PE50 plastic tube, which in turn is connected to a syringe with infusion solution. The abdominal cavity is then sutured closed. With this approach, there would be no blockage of backflow of the blood from the lower part of the body. Mice can be infused continuously with glucose (24.1 mg/kg/min) and insulin (10 mU/kg/min) at an infusion volume of 10 µL/min. Retro-orbital blood samples (70 µL each) can be taken 90, 105, 120, and 135 minutes after the start of infusion for measurement of plasma glucose and insulin concentrations. The mean of these four samples is used to estimate steady-state plasma glucose (SSPG) and insulin (SSPI) concentrations for each animal.

0550Finally, experiments to evaluate the ability of the albumin fusion proteins, the therapeutic compositions of the instant application, either alone or in combination with any one or more of the therapeutic drugs listed for the treatment of diabetes mellitus, to decrease plasma glucose can be performed in the following two groups of "NIDDM" mice models that are STZ-injected: (1) fat-fed C57BL/6J, and (2) fructose-fed C57BL/6J. Plasma glucose concentrations of the mice for these studies may range from 255 to 555 mg/dL. Mice are randomly assigned to treatment with either vehicle, albumin fusion therapeutics of the present invention either alone or in combination with any one or more of the therapeutic drugs listed for the treatment of diabetes mellitus. A total of three doses can be administered. Tail vein blood samples can be taken for measurement of the plasma glucose concentration before the first dose and 3 hours after the final dose.

0551Plasma glucose concentrations can be determined using the Glucose Diagnostic Kit from Sigma (Sigma No. 315), an enzyme colorimetric assay. Plasma insulin levels can be determined using the Rat Insulin RIA Kit from Linco Research (#RI-13K; St. Charles, MO).

<u>Example 51: Preparation of HA-hormone fusion protein (such as insulin, LH, FSH).</u>

0552The 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 (<figref idref="f0005">Figure 2</figref>), 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 1). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.

<b>Example</b> 57: <b>Preparation of targeted HA fusion proteins.</b>

0553The cDNA for the protein of interest can be isolated from cDNA library or can be 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 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 (<figref idref="f0005">Figure 2</figref>), 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 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.

<u>Example 59: Bacterial Expression of an Albumin Fusion Protein.</u>

0554A 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 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 (Amp^r), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

0555The 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^r). 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.

0556Clones containing the desired constructs are grown overnight (O/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.⁶⁰⁰) 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 lacI repressor, clearing the P/O leading to increased gene expression.

0557Cells 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., <nplcit id="ncit0199" npl-type="s"><text>Burton et al., Eur. J. Biochem. 179:379-387 (1989</text></nplcit>)). 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., <i>supra</i>). 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., <i>supra</i>).

0558Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8. The column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

0559The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. 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 NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 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 NaCl. The purified protein is stored at 4° C or frozen at -80° C.

0560In addition to the above expression vector, the present invention further includes an expression vector, called pHE4a (ATCC Accession Number 209645, deposited on February 25, 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 T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgamo sequence, and 6) the lactose operon repressor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, MD). The promoter and operator sequences are made synthetically.

0561DNA 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 ligated according to standard protocols.

0562The engineered vector may be substituted in the above protocol to express protein in a bacterial system.

<u>Example 60: Expression of an Albumin Fusion Protein in Mammalian Cells.</u>

0563The 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).

0564Suitable 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), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. 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 CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

0565Alternatively, the albumin fusion protein can be expressed in stable cell lines containing the polynucleotide encoding the albumin 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.

0566The 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., <nplcit id="ncit0200" npl-type="s"><text>Alt et al., J. Biol. Chem. 253:1357-1370 (1978</text></nplcit>); <nplcit id="ncit0201" npl-type="s"><text>Hamlin et al., Biochem. et Biophys. Acta, 1097:107-143 (1990</text></nplcit>); <nplcit id="ncit0202" npl-type="s"><text>Page et al., Biotechnology 9:64-68 (1991</text></nplcit>)). Another useful selection marker is the enzyme glutamine synthase (GS) (<nplcit id="ncit0203" npl-type="s"><text>Murphy et al., Biochem J. 227:277-279 (1991</text></nplcit>); <nplcit id="ncit0204" npl-type="s"><text>Bebbington et al., Bio/Technology 10:169-175 (1992</text></nplcit>). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.

0567Derivatives 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 (<nplcit id="ncit0205" npl-type="s"><text>Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985</text></nplcit>)) plus a fragment of the CMV-enhancer (<nplcit id="ncit0206" npl-type="s"><text>Boshart et al., Cell 41:521-530 (1985</text></nplcit>)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites 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.

0568Specifically, the plasmid 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.

0569A 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. <patcit id="pcit0117" dnum="WO9634891A"><text>WO 96/34891</text></patcit>.)

0570The 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.

0571The 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. <i>E</i>. <i>coli</i> 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.

0572Chinese hamster ovary cells lacking an active DHFR gene is used for transfection. Five µg of the expression plasmid pC6 or pC4 is cotransfected with 0.5 µg of the plasmid pSVneo using lipofectin (Felgner et al., <i>supra</i>). The plasmid pSV2-neo contains a dominant selectable marker, the <i>neo</i> gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. 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 methotrexate plus 1 mg/ml G418. 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 µM, 2 µM, 5 µM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 µM. Expression of the desired fusion protein is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.

<u>Example 69: Biological Effects of Fusion Proteins of the Invention.</u>

Astrocyte and Neuronal Assays

.

0573Albumin fusion proteins of the invention can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incorporation assay, for example, can be used to elucidate an albumin fusion protein of the invention's activity on these cells.

0574Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons <i>in vitro</i> have demonstrated increases in both neuron survival and neurite outgrowth (<nplcit id="ncit0207" npl-type="s"><text>Walicke et al., "Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension." Proc. NatL Acad Sci. USA 83:3012-3016. (1996</text></nplcit>)). However, reports from experiments done on PC-12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of an albumin fusion protein of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for examples, a thymidine incorporation assay.

Fibroblast and endothelial cell assays

.

0575Human lung fibroblasts are obtained from Clonetics (San Diego, CA) and maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, CA). For proliferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test fusion protein of the invention proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, CA) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CytoFluor fluorescence reader. For the PGE₂ assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or fusion protein of the invention with or without IL-1α for 24 hours. The supernatants are collected and assayed for PGE₂ by EIA kit (Cayman, Ann Arbor, MI). For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1 % BSA basal medium, the cells are incubated with FGF-2 or with or without an albumin fusion protein of the invention and/or IL-1α for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, MA).

0576Human lung fibroblasts are cultured with FGF-2 or an albumin fusion protein of the invention for 3 days in basal medium before the addition of Alamar Blue to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10 - 2500 ng/ml which can be used to compare stimulation with the fusion protein of the invention.

Cell proliferation based on [3H]thymidine incorporation

0577The following [3H]Thymidine incorporation assay can be used to measure the effect of a Therapeutic proteins, e.g., growth factor proteins, on the proliferation of cells such as fibroblast cells, epithelial cells or immature muscle cells.

0578Sub-confluent cultures are arrested in G1 phase by an 18 h incubation in serum-free medium. Therapeutic proteins are then added for 24 h and during the last 4 h, the cultures are labeled with [3H]thymidine, at a final concentration of 0.33 µM (25 Ci/mmol, Amersham, Arlington Heights, IL). The incorporated [3H]thymidine is precipitated with ice-cold 10% trichloroacetic acid for 24 h. Subsequently, the cells are rinsed sequentially with ice-cold 10% trichloroacetic acid and then with ice-cold water. Following lysis in 0.5 M NaOH, the lysates and PBS rinses (500 ml) are pooled, and the amount of radioactivity is NaOH, the lysates and PBS rinses (500 ml) are pooled, and the amount of radioactivity is measured.

Parkinson Models.

0579The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopaminergic projection neurons. An animal model for Parkinson' that has been extensively characterized involves the systemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized by monoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP⁺) and released. Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP⁺ is then concentrated in mitochondria by the electrochemical gradient and selectively inhibits nicotidamide adenine disphosphate: ubiquinone oxidoreductionase (complex I), thereby interfering with electron transport and eventually generating oxygen radicals.

0580It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (<nplcit id="ncit0208" npl-type="s"><text>Ferrari et al., Dev. Biol. 1989</text></nplcit>). Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (<nplcit id="ncit0209" npl-type="s"><text>Otto and Unsicker, J. Neuroscience, 1990</text></nplcit>).

0581Based on the data with FGF-2, an albumin fusion protein of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival <i>in vitro</i> and it can also be tested <i>in vivo</i> for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment. The potential' effect of an albumin fusion protein of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm² on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (N1). The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopaminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factors are also added at that time.

0582Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent an increase in the number of dopaminergic neurons surviving <i>in vitro.</i> Therefore, if a therapeutic protein of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the fusion protein may be involved in Parkinson's Disease.

<u>Example 70: The Effect of Albumin Fusion Proteins of the Invention on the Growth of Vascular Endothelial Cells.</u>

0583On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at 2-5x10⁴ cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10% FBS, 8 units/ml heparin. An albumin fusion proteins of the invention, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.

0584An increase in the number of HUVEC cells indicates that the fusion protein may proliferate vascular endothelial cells, while a decrease in the number of HUVEC cells indicates that the fusion protein inhibits vascular endothelial cells.

<u>Example 72: Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models.</u><i>Diabetic db+</i>/<i>db+ Mouse Model.</i>

0585To demonstrate that an albumin fusion protein of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+ mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epithelialization rather than contraction (<nplcit id="ncit0210" npl-type="s"><text>Gartner, M.H. et al., J. Surg. Res. 52:389 (1992</text></nplcit>); <nplcit id="ncit0211" npl-type="s"><text>Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990</text></nplcit>)).

0586The diabetic animals have many of the characteristic features observed in Type <b>II</b> diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their normal heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (<nplcit id="ncit0212" npl-type="s"><text>Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293 (1982</text></nplcit>)). Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (<nplcit id="ncit0213" npl-type="s"><text>Mandel et al., J. Immunol. 120:1375 (1978</text></nplcit>); <nplcit id="ncit0214" npl-type="s"><text>Debray-Sachs, M. et al., Clin. Exp. Immunol. 51(1):1-7 (1983</text></nplcit>); <nplcit id="ncit0215" npl-type="s"><text>Leiter et al., Am. J. of Pathol. 114:46-55 (1985</text></nplcit>)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been describe in these animals (<nplcit id="ncit0216" npl-type="s"><text>Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984</text></nplcit>); <nplcit id="ncit0217" npl-type="s"><text>Robertson et al., Diabetes 29(1):60-67 (1980</text></nplcit>); <nplcit id="ncit0218" npl-type="s"><text>Giacomelli et al., Lab Invest. 40(4):460-473 (1979</text></nplcit>); <nplcit id="ncit0219" npl-type="s"><text>Coleman, D.L., Diabetes 31 (Suppl):1-6 (1982</text></nplcit>)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (<nplcit id="ncit0220" npl-type="s"><text>Mandel et al., J. Immunol. 120:1375-1377 (1978</text></nplcit>)).

0587The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (<nplcit id="ncit0221" npl-type="s"><text>Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990</text></nplcit>)).

0588Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littermates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques, The experiments are conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.

0589Wounding protocol is performed according to previously reported methods (<nplcit id="ncit0222" npl-type="s"><text>Tsuboi, R. and Rifkin, D.B., J. Exp. Med. 172:245-251 (1990</text></nplcit>)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin (0.01 mg/mL), 2',2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

0590Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

0591An albumin fusion protein of the invention is administered using at a range different doses, from 4mg to 500mg per wound per day for 8 days in vehicle. Vehicle control groups received 50mL of vehicle solution.

0592Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

0593Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.

0594Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64mm², the corresponding size of the dermal punch. Calculations are made using the following formula: <maths id="math0001"><math display="block"><mfenced open="[" close="]"><mi>Open area on day</mi><mspace width="1em" /><mn>8</mn></mfenced><mo>-</mo><mfenced open="[" close="]"><mi>Open area on day</mi><mspace width="1em" /><mn>1</mn></mfenced><mo>/</mo><mfenced open="[" close="]"><mi>Open area on day</mi><mspace width="1em" /><mn>1</mn></mfenced></math><img file="EP1463751B1_D0002.tif" /></maths>

0595Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&amp;E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with an albumin fusion protein of the invention. This assessment included verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (<nplcit id="ncit0223" npl-type="s"><text>Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990</text></nplcit>)). A calibrated lens micrometer is used by a blinded observer.

0596Tissue sections are also stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control. Keratinocyte growth is determined by evaluating the extent of reepithelialization of the wound using a calibrated lens micrometer.

0597Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is demonstrated by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer served as a positive tissue control and human brain tissue is used as a negative tissue control. Each specimen included a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.

0598Experimental data are analyzed using an unpaired t test. A p value of &lt; 0.05 is considered significant

<i>Steroid Impaired Rat Model</i>

0599The inhibition of wound healing by steroids has been well documented in various <i>in vitro</i> and <i>in vivo</i> systems (<nplcit id="ncit0224" npl-type="s"><text>Wahl, Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989</text></nplcit>); <nplcit id="ncit0225" npl-type="s"><text>Wahlet al., J. Immunol. 115: 476-481 (1975</text></nplcit>); <nplcit id="ncit0226" npl-type="s"><text>Werb et al., J. Exp. Med. 147:1684-1694 (1978</text></nplcit>)). Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing vascular permeability (<nplcit id="ncit0227" npl-type="s"><text>Ebert et al., An. Intern. Med. 37:701-705 (1952</text></nplcit>)), fibroblasts proliferation, and collagen synthesis (<nplcit id="ncit0228" npl-type="s"><text>Beck et al., Growth Factors. 5: 295-304 (1991</text></nplcit>); <nplcit id="ncit0229" npl-type="s"><text>Haynes et al., J. Clin. Invest. 61: 703-797 (1978</text></nplcit>)) and producing a transient reduction of circulating monocytes (<nplcit id="ncit0230" npl-type="s"><text>Haynes et al., J. Clin. Invest. 61: 703-797 (1978</text></nplcit>); <nplcit id="ncit0231" npl-type="b"><text>Wahl, "Glucocorticoids and wound healing", In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989</text></nplcit>)). The systemic administration of steroids to impaired wound healing is a well establish phenomenon in rats (<nplcit id="ncit0232" npl-type="s"><text>Beck et al., Growth Factors. 5: 295-304 (1991</text></nplcit>); <nplcit id="ncit0233" npl-type="s"><text>Haynes et al., J. Clin. Invest. 61: 703-797 (1978</text></nplcit>); <nplcit id="ncit0234" npl-type="b"><text>Wahl, "Glucocorticoids and wound healing", In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989</text></nplcit>); <nplcit id="ncit0235" npl-type="s"><text>Pierce et al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989</text></nplcit>)).

0600To demonstrate that an albumin fusion protein of the invention can accelerate the healing process, the effects of multiple topical applications of the fusion protein on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of methylprednisolone is assessed.

0601Young adult male Sprague Dawley rats weighing 250-300 g (Charles River Laboratories) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water <i>ad libitum.</i> All manipulations are performed using aseptic techniques. This study is conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.

0602The wounding protocol is followed according to that described above. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wounds are left open for the duration of the experiment. Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

0603Wounds are visually examined and photographed at a fixed distance at the day of wounding and at the end of treatment. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

0604The fusion protein of the invention is administered using at a range different doses, from 4mg to 500mg per wound per day for 8 days in vehicle. Vehicle control groups received 50mL of vehicle solution.

0605Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300mg/kg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

0606Three groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups.

0607Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64mm². the corresponding size of the dermal punch. Calculations are made using the following formula: <maths id="math0002"><math display="block"><mfenced open="[" close="]"><mi>Open area on day</mi><mspace width="1em" /><mn>8</mn></mfenced><mo>-</mo><mfenced open="[" close="]"><mi>Open area on day</mi><mspace width="1em" /><mn>1</mn></mfenced><mo>/</mo><mfenced open="[" close="]"><mi>Open area on day</mi><mspace width="1em" /><mn>1</mn></mfenced></math><img file="EP1463751B1_D0003.tif" /></maths>

0608Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&amp;E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with an albumin fusion protein of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.

0609Experimental data are analyzed using an unpaired t test. A p value of &lt; 0.05 is considered significant.

<u>Example 75: Construction of GAS Reporter Construct.</u>

0610One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathways. Activated proteins in the Jaks-STATs pathway bind to gamma activation site "GAS" elements or interferon-sensitive responsive element ("ISRE"), located in the promoter of many genes. The binding of a protein to these elements alter the expression of the associated gene.

0611GAS and ISRE elements are recognized by a class of transcription factors called Signal Transducers and Activators of Transcription, or "STATs." There are six members of the STATs family. Stat1 and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and is not in many cell types though it has been found in T helper class I, cells after treatment with IL-12. Stat5 was originally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells. It can be activated in tissue culture cells by many cytokines.

0612The STATs are activated to translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase ("Jaks") family. Jaks represent a distinct family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinases display significant sequence similarity and are generally catalytically inactive in resting cells.

0613The Jaks are activated by a wide range of receptors summarized in the Table below. (Adapted from review by <nplcit id="ncit0236" npl-type="s"><text>Schidler and Darnell, Ann. Rev. Biochem. 64:621-51 (1995</text></nplcit>)). A cytokine receptor family, capable of activating Jaks, is divided into two groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF. CNTF, and thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding Trp-Ser-Xaa-Trp-Ser (SEQ ID NO: 1113)).

0614Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements. This entire process is encompassed in the Jaks-STATs signal transduction pathway. Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS or the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells. For example, growth factors and cytokines are known to activate the Jaks-STATs pathway (See Table 5, below). Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STATs pathway can be identified. <tables id="tabl0005" num="0005"><table frame="none"><title><b><u>Table 5</u></b></title><tgroup cols="6" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="42mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="14mm" /><colspec colnum="4" colname="col4" colwidth="14mm" /><colspec colnum="5" colname="col5" colwidth="14mm" /><colspec colnum="6" colname="col6" colwidth="52mm" /><thead><row><entry valign="top"><u>JAKs</u></entry><entry namest="col2" nameend="col5" align="center" valign="top"><u>STATS</u></entry><entry valign="top"><u>GAS(elements) or ISRE</u></entry></row><row><entry valign="top"><u>Ligand</u></entry><entry align="center" valign="top"><u>tyk2</u></entry><entry align="center" valign="top"><u>Jak1</u></entry><entry align="center" valign="top"><u>Jak2</u></entry><entry align="center" valign="top"><u>Jak3</u></entry><entry valign="top" /></row></thead><tbody><row><entry /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry /></row></tbody></tgroup><tgroup cols="6" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="42mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="14mm" /><colspec colnum="4" colname="col4" colwidth="14mm" /><colspec colnum="5" colname="col5" colwidth="14mm" /><colspec colnum="6" colname="col6" colwidth="52mm" /><thead><row><entry valign="top"><u>IFN family</u></entry><entry valign="top" /><entry valign="top" /><entry valign="top" /><entry valign="top" /><entry valign="top" /></row></thead><tbody><row><entry>IFN-a/B</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry><entry align="center">-</entry><entry>1,2,3 ISRE</entry></row><row><entry>IFN-g</entry><entry align="center" /><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry><entry>1 GAS (IRF1>Lys6>IFP)</entry></row><row><entry>Il-10</entry><entry align="center">+</entry><entry align="center">?</entry><entry align="center">?</entry><entry align="center">-</entry><entry>1,3</entry></row><row><entry /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry /></row></tbody></tgroup><tgroup cols="6" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="42mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="14mm" /><colspec colnum="4" colname="col4" colwidth="14mm" /><colspec colnum="5" colname="col5" colwidth="14mm" /><colspec colnum="6" colname="col6" colwidth="52mm" /><thead><row><entry valign="top"><u>gp130 family</u></entry><entry valign="top" /><entry valign="top" /><entry valign="top" /><entry valign="top" /><entry valign="top" /></row></thead><tbody><row><entry>IL-6 (Pleiotropic)</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">?</entry><entry>1,3 GAS(IRF1>Lys6>IFP)</entry></row><row><entry>Il-11(Pleiotropic)</entry><entry align="center">?</entry><entry align="center">+</entry><entry align="center">?</entry><entry align="center">?</entry><entry>1,3</entry></row><row><entry>OnM(Pleiotropic)</entry><entry align="center">?</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">?</entry><entry>1,3</entry></row><row><entry>LIF(Pleiotropic)</entry><entry align="center">?</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">?</entry><entry>1,3</entry></row><row><entry>CNTF(Pleiotropic)</entry><entry align="center">-/+</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">?</entry><entry>1,3</entry></row><row><entry>G-CSF(Pleiotropic)</entry><entry align="center">?</entry><entry align="center">+</entry><entry align="center">?</entry><entry align="center">?</entry><entry>1,3</entry></row><row><entry>IL-12(Pleiotropic)</entry><entry align="center">+</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">+</entry><entry>1,3</entry></row><row><entry /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry /></row></tbody></tgroup><tgroup cols="6" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="42mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="14mm" /><colspec colnum="4" colname="col4" colwidth="14mm" /><colspec colnum="5" colname="col5" colwidth="14mm" /><colspec colnum="6" colname="col6" colwidth="52mm" /><thead><row><entry valign="top"><u>g-C family</u></entry><entry valign="top" /><entry valign="top" /><entry valign="top" /><entry valign="top" /><entry valign="top" /></row></thead><tbody><row><entry>IL-2 (lymphocytes)</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">-</entry><entry align="center">+</entry><entry>1,3,5 GAS</entry></row><row><entry>IL-4 (lymph/myeloid)</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">-</entry><entry align="center">+</entry><entry>6GAS(IRF1=IFP>>Ly6)(IgH)</entry></row><row><entry>IL-7 (lymphocytes)</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">-</entry><entry align="center">+</entry><entry>5 GAS</entry></row><row><entry>IL-9 (lymphocytes)</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">-</entry><entry align="center">+</entry><entry>5 GAS</entry></row><row><entry>IL-13 (lymphocyte)</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">?</entry><entry align="center">?</entry><entry>6 GAS</entry></row><row><entry>IL-15</entry><entry align="center">?</entry><entry align="center">+</entry><entry align="center">?</entry><entry align="center">+</entry><entry>5 GAS</entry></row><row><entry /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry /></row><row><entry><u>gp140 family</u></entry><entry /><entry /><entry /><entry /><entry /></row><row><entry>IL-3 (myeloid)</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">-</entry><entry>5 GAS(IRF1>IFP>>Ly6)</entry></row><row><entry>IL-5 (myeloid)</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">-</entry><entry>5 GAS</entry></row><row><entry>GM-CSF (myeloid)</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">-</entry><entry>5 GAS</entry></row><row><entry /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry /></row></tbody></tgroup><tgroup cols="6" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="42mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="14mm" /><colspec colnum="4" colname="col4" colwidth="14mm" /><colspec colnum="5" colname="col5" colwidth="14mm" /><colspec colnum="6" colname="col6" colwidth="52mm" /><thead><row><entry valign="top"><u>Growth hormone family</u></entry><entry valign="top" /><entry valign="top" /><entry valign="top" /><entry valign="top" /><entry valign="top" /></row></thead><tbody><row><entry>GH</entry><entry align="center">?</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">-</entry><entry>5</entry></row><row><entry>PRL</entry><entry align="center">?</entry><entry align="center">+/-</entry><entry align="center">+</entry><entry align="center">-</entry><entry>1,3,5</entry></row><row><entry>EPO</entry><entry align="center">?</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">-</entry><entry>5 GAS (B-CAS>IRF1=IFP>>Ly6)</entry></row><row><entry /><entry align="center" /><entry align="center" /><entry align="center" /><entry align="center" /><entry /></row></tbody></tgroup><tgroup cols="6" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="42mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="14mm" /><colspec colnum="4" colname="col4" colwidth="14mm" /><colspec colnum="5" colname="col5" colwidth="14mm" /><colspec colnum="6" colname="col6" colwidth="52mm" /><thead><row><entry valign="top"><u>Receptor Tyrosine Kinases</u></entry><entry valign="top" /><entry valign="top" /><entry valign="top" /><entry valign="top" /><entry valign="top" /></row></thead><tbody><row><entry>EGF</entry><entry align="center">?</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry><entry>1,3 GAS (IRF1)</entry></row><row><entry>PDGF</entry><entry align="center">?</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry><entry>1,3</entry></row><row><entry>CSF-1</entry><entry align="center">?</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry><entry>1,3 GAS(not IRF1)</entry></row></tbody></tgroup></table></tables>

0615To construct a synthetic GAS containing promoter element, which is used in the Biological Assays described in Examples 78-80, a PCR based strategy is employed to generate a GAS-SV40 promoter sequence. The 5' primer contains four tandem copies of the GAS binding site found in the IRF1 promoter and previously demonstrated to bind STATs upon induction with a range of cytokines (<nplcit id="ncit0237" npl-type="s"><text>Rothman et al., Immunity 1:457-468 (1994</text></nplcit>).), although other GAS or ISRE elements can be used instead. The 5' primer also contains 18bp of sequence complementary to the SV40 early promoter sequence and is flanked with an XhoI site. The sequence of the 5' primer is: <img file="EP1463751B1_D0004.tif" />

0616The downstream primer is complementary to the SV40 promoter and is flanked with a Hind III site: 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO: 1115)

0617PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI/Hind III and subcloned into BLSK2-. (Stratagene.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence: <img file="EP1463751B1_D0005.tif" />

0618With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or "SEAP." Clearly, however, any reporter molecule can be instead of SEAP, in this or in any of the other Examples. Well known reporter molecules that can be used instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.

0619The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned into the pSEAP-Promoter vector obtained from Clontech using HindIII and XhoI, effectively replacing the SV40 promoter with the amplified GAS:SV40 promoter element, to create the GAS-SEAP vector. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.

0620Thus, in order to generate mammalian stable cell lines expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using SalI and NotI, and inserted into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (Clontech), using these restriction sites in the multiple cloning site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 78-80.

0621Other constructs can be made using the above description and replacing GAS with a different promoter sequence. For example, construction of reporter molecules containing EGR and NF-KB promoter sequences are described in Examples 78-82. However, many other promoters can be substituted using the protocols described in these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, II-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used to test reporter construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.

<u>Example 76: Assay for SEAP Activity.</u>

0622As a reporter molecule for the assays described in examples disclosed herein, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.

0623Prime a dispenser with the 2.5x Dilution Buffer and dispense 15 ul of 2.5x dilution buffer into Optiplates containing 35 ul of a solution containing an albumin fusion protein of the invention. Seal the plates with a plastic sealer and incubate at 65 degree C for 30 min. Separate the Optiplates to avoid uneven heating.

0624Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see the Table below). Add 50 ul Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates on a luminometer, thus one should treat 5 plates at each time and start the second set 10 minutes later.

0625Read the relative light unit in the luminometer. Set H12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity. <tables id="tabl0006" num="0006"><table frame="all"><title><b><u>Table 6</u></b></title><tgroup cols="6"><colspec colnum="1" colname="col1" colwidth="20mm" /><colspec colnum="2" colname="col2" colwidth="38mm" /><colspec colnum="3" colname="col3" colwidth="22mm" /><colspec colnum="4" colname="col4" colwidth="20mm" /><colspec colnum="5" colname="col5" colwidth="38mm" /><colspec colnum="6" colname="col6" colwidth="22mm" /><thead><row><entry valign="top"><b># of plates</b></entry><entry valign="top"><b>Rxn buffer diluent (ml)</b></entry><entry valign="top"><b>CSPD (ml)</b></entry><entry valign="top"><b># of plates</b></entry><entry valign="top"><b>Rxn buffer diluent (ml)</b></entry><entry valign="top"><b>CSPD (ml)</b></entry></row></thead><tbody><row><entry>10</entry><entry>60</entry><entry>3</entry><entry>31</entry><entry>165</entry><entry>8.25</entry></row><row><entry>11</entry><entry>65</entry><entry>3.25</entry><entry>32</entry><entry>170</entry><entry>8.5</entry></row><row><entry>12</entry><entry>70</entry><entry>3.5</entry><entry>33</entry><entry>175</entry><entry>8.75</entry></row><row><entry>13</entry><entry>75</entry><entry>3.75</entry><entry>34</entry><entry>180</entry><entry>9</entry></row><row><entry>14</entry><entry>80</entry><entry>4</entry><entry>35</entry><entry>185</entry><entry>9.25</entry></row><row><entry>15</entry><entry>85</entry><entry>4.25</entry><entry>36</entry><entry>190</entry><entry>9.5</entry></row><row><entry>16</entry><entry>90</entry><entry>4.5</entry><entry>37</entry><entry>195</entry><entry>9.75</entry></row><row><entry>17</entry><entry>95</entry><entry>4.75</entry><entry>38</entry><entry>200</entry><entry>10</entry></row><row><entry>18</entry><entry>100</entry><entry>5</entry><entry>39</entry><entry>205</entry><entry>10.25</entry></row><row><entry>19</entry><entry>105</entry><entry>5.25</entry><entry>40</entry><entry>210</entry><entry>10.5</entry></row><row><entry>20</entry><entry>110</entry><entry>5.5</entry><entry>41</entry><entry>215</entry><entry>10.75</entry></row><row><entry>21</entry><entry>115</entry><entry>5.75</entry><entry>42</entry><entry>220</entry><entry>11</entry></row><row><entry>22</entry><entry>120</entry><entry>6</entry><entry>43</entry><entry>225</entry><entry>11.25</entry></row><row><entry>23</entry><entry>125</entry><entry>6.25</entry><entry>44</entry><entry>230</entry><entry>11.5</entry></row><row><entry>24</entry><entry>130</entry><entry>6.5</entry><entry>45</entry><entry>235</entry><entry>11.75</entry></row><row><entry>25</entry><entry>135</entry><entry>6.75</entry><entry>46</entry><entry>240</entry><entry>12</entry></row><row><entry>26</entry><entry>140</entry><entry>7</entry><entry>47</entry><entry>245</entry><entry>12.25</entry></row><row><entry>27</entry><entry>145</entry><entry>7.25</entry><entry>48</entry><entry>250</entry><entry>12.5</entry></row><row><entry>28</entry><entry>150</entry><entry>7.5</entry><entry>49</entry><entry>255</entry><entry>12.75</entry></row><row><entry>29</entry><entry>155</entry><entry>7.75</entry><entry>50</entry><entry>260</entry><entry>13</entry></row><row><entry>30</entry><entry>160</entry><entry>8</entry><entry /><entry /><entry /></row></tbody></tgroup></table></tables>

<u>Example 77: Assay Identifying Neuronal Activity.</u>

0626When cells undergo differentiation and proliferation, a group of genes are activated through many different signal transduction pathways. One of these genes, EGR1 (early growth response gene 1), is induced in various tissues and cell types upon activation. The promoter of EGR1 is responsible for such induction. Using the EGR1 promoter linked to reporter molecules, the ability of fusion proteins of the invention to activate cells can be assessed.

0627Particularly, the following protocol is used to assess neuronal activity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are known to proliferate and/or differentiate by activation with a number of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). The EGR1 gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to SEAP reporter, activation of PC12 cells by an albumin fusion protein of the present invention can be assessed.

0628The EGR/SEAP reporter construct can be assembled by the following protocol. The EGR-1 promoter sequence (-633 to +1)(<nplcit id="ncit0238" npl-type="s"><text>Sakamoto K et al., Oncogene 6:867-871 (1991</text></nplcit>)) can be PCR amplified from human genomic DNA using the following primers: <ul id="ul0008" list-style="none" compact="compact"><li>First primer. 5' GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3' (SEQ ID NO: 1117)</li><li>Second primer: 5' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3' (SEQ ID NO: 1118)</li></ul>

0629Using the GAS:SEAP/Neo vector produced in Example 75, EGR1 amplified product can then be inserted into this vector. Linearize the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1promoter.

0630To prepare 96 well-plates for cell culture, two mls of a coating solution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cm plate or 50 ml per well of the 96-well plate, and allowed to air dry for 2 hr.

0631PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 ug/ml streptomycin on a precoated 10 cm tissue culture dish. One to four split is done every three to four days. Cells are removed from the plates by scraping and resuspended with pipetting up and down for more than 15 times.

0632Transfect the EGR/SEAP/Neo construct into PC12 using techniques known in the art. EGR-SEAP/PC12 stable cells are obtained by growing the cells in 300 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 300 ug/ml G418 for couple of passages.

0633To assay for neuronal activity, a 10 cm plate with cells around 70 to 80% confluent is screened by removing the old medium. Wash the cells once with PBS (Phosphate buffered saline). Then starve the cells in low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS with antibiotics) overnight.

0634The next morning, remove the medium and wash the cells with PBS. Scrape off the cells from the plate, suspend the cells well in 2 ml low serum medium. Count the cell number and add more low serum medium to reach final cell density as 5x10⁵ cells/ml.

0635Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to 1x10⁵ cells/well). Add a series of different concentrations of an albumin fusion protein of the inventon, 37 degree C for 48 to 72 hr. As a positive control, a growth factor known to activate PC 12 cells through EGR can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAP is typically seen in the positive control wells. SEAP assay may be routinely performed using techniques known in the art and/or as described in Example 76.

<u>Example 78: Assay for T-cell Activity.</u>

0636The following protocol is used to assess T-cell activity by identifying factors, and determining whether an albumin fusion protein of the invention proliferates and/or differentiates T-cells. T-cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 75. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The T-cell used in this assay is Jurkat T-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582) cells can also be used.

0637Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In order to generate stable cell lines, approximately 2 million Jurkat cells are transfected with the GAS-SEAP/neo vector using DMRIE-C (Life Technologies)(transfection procedure described below). The transfected cells are seeded to a density of approximately 20,000 cells per well and transfectants resistant to 1 mg/ml genticin selected. Resistant colonies are expanded and then tested for their response to increasing concentrations of interferon gamma. The dose response of a selected clone is demonstrated.

0638Specifically, the following protocol will yield sufficient cells for 75 wells containing 200 ul of cells. Thus, it is either scaled up, or performed in multiple to generate sufficient cells for multiple 96 well plates. Jurkat cells are maintained in RPMI + 10% serum with 1%Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C and incubate at room temperature for 15-45 mins.

0639During the incubation period, count cell concentration, spin down the required number of cells (10⁷ per transfection), and resuspend in OPTI-MEM to a final concentration of 10⁷ cells/mL Then add 1ml of 1 x 10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degree C for 6 hrs. After the incubation, add 10 ml of RPMI + 15% serum.

0640The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI + 10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated with varying concentrations of one or more fusion proteins of the present invention.

0641On the day of treatment with the fusion protein, the cells should be washed and resuspended in fresh RPMI + 10% serum to a density of 500,000 cells per ml. The exact number of cells required will depend on the number of fusion proteins and the number of different concentrations of fusion proteins being screened. For one 96 well plate, approximately 10 million cells (for 10 plates, 100 million cells) are required.

0642The well dishes containing Jurkat cells treated with the fusion protein are placed in an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35 ul samples from each well are then transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plates should be covered (using sellophene covers) and stored at -20 degree C until SEAP assays are performed according to Example 76. The plates containing the remaining treated cells are placed at 4 degree C and serve as a source of material for repeating the assay on a specific well if desired.

0643As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate Jurkat T cells. Over 30 fold induction is typically observed in the positive control wells.

0644The above protocol may be used in the generation of both transient, as well as, stable transfected cells, which would be apparent to those of skill in the art.

<u>Example 79: Assay for T-cell Activity.</u>

0645NF-KB (Nuclear Factor KB) is a transcription factor activated by a wide variety of agents including the inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by expression of certain viral gene products. As a transcription factor, NF-KB regulates the expression of genes involved in immune cell activation, control of apoptosis (NF- KB appears to shield cells from apoptosis), B and T-cell development, anti-viral and antimicrobial responses, and multiple stress responses.

0646In non-stimulated conditions, NF- KB is retained in the cytoplasm with I-KB (Inhibitor KB). However, upon stimulation, I- KB is phosphorylated and degraded, causing NF- KB to shuttle to the nucleus, thereby activating transcription of target genes. Target genes activated by NF- KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

0647Due to its central role and ability to respond to a range of stimuli, reporter constructs utilizing the NF-KB promoter element are used to screen the fusion protein. Activators or inhibitors of NF-KB would be useful in treating, preventing, and/or diagnosing diseases. For example, inhibitors of NF-KB could be used to treat those diseases related to the acute or chronic activation of NF-KB, such as rheumatoid arthritis.

0648To construct a vector containing the NF-KB promoter element, a PCR based strategy is employed. The upstream primer contains four tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO: 1119), 18 bp of sequence complementary to the 5' end of the SV40 early promoter sequence, and is flanked with an XhoI site: <img file="EP1463751B1_D0006.tif" />

0649The downstream primer is complementary to the 3' end of the SV40 promoter and is flanked with a Hind III site: <ul id="ul0009" list-style="none" compact="compact"><li>5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO: 1115)</li></ul>

0650PCR amplification is performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI and Hind III and subcloned into BLSK2-. (Stratagene) Sequencing with the T7 and T3 primers confirms the insert contains the following sequence: <img file="EP1463751B1_D0007.tif" />

0651Next, replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment using XhoI and HindIII. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.

0652In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP vector using restriction enzymes SalI and NotI, and inserted into a vector containing neomycin resistance. Particularly, the NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 with SalI and NotL

0653Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are created and maintained according to the protocol described in Example 76. Similarly, the method for assaying fusion proteins with these stable Jurkat T-cells is also described in Example 76. As a positive control, exogenous TNF alpha (0.1,1, 10 ng) is added to wells H9, H10, and H11, with a 5-10 fold activation typically observed.

<u>Example 80:_Assay Identifying Myeloid Activity.</u>

0654The following protocol is used to assess myeloid activity of an albumin fusion protein of the present invention by determining whether the fusion protein proliferates and/or differentiates myeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 75. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is U937, a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used.

0655To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced in Example 75, a DEAE-Dextran method (<nplcit id="ncit0239" npl-type="s"><text>Kharbanda et. al., 1994, Cell Growth &amp; Differentiation, 5:259-265</text></nplcit>) is used. First, harvest 2x10⁷ U937 cells and wash with PBS. The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.

0656Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaCl, 5 mM KCI, 375 uM Na₂HPO₄.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂. Incubate at 37 degrees C for 45 min.

0657Wash the cells with RPMI 1640 medium containing 10% FBS and then resuspend in 10 ml complete medium and incubate at 37 degree C for 36 hr.

0658The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 400 ug/mlG418 for couple of passages.

0659These cells are tested by harvesting 1x10⁸ cells (this is enough for ten 96-well plates assay) and wash with PBS. Suspend the cells in 200 ml above described growth medium, with a final density of 5x10⁵ cells/mL Plate 200 ul cells per well in the 96-well plate (or 1x10⁵ cells/well).

0660Add different concentrations of the fusion protein. Incubate at 37 degee C for 48 to 72 hr. As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate U937 cells. Over 30 fold induction is typically observed in the positive control wells. SEAP assay the supernatant according to methods known in the art and/or the protocol described in Example 76.

<u>Example 81: Assay Identifying Changes in_Small Molecule Concentration and Membrane Permeabitity.</u>

0661Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential. These alterations can be measured in an assay to identify fusion proteins which bind to receptors of a particular cell. Although the following protocol describes an assay for calcium, this protocol can easily be modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule which is detectable by a fluorescent probe.

0662The following assay uses Fluorometric Imaging Plate Reader ("FLIPR") to measure changes in fluorescent molecules (Molecular Probes) that bind small molecules. Clearly, any fluorescent molecule detecting a small molecule can be used instead of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.; catalog no. F-14202), used here.

0663For adherent cells, seed the cells at 10,000 -20,000 cells/well in a Co-star black 96-well plate with clear bottom. The plate is incubated in a CO₂ incubator for 20 hours. The adherent cells are washed two times in Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash.

0664A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to each well. The plate is incubated at 37 degrees C in a CO₂ incubator for 60 min. The plate is washed four times in the Biotek washer with HBSS leaving 100 ul of buffer.

0665For non-adherent cells, the cells are spun down from culture media. Cells are re-suspended to 2-5x10⁶ cells/ml with HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The tube is then placed in a 37 degrees C water bath for 30-60 min. The cells are washed twice with HBSS, resuspended to 1x10⁶ cells/ml, and dispensed into a microplate, 100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley Cell Wash with 200 ul, followed by an aspiration step to 100 ul final volume.

0666For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-4. The fusion protein of the invention is added to the well, and a change in fluorescence is detected.

0667To measure the fluorescence of intracellular calcium, the FLIPR is set for the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul. Increased emission at 530 nm indicates an extracellular signaling event caused by an albumin fusion protein of the present invention or a molecule induced by an albumin fusion protein of the present invention, which has resulted in an increase in the intracellular Ca⁺⁺ concentration.

<u>Example 82: Assay Identifying Tyrosine Kinase Activity.</u>

0668The Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine Kinase (RPTK) group are receptors for a range of mitogenic and metabolic growth factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix proteins.

0669Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated tyrosine kinases of the src-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine superfamily of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

0670Because of the wide range of known factors capable of stimulating tyrosine kinase activity, identifying whether an albumin fusion protein of the present invention or a molecule induced by a fusion proetin of the present invention is capable of activating tyrosine kinase signal transduction pathways is of interest. Therefore, the following protocol is designed to identify such molecules capable of activating the tyrosine kinase signal transduction pathways.

0671Seed target cells (e.g., primary keratinocytes) at a density of approximately 25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased from Nalge Nunc (Naperville, IL). The plates are sterilized with two 30 minute rinses with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, MO) or 10% Matrigel purchased from Becton Dickinson (Bedford,MA), or calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of alamarBlue as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, CA) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford,MA) are used to cover the Loprodyne Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in some proliferation experiments.

0672To prepare extracts, A431 cells are seeded onto the nylon membranes of Loprodyne plates (20,000/200ml/well) and cultured overnight in complete medium. Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF (60ng/ml) or a different concentrations of an albumin fusion protein of the invention, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and a cocktail of protease inhibitors (# 1836170) obtained from' Boeheringer Mannheim (Indianapolis, IN)) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 4°C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using house vacuum. Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice. To obtain extracts clarified by centrifugation, the content of each well, after detergent solubilization for 5 minutes, is removed and centrifuged for 15 minutes at 4 degree C at 16,000 x g.

0673Test the filtered extracts for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one method is described here.

0674Generally, the tyrosine kinase activity of an albumin fusion protein of the invention is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a range of tyrosine kinases and are available from Boehringer Mannheim.

0675The tyrosine kinase reaction is set up by adding the following components in order. First, add 10ul of 5uM Biotinylated Peptide, then 10ul ATP/Mg₂₊ (5mM ATP/50mM MgCl₂), then 10ul of 5x Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1mM EGTA, 100mM MgCl₂, 5 mM MaCl₂, 0.5 mg/ml BSA), then 5ul of Sodium Vanadate(1mM), and then 5ul of water. Mix the components gently and preincubate the reaction mix at 30 degree C for 2 min. Initial the reaction by adding 10ul of the control enzyme or the filtered supernatant.

0676The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120mm EDTA and place the reactions on ice.

0677Tyrosine kinase activity is determined by transferring 50 ul aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 37 degree C for 20 min. This allows the streptavidin coated 96 well plate to associate with the biotinylated peptide. Wash the MTP module with 300ul/well of PBS four times. Next add 75 ul of anti-phospotyrosine antibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5u/ml)) to each well and incubate at 37 degree C for one hour. Wash the well as above.

0678Next add 100ul of peroxidase substrate solution (Boehringer Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min). Measure the absorbance of the sample at 405 nm by using ELISA reader. The level of bound peroxidase activity is quantitated using an ELISA reader and reflects the level of tyrosine kinase activity.

<u>Example 86: Human Dermal Fibroblast and Aortic Smooth Muscle Cell Proliferation.</u>

0679An albumin fusion protein of the invention is added to cultures of normal human dermal fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC) and two co-assays are performed with each sample. The first assay examines the effect of the fusion protein on the proliferation of normal human dermal fibroblasts (NHDF) or aortic smooth muscle cells (AoSMC). Aberrant growth of fibroblasts or smooth muscle cells is a part of several pathological processes, including fibrosis, and restenosis. The second assay examines IL6 production by both NHDF and SMC. IL6 production is an indication of functional activation. Activated cells will have increased production of a number of cytokines and other factors, which can result in a proinflammatory or immunomodulatory outcome. Assays are run with and without co-TNFa stimulation, in order to check for costimulatory or inhibitory activity..

0680Briefly, on day 1, 96-well black plates are set up with 1000 cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 µl culture media. NHDF culture media contains: Clonetics FB basal media, 1mg/ml hFGF, 5mg/ml insulin, 50mg/ml geatamycin, 2%FBS, while AoSMC culture media contains Clonetics SM basal media, 0.5 µg/ml hEGF, 5mg/ml insulin, 1µg/ml hFGF, 50mg/ml gentamycin, 50 µg/ml Amphotericin B, 5%FBS. After incubation at 37°C for at least 4-5 hours culture media is aspirated and replaced with growth arrest media. Growth arrest media for NHDF contains fibroblast basal media, 50mg/ml gentamycin, 2% FBS, while growth arrest media for AoSMC contains SM basal media, 50mg/ml gentamycin, 50µg/ml Amphotericin B, 0.4% FBS. Incubate at 37°C until day 2.

0681On day 2, serial dilutions and templates of an albumin fusion protein of the invention are designed such that they always include media controls and known-protein controls. For both stimulation and inhibition experiments, proteins are diluted in growth arrest media. For inhibition experiments, TNFa is added to a final concentration of 2ng/ml (NHDF) or 5ng/ml (AoSMC). Add 1/3 vol media containing controls or an albumin fusion protein of the invention and incubate at 37 degrees C/5% CO₂ until day 5.

0682Transfer 60µl from each well to another labeled 96-well plate, cover with a plate-sealer, and store at 4 degrees C until Day 6 (for IL6 ELISA). To the remaining 100 µl in the cell culture plate, aseptically add Alamar Blue in an amount equal to 10% of the culture volume (10µl). Return plates to incubator for 3 to 4 hours. Then measure fluorescence with excitation at 530nm and emission at 590nm using the CytoFluor. This yields the growth stimulation/inhibition data.

0683On day 5, the IL6 ELISA is performed by coating a 96 well plate with 50-100 ul/well of Anti-Human IL6 Monoclonal antibody diluted in PBS, pH 7.4, incubate ON at room temperature.

0684On day 6, empty the plates into the sink and blot on paper towels. Prepare Assay Buffer containing PBS with 4% BSA. Block the plates with 200 µl/well of Pierce Super Block blocking buffer in PBS for 1-2 hr and then wash plates with wash buffer (PBS, 0.05% Tween-20). Blot plates on paper towels. Then add 50 µl/well of diluted Anti-Human IL-6 Monoclonal, Biotin-labeled antibody at 0.50 mg/ml. Make dilutions of IL-6 stock in media (30, 10, 3, 1, 0.3, 0 ng/ml). Add duplicate samples to top row of plate. Cover the plates and incubate for 2 hours at RT on shaker.

0685Plates are washed with wash buffer and blotted on paper towels. Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and add 100 µl/well. Cover the plate and incubate 1 h at RT. Plates are again washed with wash buffer and blotted on paper towels.

0686Add 100 µl/well of Enhancement Solution. Shake for 5 minutes. Read the plate on the Wallac DELFIA Fluommeter. Readings from triplicate samples in each assay were tabulated and averaged.

0687A positive result in this assay suggests AoSMC cell proliferation and that the albumin fusion protein may be involved in dermal fibroblast proliferation and/or smooth muscle cell proliferation. A positive result also suggests many potential uses of the fusion protein and polynucleotides encoding the albumin fusion protein. For example, inflammation and immune responses, wound healing, and angiogenesis, as detailed throughout this specification. Particularly, fusion proteins may be used in wound healing and dermal regeneration, as well as the promotion of vasculogenesis, both of the blood vessels and lymphatics. The growth of vessels can be used in the treatment of, for example, cardiovascular diseases. Additionally, fusion proteins showing antagonistic activity in this assay may be useful in treating diseases, disorders, and/or conditions which involve angiogenesis by acting as an anti-vascular agent (e.g., anti-angiogenesis). These diseases, disorders, and/or conditions are known in the art and/or are described herein, such as, for example, malignancies, solid tumors, 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 collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis. Moreover, albumin fusion proteins that act as antagonists in this assay may be useful in treating anti-hyperproliferative diseases and/or anti-inflammatory known in the art and/or described herein.

<u>Example 87: Cellular Adhesion Molecule (CAM) Expression on Endothelial Cells.</u>

0688The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.

0689Briefly, endothelial cells (e.g., Human Umbilical Vein Endothelial cells (HUVECs)) are grown in a standard 96 well plate to confluence, growth medium is removed from the cells and replaced with 100 µl of 199 Medium (10% fetal bovine serum (FBS)). Samples for testing (containing an albumin fusion protein of the invention) and positive or negative controls are added to the plate in triplicate (in 10 µl volumes). Plates are then incubated at 37°C for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 µl of 0.1% paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well. Plates are held at 4°C for 30 min. Fixative is removed from the wells and wells are washed 1X with PBS(+Ca,Mg) + 0.5% BSA and drained. 10 µl of diluted primary antibody is added to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 ug/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37°C for 30 min. in a humidified environment. Wells are washed three times with PBS(+Ca,Mg) + 0.5% BSA. 20 µl of diluted ExtrAvidin-Alkaline Phosphatase (1:5,000 dilution, referred to herein as the working dilution) are added to each well and incubated at 37°C for 30 min. Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. Dissolve 1 tablet of p-Nitrophenol Phosphate pNPP per 5 ml of glycine buffer (pH 10.4). 100 µl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 (10°) > 10^-0.5 > 10^-1 > 10^-1.5.5 µl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 µl of pNNP reagent is then added to each of the standard wells. The plate is incubated at 37°C for 4h. A volume of 50 µl of 3M NaOH is added to all wells. The plate is read on a plate reader at 405 nm using the background subtraction option on blank wells filled with glycine buffer only. Additionally, the template is set up to indicate the concentration of AP-conjugate in each standard well [ 5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.

<u>Example 88: Alamar Blue Endothelial Cells Proliferation Assay.</u>

0690This assay may be used to quantitatively determine protein mediated inhibition of bFGF-induced proliferation of Bovine Lymphatic Endothelial Cells (LECs), Bovine Aortic Endothelial Cells (BAECs) or Human Microvascular Uterine Myometrial Cells (UTMECs). This assay incorporates a fluorometric growth indicator based on detection of metabolic activity. A standard Alamar Blue Proliferation Assay is prepared in EGM-2MV with 10 ng /ml of bFGF added as a source of endothelial cell stimulation. This assay may be used with a variety of endothelial cells with slight changes in growth medium and cell concentration. Dilutions of protein batches to be tested are diluted as appropriate. Serum-free medium (GIBCO SFM) without bFGF is used as a non-stimulated control and Angiostatin or TSP-1 are included as a known inhibitory controls.

0691Briefly, LEC, BAECs or UTMECs are seeded in growth media at a density of 5000 to 2000 cells/well in a 96 well plate and placed at 37 degreesC overnight. After the overnight incubation of the cells, the growth media is removed and replaced with GIBCO EC-SFM. The cells are treated with the appropriate dilutions of an albumin fusion protein of the invention or control protein sample(s) (prepared in SFM) in triplicate wells with additional bFGF to a concentration of 10 ng/ ml. Once the cells have been treated with the samples, the plate(s) is/are placed back in the 37° C incubator for three days. After three days 10 ml of stock alamar blue (Biosource Cat# DAL1100) is added to each well and the plate(s) is/are placed back in the 37°C incubator for four hours. The plate(s) are then read at 530nm excitation and 590nm emission using the CytoFluor fluorescence reader. Direct output is recorded in relative fluorescence units.

0692Alamar blue is an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. As cells grow in culture, innate metabolic activity results in a chemical reduction of the immediate surrounding environment. Reduction related to growth causes the indicator to change from oxidized (non-fluorescent blue) form to reduced (fluorescent red) form (i.e., - stimulated proliferation will produce a stronger signal and inhibited proliferation will produce a weaker signal and the total signal is proportional to the total number of cells as well as their metabolic activity). The background level of activity is observed with the starvation medium alone. This is compared to the output observed from the positive control samples (bFGF in growth medium) and protein dilutions.

<u>Example 92: Ligand Binding Assays.</u>

0693The following assay may be used to assess ligand binding activity of an albumin fusion protein of the invention.

0694Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format. The purified ligand for an albumin fusion protein of the invention is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards the fusion protein. Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell polypeptide sources. For these assays, specific polypeptide binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.

<u>Example 101: Support of Chicken Embryo Neuron Survival.</u>

0695To test whether sympathetic neuronal cell viability is supported by an albumin fusion protein of the invention, the chicken embryo neuronal survival assay of Senaldi <i>et al</i> may be utilized <i>(</i><nplcit id="ncit0240" npl-type="s"><text>Proc. Natl. Acad. Sci., U.S.A., 96:11458-63 (1998</text></nplcit>)). Briefly, motor and sympathetic neurons are isolated from chicken embryos, resuspended in L15 medium (with 10% FCS, glucose, sodium selenite, progesterone, conalbumin, putrescine, and insulin; Life Technologies, Rockville, MD.) and Dulbecco's modified Eagles medium [with 10% FCS, glutamine, penicillin, and 25 mM Hepes buffer (pH 7.2); Life Technologies, Rockville, MD.], respectively, and incubated at 37°C in 5% CO₂ in the presence of different concentrations of the purified fusion protein of the invention, as well as a negative control lacking any cytokine. After 3 days, neuron survival is determined by evaluation of cellular morphology, and through the use of the colorimetric assay of Mosmann (<nplcit id="ncit0241" npl-type="s"><text>Mosmann, T., J. Immunol. Methods, 65:55-63 (1983</text></nplcit>)). Enhanced neuronal cell viability as compared to the controls lacking cytokine is indicative of the ability of the albumin fusion protein to enhance the survival of neuronal cells.

<u>Example 105: Immobillzation of biomolecules.</u>

0696This example provides a method for the stabilization of an albumin fusion protein of the invention in non-host cell lipid bilayer constucts (see, e.g., <nplcit id="ncit0242" npl-type="s"><text>Bieri et al., Nature Biotech 17:1105-1108 (1999</text></nplcit>)) which can be adapted for the study of fusion proteins of the invention in the various functional assays described above. Briefly, carbohydrate-specific chemistry for biotinylation is used to confine a biotin tag to an albumin fusion protein of the invention, thus allowing uniform orientation upon immobilization. A 50uM solution of an albumin fusion protein of the invention in washed membranes is incubated with 20 mM NaIO4 and 1.5 mg/ml (4mM) BACH or 2 mg/m1 (7.5mM) biotin-hydrazide for 1 hr at room temperature (reaction volume, 150ul). Then the sample is dialyzed (Pierce Slidealizer Cassett, 10 kDa cutoff; Pierce Chemical Co., Rockford IL) at 4C first for 5 h, exchanging the buffer after each hour, and finally for 12 h against 500 ml buffer R (0.15 M NaCl, 1 mM MgCl2, 10 mM sodium phosphate, pH7). Just before addition into a cuvette, the sample is diluted 1:5 in buffer ROG50 (Buffer R supplemented with 50 mM octylglucoside).

<u>Example 116: Activity of Construct 3070 (GLP-1 Albumin Fusion) Measured by <i>In Vitro</i> Stimulation of Insulin mRNA in INS-1 Cells</u>

0697It has recently been shown that GLP-1 increases the expression of insulin mRNA in pancreatic beta-cells (<nplcit id="ncit0243" npl-type="s"><text>Buteau et al., Diabetologia 1999 Jul;42(7):856-64</text></nplcit>). Thus, the ability of the GLP-1 albumin fusion protein encoded by CID 3070 to stimulate insulin mRNA was evaluated using the pancreatic beta-cell line INS-1 (832/13).

0698<figref idref="f0018">Figure 14</figref> illustrates the steady-state levels of insulin mRNA in INS-1 (832/13) cells after treatment with GLP-1 or GLP-1 albumin fusion protein encoded by construct ID 3070 (CID 3070 protein). Both GLP-1 and the CID 3070 protein stimulate transcription of the insulin gene. The first bar (black) represents the untreated cells. Bars 2-4 (white) represent cells treated with the indicated concentrations of GLP-1. Bars 5-7 (gray) represent cells treated with the indicated concentrations of CID 3070 protein.

SEQL

0699<ul id="ul0010" list-style="none"><li><110> Human Genome Sciences, Inc.</li><li><120> Albumin Fusion Proteins</li><li><130> PF564PCT</li><li><150> 60/341,811 <151> 2001-12-21</li><li><150> 60/360,000 <151> 2002-02-28</li><li><150> 60/378,950 <151> 2002-05-10</li><li><150> 60/398,008 <151> 2002-07-24</li><li><150> 60/411,355 <151> 2002-09-18</li><li><150> 60/414,984 <151> 2002-10-02</li><li><150> 60/417,611 <151> 2002-10-11</li><li><150> 60/420,246 <151> 2002-10-23</li><li><150> 60/423,623 <151> 2002-11-05</li><li><150> 60/351,360 <151> 2002-01-28</li><li><150> 60/382,617 <151> 2002-05-24</li><li><150> 60/383,123 <151> 2002-05-28</li><li><150> 60/385,708 <151> 2002-06-05</li><li><150> 60/394,625 <151> 2002-07-10</li><li><150> 60/411,426 <151> 2002-09-18</li><li><150> 60/350,358 <151> 2002-01-24</li><li><150> 60/359,370</li><li><151> 2002-02-26</li><li><150> 60/367,500 <151> 2002-03-27</li><li><150> 60/402,131 <151> 2002-08-09</li><li><150> 60/402,708 <151> 2002-08-13</li><li><150> 60/370,227 <151> 2002-04-08</li><li><160> 2222</li><li><170> Patentin Ver. 2.0</li><li><210> 110 <211> 1203 <212> DNA <213> Homo sapiens</li><li><400> 110 <img file="EP1463751B1_D0008.tif" /><img file="EP1463751B1_D0009.tif" /></li><li><210> 198 <211> 543 <212> DNA <213> Homo sapiens</li><li><400> 198 <img file="EP1463751B1_D0010.tif" /></li><li><210> 200 <211> 543 <212> DNA <213> Homo sapiens</li><li><400> 200 <img file="EP1463751B1_D0011.tif" /></li><li><210> 201 <211> 543 <212> DNA <213> Homo sapiens</li><li><400> 201 <img file="EP1463751B1_D0012.tif" /><img file="EP1463751B1_D0013.tif" /></li><li><210> 202 <211> 543 <212> DNA <213> Homo sapiens</li><li><400> 202 <img file="EP1463751B1_D0014.tif" /></li><li><210> 414 <211> 639 <212> PRT <213> Homo sapiens</li><li><400> 414 <img file="EP1463751B1_D0015.tif" /><img file="EP1463751B1_D0016.tif" /><img file="EP1463751B1_D0017.tif" /><img file="EP1463751B1_D0018.tif" /></li><li><210> 416 <211> 639 <212> PRT <213> Homo sapiens</li><li><400> 416 <img file="EP1463751B1_D0019.tif" /><img file="EP1463751B1_D0020.tif" /><img file="EP1463751B1_D0021.tif" /></li><li><210> 417 <211> 639 <212> PRT <213> Homo sapiens</li><li><400> 417 <img file="EP1463751B1_D0022.tif" /><img file="EP1463751B1_D0023.tif" /><img file="EP1463751B1_D0024.tif" /></li><li><210> 418 <211> 639 <212> PRT <213> Homo sapiens</li><li><400> 418 <img file="EP1463751B1_D0025.tif" /><img file="EP1463751B1_D0026.tif" /><img file="EP1463751B1_D0027.tif" /></li><li><210> 630 <211> 180 <212> PRT <213> Homo sapiens</li><li><400> 630 <img file="EP1463751B1_D0028.tif" /></li><li><210> 632 <211> 180 <212> PRT <213> Homo sapiens</li><li><400> 632 <img file="EP1463751B1_D0029.tif" /><img file="EP1463751B1_D0030.tif" /></li></ul><ul id="ul0011" list-style="none"><li><210> 633 <211> 180 <212> PRT <213> Homo sapiens</li><li><400> 633 <img file="EP1463751B1_D0031.tif" /><img file="EP1463751B1_D0032.tif" /></li><li><210> 634 <211> 180 <212> PRT <213> Homo sapiens</li><li><400> 634 <img file="EP1463751B1_D0033.tif" /></li><li><210> 988 <211> 90 <212> DNA <213> Homo sapiens</li><li><400> 988 <img file="EP1463751B1_D0034.tif" /></li><li><210> 989 <211> 75 <212> DNA <213> Homo sapiens</li><li><400> 989 <img file="EP1463751B1_D0035.tif" /></li><li><210> 992 <211> 89 <212> DNA <213> Homo sapiens</li><li><400> 992 <img file="EP1463751B1_D0036.tif" /></li><li><210> 993 <211> 49 <212> DNA <213> Homo sapiens</li><li><400> 993 ggcgcgcctc atctaccctt aaccaaccaa gcaatgaatt ccttagcag    49</li><li><210> 994 <211> 90 <212> DNA <213> Homo sapiens</li><li><400> 994 <img file="EP1463751B1_D0037.tif" /></li><li><210> 995 <211> 75 <212> DNA <213> Homo sapiens</li><li><400> 995 <img file="EP1463751B1_D0038.tif" /></li><li><210> 996 <211> 89 <212> DNA <213> Homo sapiens</li><li><400> 996 <img file="EP1463751B1_D0039.tif" /></li><li><210> 997 <211> 49 <212> DNA <213> Homo sapiens</li><li><400> 997 ggcgcgcctc atctaccctt aaccaaccaa gcaatgaatt ccttagcag    49</li><li><210> 1037 <211> 1782 <212> DNA <213> Homo sapiens</li><li><400> 1037 <img file="EP1463751B1_D0040.tif" /></li><li><210> 1038 <211> 585 <212> PRT <213> Homo Sapiens</li><li><400> 1038 <img file="EP1463751B1_D0041.tif" /><img file="EP1463751B1_D0042.tif" /><img file="EP1463751B1_D0043.tif" /></li><li><210> 1039 <211> 58 <212> DNA <213> Artificial Sequence.</li><li><220> <221> primer_bind <223> primer used to generate XhoI and ClaI site in pPPC0006</li><li><400> 1039 gcctcgagaa aagagatgca cacaagagtg aggttgctca tcgatttaaa gatttggg    58</li><li><210> 1040 <211> 59 <212> DNA <213> Artificial Sequence</li><li><220> <221> primer_bind <223> primer used in generation XhoI and ClaI site in pPPC0006</li><li><400> 1040 aatcgatgag caacctcact cttgtgtgca tctcttttct cgaggctcct ggaataagc    59</li><li><210> 1041 <211> 24 <212> DNA <213> Artificial Sequence</li><li><220> <221> primer_bind <223> primer used in generation XhoI and ClaI site in pPPC0006</li><li><400> 1041 tacaaactta agagtccaat tagc    24</li><li><210> 1042 <211> 29 <212> DNA <213> Artificial Sequence</li><li><220> <221> primer_bind <223> primer used in generation XhoI and ClaI site in pPPC0006</li><li><400> 1042 cacttctcta gagtggtttc atatgtctt    29</li><li><210> 1043 <211> 60 <212> DNA <213> Artificial Sequence</li><li><220> <221> Misc_Structure <223> Synthetic oligonucleotide used to alter restriction sites in pPPC0007</li><li><400> 1043 aagctgcctt aggcttataa taaggcgcgc cggccggccg tttaaactaa gcttaattct    60</li><li><210> 1044 <211> 60 <212> DNA <213> Artificial Sequence</li><li><220> <221> Misc_Structure <223> Synthetic oligonucleotide used to alter restriction sites in pPPC0007</li><li><400> 1044 agaattaagc ttagtttaaa cggccggccg gcgcgcctta ttataagcct aaggcagctt 60</li><li><210> 1045 <211> 32 <212> DNA <213> Artificial Sequence</li><li><220> <221> primer_bind <223> forward primer useful for generation of albumin fusion protein in which the albumin moiety is N-terminal of the Therapeutic Protein</li><li><220> <221> misc_feature <222> (18) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (19) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (20) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (21) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (22) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (23) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (24) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (25) <223> n equals a,t.g, or c</li><li><220> <221> misc_feature <222> (26) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (27) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (28) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (29) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (30) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (31) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (32) <223> n equals a,t,g, or c</li><li><400> 1045 aagctgcctt aggcttannn nnnnnnnnnn nn    32</li><li><210> 1046 <211> 51 <212> DNA <213> Artificial Sequence</li><li><220> <221> primer_bind <223> reverse primer useful for generation of albumin fusion protein in which the albumin moiety is N-terminal of the Therapeutic Protein</li><li><220> <221> misc_feature <222> (37) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (38) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (39) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (40) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (41) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (42) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (43) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (44) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (45) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (46) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (47) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (48) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (49) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (50) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (51) <223> n equals a,t,g, or c</li><li><400> 1046 gcgcgcgttt aaacggccgg ccggcgcgcc ttattannnn nnnnnnnnnn n    51</li><li><210> 1047 <211> 33 <212> DNA <213> Artificial Sequence</li><li><220> <223> forward primer useful for generation of albumin fusion protein in which the albumin moiety is c-terminal of the Therapeutic Protein</li><li><220> <221> misc_feature <222> (19) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (20) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (21) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (22) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (23) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (24) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (25) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (26) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (27) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (28) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (29) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (30) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (31) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (32) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (33) <223> n equals a,t,g, or c</li><li><400> 1047 aggagcgtcg acaaaagann nnnnnnnnnn nnn    33</li><li><210> 1048 <211> 52 <212> DNA <213> Artificial Sequence</li><li><220> <221> primer_bind <223> reverse primer useful for generation of albumin fusion protein in which the albumin moiety is c-terminal of the Therapeutic Protein</li><li><220> <221> misc_feature <222> (38) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (39) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (40) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (41) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (42) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (43) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (44) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (45) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (46) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (47) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (48) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (49) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (50) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (51) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (52) <223> n equals a,t,g, or c</li><li><400> 1048 ctttaaatcg atgagcaacc tcactcttgt gtgcatcnnn nnnnnnnnnn nn    52</li><li><210> 1049 <211> 24 <212> PRT <213> Artificial Sequence</li><li><220> <221> signal <223> signal peptide of natural human serum albumin protein</li><li><400> 1049 <img file="EP1463751B1_D0044.tif" /></li><li><210> 1052 <211> 46 <212> DNA <213> Artificial Sequence</li><li><220> <221> primer bind <223> forward primer useful for inserting Therapeutic protein into pC4:HSA vector</li><li><220> <221> misc_feature <222> (29) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (30) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (31) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (32) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (33) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (34) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (35) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (36) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (37) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (38) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (39) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (40) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (41) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (42) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (43) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (44) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (45) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (46) <223> n equals a,t,g, or c</li><li><400> 1052 ccgccgctcg aggggtgtgt ttcgtcgann nnnnnnnnnn nnnnnn    46</li><li><210> 1053 <211> 55 <212> DNA <213> Artificial Sequence</li><li><220> <221> primer_bind <223> reverse primer useful for inserting Therapeutic protein into pC4:HSA vector</li><li><220> <221> misc_feature <222> (38) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (39) <223> n equals a,t,g, or c</li></ul><ul id="ul0012" list-style="none"><li><220> <221> misc_feature <222> (40) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (41) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (42) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (43) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (44) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (45) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (46) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (47) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (48) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (49) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (50) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (51) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (52) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (53) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (54) <223> n equals a,t,g, or c</li><li><220> <221> misc_feature <222> (55) <223> n equals a,t,g, or c</li><li><400> 1053 agtcccatcg atgagcaacc tcactcttgt gtgcatcnnn nnnnnnnnnn nnnnn    55</li><li><210> 1054 <211> 17 <212> PRT <213> Artificial Sequence</li><li><220> <221> signal <223> Stanniocalcin signal peptide</li><li><400> 1054 <img file="EP1463751B1_D0045.tif" /></li><li><210> 1055 <211> 22 <212> PRT <213> Artificial Sequence</li><li><220> <221> signal <223> Synthetic signal peptide</li><li><400> 1055 <img file="EP1463751B1_D0046.tif" /></li><li><210> 1093 <211> 1830 <212> DNA <213> Homo sapiens</li><li><400> 1093 <img file="EP1463751B1_D0047.tif" /></li><li><210> 1094 <211> 609 <212> PRT <213> Homo sapiens</li><li><400> 1094 <img file="EP1463751B1_D0048.tif" /><img file="EP1463751B1_D0049.tif" /><img file="EP1463751B1_D0050.tif" /></li><li><210> 1095 <211> 19 <212> PRT <213> Homo sapiens</li><li><400> 1095 <img file="EP1463751B1_D0051.tif" /></li><li><210> 1096 <211> 29 <212> PRT <213> Homo sapiens</li><li><400> 1096 <img file="EP1463751B1_D0052.tif" /></li><li><210> 1097 <211> 22 <212> PRT <213> Homo sapiens</li><li><400> 1097 <img file="EP1463751B1_D0053.tif" /></li><li><210> 1098 <211> 21 <212> PRT <213> Homo sapiens</li><li><400> 1098 <img file="EP1463751B1_D0054.tif" /></li><li><210> 1099 <211> 18 <212> PRT <213> Artificial Sequence</li><li><220> <221> MUTAGEN <222> (14) to (18) <223> variant of HSA native leader</li><li><400> 1099 <img file="EP1463751B1_D0055.tif" /></li><li><210> 1100 <211> 18 <212> PRT <213> Artificial Sequence</li><li><220> <221> MUTAGEN <222> (14) to (18) <223> Variant of HSA native leader</li><li><400> 1100 <img file="EP1463751B1_D0056.tif" /></li><li><210> 1101 <211> 18 <212> PRT <213> Artificial Sequence</li><li><220> <221> MUTAGEN <222> (14) to (18) <223> Variant of HSA native leader</li><li><400> 1101 <img file="EP1463751B1_D0057.tif" /></li><li><210> 1102 <211> 18 <212> PRT <213> Artificial Sequence</li><li><220> <221> MUTAGEN <222> (14) to (18) <223> Variant of HSA native leader</li><li><400> 1102 <img file="EP1463751B1_D0058.tif" /><img file="EP1463751B1_D0059.tif" /></li><li><210> 1103 <211> 18 <212> PRT <213> Artificial Sequence</li><li><220> <221> MUTAGEN <222> (14) to (18) <223> Variant of HSA native leader</li><li><400> 1103 <img file="EP1463751B1_D0060.tif" /></li><li><210> 1104 <211> 18 <212> PRT <213> Artificial Sequence</li><li><220> <221> MUTAGEN <222> (14) to (18) <223> Variant of HSA native leader</li><li><400> 1104 <img file="EP1463751B1_D0061.tif" /></li><li><210> 1105 <211> 23 <212> PRT <213> Artificial Sequence</li><li><220> <221> MUTAGEN <222> (14) to (23) <223> Variant of HSA native leader</li><li><400> 1105 <img file="EP1463751B1_D0062.tif" /></li><li><210> 1106 <211> 9 <212> PRT <213> Homo sapiens</li><li><400> 1106 <img file="EP1463751B1_D0063.tif" /></li><li><210> 1107 <211> 11 <212> DNA <213> Artificial Sequence</li><li><220> <221> misc_feature <222> (1) to (11) <223> Kozak sequence</li><li><400> 1107 ccgccaccat g    11</li><li><210> 1108 <211> 19 <212> PRT <213> Homo sapiens</li><li><400> 1108 <img file="EP1463751B1_D0064.tif" /></li><li><210> 1109 <211> 86 <212> PRT <213> Homo sapiens</li><li><220> <221> SITE <222> (84) <223> Xaa equals any one of Glu or Asp</li><li><400> 1109 <img file="EP1463751B1_D0065.tif" /><img file="EP1463751B1_D0066.tif" /></li><li><210> 1110 <211> 24 <212> PRT <213> Homo sapiens</li><li><400> 1110 <img file="EP1463751B1_D0067.tif" /></li><li><210> 1111 <211> 24 <212> PRT <213> Homo sapiens</li><li><400> 1111 <img file="EP1463751B1_D0068.tif" /></li><li><210> 1113 <211> 5 <212> PRT <213> Homo sapiens</li><li><220> <221> Site <222> (3) <223> Xaa equals any of the twenty naturally ocurring L-amino acids</li><li><400> 1113 <img file="EP1463751B1_D0069.tif" /></li><li><210> 1114 <211> 86 <212> DNA <213> Artificial Sequence</li><li><220> <221> Primer_Bind <223> Synthetic sequence with 4 tandem copies of the GAS binding site found in the IRF1 promoter (<nplcit id="ncit0244" npl-type="s"><text>Rothman et al., Immunity 1:457-468 (1994</text></nplcit>)), 18 nucleotides complementary to the SV40 early promoter, and a Xho I restriction site.</li><li><400> 1114 <img file="EP1463751B1_D0070.tif" /></li><li><210> 1115 <211> 27 <212> DNA <213> Artificial Sequence</li><li><220> <221> Primer_Bind <223> Synthetic sequence complementary to the SV40 promter; includes a Hind III restriction site.</li><li><400> 1115 gcggcaagct ttttgcaaag cctaggc    27</li><li><210> 1116 <211> 271 <212> DNA <213> Artificial Sequence</li><li><220> <221> Protein_Bind <223> Synthetic promoter for use in biological assays; includes GAS binding sites found in the IRF1 promoter (<nplcit id="ncit0245" npl-type="s"><text>Rothman et al., Immunity 1:457-468 (1994</text></nplcit>)).</li><li><400> 1116 <img file="EP1463751B1_D0071.tif" /><img file="EP1463751B1_D0072.tif" /></li><li><210> 1117 <211> 32 <212> DNA. <213> Artificial Sequence</li><li><220> <221> Primer_Bind <223> Synthetic primer complementary to human genomic EGR-1 promoter sequence (<nplcit id="ncit0246" npl-type="s"><text>Sakamoto et al., Oncogene 6:867-871 (1991</text></nplcit>)); includes a Xho I restriction site.</li><li><400> 1117 gcgctcgagg gatgacagcg atagaacccc gg    32</li><li><210> 1118 <211> 31 <212> DNA <213> Artificial Sequence</li><li><220> <221> Primer_Bind <223> Synthetic primer complementary to human genomic EGR-1 promoter sequence (<nplcit id="ncit0247" npl-type="s"><text>Sakamoto et al., Oncogene 6:867-871 (1991</text></nplcit>)); includes a Hind III restriction site.</li><li><400> 1118 gcgaagcttc gcgactcccc ggatccgcct c    31</li><li><210> 1119 <211> 12 <212> DNA <213> Homo sapiens</li><li><400> 1119 ggggactttc cc    12</li><li><210> 1120 <211> 73 <212> DNA <213> Artificial Sequence</li><li><220> <221> Primer_Bind <223> Synthetic primer with 4 tandem copies of the NF-KB binding site (GGGGACTTTCCC), 18 nucleotides complementary to the 5' end of the SV40 early promoter sequence, and a XhoI restriction site.</li><li><400> 1120 <img file="EP1463751B1_D0073.tif" /></li><li><210> 1121 <211> 256 <212> DNA <213> Artificial Sequence</li><li><220> <221> Protein Bind <223> Synthetic promoter for use in biological assays; includes NP-KB binding sites.</li><li><400> 1121 <img file="EP1463751B1_D0074.tif" /></li><li><210> 1167 <211> 24 <212> PRT <213> Homo sapiens</li><li><400> 1167 <img file="EP1463751B1_D0075.tif" /></li><li><210> 1168 <211> 18 <212> PRT <213> Homo sapiens</li><li><400> 1168 <img file="EP1463751B1_D0076.tif" /></li><li><210> 1169 <211> 21 <212> PRT <213> Homo sapiens</li><li><400> 1169 <img file="EP1463751B1_D0077.tif" /></li><li><210> 1176 <211> 24 <212> PRT <213> Homo sapiens</li><li><400> 1176 <img file="EP1463751B1_D0078.tif" /></li><li><210> 1177 <211> 18 <212> PRT <213> Homo sapiens</li><li><400> 1177 <img file="EP1463751B1_D0079.tif" /></li><li><210> 1216 <211> 543 <212> DNA <213> Homo sapiens</li><li><400> 1216 <img file="EP1463751B1_D0080.tif" /></li><li><210> 1217 <211> 543 <212> DNA <213> Homo sapiens</li><li><400> 1217 <img file="EP1463751B1_D0081.tif" /><img file="EP1463751B1_D0082.tif" /></li><li><210> 1218 <211> 543 <212> DNA <213> Homo sapiens</li><li><400> 1218 <img file="EP1463751B1_D0083.tif" /></li><li><210> 1219 <211> 543 <212> DNA <213> Homo sapiens</li><li><400> 1219 <img file="EP1463751B1_D0084.tif" /></li><li><210> 1220 <211> 543 <212> DNA <213> Homo sapiens</li><li><400> 1220 <img file="EP1463751B1_D0085.tif" /></li><li><210> 1221 <211> 180 <212> DNA <213> Homo sapiens</li><li><400> 1221 <img file="EP1463751B1_D0086.tif" /></li><li><210> 1222 <211> 180 <212> DNA <213> Homo sapiens</li><li><400> 1222 <img file="EP1463751B1_D0087.tif" /></li><li><210> 1231 <211> 669 <212> PRT <213> Homo sapiens</li><li><400> 1231 <img file="EP1463751B1_D0088.tif" /><img file="EP1463751B1_D0089.tif" /><img file="EP1463751B1_D0090.tif" /></li><li><210> 1232 <211> 669 <212> PRT <213> Homo sapiens</li><li><400> 1232 <img file="EP1463751B1_D0091.tif" /><img file="EP1463751B1_D0092.tif" /><img file="EP1463751B1_D0093.tif" /></li><li><210> 1233 <211> 669 <212> PRT <213> Homo sapiens</li><li><400> 1233 <img file="EP1463751B1_D0094.tif" /><img file="EP1463751B1_D0095.tif" /><img file="EP1463751B1_D0096.tif" /><img file="EP1463751B1_D0097.tif" /></li><li><210> 1234 <211> 667 <212> PRT <213> Homo sapiens</li><li><400> 1234 <img file="EP1463751B1_D0098.tif" /><img file="EP1463751B1_D0099.tif" /><img file="EP1463751B1_D0100.tif" /></li></ul><ul id="ul0013" list-style="none"><li><210> 1235 <211> 669 <212> PRT <213> Homo sapiens</li><li><400> 1235 <img file="EP1463751B1_D0101.tif" /><img file="EP1463751B1_D0102.tif" /><img file="EP1463751B1_D0103.tif" /><img file="EP1463751B1_D0104.tif" /></li><li><210> 1236 <211> 669 <212> PRT <213> Homo sapiens</li><li><400> 1236 <img file="EP1463751B1_D0105.tif" /><img file="EP1463751B1_D0106.tif" /><img file="EP1463751B1_D0107.tif" /></li><li><210> 1237 <211> 669 <212> PRT <213> Homo sapiens</li><li><400> 1237 <img file="EP1463751B1_D0108.tif" /><img file="EP1463751B1_D0109.tif" /><img file="EP1463751B1_D0110.tif" /></li><li><210> 1246 <211> 180 <212> PRT <213> Homo sapiens</li><li><400> 1246 <img file="EP1463751B1_D0111.tif" /><img file="EP1463751B1_D0112.tif" /></li><li><210> 1247 <211> 180 <212> PRT <213> Homo sapiens</li><li><400> 1247 <img file="EP1463751B1_D0113.tif" /><img file="EP1463751B1_D0114.tif" /></li><li><210> 1248 <211> 180 <212> PRT <213> Homo sapiens</li><li><400> 1248 <img file="EP1463751B1_D0115.tif" /></li><li><210> 1249 <211> 180 <212> PRT <213> Homo sapiens</li><li><400> 1249 <img file="EP1463751B1_D0116.tif" /></li><li><210> 1250 <211> 180 <212> PRT <213> Homo sapiens</li><li><400> 1250 <img file="EP1463751B1_D0117.tif" /><img file="EP1463751B1_D0118.tif" /></li><li><210> 1251 <211> 60 <212> PRT <213> Homo sapiens</li><li><400> 1251 <img file="EP1463751B1_D0119.tif" /></li><li><210> 1252 <211> 60 <212> PRT <213> Homo sapiens</li><li><400> 1252 <img file="EP1463751B1_D0120.tif" /></li><li><210> 1261 <211> 22 <212> DNA <213> Homo sapiens</li><li><400> 1261 aggagcgtcg acaaaagaca cg    22</li><li><210> 1262 <211> 26 <212> DNA <213> Homo sapiens</li><li><400> 1262 ctttaaatcg atgagcaacc tcactc    26</li><li><210> 1263 <211> 22 <212> DNA <213> Homo sapiens</li><li><400> 1263 aggagcgtcg acaaaagaca cg    22</li><li /><li><210> 1264 <211> 26 <212> DNA <213> Homo sapiens</li><li><400> 1264 ctttaaatcg atgagcaacc tcactc    26</li><li><210> 1265 <211> 22 <212> DNA <213> Homo sapiens</li><li><400> 1265 aggagcgtcg acaaaagaca cg    22</li><li><210> 1266 <211> 26 <212> DNA <213> Homo sapiens</li><li><400> 1266 ctttaaatcg atgagcaacc tcactc    26</li><li><210> 1267 <211> 22 <212> DNA <213> Homo sapiens</li><li><400> 1267 aggagcgtcg acaaaagaga ag    22</li><li><210> 1268 <211> 26 <212> DNA <213> Homo sapiens</li><li><400> 1268 ctttaaatcg atgagcaacc tcactc    26</li><li><210> 1269 <211> 22 <212> DNA <213> Homo sapiens</li><li><400> 1269 aggagcgtcg acaaaagaca ca    22</li><li><210> 1270 <211> 26 <212> DNA <213> Homo sapiens</li><li><400> 1270 ctttaaatcg atgagcaacc tcactc    26</li><li><210> 1271 <211> 29 <212> DNA <213> Homo sapiens</li><li><400> '1271 agcgtcgaca aaagacacgc tgaaggtac    29</li><li><210> 1272 <211> 21 <212> DNA <213> Homo sapiens</li><li><400> 1272 catgatcttc aaatggacac t    21</li><li><210> 1273 <211> 22 <212> DNA <213> Homo sapiens</li><li><400> 1273 aggagcgtcg acaaaagaca cg    22</li><li><210> 1274 <211> 21 <212> DNA <213> Homo sapiens</li><li><400> 1274 catgatcttc aaatggacac t    21</li><li><210> 1280 <211> 674 <212> PRT <213> Homo sapiens</li><li><400> 1280 <img file="EP1463751B1_D0121.tif" /><img file="EP1463751B1_D0122.tif" /><img file="EP1463751B1_D0123.tif" /></li></ul><ul id="ul0014" list-style="none"><li><210> 1281 <211> 267 <212> DNA <213> Homo sapiens</li><li><400> 1281 <img file="EP1463751B1_D0124.tif" /><img file="EP1463751B1_D0125.tif" /></li><li><210> 1282 <211> 89 <212> PRT <213> Homo sapiens</li><li><400> 1282 <img file="EP1463751B1_D0126.tif" /></li><li><210> 1283 <211> 20 <212> DNA <213> Homo sapiens</li><li><400> 1283 gtcaatacct tcttgaacca    20</li><li><210> 1284 <211> 27 <212> DNA <213> Homo sapiens</li><li><400> 1284 ctcccaaatc tttaaatcga tgagcaa    27</li><li><210> 1391 <211> 543 <212> DNA <213> Homo sapiens</li><li><400> 1391 <img file="EP1463751B1_D0127.tif" /></li><li><210> 1395 <211> 42 <212> DNA <213> Homo sapiens</li><li><400> 1395 gctggttgta agaacttctt ctggaagact ttcacttctt gt    42</li><li><210> 1439 <211> 543 <212> DNA <213> Homo sapiens</li><li><400> 1439 <img file="EP1463751B1_D0128.tif" /></li><li><210> 1440 <211> 543 <212> DNA <213> Homo sapiens</li><li><400> 1440 <img file="EP1463751B1_D0129.tif" /></li><li><210> 1452 <211> 825 <212> DNA <213> Homo sapiens</li><li><400> 1452 <img file="EP1463751B1_D0130.tif" /></li><li><210> 1453 <211> 249 <212> DNA <213> Homo sapiens</li><li><400> 1453 <img file="EP1463751B1_D0131.tif" /></li><li><210> 1454 <211> 435 <212> DNA <213> Homo sapiens</li><li><400> 1454 <img file="EP1463751B1_D0132.tif" /><img file="EP1463751B1_D0133.tif" /></li><li><210> 1455 <211> 231 <212> DNA <213> Homo sapiens</li><li><400> 1455 <img file="EP1463751B1_D0134.tif" /></li><li><210> 1456 <211> 435 <212> DNA <213> Homo sapiens</li><li><400> 1456 <img file="EP1463751B1_D0135.tif" /></li><li><210> 1457 <211> 249 <212> DNA <213> Homo sapiens</li><li><400> 1457 <img file="EP1463751B1_D0136.tif" /></li><li><210> 1458 <211> 231 <212> DNA <213> Homo sapiens</li><li><400> 1458 <img file="EP1463751B1_D0137.tif" /></li><li><210> 1472 <211> 90 <212> DNA <213> Homo sapiens</li><li><400> 1472 <img file="EP1463751B1_D0138.tif" /></li><li><210> 1559 <211> 654 <212> PRT <213> Homo sapiens</li><li><400> 1559 <img file="EP1463751B1_D0139.tif" /><img file="EP1463751B1_D0140.tif" /><img file="EP1463751B1_D0141.tif" /></li><li><210> 1563 <211> 623 <212> PRT <213> Homo sapiens</li><li><400> 1563 <img file="EP1463751B1_D0142.tif" /><img file="EP1463751B1_D0143.tif" /><img file="EP1463751B1_D0144.tif" /></li><li><210> 1607 <211> 664 <212> PRT <213> Homo sapiens</li><li><400> 1607 <img file="EP1463751B1_D0145.tif" /><img file="EP1463751B1_D0146.tif" /><img file="EP1463751B1_D0147.tif" /></li><li><210> 1608 <211> 664 <212> PRT <213> Homo sapiens</li><li><400> 1608 <img file="EP1463751B1_D0148.tif" /><img file="EP1463751B1_D0149.tif" /><img file="EP1463751B1_D0150.tif" /></li></ul><ul id="ul0015" list-style="none"><li><210> 1609 <211> 663 <212> PRT <213> Homo sapiens</li><li><400> 1609 <img file="EP1463751B1_D0151.tif" /><img file="EP1463751B1_D0152.tif" /><img file="EP1463751B1_D0153.tif" /><img file="EP1463751B1_D0154.tif" /></li><li><210> 1610 <211> 663 <212> PRT <213> Homo sapiens</li><li><400> 1610 <img file="EP1463751B1_D0155.tif" /><img file="EP1463751B1_D0156.tif" /><img file="EP1463751B1_D0157.tif" /></li><li><210> 1620 <211> 700 <212> PRT <213> Homo sapiens</li><li><400> 1620 <img file="EP1463751B1_D0158.tif" /><img file="EP1463751B1_D0159.tif" /><img file="EP1463751B1_D0160.tif" /></li><li><210> 1621 <211> 668 <212> PRT <213> Homo sapiens</li><li><400> 1621 <img file="EP1463751B1_D0161.tif" /><img file="EP1463751B1_D0162.tif" /><img file="EP1463751B1_D0163.tif" /></li><li><210> 1622 <211> 730 <212> PRT <213> Homo sapiens</li><li><400> 1622 <img file="EP1463751B1_D0164.tif" /><img file="EP1463751B1_D0165.tif" /><img file="EP1463751B1_D0166.tif" /><img file="EP1463751B1_D0167.tif" /></li><li><210> 1623 <211> 662 <212> PRT <213> Homo sapiens</li><li><400> 1623 <img file="EP1463751B1_D0168.tif" /><img file="EP1463751B1_D0169.tif" /><img file="EP1463751B1_D0170.tif" /></li><li><210> 1624 <211> 730 <212> PRT <213i Homo sapiens</li><li><400> 1624 <img file="EP1463751B1_D0171.tif" /><img file="EP1463751B1_D0172.tif" /><img file="EP1463751B1_D0173.tif" /><img file="EP1463751B1_D0174.tif" /></li><li><210> 1625 <211> 668 <212> PRT <213> Homo sapiens</li><li><400> 1625 <img file="EP1463751B1_D0175.tif" /><img file="EP1463751B1_D0176.tif" /><img file="EP1463751B1_D0177.tif" /></li></ul><ul id="ul0016" list-style="none"><li><210> 1626 <211> 662 <212> PRT <213> Homo sapiens</li><li><400> 1626 <img file="EP1463751B1_D0178.tif" /><img file="EP1463751B1_D0179.tif" /><img file="EP1463751B1_D0180.tif" /><img file="EP1463751B1_D0181.tif" /></li><li><210> 1640 <211> 655 <212> PRT <213> Homo sapiens</li><li><400> 1640 <img file="EP1463751B1_D0182.tif" /><img file="EP1463751B1_D0183.tif" /><img file="EP1463751B1_D0184.tif" /></li><li><210> 1727 <211> 180 <212> PRT <213> Homo sapiens</li><li><400> 1727 <img file="EP1463751B1_D0185.tif" /></li><li><210> 1731 <211> 14 <212> PRT <213> Homo sapiens</li><li><400> 1731 <img file="EP1463751B1_D0186.tif" /></li><li><210> 1775 <211> 180 <212> PRT <213> Homo sapiens</li><li><900> 1775 <img file="EP1463751B1_D0187.tif" /></li><li><210> 1776 <211> 180 <212> PRT <213> Homo sapiens</li><li><400> 1776 <img file="EP1463751B1_D0188.tif" /><img file="EP1463751B1_D0189.tif" /></li><li><210> 1788 <211> 93 <212> PRT <213> Homo sapiens</li><li><400> 1788 <img file="EP1463751B1_D0190.tif" /><img file="EP1463751B1_D0191.tif" /></li><li><210> 1789 <211> 83 <212> PRT <213> Homo sapiens</li><li><400> 1789 <img file="EP1463751B1_D0192.tif" /></li><li><210> 1790 <211> 145 <212> PRT <213> Homo sapiens</li><li><400> 1790 <img file="EP1463751B1_D0193.tif" /><img file="EP1463751B1_D0194.tif" /></li><li><210> 1791 <211> 77 <212> PRT <213> Homo sapiens</li><li><400> 1791 <img file="EP1463751B1_D0195.tif" /></li><li><210> 1792 <211> 145 <212> PRT <213> Homo sapiens</li><li><400> 1792 <img file="EP1463751B1_D0196.tif" /><img file="EP1463751B1_D0197.tif" /></li><li><210> 1793 <211> 83 <212> PRT <213> Homo sapiens</li><li><400> 1793 <img file="EP1463751B1_D0198.tif" /></li><li><210> 1794 <211> 77 <212> PRT <213> Homo sapiens</li><li><400> 1794 <img file="EP1463751B1_D0199.tif" /></li><li><210> 1808 <211> 30 <212> PRT <213> Homo sapiens</li><li><400> 1808 <img file="EP1463751B1_D0200.tif" /></li><li><210> 1922 <211> 35 <212> DNA <213> Homo sapiens</li><li><400> 1922 aagctgcctt aggcttagct ggttgtaaga acttc    35</li><li><210> 1923 <211> 39 <212> DNA <213> Homo sapiens</li><li><400> 1923 cggccggggc gcgccttaac aagaagtgaa agtcttcca    39</li><li><210> 2004 <211> 26 <212> DNA <213> Homo sapiens</li><li><400> 2004 gcacacggtg aaggtacttt cacttc    26</li><li><210> 2005 <211> 26 <212> DNA <213> Homo sapiens</li><li><400> 2005 ctttaaatcg atgagcaacc tcactc    26</li><li><210> 2006 <211> 26 <212> DNA <213> Homo sapiens</li><li><400> 2006 gcacacggtg aaggtacttt cacttc    26</li><li><210> 2007 <211> 26 <212> DNA <213> Homo sapiens</li><li><400> 2007 ctttaaatcg atgagcaacc tcactc    26</li><li><210> 2008 <211> 28 <212> DNA <213> Homo sapiens</li><li><400> 2008 actgctgatg cagcggccgc ccgtaatg    28</li><li><210> 2009 <211> 21 <212> DNA <213> Homo sapiens</li><li><400> 2009 catgatcttc aaatggacac t    21</li><li><210> 2010 <211> 28 <212> DNA <213> Homo sapiens</li><li><400> 2010 actgctgatg cagcggccgc ccgtaatg    28</li><li><210> 2011 <211> 21 <212> RNA <213> Homo sapiens</li><li><400> 2011 catgatcttc aaatggacac t    21</li><li><210> 2030 <211> 22 <212> DNA <213> Homo sapiens</li><li><400> 2030 gcaggacctt accacccctc ag    22</li><li><210> 2031 <211> 58 <212> DNA <213> Homo sapiens</li><li><400> 2031 ctttaaatcg atgagcaacc tcactcttgt gtgcatctct acccttaacc aaccaagc    58</li><li><210> 2032 <211> 20 <212> DNA <213> Homo sapiens</li><li><400> 2032 aattaaccct cactaaaggg    20</li><li><210> 2033 <211> 27 <212> DNA <213> Homo sapiens</li><li><400> 2033 ctcccaaatc tttaaatcga tgagcaa    27</li><li><210> 2034 <211> 20 <212> DNA <213> Homo sapiens</li><li><400> 2034 gtcaatacct tcttgaacca    20</li><li><210> 2035 <211> 27 <212> DNA <213> Homo sapiens</li><li><400> 2035 ctcccaaatc tttaaatcga tgagcaa    27</li><li><210> 2036 <211> 20 <212> DNA <213> Homo sapiens</li><li><400> 2036 gtcaatacct tcttgaacca    20</li><li><210> 2037 <211> 27 <212> DNA <213> Homo sapiens</li><li><400> 2037 ctcccaaatc tttaaatcga tgagcaa    27</li><li><210> 2038 <211> 20 <212> DNA. <213> Homo sapiens</li><li><400> 2038 gtcaatacct tcttgaacca    20</li><li><210> 2039 <211> 27 <212> DNA <213> Homo sapiens</li><li><400> 2039 ctcccaaatc tttaaatcga tgagcaa    27</li><li><210> 2040 <211> 20 <212> DNA <213> Homo sapiens</li><li><400> 2040 aattaaccct cactaaaggg    20</li><li><210> 2041 <211> 27 <212> DNA <213> Homo sapiens</li><li><400> 2041 ctcccaaatc tttaaatcga tgagcaa    27</li><li><210> 2042 <211> 20 <212> DNA <213> Homo sapiens</li><li><400> 2042 gtcaatacct tcttgaacca    20</li><li><210> 2043 <211> 27 <212> DNA <213> Homo sapiens</li><li><400> 2043 ctcccaaatc tttaaatcga tgagcaa    27</li><li><210> 2064 <211> 20 <212> DNA <213> Homo sapiens</li><li><400> 2064 catacaaact taagagtcca    20</li><li><210> 2065 <211> 27 <212> DNA <213> Homo sapiens</li><li><400> 2065 ctcccaaatc tttaaatcga tgagcaa    27</li><li><210> 2128 <211> 65 <212> PRT <213> Homo sapiens</li><li><400> 2128 <img file="EP1463751B1_D0201.tif" /></li><li><210> 2129 <211> 70 <212> PRT <213> Homo sapiens</li><li><400> 2129 <img file="EP1463751B1_D0202.tif" /></li><li><210> 2132 <211> 21 <212> PRT <213> Homo sapiens</li><li><400> 2132 <img file="EP1463751B1_D0203.tif" /><img file="EP1463751B1_D0204.tif" /></li><li><210> 2133 <211> 18 <212> PRT <213> Homo sapiens</li><li><400> 2133 <img file="EP1463751B1_D0205.tif" /></li><li><210> 2134 <211> 18 <212> PRT <213> Homo sapiens</li><li><400> 2134 <img file="EP1463751B1_D0206.tif" /></li><li><210> 2135 <211> 17 <212> PRT <213> Homo sapiens</li><li><400> 2135 <img file="EP1463751B1_D0207.tif" /></li><li><210> 2136 <211> 29 <212> PRT <213> Homo sapiens</li><li><400> 2136 <img file="EP1463751B1_D0208.tif" /></li><li><210> 2137 <211> 23 <212> PRT <213> Homo sapiens</li><li><400> 2137 <img file="EP1463751B1_D0209.tif" /></li><li><210> 2138 <211> 394 <212> DNA <213> Homo sapiens</li><li><400> 2138 <img file="EP1463751B1_D0210.tif" /></li><li><210> 2139 <211> 4 <212> PRT <213> Homo sapiens</li><li><400> 2139 <img file="EP1463751B1_D0211.tif" /></li><li><210> 2140 <211> 4 <212> PRT <213> Homo sapiens</li><li><400> 2140 <img file="EP1463751B1_D0212.tif" /></li><li><210> 2160 <211> 180 <212> DNA <213> Homo sapiens</li><li><400> 2160 <img file="EP1463751B1_D0213.tif" /></li><li><210> 2170 <211> 673 <212> PRT <213> Homo sapiens</li><li><400> 2170 <img file="EP1463751B1_D0214.tif" /><img file="EP1463751B1_D0215.tif" /><img file="EP1463751B1_D0216.tif" /></li><li><210> 2180 <211> 60 <212> PRT <213> Homo sapiens</li><li><400> 2180 <img file="EP1463751B1_D0217.tif" /></li><li><210> 2194 <211> 29 <212> DNA <213> Homo sapiens</li><li><400> 2194 aaacttaaga gtccaattag cttcatcgc    29</li><li><210> 2195 <211> 24 <212> DNA <213> Homo sapiens</li><li><400> 2195 ccggaattcc ttagcagctt gacc    24</li></ul>