PEPTmE YY MODIFIED TRANSFERRIN FUSION PROTEINS

RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application 60/622,147, filed October 27, 2004, which is incorporated by reference in its entirety.

[0002] This application is related to PCT/US2003/026818, filed August 28, 2003, which is a continuation-in-part of U.S. Application 10/378,094, filed March 4, 2003, which claims the benefit of U.S. Provisional Application 60/315/745, filed August 30, 2001 and U.S. Provisional Application 60/334,059, filed November 30, 2001, all of which are herein incorporated by reference in their entirety. This application is also related to U.S. Provisional Application 60/406,977, filed August 30, 2002, U.S. Provisional Application 60/598,031, filed August 3, 2004 and PCT/US2004/006462, filed March 4, 2004, all of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to therapeutic proteins or peptides with extended serum stability or in vivo circulatory half-life fused to or inserted in a transferrin molecule modified to reduce or inhibit glycosylation, iron binding and/or transferrin receptor binding.

BACKGROUND OF THE INVENTION

[0004] Therapeutic proteins or peptides in their native state or when recombinantly produced are typically labile molecules exhibiting short periods of serum stability or short in vivo circulatory half-lives. In addition, these molecules are often extremely labile when formulated, particularly when formulated in aqueous solutions for diagnostic and therapeutic purposes.

[0005] Few practical solutions exist to extend or promote the stability in vivo or in vitro of proteinaceous therapeutic molecules. Polyethylene glycol (PEG) is a substance that can be attached to a protein, resulting in longer-acting, sustained activity of the protein. If the activity of a protein is prolonged by the attachment to PEG, the frequency that the protein

needs to be administered may be decreased. PEG attachment, however, often decreases or destroys the protein's therapeutic activity. While in some instance PEG attachment can reduce immunogenicity of the protein, in other instances it may increase immunogenicity.

[0006] Therapeutic proteins or peptides have also been stabilized by fusion to a protein capable of extending the in vivo circulatory half-life of the therapeutic protein. For instance, therapeutic proteins fused to albumin or to antibody fragments may exhibit extended in vivo circulatory half-life when compared to the therapeutic protein in the unfused state. See U.S. Patents 5,876,969 and 5,766,883.

[0007] Another serum protein, glycosylated human transferrin (Tf) has also been used to make fusions with therapeutic proteins to target delivery to the interior of cells or to carry agents across the blood-brain barrier. These fusion proteins comprising glycosylated human Tf have been used to target nerve growth factor (NGF) or ciliary neurotrophic factor (CNTF) across the blood-brain barrier by fusing full-length Tf to the agent. See U.S. Patents 5,672,683 and 5977,307. In these fusion proteins, the Tf portion of the molecule is glycosylated and binds to two atoms of iron, which is required for Tf binding to its receptor on a cell and, according to the inventors of these patents, to target delivery of the NGF or CNTF moiety across the blood-brain barrier. Transferrin fusion proteins have also been produced by inserting an HIV-I protease target sequence into surface exposed loops of glycosylated transferrin to investigate the ability to produce another form of Tf fusion for targeted delivery to the inside of a cell via the Tf receptor (AIi et al. (1999) J. Biol. Chem. 274(34):24066-24073).

[0008] Serum transferrin (Tf) is a monomelic glycoprotein with a molecular weight of 80,000 daltons that binds iron in the circulation and transports it to various tissues via the transferrin receptor (TfR) (Aisen et al. (1980) Ann. Rev. Biochem. 49: 357-393; MacGillivray et al. (1981) J. Biol. Chem. 258: 3543-3553, U.S. Patent 5,026,651). Tf is one of the most common serum molecules, comprising up to about 5-10% of total serum proteins. Carbohydrate deficient transferrin occurs in elevated levels in the blood of alcoholic individuals and exhibits a longer half life (approximately 14-17 days) than that of glycosylated transferrin (approximately 7-10 days). See van Eijk et al. (1983) Clin. Chim. Acta 132: 167-171, Stibler (1991) Clin. Chem. 37:2029-2037 (1991), Arndt (2001) Clin.

Chem. 47(1): 13-27 and Stibler et al. in "Carbohydrate-deficient consumption", Advances in the Biosciences, (Ed Nordmann et al), Pergamon, 1988, Vol. 71, pages 353-357).

[0009] The structure of Tf has been well characterized and the mechanism of receptor binding, iron binding and release and carbonate ion binding have been elucidated (U.S. Patents 5,026,651, 5,986,067 and MacGillivray et al (1983) J. Biol. Chem. 258(6):3543- 3546).

[0010] Transferrin and antibodies that bind the transferrin receptor have also been used to deliver or carry toxic agents to tumor cells as cancer therapy (Baselga and Mendelsohn, 1994), and transferrin has been used as a non- viral gene therapy vector to deliver DNA to cells (Frank et al., 1994; Wagner et al., 1992). The ability to deliver proteins to the central nervous system (CNS) using the transferrin receptor as the entry point has been demonstrated with several proteins and peptides including CD4 (Walus et al, 1996), brain derived neurotrophic factor (Pardridge et al., 1994), glial derived neurotrophic factor (Albeck et al.), a vasointestinal peptide analogue (Bickel et al., 1993), a beta-amyloid peptide (Saito et al., 1995), and an antisense oligonucleotide (Pardridge et al, 1995).

[0011] Transferrin fusion proteins have not, however, been modified or engineered to extend the in vivo circulatory half-life of a therapeutic protein or peptide nor to increase bioavailability by reducing or inhibiting glycosylation of the Tf moiety nor to reduce or prevent iron and/or Tf receptor binding.

SUMMARY OF THE INVENTION

[0012] As described in more detail below, the present invention includes modified Tf fusion proteins comprising at least one therapeutic protein, polypeptide or peptide entity, wherein the Tf portion is engineered to extend the in vivo circulatory half-life or bioavailability of the molecule. The invention also includes pharmaceutical formulations and compositions comprising the fusion proteins, methods of extending the serum stability, in vivo circulatory half-life and bioavailability of a therapeutic protein by fusion to modified transferrin, nucleic acid molecules encoding the modified Tf fusion proteins, and the like. Another aspect of the present invention relates to methods of treating a patient with a modified Tf fusion protein.

[0013] Preferably, the modified Tf fusion proteins comprise a human transferrin Tf moiety that has been modified to reduce or prevent glycosylation and/or iron and receptor binding.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows an alignment of the N and C Domains of Human (Hu) transferrin (Tf) (SEQ ID NO: 3) with similarities and identities highlighted.

[0015] Figures 2A-2B show an alignment of transferrin sequences from different species. Light shading: Similarity; Dark shading: Identity (SEQ ID NOS: 19-25).

[0016] Figure 3 shows the location of a number of Tf surface exposed insertion sites for therapeutic proteins, polypeptides or peptides.

[0017] Figure 4A-4B show the amino acid and nucleic acid sequences of PYY(I -36) (SEQ ID NO: 4 and SEQ ID NO: 26) and amino acid sequence of PYY(3-36) (amino acids 3-36 of SEQ ID NO: 4 or SEQ ID NO: 5).

[0018] Figure 5 shows the molecular models of PYY(left), [Pro ³⁴ ]PYY(middle), and PYY(3- 36) (right). The molecular models show differences in the juxtaposition fo the NH ₂ and COOH termini and changes in the COOH-terminal helix that lead to changes in the receptor subtype selectivity. The arrows on the [Pro ³⁴ ]PYY and PYY(3-36) models indicate the critical regions of the PYY sequence that are important for Y receptor subtype selectivity.

[0019] Figure 6 shows pREX0197.

[0020] Figures 7A-7B show the amino acid and nucleic acid sequences for PYY-mTf fusion proteins. Figure 7A shows the sequences of PYY(I -36)-Tf fusion protein. (Mutation C901A and C912T to prevent hairpin loop in primers does not cause amino acid change.) Figure 7B shows the sequences of PYY(l-36)(codon optimized)-mTf fusion protein. Codon optimization of DNA sequence used the Saccharomyces cerevisiae table from http://www.kazusa.or.jp/codon/

[0021] Figure 8 shows pREX0616.

[0022] Figure 9 shows pSAC35.

[0023] Figure 10 shows pREX0617.

[0024] Figures 1 IA-I IB show pREX0608 and pREX0609.

[0025] Figures 12A-12B show pREX0639 and pREX0640.

[0026] Figures 13A-13B show pREX0838 and pREX0839.

[0027] Figures 14A-14B show pREX0641 and pREX0642.

[0028] Figures 15A-15B show pREX0643 and pREX0644.

[0029] Figures 16A-16B show pREX0738 and pREX0739.

[0030] Figures 17A-17B show pREX0740 and pREX0741.

[0031] Figures 18A-18C show pREX0584, pREX0907, and pREX0908.

[0032] Figures 19A-19B show pREX0909 and pREX0910.

DETAILED DESCRIPTION General Description

[0033] The present invention is based in part on the finding by the inventors that therapeutic proteins can be stabilized to extend their serum half-life and/or activity in vivo by genetically fusing the therapeutic proteins to transferrin, modified transferrin, or a portion of transferrin or modified transferrin sufficient to extend the half-life of the therapeutic protein in serum. The modified transferrin fusion proteins include a transferrin protein or domain covalently linked to a therapeutic protein or peptide, wherein the transferrin portion is modified to contain one or more amino acid substitutions, insertions or deletions compared to a wild-type transferrin sequence. In one embodiment, Tf fusion proteins are engineered to reduce or prevent glycosylation within the Tf or a Tf domain. In other embodiments, the Tf protein or Tf domain(s) is modified to exhibit reduced or no binding to iron or carbonate ion, or to have a reduced affinity or not bind to a Tf receptor (TfR).

[0034] The therapeutic proteins contemplated by the present invention include, but are not limited to polypeptides, antibodies, peptides, or fragments or variants thereof.

[0035] The present invention therefore includes transferrin fusion proteins, therapeutic compositions comprising the fusion proteins, and methods of treating, preventing, or ameliorating diseases or disorders by administering the fusion proteins. A transferrin fusion protein of the invention includes at least a fragment or variant of a therapeutic protein and at least a fragment or variant of modified transferrin, which are associated with one another, preferably by genetic fusion (i.e., the transferrin 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 modified transferrin) or

chemical conjugation to one another. The therapeutic protein and transferrin protein, once part of the transferrin fusion protein, may be referred to as a "portion", "region" or "moiety" of the transferrin fusion protein (e.g., a "therapeutic protein portion' or a "transferrin protein portion").

[0036] In one embodiment, the invention provides a transferrin fusion protein comprising, or alternatively consisting of, a therapeutic protein and a modified serum transferrin protein. In other embodiments, the invention provides a transferrin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment of a therapeutic protein and a modified transferrin protein. In other embodiments, the invention provides a transferrin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active variant of a therapeutic protein and modified transferrin protein. In further embodiments, the invention provides a transferrin fusion protein comprising a therapeutic protein, and a biologically active and/or therapeutically active fragment of modified transferrin. In another embodiment, the therapeutic protein portion of the transferrin fusion protein is the active form of the therapeutic protein.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

Definitions

[0038] As used herein, an "amino acid corresponding to" or an "equivalent amino acid" in a transferrin sequence is identified by alignment to maximize the identity or similarity between a first transferrin sequence and at least a second transferrin sequence. The number used to identify an equivalent amino acid in a second transferrin sequence is based on the number used to identify the corresponding amino acid in the first transferrin sequence. In certain cases, these phrases may be used to describe the amino acid residues in human transferrin compared to certain residues in rabbit serum transferrin.

[0039] As used herein, the term "biological activity" refers to a function or set of activities performed by a therapeutic molecule, protein or peptide in a biological context (i.e., in an

organism or an in vitro facsimile thereof). Biological activities may include but are not limited to the functions of the therapeutic molecule portion of the claimed fusion proteins, such as, but not limited to, the induction of extracellular matrix secretion from responsive cell lines, the induction of hormone secretion, the induction of chemotaxis, the induction of mitogenesis, the induction of differentiation, or the inhibition of cell division of responsive cells. A fusion protein or peptide of the invention is considered to be biologically active if it exhibits one or more biological activities of its therapeutic protein's native counterpart.

[0040] As used herein, "binders" are agents used to impart cohesive qualities to the powdered material. Binders, or "granulators" as they are sometimes known, impart a cohesiveness to the tablet formulation, which insures the tablet remaining intact after compression, as well as improving the free-flowing qualities by the formulation of granules of desired hardness and size. Materials commonly used as binders include starch; gelatin; sugars, such as sucrose, glucose, dextrose, molasses, and lactose; natural and synthetic gums, such as acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, Veegum, microcrystalline cellulose, microcrystalline dextrose, amylose, and larch arabogalactan, and the like.

[0041] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a composition 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.

[0042] As used herein, "coloring agents" are agents that give tablets a more pleasing appearance, and in addition help the manufacturer to control the product during its preparation and help the user to identify the product. Any of the approved certified water- soluble FD&C dyes, mixtures thereof, or their corresponding lakes may be used to color tablets. A color lake is the combination by adsorption of a water-soluble dye to a hydrous oxide of a heavy metal, resulting in an insoluble form of the dye.

[0043] As used herein, "diluents" are inert substances added to increase the bulk of the formulation to make the tablet a practical size for compression. Commonly used diluents include calcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, dry starch, powdered sugar, silica, and the like.

[0044] As used herein, "disintegrators" or "disintegrants" are substances that facilitate the breakup or disintegration of tablets after administration. Materials serving as disintegrants have been chemically classified as starches, clays, celluloses, algins, or gums. Other disintegrators include Veegum HV, methylcellulose, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp, cross- linked polyvinylpyrrolidone, carboxymethylcellulose, and the like.

[0045] The term "dispersibility" or "dispersible" means a dry powder having a moisture content of less than about 10% by weight (%w) water, usually below about 5%w and preferably less than about 3%w; a particle size of about 1.0-5.0 μm mass median diameter (MMD), usually 1.0-4.0 μm MMD, and preferably 1.0-3.0 μm MMD; a delivered dose of about >30%, usually >40%, preferably >50%, and most preferred >60%; and an aerosol particle size distribution of 1.0-5.0 μm mass median aerodynamic diameter (MMAD), usually 1.5-4.5 μm MMAD, and preferably 1.5-4.0 μm MMAD.

[0046] The term "dry" means that the composition has a moisture content such that the particles are readily dispersible in an inhalation device to form an aerosol. This moisture content is generally below about 10% by weight (%w) water, usually below about 5%w and preferably less than about 3%w.

[0047] As used herein, "effective amount" means an amount of a drug or pharmacologically active agent that is sufficient to provide the desired local or systemic effect and performance at a reasonable benefit/risk ratio attending any medical treatment.

[0048] As used herein, "flavoring agents" vary considerably in their chemical structure, ranging from simple esters, alcohols, and aldehydes to carbohydrates and complex volatile oils. Synthetic flavors of almost any desired type are now available.

[0049] As used herein, the terms "fragment of a Tf protein" or "Tf protein," or "portion of a Tf protein" refer to an amino acid sequence comprising at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of a naturally occurring Tf protein or mutant thereof.

[0050] As used herein, the term "gene" refers to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression. Genes can also include non-expressed

DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0051] As used herein, a "heterologous polynucleotide" or a "heterologous nucleic acid" or a "heterologous gene" or a "heterologous sequence" or an "exogenous DNA segment" refers to a polynucleotide, nucleic acid or DNA segment that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form. A heterologous gene in a host cell includes a gene that is endogenous to the particular host cell, but has been modified. Thus, the terms refer to a DNA segment which is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. As an example, a signal sequence native to a yeast cell but attached to a human Tf sequence is heterologous.

[0052] As used herein, an "isolated" nucleic acid sequence refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by agarose gel electrophoresis. For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semi-synthetic, synthetic origin, or any combinations thereof.

[0053] As used herein, two or more DNA coding sequences are said to be "joined" or "fused" when, as a result of in-frame fusions between the DNA coding sequences, the DNA coding sequences are translated into a fusion polypeptide. The term "fusion" in reference to Tf fusions includes, but is not limited to, attachment of at least one therapeutic protein,

polypeptide or peptide to the N -terminal end of Tf, attachment to the C-terminal end of Tf, and/or insertion between any two amino acids within Tf.

[0054] As used herein, "lubricants" are materials that perform a number of functions in tablet manufacture, such as improving the rate of flow of the tablet granulation, preventing adhesion of the tablet material to the surface of the dies and punches, reducing interparticle friction, and facilitating the ejection of the tablets from the die cavity. Commonly used lubricants include talc, magnesium stearate, calcium stearate, stearic acid, and hydrogenated vegetable oils. Typical amounts of lubricants range from about 0.1% by weight to about 5% by weight.

[0055] As used herein, "Modified transferrin" refers to a transferrin molecule that exhibits at least one modification of its amino acid sequence, compared to wild-type transferrin. Such modifications may include, but not limited to, modifications that reduce glycosylation compared to fully glycosylated Tf protein. Modified Tf may also include Tf that has reduced glycosylation via enzymatic removal of carbohydrate residues.

[0056] As used herein, "Modified transferrin fusion protein" as used herein refers to a protein formed by the fusion of at least one molecule of modified transferrin (or a fragment or variant thereof) to at least one molecule of a therapeutic protein (or fragment or variant thereof).

[0057] As used herein, the terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. Unless specifically limited, the terms encompass nucleic acids containing analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J! Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) MoI. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0058] As used herein, a DNA segment is referred to as "operably linked" when it is placed into a functional relationship with another DNA segment. For example, DNA for a signal sequence is operably linked to DNA encoding a fusion protein of the invention if it is expressed as a preprotein that participates in the secretion of the fusion protein; a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence. Generally, DNA sequences that are operably linked are contiguous, and in the case of a signal sequence or fusion protein both contiguous and in reading phase. However, enhancers need not be contiguous with the coding sequences whose transcription they control. Alternatively, DNA sequences that are operably linked may be separated by one or more intron sequences wherein splicing of the intron sequences results in the sequences being contiguous in the resulting mature mRNA. Linking, in this context, is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof.

[0059] As used herein, "pharmaceutically acceptable" refers to materials and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Typically, as used herein, the term "pharmaceutically acceptable" means approvable 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.

[0060] As used herein, "physiologically effective amount" is that amount delivered to a subject to give the desired palliative or curative effect. This amount is specific for each drug and its ultimate approved dosage level.

[0061] As used herein, the term "powder" means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli. Thus, the powder is said to be "respirable." Preferably the average particle size is less than about 10 microns (μm) in diameter with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 μm and most preferably less than about 5.0 μm. Usually the particle size distribution is between about 0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5 μm.

[0062] As used herein, the term "promoter" refers to a region of DNA involved in binding RNA polymerase to initiate transcription.

[0063] As used herein, the term "recombinant" refers to a cell, tissue or organism that has undergone transformation with a new combination of genes or DNA.

[0064] As used herein, the term "subject" can be a human, a mammal, or an animal. The subject being treated is a patient in need of treatment.

[0065] As used herein, a targeting entity, protein, polypeptide or peptide refers to a molecule that binds specifically to a particular cell type [normal (e.g., lymphocytes) or abnormal e.g., (cancer cell)] and therefore may be used to target a Tf fusion protein or compound (drug, or cytotoxic agent) to that cell type specifically.

[0066] As used herein, "tablets" are solid pharmaceutical dosage forms containing drug substances with or without suitable diluents and prepared either by compression or molding methods well known in the art. Tablets have been in widespread use since the latter part of the 19 ^th century and their popularity continues. Tablets remain popular as a dosage form because of the advantages afforded both to the manufacturer (e.g., simplicity and economy of preparation, stability, and convenience in packaging, shipping, and dispensing) and the patient (e.g., accuracy of dosage, compactness, portability, blandness of taste, and ease of administration). Although tablets are most frequently discoid in shape, they may also be round, oval, oblong, cylindrical, or triangular. They may differ greatly in size and weight depending on the amount of drug substance present and the intended method of administration. They are divided into two general classes, (1) compressed tablets, and (2) molded tablets or tablet triturates. In addition to the active or therapeutic ingredient or ingredients, tablets contain a number or inert materials or additives. A first group of such additives includes those materials that help to impart satisfactory compression characteristics to the formulation, including diluents, binders, and lubricants. A second group of such additives helps to give additional desirable physical characteristics to the finished tablet, such as disintegrators, colors, flavors, and sweetening agents.

[0067] As used herein, the term "therapeutically effective amount" refers to that amount of the transferrin fusion protein comprising a therapeutic molecule which, when administered to a subject in need thereof, is sufficient to effect treatment. The amount of transferrin fusion protein which constitutes a "therapeutically effective amount" will vary depending on the

therapeutic protein used, the severity of the condition or disease, and the age and body weight of the subject to be treated, but can be determined routinely by one or ordinary skill in the art having regard to his/her own knowledge and to this disclosure.

[0068] As used herein, "therapeutic protein" refers to proteins, polypeptides, peptides or fragments or variants thereof, having one or more therapeutic, prophylactic and/or biological activities. Therapeutic proteins encompassed by the invention include but are not limited to proteins, polypeptides, peptides, antibodies, and biologies. A preferred therapeutic protein is a Peptide YY or a GLP-I peptide. The terms peptides, proteins, and polypeptides are used interchangeably herein. Additionally, the term "therapeutic protein" may refer to the endogenous or naturally occurring correlate of a therapeutic protein. By a polypeptide displaying a "therapeutic activity" or a protein that is "therapeutically active" is meant a polypeptide that possesses one or more known biological and/or therapeutic activities associated with a therapeutic protein such as one or more of the therapeutic proteins described herein or otherwise known in the art. As a non-limiting example, a "therapeutic protein" is a protein that is useful to treat, prevent or ameliorate a disease, condition or disorder. Such a disease, condition or disorder may be in humans or in a non-human animal, e.g., veterinary use.

[0069] As used herein, the term "transformation" refers to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell. As used herein, the term "genetic transformation" refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell.

[0070] As used herein, the term "transformant" refers to a cell, tissue or organism that has undergone transformation.

[0071] As used herein, the term "transgene" refers to a nucleic acid that is inserted into an organism, host cell or vector in a manner that ensures its function.

[0072] As used herein, the term "transgenic" refers to cells, cell cultures, organisms, bacteria, fungi, animals, plants, and progeny of any of the preceding, which have received a foreign or modified gene and in particular a gene encoding a modified Tf fusion protein by one of the various methods of transformation, wherein the foreign or modified gene is from the same or different species than the species of the organism receiving the foreign or modified gene.

[0073] "Variants or variant" refers to a polynucleotide or nucleic acid differing from a reference nucleic acid or polypeptide, but retaining essential properties thereof. Generally,

variants are overall closely similar, and, in many regions, identical to the reference nucleic acid or polypeptide. As used herein, "variant" refers to a therapeutic protein portion of a transferrin fusion protein of the invention, differing in sequence from a native therapeutic protein but retaining at least one functional and/or therapeutic property thereof as described elsewhere herein or otherwise known in the art.

[0074] As used herein, the term "vector" refers broadly to any plasmid, phagemid or virus encoding an exogenous nucleic acid. The term is also be construed to include non-plasmid, non-phagemid and non- viral compounds which facilitate the transfer of nucleic acid into virions or cells, such as, for example, polylysine compounds and the like. The vector may be a viral vector that is suitable as a delivery vehicle for delivery of the nucleic acid, or mutant thereof, to a cell, or the vector may be a non-viral vector which is suitable for the same purpose. Examples of viral and non- viral vectors for delivery of DNA to cells and tissues are well known in the art and are described, for example, in Ma et al. (1997, Proc. Natl. Acad. ScL U.S.A. 94:12744-12746). Examples of viral vectors include, but are not limited to, a recombinant vaccinia virus, a recombinant adenovirus, a recombinant retrovirus, a recombinant adeno-associated virus, a recombinant avian pox virus, and the like (Cranage et al, 1986, EMBO J. 5:3057-3063; International Patent Application No. WO 94/17810, published August 18, 1994; International Patent Application No. WO 94/23744, published October 27, 1994). Examples of non- viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA, and the like.

[0075] As used herein, the term "wild type" refers to a polynucleotide or polypeptide sequence that is naturally occurring.

Transferrin and Transferrin Modifications

[0076] The present invention provides fusion proteins comprising therapeutic protein and transferrin. Any transferrin may be used to make modified Tf fusion proteins of the invention.

[0077] Any transferrin may be used to make modified Tf fusion proteins of the invention. As an example, the wild-type human Tf(Tf) is a 679 amino acid protein of approximately 75kDa (not accounting for glycosylation), with two main domains, or lobes, N (about 330 amino acids) and C (about 340 amino acids), which appear to originate from a gene duplication. See

GenBank accession numbers NM_001063, XM_002793, M12530, XM 039845, XM 039847 and S95936 (www.ncbi.nlm.nih.gov/), all of which are herein incorporated by reference in their entirety, as well as SEQ ID NOS 1, 2 and 3. The two domains have diverged over time but retain a large degree of identity/similarity (Fig. 1).

[0078] Each of the N and C domains is further divided into two subdomains, Nl and N2, Cl and C2. The function of Tf is to transport iron to the cells of the body. This process is mediated by the Tf receptor (TfR), which is expressed on all cells, particularly actively growing cells. TfR recognizes the iron bound form of Tf (two molecules of which are bound per receptor), endocytosis then occurs whereby the TfR/Tf complex is transported to the endosome, at which point the localized drop in pH results in release of bound iron and the recycling of the TfR/Tf complex to the cell surface and release of Tf (known as apoTf in its iron-unbound form). Receptor binding is through the C domain of Tf. The two glycosylation sites in the C domain do not appear to be involved in receptor binding as unglycosylated iron bound Tf does bind the receptor.

[0079] Each Tf molecule can carry two iron ions (Fe ³⁺ ). These are complexed in the space between the Nl and N2, Cl and C2 sub domains resulting in a conformational change in the molecule. Tf crosses the blood brain barrier (BBB) via the Tf receptor.

[0080] In human transferrin, the iron binding sites comprise at least amino acids Asp 63 (Asp 82 of SEQ ID NO: 2 which includes the native Tf signal sequence), Asp 392 (Asp 411 of SEQ ID NO: 2), Tyr 95 (Tyr 114 of SEQ ID NO: 2), Tyr 426 (Tyr 445 of SEQ ID NO: 2), Tyr 188 (Tyr 207 of SEQ ID NO: 2), Tyr 514 or 517 (Tyr 533 or Tyr 536 SEQ ID NO: 2), His 249 (His 268 of SEQ ID NO: 2), and His 585 (His 604 of SEQ ID NO: 2) of SEQ ID NO: 3. The hinge regions comprise at least N domain amino acid residues 94-96, 245- 247 and/or 316-318 as well as C domain amino acid residues 425-427, 581-582 and/or 652-658 of SEQ ID NO: 3. The carbonate binding sites comprise at least amino acids Thr 120 (Thr 139 of SEQ ID NO: 2), Thr 452 (Thr 471 of SEQ ID NO: 2), Arg 124 (Arg 143 of SEQ ID NO: 2), Arg 456 (Arg 475 of SEQ ID NO: 2), Ala 126 (Ala 145 of SEQ ID NO: 2), Ala 458 (Ala 477 of SEQ ID NO: 2), GIy 127 (GIy 146 of SEQ ID NO: 2), and GIy 459 (GIy 478 of SEQ ID NO: 2) of SEQ ID NO: 3.

[0081] In one embodiment of the invention, the modified transferrin fusion protein includes a modified human transferrin, although any animal Tf molecule may be used to produce the

fusion proteins of the invention, including human Tf variants, cow, pig, sheep, dog, rabbit, rat, mouse, hamster, echnida, platypus, chicken, frog, hornworm, monkey, as well as other bovine, canine and avian species. All of these Tf sequences are readily available in GenBank and other public databases. The human Tf nucleotide sequence is available (see SEQ ID NOS 1 , 2 and 3 and the accession numbers described above and available at www.ncbi.nlm.nih.gov/) and can be used to make genetic fusions between Tf or a domain of Tf and the therapeutic molecule of choice. Fusions may also be made from related molecules such as lacto transferrin (lactoferrin) GenBank Ace: NM_002343) or melanotransferrin (GenBank Ace. NM_013900, murine melanotransferrin).

[0082] Melanotransferrin is a glycosylated protein found at high levels in malignant melanoma cells and was originally named human melanoma antigen p97 (Brown et ah, 1982, Nature, 296: 171-173). It possesses high sequence homology with human serum transferrin, human lactoferrin, and chicken transferrin (Brown et ah, 1982, Nature, 296: 171-173; Rose et ah, Proc. Natl. Acad. Sci. USA, 1986, 83: 1261-1265). However, unlike these receptors, no cellular receptor has been identified for melanotransferrin. Melanotransferrin reversibly binds iron and it exists in two forms, one of which is bound to cell membranes by a glycosyl phosphatidylinositol anchor while the other form is both soluble and actively secreted (Baker et ah, 1992, FEBS Lett, 298: 215-218; Alemany et ah, 1993, J. Cell Sci., 104: 1155-1162; Food et ah, 1994, J. Biol. Chem. 274: 701 1-7017).

[0083] Lactoferrin (Lf), a natural defense iron-binding protein, has been found to possess antibacterial, antimycotic, antiviral, antineoplastic and anti-inflammatory activity. The protein is present in exocrine secretions that are commonly exposed to normal flora: milk, tears, nasal exudate, saliva, bronchial mucus, gastrointestinal fluids, cervico-vaginal mucus and seminal fluid. Additionally, Lf is a major constituent of the secondary specific granules of circulating polymorphonuclear neutrophils (PMNs). The apoprotein is released on degranulation of the PMNs in septic areas. A principal function of Lf is that of scavenging free iron in fluids and inflamed areas so as to suppress free radical-mediated damage and decrease the availability of the metal to invading microbial and neoplastic cells. In a study that examined the turnover rate of ¹²⁵ I Lf in adults, it was shown that Lf is rapidly taken up by the liver and spleen, and the radioactivity persisted for several weeks in the liver and spleen (Bennett et a (1979), Clin. Sci. (Lond.) 57: 453-460).

[0084] In one embodiment, the transferrin portion of the transferrin fusion protein of the invention includes a transferrin splice variant. In one example, a transferrin splice variant can be a splice variant of human transferrin. In one specific embodiment, the human transferrin splice variant can be that of Genbank Accession AAA61140.

[0085] In another embodiment, the transferrin portion of the transferrin fusion protein of the invention includes a lactoferrin splice variant. In one example, a human serum lactoferrin splice variant can be a novel splice variant of a neutrophil lactoferrin. In one specific embodiment, the neutrophil lactoferrin splice variant can be that of Genbank Accession AAA59479. In another specific embodiment, the neutrophil lactoferrin splice variant can comprise the following amino acid sequence EDCIALKGEADA (SEQ ID NO: 18), which includes the novel region of splice-variance.

[0086] In another embodiment, the transferrin portion of the transferrin fusion protein of the invention includes a melanotransferrin variant.

[0087] Modified Tf fusions may be made with any Tf protein, fragment, domain, or engineered domain. For instance, fusion proteins may be produced using the full-length Tf sequence, with or without the native Tf signal sequence. Tf fusion proteins may also be made using a single Tf domain, such as an individual N or C domain or a modified form of Tf comprising 2N or 2C domains (see U.S. Provisional Application 60/406,977, filed August 30, 2002, which is herein incorporated by reference in its entirety). In some embodiments, fusions of a therapeutic protein to a single C domain may be produced, wherein the C domain is altered to reduce, inhibit or prevent glycosylation. In other embodiments, the use of a single N domain is advantageous as the Tf glycosylation sites reside in the C domain and the N domain, on its own. A preferred embodiment is the Tf fusion protein having a single N domain which is expressed at a high level.

[0088] As used herein, a C terminal domain or lobe modified to function as an N-like domain is modified to exhibit glycosylation patterns or iron binding properties substantially like that of a native or wild-type N domain or lobe. In a preferred embodiment, the C domain or lobe is modified so that it is not glycosylated and does not bind iron by substitution of the relevant C domain regions or amino acids to those present in the corresponding regions or sites of a native or wild-type N domain.

[0089] As used herein, a Tf moiety comprising "two N domains or lobes" includes a Tf molecule that is modified to replace the native C domain or lobe with a native or wild-type N domain or lobe or a modified N domain or lobe or contains a C domain that has been modified to function substantially like a wild-type or modified N domain.

[0090] Analysis of the two domains by overlay of the two domains (Swiss PDB Viewer 3.7b2, Iterative Magic Fit) and by direct amino acid alignment (ClustalW multiple alignment) reveals that the two domains have diverged over time. Amino acid alignment shows 42% identity and 59% similarity between the two domains. However, approximately 80% of the N domain matches the C domain for structural equivalence. The C domain also has several extra disulfide bonds compared to the N domain.

[0091] Alignment of molecular models for the N and C domain reveals the following structural equivalents:

The disulfide bonds for the two domains align as follows:

Bold aligned disulfide bonds Italics bridging peptide

[0092] In one embodiment, the transferrin portion of the transferrin fusion protein includes at least two N terminal lobes of transferrin. In further embodiments, the transferrin portion of the transferrin fusion protein includes at least two N terminal lobes of transferrin derived from human serum transferrin.

[0093] In another embodiment, the transferrin portion of the transferrin fusion protein includes, comprises, or consists of at least two N terminal lobes of transferrin having a mutation in at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyrl88, and His249 of SEQ ID NO: 3.

[0094] In another embodiment, the transferrin portion of the modified transferrin fusion protein includes a recombinant human serum transferrin N-terminal lobe mutant having a mutation at Lys206 or His207 of SEQ ID NO: 3.

[0095] In another embodiment, the transferrin portion of the transferrin fusion protein includes, comprises, or consists of at least two C terminal lobes of transferrin. In further embodiments, the transferrin portion of the transferrin fusion protein includes at least two C terminal lobes of transferrin derived from human serum transferrin.

[0096] In a further embodiment, the C terminal lobe mutant further includes a mutation of at least one of Asn413 and Asnβl 1 of SEQ ID NO: 3 or equivalent amino acids corresponding to these positions,which does not allow glycosylation.

[0097] In another embodiment, the transferrin portion of the transferrin fusion protein includes at least two C terminal lobes of transferrin having a mutation in at least one amino acid residue selected from the group consisting of Asp392, Tyr426, Tyr514, Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant retains the ability to bind metal. In an alternate embodiment, the transferrin portion of the transferrin fusion protein includes at least two C terminal lobes of transferrin having a mutation in at least one amino acid residue selected from the group consisting of Tyr426, Tyr514, Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant has a reduced ability to bind metal. In another embodiment, the transferrin portion of the transferrin fusion protein includes at least two C terminal lobes of transferrin having a mutation in at least one amino acid residue selected from the group consisting of Asp392, Tyr426, Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant does not retain the ability to bind metal and functions substantially like an N domain.

[0098] In some embodiments, the Tf or Tf portion will be of sufficient length to increase the in vivo circulatory half-life, serum stability, in vitro solution stability or bioavailability of the therapeutic protein or peptide compared to the in vivo circulatory half-life, serum stability, in vitro solution stability or bioavailability of the therapeutic protein or peptide in an unfused state. Such an increase in stability, serum half-life or bioavailability may be about a 30%, 50%, 70%, 80%, 90% or more increase over the unfused therapeutic protein or peptide. In some cases, the transferrin fusion proteins comprising modified transferrin exhibit a serum half-life of about 10-20 or more days, about 12-18 days or about 14-17 days.

[0099] When the C domain of Tf is part of the fusion protein, the two N-linked glycosylation sites, amino acid residues corresponding to N413 and N611 of SEQ ID NO: 3 may be mutated to prevent glycosylation or hypermannosylationn and extend the serum half-life of the fusion protein and/or therapeutic protein (to produce asialo-, or in some instances, monosialo-Tf or disialo-Tf). In addition to Tf amino acids corresponding to N413 and N611, mutations may be to the adjacent residues within the N-X-S/T glycosylation site to prevent or substantially reduce glycosylation. See U.S. Patent 5,986,067 of Funk et al. It has also been reported that the N domain of Tf expressed in Pichia pastoris becomes O-linked glycosylated with a single hexose at S32 which also may be mutated or modified to prevent such glycosylation.

[00100] Accordingly, in one embodiment of the invention, the transferrin fusion protein includes a modified transferrin molecule wherein the transferrin exhibits reduced glycosylation, including but not limited to asialo- monosialo- and disialo- forms of Tf. In another embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant transferrin mutant that is mutated to prevent glycosylation. In another embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant transferrin mutant that is fully glycosylated. In a further embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant human serum transferrin mutant that is mutated to prevent glycosylation, wherein at least one of Asn413 and Asn611 of SEQ ID NO: 3 are mutated to an amino acid which does not allow glycosylation. In another embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant human serum transferrin mutant that is mutated to prevent or substantially reduce glycosylation, wherein mutations may be to the adjacent residues within the N-X-S/T glycosylation site. Moreover, glycosylation may be reduced or prevented by mutating the

serine or threonine residue. Further, changing the X to proline is known to inhibit glycosylation.

[00101] As discussed below in more detail, modified Tf fusion proteins of the invention may also be engineered to not bind iron and/or bind the Tf receptor. In other embodiments of the invention, the iron binding is retained and the iron binding ability of Tf may be used to deliver a therapeutic protein or peptide(s) to the inside of a cell, across an epithelial or endothelial cell membrane and/or across the BBB. These embodiments that bind iron and/or the Tf receptor will often be engineered to reduce or prevent glycosylation to extend the serum half-life of the therapeutic protein. The N domain alone will not bind to TfR when loaded with iron, and the iron bound C domain will bind TfR but not with the same affinity as the whole molecule.

[00102] In another embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant does not retain the ability to bind metal ions. In an alternate embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant has a weaker binding avidity for metal ions than wild-type serum transferrin. In an alternate embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant has a stronger binding avidity for metal ions than wild-type serum transferrin.

[00103] In another embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant does not retain the ability to bind to the transferrin receptor. In an alternate embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant has a weaker binding avidity for the transferrin receptor than wild-type serum transferrin. In an alternate embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant has a stronger binding avidity for the transferrin receptor than wild-type serum transferrin.

[00104] In another embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant does not retain the ability to bind to carbonate ions. In an alternate embodiment, the transferrin

portion of the transferrin fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant has a weaker binding avidity for carbonate ions than wild-type serum transferrin. In an alternate embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant has a stronger binding avidity for carbonate ions than wild-type serum transferrin.

[00105] In another embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant human serum transferrin mutant having a mutation in at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyrl88, His249, Asp392, Tyr426, Tyr514, Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant retains the ability to bind metal ions. In an alternate embodiment, a recombinant human serum transferrin mutant having a mutation in at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyrl88, His249, Asp392, Tyr426, Tyr514, Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant has a reduced ability to bind metal ions. In another embodiment, a recombinant human serum transferrin mutant having a mutation in at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyrl88, His249, Asp392, Tyr426, Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant does not retain the ability to bind metal ions.

[00106] In another embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant human serum transferrin mutant having a mutation at Lys206 or His207 of SEQ ID NO: 3, wherein the mutant has a stronger binding avidity for metal ions than wild-type human serum transferrin (see U.S. Patent 5,986,067, which is herein incorporated by reference in its entirety). In an alternate embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant human serum transferrin mutant having a mutation at Lys206 or His207 of SEQ ID NO: 3, wherein the mutant has a weaker binding avidity for metal ions than wild-type human serum transferrin. In a further embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant human serum transferrin mutant having a mutation at Lys206 or His207 of SEQ ID NO:3, wherein the mutant does not bind metal ions.

[00107] Any available technique may be used to produce the transferrin fusion proteins of the invention, including but not limited to molecular techniques commonly available, for instance, those disclosed in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd

Ed., Cold Spring Harbor Laboratory Press, 1989. When carrying out nucleotide substitutions using techniques for accomplishing site-specific mutagenesis that are well known in the art, the encoded amino acid changes are preferably of a minor nature, that is, conservative amino acid substitutions, although other, non-conservative, substitutions are contemplated as well, particularly when producing a modified transferrin portion of a Tf fusion protein, e.g., a modified Tf protein exhibiting reduced glycosylation, reduced iron binding and the like. Specifically contemplated are amino acid substitutions, small deletions or insertions, typically of one to about 30 amino acids; insertions between transferrin domains; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, or small linker peptides of less than 50, 40, 30, 20 or 10 residues between transferrin domains or linking a transferrin protein and therapeutic protein or peptide or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain.

[00108] Examples of conservative amino acid substitutions are substitutions made within the same group such as within the group of basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine, threonine, methionine).

[00109] Non-conservative substitutions encompass substitutions of amino acids in one group by amino acids in another group. For example, a non-conservative substitution would include the substitution of a polar amino acid for a hydrophobic amino acid. For a general description of nucleotide substitution, see e.g. Ford et αl. (1991), Prot. Exp. Pur. 2: 95-107. Non-conservative substitutions, deletions and insertions are particularly useful to produce Tf fusion proteins of the invention that exhibit no or reduced binding of iron, no or reduced binding of the fusion protein to the Tf receptor and/or no or reduced glycosylation.

[00110] Iron binding and/or receptor binding may be reduced or disrupted by mutation, including deletion, substitution or insertion into, amino acid residues corresponding to one or more of Tf N domain residues Asp63, Tyr95, Tyrl88, His249 and/or C domain residues Asp 392, Tyr 426, Tyr 514 and/or His 585 of SEQ ID NO: 3. Iron binding may also be affected by mutation to amino acids Lys206, His207 or Arg632 of SEQ ID NO: 3. Carbonate binding may be reduced or disrupted by mutation, including deletion, substitution or insertion into,

amino acid residues corresponding to one or more of Tf N domain residues Thrl20, Argl24, Alal26, GIy 127 and/or C domain residues Thr 452, Arg 456, Ala 458 and/or GIy 459 of SEQ ID NO: 3. A reduction or disruption of carbonate binding may adversely affect iron and/or receptor binding.

[00111] Binding to the Tf receptor may be reduced or disrupted by mutation, including deletion, substitution or insertion into, amino acid residues corresponding to one or more of TfN domain residues described above for iron binding.

[00112] As discussed above, glycosylation may be reduced or prevented by mutation, including deletion, substitution or insertion into, amino acid residues corresponding to one or more of Tf C domain residues around the N-X-S/T sites corresponding to C domain residues N413 and/or N611 (See U.S. Patent No. 5,986,067). For instance, the N413 and/or N611 may be mutated to GIu residues.

[00113] In instances where the Tf fusion proteins of the invention are not modified to prevent glycosylation, iron binding, carbonate binding and/or receptor binding, glycosylation, iron and/or carbonate ions may be stripped from or cleaved off of the fusion protein. For instance, available deglycosylases may be used to cleave glycosylation residues from the fusion protein, in particular the sugar residues attached to the Tf portion, yeast deficient in glycosylation enzymes may be used to prevent glycosylation and/or recombinant cells may be grown in the presence of an agent that prevents glycosylation, e.g., tunicamycin.

[00114] The carbohydrates on the fusion protein may also be reduced or completely removed enzymatically by treating the fusion protein with deglycosylases. Deglycosylases are well known in the art. Examples of deglycosylases include but are not limited to galactosidase, PNGase A, PNGase F, glucosidase, mannosidase, fucosidase, and Endo H deglycosylase.

[00115] Nevertheless, in certain circumstances, it may be preferable for oral delivery that the Tf portion of the fusion protein be fully glycosylated

[00116] Additional mutations may be made with Tf to alter the three dimensional structure of Tf, such as modifications to the hinge region to prevent the conformational change needed for iron binding and Tf receptor recognition. For instance, mutations may be made in or around N domain amino acid residues 94-96, 245-247 and/or 316-318 as well as C domain

amino acid residues 425-427, 581-582 and/or 652-658. In addition, mutations may be made in or around the flanking regions of these sites to alter Tf structure and function.

[00117] In one aspect of the invention, the transferrin fusion protein can function as a carrier protein to extend the half life or bioavailability of the therapeutic protein as well as, in some instances, delivering the therapeutic protein inside a cell and/or across the blood brain barrier. In an alternate embodiment, the transferrin fusion protein includes a modified transferrin molecule wherein the transferrin does not retain the ability to cross the blood brain barrier.

[00118] In another embodiment, the transferrin fusion protein includes a modified transferrin molecule wherein the transferrin molecule retains the ability to bind to the transferrin receptor and transport the therapeutic peptide inside cells. In an alternate embodiment, the transferrin fusion protein includes a modified transferrin molecule wherein the transferrin molecule does not retain the ability to bind to the transferrin receptor and transport the therapeutic peptide inside cells.

[00119] In further embodiments, the transferrin fusion protein includes a modified transferrin molecule wherein the transferrin molecule retains the ability to bind to the transferrin receptor and transport the therapeutic peptide inside cells and retains the ability to cross the blood brain barrier. In an alternate embodiment, the transferrin fusion protein includes a modified transferrin molecule wherein the transferrin molecule retains the ability to cross the blood brain barrier, but does not retain the ability to bind to the transferrin receptor and transport the therapeutic peptide inside cells.

Modified Transferrin Fusion Proteins

[00120] The fusion of proteins of the invention may contain one or more copies of the therapeutic protein or polypeptide attached to the N-terminus and/or the C-terminus of the Tf protein. In some embodiments, the therapeutic protein or polypeptide is attached to both the N- and C-terminus of the Tf protein and the fusion protein may contain one or more equivalents of the therapeutic protein or polypeptide on either or both ends of Tf. In other embodiments, the therapeutic protein or polypeptide is inserted into known domains of the Tf protein, for instance, into one or more of the surface loops of Tf (see AIi et al. (1999) J. Biol. Chem. 274(34):24066-24073). In fact, the therapeutic protein or polypeptide may be inserted into multiple loops of transferrin to create a multivalent molecule with increased avidity for the antigen, receptor, or targeting molecule, which the therapeutic protein binds. In other

embodiments, the therapeutic protein or polypeptide is inserted between the N and C domains of Tf. Alternatively, the therapeutic protein or polypeptide is inserted anywhere in the transferrin molecule.

[00121] Generally, the transferrin fusion protein of the invention may have one modified transferrin-derived region and one therapeutic protein region. Multiple regions of each protein, however, may be used to make a transferrin fusion protein of the invention. Similarly, more than one therapeutic protein may be used to make a transferrin fusion protein of the invention, thereby producing a multi-functional modified Tf fusion protein.

[00122] In one embodiment, the transferrin fusion protein of the invention contains a therapeutic protein or polypeptide or portion thereof fused to a transferrin molecule or portion thereof. In another embodiment, the transferrin fusion protein of the inventions contains a therapeutic protein or polypeptide fused to the N terminus of a transferrin molecule. In an alternate embodiment, the transferrin fusion protein of the invention contains a therapeutic protein or polypeptide fused to the C terminus of a transferrin molecule. In a further embodiment, the transferrin fusion protein of the invention contains a transferrin molecule fused to the N terminus of a therapeutic protein or polypeptide. In an alternate embodiment, the transferrin fusion protein of the invention contains a transferrin molecule fused to the C terminus of a therapeutic protein or polypeptide.

[00123] In other embodiments, the transferrin fusion protein of the inventions contains a therapeutic protein fused to both the N-terminus and the C-terminus of modified transferrin. In another embodiment, the therapeutic proteins fused at the N- and C- termini bind the same therapeutic proteins. In an alternate embodiment, the therapeutic proteins fused at the N- and C- termini are different therapeutic proteins. In another alternate embodiment, the therapeutic proteins fused to the N- and C- termini bind different therapeutic proteins which may be used to treat or prevent the same disease, disorder, or condition. In another embodiment, the therapeutic proteins fused at the N- and C- termini are different therapeutic proteins which may be used to treat or prevent diseases or disorders which are known in the art to commonly occur in patients simultaneously.

[00124] In addition to modified transferrin fusion protein of the invention in which the modified transferrin portion is fused to the N terminal and/or C-terminal of the therapeutic protein portion, transferrin fusion protein of the invention may also be produced by inserting

the therapeutic protein or peptide of interest (e.g., a therapeutic protein or peptide as disclosed herein, or a fragment or variant thereof) into an internal region of the modified transferrin. Internal regions of modified transferrin include, but are not limited to, the iron binding sites, the hinge regions, the bicarbonate binding sites, or the receptor binding domain.

[00125] Within the protein sequence of the modified transferrin molecule a number of loops or turns exist, which are stabilized by disulfide bonds. 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 generate a modified transferrin molecule with specific biological activity.

[00126] When therapeutic proteins are inserted into or replace at least one loop of a Tf molecule, insertions may be made within any of the surface exposed loop regions, in addition to other areas of Tf. For instance, insertions may be made within the loops comprising Tf amino acids 32-33, 74-75, 256-257, 279-280 and 288-289 (AIi et al, supra) (See Figure 3). As previously described, insertions may also be made within other regions of Tf such as the sites for iron and bicarbonate binding, hinge regions, and the receptor binding domain as described in more detail below. The loops in the Tf protein sequence that are amenable to modification/replacement for the insertion of proteins or peptides may also be used for the development of a screenable library of random peptide inserts. Any procedures may be used to produce nucleic acid inserts for the generation of peptide libraries, including available phage and bacterial display systems, prior to cloning into a Tf domain and/or fusion to the ends of Tf.

[00127] The N-terminus of Tf is free and points away from the body of the molecule. Fusions of proteins or peptides on the N-terminus may therefore be a preferred embodiment. Such fusions may include a linker region, such as but not limited to a poly-glycine stretch, to separate the therapeutic protein from Tf. Attention to the junction between the leader sequence, the choice of leader sequence, and the structure of the mRNA by codon manipulation/optimization (no major stem loops to inhibit ribosome progress) will increase secretion and can be readily accomplished using standard recombinant DNA techniques.

[00128] The C-terminus of Tf appears to be more buried and secured by a disulfide bond 6 amino acids from the C-terminus. In human Tf, the C-terminal amino acid is a proline which, depending on the way that it is orientated, will either point a fusion away or into the body of

the molecule. A linker or spacer moiety at the C-terminus may be used in some embodiments of the invention. There is also a proline near the N-terminus. In one aspect of the invention, the proline at the N- and/or the C- termini may be changed. In another aspect of the invention, the C-terminal disulfide bond may be eliminated to untether the C-terminus.

[00129] In yet other embodiments, small molecule therapeutics may be complexed with iron and loaded on a modified Tf protein fusion for delivery to the inside of cells and across the BBB. The addition of a targeting peptide or, for example, a single chain antibody (SCA) can be used to target the payload to a particular cell type, e.g., a cancer cell.

Therapeutic Proteins and Peptides

[00130] Any therapeutic molecule may be used as the fusion partner to Tf according to the methods and compositions of the present invention. As used herein, a therapeutic molecule is typically a protein or peptide capable of exerting a beneficial biological effect in vitro or in vivo and includes proteins or peptides that exert a beneficial effect in relation to normal homeostasis, physiology or a disease state. Therapeutic molecules do not include fusion partners commonly used as markers or protein purification aids, such as bacterial galactosidases (see for example, U.S. Patent 5, 986, 067 and Aldred et al. (1984) Biochem. Biophys. Res. Commun. 122: 960-965). For instance, a beneficial effect as related to a disease state includes any effect that is advantageous to the treated subject, including disease prevention, disease stabilization, the lessening or alleviation of disease symptoms or a modulation, alleviation or cure of the underlying defect to produce an effect beneficial to the treated subject.

[00131] A modified transferrin fusion protein of the invention includes at least a fragment or variant of a therapeutic protein and at least a fragment or variant of modified serum transferrin, which are associated with one another, preferably by genetic fusion.

[00132] In one embodiment, the transferrin fusion protein includes a modified transferrin molecule linked to a neuropharmaceutical agent. In another embodiment, the modified transferrin fusion protein includes transferrin at the carboxyl terminus linked to a neuropharmaceutical agent at the amino terminus. In an alternate embodiment, the modified transferrin fusion protein includes transferrin at the amino terminus linked to a

neuropharmaceutical agent at the carboxy terminus. In specific embodiments, the neuropharmaceutical agent is either nerve growth factor or ciliary neurotrophic factor.

[00133] In further embodiments, a modified transferrin fusion protein of the invention may contain at least a fragment or variant of a therapeutic protein. In a further embodiment, the transferrin fusion proteins can contain peptide fragments or peptide variants of proteins or antibodies wherein the variant or fragment retains at least one biological or therapeutic activity. The transferrin fusion proteins can contain therapeutic proteins that can be peptide fragments or peptide variants at least about 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, 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 about 40, at least about 50, at least about 55, at least about 60 or at least about 70 or more amino acids in length fused to the N and/or C termini, inserted within, or inserted into a loop of a modified transferrin.

[00134] The modified transferrin fusion proteins of the present invention may contain one or more peptides. Increasing the number of peptides enhances the function of the peptides fused to transferrin and the function of the entire transferrin fusion protein. The peptides may be used to make a bi- or multi-functional fusion protein by including peptide or protein domains with multiple functions. For instance, a multi-functional fusion protein can be made with a therapeutic protein and a second protein to target the fusion protein to a specific target. Other peptides may be used to induce the immune response of a cellular system, or induce an antiviral, antibacterial, or anti-pathogenic response.

[00135] In another embodiment, the modified transferrin fusion molecules contain a therapeutic protein portion that can be fragments of a therapeutic protein that include the full length protein as well as polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence.

[00136] In another embodiment, the modified transferrin fusion molecules contain a therapeutic protein portion that can be fragments of a therapeutic protein that include the full length protein as well as polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence.

[00137] In another embodiment, the modified transferrin fusion molecules contain a therapeutic protein portion that can have one or more amino acids deleted from both the amino and the carboxy termini.

[00138] In another embodiment, the modified transferrin fusion molecules contain a therapeutic protein portion that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference therapeutic protein set forth herein, or fragments thereof. In further embodiments, the transferrin fusion molecules contain a therapeutic protein portion that is at least about 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.

[00139] In another embodiment, the modified transferrin fusion molecules contain the therapeutic protein portion that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, the native or wild-type amino acid sequence of a therapeutic protein. Fragments, of these polypeptides are also provided.

[00140] The therapeutic proteins corresponding to a therapeutic protein portion of a modified transferrin fusion protein of the invention, such as cell surface and secretory proteins, can be modified by the attachment or removal of one or more oligosaccharide groups. The modification referred to as glycosylation can significantly 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 or threonine residues; and glycosylation characterized by N-linked oligosaccharides, which are attached to asparagine residues in an Asn-X-Ser/Thr sequence, where X can be an amino acid except proline. 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. For example, several types of human interferon are glycosylated.

[00141] Therapeutic proteins corresponding to a therapeutic protein portion of a transferrin 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, e.g., by substitution or deletion of amino acid residues, such as substitution of glutamine for asparagine, or unglycosylated recombinant proteins may be

produced by expressing the proteins in host cells that will not glycosylate them, e.g. in glycosylation-deficient yeast. These approaches are known in the art.

[00142] Therapeutic proteins and their nucleic 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 GenSeq. The Accession Numbers and sequences referred to below are herein incorporated by reference in their entirety.

[00143] In other embodiments, the transferrin 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 described elsewhere in this application. In further embodiments, the therapeutically active protein portions of the transferrin fusion proteins of the invention are fragments or variants of the reference sequences cited herein.

[00144] The present invention is further directed to modified Tf fusion proteins comprising fragments of the therapeutic proteins herein described. Even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the therapeutic protein portion, 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 with less than the majority of the residues of the complete polypeptide removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can be assayed by routine methods described herein and otherwise known in the art. It is not unlikely that a mutant 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.

[00145] Also as mentioned above, even if deletion of one or more amino acids from the N- terminus or C-terminus of a therapeutic protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) 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.

[00146] Peptide fragments of the therapeutic proteins can be 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 of which the amino acid sequence is a fragment.

[00147] The peptide fragments of the therapeutic protein may comprise only the N- and C- termini of the protein, i.e., the central portion of the therapeutic protein has been deleted. Alternatively, the peptide fragments may comprise non-adjacent and/or adjacent portions of the central part of the therapeutic protein.

[00148] Other polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of a therapeutic protein used in the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.

[00149] Generally, variants of proteins are overall very similar, and, in many regions, identical to the amino acid sequence of the therapeutic protein corresponding to a therapeutic protein portion of a transferrin fusion protein of the invention. Nucleic acids encoding these variants are also encompassed by the invention.

[00150] Further therapeutic polypeptides that may be used in the invention are polypeptides encoded by polynucleotides which hybridize to the complement of a nucleic acid molecule encoding an amino acid sequence of a therapeutic protein under stringent hybridization conditions which are known to those of skill in the art. (see, for example, Ausubel, F.M. et al, eds., 1989 Current protocol in Molecular Biology, Green Publishing Associates, Inc., and John Wiley & Sons Inc., New. York). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[00151] By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that

the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence, or in one or more contiguous groups within the reference sequence.

[00152] As a practical matter, whether any particular polypeptide is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a transferrin fusion protein of the invention or a fragment thereof (such, as the therapeutic protein portion of the transferrin fusion protein or the transferrin portion of the transferrin 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 Brufiag et al. (Comp. App. Biosci 245 (1990)).

[00153] The polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide may be used to produce modified Tf fusion proteins. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code can be utilized. Moreover, polypeptide variants in which less than about 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 can also be utilized. 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 host, such as, yeast or E. coli as described above).

[00154] In other embodiments, the therapeutic protein moiety has conservative substitutions compared to the wild-type sequence. By "conservative substitutions" is intended swaps within groups such as replacement of the aliphatic or hydrophobic amino acids Ala, VaI, Leu

and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and GIu; replacement of the amide residues Asn and GIn, 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 GIy. Guidance concerning how to make phenotypically silent amino acid substitutions is provided, for example, in Bowie et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990). In specific embodiments, the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of the amino acid sequence of a therapeutic protein described herein and/or serum transferrin, and/ modified transferrin protein of the invention, wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150 amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence. In further embodiments, the amino acid substitutions are conservative. Nucleic acids encoding these polypeptides are also encompassed by the invention.

[00155] The modified fusion proteins of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds 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.

[00156] Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxy 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, glycosylation, GPI

anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York(1993); POST- TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et at. (1990) Meth. Enzymol. 182:626- 646; Rattan et al., Ann. N. Y. Acad. Sci. 663:48-62.

[00157] The therapeutic proteins of the present invention include, but are not limited to polypeptide, peptide, antibody, or fragments and variants thereof. Preferably, the therapeutic proteins of the present invention include glucagon-like peptide- 1 (GLP-I) and peptide YY (PYY).

Glucagon-Like Peptide-1 (GLP-I)

[00158] Glucagon-Like Peptide-1 (GLP-I) is a gastrointestinal hormone that regulates insulin secretion belonging to the so-called enteroinsular axis. The enteroinsular axis designates a group of hormones, released from the gastrointestinal mucosa in response to the presence and absorption of nutrients in the gut, which promote an early and potentiated release of insulin. The incretin effect which is the enhancing effect on insulin secretion is probably essential for a normal glucose tolerance. GLP-I is a physiologically important insulinotropic hormone because it is responsible for the incretin effect.

[00159] GLP-I is a product of proglucagon (Bell, et al, Nature, 1983, 304: 368-371). It is synthesized in intestinal endocrine cells in two principal major molecular forms, as GLP- 1(7- 36)amide and GLP-l(7-37). The peptide was first identified following the cloning of cDNAs and genes for proglucagon in the early 1980s.

[00160] Initial studies done on the full length peptide GLP-l(l-37) and GLP-l(l-36 ^amide ) concluded that the larger GLP-I molecules are devoid of biological activity. In 1987, three independent research groups demonstrated that removal of the first six amino acids resulted in a GLP-I molecule with enhanced biological activity.

[00161] The amino acid sequence of GLP-I is disclosed by Schmidt et al. (1985 Diabetologia 28 704-707). Human GLP-I is a 37 amino acid residue peptide originating from preproglucagon which is synthesized in the L-cells in the distal ileum, in the pancreas, and in the brain. Processing of preproglucagon to GLP- 1(7-36 ^amide ), GLP- 1(7-37) and GLP- 2 occurs mainly in the L-cells. The amino acid sequence of GLP- 1(7-36 ^amide ) and GLP- 1(7- 37) is (SEQ ID NO: 6):

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Gly-Gln-Ala-Ala- Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-X wherein X is NH ₂ for GLP-I (7-36 ^amide ) and X is GIy for GLP-l(7-37).

[00162] GLP-I like molecules possesses anti-diabetic activity in human subjects suffering from Type II (non-insulin-dependent diabetes mellitus (NIDDM)) and, in some cases, even Type I diabetes. Treatment with GLP-I elicits activity, such as increased insulin secretion and biosynthesis, reduced glucagon secretion, delayed gastric emptying, only at elevated glucose levels, and thus provides a potentially much safer therapy than insulin or sulfonylureas. Post-prandial and glucose levels in patients can be moved toward normal levels with proper GLP-I therapy. There are also reports suggesting GLP-I -like molecules possess the ability to preserve and even restore pancreatic beta cell function in Type-II patients.

[00163] Any GLP-I sequence may be used to make Tf fusion proteins of the present invention, including GLP-l(7-35), GLP-l(7-36), and GLP-l(7-37). A perferred GLP-I sequence comprises GLP-I (7-36)(A8G;K34A) which comprises amino acid substitutions at A8 (Ala at position 2 in SEQ ID NO: 6) and K34 (Lys at position 31 in SEQ ID NO: 6). GLP-I also has powerful actions on the gastrointestinal tract. Infused in physiological amounts, GLP-I potently inhibits pentagastrin-induced as well as meal-induced gastric acid secretion (Schjoldager et al, Dig. Dis. Sci. 1989, 35:703-708; Wettergren et al, Dig Dis Sci 1993; 38:665-673). It also inhibits gastric emptying rate and pancreatic enzyme secretion (Wettergren et al, Dig Dis Sci 1993; 38:665-673). Similar inhibitory effects on gastric and pancreatic secretion and motility may be elicited in humans upon perfusion of the ileum with carbohydrate- or lipid-containing solutions (Layer et al, Dig Dis Sci 1995, 40: 1074-1082; Layer et al, Digestion 1993, 54: 385-38). Concomitantly, GLP-I secretion is greatly stimulated, and it has been speculated that GLP-I may be at least partly responsible for this

so-called "ileal-brake" effect (Layer et al, Digestion 1993; 54: 385-38). In fact, recent studies suggest that, physiologically, the ileal-brake effects of GLP-I may be more important than its effects on the pancreatic islets. Thus, in dose response studies GLP-I influences gastric emptying rate at infusion rates at least as low as those required to influence islet secretion (Nauck et al, Gut 1995; 37 (suppl. 2): Al 24).

[00164] GLP-I seems to have an effect on food intake. Intraventricular administration of GLP-I profoundly inhibits food intake in rats (Schick et al. in Ditschuneit et al. (eds.), Obesity in Europe, John Libbey & Company ltd, 1994; pp. 363-367; Turton et al, Nature 1996, 379: 69-72). This effect seems to be highly specific. Thus, N-terminally extended GLP-I(PG 72-107) amide is inactive and appropriate doses of the GLP-I antagonist, exendin 9-39, abolish the effects of GLP-I (Tang-Christensen et al, Am. J. Physiol., 1996, 271(4 Pt 2):R848-56). Acute, peripheral administration of GLP-I does not inhibit food intake acutely in rats (Tang-Christensen et al, Am. J. Physiol., 1996, 271(4 Pt 2):R848-56; Turton et al, Nature 1996, 379: 69-72). However, it remains possible that GLP-I secreted from the intestinal L-cells may also act as a satiety signal.

[00165] In diabetic and pre-diabetic patients, GLP's insulinotropic effects and the effects of GLP-I on the gastrointestinal tract are preserved (Willms et al, Diabetologia 1994; 37, suppl.1: Al 18), which may help curtail meal-induced glucose excursions, but, more importantly, may also influence food intake. Administered intravenously, continuously for one week, GLP-I at 4 ng/kg/min has been demonstrated to dramatically improve glycaemic control in NIDDM patients without significant side effects (Larsen et al, Diabetes 1996; 45, suppl. 2: 233A.).

[00166] GLP-I /transferrin fusion proteins comprising at least one analog of GLP-I and fragments thereof are useful in the treatment of Type 1 and Type 2 diabetes and obesity.

[00167] As used herein, the term "GLP-I molecule" means GLP-I, a GLP-I analog, or GLP-I derivative.

[00168] As used herein, the term "GLP-I analog" is defined as a molecule having one or more amino acid substitutions, deletions, inversions, or additions compared with GLP-I . Many GLP-I analogs are known in the art and include, for example, GLP-l(7-34), GLP-1(7- 35), GLP-l(7-36), VaI ⁸ -GLP-l(7-37), Gly ⁸ -GLP- 1(7-37), Ser ⁸ -GLP- 1(7-37), Gln ⁹ -GLP1(7- 37), D-Gln ⁹ -GLP-l(7-37), Thr ¹⁶ -Lys ¹⁸ -GLP-l(7-37), and Lys ¹⁸ -GLP-l(7-37). Other analogs

include dipeptidyl-peptidase resistant versions of GLP-I, wherein the N-terminal end of the peptide is protected. Such analogs include, but are not limited to GLP-I with additional amino acids, such as histidine residue added to the N-terminal end or substituted into the N- terminal amino acids (amino acid 7 or 8 in GLP-l(7-36) or GLP-l(7-37). In these analogs, the N-terminal end may comprise the residues His-His-Ala, Gly-His-Ala, His-Gly-Glu, His- Ser-Glu, His-Ala-Glu, His-Gly-Glu, His-Ser-Glu, His-His-Ala-Glu, His-His-Gly-Glu, His- His-Ser-Glu, Gly-His-Ala-Glu, Gly-His-Gly-Glu, Gly-His-Ser-Glu, His-X-Ala-Glu, His-X- Gly-Glu, His-X-Ser-Glu, wherein X is any amino acid. U.S. Patent 5,118,666 discloses examples of GLP-I analogs such as GLP-l(7-34) and GLP-l(7-35).

[00169] The term "GLP-I derivative" is defined as a molecule having the amino acid sequence of GLP-I or a GLP-I analog, but additionally having chemical modification of one or more of its amino acid side groups, α-carbon atoms, terminal amino group, or terminal carboxylic acid group. A chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties.

[00170] As used herein, the term "GLP-I related compound" refers to any compound falling within the GLP-I, GLP-I analog, or GLP-I derivative definition.

[00171] WO 91/11457 discloses analogs of the active GLP-I peptides 7-34, 7-35, 7-36, and 7-37 which can also be useful as GLP-I moieties.

[00172] EP 0708179-A2 (Eli Lilly & Co.) discloses GLP-I analogs and derivatives that include an N-terminal imidazole group and optionally an unbranched C ₆ -Ci _O acyl group in attached to the lysine residue in position 34.

[00173] EP 0699686-A2 (Eli Lilly & Co.) discloses certain N-terminal truncated fragments of GLP-I that are reported to be biologically active.

[00174] U.S. Patent 5,545,618 discloses GLP-I molecules consisting essentially of GLP- 1(7-34), GLPl(7-35), GLP-l(7-36), or GLP-l(7-37), or the amide forms thereof, and pharmaceutically-acceptable salts thereof, having at least one modification selected from the group consisting of: (a) substitution of glycine,serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, arginine, or D-lysine for lysine at position 26 and/or position 34; or substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, lysine, or a D-arginine for arginine at position 36; (b) substitution

of an oxidation-resistant amino acid for tryptophan at position 31 ; (c) substitution of at least one of: tyrosine for valine at position 16; lysine for serine at position 18; aspartic acid for glutamic acid at position 21; serine for glycine at position 22; arginine for glutamine at position 23; arginine for alanine at position 24; and glutamine for lysine at position 26; and (d) substitution of at least one of: glycine, serine, or cysteine for alanine at position 8; aspartic acid, glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine for glutamic acid at position 9; serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine for glycine at position 10; and glutamic acid for aspartic acid at position 15; and (e) substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine, or the D- or N- acylated or alkylated form of histidine for histidine at position 7; wherein, in the substitutions is (a), (b), (d), and (e), the substituted amino acids can optionally be in the D-form and the amino acids substituted at position 7 can optionally be in the N-acylated or N-alkylated form.

[00175] U.S. Pat. No. 5,118,666 discloses a GLP-I molecule having insulinotropic activity. Such molecule is selected from the group consisting of a peptide having the amino acid sequence His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Gly-Gln-Ala- Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys (SEQ ID NO: 7) or His-Ala-Glu-Gly-Thr-Phe- Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile-Ala-Trp-Leu-Val- Lys-Gly (SEQ ID NO: 8); and a derivative of said peptide and wherein said peptide is selected from the group consisting of: a pharmaceutically-acceptable acid addition salt of said peptide; a pharmaceutically-acceptable carboxylate salt of said peptide; a pharmaceutically- acceptable lower alkylester of said peptide; and a pharmaceutically-acceptable amide of said peptide selected from the group consisting of amide, lower alkyl amide, and lower dialkyl amide.

[00176] U.S. Patent 6,277,819 teaches a method of reducing mortality and morbidity after myocardial infarction comprising administering GLP-I, GLP-I analogs, and GLP-I derivatives to the patient. The GLP-I analog being represented by the following structural formula (SEQ ID NO: 9): R _r Xi-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-X ₂ - Gly-Gln-Ala-Ala-Lys- Xs-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R^ and pharmaceutically- acceptable salts thereof, wherein: Ri is selected from the group consisting of L-histidine, D- histidine, desamino-histidine, 2-amino-histidine, .beta.-hydroxy-histidine, homohistidine,

alpha-fluoromethyl-histidine, and alpha-methyl-histidine; Xi is selected from the group consisting of Ala, GIy, VaI, Thr, lie, and alpha-methyl-Ala; X ₂ is selected from the group consisting of GIu, Gin, Ala, Thr, Ser, and GIy; X ₃ is selected from the group consisting of GIu, Gin, Ala, Thr, Ser, and GIy; R ₂ is selected from the group consisting OfNH ₂ , and GIy-- OH; provided that the GLP-I analog has an isoelectric point in the range from about 6.0 to about 9.0 and further providing that when Ri is His, Xi is Ala, X ₂ is GIu, and X ₃ is GIu, R ₂ must be NH ₂ .

[00177] Ritzel et al. (Journal of Endocrinology, 1998, 159: 93-102) disclose a GLP-I analog, [Ser ⁸ ]GLP-l, in which the N-terminal second amino acid, alanine, is replaced with serine. The modification did not impair the insulinotropic action of the peptide but produced an analog with increased plasma stability as compared to GLP-I .

[00178] U.S. Patent 6,429,197 teaches that GLP-I treatment after acute stroke or hemorrhage, preferably intravenous administration, can be an ideal treatment because it provides a means for optimizing insulin secretion, increasing brain anabolism, enhancing insulin effectiveness by suppressing glucagon, and maintaining euglycemia or mild hypoglycemia with no risk of severe hypoglycemia or other adverse side effects. The present invention provides a method of treating the ischemic or reperfused brain with GLP-I or its biologically active analogues after acute stroke or hemorrhage to optimize insulin secretion, to enhance insulin effectiveness by suppressing glucagon antagonism, and to maintain euglycemia or mild hypoglycemia with no risk of severe hypoglycemia.

[00179] U.S. Patent 6,277,819 provides a method of reducing mortality and morbidity after myocardial infarction, comprising administering to a patient in need thereof, a compound selected from the group consisting of GLP-I, GLP-I analogs, GLP-I derivatives and pharmaceutically-acceptable salts thereof, at a dose effective to normalize blood glucose.

[00180] U.S. Patent 6,191,102 discloses a method of reducing body weight in a subject in need of body weight reduction by administering to the subject a composition comprising a glucagon- like peptide- 1 (GLP-I), a glucagon-like peptide analog (GLP-I analog), a glucagon-like peptide derivative (GLP-I derivative) or a pharmaceutically acceptable salt thereof in a dose sufficient to cause reduction in body weight for a period of time effective to produce weight loss, said time being at least 4 weeks.

[00181] GLP-I is fully active after subcutaneous administration (Ritzel et al, Diabetologia 1995; 38: 720-725), but is rapidly degraded mainly due to degradation by dipeptidyl peptidase IV-like enzymes (Deacon et al, J Clin Endocrinol Metab 1995, 80: 952-957; Deacon et α/.,1995, Diabetes 44: 1126-1131). Thus, unfortunately, GLP-I and many of its analogues have a short plasma half-life in humans (Orskov et al, Diabetes 1993; 42:658- 661). Accordingly, it is an objective of the present invention to provide transferrin fusion proteins comprising GLP-I or analogues thereof which have a protracted profile of action relative to GLP-l(7-37). It is a further object of the invention to provide derivatives of GLP- 1 and analogues thereof which have a lower clearance than GLP-l(7-37). Moreover, it is an object of the invention to provide pharmaceutical compositions comprising GLP-I /transferrin fusion proteins or GLP-I analog/transfeπϊn fusion proteins with improved stability. Additionally, the present invention includes the use of GLP-I /transferrin fusion proteins or GLP-I analog/transferrin fusion proteins to treat diseases associated with GLP-I such as but not limited to those described above. In particular, such fusion proteins may be used to treat diabetes or pre-diabetes in normal weight, over-weight or obese patients.

[00182] In one aspect of the present invention, the pharmaceutical compositions comprising the GLP-I peptide/transferrin fusion proteins and GLP-I analog/transferrin fusion proteins may be formulated by any of the established methods of formulating pharmaceutical compositions, e.g. as described in Remington's Pharmaceutical Sciences, 1985. The composition may be in a form suited for systemic injection or infusion and may, as such, be formulated with a suitable liquid vehicle such as sterile water or an isotonic saline or glucose solution. The compositions may be sterilized by conventional sterilization techniques which are well known in the art. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration. The composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

[00183] The GLP-I /transferrin fusion proteins and GLP-I analog/transferrin fusion proteins of the present invention may also be adapted for nasal, transdermal, pulmonal or rectal administration. The pharmaceutically acceptable carrier or diluent employed in the composition may be any conventional solid carrier. Examples of solid carriers are lactose,

terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

[00184] It may be of particular advantage to provide the composition of the invention in the form of a sustained release formulation. As such, the composition may be formulated as microcapsules or microparticles containing the GLP-I /transferrin fusion protein or GLP-I analog/transferrin fusion protein encapsulated by or dispersed in a suitable pharmaceutically acceptable biodegradable polymer such as polylactic acid, polyglycolic acid or a lactic acid/glycolic acid copolymer.

[00185] For nasal administration, the preparation may contain GLP-I /transferrin fusion proteins or GLP-I analog/transferrin fusion proteins dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application. The carrier may contain additives such as solubilizing agents, e.g. propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabenes.

[00186] Generally, the compounds of the present invention are dispensed in unit dosage form comprising 0.5-500 mg of the fusion protein together with a pharmaceutically acceptable carrier per unit dosage.

[00187] Moreover, the present invention contemplates the use of the GLP-I /transferrin and GLP-I analog/transferrin fusion proteins for the manufacture of a medicinal product which can be used in the treatment of diseases associated with elevated glucose level, such as but not to limited to those described above. Specifically, the present invention contemplates the use of GLP-I /transferrin fusion protein for the treatment of diabetes including type II diabetes, pre-diabetes, obesity, severe burns, and heart failure, including congestive heart failure and acute coronary syndrome.

[00188] The N-terminus of GLP-I is normally amidated. In yeast, amidation does not occur. In one aspect of the invention, in order to compensate for amidation on the N-terminus which does not occur in yeast, an extra amino acid is added on the N-terminus of GLP-I. The addition of an amino acid to the N-terminus of GLP-I may prevent dipeptidyl peptidase from cleaving at the second amino acid of GLP-I due to steric hindrance. Therefore, GLP-I will remain functionally active. Any one of the 20 amino acids may be added to the N-terminus of GLP-I. In some instances, an uncharged or positively charged amino acid may be used

and preferably, a smaller amino acid such as Glycine is added. The GLP-I with the extra amino acid is then fused to transferrin. Accordingly, the GLP-I with the added amino acid will be fused at the N-terminus of the GLP-I /transferrin fusion protein.

[00189] In one embodiment of making the GLP-l(7-36) or GLP-l(7-37) peptide more resistant to cleavage by dipeptidyl peptidase, a His residue is added at the N-terminus of GLP-I or is inserted after the His residue at the N-terminus of GLP-I, so that the N-terminus of GLP-I begins with His-His.

[00190] In another embodiment of the invention, the second residue from the N-terminus in the GLP-l(7-36) or GLP-l(7-37) peptide (SEQ ID NO: 6) is substituted with another amino acid. For example, the Ala residue at the second residue from the N-terminus in the GLP- 1(7-36) or GLP-l(7-37) peptide may be substituted with Ser, GIy, VaI, or another amino acid.

GLP-mTf Fusion Protein for Treating Type 2 Diabetes or Pre-Diabetes

[00191] As discussed above, GLP-I activates and regulates important endocrine hormone systems in the body and plays a critical management role in the metabolism of glucose. Unlike all other diabetic treatments on the market GLP-I has the potential to be restorative by acting as a growth factor for beta cells thus improving the ability of the pancreas to secrete insulin and also, to make the existing insulin levels act more efficiently by improving sensitivity and better stabilizing glucose levels. This reduces the burden on daily monitoring of glucose levels and potentially offers a delay in the serious long term side effects caused by fluctuations in blood glucose due to diabetes. Furthermore, GLP-I can reduce appetite and reduce weight. Obesity is an inherent consequence of poor control of glucose metabolism and this only serves to aggravate the diabetic condition.

[00192] Clinical application of natural GLP-I is limited because it is rapidly degraded in the circulation (half-life is several minutes). To maintain therapeutic levels in the circulation requires constant administration of high doses using pumps or patch devices which adds to the cost of treatment. This is inconvenient for long term chronic use especially in conjunction with all the other medications for treating diabetes and monitoring of glucose levels. The GLP-I fusion proteins retain the activity of GLP-I but have the long half-life (14-17 days), solubility, and biodistribution properties of transferrin (mTf). These properties

could provide for a low cost, small volume, monthly s.c. (subcutaneous) injection and this type of product is absolutely needed for long term chronic use.

GLP-I Fusion Proteins in Combination with Other Therapeutic Agents

[00193] In one aspect of the invention, a GLP-I fusion protein, for example, GLP/mTf fusion protein, of the present invention are used in combination with at least one second therapeutic molecule such as Glucophage® (metformin hydrochloride tablets) or Glucophage® XR (metformin hydrochloride extended-release tablets) to treat type II diabetes, obesity, and other diseases or conditions associated with abnormal glucose levels.

[00194] Glucophage® and Glucophage® XR are oral antihyperglycemic drugs for the mangagement of type II diabetes. Glucophage® XR is an extended release formulation of Glucophage. Accordingly, Glucophage® XR may be taken once daily because the drug is released slowly from the dosage form. Glucophage® helps the body produce less glucose from the liver. Accordingly, Glucophage® is effective in controlling blood sugar level in a patient. Glucophage® rarely causes low blood glucose (hypoglycemia) because it does not cause the body to make more insulin.

[00195] Glucophage® also helps lower the fatty blood components, triglycerides and cholesterol, that are often high in people with Type II diabetes. Metformin has been shown to decrease the appetite and help people lose a few pounds when they starting taking the medicine.

[00196] Metformin has been approved for treatment with sulfonylureas, or with insulin, or as monotherapy (by itself)- Metformin has been suggested for use in treating various cardiovascular diseases such as hypertension in insulin resistant patients (WO 9112003- Upjohn), for dissolving blood clots (in combination with a t-PA-derivative) (WO 9108763, WO 9108766, WO 9108767 and WO 9108765-Boehringer Mannheim), ischemia and tissue anoxia (EP 283369-Lipha), atherosclerosis (DE 1936274-Brunnengraber & Co., DE 2357875-Hurka, and U.S. Pat. No. 4,205,087-ICI). In addition, it has been suggested to use metformin in combination with prostaglandin-analogous cyclopentane derivatives as coronary dilators and for blood pressure lowering (U.S. Pat. No. 4,182,772-Hoechst). Metformin has also been suggested for use in cholesterol lowering when used in combination with 2-hydroxy-3,3,3-trifluoropropionic acid derivatives (U.S. Pat. No. 4,107,329-ICI), 1,2-

diarylethylene derivatives (U.S. Pat. No. 4,061,772-Hoechst), substituted aryloxy-3,3,3- trifluoro-2-propionic acids, esters and salts (U.S. Pat. No. 4,055,595-ICI), substituted hydroxyphenyl-piperidones (U.S. Pat. No. 4,024,267-Hoechst), and partially hydrogenated lH-indeno-[l,2B]-pyridine derivatives (U.S. Pat. No. 3,980,656-Hoechst).

[00197] Montanari et al. (Pharmacological Research, Vol. 25, No. 1, 1992) disclose that use of metformin in amounts of 500 mg twice a day (b.i.d.) increased post-ischemia blood flow in a manner similar to 850 mg metformin three times a day (t.i.d.). Sirtori et al. (J. Cardiovas. Pharm., 6:914-923, 1984), disclose that metformin in amounts of 850 mg three times a day (t.i.d) increased arterial flow in patients with peripheral vascular disease.

[00198] The present invention provides the treatment of various diseases comprising modified GLP-I of the present invention or its fusion protein in combination with one or more therapeutic agents such as metformin. In one embodiment, the modified GLP-I or its fusion protein in combination with metformin is used to treat diseases and conditions associated with abnormal blood glucose level, such as diabetes. Preferably, the GLP-1/mTf fusion protein in combination with metformin is used to treat type II diabetes or obesity.

[00199] Other therapeutic agents that may be used in combination with modified GLP-I of the present invention and its fusion proteins include but are not limited to PYY, mTF-PYY fusion proteins, sulfonylurea and sulfonylurea-like agents, thiazolidinediones, Peroxisome Proliferator- Activated Receptor (PPAR) gamma modulators, PPAR alpha modulators, Protein Tyrosine Phosphatase- IB inhibitors, Insulin Receptor Tyrosine Kinase activators, 1 lbeta- hydroxysteroid dehydrogenase inhibitors, glycogen phosphorylase inhibitors, glucokinase activators, beta-3 adrenergic agonists, and glucagon receptor agonists.

Peptide YY (PYY)

[00200] Peptide YY (PYY) (Tatemoto et al, PNAS 79:2514 (1982), see also. U.S. Patents 6,734,166; 6,558,708; 5,912,227; 5,574,010; and published applications WO03/026591, WO03/057235, and U.S. 2004/0157777, all of which are herein incorporated by reference in their entirety) may be used to produce modified transferrin fusion proteins of the invention that exhibit PYY or PYY-like activity.

[00201] PYY is produced by neuroendocrine cells of the intestine in response to ingestion of food. It exists in two forms, PYY( _I-36 ) and PYYo _-36) , the latter being generated by the action of DPP-IV. PYY _(3-36) is the dominant form in the circulation and is a neuropeptide Y (NPY) Y ₂ receptor agonist. Activation of Y ₂ receptors on NPY neurons reduces NPY release, thereby restraining food intake. Infusion of PYYo _-36) in humans results in reduced food intake (Batterham, RL et al. [2002] Nature, 418, 650-654), making it a potential weight-loss treatment. Various forms of PYY, including PYY( _I-36 ), PYY(3-36) and the analog [Pro ³⁴ ]PYY are also able to bind and activate the Yi and Y5 receptors.

[00202] Any PYY peptide may be used to make mTf fusion proteins, in particular, amino acids 1-36 or 3-36 of human PYY (1 -36^PIKPEAPGEDASPEELNRYYA- SLRHYLNLVTRQRY (SEQ ID NO: 4); 3-36: IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 5)).

[00203] mTf-PYY fusions of the invention may be used alone, or in combination with a second agent, such as a GLP-I -mTf fusion, to treat obesity, diabetes or pre-diabetes or may be used to regulate or control weight or blood glucose levels. mTF-PYY fusions may also be used to treat other disorders for which unfused PYY or PYY (NPY or Y2) receptor agonists may be used.

[00204] Particularly advantageous is a fusion protein in which GLP-I is fused to the N- terminus of mTf and PYY( _3-36 ) is fused to the C-terminus to produce a molecule able to activate both the GLP-I and Y ₂ receptors.

Nucleic Acids

[00205] The present invention also provides nucleic acid molecules encoding transferrin fusion proteins comprising a transferrin protein or a portion of a transferrin protein covalently linked or joined to a therapeutic protein, preferably a therapeutic protein. As discussed in more detail above, any therapeutic protein may be used. The fusion protein may further comprise a linker region, for instance a linker less than about 50, 40, 30, 20, or 10 amino acid residues. The linker can be covalently linked to and between the transferrin protein or portion thereof and the therapeutic protein, preferably the therapeutic protein. Nucleic acid molecules of the invention may be purified or not.

[00206] Host cells and vectors for replicating the nucleic acid molecules and for expressing the encoded fusion proteins are also provided. Any vectors or host cells may be used, whether prokaryotic or eukaryotic, but eukaryotic expression systems, in particular yeast expression systems, may be preferred. Many vectors and host cells are known in the art for such purposes. It is well within the skill of the art to select an appropriate set for the desired application.

[00207J DNA sequences encoding transferrin, portions of transferrin and therapeutic proteins of interest may be cloned from a variety of genomic or cDNA libraries known in the art. The techniques for isolating such DNA sequences using probe-based methods are conventional techniques and are well known to those skilled in the art. Probes for isolating such DNA sequences may be based on published DNA or protein sequences (see, for example, Baldwin, G.S. (1993) Comparison of Transferrin Sequences from Different Species. Comp. Biochem. Physiol. 106B/l :203-218 and all references cited therein, which are hereby incorporated by reference in their entirety). Alternatively, the polymerase chain reaction (PCR) method disclosed by Mullis et al. (U.S. Pat. No. 4,683,195) and Mullis (U.S. Pat. No. 4,683,202), incorporated herein by reference may be used. The choice of library and selection of probes for the isolation of such DNA sequences is within the level of ordinary skill in the art.

[00208] As known in the art "similarity" between two polynucleotides or polypeptides is determined by comparing the nucleotide or amino acid sequence and its conserved nucleotide or amino acid substitutes of one polynucleotide or polypeptide to the sequence of a second polynucleotide or polypeptide. Also known in the art is "identity" which means the degree of sequence relatedness between two polypeptide or two polynucleotide sequences as determined by the identity of the match between two strings of such sequences. Both identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).

[00209] While there exist a number of methods to measure identity and similarity between two polynucleotide or polypeptide sequences, the terms "identity" and "similarity" are well known to skilled artisans (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipman, D., SIAM J. Applied Math. 48: 1073 (1988).

[00210] Preferred methods to determine identity are designed to give the largest match between the two sequences tested. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, et al, Nucl. Acid Res. 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, et al, J. MoI. Biol. 215:403 (1990)). The degree of similarity or identity referred to above is determined as the degree of identity between the two sequences, often indicating a derivation of the first sequence from the second. The degree of identity between two nucleic acid sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch J. MoI. Biol. 48:443-453 (1970)). For purposes of determining the degree of identity between two nucleic acid sequences for the present invention, GAP is used with the following settings: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

Codon Optimization

[00211] The degeneracy of the genetic code permits variations of the nucleotide sequence of a transferrin protein and/or therapeutic protein of interest, while still producing a polypeptide having the identical amino acid sequence as the polypeptide encoded by the native DNA sequence. The procedure, known as "codon optimization" (described in U.S. Patent 5,547,871 which is incorporated herein by reference in its entirety) provides one with a means of designing such an altered DNA sequence. The design of codon optimized genes should take into account a variety of factors, including the frequency of codon usage in an organism, nearest neighbor frequencies, RNA stability, the potential for secondary structure

formation, the route of synthesis and the intended future DNA manipulations of that gene. In particular, available methods may be used to alter the codons encoding a given fusion protein with those most readily recognized by yeast when yeast expression systems are used.

[00212] The degeneracy of the genetic code permits the same amino acid sequence to be encoded and translated in many different ways. For example, leucine, serine and arginine are each encoded by six different codons, while valine, proline, threonine, alanine and glycine are each encoded by four different codons. However, the frequency of use of such synonymous codons varies from genome to genome among eukaryotes and prokaryotes. For example, synonymous codon-choice patterns among mammals are very similar, while evolutionarily distant organisms such as yeast (such as S. cerevisiae), bacteria (such as E. coli) and insects (such as D. melanogaster) reveal a clearly different pattern of genomic codon use frequencies (Grantham, R., et al, Nucl. Acid Res., 8, 49-62 (1980); Grantham, R., et al, Nucl. Acid Res., 9, 43-74 (1981); Maroyama, T., et al, Nucl. Acid Res., 14, 151-197 (1986); Aota, S., et al, Nucl. Acid Res., 16, 315-402 (1988); Wada, K., et al, Nucl. Acid Res., 19 Supp., 1981-1985 (1991); Kurland, C. G., FEBS Lett., 285, 165-169 (1991)). These differences in codon-choice patterns appear to contribute to the overall expression levels of individual genes by modulating peptide elongation rates. (Kurland, C. G., FEBS Lett., 285, 165-169 (1991); Pedersen, S., EMBO J., 3, 2895-2898 (1984); Sorensen, M. A., J. MoI. Biol., 207, 365-377 (1989); Randall, L. L., et al, Eur. J. Biochem., 107, 375-379 (1980); Curran, J. F., and Yarus, M., J. MoI. Biol., 209, 65-77 (1989); Varenne, S., et al, J. MoI. Biol., 180, 549-576 (1984), Varenne, S., et al, J. MoI, Biol., 180, 549-576 (1984); Garel, J.-P., J. Theor. Biol., 43, 21 1-225 (1974); Ikemura, T., J. MoI. Biol., 146, 1-21 (1981); Ikemura, T., J. MoI. Biol., 151, 389-409 (1981)).

[00213] The preferred codon usage frequencies for a synthetic gene should reflect the codon usages of nuclear genes derived from the exact (or as closely related as possible) genome of the cell/organism that is intended to be used for recombinant protein expression, particularly that of yeast species. As discussed above, in one preferred embodiment the human Tf sequence is codon optimized, before or after modification as herein described for yeast expression as may be the therapeutic protein nucleotide sequence(s).

Vectors

[00214] Expression units for use in the present invention will generally comprise the following elements, operably linked in a 5' to 3' orientation: a transcriptional promoter, a secretory signal sequence, a DNA sequence encoding a modified Tf fusion protein comprising transferrin protein or a portion of a transferrin protein joined to a DNA sequence encoding a therapeutic protein or peptide of interest and a transcriptional terminator. As discussed above, any arrangement of the therapeutic protein or peptide fused to or within the Tf portion may be used in the vectors of the invention. The selection of suitable promoters, signal sequences and terminators will be determined by the selected host cell and will be evident to one skilled in the art and are discussed more specifically below.

[00215] Suitable yeast vectors for use in the present invention are described in U.S. Patent 6,291,212 and include YRp7 (Struhl et al, Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978), YEpl3 (Broach et al, Gene 8: 121-133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978), pPPC0005, pSeCHSA, pScNHSA, ρC4, pSAC35 (Sleep et al, 1991, Bio/Technology 9,183-187 and EP 431 880 B) and derivatives thereof. Useful yeast plasmid vectors also include pRS403-406, pRS413-416 and the Pichia vectors 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, TRPl, LEU2 and URAS. PlasmidspRS413-41.6 are Yeast Centromere plasmids (YCps).

[00216] Such vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected. Preferred selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources, and include LEU2 (Broach et al. ibid.), URA3 (Botstein et al., Gene 8: 17, 1979), HIS3 (Struhl et al, ibid.) or POTl (Kawasaki and Bell, EP 171,142). Other suitable selectable markers include the CAT gene, which confers chloramphenicol resistance on yeast cells. Preferred promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al, J Biol. Chem. 225: 12073-12080, 1980; Alber and Kawasaki, J. MoI. Appl. Genet. 1: 419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or alcohol dehydrogenase genes (Young et al, in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al, (eds.), p. 355, Plenum, N.Y., 1982; Ammerer, Meth. Enzymol. 101: 192-201, 1983). In

this regard, particularly preferred promoters are the TPIl promoter (Kawasaki, U.S. Pat. No. 4,599,311) and the ADH2-4 ⁰ (see U.S. Patent 6,291,212 promoter (Russell et al, Nature 304: 652-654, 1983). The expression units may also include a transcriptional terminator. A preferred transcriptional terminator is the TPIl terminator (Alber and Kawasaki, ibid.). Other preferred vectors and preferred components such as promoters and terminators of a yeast expression system are disclosed in European Patents EP 0258067, EP 0286424, EP0317254, EP 0387319, EP 0386222, EP 0424117, EP 0431880, and EP 1002095; European Patent Publications EP 0828759, EP 0764209, EP 0749478, and EP 0889949; PCT Publication WO 00/44772 and WO 94/04687; and U.S. Patents 5,739,007; 5,637,504; 5,302,697; 5,260,202; 5,667,986; 5,728,553; 5,783,423; 5,965,386; 6150,133; 6,379,924; and 5,714,377; which are herein incorporated by reference in their entirety.

[00217] In addition to yeast, modified fusion proteins of the present invention can be expressed in filamentous fungi, for example, strains of the fungi Aspergillus. Examples of useful promoters include those derived from Aspergillus nidulans glycolytic genes, such as the adh3 promoter (McKnight et al, EMBO J. 4: 2093-2099, 1985) and the tpiA promoter. An example of a suitable terminator is the adh3 terminator (McKnight et al, ibid.). The expression units utilizing such components may be cloned into vectors that are capable of insertion into the chromosomal DNA of Aspergillus, for example.

[00218] Mammalian expression vectors for use in carrying out the present invention will include a promoter capable of directing the transcription of the modified Tf fusion protein. Preferred promoters include viral promoters and cellular promoters. Preferred viral promoters include the major late promoter from adenovirus 2 (Kaufman and Sharp, MoI. Cell. Biol. 2: 1304-13199, 1982) and the SV40 promoter (Subramani et al, MoI. Cell. Biol. 1 : 854-864, 1981). Preferred cellular promoters include the mouse metallothionein 1 promoter (Palmiter et al, Science 222: 809-814, 1983) and a mouse VK (see U.S. Patent 6,291,212) promoter (Grant et al, Nuc. Acids Res. 15: 5496, 1987). A particularly preferred promoter is a mouse V _H (see U.S. Patent 6,291,212) promoter (Loh et al, ibid.). Such expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the DNA sequence encoding the transferrin fusion protein. Preferred RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes.

[00219] Also contained in the expression vectors is a polyadenylation signal located downstream of the coding sequence of interest. Polyadenylation signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the adenovirus 5 ElB region and the human growth hormone gene terminator (DeNoto et al., Nucl. Acid Res. 9: 3719-3730, 1981). A particularly preferred polyadenylation signal is the V _H (see U.S. Patent 6,291,212) gene terminator (Loh et al, ibid.)- The expression vectors may include a noncoding viral leader sequence, such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites. Preferred vectors may also include enhancer sequences, such as the SV40 enhancer and the mouse μ (see U.S. Patent 6,291,212) enhancer (Gillies, Cell 33: 717-728, 1983). Expression vectors may also include sequences encoding the adenovirus VA RNAs.

Transformation

[00220] Techniques for transforming fungi are well known in the literature, and have been described, for instance, by Beggs (ibid.), Hinnen et al. (Proc. Natl. Acad. Sci. USA 75: 1929- 1933, 1978), Yelton et al., (Proc. Natl. Acad. Sci. USA 81 : 1740-1747, 1984), and Russell (Nature 301 : 167-169, 1983). Other techniques for introducing cloned DNA sequences into fungal cells, such as electroporation (Becker and Guarente, Methods in Enzymol. 194: 182- 187, 1991) may be used. The genotype of the host cell will generally contain a genetic defect that is complemented by the selectable marker present on the expression vector. Choice of a particular host and selectable marker is well within the level of ordinary skill in the art.

[00221] Cloned DNA sequences comprising modified Tf fusion proteins of the invention may be introduced into cultured mammalian cells by, for example, calcium phosphate- mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973.) Other techniques for introducing cloned DNA sequences into mammalian cells, such as electroporation (Neumann et al, EMBO J. 1: 841-845, 1982), or lipofection may also be used. In order to identify cells that have integrated the cloned DNA, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. A preferred amplifiable selectable marker is the DHFR gene.

A particularly preferred amplifiable marker is the DHFR ^r (see U.S. Patent 6,291,212) cDNA (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80: 2495-2499, 1983). Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass.) and the choice of selectable markers is well within the level of ordinary skill in the art.

Host Cells

[00222] The present invention also includes a cell, preferably a yeast cell transformed to express a modified transferrin fusion protein of the invention. In addition to the transformed host cells themselves, the present invention also includes 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.

[00223] Host cells for use in practicing the present invention include eukaryotic cells, and in some cases prokaryotic cells, capable of being transformed or transfected with exogenous DNA and grown in culture, such as cultured mammalian, insect, fungal, plant and bacterial cells.

[00224] Fungal cells, including species of yeast (e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp.) may be used as host cells within the present invention. Examples of fungi including yeasts contemplated to be useful in the practice, of the present invention as hosts for expressing the, transferrin fusion protein of the inventions are Pichia (some species of which were formerly classified as Hansenulά), Saccharomyces, Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Zygosaccharomyces, Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, and the like. Examples of Saccharomyces spp. are S. cerevisiae, S. italicus and S. rouxii. Examples of Kluyveromyces spp. are K. fragilis, K. lactis and K. marxianus. A suitable Torulaspora species is T. delbrueckii. Examples of Pichia spp. are P. angusta (formerly H. polymorpha), P. anomala (formerly H. anomala) and P. pastoris.

[00225] Particularly useful host cells to produce the Tf fusion proteins of the invention are the methylotrophic Pichia pastoris (Steinlein et al. (1995) Protein Express. Purif. 6:619- 624). Pichia pastoris has been developed to be an outstanding host for the production of foreign proteins since its alcohol oxidase promoter was isolated and cloned; its transformation was first reported in 1985. P. pastoris can utilize methanol as a carbon source in the absence of glucose. The P. pastoris expression system can use the methanol-induced alcohol oxidase (AOXl) promoter, which controls the gene that codes for the expression of alcohol oxidase, the enzyme which catalyzes the first step in the metabolism of methanol. This promoter has been characterized and incorporated into a series of P. pastoris expression vectors. Since the proteins produced in P. pastoris are typically folded correctly and secreted into the medium, the fermentation of genetically engineered P. pastoris provides an excellent alternative to E. coli expression systems. A number of proteins have been produced using this system, including tetanus toxin fragment, Bordatella pertussis pertactin, human serum albumin and lysozyme.

[00226] Strains of the yeast Saccharomyces cerevisiae are another preferred host. In a preferred embodiment, a yeast cell, or more specifically, a Saccharomyces cerevisiae host cell that contains a genetic deficiency in a gene required for asparagine-linked glycosylation of glycoproteins is used. S. cerevisiae host cells having such defects may be prepared using standard techniques of mutation and selection, although many available yeast strains have been modified to prevent or reduce glycosylation or hypermannosylation. Ballou et al. (J. Biol. Chem. 255: 5986-5991, 1980) have described the isolation of mannoprotein biosynthesis mutants that are defective in genes which affect asparagine-linked glycosylation. Gentzsch and Tanner (Glycobiology 7:481-486, 1997) have described a family of at least six genes (PMTl -6) encoding enzymes responsible for the first step in O-glycosylation of proteins in yeast. Mutants defective in one or more of these genes show reduced O-linked glycosylation and/or altered specificity of O-glycosylation.

[00227] To optimize production of the heterologous proteins, it is also preferred that the host strain carries a mutation, such as the S. cerevisiae pep4 mutation (Jones, Genetics 85: 23-33, 1977), which results in reduced proteolytic activity. Host strains containing mutations in other protease encoding regions are particularly useful to produce large quantities of the Tf fusion proteins of the invention.

[00228] Host cells containing DNA constructs of the present invention are grown in an appropriate growth medium. As used herein, the term "appropriate growth medium" means a medium containing nutrients required for the growth of cells. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors. The growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct. Yeast cells, for example, are preferably grown in a chemically defined medium, comprising a carbon source, e.g. sucrose, a non-amino acid nitrogen source, inorganic salts, vitamins and essential amino acid supplements. The pH of the medium is preferably maintained at a pH greater than 2 and less than 8, preferably at pH 5.5-6.5. Methods for maintaining a stable pH include buffering and constant pH control. Preferred buffering agents include succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, Mo.). Yeast cells having a defect in a gene required for asparagine-linked glycosylation are preferably grown in a medium containing an osmotic stabilizer. A preferred osmotic stabilizer is sorbitol supplemented into the medium at a concentration between 0.1 M and 1.5 M., preferably at 0.5 M or 1.0 M.

[00229] Cultured mammalian cells are generally grown in commercially available serum- containing or serum-free media. Selection of a medium appropriate for the particular cell line used is within the level of ordinary skill in the art. Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, to begin expressing the DNA sequence(s) of interest. Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drug concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels.

[00230] Baculovirus/insect cell expression systems may also be used to produce the modified Tf fusion proteins of the invention. The BacPAK™ Baculovirus Expression System (BD Biosciences (Clontech)) expresses recombinant proteins at high levels in insect host cells. The target gene is inserted into a transfer vector, which is cotransfected into insect host cells with the linearized BacPAKό viral DNA. The BacPAKό DNA is missing an essential portion of the baculovirus genome. When the DNA recombines with the vector, the essential element is restored and the target gene is transferred to the baculovirus genome. Following

recombination, a few viral plaques are picked and purified, and the recombinant phenotype is verified. The newly isolated recombinant virus can then be amplified and used to infect insect cell cultures to produce large amounts of the desired protein.

[00231] Tf fusion proteins of the present invention may also be produced using transgenic plants and animals. For example, sheep and goats can make the therapeutic protein in their milk. Tobacco plants can include the protein in their leaves. Both transgenic plant and animal production of proteins comprises adding a new gene coding the fusion protein into the genome of the organism. Not only can the transgenic organism produce a new protein, but it can also pass this ability onto its offspring.

Secretory Signal Sequences

[00232] The terms "secretory signal sequence" or "signal sequence" or "secretion leader sequence" are used interchangeably and are described, for example in U.S. Pat. 6,291,212 and U.S. Pat 5,547,871, both of which are herein incorporated by reference in their entirety. Secretory signal sequences or signal sequences or secretion leader sequences encode secretory peptides. A secretory peptide is an amino acid sequence that acts to direct the secretion of a mature polypeptide or protein from a cell. Secretory peptides are generally characterized by a core of hydrophobic amino acids and are typically (but not exclusively) found at the amino termini of newly synthesized proteins. Very often the secretory peptide is cleaved from the mature protein during secretion. Secretory peptides may contain processing sites that allow cleavage of the signal peptide from the mature protein as it passes through the secretory pathway. Processing sites may be encoded within the signal peptide or may be added to the signal peptide by, for example, in vitro mutagenesis.

[00233] Secretory peptides may be used to direct the secretion of modified Tf fusion proteins of the invention. One such secretory peptide that may be used in combination with other secretory peptides is the alpha mating factor leader sequence. Secretory signal sequences or signal sequences or secretion leader sequences are required for a complex series of post-translational processing steps which result in secretion of a protein. If an intact signal sequence is present, the protein being expressed enters the lumen of the rough endoplasmic reticulum and is then transported through the Golgi apparatus to secretory vesicles and is finally transported out of the cell. Generally, the signal sequence immediately follows the initiation codon and encodes a signal peptide at the amino-terminal end of the protein to be

secreted. In most cases, the signal sequence is cleaved off by a specific protease, called a signal peptidase. Preferred signal sequences improve the processing and export efficiency of recombinant protein expression using viral, mammalian or yeast expression vectors. In some cases, the native Tf signal sequence may be used to express and secrete fusion proteins of the invention.

Linkers

[00234] The Tf moiety and the therapeutic protein of the modified transferrin fusion proteins of the invention can be fused directly or using a linker peptide of various lengths to provide greater physical separation and allow more spatial mobility between the fused proteins and thus maximize the accessibility of the therapeutic protein, for instance, for binding to its cognate receptor. The linker peptide may consist of amino acids that are flexible or more rigid. For example, a linker such as but not limited to a poly-glycine stretch. The linker can be less than about 50, 40, 30, 20, or 10 amino acid residues. The linker can be covalently linked to and between the transferrin protein or portion thereof and the therapeutic protein.

Detection of Tf Fusion Proteins

[00235] Assays for detection of biologically active modified transferrin-fusion protein may include Western transfer, protein blot or colony filter as well as activity based assays that detect the fusion protein comprising transferrin and therapeutic protein. A Western transfer filter may be prepared using the method described by Towbin et al. (Proc. Natl. Acad. ScL USA 16: 4350-4354, 1979). Briefly, samples are electrophoresed in a sodium dodecylsulfate polyacrylamide gel. The proteins in the gel are electrophoretically transferred to nitrocellulose paper. Protein blot filters may be prepared by filtering supernatant samples or concentrates through nitrocellulose filters using, for example, a Minifold (Schleicher & Schuell, Keene, N.H.). Colony filters may be prepared by growing colonies on a nitrocellulose filter that has been laid across an appropriate growth medium. In this method, a solid medium is preferred. The cells are allowed to grow on the filters for at least 12 hours. The cells are removed from the filters by washing with an appropriate buffer that does not remove the proteins bound to the filters. A preferred buffer comprises 25 mM Tris-base, 19 mM glycine, pH 8.3, 20% methanol.

[00236] Transferrin fusion proteins of the present invention may be labeled with a radioisotope or other imaging agent and used for in vivo diagnostic purposes. Preferred radioisotope imaging agents include iodine- 125 and technetium-99, with technetium-99 being particularly preferred. Methods for producing protein-isotope conjugates are well known in the art, and are described by, for example, Eckelman et al. (U.S. Pat. No. 4,652,440), Parker et al. (WO 87/05030) and Wilber et al. (EP 203,764). Alternatively, the transferrin fusion proteins may be bound to spin label enhancers and used for magnetic resonance (MR) imaging. Suitable spin label enhancers include stable, sterically hindered, free radical compounds such as nitroxides. Methods for labeling ligands for MR imaging are disclosed by, for example, Coffman et al. (U.S. Pat. No. 4,656,026).

[00237] Detection of a transferrin fusion protein of the present invention can be facilitated by coupling {i.e., physically linking) the therapeutic protein to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β- galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵ I, ¹³¹ I ₅ ³⁵ S Or ³ H.

[00238] In one embodiment where one is assaying for the ability of a transferrin fusion protein of the invention to bind or compete with an antigen for binding to an 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, en∑yme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, the binding of the transferrin fusion protein is detected by detecting a label

on the transferrin fusion protein. In another embodiment, the transferrin fusion protein is detected by detecting binding of a secondary antibody or reagent that interacts with the transferrin fusion protein. In a further embodiment, the secondary antibody or reagent is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

[00239] Fusion proteins of the invention may also be detected by assaying for the activity of the therapeutic protein moiety. Specifically, transferrin fusion proteins of the invention may be assayed for functional activity (e.g., biological activity or therapeutic activity) using assays known to one of ordinary skill in the art. Additionally, one of skill in the art may routinely assay fragments of a therapeutic protein corresponding to a therapeutic protein portion of a fusion protein of the invention, for activity using well-known assays. Further, one of skill in the art may routinely assay fragments of a modified transferrin protein for activity using assays known in the art.

[00240] For example, in one embodiment where one is assaying for the ability of a transferrin fusion protein of the invention to bind or compete with a therapeutic protein for binding to an anti-therapeutic polypeptide antibody and/or anti-transferrin 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), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

[00241] In a further embodiment, where a binding partner (e.g., a receptor or a ligand) of a therapeutic protein is identified, binding to that binding partner by a transferrin fusion protein containing that therapeutic protein as the therapeutic protein portion of the fusion can be

assayed, e.g., by means well-known in the art, such as, for example, reducing and non- reducing gel chromatography, protein affinity chromatography, and affinity blotting. Other methods will be known to the skilled artisan and are within the scope of the invention.

Production of Fusion Proteins

[00242] The present invention further provides methods for producing a modified fusion protein of the invention using nucleic acid molecules herein described. In general terms, the production of a recombinant form of a protein typically involves the following steps.

[00243] A nucleic acid molecule is first obtained that encodes a transferrin fusion protein of the invention. The nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame. The expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein. Optionally the recombinant protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.

[00244] Each of the foregoing steps can be accomplished in a variety of ways. For example, the construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above. The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier and are otherwise known to persons skilled in the art. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce a desired recombinant protein.

[00245] As discussed above, any expression system may be used, including yeast, bacterial, animal, plant, eukaryotic and prokaryotic systems. In some embodiments, yeast, mammalian cell culture and transgenic animal or plant production systems are preferred. In other embodiments, yeast systems that have been modified to reduce native yeast glycosylation, hyper-glycosylation or proteolytic activity may be used.

Isolation/Purification of Modified Transferrin Fusion Proteins

[00246] Secreted, biologically active, modified transferrin fusion proteins may be isolated from the medium of host cells grown under conditions that allow the secretion of the biologically active fusion proteins. The cell material is removed from the culture medium, and the biologically active fusion proteins are isolated using isolation techniques known in the art. Suitable isolation techniques include precipitation and fractionation by a variety of chromatographic methods, including gel filtration, ion exchange chromatography and affinity chromatography.

[00247] A particularly preferred purification method is affinity chromatography on an iron binding or metal chelating column or an immunoaffinity chromatography using an antibody directed against the transferrin or therapeutic protein of the polypeptide fusion. The antibody is preferably immobilized or attached to a solid support or substrate. A particularly preferred substrate is CNBr-activated Sepharose (Pharmacia LKB Technologies, Inc., Piscataway, N.J.). By this method, the medium is combined with the antibody/substrate under conditions that will allow binding to occur. The complex may be washed to remove unbound material, and the transferrin fusion protein is released or eluted through the use of conditions unfavorable to complex formation. Particularly useful methods of elution include changes in pH, wherein the immobilized antibody has a high affinity for the transferrin fusion protein at a first pH and a reduced affinity at a second (higher or lower) pH; changes in concentration of certain chaotropic agents; or through the use of detergents; or through the use of imidazole.

Delivery of a Drug or Therapeutic Protein to the inside of a Cell and/or across the Blood Brain Barrier (BBB)

[00248] Within the scope of the invention, the modified transferrin fusion proteins may be used as a carrier to deliver a molecule or small molecule therapeutic complexed to the ferric ion of transferrin to the inside of a cell or across the blood brain barrier or other barriers including across the cell membrane of any cell type that naturally or engineered to express a Tf receptor. In these embodiments, the Tf fusion protein will typically be engineered or modified to inhibit, prevent or remove glycosylation to extend the serum half-life of the fusion protein and/or therapeutic protein portion. The addition of a targeting peptide is

specifically contemplated to further target the Tf fusion protein to a particular cell type, e.g., a cancer cell.

[00249] In one embodiment, the iron-containing, anti-anemic drug, ferric-sorbitol-citrate complex is loaded onto a modified Tf fusion protein of the invention. Ferric-sorbitol-citrate (FSC) has been shown to inhibit proliferation of various murine cancer cells in vitro and cause tumor regression in vivo, while not having ^" any effect on proliferation of non-malignant cells (Poljak-Blazi et al. (June 2000) Cancer Biotherapy and Radiopharmaceuticals (United States), 15/3:285-293).

[00250] In another embodiment, the antineoplastic drug Adriamycin® (doxorubicin) and/or the chemotherapeutic drug bleomycin, both of which are known to form complexes with ferric ion, is loaded onto a Tf fusion protein of the invention. In other embodiments, a salt of a drug, for instance, a citrate or carbonate salt, may be prepared and complexed with the ferric iron that is then bound to Tf. As tumor cells often display a higher turnover rate for iron; transferrin modified to carry at least one anti-tumor agent, may provide a means of increasing agent exposure or load to the tumor cells. (Demant, E.J., (1983) Eur. J. Biochem. 137/(1-2): 113-118; Padbury et al. (1985) J. Biol. Chem. 260/13:7820-7823).

Pharmaceutical Formulations and Treatment Methods

[00251] The modified fusion proteins comprising a modified transferrin of the invention may be administered to a patient in need thereof using standard administration protocols. For instance, the modified Tf fusion proteins of the present invention can be provided alone, or in combination, or in sequential combination with other agents that modulate a particular pathological process. As used herein, two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same or near the same time.

[00252] The fusion proteins of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebrovascular, transdermal and buccal routes. For example, an agent may be administered locally to a site of injury via microinfusion. Alternatively, or concurrently, administration may be noninvasive by either the oral, inhalation, nasal, or pulmonary route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[00253] While any method of administration may be used to deliver the mTF fusion proteins of the invention, administration or delivery orally may be a preferred embodiment for certain classes of fusion proteins or to treat certain conditions.

[00254] The present invention further provides compositions containing one or more fusion proteins of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise about 1 pg/kg to about 100 mg/kg body weight. The preferred dosages for systemic administration comprise about 100 ng/kg to about 100 mg/kg body weight. The preferred dosages for direct administration to a site via microinfusion comprise about 1 ng/kg to about 1 mg/kg body weight. When administered via direct injection or microinfusion, modified fusion proteins of the invention may be engineered to exhibit reduced or no binding of iron to prevent, in part, localized iron toxicity.

[00255] In addition to the pharmacologically active fusion protein, the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water- soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol and dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.

[00256] The pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient. Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

[00257] The pharmaceutical composition of the present invention can be in unit dosage form, e.g. as tablets or capsules. In such form, the composition is sub-divided in unit dose containing appropriate quantities of the active ingredient; the unit dosage forms can be packaged compositions, for example, packeted powders, vials, ampoules, prefϊlled syringes or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form. The dosage to be used in the treatment must be subjectively determined by the physician.

[00258] In practicing the methods of this invention, the fusion proteins of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In certain preferred embodiments, the compounds of this invention may be co¬ administered along with other compounds typically prescribed for these conditions according to generally accepted medical practice. The compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.

Oral Pharmaceutical Compositions and Delivery Methods

[00259] In the present invention, Tf fusion proteins, including but not limited to modified Tf fusion proteins, may be formulated for oral delivery. In particular, certain fusion proteins of the invention that are used to treat certain classes of diseases or medical conditions may be particularly amenable for oral formulation and delivery. Such classes of diseases or conditions include, but are not limited to, acute, chronic and recurrent diseases. Chronic or recurrent diseases include, but are not limited to, viral disease or infections, cancer, a metabolic diseases, obesity, autoimmune diseases, inflammatory diseases, allergy, graft-vs.- host disease, systemic microbial infection, anemia, cardiovascular disease, psychosis, genetic diseases, neurodegenerative diseases, disorders of hematopoietic cells, diseases of the endocrine system or reproductive systems, gastrointestinal diseases. Examples of these classes of disease include diabetes, multiple sclerosis, asthma, HCV or HIV infections, hypertension, hypercholesterolemia, arterial scherosis, arthritis, and Alzheimer's disease. In many chronic diseases, oral formulations of Tf fusion proteins of the invention and methods of administration are particularly useful because they allow long-term patient care and therapy via home oral administration without reliance on injectable treatment or drug protocols.

[00260] Oral formulations and delivery methods comprising Tf fusion proteins of the invention take advantage of, in part, transferrin receptor mediated transcytosis across the gastrointestinal (GI) epithelium. The Tf receptor is found at a very high density in the human GI epithelium, transferrin is highly resistant to tryptic and chymotryptic digestion and Tf chemical conjugates have been used to successfully deliver proteins and peptides across the GI epithelium (Xia et al, (2000) J. Pharmacol. Experiment. Therap., 295:594-600; Xia et al. (2001) Pharmaceutical Res., 18(2): 191-195; and Shah et al. (1996) J. Pharmaceutical Sci., 85(12): 1306-1311, all of which are herein incorporated by reference in their entirety). Once transported across the GI epithelium, Tf fusion proteins of the invention exhibit extended half-life in serum, that is, the therapeutic protein or peptide(s) attached or inserted into Tf exhibit an extended serum half-life compared to the protein or peptide in its non-fused state.

[00261] Oral formulations of Tf fusion proteins of the invention may be prepared so that they are suitable for transport to the GI epithelium and protection of the Tf fusion protein component and other active components in the stomach. Such formulations may include carrier and dispersant components and may be in any suitable form, including aerosols (for oral or pulmonary delivery), syrups, elixirs, tablets, including chewable tablets, hard or soft capsules, troches, lozenges, aqueous or oily suspensions, emulsions, cachets or pellets granulates, and dispersible powders. Preferably, Tf fusion protein formulations are employed in solid dosage forms suitable for simple, and preferably oral, administration of precise dosages. Solid dosage forms for oral administration are preferably tablets, capsules, or the like.

[00262] For oral administration in the form of a tablet or capsule, care should be taken to ensure that the composition enables sufficient active ingredient to be absorbed by the host to produce an effective response. Thus, for example, the amount of Tf fusion protein may be increased over that theoretically required or other known measures such as coating or encapsulation may be taken to protect the polypeptides from enaymatic action in the stomach.

[00263] Traditionally, peptide and protein drugs have been administered by injection because of the poor bioavailability when administered non-parenterally, and in particular orally. These drugs are prone to chemical and conformational instability and are often degraded by the acidic conditions in the stomach, as well as by enzymes in the stomach and gastrointestinal tract. In response to these delivery problems, certain technologies for oral

delivery have been developed, such as encapsulation in nanoparticles composed of polymers with a hydrophobic backbone and hydrophilic branches as drug carriers, encapsulation in microparticles, insertion into liposomes in emulsions, and conjugation to other molecules. All of which may be used with the Tf fusion molecules of the present invention.

[00264] Examples of nanoparticles include mucoadhesive nanoparticles coated with chitosan and Carbopol (Takeuchi et al, Adv. Drug Deliv. Rev. 47(l):39-54, 2001) and nanoparticles containing charged combination polyesters, poly(2-sulfobutyl-vinyl alcohol) and poly(D,L- lactic-co-glycolic acid) (Jung et al, Eur. J. Pharm. Biopharm. 50(1): 147-160, 2000). Nanoparticles containing surface polymers with poly-N-isopropylacrylamide regions and cationic poly-vinylamine groups showed improved absorption of salmon calcitonin when administered orally to rats.

[00265] Drug delivery particles composed of alginate and pectin, strengthened with polylysine, are relatively acid and base resistant and can be used as a carrier for drugs. These particles combine the advantages of bioadhesion, enhanced absorption and sustained release (Liu et al, J. Pharm. Pharmacol. 51(2): 141 -149, 1999).

[00266] Additionally, lipoamino acid groups and liposaccharide groups conjugated to the N- and C-termini of peptides such as synthetic somatostatin, creating an amphipathic surfactant, were shown to produce a composition that retained biological activity (Toth et al, J. Med. Chem. 42(19):4010-4013, 1999).

[00267] Examples of other peptide delivery technologies include carbopol-coated mucoadhesive emulsions containing the peptide of interest and either nitroso-N-acetyl-D,L- penicillamine and carbolpol or taurocholate and carbopol. These were shown to be effective when orally administered to rats to reduce serum calcium concentrations (Ogiso et al, Biol. Pharm. Bull. 24(6):656-661, 2001). Phosphatidylethanol, derived from phosphatidylcholine, was used to prepare liposomes containing phosphatidylethanol as a carrier of insulin. These liposomes, when administered orally to rats, were shown to be active (Kisel et al, Int. J. Pharm. 216(1-2): 105-114, 2001).

[00268] Insulin has also been formulated in poly( vinyl alcohol)-gel spheres containing insulin and a protease inhibitor, such as aprotinin or bacitracin. The glucose-lowering properties of these gel spheres have been demonstrated in rats, where insulin is released largely in the lower intestine (Kimura et al, Biol. Pharm. Bull. 19(6): 897-900, 1996.

[00269] Oral delivery of insulin has also been studied using nanoparticles made of poly(alkyl cyanoacrylate) that were dispersed with a surfactant in an oily phase (Damge et al., J. Pharm. Sci. 86(12):1403-1409, 1997) and using calcium alginate beads coated with chitosan (Onal et al, Artif. Cells Blood Substit. Immobil. Biotechnol. 30(3):229-237, 2002).

[00270] In other methods, the N- and C-termini of a peptide are linked to polyethylene glycol and then to allyl chains to form conjugates with improved resistance to enzymatic degradation and improved diffusion through the GI wall (www.nobexcorp.com).

[00271] BioPORTER® is a cationic lipid mixture, which interacts non-covalently with peptides to create a protective coating or layer. The peptide-lipid complex can fuse to the plasma membrane of cells, and the peptides are internalized into the cells (www.genetherapysystems.com).

[00272] In a process using liposomes as a starting material, cochleate-shaped particles have been developed as a pharmaceutical vehicle. A peptide is added to a suspension of liposomes containing mainly negatively charged lipids. The addition of calcium causes the collapse and fusion of the liposomes into large sheets composed of lipid bilayers, which then spontaneously roll up or stack into cochleates (U.S. Patent 5,840,707; http ://www.biodeliverysciences .com) .

[00273] Compositions comprising Tf fusion protein intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents in order to provide a pharmaceutically elegant and palatable preparation. For example, to prepare orally deliverable tablets, Tf fusion protein is mixed with at least one pharmaceutical excipient, and the solid formulation is compressed to form a tablet according to known methods, for delivery to the gastrointestinal tract. The tablet composition is typically formulated with additives, e.g. a saccharide or cellulose carrier, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, or other additives typically usually used in the manufacture of medical preparations. To prepare orally deliverable capsules, DHEA is mixed with at least one pharmaceutical excipient, and the solid formulation is placed in a capsular container suitable for delivery to the gastrointestinal tract. Compositions comprising Tf fusion protein may be prepared as described generally in

Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which is herein incorporated by reference.

[00274] As described above, many of the oral formulations of the invention may contain inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine. Such formulations, or enteric coatings, are well known in the art. For example, tablets containing Tf fusion protein in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for manufacture of tablets may be used. These excipients may be inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid, or talc.

[00275] The tablets may be uncoated or they may be coated with known techniques to delay disintegration and absorption in the gastrointestinal track and thereby provide a sustained action over a longer period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

[00276] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin or as soft gelatin capsules wherein the active ingredient is mixed with an aqueous or an oil medium, for example, arachis oil, peanut oil, liquid paraffin or olive oil.

[00277] Aqueous suspensions may contain Tf fusion protein in the admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecylethyloxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan

monooleate. The aqueous suspensions may also contain one or more preservatives for example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose or saccharin.

[00278] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oil suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[00279] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient and admixture with dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavoring and coloring agents, may also be present.

[00280] The pharmaceutical compositions containing Tf fusion protein may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil for example, gum acacia or gum tragacanth, naturally- occurring phosphotides, for example soybean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, and condensation products of the same partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

[00281] Syrups and elixirs containing Tf fusion protein may be formulated with sweetening agents, for example, glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparations may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvate, for example as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that

may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this period any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[00282] Pharmaceutical compositions may also be formulated for oral delivery using polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid-based systems (see, for example, DiBase and Morrel, Oral Delivery of Microencapsulated Proteins, in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)).

[00283] The proportion of pharmaceutically active Tf fusion protein to carrier and/or other substances may vary from about 0.5 to about 100 wt. % (weight percent). For oral use, the pharmaceutical formulation will generally contain from about 5 to about 100% by weight of the active material. For other uses, the formulation will generally have from about 0.5 to about 50 wt. % of the active material.

[00284] Tf fusion protein formulations employed in the invention provide an effective amount of Tf fusion protein upon administration to an individual. As used in this context, an "effective amount" of Tf fusion is an amount that is effective to ameliorate a symptom of a disease.

[00285] The Tf fusion protein composition of the present invention may be, though not necessarily, administered daily, in an effective amount to ameliorate a symptom. Generally, the total daily dosage will be at least about 50 mg, preferably at least about 100 mg, and more preferably at least about 200 mg, and preferably not more than 500 mg per day, administered orally, e.g., in 4 capsules or tablets, each containing 50 mg Tf fusion protein. Capsules or tablets for oral delivery can conveniently contain up to a full daily oral dose, e.g., 200 mg or more.

[00286] In a particularly preferred embodiment, oral pharmaceutical compositions comprising Tf fusion protein are formulated in buffered liquid form which is then encapsulated into soft or hard-coated gelatin capsules which are then coated with an appropriate enteric coating. For the oral pharmaceutical compositions of the invention, the

location of release may be anywhere in the GI system, including the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine.

[00287] In other embodiments, oral compositions of the invention are formulated to slowly release the active ingredients, including the Tf fusion proteins of the invention, in the GI system using known delayed release formulations.

[00288] Tf fusion proteins of the invention for oral delivery are capable of binding the Tf receptor found in the GI epithelium. To facilitate this binding and receptor mediated transport, Tf fusion proteins of the invention are typically produced with iron and in some instances carbonate, bound to the Tf moiety. Processes and methods to load the Tf moiety of the fusion protein compositions of the invention with iron and carbonate are known in the art

[00289] In some pharmaceutical formulations of the invention, the Tf moiety of the Tf fusion protein may be modified to increase the affinity or avidity of the Tf moiety to iron. Such methods are known in the art. For instance, mutagenesis can be used to produce mutant transferrin moieties that bind iron more avidly than natural transferrin. In human serum transferrin, the amino acids which are ligands for metal ion chelation include, but are not limited to N lobe amino acids Asp63, Tyr 95, Tyrl88, Lys206, His207 and His249; and C lobe amino acids Asp392, Tyr426, Tyr517 and His585 of SEQ ID NO: 3 (the number beside the amino acid indicates the position of the amino acid residue in the primary amino acid sequence where the valine of the mature protein is designated position 1). See U.S. Patent 5,986,067, which is herein incorporated be reference. In one embodiment, the Lys206 and His207 residues within the N lobe are replaced with GIn and GIu, respectively.

[00290] In some pharmaceutical formulations of the invention, the Tf fusion protein is engineered to contain a cleavage site between the therapeutic protein or peptide and the Tf moiety. Such cleavable sites or linkers are known in the art.

[00291] Pharmaceutical compositions of the invention and methods of the invention may include the addition of a transcytosis enhancer to facilitate transfer of the fusion protein across the GI epithelium. Such enhancers are known in the art. See Xia et ah, (2000) J. Pharmacol. Experiment. Therap., 295:594-600; and Xia et al. (2001) Pharmaceutical Res., 18(2): 191-195.

[00292] In preferred embodiments of the invention, oral pharmaceutical formulations include Tf fusion proteins comprising a modified Tf moiety exhibiting reduced or no glycosylation

fused at the N terminal end to an insulin or GLP-I protein or peptide as described above. Such pharmaceutical compositions may be used to treat glucose imbalance disorders such as diabetes by oral administration of the pharmaceutical composition comprising an effective dose of fusion protein.

[00293] The effective dose of fusion protein may be measured in a numbers of ways, including dosages calculated to alleviate symptoms associated with a specific disease state in a patient, such as the symptoms of diabetes. In other formulations, dosages are calculated to comprise an effective amount of fusion protein to induce a detectable change in blood glucose levels in the patient. Such detectable changes in blood glucose may include a decrease in blood glucose levels of between about 1% and 90%, or between about 5% and about 80%. These decreases in blood glucose levels will be dependent on the disease condition being treated and pharmaceutical compositions or methods of administration may be modified to achieve the desired result for each patient. In other instances, the pharmaceutical compositions are formulated and methods of administration modified to detect an increase in the activity level of the therapeutic protein or peptide in the patient, for instance, detectable increases in the activities of insulin or GLP-I. Such formulations and methods may deliver between about 1 pg to about 100 mg /kg body weight of fusion protein, about 100 ng to about 100 μg/kg body weight of fusion protein, about 100 μg/ to about 100 mg/kg body weight of fusion protein, about 1 μg to about 1 g of fusion protein, about 10 μg to about 100 mg of fusion protein or about 10 mg to about 50 mg of fusion protein. Formulations may also be calculated using a unit measurement of therapeutic protein activity, such as about 5 to about 500 units of human insulin or about 10 to about 100 units of human insulin. The measurements by weight or activity can be calculated using known standards for each therapeutic protein or peptide fused to Tf.

[00294] The invention also includes methods of orally administering the pharmaceutical compositions of the invention. Such methods may include, but are not limited to, steps of orally administering the compositions by the patient or a caregiver. Such administration steps may include administration on intervals such as once or twice per day depending on the Tf fusion protein, disease or patient condition or individual patient. Such methods also include the administration of various dosages of the individual Tf fusion protein. For instance, the initial dosage of a pharmaceutical composition may be at a higher level to induce a desired effect, such as reduction in blood glucose levels. Subsequent dosages may then be decreased

once a desired effect is achieved. These changes or modifications to administration protocols may be done by the attending physician or health care worker. In some instances, the changes in the administration protocol may be done by the individual patient, such as when a patient is monitoring blood glucose levels and administering a mTf-insulin or mTf-GLP-1 oral composition of the invention.

[00295] The invention also includes methods of producing oral compositions or medicant compositions of the invention comprising formulating a Tf fusion protein of the invention into an orally administerable form. In other instances, the invention includes methods of producing compositions or medicant compositions of the invention comprising formulating a Tf fusion protein of the invention into a form suitable for oral administration.

[00296] Moreover, the present invention includes pulmonary delivery of the Tf fusion protein formulations. Pulmonary delivery is particularly promising for the delivery of macromolecules which are difficult to deliver by other routes of administration. Such pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs, since drugs delivered to the lung are readily absorbed through the alveolar region directly into the blood circulation.

[00297] The present invention provides compositions suitable for forming a drug dispersion for oral inhalation (pulmonary delivery) to treat various conditions or diseases. The Tf fusion protein formulation could be delivered by different approaches such as liquid nebulizers, aerosol-based metered dose inhalers (MDFs), and dry powder dispersion devices. In formulating compositions for pulmonary delivery, pharmaceutically acceptable carriers including surface active agents or surfactants and bulk carriers are commonly added to provide stability, dispersibility, consistency, and/or bulking characteristics to enhance uniform pulmonary delivery of the composition to the subject.

[00298] Surface active agents or surfactants promotes absorption of polypeptide through mucosal membrane or lining. Useful surface active agents or surfactants include fatty acids and salts thereof, bile salts, phospholipid, or an alkyl saccharide. Examples of fatty acids and salts thereof include sodium, potassium and lysine salts of caprylate (C ₈ ), caprate (C io), laurate (C12) and myristate (CH). Examples of bile salts include cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic

acid, lithocholic acid, and ursodeoxycholic acid. Examples of phospholipids include single-chain phospholipids, such as lysophosphatidylcholine, lysophosphatidylglycerol, lysophosphatidylethanolamine, lysophosphatidylinositol and lysophosphatidylserine; or double-chain phospholipids, such as diacylphosphatidylcholines, diacylphosphatidylglycerols, diacylphosphatidylethanolamines, diacylphosphatidylinositols and diacylphosphatidylserines. Examples of alkyl saccharides include alkyl glucosides or alkyl maltosides, such as decyl glucoside and dodecyl maltoside.

[00299] Pharmaceutical excipients that are useful as carriers include stabilizers such as human serum albumin (HSA); bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.

[00300] Examples of carbohydrates for use as bulking agents include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. Examples of polypeptides for use as bulking agents include aspartame. Amino acids include alanine and glycine, with glycine being preferred.

[00301] Additives, which are minor components of the composition, may be included for conformational stability during spray drying and for improving dispersibility of the powder. These additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, and the like.

[00302] Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.

[00303] The Tf fusion compositions for pulmonary delivery may be packaged as unit doses where a therapeutically effective amount of the composition is present in a unit dose receptacle, such as a blister pack, gelatin capsule, or the like. The manufacture of blister packs or gelatin capsules is typically carried out by methods that are generally well known in the packaging art.

[00304] U.S. Patent 6,524,557 discloses a pharmaceutical aerosol formulation comprising (a) a HFA propellant; (b) a pharmaceutically active polypeptide dispersible in the propellant; and (c) a surfactant which is a C ₈ -Ci6 fatty acid or salt thereof, a bile salt, a phospholipid, or an

alkyl saccharide, which surfactant enhances the systemic absorption of the polypeptide in the lower respiratory tract. The invention also provides methods of manufacturing such formulations and the use of such formulations in treating patients.

[00305] One approach for the pulmonary delivery of dry powder drugs utilizes a hand-held device with a hand pump for providing a source of pressurized gas. The pressurized gas is abruptly released through a powder dispersion device, such as a venturi nozzle, and the dispersed powder made available for patient inhalation.

[00306] Dry powder dispersion devices are described in several patents. U.S. Pat. No. 3,921,637 describes a manual pump with needles for piercing through a single capsule of powdered medicine. The use of multiple receptacle disks or strips of medication is described in European Patent Application No. EP 0 467 172; International Patent Publication Nos. WO 91/02558; and WO 93/09832; U.S. Pat. Nos. 4,627,432; 4,811,731; 5,035,237; 5,048,514; 4,446,862; 5,048,514, and 4,446,862.

[00307] The aerosolization of protein therapeutic agents is disclosed in European Patent Application No. EP 0 289 336. Therapeutic aerosol formulations are disclosed in International Patent Publication No. WO 90/09781.

[00308] The present invention provides formulating Tf fusion protein for oral inhalation. The formulation comprises Tf fusion protein and suitable pharmaceutical excipients for pulmonary delivery. The present invention also provides administering the Tf fusion protein composition via oral inhalation to subjects in need thereof.

Transgenic Animals

[00309] The production of transgenic non-human animals that contain a modified transferrin fusion construct with increased serum half-life increased serum stability or increased bioavailability of the instant invention is contemplated in one embodiment of the present invention. In some embodiments, lactoferrin may be used as the Tf portion of the fusion protein so that the fusion protein is produced and secreted in milk.

[00310] The successful production of transgenic, non-human animals has been described in a number of patents and publications, such as, for example U.S. Patent 6,291,740 (issued September 18, 2001); U.S. Patent 6,281,408 (issued August 28, 2001); and U.S. Patent 6,271,436 (issued August 7, 2001) the contents of which are hereby incorporated by reference in their entireties.

[00311] The ability to alter the genetic make-up of animals, such as domesticated mammals including cows, pigs, goats, horses, cattle, and sheep, allows a number of commercial applications. These applications include the production of animals which express large quantities of exogenous proteins in an easily harvested form (e.g., expression into the milk or blood), the production of animals with increased weight gain, feed efficiency, carcass composition, milk production or content, disease resistance and resistance to infection by specific microorganisms and the production of animals having enhanced growth rates or reproductive performance. Animals which contain exogenous DNA sequences in their genome are referred to as transgenic animals.

[00312] The most widely used method for the production of transgenic animals is the microinjection of DNA into the pronuclei of fertilized embryos (Wall et al., J. Cell. Biochem. 49:113 [1992]). Other methods for the production of transgenic animals include the infection of embryos with retroviruses or with retroviral vectors. Infection of both pre- and post- implantation mouse embryos with either wild-type or recombinant retroviruses has been reported (Janenich, Proc. Natl. Acad. Sci. USA 73: 1260 (1976); Janenich et al, Cell 24:519 (1981); Stuhlmann ef α/., Proc. Natl. Acad. Sci. USA 81 :7151 (1984); Jahner et al, Proc. Natl. Acad Sci. USA 82:6927 (1985); Van der Putten et al, Proc. Natl. Acad Sci. USA 82:6148-6152 (1985); Stewart et al, EMBO J. 6:383-388 (1987)).

[00313] An alternative means for infecting embryos with retroviruses is the injection of virus or virus-producing cells into the blastocoele of mouse embryos (Jahner, D. et al., Nature 298:623 (1982)). The introduction of transgenes into the germline of mice has been reported using intrauterine retroviral infection of the midgestation mouse embryo (Jahner et al., supra (1982)). Infection of bovine and ovine embryos with retroviruses or retroviral vectors to create transgenic animals has been reported. These protocols involve the micro-injection of retroviral particles or growth arrested (i.e., mitomycin C-treated) cells which shed retroviral particles into the perivitelline space of fertilized eggs or early embryos (PCT International Application WO 90/08832 [1990]; and Haskell and Bowen, MoI. Reprod. Dev., 40:386 [1995]. PCT International Application WO 90/08832 describes the injection of wild-type feline leukemia virus B into the perivitelline space of sheep embryos at the 2 to 8 cell stage. Fetuses derived from injected embryos were shown to contain multiple sites of integration.

[00314] U.S. Patent 6,291,740 (issued September 18, 2001) describes the production of transgenic animals by the introduction of exogenous DNA into pre-maturation oocytes and mature, unfertilized oocytes (i.e., pre-fertilization oocytes) using retroviral vectors which transduce dividing cells (e.g., vectors derived from murine leukemia virus [MLV]). This patent also describes methods and compositions for cytomegalovirus promoter-driven, as well as mouse mammary tumor LTR expression of various recombinant proteins.

[00315] U.S. Patent 6,281,408 (issued August 28, 2001) describes methods for producing transgenic animals using embryonic stem cells. Briefly, the embryonic stem cells are used in a mixed cell co-culture with a morula to generate transgenic animals. Foreign genetic material is introduced into the embryonic stem cells prior to co-culturing by, for example, electroporation, microinjection or retroviral delivery. ES cells transfected in this manner are selected for integrations of the gene via a selection marker such as neomycin.

[00316] U.S. Patent 6,271,436 (issued August 7, 2001) describes the production of transgenic animals using methods including isolation of primordial germ cells, culturing these cells to produce primordial germ cell-derived cell lines, transforming both the primordial germ cells and the cultured cell lines, and using these transformed cells and cell lines to generate transgenic animals. The efficiency at which transgenic animals are generated is greatly increased, thereby allowing the use of homologous recombination in producing transgenic non-rodent animal species.

Gene Therapy

[00317] The use of modified transferrin fusion constructs for gene therapy wherein a modified transferrin protein or transferrin domain is joined to a therapeutic protein or peptide is contemplated in one embodiment of this invention. The modified transferrin fusion constructs with increased serum half-life or serum stability of the instant invention are ideally suited to gene therapy treatments.

[00318] The successful use of gene therapy to express a soluble fusion protein has been described. Briefly, gene therapy via injection of an adenovirus vector containing a gene encoding a soluble fusion protein consisting of cytotoxic lymphocyte antigen 4 (CTLA4) and the Fc portion of human immunoglubulin Gl was recently shown in Ijima et al. (June 10, 2001) Human Gene Therapy (United States) 12/9:1063-77. In this application of gene

therapy, a murine model of type II collagen-induced arthritis was successfully treated via intraarticular injection of the vector.

[00319] Gene therapy is also described in a number of U.S. patents including U.S. Pat. 6,225,290 (issued May 1, 2001); U.S. Pat. 6,187,305 (issued February 13, 2001); and U.S. Pat. 6,140,111 (issued October 31, 2000).

[00320] U.S. Patent 6,225,290 provides methods and constructs whereby intestinal epithelial cells of a mammalian subject are genetically altered to operatively incorporate a gene which expresses a protein which has a desired therapeutic effect. Intestinal cell transformation is accomplished by administration of a formulation composed primarily of naked DNA, and the DNA may be administered orally. Oral or other intragastrointestinal routes of administration provide a simple method of administration, while the use of naked nucleic acid avoids the complications associated with use of viral vectors to accomplish gene therapy. The expressed protein is secreted directly into the gastrointestinal tract and/or blood stream to obtain therapeutic blood levels of the protein thereby treating the patient in need of the protein. The transformed intestinal epithelial cells provide short or long term therapeutic cures for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein.

[00321] U.S. Pat. 6,187,305 provides methods of gene or DNA targeting in cells of vertebrate, particularly mammalian, origin. Briefly, DNA is introduced into primary or secondary cells of vertebrate origin through homologous recombination or targeting of the DNA, which is introduced into genomic DNA of the primary or secondary cells at a preselected site.

[00322] U.S. Pat. 6,140,111 (issued October 31, 2000) describes retroviral gene therapy vectors. The disclosed retroviral vectors include an insertion site for genes of interest and are capable of expressing high levels of the protein derived from the genes of interest in a wide variety of transfected cell types. Also disclosed are retroviral vectors lacking a selectable marker, thus rendering them suitable for human gene therapy in the treatment of a variety of disease states without the co-expression of a marker product, such as an antibiotic. These retroviral vectors are especially suited for use in certain packaging cell lines. The ability of retroviral vectors to insert into the genome of mammalian cells has made them particularly promising candidates for use in the genetic therapy of genetic diseases in humans and

animals. Genetic therapy typically involves (1) adding new genetic material to patient cells in vivo, or (2) removing patient cells from the body, adding new genetic material to the cells and reintroducing them into the body, i.e., in vitro gene therapy. Discussions of how to perform gene therapy in a variety of cells using retroviral vectors can be found, for example, in U.S. Pat. Nos. 4,868,116, issued Sep. 19, 1989, and 4,980,286, issued Dec. 25, 1990 (epithelial cells), WO 89/07136 published Aug. 10, 1989 (hepatocyte cells) , EP 378,576 published JuI. 25, 1990 (fibroblast cells), and WO 89/05345 published Jun. 15, 1989 and WO/90/06997, published Jun. 28, 1990 (endothelial cells), the disclosures of which are incorporated herein by reference.

Kits Containing Transferrin Fusion Proteins

[00323] In a further embodiment, the present invention provides kits containing transferrin fusion proteins, which can be used, for instance, for the therapeutic or non-therapeutic applications. The kit comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which includes a transferrin fusion protein that is effective for therapeutic or non-therapeutic applications, such as described above. The active agent in the composition is the therapeutic protein. The label on the container indicates that the composition is used for a specific therapy or non- therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those described above.

[00324] The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[00325] Without further description, it is believed that a person of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. For example, a skilled artisan would readily be able to determine the biological activity, both in vitro and in vivo, for the fusion protein constructs of the present invention as compared with the comparable activity of the therapeutic moiety in its unfused state. Similarly, a person skilled in the art could readily

determine the serum half life and serum stability of constructs according to the present invention. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLES Example 1 : Peptide YY/mTf fusion proteins

[00326] Peptide YY (PYY) regulates food intake. PYY is produced by neuroendocrine cells of the intestine in response to the ingestion of food. Like other peptides, PYY has a short plasma half-life in humans. The present invention provides fusion proteins with PYY fused to mTf with extended serum stability and in vivo circulatory half-life and pharmaceutical compositions of such fusion proteins for treating patients in need thereof.

[00327] In this example, the steps for producing PYY/mTf fusion protein are described. The same steps may be used to generate transferrin fusion proteins with other PYYs, PYY analogs or derivatives, etc.

[00328] The amino acid sequence for PYY(I -36) and PYY(3-36) is given below and is derived from NM 004160 (Figs. 4A-D).

[00329] 1-36 YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 4) [00330] 3-36 IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 5)

[00331] PYY targets a number of receptors, most notably Yi and Y ₂ in the hypothalamus. The functions of PYY are believed to include 'inhibition of gastric secretion, inhibition of pancreatic secretion, inhibition of intestinal secretion, and inhibition of gastrointestinal motility (Keire et al. (2000) Am. J. Physiol. Gastrointest Liver Physiol. 279:G126-131) and different actions are mediated by different receptors. mTf-PYY fusion proteins of the invention may be used to modulate one or more of these biological functions. The conformation, dictated by the sequence of the peptide, determines the receptor targeted (Fig. 5). PYY(I -36) and PYY(3-36) both target the Y ₂ receptor with approximately equal affinity, however, PYY(3-36) has 250Ox lower affinity for the Yi receptor than PYY(l-36). The 1-36 peptide is processed in vivo to the 3-36 peptide by DPP IV. Loss of the N-terminal two

amino acids results in the inability to form the loop structure of PYY(I -36) resulting in the more elongated structure of PYY(3-36) (Fig. 5).

Construction of Vectors.

[00332] To make PYY(l-36) mTf fusion protein, the plasmid pREX0197 (Fig.6) was used as the template for overlapping PCR mutagenesis. Two sets of PCR reactions were performed using primers P0025 with P0625 and P0627 with POO 12 (Fig. 7A). The products from these reactions were gel purified and used in a second round of PCR with just the outer primers P0025 and P0012 to join the two initial PCR products together. The resulting PCR product was digested with the restriction enzymes Aflll and EcoBl and ligated into AβωJEcoBl digested pREXO 197. The insert was DNA sequenced to check for a clone free of any PCR induced errors and the resulting plasmid designated pREX0616 (Fig. 8).

[00333] Once the DNA sequence had been confirmed the expression cassette was recovered by NotVPvul digestion of pREXOόl 6 and ligated into Notl digested pSAC35 (Fig. 9). The resulting plasmid was designated pREX0617 (Fig. 10).

[00334] In a similar manner, PYY(3-36) mTf fusion protein constructs were made using P0025 with P0626 and P0627 with P0012 (Fig. 7A) as the primer pairs for the first round PCR. The products of these reactions were joined in a second reaction with just the outer primers and the product ligated into pREX0197 as described above to create pREX0608 (Fig. 1 IA) the Notl expression cassette from which was subsequenctly cloned into Notl cut pSAC35 to create pREX0609 (Fig. 1 IB).

Codon Optimized Version

[00335] To make PYY(I -36) mTf or PYY(3-36) mTf fusion protein in which the DNA sequence of PYY has been optimized for expression in a Saccharomyces host strain, the same methodology would be used except the primer pairs would be P0025 with P0628 and P0630 with POO 12 for PYY(I -36) and P0025 with P0629 and P0630 with POO 12 for PYY(3-36) (Fig. 7B).

Options for Making PYY/m Tf fusion proteins

[00336] Examples of possible options include but are not limited to: Leader Sequence- PYY(I -36 or 3-36; R33X)-Linker-mTf. Preferably, the leader sequence nL (natural leader)

having the amino acid sequence MRLA VGALLVCA VLGLCLA (Genbank Accession No. NM_001063; SEQ ID NO: 1 (residues 1-19)) is used. However, the leader sequence could be any other leader that gives efficient cleavage and productivity.

[00337] The PYY fragment may contain one or more substitutions. In one embodiment, R33 could be substituted with X, wherein X is A or X is some similar conservative change. In another embodiment, the PYY fragment could be additionally substituted at Rl 9 and/or R25 with X, wherein X is A or X is some similar conservative change.

[00338] The linker could be:

(PEAPTD) _n, (SEQ ID NO: 10) where n is 1 to 4 or more repeats;

(IgG hinge) _n> where n is 1 to 4 or more repeats;

(SGAPPPS) _n (SEQ ID NO: 11) where n is 1 to 4 or more repeats; or combinations thereof e.g. PEAPTD IgG hinge.

[00339] As examples, the following contructs were made: pREX0616/0617 PYY(l-36) mTf (Figs 8 and 10) pREX0608/0609 PYY(3-36) mTf (Figs 1 IA and 1 IB) pREX0639/0640 PYY(3-36;R33A) mTf (Figs 12A and 12B) pREX0838/0839 PYY(3-36) (PEAPTD) ₂ mTf (Figs 13A and 13B) pREX0641/0642 PYY(3-36;R33A) (PEAPTD) ₂ mTf (Figs 14A and 14B) pREX0643/0644 PYY(3-36;R19A,R25A,R33A) (PEAPTD) ₂ mTf (Figs 15A and 15B).

[00340] The first pREX number is the base plasmid for construction and the second number is the pSAC35 based expression vector.

1 2 3 3

1 3 9 5 3 6 Linker mTf pREX0617 : j||]ikpeapgedaspeelnryyaslrhylnlvtrqry vpdktvrwcav pREX0609 : - -ikpeapgedaspeelnryyaslrhylnlvtrqry vpdktvrwcav pREX0640 : - -ikpeapgedaspeelnryyaslrhylnlvtjiqry vpdktvrwcav pREX0642 : - -ikpeapgedaspeelnryyaslrhylnlvtaqry^^^^^^^^PSSvpdktvrwcav pREX0644 : - -ikpeapqedaspeelnlyyaslllhvlnlvtlqrvmiB^^^Svpdktvrwcav pREX00617 (SEQ ID NO: 12) pREX0609 (SEQ ID NO: 13) pREX0640 (SEQ ID NO: 14) pREX00642 (SEQ ID NO: 15) pREX0644 (SEQ ID NO: 16)

[00341] R to A changes in pREX0640 and 0642 were designed to prevent proteolytic cleavage when produced in yeast. N-terminal sequencing of pREX0609 indicated the major species to be cleaved between residues 33 and 34. Additional R to A changes could also be made as in pREX0644 to prevent cleavage in yeast.

[00342] The linker between PYY and mTf is a repeat unit of the linker found between the N and C lobes of Tf (residues 332-337). This repeat linker has been found to increase the productivity and activity of GLP-I constructs. The hinge region from human IgGl is also a good linker for GLP-I . Preferably, the hinge region derived from human IgGl is VEPKSSDKTHTSPPSPAPELLGGPS (SEQ ID NO: 17), wherein the undelined S is changed from C in its natural sequence. This change could also be a conserved change to some other similar amino acid e.g. A or G. These linkers could also be used in combination with the PEAPTD linker. The PEAPTD IgG hinge linker was shown to give high productivity. Further, the C-terminal extension of Exendin-4 (SSGAPPPS (SEQ ID NO: 1 1)) could also be used as a possible linker by itself or in combination with the human IgGl hinge region or the PEAPTD linker.

[00343] As a further embodiment, GLP-I could be fused to a cleavable linker which is fused to the PYY-mTf fusion protein. These constructs allow for releasing GLP-I from the PYY-mTf fusion protein with ease. Also, these constructs allow for releasing GLP-I at a certain rate from the PYY mTf fusion protein.

[00344] Fusions could also be at the C-terminus of mTf using the methodology detailed above, for example: pREX0738/0739 mTf PYY(3-36) (Figs 16A and 16B) pREX0740/0741 mTf (PEAPTD) ₂ PYY(3-36) (Figs 17A and 17B)

Example 2: GLP-1/Peptide YY/mTf fusion proteins

[00345] In this example, the steps for producing GLP-I/ PYY/mTf fusion protein are described. nL GLP-I (7-36; A8G,K34A) mTf pYY(3-36)

[00346] The amino acid sequence for GLP-I (7-36) is amino acids 1-30 of SEQ ID NO: 6. A8 is Ala at position 2 in SEQ ID NO: 6. K34 is Lys at position 31 in SEQ ID NO: 6. nL is the natural leader sequence (amino acids 1-19 of SEQ ID NO: 2).

[00347] To make nL GLP-1(7-36;A8G,K34A) mTf pYY(3-36) fusion protein, pREX0585 was digested with SaWHinάlll as was pREX0738. From pREX0584 (Fig. 18A), the 6587bp fragment was recovered, and from pREX0738, the 114bp fragment was recovered. The two fragments were then ligated together to make pREX0907 (Fig. 18B). The resulting expression cassette was then excised from pREX0907 as a Notl fragment and ligated into Notl cut pSAC35 to give pREX0908 (Fig. 18C). nL GLP-1(7-36;A8G,K34A) mTf (PEAPTD) ₂ pYY(3-36)

[00348] To make the nL GLP-1(7-36;A8G,K34A) mTf (PEAPTD) ₂ pYY(3-36) fusion protein, pREX0585 was digested with SaH/Hindlll as was pREX0740. From pREX0584, the 6587bp fragment was recovered and from pREX0740 thel50bp fragment was recovered. The two fragments were then ligated together to make pREX0909 (Fig. 19A). The resulting expression cassette was then excised from pREX0909 as a Notl fragment and ligated into Notl cut pSAC35 to give pREX0910 (Fig. 19B).

[00349] Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents, patent applications and publications referred to in this application are herein incorporated by reference in their entirety.