JP2008531059A - Modified transferrin fusion protein - Google Patents

Abstract

  Disclosed are modified fusion proteins of transferrin, GLP-1 and linker moieties that have high productivity, biological activity, and long serum half-life. Preferred fusion proteins include those in which the transferrin moiety has been modified to exhibit no glycosylation or to exhibit reduced glycosylation. The fusion proteins of the invention are useful for treating type II diabetes, type I diabetes, obesity, congestive heart failure, and non-fatty liver disease.

Description

Related Applications This application is a priority benefit of US Provisional Application No. 60 / 658,140, filed March 4, 2005, and No. 60 / 663,757, filed March 22, 2005. All of these applications are hereby incorporated by reference in their entirety.

  However, the present application does not claim priority benefits, but is an international application PCT / US03 / 26818 filed on August 28, 2003 (this is a US application filed March 4, 2003). No. 10 / 378,094 claims the benefit of U.S. Patent Application No. 10 / 378,094) and U.S. Patent Application No. 10 / 231,494 filed on August 30, 2002 (which is dated August 30, 2001). And claims the benefit of US Provisional Application No. 60 / 315,745, filed November 30, 2001, and US Provisional Application No. 60 / 334,059, filed November 30, 2001). All of which are hereby incorporated by reference in their entirety. This application does not claim priority benefit, but is related to US Provisional Application No. 60 / 406,977 filed Aug. 30, 2002, which is hereby incorporated by reference in its entirety. To join.

  The present invention relates to a fusion protein of GLP-1 and transferrin and the use thereof for treating diseases associated with high serum glucose levels, such as type II diabetes. The fusion proteins of the present invention may also be useful for treatment with GLP-1 such as obesity, type I diabetes, congestive heart failure and non-alcoholic, non-fatty liver disease. It can also be used to treat diseases.

  Therapeutic proteins or peptides, generally in their native state or when produced recombinantly, are unstable with a short stability period in the indicated serum or a short half-life in circulation in vivo. Is a molecule. In addition, these molecules are often quite unstable when formulated, especially when formulated into aqueous solutions for diagnosis and therapy.

  There are few practical solutions that enhance or promote the in vivo or in vitro stability of proteinaceous therapeutic molecules. Polyethylene glycol (PEG) is a substance that can bind to proteins and provide long acting, sustained protein activity. When the protein activity is prolonged due to the coupling with PEG, the number of necessary protein administrations can be reduced. However, PEG linkages often reduce or destroy the therapeutic activity of proteins. In one example, PEG conjugation can reduce the immunogenicity of a protein, while in other examples it may increase immunogenicity.

  A therapeutic protein or peptide is also stabilized by fusion to a protein that can increase the half-life of the therapeutic protein in circulation in vivo. For example, a therapeutic protein fused to albumin or an antibody fragment may exhibit an extended in vivo circulation half-life compared to an unfused therapeutic protein. See U.S. Patent Nos. 5,876,969 and 5,766,883.

  In addition, other serum proteins, glycosylated human transferrin (Tf), can be used to create fusions with therapeutic proteins to target delivery to the interior of cells, or to pass substances across the blood brain barrier. It can also be transported. Using these fusion proteins containing glycosylated human Tf, nerve growth factor (NGF) or ciliary neurotrophic factor (CNTF) crosses the blood brain barrier by fusing full-length Tf to such substances. Targeting is also done. See U.S. Pat. Nos. 5,672,683 and 5977,307. In these fusion proteins, the Tf portion of the molecule is glycosylated and binds to two iron atoms, which are required for Tf to bind to receptors on the cell, and these According to the inventors of the patent, NGF or CNTF moieties are required to target delivery across the blood brain barrier. Transferrin fusion proteins have also been produced by inserting HIV-1 protease target sequences into loops exposed on the surface of glycosylated transferrin, and such fusion proteins can enter cells through the Tf receptor. The ability to produce other forms of Tf fusion for targeted delivery of (Ali et al. (1999) J. Biol. Chem. 274 (34): 24066-24073) can be investigated.

  Serum transferrin (Tf) is a monomeric glycoprotein with a molecular weight of 80,000 daltons that binds iron in the circulation and transports iron 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, US Pat. No. 5,026,651). Tf is one of the most common serum molecules and constitutes up to about 5-10% of total serum protein. Glycodeficient transferrin occurs at elevated levels in the blood of alcoholic individuals and exhibits a longer half-life (approximately 14-17 days) than the half-life of glycosylated transferrin (approximately 7-10 days). van Eijk et al. (1983) Clin. Chim. Acta 132: 167-171, Stivler (1991) Clin. Chem. 37: 2029-2037 (1991), Arndt (2001) Clin. Chem. 47 (1): 13-27 and Stivler et al. in "Carbohydrate-defective composition", Advances in the Biosciences, (Ed Nordmann et al.), Pergamon, 1988, 71, 353-357.

  The structure of Tf is well characterized, and the mechanisms of receptor binding, iron binding and release, and carbonate ion binding have also been elucidated (US Pat. Nos. 5,026,651, 5,986,067). And MacGillivray et al. (1983) J. Biol. Chem. 258 (6): 3543-3546).

  Antibodies that bind to transferrin and transferrin receptor are also used to deliver or carry toxicants to tumor cells as a cancer treatment (Baselga and Mendelsohn, 1994), and transferrin is used for non-viral gene therapy. It has been used as a 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 transferrin receptors as entry points is CD4 (Walus et al., 1996), brain-derived neurotrophic factor (Pardridge et al., 1994), glial cell-derived neurotrophic factor (Albeck et al.), Several vasoactive intestinal peptide analogues (Bickel et al., 1993), beta-amyloid peptides (Saito et al., 1995), and antisense oligonucleotides (Pardridge et al., 1995). It has been demonstrated using proteins and peptides.

  GLP-1 is a therapeutic peptide consisting of 30/31 amino acids derived from post-translational processing of the proglucagon gene product in small intestinal secretory L cells. Infusion of GLP-1 in humans with type II diabetes stimulates insulin secretion and lowers blood glucose in a glucose-dependent manner (Nuck, M., 2004, Horm. Metab. Res. 36: 852-858; Vilsboll et al., 2004, Diabetologia.47: 357-366; and Zander et al., 2002, Lancet.359: 824-830). GLP-1 has also been found to promote satiety and inhibit gastric emptying (Zander et al., 2002, Lancet. 359: 824-830; Gutzwiller et al., 1999, Gut. 44: 81-86; Meier et al., 2002, Eur. J. Pharmacol. 440: 269-279; and Flint et al., 2001, Int. J. Obes. Relat. Metab. Dis .. 25: 781-792). Infusion of GLP-1 causes increased insulin biosynthesis and increased β-cell proliferation and β-cell mass in rodent islets of Langerhans (Perfetti et al., 2000, Endocrinology. 141: 4600-4605; Wang et al., 1997, J. Clin. Invest. 99: 2883-2889; and Stoffers et al., 2000, Diabetes.49: 741-748).

  Full-length active GLP-1 is due to rapid enzymatic inactivation by dipeptidyl peptidase IV (DPPIV) cleaving between alanine and glutamic acid at the second and third positions, respectively, at the N-terminus of the peptide Furthermore, the half-life in circulation is as short as 1-2 minutes (Kieffer et al., 1995, Endocrinology. 136: 3585-3596). The remaining fragment of GLP-1 containing 28/29 amino acids is not insulinotropic and is further cleaved by neutral endopeptidase (NEP) (Knudsen and Pridel, 1996, Eur. J. Pharmacol. 318). : 429-435, and Plumboeck et al., 2005, Diabetologia 48: 1882-1890). A single parenteral injection of GLP-1 disappears from the circulation within minutes, so there is a need for long-term degradation resistant GLP-1.

  Recently, exendin-4, a potent GLP-1 receptor agonist in the salivary glands of the American monster monster, has been approved for the treatment of patients with type II diabetes (Parks et al., 2001, Metabolism. 50: 583-589; Aziz and Anderson, 2002, J. Nutr. 132: 990-995; and Egan et al., 2002, J. Clin. Endocrinol. Metab. 87: 1282-1290). Exendin-4, like GLP-1, is insulin secretagogue, inhibits food intake and gastric emptying, and nourishes β cells in rodents. Furthermore, exendin-4 is not a substrate for DPPIV due to the presence of glycine at position 2 at its N-terminus. The downside of using exendin-4 is that it must be injected twice a day because exendin-4 has a half-life of only 2-4 hours (Kolterman et al., 2003, J. Clin. Endocrinol). Metab. 88: 3082-3089 and Fineman et al., 2003, Diabetes Care. 26: 2370-2377).

  Thus, there is still a need for long-term degradation resistant GLP-1 molecules. The present invention meets this need by providing transferrin and GLP-1 fusion proteins that extend the in vivo circulating half-life of GLP-1 protein while maintaining or increasing biological activity.

SUMMARY OF THE INVENTION As explained in more detail below, the present invention includes a fusion protein comprising a GLP-1 peptide, a substantially non-helical polypeptide linker, and a modified transferrin (mTf) molecule. In one embodiment, the fusion protein exhibits reduced glycosylation as compared to a native transferrin protein.

  The linker moiety of the present invention links the GLP-1 moiety to the modified transferrin moiety. In one embodiment, the linker is from PEAPTD (SEQ ID NO: 13), PEAPTDPEAPTD (SEQ ID NO: 10), PEAPTD (SEQ ID NO: 118-123 and 126-129) in combination with an IgG hinge linker, and PEAPTDPEAPTD in combination with an IgG hinge linker. Selected from the group consisting of

  The GLP-1 moiety of the present invention may be modified. In one embodiment, the GLP-1 moiety may be modified so that cleavage by a protease is inhibited. For example, the GLP-1 (7-36) or (7-36) moiety is a mutation from A8 to S, G or V (corresponding to A2 of SEQ ID NO: 6) and / or K34 to Q, A or N May be modified by a mutation to (corresponding to K28 of SEQ ID NO: 6).

  The GLP-1 / substantially non-helical polypeptide linker / mTf fusion protein of the invention exhibits high expression productivity compared to a similar fusion protein without a substantially non-helical linker. . Furthermore, GLP-1 / substantially non-helical polypeptide linker / mTf fusion protein of the invention exhibits high expression productivity compared to similar fusion proteins with flexible polypeptide linkers.

  In other embodiments, a GLP-1 / substantially non-helical polypeptide linker / mTf fusion protein is compared to a similar GLP-1 / mTf fusion protein without a linker as a result of such a linker. Or a substantial increase in at least one activity compared to a similar GLP-1 / mTf fusion protein with a flexible linker. For example, the fusion protein of the present invention substantially reduces blood glucose and further substantially secretes insulin as compared to a similar GLP-1 / mTf fusion protein with no linker or with a flexible linker. Can be induced.

  The fusion proteins of the invention also exhibit an extended half-life and sustained biological activity compared to the unfused GLP-1 molecule.

  Considered further, the fusion proteins of the invention may contain further modifications. For example, prior to cleavage, the N-terminus of the fusion protein may include a secretory signal sequence. The Tf portion may be modified so that the Tf portion does not bind to iron. Furthermore, the Tf moiety may be modified such that the Tf moiety does not exhibit N-linked or O-linked glycosylation. For example, the mTf moiety may contain a mutation within or adjacent to an N-linked glycosylation site comprising the sequence NXS / T. In one embodiment, the mTf moiety comprises a mutation at N413 and / or N611 or at an adjacent S / T residue.

  The nucleic acid molecule encoding the fusion protein of the present invention may be cloned into a vector and expressed in a host cell. In one embodiment of the invention, host cells are cultured to express the fusion protein and the resulting protein is isolated. For example, the fusion proteins of the invention can be expressed in yeast and then isolated and purified. The fusion protein of the present invention can also be isolated from a transgenic animal produced using the nucleic acid molecule encoding the fusion protein of the present invention.

  The invention includes a pharmaceutical composition of GLP-1 / polypeptide linker / mTf fusion protein that is not substantially helical. The present invention relates to type II diabetes, type I diabetes, obesity, congestive heart failure, non-fatty, by administering a pharmaceutical composition comprising a GLP-1 / substantially non-helical polypeptide linker / mTf fusion protein. Treatment methods for patients suffering from other liver diseases and other suitable diseases. Administration of this fusion protein can effectively reduce blood glucose levels, induce β-cell proliferation and β-cell mass increase, induce insulin secretion, decrease gastric emptying, and enhance satiety.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the alignment of the N and C domains of human (Hu) transferrin (Tf) (SEQ ID NO: 3), highlighting similarity and identity.
Figures 2A-2B show alignments of transferrin sequences from various species. Light shading: similarity; dark shading: identity (SEQ ID NOs 84-90).
FIG. 3 shows the location of multiple insertion sites exposed on the surface of Tf for therapeutic proteins, polypeptides or peptides.
FIG. 4 shows various variants of GLP-1, GLP-2 linkers and mTf used to construct various GLP-1 / GLP-2 / mTf fusion proteins. In variants 2-10, GLP-1 (-37) (variant 1, SEQ ID NO: 71) is modified with an amino acid deletion, N-terminal amino acid addition, and / or amino acid substitution. Mutant 2 (amino acids 1 to 30 of SEQ ID NO: 71). Mutant 3 (amino acids 1 to 30 of SEQ ID NO: 71). Variant 4 (SEQ ID NO: 72). Variants 5-8 (SEQ ID NO: 73). Mutant 9 (amino acids 1-27). Mutant 10 (amino acids 1 to 23 of SEQ ID NO: 71). In variants b to h, GLP-2 (peptide a, SEQ ID NO: 74) is modified. Mutant b (amino acids 2 to 24 of SEQ ID NO: 74). Variants cf (SEQ ID NO: 75). Mutant g (amino acids 1 to 29 of SEQ ID NO: 74). Mutant h (amino acids 9-34 of SEQ ID NO: 74). As an example, the N-terminal part of the fusion protein HGLP-1 (7-36) K34Q-GLP-2 (2-34) K30A-mTf is shown (SEQ ID NO: 75). In this example, the second residue of mTf is also modified from Pro to Ser.
FIG. 5 shows the relative potency in cAMP analysis using rat GLP-1 receptor cloned into CHO cells.
FIG. 6 shows a comparison of plasma concentrations of GLP-1 / mTf in monkeys as measured by ELISA and in vitro bioassay. Each point is the average of triplicate measurements. The GLP-1 / mTf fusion protein used was GLP-1 (A8G, K34A) -PEAPTDPEAPTD-mTf.
FIG. 7 is a restriction map of pREX0585 (SEQ ID NO: 11) encoding GLP-1-Tf (SEQ ID NO: 12).
FIG. 8a shows the percentage of maximal response of cAMP for various concentrations of GLP-1-Tf (SEQ ID NO: 12), GLP-1 and exendin-4 in CHO cells transfected with GLP-1R.
FIG. 8b shows the percent of maximal insulin secretion for various concentrations of GLP-1 and GLP-1-Tf (SEQ ID NO: 12) in RIN 1046.38 cells.
FIG. 8c shows the percent of the maximal response of insulin at various concentrations of GLP-1 and GLP-1-Tf in isolated rat islets.
FIG. 8d shows plasma GLP-1-Tf (SEQ ID NO: 12) over time in cynomolgus monkeys treated with GLP-1-Tf by the subcutaneous and intravenous routes of administration.
FIGS. 9a and b show non-diabetic treated with hTf, 1 mg / kg GLP-1-Tf or 10 mg / kg GLP-1-Tf by intraperitoneal (FIG. 9a) and subcutaneous (FIG. 9b) routes of administration. 2 shows blood glucose over time in mice.
Figures 10a-d show blood glucose and plasma insulin secretion levels in db / db mice treated with hTf or GLP-1-Tf (SEQ ID NO: 12).
FIGS. 11a-d show blood glucose (FIGS. 11a and c) in db / db and non-diabetic mice fed once daily for 5 days after treatment with hTf or GLP-1-Tf (SEQ ID NO: 12). Food intake (Figures 11b and d) is shown. FIG. 11a shows blood glucose over time in normal mice treated with hTf or GLP-1. FIG. 11c shows blood glucose over time in db / db mice treated with hTf or GLP-1. FIG. 11b shows food intake over time in non-diabetic mice treated with hTf or GLP-1-Tf. FIG. 11d shows food intake over time in db / db mice treated with hTf, GLP-1-Tf or exendin-4.
Figures 12a-c show BrdU + nuclei in pancreas of db / db mice treated with hTf or GLP-1-Tf (SEQ ID NO: 12).
FIG. 13a is a Western blot showing fragments corresponding to Tf and GLP-1-Tf in plasma from rats treated with Tf or GLP-1-Tf (SEQ ID NO: 12).
FIGS. 13b-c show the final neuronal, solitary nucleus and hypothalamic chamber of rats treated with hTf or GLP-1-Tf (SEQ ID NO: 12) by intraperitoneal injection (FIG. 13b) and intraventricular injection (FIG. 13c). It is an image of cFos activation in the paranucleus.
FIG. 14 is an image using immunofluorescence and confocal microscopy 24 hours after treatment with GLP-1-Tf in islets from fixed pancreas of mice treated with GLP-1-Tf or Tf. Indicates.

General Description The present invention relates, in part, to a transferrin, modified transferrin, or transferrin or modified transferrin portion that is sufficient for the GLP-1 therapeutic protein to extend the half-life of the therapeutic protein in serum. Based on the discovery by the inventors that GLP-1 therapeutic proteins can be stabilized by genetically fusing their serum half-life and / or in vivo activity. A modified transferrin fusion protein comprises a transferrin protein or domain covalently linked to a therapeutic protein or peptide, wherein the transferrin moiety has one or more amino acid substitutions, insertions or deletions compared to the wild-type transferrin sequence. Modified to include loss. In one embodiment, the Tf fusion protein is engineered to reduce or inhibit glycosylation into the Tf or Tf domain. In other embodiments, the Tf protein or Tf domain has reduced binding to iron or carbonate ions, or such binding does not occur, or has reduced affinity for the Tf receptor (TfR). Or modified so that binding to them does not occur.

The present invention is also based on the discovery that inserting a linker between GLP-1 and a transferrin molecule increases the stability and availability of the GLP-1 molecule for binding to its receptor. Also based. Specifically, it has been found that using GLP-2 or a derivative thereof as a linker between GLP-1 and mTf improves the presentation of GLP-1 to its receptor. Also, linkers that are not substantially helical, such as, but not limited to, PEAPTD, (PEAPTD) ₂ , PEAPTD (SEQ ID NOs: 118-123 and 126-129) in combination with IgG hinge linkers, and combinations with IgG hinge linkers Using (PEAPTD) ₂ has also been found to substantially increase the serum half-life, productivity of fusion protein expression, and / or activity of GLP-1. In one embodiment of the invention, a second GLP-1 or derivative thereof may be used as a linker between the GLP-1 therapeutic protein and mTf.

Therefore, the present invention includes a transferrin fusion protein, a therapeutic composition containing the fusion protein, and further includes a method for treating, preventing or ameliorating a disease or disorder by administering a fusion protein of GLP-1 and transferrin. . The GLP-1 and transferrin fusion proteins of the present invention comprise at least a fragment or variant of a GLP-1 therapeutic protein, at least a fragment or variant of a modified transferrin, and a linker, preferably associated with each other, preferably Genetic fusions (ie, such transferrin fusion proteins are nucleic acids in which a polynucleotide encoding all or part of a therapeutic protein is linked in frame to a polynucleotide encoding all or part of a modified transferrin. Produced by translating), or accompanied by a chemical bond between each other. For some GLP-1 therapeutic proteins of the transferrin fusion protein, it may be referred to as a GLP-1 “portion”, “region” or “moiety” of the transferrin fusion protein (eg, “GLP -1 therapeutic protein moiety "or" transferrin protein moiety "). Similarly, a substantially non-helical linker or GLP-2 linker of a portion of the transferrin fusion protein may be referred to as a “linker” or linker “part”, “region” or “part” of the transferrin fusion protein. is there.

  In one embodiment, the present invention comprises a transferrin fusion protein, eg, a fusion corresponding to SEQ ID NO: 12, comprising or alternatively consisting of a GLP-1 therapeutic protein, a linker protein and a modified serum transferrin protein Provide protein. In other embodiments, the present invention comprises or alternatively consists of biologically active and / or therapeutically active fragments of GLP-1 therapeutic proteins, linker proteins and modified transferrin proteins A transferrin fusion protein is provided. In other embodiments, the present invention includes or substitutes for biologically active and / or therapeutically active variants of GLP-1 therapeutic proteins, linker proteins and modified transferrin proteins. A transferrin fusion protein is provided. In a further embodiment, the present invention provides a transferrin fusion protein comprising a biologically active and / or therapeutically active fragment of GLP-1 protein, linker protein and modified transferrin. In other embodiments, the GLP-1 therapeutic protein portion of the transferrin fusion protein is an active form of the therapeutic protein.

  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 preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

Definitions As used herein, an “amino acid corresponding to” or “equal amino acid” in a transferrin sequence maximizes the identity or similarity between the first transferrin sequence and at least the second transferrin sequence. Are identified by aligning as follows. The number used to identify equal amino acids in the second transferrin sequence is based on the number used to identify the corresponding amino acid in the first transferrin sequence. In some cases, these phrases may be used to describe amino acid residues in human transferrin as compared to certain residues in rabbit serum transferrin.

  The term “biological activity” or “activity” as used herein is defined by a GLP-1 therapeutic molecule, protein or peptide in a biological environment (ie, in vivo, or a simulated environment thereof in vitro). Function or a series of activities. Biological activity includes, but is not limited to, the function of the GLP-1 therapeutic molecule portion of the claimed fusion protein, including but not limited to induction of insulin secretion, hypoglycemia, suppression of food intake Suppression of gastric emptying, beta cell proliferation and beta cell mass increase, and cFos activation in the nervous system. If the fusion proteins or peptides of the invention exhibit one or more biological activities of their natural counterparts of the therapeutic protein, i.e. non-fused counterparts, they are biologically active. Is done.

  A fusion protein comprising or not comprising GLP-2 and / or a substantially non-helical linker has one or more biological activities, at least about 10%, at least about 20 either in vivo or in vitro. %, At least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, or at least about When present at a rate of 300%, or greater, for a longer duration (ie, longer) than the same one or more biological activities in the natural counterpart of the therapeutic protein, the fusion protein is substantially Long-term biological activity. In other embodiments, a fusion protein having a GLP-2 linker and / or a substantially non-helical linker has at least about 10% one or more biological activities, either in vivo or in vitro, At least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, Or a GLP-1 and Tf fusion protein with a linker sequence deleted or a GLP-1 and Tf fusion protein having a flexible linker at least about 300%, or more With a long duration (ie long term) If you, the fusion protein shows a substantial long-term activity.

  As used herein, a “binder” is a substance used to impart tackiness to a powdered material. Binders, sometimes known as “granulating agents”, are those that impart stickiness to the tablet formulation and compress the tablet by formulating granules of the desired hardness and size. Be sure to leave it intact afterwards, in addition to improving the ease of flow. Commonly used materials as binders include starch; gelatin; sugars such as sucrose, glucose, dextrose, molasses, and lactose; natural and synthetic gums such as gum arabic, sodium alginate, Irish moss , Panwar gum, gati gum, isapol shell mucus, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, veegum, microcrystalline cellulose, microcrystalline dextrose, amylose, and larch arabogalactan arabogalactan) and the like.

  The term “carrier” as used herein means a diluent, adjuvant, excipient or vehicle with which it is administered as a composition. Such pharmaceutical carriers can be sterile liquids and include, for example, water and oils such as petroleum, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

  As used herein, a “colorant” is a substance that imparts a more pleasant appearance to the tablet and, in addition, makes it easier for the manufacturer to control the product during manufacture and to make it easier for the user to recognize the product. Any of the approved and certified water-soluble FD & C dyes, mixtures thereof, or their corresponding lakes can be used for colored tablets. A dye lake is a combination material by adsorbing a water-soluble dye to a hydrated oxide of a heavy metal to produce an insoluble form of the dye.

  As used herein, a “diluent” is an inert substance added to increase the bulk of a formulation and can be sized practically to compress tablets. Commonly used diluents include calcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, dry starch, powdered sugar, silica and the like.

  As used herein, “degradable” or “disintegrant” is a substance that promotes the disintegration or disintegration of a tablet after administration. Substances that function as disintegrants are chemically classified as starches, clays, celluloses, algins or rubbers. Other disintegrants include Veegum HV, methylcellulose, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, alginic acid, guar gum, citrus pulp, crosslinked polyvinylpyrrolidone, carboxymethylcellulose and the like.

  The term “dispersible” or “dispersible” is moisture having a water content of less than about 10% by weight (% w), usually less than about 5% w and preferably less than about 3% w; 1.0 to 5.0 μm mass median diameter (MMD), generally 1.0 to 4.0 μm MMD, and preferably 1.0 to 3.0 μm MMD; Approximately> 30%, generally> 40%, preferably> 50%, most preferably> 60%; and the aerodynamic mass median diameter of the aerosol particle size distribution of 1.0 to 5.0 μm (MMAD), generally a dry powder that is a MMAD of 1.5 to 4.5 μm, and preferably a MMAD of 1.5 to 4.0 μm.

  The term “dry” means that the composition has a water content such that the particles can be easily dispersed in an inhalation device to form an aerosol. This water content is generally less than about 10% by weight (% w) water, usually less than about 5% w, preferably less than about 3% w.

  As used herein, an “effective amount” is an amount of a drug or pharmacologically active agent sufficient to provide the desired local or systemic effect and performance at a reasonable benefit-risk ratio associated with any drug therapy. Means.

  As used herein, “flavoring agents” vary greatly in their chemical structure, ranging from simple esters, alcohols and aldehydes to carbohydrates and complex volatile oils. Almost all desired types of synthetic flavors are currently available.

  As used herein, the term “fragment of Tf protein” or “Tf protein” or “portion of Tf protein” refers to at least about 5%, 10%, 20 of a naturally occurring Tf protein, or a mutant thereof. Amino acid sequence comprising%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

  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 regulatory sequences necessary for their expression. A gene may also include DNA segments that form recognition sequences for other proteins but are not expressed, for example. Genes can be obtained from a variety of sources, for example cloned from the source of interest, or synthesized from known or predicted sequence information, and sequences designed to have the desired parameters May be included.

  As used herein, a “heterologous polynucleotide” or “heterologous nucleic acid” or “heterologous gene” or “heterologous sequence” or “exogenous DNA segment” is a polynucleotide that originates from a source that is foreign to a particular host cell. , Means a nucleic acid or DNA segment, or, if of the same origin, means a polynucleotide, nucleic acid or DNA segment 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, this term refers to a DNA segment that is foreign or heterologous to the cell, or that is homologous to the cell, but usually present at a location within the nucleic acid of the host cell where the element is not found. As an example, the signal sequence that is native to the yeast cell but bound to the human Tf sequence is heterologous.

  As used herein, a “separated” nucleic acid sequence refers to a nucleic acid sequence that is substantially free of other nucleic acid sequences, preferably at least about 20% pure, preferably as determined by agarose gel electrophoresis. Means 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 nucleic acid sequences To do. For example, isolated nucleic acid sequences can be obtained by standard cloning methods used in genetic engineering, and the nucleic acid sequences can be transferred from their natural location to different sites where they are expected to be reproduced. . Cloning methods include excising and isolating a desired nucleic acid fragment containing a nucleic acid sequence encoding a polypeptide, inserting the fragment into a vector molecule, and converting the recombinant vector into multiple copies or clones of the nucleic acid sequence. Incorporating into a host cell that is expected to replicate. Such nucleic acid sequences can be genomic, cDNA, RNA, semi-synthetic, synthetic nucleic acid sequences, or any combination thereof.

  As used herein, when a DNA coding sequence is translated into a fusion polypeptide as a result of the fusion of two or more DNA coding sequences in frame, the two or more DNA coding sequences Are said to be “linked” or “fused”. The term “fusion”, when used in reference to a Tf fusion, includes, but is not limited to, at least one therapeutic protein, polypeptide or peptide binding to the N-terminus of Tf, binding to the C-terminus of Tf, and / or Or insertion between any two amino acids within Tf.

  As used herein, a “lubricant” improves the flow rate of tablet granules, prevents the tablet material from sticking to the surface of a die or punch, reduces friction between particles, And a material that exhibits numerous performance such as facilitating the discharge of tablets from the cavity of the die. Commonly used lubricants include talc, magnesium stearate, calcium stearate, stearic acid, and hydrogenated vegetable oil. Typical amounts of lubricant range from about 0.1% to about 5% by weight.

  As used herein, “modified transferrin”, as used herein, refers to a transferrin molecule that exhibits at least one modification of its amino acid sequence as compared to wild-type transferrin.

  As used herein, a “modified transferrin fusion protein”, as used herein, means that at least one molecule of modified transferrin (or a fragment or variant thereof) is a therapeutic protein (or fragment or variant thereof). A protein formed by fusing to at least one molecule.

  The term “nucleic acid” or “polynucleotide” as used herein refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, these terms include nucleic acids containing analogs of natural nucleotides that have similar binding properties as the nucleic acid of reference and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, certain nucleic acid sequences are also conservatively modified variants (eg, degenerate codon substitutes) that are not explicitly specified as well as explicitly specified sequences. And also complementary sequences. In particular, degenerate codon substitutes result in sequences in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (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) Mol. Cell. Probes 8: 91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

  As used herein, a DNA segment is said to be “operably linked” when it is placed in functional association with another DNA segment. For example, if the DNA of the signal sequence is expressed as a precursor protein involved in the secretion of the fusion protein, they are operably linked to the DNA encoding the fusion protein of the invention; the promoter or enhancer When stimulating the transcription of, it is operably linked to a coding sequence. In general, functionally linked DNA sequences are contigs, and in the case of signal sequences or fusion proteins, both are contigs and within the reading phase. However, enhancers need not be contiguous with the coding sequences that they control transcription. In such situations, ligation is accomplished by ligation with convenient restriction sites or adapters or linkers inserted in their place.

  As used herein, “pharmaceutically acceptable” is physiologically acceptable and typically produces allergic or similar adverse reactions (eg, stomach discomfort, dizziness, etc.) when administered to humans. It means substances and compositions that do not occur. Typically, the term “pharmaceutically acceptable” as used herein is approved by a federal or state government regulatory agency, or for use in animals, more specifically humans, Means listed on the United States Pharmacopeia or other generally recognized pharmacopoeia.

  As used herein, a “physiologically effective amount” is an amount that is delivered to a subject to provide the desired palliative or therapeutic effect. This amount is specific to each drug and also to their approved final dosage level.

  “Efficacy” as used herein refers to the ability of GLP-1 to activate one or more receptors. For example, a GLP-1 / substantially non-helical linker / mTF fusion protein of the invention can be compared to a fusion protein that does not have a substantially non-helical linker or a fusion protein that has a flexible linker and In comparison, at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least A potency of about 10 times, at least about 20 times or at least about 50 times or more can be exhibited.

  The term “powder” as used herein is a composition consisting of finely dispersed solid particles that are easy to flow in an inhalation device and can be easily dispersed, after which the subject inhales the particles Means something that reaches the lungs and can penetrate the alveoli. Therefore, the powder is referred to as “respirable”. Preferably, the average particle size is less than about 10 microns (μm) in diameter and has a relatively uniform spherical 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 of such particles is about 0.1 μm to about 5 μm in diameter, specifically about 0.3 μm to about 5 μm.

  As used herein, “productivity” of expression of the fusion protein refers to the ability of the protein to be expressed in the host cell system. For example, GLP-1 and Tf fusion proteins with GLP-2 linkers or linkers that are not substantially helical can be expressed in yeast cells. As used herein, “substantially increase productivity” means that the expression of the fusion protein in the host is a similar fusion protein in which the linker is deleted or a flexible polypeptide linker. Compared to protein expression, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about It means increasing at a rate of 90%, at least about 100%, at least about 200% or at least about 300% or higher.

  The term “promoter” as used herein refers to a DNA region involved in RNA polymerase binding to initiate transcription.

  As used herein, “cleavage by a protease” means cleavage of GLP-1 by a protease. For example, DPP-IV is a protease that cleaves GLP-1 in its natural state. The amino acid substitution A8G of GLP-1 (7-37) can inhibit GLP-1 protein cleavage at these amino acids by DPP-IV. As used herein, a GLP-1 protein that has been modified to “substantially reduce cleavage by a protease” is at least about 1.5 compared to a native GLP-1 protein. Times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times A GLP-1 protein having one or more amino acid substitutions that reduces cleavage by proteases by a factor of at least about 12 times, at least about 13 times, at least about 14 times or at least about 15 times more or more.

  As used herein, the term “recombinant” means a cell, tissue or organism that has been transformed with a new combination of genes or DNA.

  The term “serum half-life” or “plasma half-life” as used herein refers to the time required for a 50% reduction in serum GLP-1 concentration in vivo. The shorter the serum half-life of GLP-1, the shorter the period during which the protein can exert a therapeutic effect. For example, if a Tf fusion peptide with or without GLP-1 and a GLP-2 linker and / or a substantially non-helical linker exhibits a measurable increase in half-life, for example, is not fused Compared to the GLP-1 molecule, it has at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400 in the medium half-life in serum. Times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1,000 times, at least about 5,000 times, at least about 10,000, at least about 25 times 5,000 times, at least about 50,000 times, at least about 75,000 times or less Again showing an increase in the proportion of more than about 100,000 times, or it they exhibit prolonged serum half-life.

  As used herein, the term “subject” can be a human, mammal or animal. The subject to be treated is a patient in need of treatment.

  As used herein, the term “substantially non-spiral linker” or “rigid linker” refers to a linker that physically separates the GLP-1 and transfer moiety of the fusion protein. “Substantially non-helical” means that the linker peptide exhibits little or no helical shape or spiral shape or secondary structure. . For example, a substantially non-helical structure may include less than about 20% helical or helical shape, or secondary structure. A typical alpha-helical peptide is right-handed (twisted clockwise) and contains an amino acid R group that extends outside the helix, which makes one complete rotation for every 3.6 amino acids. The carbonyl group of each peptide bond extends parallel to the axis of the helix, and within the helix, directly toward the four amino acids below it in the NH group of the peptide bond, A hydrogen bond is formed between them. Typically, the non-spiral linker will contain at least about 5%, at least about 10%, at least about 20% amino acids that disrupt any alpha helix formation (eg, amino acids that induce kinks in the polypeptide chain), At least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or about 100%.

  A kink can be introduced into a naturally occurring peptide by modifying amino acid residues. Examples of amino acids that cause kink in the polypeptide chain include proline and glycine amino acid residues. For example, the addition of proline or glycine at or near the middle of the barrel of a linear alpha helix is expected to bend the protein, i.e., to produce a kink. The introduction of proline residues generally causes a larger kink than the introduction of glycine residues. One skilled in the art will appreciate that proline or glycine residues can be kinked wherever they are introduced into the linker peptide. The linker peptide may contain one or more amino acid residues that induce kink. For example, a substantially non-spiral linker may comprise at least about 5% proline content, at least about 10% proline content, at least about 20% proline content, at least about 30% proline content, at least about 40% proline. Content, at least about 50% proline content, at least about 60% proline content, at least about 70% proline content, at least about 80% proline content, at least about 90% proline content, at least about 95% proline content or It may have a proline content of about 100%.

  Substantially non-spiral linkers include, but are not limited to, PEAPTD (SEQ ID NO: 13), PEAPTDPEAPTD (SEQ ID NO: 10), PEAPTDPEAPTPDPAPTD (SEQ ID NO: 14), IgG hinge (SEQ ID NO: 88, 89 and 117), PEAPTD + IgG hinge (SEQ ID NO: 118-123 and 126-129), PPPPPPPPPPPPP (SEQ ID NO: 17), GEAPTDPEAPTD (SEQ ID NO: 18), PEAGTDPEAPTD (SEQ ID NO: 19), PEAPTDGEAPTD (SEQ ID NO: 20), PEAPTDPEAGTD (SEQ ID NO: 21), PQAPTNQ (SEQ ID NO: 22), and PEAPEAPEAPEA (SEQ ID NO: 23). Typically, the substantially non-spiral linker is at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13. Having at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20 or at least about 21 amino acids or more. However, there is no upper limit on the length of the linker.

  A “tablet” as used herein is a solid pharmaceutical dosage form containing a pharmaceutical substance, which may or may not contain a suitable diluent, as is well known in the art. Manufactured by either compression or molding methods. Tablets have been used extensively since the late 19th century, and their popularity continues. Tablets are manufactured by the manufacturer (eg, ease of manufacture, economy, stability, and convenience in packaging, transportation, and dispensing) and the patient (eg, dosage accuracy, compactness, portability, mouthfeel) It is still popular as a dosage form because it provides both benefits and ease of administration. Tablets are most often disk-shaped, but may be round, oval, rectangular, cylindrical or triangular. Tablets can vary widely in size and mass depending on the amount of pharmaceutical substance present and the intended method of administration. Tablets are classified into two general types: (1) compressed tablets, and (2) wet tablets or powder tablets. In addition to the active ingredient or therapeutic ingredient or group of ingredients, tablets contain a number of inert substances or additives. A first group of such additives are materials that help to impart sufficient compression properties to the formulation, including, for example, diluents, binders, and lubricants. A second group of such additives serves to impart additional desirable physical properties to the finished tablet, such as disintegrants, colorants, flavors, and sweeteners.

  As used herein, the term “therapeutically effective amount” means the amount of transferrin fusion protein comprising a GLP-1 therapeutic molecule sufficient to effect treatment when administered to a subject in need thereof. To do. The amount of transferrin fusion protein that constitutes a “therapeutically effective amount” is expected to vary depending on the therapeutic protein used, the physical condition or severity of the disease, and the age and weight of the subject to be treated. However, one of ordinary skill in the art can determine routinely in light of his / her own knowledge and the present disclosure.

  As used herein, “therapeutic protein” or “therapeutic molecule” refers to GLP-1, GLP-1 fragments or variants or analogs thereof having one or more therapeutic and / or biological activities. Means. In this specification, the terms peptide, protein, and polypeptide are used interchangeably. As used herein, polypeptides that exhibit “therapeutic activity” or “therapeutically active” proteins are well known in the art as described herein or otherwise. GLP-1, GLP-1 fragments or variants or analogs thereof having one or more known biological and / or therapeutic activities related to GLP-1. A “therapeutic protein” is a GLP-1 protein or analog useful for treating, preventing or ameliorating a disease, physical condition or disorder. Such diseases, physical conditions or disorders may be in humans or in non-human animals, for example in veterinary use.

  The term “transformation” as used herein refers to the introduction of a nucleic acid (ie, a high molecular weight nucleotide) into a cell. The term “genetic transformation” as used herein refers to the introduction and uptake of DNA, particularly recombinant DNA, into cells.

  The term “transformant” as used herein means a cell, tissue or organism that has undergone transformation.

  As used herein, the term “transgene” means a nucleic acid that is inserted into an organism, host cell or vector to ensure that it functions.

  As used herein, the term “transgenic” refers to a cell, cell culture, or a cell culture that has received a foreign or modified gene, particularly a gene encoding a modified Tf fusion protein, in any one of a variety of transformation methods. Means an organism, organism, bacterium, fungus, animal, plant, and their progeny, wherein the foreign or modified gene is from the same species as the biological species that accepted the foreign or modified gene Or from a different species.

  “Variant group or variant” means a polynucleotide or nucleic acid that differs from a reference nucleic acid or polypeptide but retains their essential properties. In general, variants are very similar overall to a reference nucleic acid or polypeptide and are identical in many regions. As used herein, a “GLP-1 variant” or “GLP-1 analog” is a sequence that differs from a natural therapeutic protein, as described elsewhere herein, or It refers to the GLP-1 peptide portion of the transferrin fusion protein of the present invention that retains at least one functional and / or therapeutic property of other GLP-1 peptides known in the art. GLP-1 variants and analogs include GLP-1 (7-37), which includes one or more modifications that inhibit cleavage by proteases, such modifications include, but are not limited to, Examples include amino acid modifications A8G and K34A.

  The term “vector” as used herein generally refers to any plasmid, phagemid or virus that encodes an exogenous nucleic acid. The term should also be understood to include non-plasmids, non-phagemids and non-viral compounds that facilitate the introduction of nucleic acids into virions or cells, such as polylysine compounds. The vector may be a viral vector suitable as a vehicle for delivering nucleic acids, or mutants thereof, to a cell, or the vector may be a non-viral vector suitable for the same purpose. Examples of viral and non-viral vectors for delivering DNA to cells and tissues are well known in the art, for example, Ma et al. (1997, Proc. Natl. Acad. Sci. USA 94). 12744-12746). Examples of viral vectors include, but are not limited to, recombinant vaccinia virus, recombinant adenovirus, recombinant retrovirus, recombinant adeno-associated virus, recombinant avian pox virus (Cranage), and the like. Et al., 1986, EMBO J. 5: 3057-3063; International Patent Application Publication WO94 / 17810, published August 18, 1994; International Patent Application Publication WO94 / 23744, published October 27, 1994). Examples of non-viral vectors include, but are not limited to, liposomes, DNA polyamine derivatives, and the like.

  As used herein, the term “wild type” refers to a naturally occurring polynucleotide or polypeptide sequence.

Transferrin and Transferrin Modifications The present invention provides GLP-1 or GLP-1 analogs and fusion proteins comprising transferrin or modified transferrin. Any transferrin can be used to make the modified Tf fusion proteins of the invention. In one embodiment, the Tf fusion protein also includes GLP-2 and / or a substantially non-helical linker. In other embodiments, the Tf fusion protein comprises a GLP-1 linker in addition to the therapeutic molecule GLP-1 or a GLP-1 analog.

Any transferrin can be used to make the modified Tf fusion proteins of the invention. As an example, wild-type human Tf (Tf) is a protein of 679 amino acids of about 75 kDa (considering glycosylation), which is composed of two major domains, N (about 330 amino acids). ) And C (about 340 amino acids), which appear to be caused by gene duplication. GenBank accession numbers NM_001063, XM_002793, M12530, XM_039845, XM_039847, and S95936 (www.ncbi.nlm.nih.gov/) (these are both in their entirety by adding SEQ ID NO: 1, 2 and 3 by reference herein ). The two domains diverge over time but retain a significant degree of identity / similarity (FIG. 1).

  Each of the N and C domains is further divided into two subdomains, N1 and N2, C1 and C2. The function of Tf is to transport iron to the body's cells. This process is mediated by the Tf receptor (TfR), which is expressed in all cells, especially in actively proliferating cells. TfR recognizes the iron-bound form of Tf (two molecules bound per receptor) and then endocytosis occurs, thereby transporting the TfR / Tf complex to the endosome, at which point Bound iron is released by a general pH drop and the TfR / Tf complex is recycled to the cell surface, releasing Tf (the iron unbound form is known as apo Tf). 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 since non-glycosylated iron-bound Tf binds to the receptor.

Each Tf molecule can carry two iron ions (Fe ³⁺ ). These are complexed in the space between the N1 and N2 and C1 and C2 subdomains, causing a change in the conformation of the molecule. Tf crosses the blood brain barrier (BBB) via the Tf receptor.

  In human transferrin, the iron-binding site is at least the amino acid Asp63 of SEQ ID NO: 3 (Asp82 of SEQ ID NO: 2, which includes the natural Tf signal sequence), Asp392 (Asp411 of SEQ ID NO: 2), Tyr95 (SEQ ID NO: 2). 2 Tyr114), Tyr426 (Tyr445 of SEQ ID NO: 2), Tyr188 (Tyr207 of SEQ ID NO: 2), Tyr514 or 517 (Tyr533 or Tyr536 of SEQ ID NO: 2), His249 (His268 of SEQ ID NO: 2), and His585 (SEQ ID NO: 2) 2 His604). The hinge region is at least amino acid residues 94-96, 245-247 and / or 316-318 of the N domain of SEQ ID NO: 3 and amino acid residues 425-427, 581-582 and / or 652-658 of the C domain. including. Carbonic acid binding sites are at least amino acids Thr120 (SEQ ID NO: 2 Thr139), Thr452 (SEQ ID NO: 2 Thr471), Arg124 (SEQ ID NO: 2 Arg143), Arg456 (SEQ ID NO: 2 Arg475), Ala126 (SEQ ID NO: 2). No. 2 Ala145), Ala458 (Ala477 of SEQ ID NO: 2), Gly127 (Gly146 of SEQ ID NO: 2), and Gly459 (Gly478 of SEQ ID NO: 2).

In one embodiment of the invention, the modified transferrin fusion protein comprises a modified human transferrin, but any animal Tf molecule can be used to produce a fusion protein of the invention comprising a human Tf variant. Such animals include cattle, pigs, sheep, dogs, rabbits, rats, mice, hamsters, spiders, platypus, chickens, frogs, caterpillars, monkeys, as well as other bovine, canine and avian species Can also be mentioned. All of these Tf sequences are readily available in GenBank and other public databases. Human Tf nucleotide sequence is available (SEQ ID NO: S1,2 and 3 and referring to the registration number mentioned above, available www.ncbi.nlm.nih.gov/), using these, the Tf or Tf Genetic fusions between the domain and the selected therapeutic molecule can be made. Fusions may also be made from related molecules such as lactotransferrin (lactoferrin) GenBank accession number: NM — 002343) or melanotransferrin (GenBank accession number: NM — 013900, mouse melanotransferrin).

  Melanotransferrin is a glycosylated protein found at high levels in malignant melanoma cells and was initially named human melanoma antigen p97 (Brown et al., 1982, Nature, 296: 171-173). It has high sequence homology with human serum transferrin, human lactoferrin and chicken transferrin (Brown et al., 1982, Nature, 296: 171-173; Rose et al., Proc. Natl. Acad. Sci. USA, 1986, 83: 1261-1265). However, unlike these receptors, no cellular receptors for melanotransferrin have been identified. Melanotransferrin binds reversibly to iron and exists in two forms, one of which is bound to the cell membrane by an anchor of glycosylphosphatidylinositol, the other is soluble and actively secreted (Baker et al., 1992, FEBS Lett, 298: 215-218; Alemany et al., 1993, J. Cell Sci., 104: 1155-1116; Food et al., 1994, J. Biol. Chem. 274: 7011-1701) .

Lactoferrin (Lf) is a natural iron-binding protein with a defensive action and has been found to have antibacterial, antifungal, antiviral, antitumor and anti-inflammatory activities. This protein is normally present in exocrine secretions exposed to normal flora: milk, tears, nasal exudates, saliva, bronchial mucus, gastrointestinal fluid, cervical vaginal mucosa, and semen. In addition, Lf is the main component of secondary special granules of circulating polymorphonuclear neutrophils (PMN). The apoprotein is released when PMN degranulates in the suppurated area. The main function of Lf is to capture free iron in fluids and inflamed areas, thereby suppressing free radical mediated damage and introducing metal into invading microorganisms and neoplastic cells. The availability can be reduced. In studies examining the turnover rate of ¹²⁵ ILf in adults, Lf has been shown to be rapidly taken up by the liver and spleen, with radioactivity remaining in the liver and spleen for several weeks (Bennett et al. (1979), Clin. Sci. (Lond.) 57: 453-460).

  In one embodiment, the transferrin portion of the transferrin fusion protein of the invention comprises a transferrin splice variant. In one example, the transferrin splice variant may be a human transferrin splice variant. In one particular embodiment, the human transferrin splice variant may be a variant of Genbank accession number AAA61140.

  In other embodiments, the transferrin portion of the transferrin fusion protein of the invention comprises a lactoferrin splice variant. In one example, the human serum lactoferrin splice variant may be a novel splice variant of neutrophil lactoferrin. In one particular embodiment, the neutrophil lactoferrin splice variant may be a variant of Genbank accession number AAA59479. In other specific embodiments, the lactoferrin splice variant of neutrophils may comprise the following amino acid sequence EDCIALKGEADA (SEQ ID NO: 5), which includes a novel variant of the splice variant. An area is included.

  In other embodiments, the transferrin portion of the transferrin fusion protein of the invention comprises a melanotransferrin variant.

  Modified Tf fusions may be made using any Tf protein, fragment, domain, or engineered domain. For example, a fusion protein may be produced using a full length Tf sequence with or without the native Tf signal sequence. Tf fusion proteins may also be produced using a single Tf domain, such as an individual N or C domain, or a modified form of Tf that includes a 2N or 2C domain (August 30, 2002). See US Provisional Application No. 60 / 406,977 filed with the Applicant, which is hereby incorporated by reference in its entirety). In some embodiments, a fusion may be produced in which a therapeutic protein is fused to a single C domain, wherein the C domain is modified to reduce, suppress or prevent glycosylation. ing. In other embodiments, the use of a single N domain is advantageous because the Tf glycosylation site is intact in the C and N domains. A preferred embodiment is a Tf fusion protein with a single N domain expressed at high levels.

  As used herein, a C-terminal domain or lobe that has been modified to function as an N-like domain substantially resembles the glycosylation pattern or iron binding properties of a native or wild-type N domain or lobe. Modified to exhibit glycosylation patterns or iron binding properties. In a preferred embodiment, the C domain or lobe is glycosylated by replacing the associated C domain region or amino acid with the corresponding C domain region or amino acid present in the native or wild type N domain region or site. It is modified so that it does not bind to iron.

  As used herein, a Tf moiety that includes “two N domains or lobes” refers to a native C domain or lobe, a native or wild type N domain or lobe, or a modified N domain or lobe. , Or a C domain modified to function in a manner similar to a substantially wild type or modified N domain.

  Analysis of the two domains over time by superimposing the two domains (Swiss PDB Viewer 3.7b2, Iterative Magic Fit) and direct amino acid alignment (ClustalW multiple alignment) It is clear that there is a branch. Amino acid alignment shows 42% identity and 59% similarity between the two domains. However, for structural equivalence, about 80% of the N domain matches the C domain. The C domain also has some extra disulfide bonds compared to the N domain.

According to the alignment of the molecular models for the N and C domains, the following structural equivalence is evident:

The disulfide bonds of the two domains are arranged as follows:

  In one embodiment, the transferrin portion of the transferrin fusion protein comprises at least two N-terminal lobes of transferrin. In a further embodiment, the transferrin portion of the transferrin fusion protein comprises at least two N-terminal lobes of transferrin derived from human serum transferrin.

  In another embodiment, the transferrin portion of the transferrin fusion protein comprises at least one transferrin having a mutation in at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyr188, and His249 of SEQ ID NO: 3. Includes, consists of, or consists of two N-terminal lobes.

  In other embodiments, the transferrin portion of the modified transferrin fusion protein comprises a mutant of the N-terminal lobe of recombinant human serum transferrin having a mutation in Lys206 or His207 of SEQ ID NO: 3.

  In other embodiments, the transferrin portion of the transferrin fusion protein comprises, includes or consists of at least two C-terminal lobes of transferrin. In a further embodiment, the transferrin portion of the transferrin fusion protein comprises at least two C-terminal lobes of transferrin derived from human serum transferrin.

  In a further embodiment, the C-terminal lobe mutant further comprises at least one mutation of Asn413 and Asn611 of SEQ ID NO: 3, which renders glycosylation impossible.

In another embodiment, the transferrin portion of the transferrin fusion protein comprises at least 2 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. Contains one C-terminal lobe, where the mutant retains the ability to bind to metal. In an alternative embodiment, the transferrin portion of the transferrin fusion protein comprises at least two 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. Contains a C-terminal lobe, where the mutant has a reduced ability to bind to metal. In another embodiment, the transferrin portion of the transferrin fusion protein comprises at least two C 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. It contains a terminal lobe, where the mutant does not retain the ability to bind metal and functions substantially like the N domain.

  In some embodiments, the Tf or Tf moiety comprises an in vivo circulating half-life, serum stability, in vitro solution stability or bioavailability of the unfused GLP-1 therapeutic protein. In comparison, GLP-1 therapeutic protein of sufficient length to increase in vivo circulation half-life, serum stability, in vitro solution stability or bioavailability It becomes. Such increases in stability, serum half-life or bioavailability are about 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold 800 times, 900 times, 1,000 times, 5,000 times, 10,000 times, 25,000 times, 50,000 times, 75,000 times or 100,000 times or more. In some cases, the transferrin fusion protein comprising modified transferrin and GLP-1, and optionally GLP-2 and / or a substantially rigid linker is about 12-24 hours, about 18-24 hours, about 1 About 30 hours, about 38 hours, about 40 hours, about 42 hours, about 45 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, Serum half-life of about 9 days, about 10-20 days or more, about 12-18 days or about 14-17 days.

  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, are mutated for expression in yeast systems. May thereby prevent glycosylation or excessive mannosylation and increase the serum half-life of the fusion protein and / or therapeutic protein (Asialo- or, in some cases, Monosialo-Tf or Disialo) -To produce Tf). In addition to the Tf amino acids corresponding to N413 and N611, adjacent residues within the NXS / T glycosylation site may be mutated to prevent or substantially reduce glycosylation. . See U.S. Pat. No. 5,986,067 to Funk et al. It has also been reported that the N domain of Tf expressed in Pichia pastoris is an O-linked glycosylation with a single hexose at S32, which is also known as such glycosylation. May be mutated or modified to prevent.

  Thus, in one embodiment of the invention, the transferrin fusion protein comprises a modified transferrin molecule, wherein transferrin has reduced glycosylation such as, but not limited to, the asialo-, monosialo- and disialo forms of Tf. Show. In other embodiments, the transferrin portion of the transferrin fusion protein comprises a recombinant transferrin mutant mutated to prevent glycosylation. In other embodiments, the transferrin portion of the transferrin fusion protein comprises a fully glycosylated recombinant transferrin mutant. In a further embodiment, the transferrin portion of the transferrin fusion protein comprises a recombinant human serum transferrin mutant mutated to prevent N-linked glycosylation, wherein at least one of Asn413 and Asn611 of SEQ ID NO: 3. One is mutated to an amino acid that cannot be glycosylated. In other embodiments, the transferrin portion of the transferrin fusion protein comprises a recombinant human serum transferrin mutant mutated to prevent or substantially reduce glycosylation, wherein the mutation is N -X-S / T may be in an adjacent residue within the glycosylation site, for example a mutation of the S / T residue. Moreover, glycosylation can be reduced or prevented by mutating the serine or threonine residues. Furthermore, it is known to change X to proline in order to prevent glycosylation.

  As discussed in more detail below, the modified Tf fusion proteins of the invention may also be designed to not bind iron and / or bind to the Tf receptor. In other embodiments of the invention, the iron binding capacity is retained and the iron binding ability of Tf is passed through the epithelial or endothelial cell membrane and / or through the blood brain barrier to the inside of the cell for a therapeutic protein or It may be used to deliver peptides. These embodiments that bind to iron and / or Tf receptors are often designed to reduce or prevent glycosylation and increase the serum half-life of therapeutic proteins. A single N domain is expected not to bind to TfR when iron is added, and a C domain bound to iron is expected to bind TfR, but does not have the same affinity as the complete molecule.

  In other embodiments, the transferrin portion of the transferrin fusion protein comprises a recombinant transferrin mutant having a mutation, wherein the mutant does not retain the ability to bind metal ions. In an alternative embodiment, the transferrin portion of the transferrin fusion protein comprises a recombinant transferrin mutant having a mutation, wherein the mutant has a binding affinity for a metal ion that is weaker than wild-type serum transferrin. Have sex. In an alternative embodiment, the transferrin portion of the transferrin fusion protein comprises a recombinant transferrin mutant having a mutation, wherein the mutant has a stronger binding affinity for metal ions than wild type serum transferrin. Have sex.

  In other embodiments, the transferrin portion of the transferrin fusion protein comprises a recombinant transferrin mutant having a mutation, wherein the mutant does not retain the ability to bind to a transferrin receptor. For example, the modified GLP-1 and Tf fusion proteins of the present invention may bind to the cell surface GLP-1R receptor but not the Tf receptor. Such a fusion protein may have therapeutic activity on the cell surface, ie without entering the cell.

  In an alternative embodiment, the transferrin portion of the transferrin fusion protein comprises a recombinant transferrin mutant having a mutation, wherein the mutant binds to a transferrin receptor that is weaker than wild-type serum transferrin. Has affinity. In an alternative embodiment, the transferrin portion of the transferrin fusion protein comprises a recombinant transferrin mutant having a mutation, wherein the mutant has stronger binding to the transferrin receptor than wild type serum transferrin. Has affinity.

  In other embodiments, the transferrin portion of the transferrin fusion protein comprises a recombinant transferrin mutant having a mutation, wherein the mutant does not retain the ability to bind carbonate ions. In an alternative embodiment, the transferrin portion of the transferrin fusion protein comprises a recombinant transferrin mutant having a mutation, wherein the mutant has a binding affinity for carbonate ions that is weaker than wild-type serum transferrin. Have sex. In an alternative embodiment, the transferrin portion of the transferrin fusion protein comprises a recombinant transferrin mutant having a mutation, wherein the mutant has a stronger binding affinity for carbonate ions than wild type serum transferrin. Have sex.

  In another embodiment, the transferrin portion of the transferrin fusion protein comprises at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyr188, His249, Asp392, Tyr426, Tyr514, Tyr517 and His585 of SEQ ID NO: 3. A recombinant human serum transferrin mutant having a mutation in the group, wherein the mutant retains the ability to bind metal ions. In an alternative embodiment, recombination having a mutation in at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyr188, His249, Asp392, Tyr426, Tyr514, Tyr517 and His585 of SEQ ID NO: 3 Including a human serum transferrin mutant, where the mutant has the ability to bind to reduced metal ions. In another embodiment, recombinant human serum transferrin having a mutation in at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyr188, His249, Asp392, Tyr426, Tyr517 and His585 of SEQ ID NO: 3 Including mutants, wherein the mutant does not retain the ability to bind to metal ions.

  In other embodiments, the transferrin portion of the transferrin fusion protein comprises a recombinant human serum transferrin mutant having a mutation in Lys206 or His207 of SEQ ID NO: 3, wherein the mutant is a wild type human Has a stronger binding affinity for metal ions than serum transferrin (see US Pat. No. 5,986,067, which is incorporated herein by reference in its entirety). In an alternative embodiment, the transferrin portion of the transferrin fusion protein comprises a recombinant human serum transferrin mutant having a mutation in Lys206 or His207 of SEQ ID NO: 3, wherein the mutant is a wild type It has a binding affinity for metal ions that is weaker than human serum transferrin. In a further embodiment, the transferrin portion of the transferrin fusion protein comprises a recombinant human serum transferrin mutant having a mutation in Lys206 or His207 of SEQ ID NO: 3, wherein the mutant binds to a metal ion. do not do.

  Any technique can be used to produce the transferrin fusion protein of the present invention as long as it is available. Examples of such techniques include, but are not limited to, generally available molecular techniques. For example, the technology disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989. When nucleotide substitutions are performed using techniques that are well known in the art for site-directed mutagenesis, the encoded amino acid change is mild, ie, conservative amino acid substitutions are preferred, but in particular Tf Other non-conservative substitutions are similarly considered when producing modified transferrin portions of the fusion protein, eg, modified Tf proteins that exhibit reduced glycosylation, reduced iron binding, and the like. Specifically contemplated amino acid substitutions include short deletions or insertions, typically deletions or insertions of 1 to about 30 amino acids; insertions between transferrin domains; short amino or carboxyl terminal extensions, such as An amino-terminal methionine residue, or a short linker peptide of less than 50, 40, 30, 20 or 10 residues between transferrin domains, or a transferrin protein and a therapeutic protein or peptide, or a short extension that facilitates purification, For example, a poly-histidine tract, an antigenic epitope, or a linkage with a binding domain.

  Examples of conservative amino acid substitutions are substitutions made in a homogeneous group, such as basic amino acids (eg, arginine, lysine, histidine), acidic amino acids (eg, glutamic acid and aspartic acid), polar amino acids (eg, , Glutamine and asparagine), hydrophobic amino acids (eg leucine, isoleucine, valine), aromatic amino acids (eg phenylalanine, tryptophan, tyrosine), and low molecular weight amino acids (eg glycine, alanine, serine, threonine, methionine) A substitution made within a group.

  Non-conservative substitutions include substituting amino acids belonging to one group with amino acids belonging to another group. For example, non-conservative substitutions include substitution of polar amino acids with hydrophobic amino acids. For a general description of nucleotide substitution, see, eg, Ford et al. (1991), Prot. Exp. Pur. 2: See 95-107. No binding to iron or they are reduced, there is no binding to the Tf receptor of the fusion protein or they are reduced and / or there is no glycosylation or they are reduced Non-conservative substitutions, deletions and insertions are particularly useful for producing the Tf fusion proteins of the invention.

  Iron binding and / or receptor binding may be reduced or destroyed by mutation, such as the N domain residues Asp63, Tyr95, Tyr188, His249 of Tf of SEQ ID NO: 3 and Deletion, substitution or insertion into one or more amino acid residues corresponding to one or more of the C domain residues Asp392, Tyr426, Tyr514 and / or His585. Iron binding may also be affected by mutations to amino acids Lys206, His207 or Arg632 of SEQ ID NO: 3. Carbonate bonds may be reduced or destroyed by mutations, such as Nf residues Thr120, Arg124, Ala126, Gly127 and / or C domain residues Thr452, Arg456 of Tf of SEQ ID NO: 3. , Deletions, substitutions or insertions into amino acid residues corresponding to one or more of Ala458 and / or Gly459. Reduction or destruction of carbonate bonds can adversely affect iron and / or receptor binding.

  Binding to the Tf receptor may be reduced or disrupted by mutation, such as amino acid residues corresponding to one or more of the N domain residues of Tf described above for iron binding. Examples include group deletion, substitution or insertion into them.

  As discussed above, glycosylation is an amino acid residue corresponding to one or more of the C domain residues of Tf around the NXS / T site corresponding to C domain residues N413 and / or N611. May be reduced or prevented by mutations such as deletions, substitutions or insertions into them (see US Pat. No. 5,986,067). For example, N413 and / or N611 may be mutated to a Glu residue.

  In cases where the Tf fusion protein of the invention has not been modified to prevent glycosylation, iron binding, carbonate binding and / or receptor binding, the glycosylation, iron and / or carbonate ions are derived from the fusion protein. It can be detached or cleaved. For example, available deglycosylases may be used to cleave glycosylated residues, particularly sugar residues attached to the Tf moiety, from the fusion protein, or using yeast lacking glycosylases. Glycosylation may be prevented and / or recombinant cells may be grown in the presence of a substance that prevents glycosylation (eg, tunicamycin).

  Alternatively, the carbohydrate on the fusion protein may be reduced, or it may be completely removed enzymatically by treating the fusion protein with deglycosylase. 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.

  Nevertheless, in certain circumstances it may be preferred for oral delivery that the Tf portion of the fusion protein is fully glycosylated.

  Additional mutations may be made in Tf to alter the three-dimensional structure of Tf, for example, by modification to the hinge region to prevent conformational changes necessary for iron binding and Tf receptor recognition. is there. For example, the mutation is at amino acid residues 94-96, 245-247 and / or 316-318 of the N domain plus amino acid residues 425-427, 581-582 and / or 652-658 of the C domain, or It may be made around them. In addition, mutations may be made at or around the flanking regions of these sites to alter the structure and function of Tf.

  In one form of the invention, the transferrin fusion protein extends the half-life or bioavailability of the therapeutic protein, or in addition, optionally places the therapeutic protein inside the cell and / or the blood brain barrier. It can function as a carrier protein for passing and transporting. In an alternative embodiment, the transferrin fusion protein comprises a modified transferrin molecule, wherein the transferrin does not retain the ability to cross the blood brain barrier.

  In other embodiments, the transferrin fusion protein comprises a modified transferrin molecule, wherein the transferrin molecule retains the ability to bind to a transferrin receptor and transports a therapeutic peptide inside the cell. In an alternative embodiment, the transferrin fusion protein comprises a modified transferrin molecule, wherein the transferrin molecule does not retain the ability to bind to the transferrin receptor and transports the therapeutic peptide inside the cell.

  In a further embodiment, the transferrin fusion protein comprises a modified transferrin molecule, wherein the transferrin molecule retains the ability to bind to a transferrin receptor, transports a therapeutic peptide inside the cell, and crosses the blood brain barrier. Hold the ability. In an alternative embodiment, the transferrin fusion protein comprises 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 Transports therapeutic peptides inside.

Modified Transferrin Fusion Proteins Fusions of proteins of the invention may comprise one or more GLP-1 therapeutic proteins, such as GLP-1 or analogs thereof, linked to the N-terminus and / or C-terminus of the Tf protein. It may contain a copy. In some embodiments, the GLP-1 therapeutic protein is bound to both the N and C termini of the Tf protein, and the fusion protein has GLP-1 at either or both of the Tf termini. One or more equivalents of a therapeutic protein or polypeptide may be included. In other embodiments, the GLP-1 therapeutic protein or polypeptide is inserted into a known domain of the Tf protein, eg, into one or more Tf loops (Ali et al. (1999) J. Biol. Chem. 274 (34): 24066-24073). Indeed, the GLP-1 therapeutic protein or polypeptide may be inserted into all five loops of transferrin, thereby creating a pentavalent molecule with high affinity for the GLP-1 receptor. Can do. In other embodiments, the GLP-1 therapeutic protein or polypeptide is inserted between the N and C domains of Tf. Alternatively, the GLP-1 therapeutic protein or polypeptide is inserted somewhere in the transferrin molecule.

  The fusion proteins of the invention include the use of a GLP-2 linker and / or a substantially non-helical amino acid linker to separate the transferrin and GLP-1 portions of the protein. For example, in one embodiment, GLP-1 is linked to the N-terminus of Tf via a linker peptide. In other embodiments, GLP-1 is attached to the C-terminus of Tf via a linker peptide. As will be appreciated by those skilled in the art, the present invention contemplates linkers placed in various ways in the molecule to separate GLP-1 and transferrin.

  In general, the transferrin fusion protein of the invention may have a region derived from one modified transferrin and one GLP-1 therapeutic protein region. However, the transferrin fusion protein of the present invention may be prepared using a plurality of regions of each protein. Similarly, more than one GLP-1 therapeutic protein may be used to make the transferrin fusion protein of the invention, thereby producing a multifunctional modified Tf fusion protein.

  In one embodiment, the transferrin fusion protein of the invention comprises a GLP-1 therapeutic protein fused to a transferrin molecule, or portion thereof. In other embodiments, the transferrin fusion protein of the invention comprises a GLP-1 therapeutic protein or polypeptide fused to the N-terminus of the transferrin molecule. In an alternative embodiment, the transferrin fusion protein of the invention comprises a GLP-1 therapeutic protein or polypeptide fused to the C-terminus of the transferrin molecule. In a further embodiment, the transferrin fusion protein of the invention comprises a transferrin molecule fused to the N-terminus of a GLP-1 therapeutic protein or polypeptide. In an alternative embodiment, the transferrin fusion protein of the invention comprises a transferrin molecule fused to the C-terminus of a GLP-1 therapeutic protein or polypeptide.

  In other embodiments, the transferrin fusion protein of the invention comprises a GLP-1 therapeutic protein fused to both the N-terminus and C-terminus of the modified transferrin. In an alternative embodiment, such N-terminal and C-terminal fusion therapeutic proteins are used to treat or prevent diseases or disorders known in the art to be treatable with GLP-1. Including one or more GLP-1 therapeutic proteins and one or more different therapeutic proteins. In other embodiments, the therapeutic protein fused at the N-terminus and C-terminus is one or more that can be used to treat or prevent a disease or disorder known in the art to generally develop simultaneously in a patient. More GLP-1 therapeutic proteins and one or more different therapeutic proteins.

  In addition to the modified transferrin fusion protein of the present invention in which the modified transferrin portion is fused to the N-terminus and / or C-terminus of the GLP-1 therapeutic protein portion, the transferrin fusion protein of the present invention also comprises a subject GLP- One therapeutic protein or peptide (eg, a therapeutic protein or peptide disclosed herein, or a fragment or variant thereof) may be produced by insertion into the internal region of the modified transferrin. The internal region of the modified transferrin includes, but is not limited to, an iron binding site, a hinge region, a bicarbonate binding site, or a receptor binding domain.

  There are numerous loops or turns within the protein sequence of the modified transferrin molecule that are stabilized by disulfide bonds. These loops are peptides that have therapeutic activity, particularly those that require secondary structure to become functional, or therapeutic protein insertions or to generate modified transferrin molecules with specific biological activity. Useful for internal fusion.

  If the therapeutic protein is inserted into or replaced in at least one loop of the Tf molecule, the insertion can be made either in the loop region exposed on the surface as well as in other regions of Tf. For example, insertions may be made in a loop containing Tf amino acids 32-33, 74-75, 256-257, 279-280 and 288-289 (Ali et al., Supra) (see FIG. 3). As noted above, insertions may also be made in other regions of Tf, for example, sites for iron and bicarbonate binding, hinge regions and receptor binding domains as described in more detail below. May be made. In addition, loops in the Tf protein sequence that can be modified / replaced to insert proteins or peptides may be used to develop a screenable library of random peptide inserts. Any technique for producing nucleic acid inserts can be used to generate peptide libraries containing available phage and bacterial display systems prior to cloning into the Tf domain and / or fusion to the Tf terminus. is there.

  The N-terminus of Tf is free and faces outward from the molecular body. Therefore, a preferred embodiment may be a protein or peptide fusion at the N-terminus. Such fusions may include GLP-2 and / or a substantially non-helical linker to separate the GLP-1 therapeutic protein from Tf.

  The C-terminus of Tf appears to be protected with 6 amino acids deeper embedded by disulfide bonds from the C-terminus. In human Tf, the C-terminal amino acid is proline, depending on how the proline is oriented, either to direct the fusion outward or to the molecular body. Proline is also present near the N-terminus. In one form of the invention, the proline at the N and / or C terminus can also be altered. In other forms of the invention, the C-terminal disulfide bond may be removed to open the C-terminal.

In GLP-1 therapeutic proteins and peptide mammals, the glucagon gene encodes the precursor proglucagon and is processed to produce a variety of tissue-determined peptide products. Specifically, tissue-specific processing of proglucagon produces glucagon in the brain, and in the intestine, glicentin, oxyntomodulin, and glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP- 2) is generated. The organization of these peptides in the proglucagon precursor has been elucidated by the molecular clonin of cDNA from rat, hamster and bovine pancreas. Analysis of proglucagon precursors shows that the GLP-1 and GLP-2 peptides are separated from each other by a spacer or intervening peptide.

  Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that regulates insulin secretion belonging to the so-called enteroinsular axis. Enteroinsular axis refers to a group of hormones released from the gastrointestinal mucosa in response to the presence and absorption of nutrients in the gastrointestinal tract, which promotes early enhanced insulin release. Incretin action, which is an enhancing action on insulin secretion, is probably essential for normal glucose tolerance. Since GLP-1 is involved in incretin action, GLP-1 is a physiologically important insulinotropic hormone.

  GLP-1 is a product of proglucagon (Bell et al., Nature, 1983, 304: 368-371). GLP-1 is an enteroendocrine cell and is synthesized in two major molecular forms as GLP-1 (7-36) amide and GLP-1 (7-37). For the first time in the early 1980s, these peptides were identified, followed by the cloning and gene for proglucagon.

Initial studies conducted on the full-length peptides GLP-1 (1-37) and GLP-1 (1-36 ^amide ) concluded that larger GLP-1 molecules lack biological activity. It has been. In 1987, three separate research groups demonstrated that removal of the first 6 amino acids resulted in a GLP-1 molecule with enhanced biological activity.

式中、ＸはＧＬＰ−１（７〜３６^アミド）ではＮＨ₂であり、ＸはＧＬＰ−１（７〜３７）ではＧｌｙである。従って、ＧＬＰ−１（７〜３６）および（７〜３７）のＡ８は、配列番号６のＡ２に相当し、ＧＬＰ−１（７〜３６）および（７〜３７）のＫ３４は、Ｋ２８に相当する、等々である。 The amino acid sequence of GLP-1 was disclosed by Schmidt et al. (1985 Diabetologia 28 704-707). Human GLP-1 is a peptide consisting of 37 amino acid residues derived from preproglucagon synthesized in the L cell, pancreas and brain of the terminal ileum. Processing of preproglucagon into GLP-1 (7-36 ^amide ), GLP-1 (7-37) and GLP-2 occurs primarily in L cells. The amino acid sequences of GLP-1 (7-36 ^amide ) and GLP-1 (7-37) are as follows (SEQ ID NO: 6):

In the formula, X is NH ₂ in GLP-1 (7-36 ^amide ), and X is Gly in GLP-1 (7-37). Therefore, A8 of GLP-1 (7-36) and (7-37) corresponds to A2 of SEQ ID NO: 6, and K34 of GLP-1 (7-36) and (7-37) corresponds to K28. Do, and so on.

  GLP-1-like molecules have anti-diabetic activity in human subjects with Type II (Non-Insulin Dependent Diabetes Mellitus (NIDDM)) diabetes, and in some cases with Type I diabetes. Treatment with GLP-1 is more effective than insulin or sulfonylureas because only high blood glucose levels trigger activities such as increased insulin secretion and biosynthesis, decreased glucagon secretion, and slow gastric emptying. It may provide a much safer treatment. With appropriate GLP-1 treatment, the patient's postprandial blood glucose levels can be transferred to normal levels. There are also reports suggesting that GLP-1-like molecules have the ability to preserve and even restore pancreatic beta cell function in type II patients.

  Any GLP-1 sequence may be used to make a Tf fusion protein of the invention comprising GLP-1 (7-35), GLP-1 (7-36), and GLP-1 (7-37) . GLP-1 also has a strong action on the digestive tract. GLP-1 strongly inhibits pentagastrin-induced and diet-induced gastric acid secretion when injected in physiological amounts (Schjordager et al., Dig. Dis. Sci. 1989, 35: 703-708; Wettergren et al., Dig Dis Sci 1993; 38: 665-673). GLP-1 also suppresses gastric emptying rate and pancreatic enzyme secretion (Wettergren et al., Dig Dis Sci 1993; 38: 665-673). In humans, perfusion of solutions containing carbohydrates or lipids into the ileum may cause similar inhibitory effects on secretion and motility in the stomach and pancreas (Layer et al., Dig Dis Sci 1995, 40: 1074-1082; Layer et al., Digestion 1993, 54: 385-38). Concomitantly, GLP-1 secretion is significantly stimulated, and it has been speculated that GLP-1 may be at least partially involved in this so-called “ileal-brake” action. (Layer et al., Digestion 1993; 54: 385-38). Indeed, recent studies suggest that physiologically, the ileal braking action of GLP-1 may be more important than its action on the islets. Thus, in dose-response studies, GLP-1 affects gastric emptying rates at least at the minimum rate required to affect islet secretion (Nuck et al., Gut 1995; 37 (Suppl .2): A124).

  GLP-1 appears to have an effect on food intake. In rats, intracerebroventricular administration of GLP-1 significantly reduces food intake (Schick et al. In Ditschenet et al. (Eds), Obesity in Europe, John Libbey & Company Ltd, 1994; pages 363-367; Turton et al., Nature 1996, 379: 379. ~ 72). This effect appears to be highly specific. Thus, GLP-1 (PG72-107) amide extended at the N-terminus is inactive and the appropriate dose of GLP-1 antagonist exendin 9-39 abolishes the action of GLP-1 (Tang- Christensen et al., Am. J. Physiol., 1996, 271 (4 Pt 2): R848-56). In rats, acute peripheral administration of GLP-1 does not rapidly reduce food intake (Tang-Christensen et al., Am. J. Physiol., 1996, 271 (4Pt2): R848-56; Turton et al., Nature 1996, 379: 69-72). However, there remains the possibility that GLP-1 secreted from intestinal L cells can act as a satiety signal.

  In diabetic patients, maintaining the insulin secretion stimulating action of GLP-1 and the action of GLP-1 on the gastrointestinal tract (Willms et al., Diabetologia 1994; 37, suppl. 1: A118), thereby reducing diet-induced glucose surge It can help control, but more importantly, it can affect food intake. Intravenous administration of GLP-1 at 4 ng / kg / min for 1 week has been shown to dramatically improve blood glucose control without causing significant side effects in NIDDM patients (Larsen et al., Diabetes). 1996; 45, suppl.2: 233A).

  In the treatment of type I and type II diabetes and obesity, GLP-1 / transferrin fusion proteins comprising at least one GLP-1 analog and fragments thereof are useful.

  Administration of GLP-1 and fragments thereof may also be useful for treating congestive heart failure and non-alcoholic, non-fatty liver disease.

  As used herein, the term “GLP-1 molecule” means GLP-1, a GLP-1 analog, or a GLP-1 derivative.

The term “GLP-1 analog” as used herein is defined as a molecule having one or more amino acid substitutions, deletions, inversions or additions compared to GLP-1. Many GLP-1 analogs are known in the art, eg, GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36), Val ⁸ -GLP- ^{1 (7~37), Gly 8 -GLP} -1 (7~37), Ser 8 -GLP-1 (7~37), Gln 9 -GLP1 (7~37), D-Gln 9 -GLP-1 ( 7-37), Thr ¹⁶ -Lys ¹⁸ -GLP-1 (7-37), and Lys ¹⁸ -GLP-1 (7-37) (see SEQ ID NO: 96). Preferred analogs include GLP-1 (7-37; A8X, K34X), where X is the native GLP-1 sequence or any amino acid other than GLP-1 (7-37; A8G, K34A) is there. Other analogs include the dipeptidyl-peptidase resistant form of GLP-1, where the N-terminus of this peptide is protected. Such analogs include, but are not limited to, GLP-1, including additional amino acids, eg, added to the N-terminus or N-terminal amino acids (GLP-1 (7-36) or A histidine residue substituted with amino acids 7 or 8 in GLP-1 (7-37), which correspond to amino acids 1 or 2 of SEQ ID NO: 6. In these analogs, the N-terminus may contain the following 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 (refer to SEQ ID NOs: 77 to 82, respectively), His-X-Ala-Glu, His-X-Gly-Glu, His-X-Ser-Glu (where X is Any amino acid). US Pat. No. 5,118,666 describes examples of GLP-1 analogs, such as GLP-1 (7-34) and GLP-1 (7-35).

  In other embodiments, the GLP-1 peptide has a mutation that renders it less susceptible to cleavage by neutral endopeptidase (NEP). The inventors of the present invention have found that BRX0585 (SEQ ID NO: 12) is less susceptible to NEP cleavage compared to unfused GLP-1 protein.

  Several mutations have the potential to prevent NEP cleavage of GLP-1. For example, mutations at or near the N-terminal side of hydrophobic amino acids can reduce NEP's ability to cleave GLP-1. GLP-1 (7-36) and GLP-1 (7-37) are rich in hydrophobic amino acids in E27-L32 (corresponding to E21-L26 of SEQ ID NO: 6). The sites most susceptible to NEP cleavage in GLP-1 are E27 ↓ F28 (corresponding to E21F22 and W25 ↓ L26 of SEQ ID NO: 6) and W31 ↓ L32 (corresponding to E21 to L26 of SEQ ID NO: 6).

  Of note, however, residues F28 and L32 may be important for receptor binding. Loss of receptor binding is acceptable if GLP-1's ability to activate the receptor is maintained. For example, A30Q appears to cause loss of receptor binding, but receptor activation results in an increase. Thus, the present invention involves mutating one or more hydrophobic amino acids and / or amino acids in the E27-L32 region to prevent NEP cleavage and at the same time maintain GLP-1 receptor activation. The present invention also prevents NEP cleavage and at the same time maintains GLP-1 receptor activation in amino acids near hydrophobic amino acids (ie, preferably within 5 amino acids) and / or E27-L32 It also includes mutating in the amino acids of the region. For example, one or more mutations selected from the group consisting of E27X, F28X, I29X, A30Q, W31X, L32X, and / or V33X can be used to prevent NEP cleavage, where X is Any amino acid except glycine (G), proline (P), or cystine (C). In one embodiment, such a mutation is A30Q (corresponding to A24Q of SEQ ID NO: 6). Hupe-Sodmann et al., 1995, Regulatory Peptides. 58: 149-156 and Hupe-Sodmann et al., 1997, Peptides. 18: 625-632. In other embodiments, the invention includes the use of the C-terminus of exendin 4 or a derivative thereof (CEx; SEQ ID NO: 15) to reduce sensitivity to NEP.

  The term “GLP-1 derivative” has the amino acid sequence of GLP-1 or a GLP-1 analog, but in addition to one or more of its amino acid side groups, α-carbon atoms, terminal amino groups, or terminal Defined as a molecule with a chemical modification of the carboxylic acid group. Chemical modifications include, but are not limited to, adding chemical moieties, forming new bonds, and removing chemical moieties.

  As used herein, the term “GLP-1 related compound” means any compound that falls within the definition of GLP-1, a GLP-1 analog, or a GLP-1 derivative.

  WO 91/11457 describes analogs of active GLP-1 peptides 7-34, 7-35, 7-36, and 7-37, which may also be useful as GLP-1 components.

EP 0708179-A2 (Eli Lilly & Co.) GLP-1 containing an N-terminal imidazole group and optionally containing an unbranched C ₆ -C ₁₀ acyl group attached to a lysine residue at position 34. Analogues and derivatives are described.

  EP0699686-A2 (Eli Lilly & Company) describes a fragment in which a specific N-terminus of GLP-1 has been truncated, which is reported to be biologically active.

  U.S. Pat. No. 5,545,618 substantially comprises GLP-1 (7-34), GLP1 (7-35), GLP-1 (GLP-1 (GLP-1) having at least one modification selected from the group consisting of 7-36) or GLP-1 (7-37), or their amide forms, and pharmaceutically acceptable salts thereof, GLP-1 molecules are described: (a) position 26 and / or position 34 In place of lysine, substitution with glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, arginine, or D-lysine; or at position 36, arginine Instead of glycine, serine, cysteine, threonine, asparagine, glutamine, Substitution with syn, alanine, valine, isoleucine, leucine, methionine, phenylalanine, lysine, or D-arginine; (b) substitution with an amino acid having oxidation resistance instead of tryptophan at position 31; (c) At position 16: tyrosine instead of valine; lysine instead of serine at position 18; aspartic acid instead of glutamic acid at position 21; serine instead of glycine at position 22 Arginine instead of glutamine at position 23; arginine instead of alanine at position 24; and glutamine instead of lysine at position 26; and (d) at least one substitution: Glyci instead of alanine , Serine or cysteine; instead of glutamic acid at position 9, aspartic acid, glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine or phenylalanine; Instead, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine or phenylalanine; and at position 15, instead of aspartic acid, glutamic acid; and (e) instead of histidine at position 7 Glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine or Substitution in the D- or N-acylated or alkylated form of phenylalanine, or histidine (see SEQ ID NOs: 83-87, respectively); where the substitutions are (a), (b), (d) and ( When e), the substituted amino acid may be in the D form and the amino acid substituted at the 7-position may be in the N-acylated or N-alkylated form.

  US Pat. No. 5,118,666 describes GLP-1 molecules having insulin secretion stimulating activity. Such molecules are selected from the group consisting of peptides having the following amino acid sequences: 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 derivatives of said peptides Wherein the peptide is selected from the group consisting of: A pharmaceutically acceptable acid addition salt of the peptide; a pharmaceutically acceptable carboxylate salt of the peptide; a pharmaceutically acceptable lower alkyl ester of the peptide; and an amide, a lower alkylamide, and a lower dialkylamide. A pharmaceutically acceptable amide of the peptide.

US Pat. No. 6,277,819 teaches a method of reducing mortality and morbidity following myocardial infarction, which method involves the patient in GLP-1, GLP-1 analogs, and GLP-1 Administering a derivative. The GLP-1 analog is represented by the following structural formula (SEQ ID NO: 9): R ₁ -X ₁ -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu- _{X 2 -Gly-Gln-Ala-} Ala-Lys-X 3 -Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R 2, and acceptable salts their pharmaceutically, wherein: R ₁ is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine,. X ₁ is selected from the group consisting of beta-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine; X ₁ is from Ala, Gly, Val, Thr, Ile, and alpha-methyl-Ala X ₂ is selected from the group consisting of Glu, Gin, Ala, Thr, Ser, and Gly; X ₃ is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly R ₂ is selected from the group consisting of NH ₂ and Gly-OH; provided that the GLP-1 analog has an isoelectric point in the range of about 6.0 to about 9.0. Furthermore, when R ₁ is His, X ₁ is Ala, X ₂ is Glu, and X ₃ is Glu, R ₂ must be NH _2. On the condition.

Ritzel et al. (Journal of Endocrinology, 1998, 159: 93-102) describe a GLP-1 analog, [Ser ⁸ ] GLP-1, where the second amino acid at the N-terminus is alanine. It has been replaced with serine. This modification did not impair the insulinotropic effect of the peptide, but produced an analog with higher plasma stability compared to GLP-1.

  US Pat. No. 6,429,197 teaches treatment with GLP-1 after acute stroke or bleeding, preferably intravenous administration, which allows ideal treatment because it is insulin secretion. , Increases assimilation in the brain, suppresses glucagon, enhances insulin effectiveness, and reduces normoglycemia or mild hypoglycemia without the risk of severe hypoglycemia or other adverse side effects This is to provide a means to maintain. The present invention optimizes insulin secretion, enhances the effectiveness of insulin by inhibiting glucagon antagonism, and mild hypoglycemia without the risk of normoglycemia or severe hypoglycemia A method of treating ischemic or reperfused brain after acute cerebral infarction or hemorrhage using GLP-1 or a biologically active analog thereof is provided.

  US Pat. No. 6,277,819 provides a method of reducing mortality and morbidity after myocardial infarction, which can be used in patients who need it to GLP-1, GLP-1 like Administration of a compound selected from the group consisting of the body, GLP-1 derivatives, and pharmaceutically acceptable salts thereof in a dosage effective to normalize blood glucose.

  U.S. Pat. Nos. 6,191,102 and 6,583,111 describe a method for reducing the weight of a patient in need of weight loss, which involves the patient glucagon-like peptide-1 ( GLP-1), glucagon-like peptide analogue (GLP-1 analogue), glucagon-like peptide derivative (GLP-1 derivative), or a composition comprising a pharmaceutically acceptable salt thereof, to cause weight loss A sufficient dose is made by administering for a period effective to cause weight loss, said period being at least 4 weeks.

  GLP-1 has sufficient activity 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 al., 1995, Diabetes 44: 1126-1131). Unfortunately, many of GLP-1 and its analogs have a short plasma half-life in humans (Orskov et al., Diabetes 1993; 42: 658-661). Accordingly, it is an object of the present invention to provide a transferrin fusion protein comprising GLP-1 or an analog thereof having a sustained profile of action compared to GLP-1 (7-37). A further object of the present invention is to provide derivatives of GLP-1 having lower clearance than GLP-1 (7-37), and analogs thereof. Moreover, it is an object of the present invention to provide a pharmaceutical composition comprising a GLP-1 / transferrin fusion protein or a GLP-1 analog / transferrin fusion protein with improved stability. In addition, the present invention is for treating diseases such as, but not limited to, type II diabetes, obesity, metabolic syndrome, pre-diabetes, non-fatty liver disease or congestive heart failure, and the aforementioned diseases Use of a GLP-1 / transferrin fusion protein or a GLP-1 analog / transferrin fusion protein.

  Any GLP-1 therapeutic molecule can be used as a fusion partner with Tf according to the methods and compositions of the invention. As used herein, a “therapeutic molecule” is GLP-1 or a variant or analog thereof that can exert a beneficial biological effect in vitro or in vivo. For example, a beneficial effect associated with a medical condition includes any effect that is beneficial to the treated subject, such as prevention of disease, stabilization of disease, disease to produce a beneficial effect in the treated subject. Minor mitigation or alleviation, or adjustment, mitigation or healing of fundamental defects.

  The modified transferrin fusion proteins of the present invention include at least a fragment or variant of a therapeutic protein and at least a modified fragment or variant of serum transferrin, which are preferably linked together by genetic fusion. Yes.

  In a further embodiment, the modified transferrin fusion protein of the invention may comprise at least a fragment or variant of a GLP-1 therapeutic protein. In further embodiments, the transferrin fusion protein may comprise peptide fragments or peptide variants of the protein, wherein these variants or fragments retain at least one biological or therapeutic activity. The transferrin fusion protein may comprise a GLP-1 therapeutic protein, wherein the GLP-1 therapeutic protein is at least about 4, at least about 5, at least about 6, at least about 7, at least about 8 in length. At least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least A peptide fragment of about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, or at least about 31 amino acids or Petti Are possible variants, which are fused to the N and / or C-terminus, inserted into the modified transferrin, also be inserted into the loop of a modified transferrin.

  The modified transferrin fusion protein of the present invention may contain one or more peptides. Increasing the number of peptides may enhance the function of the peptide fused to transferrin and the function of the entire transferrin fusion protein. The peptide may be used to make a bifunctional or multifunctional fusion protein, by including a peptide or protein domain with multiple functions. For example, a multifunctional fusion protein can be made using a GLP-1 therapeutic protein and a second protein that targets the fusion protein to one or more specific targets.

  The transferrin fusions of the invention can include a linker that links transferrin to the GLP-1 therapeutic peptide. Preferably, the linker is GLP-2 or one or more copies of the sequence PEAPTD (SEQ ID NO: 13). There is one or more GLP-1 peptides at the amino terminus of the fusion protein.

  In other embodiments, the modified transferrin fusion molecule comprises a GLP-1 therapeutic protein moiety, eg, a polypeptide having one or more residues deleted from the amino terminus of the amino acid sequence, for example. It may also be a fragment of a GLP-1 therapeutic protein containing its full-length protein.

  In other embodiments, the modified transferrin fusion molecule comprises a GLP-1 therapeutic protein portion that is only a polypeptide having, for example, one or more residues deleted from the carboxy terminus of the amino acid sequence. Rather, it may be a fragment of a GLP-1 therapeutic protein containing its full-length protein.

  In other embodiments, the modified transferrin fusion molecule comprises a GLP-1 therapeutic protein moiety that may have one or more amino acids deleted from both the amino and carboxy termini. Good.

  In other embodiments, the modified transferrin fusion molecule comprises a GLP-1 therapeutic protein moiety that is at least about 80 with the reference GLP-1 therapeutic protein or fragment thereof set forth herein. %, 85%, 90%, 95%, 96%, 97%, 98% or 99% are identical. In a further embodiment, the transferrin fusion molecule comprises a GLP-1 therapeutic protein portion that is at least about 80 and a reference polypeptide having an amino acid sequence with deletions at the N and C termini as described above. %, 85%, 90%, 95%, 96%, 97%, 98% or 99% are identical.

  In other embodiments, the modified transferrin fusion molecule comprises a therapeutic protein moiety that is at least about 80%, 85%, 90%, for example, with a native or wild type amino acid sequence of a GLP-1 therapeutic protein. %, 95%, 96%, 97%, 98%, 99% or 100% identical. Also provided are fragments of these polypeptides.

  GLP-1 therapeutic proteins corresponding to the GLP-1 therapeutic protein portion of the modified transferrin fusion protein of the invention, such as cell surface and secreted proteins, are modified by linking one or more oligosaccharide groups Can do. Modifications termed glycosylation can significantly affect the physical properties of proteins and can be important for protein stability, secretion and localization. Glycosylation occurs at specific locations along the polypeptide backbone. In general, there are two main types of glycosylation: glycosylation characterized by O-linked oligosaccharides, where the oligosaccharides bind to serine or threonine residues; and N-linked Glycosylation characterized by oligosaccharides, in which case the oligosaccharides bind to asparagine residues in the Asn-X-Ser / Thr sequence, where X can be an amino acid other than proline. Variables such as protein structure and cell type affect the number and nature of carbohydrate units in the chain at various glycosylation sites. Also, within a given cell type, glycosylated isomers often occur at the same site. For example, it is glycosylated with several types of human interferon.

  The therapeutic proteins corresponding to the therapeutic protein portions of the transferrin fusion proteins of the present invention, and analogs and variants thereof, may result from manipulation of nucleic acid sequences by host cells expressing them or other conditions for their expression. May be modified to alter glycosylation at one or more sites. For example, glycosylated isomers can be obtained by deactivating the glycosylation site or introducing a glycosylation site, e.g. by substitution or deletion of amino acid residues, e.g. by replacing asparagine with glutamine. Recombinant proteins that can be produced or are not glycosylated can be produced by expressing the proteins in host cells (eg, glycosylation deficient yeast) that are not expected to glycosylate them. These approaches are known in the art.

  Therapeutic proteins, and their nucleic acid sequences, are well known in the art and are available in public databases such as Chemical Abstracts Services Databases (eg CAS Registry), GenBank and GenSeq. is there. The registration numbers and sequences referred to below are hereby incorporated by reference in their entirety.

  In other embodiments, the transferrin fusion protein of the invention exhibits a therapeutic and / or biological activity corresponding to the therapeutic and / or biological activity of a therapeutic protein described elsewhere in this application. It may have. In a further embodiment, the protein portion having therapeutic activity of the transferrin fusion protein of the invention is a fragment or variant of the reference sequence cited herein.

  The present invention is further directed to a modified Tf fusion protein comprising a fragment of the GLP-1 therapeutic protein described herein. Even if one or more amino acids are deleted from the N-terminus of the protein and one or more of the biological functions of the therapeutic protein moiety are altered or lost, other therapeutic activities and / or Or functional activity (eg, biological activity, ability to multimerize, ability to bind ligand) may still be retained. For example, the ability of a polypeptide with a deletion at the N-terminus to induce and / or bind to an antibody that recognizes a complete or mature form of the polypeptide generally remains removed from the N-terminus. It is expected that a group will be retained if it is less than half of the residues of the complete polypeptide. Whether a particular polypeptide lacking the N-terminal residue of the complete polypeptide retains such immunological activity can be determined by routine methods described herein, Other than those known in the art. It cannot be said that a mutant lacking many N-terminal amino acid residues may retain some biological or immunogenic activity. In practice, peptides composed of as few as six amino acid residues can often elicit an immune response.

  In addition, as described above, one or more amino acids are deleted from the N-terminus or C-terminus of the therapeutic protein, and one or more of the biological functions of the protein are altered or lost. Even so, other functional activities (eg, biological activity, ability to multimerize, ability to bind ligand) and / or therapeutic activity may still be retained. For example, the ability of a polypeptide with a deletion at the C-terminus to induce and / or bind to an antibody that recognizes a complete or mature polypeptide form is generally a complete or mature polypeptide. If fewer than half of the residues are removed from the C-terminus, they are expected to be retained. Whether a particular polypeptide lacking a N-terminal and / or C-terminal residue of a reference polypeptide retains therapeutic activity can be determined by routine methods and / or methods described herein. It can be easily measured by other methods known in the art.

  A peptide fragment of a GLP-1 therapeutic protein comprises or comprises an amino acid sequence that exhibits therapeutic and / or functional activity (eg, biological activity) of a polypeptide sequence of a therapeutic protein whose amino acid sequence is a fragment. It may be a fragment.

  A peptide fragment of a GLP-1 therapeutic protein may include only the N-terminus and C-terminus of the protein, ie, in this case, the central portion of the therapeutic protein is deleted. Alternatively, such peptide fragments may include portions that are not adjacent to the central portion of the therapeutic protein and / or may include portions that are adjacent to the central portion of the therapeutic protein.

  Other polypeptide fragments are biologically active fragments. Biologically active fragments are those that exhibit activity that is similar but not necessarily identical to that of the therapeutic protein used in the present invention. The biological activity of such a fragment may include an improved desired activity or may include a reduced undesirable activity.

  In general, protein variants are generally very similar to the amino acid sequence of the therapeutic protein corresponding to the GLP-1 therapeutic protein portion of the transferrin fusion protein of the invention and are identical in many regions. . In addition, nucleic acids encoding these variants are also encompassed by the present invention.

  Further therapeutic polypeptides that can be used in the present invention are obtained by polynucleotides that hybridize under stringent hybridization conditions known to those skilled in the art to the complement of a nucleic acid molecule encoding the amino acid sequence of a GLP-1 therapeutic protein. The encoded polypeptide. (For example, Ausubel, FM et al., 1989 Current protocol in Molecular Biology, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, Wis.) See). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  If a polypeptide has an amino acid sequence that is at least 95% “identical” to the query amino acid sequence of the present invention, the polypeptide sequence of the agenda will have no more than 5 amino acid changes per 100 amino acids of the query amino acid sequence. It may contain, but otherwise it is identical to the query sequence. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence, insertions, deletions or substitutions of other amino acids that are allowed in the sequence of the subject are 5 amino acid residues. % Or less. These reference sequence changes may occur at the amino or carboxy terminal position of the reference amino acid sequence or may occur somewhere between the amino or carboxy terminal positions, in the latter case The positions are either interspersed individually between residues in the reference sequence, or interspersed with one or more contig groups within the reference sequence.

  In practice, any particular polypeptide is present, for example, at least in the amino acid sequence of the transferrin fusion protein of the invention or fragment thereof (eg, the therapeutic protein portion of the transferrin fusion protein or the transferrin portion of the transferrin fusion protein). Whether about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% are identical can be determined conventionally using known computer programs. A preferred method (also referred to as global sequence alignment) for determining the best overall match between a query sequence (sequence of the present invention) and an agenda sequence is Brufiag et al. (Comp. App. Biosci 245 (1990)). ) Using the FASTDB computer program based on the above algorithm.

  The polynucleotide variants of the invention may contain changes in the coding region, non-coding region, or both. Polynucleotide variants containing alterations that result in silent substitutions, additions or deletions, but do not alter the properties or activities of the encoded polypeptide can also be used to produce modified Tf fusion proteins. Nucleotide variants produced by silent substitution due to the degeneracy of the genetic code are available. Further, 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 or deleted in any combination Alternatively, polypeptide variants with additions can also be used. Polynucleotide variants can be produced for a variety of reasons, eg, to optimize codon expression for a particular host (codons in human mRNA are preferred for hosts such as yeast or E. coli described above. To codon).

  In other embodiments, the GLP-1 therapeutic protein moiety has conservative substitutions compared to the wild-type sequence. “Conservative substitution” means within-group exchange, eg, exchange of aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; exchange of hydroxyl residues Ser and Thr; acidic residues Asp and Glu Exchange of amide residues Asn and Gln; exchange of basic residues Lys, Arg and His; exchange of aromatic residues Phe, Tyr and Trp; and small amino acids Ala, Ser, Thr, Met and Gly Is an exchange. Guidance on 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 certain embodiments, the polypeptides of the invention comprise or are fragments or variants of the amino acid sequences of the therapeutic proteins and / or serum transferrin and / or modified transferrin proteins of the invention described herein. Wherein the fragment or variant is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150 compared to the reference amino acid sequence. With additions, substitutions and / or deletions of amino acid residues. In further embodiments, such amino acid substitutions are conservative. Nucleic acids encoding these polypeptides are also encompassed by the present invention.

  The modified fusion protein of the present invention may be composed of amino acids linked to each other by peptide bonds or modified peptide bonds, and includes amino acids other than the amino acids encoded by 20 genes. Also good. The polypeptide can be altered either by natural processes such as post-translational processing or by chemical modification techniques well known in the art. Such modifications are basic textbooks, well documented in more detailed books, and are also described in vast research literature.

  Modifications can occur anywhere in the polypeptide, such as at the peptide backbone, amino acid side chains, and the amino or carboxy terminus. Of course, the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Many types of modifications may also be included in a given polypeptide. A polypeptide may be branched (eg, as a result of ubiquitination) or cyclic, in which case it may or may not contain branches. Cyclic polypeptides, branched polypeptides, and branched cyclic polypeptides may be produced by post-translation natural processes or may be produced by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol Covalent bond, crosslinking, cyclization, disulfide bond formation, demethylation, covalent bridge formation, cysteine formation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation Proteolytic processing, phosphorylation, prenylation, racemization, sulfation, amino acid addition to proteins mediated by transfer RNA, such as arginylation, and ubiquitination. (For example, PROTEINS-STRUCTURE AND MOLECULAR PROPERIES, 2nd edition, T.E. Creighton, WH Freeman and Company, New York (1993); (Academic Press), New York, p. 1-12 (1983); Seeifter et al. (1990) Meth. Enzymol. 182: 626-646; Rattan et al., Ann. N. A. Cid. 663: 48-62. ).

GLP-2
GLP-2 is a peptide consisting of 33 amino acids expressed from the glucagon gene in a tissue-specific manner. GLP-2 has the following sequence: His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Thr-Arg -Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-Arg, SEQ ID NO: 96). GLP-2 is related to GLP-1. However, unlike GLP-1, the physiological role of GLP-2 is unknown.

  In the present invention, GLP-2 is used as a linker for linking GLP-1 to transferrin or modified transferrin. We have found that when GLP-2 is used as a linker in a GLP / mTf fusion protein, the fusion protein is stabilized and produces a more potent GLP-1 peptide. It appears that the peptide is more accessible due to its binding to the receptor. Accordingly, the present invention provides a transferrin fusion protein comprising GLP-1 linked to GLP-2 linked to transferrin or a modified transferrin molecule. Such transferrin molecules can be serum transferrin, lactoferrin, or melanotransferrin molecules.

  There is at least one GLP-1 molecule at the N-terminus of the transferrin fusion protein. In one embodiment, there are 2-5 GLP-1 molecules. In other embodiments, there are 3 or 4 GLP-1 molecules.

  In a preferred embodiment, the GLP-2 linker sequence is modified such that the amino acid corresponding to the N-terminal amino acid of GLP-2 (ie, histidine) is either absent or changed to another amino acid. Is done.

Nucleic Acids The present invention also provides nucleic acid molecules that encode a transfer protein or portion of a transferrin protein covalently linked or bound to a GLP-1 therapeutic protein, preferably a fusion protein comprising a therapeutic protein. Any therapeutic protein can be used, as described in more detail below. The fusion protein may further comprise a linker region, eg, a linker of less than about 50, less than 40, less than 30, less than 20, or less than 10 amino acid residues. This linker can be covalently linked to the transferrin protein or portion thereof, and can further be covalently linked between the transferrin protein or portion thereof and the therapeutic protein, preferably the therapeutic protein. The nucleic acid molecule of the present invention may be purified or may not be purified.

  Also provided are host cells and vectors for replicating nucleic acid molecules and expressing the encoded fusion protein. Any vector or host cell can be used, whether prokaryotic or eukaryotic, but eukaryotic expression systems, particularly yeast expression systems, may be preferred. Many vectors and host cells for such purposes are known in the art. It is well within the skill of the art to select the appropriate combination suitable for the desired application.

  DNA sequences encoding transferrin, portions of transferrin and GLP-1 and linkers can be cloned from a wide variety of genomic or cDNA libraries known in the art. The prior art of techniques for separating such DNA sequences using probe-based methods is well known to those skilled in the art. Probes for separating such DNA sequences may be based on published DNA or protein sequences (e.g., Baldwin, GS (1993) Comparison of Transfer Sequences Different Specs. Comp. Biochem. Physiol). .106B / 1: 203-218, and any documents cited therein are hereby incorporated by reference in their entirety). Alternatively, the polymerase chain reaction (PCR) method described in Mullis et al. (US Pat. No. 4,683,195) and Mullis (US Pat. No. 4,683,202), incorporated herein by reference. It may be used. Library selection and probe selection for separating such DNA sequences is within the ordinary skill in the art.

  As is known in the art, “similarity” between two polynucleotides or polypeptides refers to the nucleotide or amino acid sequence of one polynucleotide or polypeptide, as well as its conserved nucleotide or amino acid substitutions. Determined by comparison with the sequence of two polynucleotides or polypeptides. Also, as is known in the art, “identity” is determined by the identity of a match between two strings of such a sequence, as determined between two polypeptide sequences, or two Means the degree of sequence relatedness between polynucleotide sequences. Identity and similarity can be easily calculated (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatives and Gen. Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, Ed., H.G. Press (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., M. S Stockton ck (M)

  There are numerous ways to measure identity and similarity between two polynucleotide or polypeptide sequences, but the terms “identity” and “similarity” are well known to those skilled in the art (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J. Ed., M Stockton Press, New York, 1991; J. Applied Math., 48: 1073 (1988) Methods commonly used to determine the identity or similarity of two sequences include, but are not limited to, Guid. 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). .

  Preferred methods for determining identity are designed to give the maximum match between the two sequences tested. Methods for determining identity and similarity are organized into computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, et al., Nucl. Acid Res. 12 (1): 387 (1984). ), BLASTP, BLASTN, FASTA (Atschul, et al., J. Mol. Biol. 215: 403 (1990)). The degree of similarity or identity described above is determined as the degree of identity between two sequences, but often indicates a derivation of the first sequence from the second sequence. The degree of identity between two nucleic acid sequences may be determined by computer programs known in the art, such as GAP included in the GCG program package (Needleman). And Wunsch J. Mol. Biol. 48: 443-453 (1970)). In order to determine the degree of identity between two nucleic acid sequences according to the present invention, GAP is used with the following settings: GAP creation penalty of 5.0 and gap extension penalty of 0.3.

The degeneracy of the codon-optimized gene code allows for variations in the nucleotide sequence of the transferrin protein and / or therapeutic protein of interest, but such a variation was nevertheless encoded by the native DNA sequence. A polypeptide having the same amino acid sequence as the polypeptide is produced. Such an approach is known as “codon optimization” (described in US Pat. No. 5,547,871, which is hereby incorporated by reference in its entirety), thereby One is provided with means for designing such modified DNA sequences. The design of codon-optimized genes should take into account a wide variety of factors, including the frequency of codon usage in organisms, nearest neighbor frequencies, RNA stability, Possible secondary structure formation, synthetic pathways and further DNA manipulations of interest for the gene. In particular, when a yeast expression system is used, methods available for changing the codon encoding a given fusion protein to the codon most easily recognized by yeast can be used.

  The degeneracy of the genetic code allows 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, and 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 in eukaryotes and prokaryotes. For example, synonymous codon selection patterns between mammals are very similar, but evolutionarily distant organisms such as yeast (eg S. cerevisiae), bacteria (eg E. coli) and insects (eg D. melanogaster) ) Shows distinct patterns in the frequency of genomic codon usage (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 upp, 1981~1985 (1991);.. Kurland, C.G., FEBS Lett, 285,165~169 (1991)). These differences in codon selection patterns appear to contribute to the overall expression level of individual genes by regulating the rate of peptide elongation. (Kurland, CG, FEBS Lett., 285, 165-169 (1991); Pedersen, S., EMBO J., 3, 2895-2898 (1984); Sorensen, MA, J. Mol. Biol., 207, 365-377 (1989); Randall, L.L., et al., Eur.J.Biochem., 107,375-379 (1980); Curran, J.F. and Yarus, M., J. Mol. Biol., 209, 65-77 (1989); Varenne, S., et al., J. Mol. Biol., 180, 549-576 (1984), Varenne, S., et al., J. Mol, Biol. , 180, 549-576 (1984); Garel, J.-P., J. Theor. Ikemura, T., J. Mol. Biol., 146, 1-21 (1981); Ikemura, T., J. Mol. Biol., 151, 389-409 (1974); 1981)).

  Preferred codon usage in the case of synthetic genes was derived from the exact (or as closely related as possible) genome of the cell / organism intended for use in recombinant protein expression, particularly the genome of the yeast species. Should reflect the use of codons in the nuclear gene. As discussed above, in one preferred embodiment, the human Tf sequence is optimally codoned before or after modification, as well as the therapeutic protein nucleotide sequence, as described herein for yeast expression. It becomes.

Vectors An expression unit for use in the present invention will generally comprise the following components linked to function in the 5 'to 3' direction: a transcriptional promoter, a secretion signal sequence, a linker, A DNA sequence encoding a modified Tf fusion protein comprising a transferrin protein or a portion of a transferrin protein linked to a DNA sequence encoding GLP-1 and a transcription terminator. As discussed above, any sequence of GLP-1 and linker fused to the Tf moiety can be used in the vectors of the present invention. The selection of the appropriate promoter, signal sequence and terminator will be determined depending on the selected host cell, which will be apparent to those skilled in the art and are discussed more specifically below.

  Suitable yeast vectors for use in the present invention are described in US Pat. No. 6,291,212, for example, YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978). YEp13 (Broach et al., Gene 8: 121-133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275: 104-108, 1978), pPPC0005, pSeCHSA, pScNHSA, pC4, and derivatives thereof. Useful yeast plasmid vectors also include pRS403-406, pRS413-416 and Pichia vectors, which are Stratagene Cloning Systems, La Jolla, CA 92037, USA. More available. Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integration plasmids (YIp) and incorporate the yeast selection markers HIS3, TRP1, LEU2 and URA3. Plasmid pRS413-41.6 is a yeast centromere plasmid (YCp).

Such vectors will generally contain a selectable marker, which can be one of a number of genes exhibiting a dominant phenotype, for which phenotypic analysis exists and transformants Allows selection of Preferred selectable markers are those that complement the auxotrophy of the host cell, those that provide antibiotic resistance, or that allow the use of a cell specific carbon source, such selectable markers include: Examples include LEU2 (Broach et al., Ibid.), URA3 (Botstein et al., Gene 8:17, 1979), HIS3 (Struhl et al. Ibid.) Or POT1 (Kawasaki and Bell, EP171, 142). Other suitable selectable markers include CAT genes that confer chloramphenicol resistance to yeast cells. Preferred promoters for use in yeast include promoters derived from yeast glycolytic genes (Hitzeman et al., J Biol. Chem. 225: 12073-12080, 1980; Albert and Kawasaki, J. Mol. Appl. Genet. 1). : 419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or the gene of alcohol dehydrogenase (Young et al., In Genetic Engineering of Chemicals for Chemicals, Hollander et al., Ed. Y., 1982; Ammerer, Meth. Enzymol. 101: 192-201, 1983). In this regard, particularly preferred promoters are the TPI1 promoter (Kawasaki, US Pat. No. 4,599,311) and the ADH2-4 ^C (see US Pat. No. 6,291,212) promoter (Russell et al., Nature 304: 652-654). 1983). The expression unit may include a transcription terminator. A preferred transcription terminator is the TPI1 terminator (Alber and Kawasaki, ibid). Other preferred vectors and preferred components, such as promoters and terminators of yeast expression systems, are described in European Patents EP0258067, EP0286424, EP0317254, EP0387319, EP0386222, EP04424117, EP0432180, and EP1002095; PCT Publications WO 00/44772 and WO 94/04687; and US Pat. Nos. 5,739,007; 5,637,504; 5,302,697; 5,260,202; 5,667,986; 5,728,553 5,785,423; 5,965,386; 6150,133; 6,379,924; and 5,714,377, which are The entire contents are hereby incorporated by reference.

  In addition to yeast, the modified fusion proteins of the present invention can be expressed in filamentous fungi, for example, strains of the fungus Aspergillus. Examples of useful promoters include those derived from the glycolytic genes of Aspergillus nidulans, such as the adh3 promoter (McKnight et al., EMBO J. 4: 2093-2099, 1985), and the tpiA promoter. Can be mentioned. An example of a suitable terminator is the adh3 terminator (McKnight et al., Ibid). An expression unit utilizing such a component may be cloned into a vector that can be inserted into Aspergillus chromosomal DNA, for example.

Mammalian expression vectors for use in the practice of the present invention include promoters that can be specific for transcription of modified Tf fusion proteins. Preferred promoters include viral promoters and cellular promoters. Preferred viral promoters include the major late promoter from adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2: 1304-1199, 1982) and the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1: 854). -864, 1981). Preferred cellular promoters include the mouse metallothionein 1 promoter (Palmiter et al., Science 222: 809-814, 1983) and the mouse V6 (see US Pat. No. 6,291,212) promoter (Grant et al., Nuc. Acids Res. 15: 5496, 1987). A particularly preferred promoter is the mouse V _H (see US Pat. No. 6,291,212) promoter (Loh et al., Ibid). Such expression vectors may also contain a series of RNA splicing sites, which are located downstream of the promoter and upstream of the DNA sequence encoding the transferrin fusion protein. Preferred RNA splicing sites can be obtained from adenovirus and / or immunoglobulin genes.

Also included in the expression vector is a polyadenylation signal located downstream of the coding sequence of interest. Polyadenylation signals include SV40-derived early or late polyadenylation signals (Kaufman and Sharp, ibid.), Adenovirus 5E1B region and human growth hormone gene terminator-derived polyadenylation signals (DeNoto et al., Nucl. Acid Res. 9). : 3719-3730, 1981). A particularly preferred polyadenylation signal is the V _H (see US Pat. No. 6,291,212) gene terminator (Loh et al., Ibid). The expression vector may contain a non-coding viral leader sequence, for example, the adenovirus 2 tripartite leader located between the promoter and the RNA splicing site. Preferred vectors may also include enhancer sequences, such as the SV40 enhancer and the mouse (see US Pat. No. 6,291,212) enhancer (Gillies, Cell 33: 717-728, 1983). The expression vector may also contain a sequence encoding an adenovirus VA RNA.

Techniques for transforming transformed fungi are well known in the literature. For example, Beggs (in the same book), Hinnen et al. (Proc. Natl. Acad. Sci. USA 75: 1929-1933, 1978), Yelton et al. (Proc. Natl.Acad.Sci.USA81: 1740-1747, 1984), and Russell (Nature 301: 167-169, 1983). Other techniques for introducing cloned DNA sequences into fungal cells may be used, such as electroporation (Becker and Guarente, Methods in Enzymol. 194: 182-187, 1991). The genotype of the host cell is generally expected to include a genetic defect that is complemented by a selectable marker present in the expression vector. The selection of a particular host and selectable marker is well within the level of ordinary skill in the art.

A cloned DNA sequence containing the fusion protein of the invention may be introduced into cultured mammalian cells, for example by 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 a cloned DNA sequence into mammalian cells, such as electroporation (Neumann et al., EMBO J. 1: 841-845, 1982) or lipofection can also be used. 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 drug resistance such as neomycin, hygromycin and methotrexate. The selectable marker can be an amplifiable selectable marker. A preferred amplifiable selectable marker is the DHFR gene. A particularly preferred amplifiable marker is DHFR ^r (see US Pat. No. 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 selection of selectable markers is well within the level of ordinary skill in the art.

Host cells The invention also includes cells, preferably yeast cells, that have been transformed to express a modified transferrin fusion protein of the invention. In addition to the transformed host cell itself, the present invention also includes a culture of said cell, preferably a monoclonal (clonally uniform) culture, or a culture derived from a monoclonal culture in a nutrient medium. . Once the polypeptide is secreted, the medium will contain the polypeptide with the cells, or will contain the polypeptide without cells if the medium is filtered or centrifuged.

  Host cells for use in the practice of the present invention include eukaryotic cells, in some cases prokaryotic cells, which can be transformed or transfected with exogenous DNA and grown in culture, eg Cultured mammalian, insect, fungal, plant and bacterial cells.

  Fungal cells, such as yeast species (eg, Saccharomyces sp., Schizosaccharomyces sp., Pichia sp.) Can be used as host cells within the scope of the present invention. In the practice of the present invention, examples of fungi such as yeast that may be useful as hosts for expressing the transferrin fusion protein of the present invention include the genus Pichia (of which some species were previously classified as Hansenula) Saccharomyces genus, Kluyveromyces genus, Aspergillus genus, Candida spp. ), Trichoderma, Cephalosporium, Humicola, Mucor, Acapangabi, Yarrowia, Metschnikowia, Rhodosporidium, Leukospo Indium genus Botorioasukasu genus (Botryoascus), the genus Sporidiobolus (Sporidiobolus), and the like Endomikopushisu genus (Endomycopsis). Examples of Saccharomyces spp. Cerebjee, S. Italicus and S. R. rouxii. Examples of Kluyveromyces spp. Fragilis, K. K. lactis, and K. lactis. K. marxianus. Suitable Torlaspora species are T. pylori. T. delbrueckii. Examples of Pichia spp. Angsta (P. angustta, formerly H. polymorpha), P. a. P. anomala (formerly H. anomala), and P. anomala. P. pastoris.

  A particularly useful host cell for producing the Tf fusion proteins of the invention is methylotrophic Pichia pastoris (Steinlein et al. (1995) Protein Express. Purif. 6: 619-624). Pichia pastoris has been developed as an excellent host for foreign protein production 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. P. The Pastoris expression system can use an alcohol oxidase (AOX1) promoter induced by methanol, which controls the gene encoding the expression of alcohol oxidase, an enzyme that catalyzes the first step in methanol metabolism. This promoter has been characterized and a series of P. pyloris. It is incorporated into an expression vector for Pastoris. P. Proteins produced in Pastoris are typically correctly folded and secreted into the medium, so that genetically engineered P. coli is Pastoris fermentation is described in E. An excellent alternative to the E. coli expression system is provided. Using this system, a number of proteins have been produced, such as tetanus toxin fragments, Bordatella pertussis pertactin, human serum albumin and lysozyme.

  Another preferred host is the yeast Saccharomyces cerevisiae strain. In preferred embodiments, yeast cells, or more specifically, Saccharomyces cerevisiae host cells that contain a genetic defect in a gene required for asparagine-linked glycosylation of a glycoprotein are used. S. having such a defect. Although cerevisiae host cells can be prepared using standard mutation and selection techniques, many available yeast strains have been modified to prevent or reduce glycosylation or excessive mannosylation. Yes. Ballou et al. (J. Biol. Chem. 255: 5986-5991, 1980) describe the isolation of mannoprotein biosynthetic mutants that lack genes that affect asparagine-linked glycosylation. Gentzsch and Tanner (Glycobiology 7: 481-486, 1997) describe a family of at least six genes (PMT1-6) encoding enzymes involved in the first step in protein O-glycosylation in yeast. ing. Mutants lacking one or more of these genes exhibit reduced O-linked glycosylation and / or altered O-glycosylation specificity.

  In one embodiment, the host is an S. cerevisiae cell as described in WO 05/061718 (incorporated herein by reference in its entirety). A cerevisiae strain. For example, the host may comprise a pSAC35-based plasmid carrying a copy of the PDI1 gene or any other chaperone gene in a strain having a host version in which PDI1 or other chaperone is knocked out, respectively. Such a construct provides enhanced stability. In the Examples section of this specification, the strains referred to as “control strain” and “strain A” are strains of the same name described in WO05 / 061718.

  In order to optimize heterologous protein production, it is also preferred that the host strain has a mutation, e.g. Cerevisiae pep4 mutation (Jones, Genetics 85: 23-33, 1977). Host strains containing mutations in regions encoding other proteases are particularly useful for producing large quantities of the Tf fusion protein of the invention.

  Host cells containing the DNA constructs of the present invention are grown in a suitable growth medium. The term “appropriate growth medium” as used herein means a medium containing nutrients necessary for cell growth. Nutrients required for cell growth can include carbon sources, nitrogen sources, essential amino acids, vitamins, inorganic substances and growth factors. Growth media generally selects for cells that contain the DNA construct, eg, by drug selection or lack of essential nutrients, which are supplemented by selectable markers present on the DNA construct or transfected with the DNA construct. Is done. For example, yeast cells are preferably grown in a chemically defined medium containing a carbon source such as sucrose, nitrogen sources other than amino acids, inorganic salts, vitamins, and supplements of essential amino acids. The pH of the medium is preferably maintained at a pH greater than 2 and less than 8, and preferably maintained at pH 5.5-6.5. Methods for maintaining a stable pH include buffering and control to a constant pH. Preferred buffering agents include succinic acid and bis-Tris (Sigma Chemical Co., St. Louis, Mo.) Yeast cells lacking a gene required for asparagine-linked glycosylation are preferred. Is grown in a medium containing an osmotic stabilizer, a preferred osmotic stabilizer is sorbitol, which is at a concentration of 0.1M to 1.5M, preferably at a concentration of 0.5M or 1.0M. Supplemented to the medium.

  Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media. The selection of a suitable medium for the particular cell line used is within the ordinary skill level in the art. Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, in order to initiate expression of the DNA sequence of interest. Next, drug selection is performed to select the growth of cells that stably express the selectable marker. For cells transfected with an amplifiable selectable marker, the drug concentration may be increased stepwise to select those that have a high copy number of the cloned sequence and thus a high expression level.

The modified Tf fusion protein of the present invention may also be produced using a baculovirus / insect cell expression system. 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 inserted into the insect host cell. Are transfected with linearized BacPAK6 viral DNA, which lacks an essential part of the baculovirus genome, and when the DNA recombines with the vector, the essential components are restored, The target gene is transferred to the baculovirus genome, and after recombination, 2-3 viral plaques are picked up and purified to verify the recombinant phenotype, and then the newly isolated recombinant virus is amplified. Insect cell culture using it Infects nourishment and can produce large quantities of the desired protein.

  The Tf fusion protein of the present invention may be produced using transgenic plants and animals. For example, sheep and goats can make therapeutic proteins in their milk. Alternatively, tobacco plants can include proteins in their leaves. Both protein production in transgenic plants and animals involves the addition of a gene encoding the fusion protein to the genome of the organism. Transgenic organisms can not only produce new proteins, but also pass this ability to their offspring.

The secretory signal sequence terms “secretory signal sequence” or “signal sequence” or “secretory leader sequence” are used interchangeably and further include, for example, US Pat. No. 6,291,212 and US Pat. No. 5,547,871. (All of which are incorporated herein by reference in their entirety). The secretory signal sequence or signal sequence or secretory leader sequence encodes a secretory peptide. A secretory peptide is an amino acid sequence that acts to direct secretion of a mature polypeptide or protein from a cell. Secreted peptides are generally characterized by a core of hydrophobic amino acids and are typically (but not necessarily) found at the amino terminus of newly synthesized proteins. Secreted peptides are very often cleaved from the mature protein during secretion. The secretory peptide may contain processing sites that allow the signal peptide to cleave the mature protein as it passes through the secretory pathway. The processing site may be encoded within the signal peptide or may be added to the signal peptide, for example by in vitro mutagenesis.

  Secreted peptides may be used to direct secretion of the GLP-1 / mTf and GLP-1 / linker / GLP-1 fusion proteins of the invention. One such secretory peptide that can be used in combination with other secreted peptides is the alpha mating factor leader sequence. Secretory signal sequences or signal sequences or secretory leader sequences are required for a complex series of post-translational processing steps that cause secretion of the protein. In the presence of an intact signal sequence, the expressed protein enters the lumen of the rough endoplasmic reticulum, then is transported through the Golgi apparatus to the secretory vesicle and finally out of the cell. . In general, a signal sequence is present immediately after the start codon and encodes a signal peptide at the amino terminus of the protein to be secreted. In most cases, this signal sequence is cleaved by a specific protease called a signal peptidase. Preferred signal sequences improve recombinant protein processing and transport efficiency of recombinant protein expression using viral, mammalian or yeast expression vectors.

In one embodiment, the native Tf signal sequence may be used to express and secrete the fusion protein of the invention. Since transferrin molecules are present in various types of secretions such as blood, tears and milk, there are many different transferrin signal peptides. For example, the transferrin signal peptide may be derived from serum transferrin, lactotransferrin, or melanotransferrin. The natural transferrin signal peptide may be derived from various species such as insects, mammals, fish, frogs, ducks, chickens, or may be derived from other species. Preferably, such a signal peptide is derived from a mammalian transferrin molecule. More preferably, such signal peptide is derived from human serum transferrin. The following table summarizes the signal peptide sequences from various mammalian transferrin molecules ( www.chatham.edu/undergraduate/bio/lambert/transferrin/signal.htm ).

  In other embodiments, the signal peptide is derived from a mutant or modified transferrin molecule having a functionally active signal peptide. In addition, the signal peptide is a variant or modified form of the transferrin signal peptide that retains the ability to transport the transferrin fusion protein of the invention across the cell membrane and process the fusion protein.

  In other embodiments, the signal sequence derived by transfer may be used to secrete a heterologous protein, for example, any protein of interest that is heterologous to the Tf signal sequence is expressed using the Tf signal, It can also be secreted. In particular, the Tf signal sequence may be used to secrete proteins from recombinant yeast. Preferably, the signal peptide is derived from human serum transferrin (nL, amino acids 1-19 of SEQ ID NO: 2).

  In some cases it may be preferred to include a short propeptide sequence between the signal sequence and the mature protein to ensure efficient removal of the signal sequence, in which case the C-terminal portion of the propeptide is It contains a recognition site for a protease (eg yeast kex2p protease). Preferably, the propeptide sequence is about 2-12 amino acids in length, more preferably about 4-8 amino acids in length. Examples of such propeptides are Arg-Ser-Leu-Asp-Lys-Arg (SEQ ID NO: 113), Arg-Ser-Leu-Asp-Arg-Arg (SEQ ID NO: 114), Arg-Ser-Leu-Glu. -Lys-Arg (SEQ ID NO: 115) and Arg-Ser-Leu-Glu-Arg-Arg (SEQ ID NO: 116).

Linker The Tf portion of the modified transferrin fusion protein of the invention and the therapeutic protein use linker peptides of various lengths to provide greater physical separation and greater spatial movement between the fused proteins. Which, for example, maximizes the accessibility of the therapeutic protein to bind to its cognate receptor, for example. In one embodiment, as discussed above, GLP-2, or a fragment thereof, can be used as a linker, preferably when GLP-1 is the therapeutic protein moiety. Such linkers can be less than about 50, less than 40, less than 30, less than 20, less than 10, or less than 5 amino acid residues. Such linkers can be covalently linked to the transferrin protein or portion thereof and between it and a therapeutic protein (eg, GLP-1). These linkers may be used to link GLP-1 to transferrin.

In a preferred embodiment of the invention, GLP-1 is linked to mTf via a substantially non-helical linker. Examples of such rigid linkers include PE, PEA, PEAPTD (SEQ ID NO: 13), (PEAPTD) ₂ (SEQ ID NO: 10), (PEAPTD) ₃ (SEQ ID NO: 14), or (PEAPTD) _n. Where n is an integer. The invention also provides an IgG hinge linker, a CEx linker (SSGAPPPS; SEQ ID NO: 15 (C-terminal extension to exendin-4)), an IgG hinge linker linked to a PEAPTD linker, and an IgG hinge linker linked to a CEx linker. .

Detection of GLP-1 / Tf fusion proteins Analysis to detect biologically active modified transferrin-fusion proteins may include Western transfer, protein blot or colony filters, plus transferrin and therapeutic Analysis based on activity to detect fusion proteins including proteins may be included. The western transfer filter may be prepared using the method described by Towbin et al. (Proc. Natl. Acad. Sci. USA 76: 4350-4354, 1979). Briefly, samples are electrophoresed in sodium dodecyl sulfate polyacrylamide gels. Proteins in the gel are transferred to nitrocellulose paper by electrophoresis. Protein blot filters can be prepared, for example, by filtering supernatant samples or concentrates through nitrocellulose filters using Minifold (Schleicher & Schuell, Keene, NH). A colony filter can be prepared by growing colonies on a nitrocellulose filter with an appropriate growth medium. In this method, a solid medium is preferred. Cells are grown on the filter for at least 12 hours. Cells are removed from the filter by washing with an appropriate buffer that does not remove the protein bound to the filter. A preferred buffer comprises 25 mM Tris base, 19 mM glycine, pH 8.3, 20% methanol.

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

Detection of the fusion protein of the invention can be facilitated by coupling (ie, physically linking) the therapeutic protein and 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; examples of luminescent materials include luminol; Examples of such include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

  In one embodiment, various immunoassays known in the art can be used to analyze the ability of the transferrin fusion protein of the invention to bind to an antibody or to compete with an antigen for binding to an antibody, and this Examples of such immunoassays include, but are not limited to, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, immunoradiometric assay, gel diffusion precipitation reaction, immunodiffusion analysis, in situ immunoassay (eg, colloidal gold, Competition using techniques such as Western blots, precipitation reactions, agglutination analysis (eg gel aggregation analysis), complement binding analysis, immunofluorescence analysis, protein A analysis, and immunoelectrophoresis analysis And non-competitive System, and the like. In one embodiment, transferrin fusion protein binding is detected by detecting a label on the transferrin fusion protein. In other embodiments, 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.

  The fusion protein of the invention can also be detected by analyzing for the activity of the therapeutic protein moiety. Specifically, the transferrin fusion protein of the invention may be analyzed for functional activity (eg, biological activity or therapeutic activity) using assays known in the art. In addition, those skilled in the art can use well-known analyzes to analyze fragments of therapeutic proteins corresponding to the therapeutic protein portion of the fusion proteins of the invention for activity in a routine manner. In addition, one of ordinary skill in the art may analyze the activity of the modified transferrin protein fragments using routine procedures and using assays known in the art.

  For example, in one embodiment, known in the art when analyzing 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. A variety of immunoassays can be used, including but not limited to, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunizations Diffusion analysis, in situ immunoassay (eg using colloidal gold, enzyme or radioisotope labeling), Western blot, precipitation reaction, agglutination analysis (eg gel aggregation analysis), complement binding analysis, immunofluorescence analysis, protein A analysis, and immunization Electrical Kinematic analysis competitive and non-competitive assay system using the techniques and the like. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In other embodiments, the primary antibody is detected by detecting binding of the secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

  In a further embodiment, when identifying a therapeutic protein binding partner (eg, a receptor or ligand), binding to the binding partner by a transferrin fusion protein comprising the therapeutic protein as the therapeutic protein portion of the fusion. It can be analyzed by means well known in the art, for example by reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. Other methods will be known to those skilled in the art and are within the scope of the present invention.

Production of Fusion Proteins The present invention further provides a method for producing a modified fusion protein of the present invention using the nucleic acid molecules described herein. Generally speaking, recombinant production of a protein typically includes the following steps.

  First, a nucleic acid molecule encoding the transferrin fusion protein of the present invention is obtained. This nucleic acid molecule is then preferably operably linked to an appropriate control sequence as described above to form an expression unit containing the protein open reading frame. The expression unit is used to transform an appropriate host, and the transformed host is cultured under conditions that produce a recombinant protein. Optionally, the recombinant protein may be isolated from the medium or cells; recovery and purification of this protein is not essential in cases where some impurities can be tolerated.

  Each of the foregoing steps can be accomplished in various ways. For example, the construction of expression vectors operable in various hosts is accomplished using appropriate replicons and control sequences as described above. The control sequences, expression vectors and transformation methods vary depending on the type of host cell used to express the gene and have been discussed in detail so far or are known to those skilled in the art. In general, if not available, an appropriate restriction site can be added to the end of the coding sequence to provide a excisable gene for insertion into these vectors. One skilled in the art can readily adapt any host / expression system known in the art for use with the nucleic acid molecules of the invention so that the desired recombinant protein is produced.

  As discussed above, any expression system can be used, including yeast, bacteria, animals, plants, 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, excessive glycosylation, or proteolytic activity may be used.

Isolation / purification of the modified transferrin fusion protein The secreted biologically active modified transferrin fusion protein can be separated from the medium in which the host cells are grown under conditions that secrete the biologically active fusion protein. . Cell-derived material is removed from the cultured medium and the biologically active fusion protein is isolated using separation techniques known in the art. Suitable separation techniques include precipitation and fractionation by a wide variety of chromatographic methods, including gel filtration, ion exchange chromatography, and affinity chromatography.

  Particularly preferred purification methods are affinity chromatography for iron binding, or immunoaffinity chromatography using a metal chelate column or an antigen directed against a polypeptide fusion transferrin or therapeutic protein. The antigen is preferably immobilized on or bound to a solid support, or substrate. A particularly preferred substrate is CNBr-activated Sepharose (Pharmacia LKB Technologies, Inc., Piscataway, NJ). By this method, the medium is mixed with the antigen / substrate under conditions that allow binding to occur. The complex can be washed to remove unbound material, or the transferrin fusion protein can be released or eluted by use of conditions unsuitable for complex formation. A particularly useful elution method is a pH change, where the immobilized antigen has a high affinity for the transferrin fusion protein at the first pH but a second (higher or lower) pH. Will decrease the affinity; a change in the concentration of a given chaotropic reagent may be mentioned, or a method by the use of a surfactant.

Delivery of drugs or therapeutic proteins that pass through the interior of the cell and / or the blood brain barrier (BBB) Within the scope of the present invention, the modified transferrin fusion protein may be used as a carrier, by means of such a carrier Any cell type that expresses a Tf receptor, eg, naturally or artificially, with a molecule or small molecule therapeutic agent complexed with a ferric ion inside the cell or through the blood brain barrier or other barrier. Can be delivered across the cell membrane. In these embodiments, the Tf fusion protein is typically designed such that glycosylation is suppressed, prevented or eliminated to increase the serum half-life of the fusion protein and / or therapeutic protein moiety. Or it will be modified. The addition of the target peptide is particularly contemplated to further target the Tf fusion protein to a specific cell type (eg, cancer cell).

  In one embodiment, ferric iron-sorbitol-citrate complex, an iron-containing anti-anemic agent, is loaded onto the modified Tf fusion protein of the invention. Ferric-sorbitol-citrate (FSC) inhibits the growth of various mouse cancer cells in vitro and causes tumor regression in vivo, but has no effect on the growth of non-malignant cells (Poljak-Blazi et al. (June 2000) Cancer Biotherapy and Radiopharmaceuticals (USA), 15/3: 285-293).

In other embodiments, the anti-neoplastic agent adriamycin® ⁽ doxorubicin) and / or the chemotherapeutic agent bleomycin, both of which are known to complex with ferric ions, are disclosed herein. Of the Tf fusion protein. In other embodiments, a salt of the drug (eg, citrate or carbonate) may be prepared, complexed with ferric iron, and subsequently bound to Tf. Because tumor cells often exhibit higher turnover rates of iron, transferrins modified to carry at least one anti-tumor agent provide a means to increase drug exposure or burden on tumor cells. There is a possibility to provide. (Demant, EJ, (1983) Eur. J. Biochem. 137 / (1-2): 113-118; Padbury et al. (1985) J. Biol. Chem. 260/13: 7820-7823).

Pharmaceutical formulations and methods of treatment In one form of the invention, a pharmaceutical composition comprising GLP-1 / linker / mTf protein can be formulated by any established method of formulating a pharmaceutical composition, eg, Remington It may be formulated as described in 's Pharmaceutical Sciences, 1985. In one embodiment, the pharmaceutical composition comprises the fusion protein set forth in SEQ ID NO: 12. The composition can be in a form suitable for systemic injection or infusion, as such using a suitable liquid vehicle such as sterile water or isotonic saline or glucose solution, It may be formulated. The composition can also be sterilized by conventional sterilization techniques well known in the art. The resulting aqueous solution may be packaged for use or filtered under aseptic conditions and further lyophilized, such lyophilized preparations being sterilized prior to administration. Mixed with aqueous solution. The composition may contain pharmaceutically acceptable adjuvants as necessary to approximate physiological conditions, and examples of such adjuvants include buffers and tonicity adjusting agents, For example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and the like.

  The GLP-1 / linker / mTf fusion proteins of the present invention are also suitable for oral, nasal, transdermal, pulmonary or rectal administration (see PCT / US03 / 26778, which is hereby incorporated by reference in its entirety. Join the statement). The pharmaceutically acceptable carrier or diluent used in the present composition can be any conventional solid carrier. Examples of solid carriers are lactose, clay, sucrose, talc, gelatin, agar, pectin, gum arabic, 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, which may be alone or with a wax. You may mix.

  It may be particularly advantageous to provide the composition of the invention in the form of a sustained release formulation. As such, the composition is encapsulated by or dispersed in a suitable pharmaceutically acceptable biodegradable polymer such as polylactic acid, polyglycolic acid or lactic acid / glycolic acid copolymer. They may be formulated as microcapsules or microparticulates containing 1 / linker / mTf.

  For nasal administration, such formulations may comprise GLP-1 / linker / mTf dissolved or suspended in a liquid carrier, particularly an aqueous carrier for application to an aerosol. Such carriers may contain additives such as solubilizers such as propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrins, or preservatives such as parabens.

  In general, the compounds of the invention are formulated in unit dosage form, which comprises 0.5 to 500 mg of the fusion protein per unit dosage together with a pharmaceutically acceptable carrier.

  Moreover, the present invention contemplates the use of GLP-1 / linker / mTf to produce a medicament that can be used in the treatment of diseases associated with high blood glucose levels, such as, but not limited to, those mentioned above To do. Specifically, the present invention uses GLP-1 / transferrin fusion protein to treat diabetes (such as type II diabetes), obesity, severe burns, and heart failure such as congestive heart failure and acute coronary syndrome. Intended.

The N-terminus of GLP-1 is usually amidated. In yeast, amidation does not occur. In one form of the invention, an additional amino acid is added to the N-terminus of GLP-1 to compensate for the N-terminus amidation that does not occur in yeast. The addition of an amino acid to the N-terminus of GLP-1 can prevent dipeptidyl peptidase from cleaving at the second amino acid of GLP-1 due to steric hindrance. To that end, GLP-1 is still functionally active. Any of the 20 amino acids may be added to the N-terminus of GLP-1. In some cases, an uncharged or positively charged amino acid may be used, preferably histidine is added. Next, GLP-1 with additional amino acids is fused to transferrin. Therefore, GLP-1 to which an amino acid is added is fused to the N-terminus of the transferrin moiety, and the free GLP-1 N-terminus is left as it is.

  In one embodiment that makes GLP-1 (7-36) or GLP-1 (7-37) peptides more resistant to cleavage by dipeptidyl peptidases, a His residue is added to the N-terminus of GLP-1. Or inserted after the His residue at the N-terminus of GLP-1 such that the N-terminus of GLP-1 begins with His-His.

  In another embodiment of the invention, the second residue from the N-terminus in GLP-1 (7-36) or GLP-1 (7-37) peptide (SEQ ID NO: 6) is substituted with another amino acid. . For example, in GLP-1 (7-36) or GLP-1 (7-37) peptide, at the second residue from the N-terminus, the Ala residue is replaced with Ser, Gly, Val, or other amino acid It may be.

  The GLP-1 / linker / mTf fusion proteins of the invention can be administered to patients in need thereof using standard administration protocols. For example, the fusion proteins of the invention may be provided alone or provided in combination with, or in continuous with, other substances that modulate a particular pathological process. As used herein, when two drugs are administered simultaneously, or are administered independently in a manner such that the two drugs act simultaneously or nearly simultaneously, the two substances are Said to be administered in combination.

  The fusion proteins of the invention can be administered by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, and buccal routes. For example, the drug may be administered locally to the site of injury by microinjection. Alternatively or simultaneously, administration may be non-invasive administration by either oral, inhalation, nasal or pulmonary route. The dose to be administered will depend on the age, health and weight of the recipient, the type of combination therapy, the frequency of treatment as required and the nature of the desired effect.

  Although any administration method for delivering the fusion protein of the invention can be used, oral administration or delivery is preferred for a given class of fusion protein or for treating a given body condition. It can be.

  The invention further provides a composition comprising one or more fusion proteins of the invention. While individual needs vary, determination of optimal effective dosage ranges for each component is within the skill of the art. Typical dosages include about 1 pg / kg to about 150 mg / kg body weight. In one embodiment, dosages for systemic administration include about 100 ng / kg to about 100 mg / kg body weight. In other embodiments, the dose ranges from 300 μg / kg to 900 μg / kg. The invention also includes once a week administration at a total dose of about 50 mg, 100 mg, or 150 mg. Further, dosages for direct administration to the site by microinjection include about 1 ng / kg to about 1 mg / kg body weight. When administered by direct injection or microinfusion, the modified fusion proteins of the invention exhibit reduced binding to or in combination with iron to prevent, in part, localized iron toxicity. It may be designed so that no coupling occurs.

  In addition to pharmacologically active fusion proteins, the compositions of the present invention deliver suitable pharmaceutically acceptable carriers such as excipients and auxiliaries that facilitate the processing of the active compound to the site of action. Therefore, it may be included in a pharmaceutically usable preparation. Formulations suitable for parenteral administration include aqueous solutions of the active compounds in water-soluble form (eg, water-soluble salts). In addition, suspensions of the active compounds may be administered, if necessary, oily suspension injections. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides. Aqueous suspension injections may contain substances which increase the viscosity of the suspension, and examples of such substances include sodium carboxymethylcellulose, sorbitol and dextran. In some cases, the suspension may contain a stabilizer. Liposomes can also be used to encapsulate drugs for delivery to cells.

  The pharmaceutical preparation for systemic administration according to the present invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations can be used to achieve systemic administration of the active ingredient simultaneously. Formulations suitable for oral administration include hard or soft gelatin capsules, pills, tablets such as coated tablets, elixirs, suspensions, syrups, or inhalants, and controlled release forms thereof.

  The pharmaceutical composition of the present invention can be in unit dosage form, such as a tablet or a capsule. In such form, the composition is further subdivided into unit doses containing appropriate quantities of the active component; the unit dosage form can be a packaged composition, eg, packaged, Examples include powders, vials, ampoules, prefilled syringes, or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be a suitable number of such compositions packaged. The dosage used in treatment must be determined subjectively by the physician.

  In practicing the methods of the invention, the fusion proteins of the invention may be used alone or in combination, or may be used in combination with other therapeutic or diagnostic agents. In preferred specific embodiments, the compounds of the invention are typically co-administered with other compounds prescribed for these physical conditions in accordance with generally accepted medical practice. May be. The compounds of the invention may be utilized in vivo, usually in mammals such as humans, sheep, horses, cows, pigs, dogs, cats, rats and mice, or in vitro.

  In the present invention, fusion proteins, including but not limited to modified Tf fusion proteins, can be formulated for oral delivery. In particular, certain fusion proteins of the invention used to treat certain classes of diseases or conditions are particularly suitable 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 diseases or infections, cancer, metabolic diseases, obesity, autoimmune diseases, inflammatory diseases, allergies, graft-versus-host diseases, systemic microorganisms Examples include infection, anemia, cardiovascular disease, psychosis, genetic disease, neurodegenerative disease, hematopoietic cell disorder, endocrine or reproductive system disease, and gastrointestinal disease. Examples of these classes of diseases include diabetes, multiple sclerosis, asthma, HCV or HIV infection, hypertension, hypercholesterolemia, arteriosclerosis, arthritis, and Alzheimer's disease. In many chronic diseases, the oral formulation and method of administration of the Tf fusion protein of the present invention enables long-term patient care and treatment by oral administration at home, without resorting to treatment by injection or drug protocol. It is particularly useful.

  The oral formulations and delivery methods comprising the fusion proteins of the present invention take part in transferrin receptor-mediated transcytosis that passes through the gastrointestinal (GI) epithelium. Tf receptors are found at very high density in the human GI epithelium, and transferrin is highly resistant to trypsin and chymotrypsin digestion, so using Tf chemical conjugates , Proteins and peptides can be successfully delivered 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 incorporated herein by reference in their entirety). The fusion protein of the invention, once transported across the GI epithelium, exhibits an extended half-life in serum, ie, a therapeutic protein or peptide bound to or inserted into Tf Shows an extended serum half-life compared to the unfused protein or peptide.

  Oral formulations of the fusion proteins of the invention can be prepared to be suitable for transport to the GI epithelium and protection of the fusion protein component and other active ingredients in the stomach. Such formulations may contain a carrier and dispersant component and can be in any suitable form, such as an aerosol (for oral or pulmonary delivery), syrup, elixir Agents, tablets (including chewable tablets), hard or soft capsules, troches, lozenges, aqueous or oil suspensions, emulsions, cachets or pellets, granules, and dispersible powders. Preferably, the fusion protein formulation is used in a solid dosage form suitable for simple administration of precise dosages, and preferably oral administration. Solid dosage forms for oral administration are preferably tablets, capsules and the like.

  For oral administration in the form of tablets or capsules, care should be taken to ensure that the composition is able to absorb sufficient active ingredient into the host to cause an effective reaction. Thus, for example, the amount of fusion protein may be greater than is logically necessary, or other known means such as coating or encapsulation may be used to protect the polypeptide from enzymatic action in the stomach. May be.

  Traditionally, peptide and protein drugs are administered by injection because of low bioavailability when administered orally. These drugs tend to be chemically and conformally unstable and are often degraded by acidic conditions in the stomach, as well as by enzymes in the stomach and gastrointestinal tract. In response to these delivery problems, certain types of oral delivery technologies have been developed. For example, encapsulation into nanoparticles composed of a polymer containing a hydrophobic main chain and a hydrophilic branch as a drug carrier, a microparticulate agent Encapsulation, insertion into liposomes in emulsions, and conjugates with other molecules. Any of these can be used with the fusion molecules of the present invention.

  Examples of nanoparticles include mucoadhesive nanoparticles coated with chitosan or carbopol (Takeuchi et al., Adv. Drug Deliv. Rev. 47 (1): 39-54, 2001), and combinations of charged Nanoparticles comprising polyester, poly (2-sulfobutyl-vinyl alcohol) and poly (D, L-lactic acid-co-glycolic acid) (Jung et al., Eur. J. Pharm. Biopharm. 50 (1): 147-160, 2000). Nanoparticles containing a polymer on the surface with a poly-N-isopropylacrylamide region and cationic poly-vinylamine groups showed improvement in salmon calcitonin absorption when administered orally to rats.

  Particles for drug delivery composed of alginate and pectin, reinforced with polylysine, have relatively acid and base resistance and can be used as a carrier for drugs. These particles combine the advantages of bioadhesion, enhanced absorbency and sustained release (Liu Liu et al., J. Pharm. Pharmacol. 51 (2): 141-149, 1999).

  In addition, lipoamino acid groups and liposaccharide groups conjugated to the N-terminus and C-terminus of peptides such as synthetic somatostatin form amphiphilic surfactants, which are compositions that retain biological activity. It has been shown to produce (Toth et al., J. Med. Chem. 42 (19): 4010-4013, 1999).

  Examples of techniques for delivering other peptides include peptides of interest and carbo, including nitroso-N-acetyl-D, L-penicillamine and either carbopol, or taurocholate, and carbopol. Examples include mucoadhesive emulsions coated with poles. These have been shown to be effective when administered orally to rats since they reduced serum calcium levels (Ogiso et al., Biol. Pharm. Bull. 24 (6): 656-661,2001). Liposomes containing phosphatidylethanol as an insulin carrier were prepared using phosphatidylethanol derived from phosphatidylcholine. These liposomes have been shown to be active when administered orally to rats (Kisel et al., Int. J. Pharm. 216 (1-2): 105-114, 2001).

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

  Oral delivery of insulin using nanoparticles made of poly (alkyl cyanoacrylate) dispersed in an oil phase using a surfactant (Damge et al., J. Pharm. Sci. 86 (12): 1403 And 1409, 1997) and oral delivery of insulin using chitosan-coated calcium alginate beads (Onal et al., Artif. Cells Blood Subst. Immobil. Biotechnol. 30 (3): 229-237, 2002) Has been.

In other methods, the N- and C-termini of the peptide are linked to polyethylene glycol and then linked to the allyl chain, thereby improving resistance to enzymatic degradation and further improving diffusion through the digestive tract wall. A bonded body is formed ( www.nobexcorp.com ).

Bio Porter (BioPORTER ^(R)) is a cationic lipid mixture, interact to form a protective coating or layer by non-covalent bond with the peptide. This peptide-lipid complex can be fused to the plasma membrane of the cell to incorporate the peptide into the cell ( www.genetherapysystems.com ).

In processes using liposomes as starting materials, spiral particles have been developed as pharmaceutical vehicles. Peptides are added to a suspension of liposomes containing mainly negatively charged lipids. The addition of calcium causes liposome disintegration and fusion into a large sheet composed of lipid bilayers, which subsequently rolls up or overlaps in a spiral shape (US Pat. No. 5,840,707; www.biodeliverysciences.com ).

  Compositions comprising fusion proteins intended for oral use can be prepared according to any method known in the art for producing pharmaceutical compositions, such compositions being pharmaceutically elegant and palatable. In order to provide a good formulation, it may contain one or more substances selected from the group consisting of sweeteners. For example, to prepare an orally deliverable tablet, the Tf fusion protein and at least one pharmaceutical excipient are mixed and the solid formulation is compressed and delivered to the gastrointestinal tract according to known methods. To form tablets. Compositions for tablets are typically additives such as sugar or cellulose carriers, binders such as starch paste or methylcellulose, fillers, disintegrants, or other additives commonly used in the manufacture of pharmaceutical formulations. It is formulated using an agent. To prepare an orally deliverable capsule, DHEA and at least one pharmaceutical excipient are mixed and the solid formulation is put into a capsule container suitable for delivery to the gastrointestinal tract. Put in. Compositions comprising fusion proteins are generally described in Remington's Pharmaceutical Sciences, 18th Edition, 1990 (Mack Publishing Co. Easton Pa. 18042), Chapter 89 (incorporated herein by reference). May be manufactured as shown.

  As noted above, many of the oral formulations of the present invention may contain inert ingredients that allow protection against the gastric environment and release biologically active substances in the intestine. Such formulations or enteric coatings are well known in the art. For example, a tablet containing a Tf fusion protein mixed with a non-toxic pharmaceutically acceptable excipient suitable for tablet manufacture may be used. These excipients include inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as maize starch, gelatin or gum arabic, and lubricants such as Examples include magnesium stearate, stearic acid, or talc.

  Tablets may be uncoated or may be coated by known techniques, thereby delaying disintegration and absorption in the gastrointestinal tract and providing a sustained action over a long period of time Is possible. For example, a time delay substance such as glyceryl monostearate or glyceryl distearate may be used alone, or a wax may be used in combination.

  Formulations for oral use may also be provided as hard gelatin capsules, in which case the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or soft gelatin capsules In which case the active ingredient is mixed with an aqueous or oily medium, for example arachis oil, arachis oil, liquid paraffin, or olive oil.

  Aqueous suspensions may contain the fusion protein in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic; naturally occurring as dispersants or wetting agents Phosphatides such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, such as heptadecylethyloxycetanol, or ethylene oxide A condensation product of a fatty acid and a partial ester derived from hexitol, such as polyoxyethylene sorbitol monooleate or ethylene oxide, and a fatty acid and hexitol. Condensation products of partial esters derived from Shitoru anhydrides, for example polyoxyethylene sorbitan monooleate. Aqueous suspensions also contain one or more preservatives, such as ethyl or n-propyl-p-hydroxybenzoate, one or more colorants, one or more flavors, and one or more It may contain further sweeteners such as sucrose or saccharin.

  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 oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those mentioned above and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

  Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient and a mixture with the dispersing or wetting agent, suspending agent and one or more preservatives. To do. Examples of suitable dispersing or wetting agents and suspending agents are those already mentioned above. Additional excipients such as sweetening, flavoring and coloring agents may also be included.

  The pharmaceutical composition containing the fusion protein may be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as gum arabic or tragacanth, a naturally occurring phosphatide, such as soybean lecithin, and an ester or partial ester derived from fatty acids and hexitol anhydrides; For example, sorbitan monooleate and condensation products of the same partial ester with ethylene oxide are possible, for example polyoxyethylene sorbitan monooleate. Such an emulsion may contain a sweetener and a flavoring agent.

  Syrups containing fusion proteins and elixirs can 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, such as a sterile aqueous or oily suspension for injection. 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 preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (eg, 1,3-butanediol solution). Among the acceptable vehicles and solvents that can 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. During this period, any non-irritating non-volatile oil can be used, for example, synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables.

  Alternatively, pharmaceutical compositions may be formulated for oral delivery using polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid-based systems (eg, DiBase). And Morrel, Oral Delivery of Microencapsulated Proteins, in Protein Delivery: Physical Systems, Sanders and Hendren (edit), pages 255-288 (see Plenum Press) 97.

  The ratio of fusion protein having pharmacological activity to carrier and / or other substances may vary in the range of about 0.5 to about 100 weight percent (weight percent). For oral use, pharmaceutical formulations are generally expected to contain from about 5 to about 100% by weight of the active substance. For other uses, pharmaceutical formulations are generally expected to contain from about 0.5 to about 50% by weight of the active substance.

  The fusion protein formulation used in the present invention provides an effective amount of fusion protein when administered to an individual. When used in such situations, an “effective amount” of the fusion is an amount effective to ameliorate the symptoms of the disease.

  The fusion protein composition of the present invention may be administered daily in an effective amount that can ameliorate symptoms, although not necessarily. In general, the total daily dose is expected to be at least about 50 mg, preferably at least about 100 mg, more preferably at least about 200 mg per day, preferably 4 capsules each containing eg 50 mg of Tf fusion protein Or 500 mg or less per day is orally administered with a tablet. Capsules or tablets for oral delivery may conveniently contain a daily oral dose (eg 200 mg or more) or less.

  In a particularly preferred embodiment, an oral pharmaceutical composition comprising the fusion protein is formulated in the form of a buffered liquid, which is then encapsulated in a soft or hard coated gelatin capsule and then the appropriate enteric solution. Coat with coating. For the oral pharmaceutical composition of the present invention, the release position can be anywhere in the digestive tract system such as the small intestine (duodenum, jejunum or ileum) or the large intestine.

  In other embodiments, the oral composition of the present invention is formulated in the gastrointestinal system such that the active ingredient comprising the fusion protein of the present invention is sustained-released using a known delayed release formulation.

  The Tf fusion protein of the present invention for oral delivery can bind to a receptor present in the GI epithelium. In order to facilitate this binding and receptor-mediated transport, fusion proteins of the invention are typically produced in which iron, and optionally carbonate, is bound to the moiety. The steps and methods of loading portions of the fusion protein composition of the present invention with iron or carbonate are known in the art.

  In some pharmaceutical formulations of the invention, the portion of the fusion protein may be modified to increase the affinity or affinity of the portion for iron. Such methods are known in the art. For example, mutagenesis can be used to produce a mutant transferrin moiety that binds iron more strongly than natural transferrin. In human serum transferrin, amino acids that are ligands for metal ion chelation include, but are not limited to, N-lobe amino acids Asp63, Tyr95, Tyr188, Lys206, His207, and His249; and C-lobe amino acids. Examples include Asp392, Tyr426, Tyr517, and His585 of SEQ ID NO: 3 (the number next to the amino acid indicates the position of the amino acid residue in the primary amino acid sequence, where the mature protein valine is designated as position 1. Have been). See US Pat. No. 5,986,067 (incorporated herein by reference). In one embodiment, the Lys206 and His207 residues in the N lobe are replaced with Gln and Glu, respectively.

  In some pharmaceutical formulations of the invention, the fusion protein is designed to include a cleavage site between the therapeutic protein or peptide and the moiety. Such cleavable sites or linkers are known in the art.

  The pharmaceutical composition of the present invention and the method of the present invention may comprise the addition of a transcytosis enhancer to promote the translocation of the fusion protein through the GI epithelium. Such enhancers are known in the art. Xia et al. (2000) J. MoI. Pharmacol. Experiment. Therap. , 295: 594-600; and Xia et al. (2001) Pharmaceutical Res. , 18 (2): 191-195.

  In a preferred embodiment of the invention, the oral pharmaceutical formulation is a fusion comprising a modified moiety that exhibits reduced or no glycosylation, fused to a GLP-1 protein or peptide at the N-terminus as described above. Contains protein. Such pharmaceutical compositions can also be used to treat glucose imbalance disorders such as diabetes by orally administering a pharmaceutical composition comprising an effective amount of the fusion protein.

  An effective amount of the fusion protein can be measured in a number of ways, for example, a dose calculated to relieve symptoms associated with a particular condition in a patient (eg, symptoms of diabetes). In other formulations, the dosage is calculated to include an amount of the fusion protein effective to cause a detectable change in blood glucose level in the patient. Such detectable changes in blood glucose include a decrease in blood glucose level between about 1% and 90%, or between about 5% and about 80%. Such a decrease in blood glucose level is expected to depend on the disease state being treated, and the pharmaceutical composition or method of administration can also be adjusted to achieve the desired results for each patient. In other examples, the pharmaceutical composition is formulated to adjust the method of administration such that an increased level of therapeutic protein or peptide activity in the patient, eg, a detectable increase in insulin or GLP-1 activity, is detected. . Such formulations and methods result in about 1 pg to about 150 mg / kg body weight fusion protein, about 100 ng to about 100 μg / kg body weight fusion protein, about 100 μg / kg to about 100 mg / kg body weight fusion protein, about 1 μg to about 100 μg / kg body weight fusion protein. It is possible to deliver 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. In one embodiment, the effective amount is from 300 μg / kg to 900 μg / kg. In other embodiments, the effective amount is administered once a week at a total dose of about 50 mg, 100 mg or 150 mg.

  An effective amount of the formulation can also be calculated by measuring units of therapeutic protein activity, for example, about 5 to about 500 units of human insulin, or about 10 to about 100 units of human insulin. . Measurements by mass or activity can be calculated using known standards for each therapeutic protein or peptide fused to Tf.

  When the fusion protein of the invention is used to treat diabetes, efficacy can be measured by a decrease in glycated hemoglobin (HbA1c), which is a measure of glycemic control in long-term administration. For example, an effective amount is at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, as measured by methods known in the art. At least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, or at least about It can be a dose that exhibits a 100-fold reduction or more.

  The present invention also includes a method of orally administering the pharmaceutical composition of the present invention. Such methods may include, but are not limited to, orally administering the composition by a patient or caregiver. Such administration steps include administering at intervals, eg, once or twice per day, depending on the fusion protein, the disease or the physical condition of the patient, or the individual patient. Also good. Such methods may also include administering different types of fusion proteins at various dosages. For example, the initial dose of the pharmaceutical composition may be at a relatively high level that can induce a desired effect, such as a decrease in blood glucose level. Once the desired effect is reached, the dosage may then be reduced. Such changes or adjustments to the administration protocol can be made by the attending physician or healthcare professional. In some cases, changes in the administration protocol may be made by individual patients, for example when the patient monitors blood glucose levels and administers the mTf-GLP-1 oral composition of the invention.

  The present invention also includes a method for producing the oral or pharmaceutical composition of the present invention, which comprises formulating the Tf fusion protein of the present invention into an orally administrable form. In other examples, the invention includes a method of making a composition or pharmaceutical composition of the invention, the method comprising formulating a Tf fusion protein of the invention into a form suitable for oral administration.

  Moreover, the present invention includes pulmonary delivery of Tf fusion protein formulations. Pulmonary delivery is particularly promising for the delivery of macromolecules that are difficult to deliver by other routes of administration. Since pulmonary delivered drugs are readily absorbed directly into the blood circulation via the alveolar region, such pulmonary delivery is a systemic and local delivery for treating lung disease. May be valid for both.

  The present invention provides compositions suitable for forming drug dispersions for oral inhalation (pulmonary delivery) to treat various physical conditions or diseases. The fusion protein formulation can be delivered by a variety of approaches, such as by liquid nebulizers, aerosol-based metered dose inhalers (MDI), and dry powder dispersion devices. In formulating a composition for pulmonary delivery, pharmaceutically acceptable carriers include surfactants or surfactants, and bulk carriers are generally one of the compositions of interest to a subject. To enhance stability, dispersibility, consistency and / or bulking properties to enhance pulmonary delivery.

Surfactants or surfactants promote the absorption of polypeptides through the mucosa and mucosal lining. Useful surfactants or surfactants include fatty acids and their salts, bile salts, phospholipids, or alkyl saccharides. Examples of fatty acids and their salts include the sodium, potassium and lysine salts of caprylic acid (C ₈ ), caprate (C ₁₀ ), laurate (C ₁₂ ) and myristate (C ₁₄ ). . 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 Examples include acids.

  Examples of phospholipids include single chain phospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, lysophosphatidylethanolamine, lysophosphatidylinositol and lysophosphatidylserine; or double chain phospholipids such as diacylphosphatidylcholine, diacylphosphatidylglycerol, Examples include diacylphosphatidylethanolamine, diacylphosphatidylinositol and diacylphosphatidylserine. Examples of alkyl saccharides include alkyl glucosides or alkyl maltosides such as decyl glucoside and dodecyl maltoside.

  Pharmaceutical excipients useful as carriers include stabilizers such as human serum albumin (HSA); bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusting agents or buffers; salts such as sodium chloride; It is done. These carriers may be in crystalline form, may be in amorphous form, or may be a mixture of these two forms.

  Examples of carbohydrates for use as bulking agents include monosaccharides such as galactose, D-mannose, sorbose, etc .; disaccharides such as lactose, trehalose, etc .; cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin; And polysaccharides such as raffinose, maltodextrin, dextran and the like; alditols such as mannitol, xylitol and the like. An example of a polypeptide for use as a bulking agent includes aspartame. Amino acids include alanine and glycine, with glycine being preferred.

  For conformational stability during spray drying, additives that are minor components of the composition may be included to further improve the dispersibility of the powder. These additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine and the like.

  Suitable pH adjusting or buffering agents include organic salts made from organic acids and bases, such as sodium citrate, sodium ascorbate, and preferably sodium citrate.

  A Tf fusion composition for pulmonary delivery may be packaged as a unit dosage, in which case a therapeutically effective amount of the composition is contained in a single unit dosage container, such as a blister pack, gelatin capsule, etc. Etc. The production of blister packs or gelatin capsules is typically performed by methods generally well known in the packaging art.

US Pat. No. 6,524,557 discloses an aerosol pharmaceutical formulation comprising (a) an HFA propellant; (b) a polypeptide having pharmaceutical activity dispersible in the propellant; and (c) a surfactant. And such surfactants are C ₈ -C ₁₆ fatty acids or their salts, bile salts, phospholipids, or alkyl saccharides, and such surfactants are systemic absorption of polypeptides in the lower respiratory tract. To strengthen. The present invention also provides a method for producing such formulations and the use of such formulations in the treatment of patients.

  One approach to pulmonary delivery of dry powder drugs is to utilize a handheld device with a manual pump to provide a source of pressurized gas. The pressurized gas is suddenly released through a powder dispersion device such as a venturi nozzle, and the dispersed powder becomes available for patient inhalation.

  Dry powder dispersion devices are described in numerous patents. U.S. Pat. No. 3,921,637 describes a manual pump with a needle for piercing a single capsule of powdered drug. The use of discs or strips with a plurality of containers for dosing is described in European Patent Application Publication No. EP 0467172; International Patent Application Publication Nos. WO 91/02558; and WO 93/09832; US Pat. No. 4,627,432; No. 731; 5,035,237; 5,048,514; 4,446,862; 5,048,514, and 4,446,862.

  Aerosolization of protein therapeutics is described in European Patent Application Publication No. EP 0 289 336. Aerosol therapeutic formulations are described in International Patent Application Publication No. WO 90/09781.

  The present invention provides for formulating a Tf fusion protein for oral inhalation. Such formulations comprise a Tf fusion protein and a pharmaceutical excipient suitable for pulmonary delivery. The present invention also provides for administering Tf fusion protein compositions to a subject in need thereof by oral inhalation.

GLP-1-mTf fusion protein for treating type II diabetes As discussed above, GLP-1 activates and regulates an important endocrine hormone system in the body and is also important in glucose metabolism. Play an administrative role. Unlike all other diabetes treatments on the market, treatment with GLP-1 can be restored by improving the ability of the pancreas to secrete insulin by acting as a β-cell growth factor, further improving sensitivity, Has the potential to make existing insulin levels work more efficiently. This alleviates the burden of daily monitoring of blood glucose levels and may also delay serious long-term side effects caused by blood glucose fluctuations due to diabetes. Moreover, GLP-1 can reduce appetite and lose weight. Obesity is an inherent consequence of poor control of glucose metabolism, which only has the effect of exacerbating the physical condition of diabetes.

  Since native GLP-1 is rapidly degraded in the circulation (half-life is a few minutes), its clinical application is limited. In order to maintain a therapeutic level in the circulation, it is necessary to administer a constant high dose with a pump or patch device, which increases the cost of treatment. This is inconvenient for long-term continuous use, especially when combined with any other medication that treats diabetes or monitors blood glucose levels. The GLP-1 / linker / mTf fusion protein retains GLP-1 activity, but has the long half-life, solubility and biodistribution properties of modified transferrin (mTf). These properties allow low cost, small volume, monthly s. c. (Subcutaneous) injection can be realized and this type of product is absolutely necessary for long-lasting long-lasting use.

  Preferably, the GLP-1 / linker / mTf fusion protein of the invention is used to treat diabetes or obesity. The linker may be a substantially non-spiral linker, i.e., a rigid linker, including but not limited to PEAPTD (SEQ ID NO: 13), PEAPTDPEAPTD (SEQ ID NO: 10), PEAPTDPEAPTPDPAPTD (sequence) No. 14), IgG hinge region (SEQ ID NOs: 88, 89, and 117), and IgG hinge region and PEAPTD (SEQ ID NOs: 118-123 and 126-129). In one embodiment, the GLP-1 / linker / mTf fusion protein is BRX0585 (SEQ ID NO: 12) described in Example 4, which includes GLP-1 (7-37), including modifications of A8G and K34A. ), A PEAPTDPEAPTD linker and mTf lacking N-glycosylation at positions 413 and 611 by substitution of adjacent S / T residues.

  GLP-2 can also be used as a linker that links GLP-1 to mTf. In one embodiment, the GLP-2 peptide in the fusion protein is modified to form a more stable fusion protein resulting in a more potent GLP-1. For example, the GLP-2 peptide could be modified to have a substantially reduced GLP-2 activity. The GLP-2 peptide could be modified by deleting the amino acid corresponding to H1 of the GLP-2 peptide. The GLP-2 peptide can also be modified so that cleavage by the protease is substantially reduced, where the cleavage by the protease is mediated by Yap3p. For example, an amino acid corresponding to K30 of GLP-2 could be mutated to A, for example. Alternatively, GLP-2 could be modified by deleting amino acids corresponding to K30-R34 or by deleting amino acids corresponding to H1-D8.

  In other embodiments, the fusion protein comprises at least two GLP-1 peptides at the N-terminus. Moreover, GLP-1 could be modified, for example, by adding an amino acid to the N-terminus of GLP-1. Preferably, the added amino acid is H or G. Alternatively, GLP-1 could be modified by mutating A8 to S. GLP-1 could be modified by mutating K34 to Q, A or N. GLP-1 could also be modified by deleting V33-R36.

GLP-1-mTf fusion protein in combination with other therapeutic agents In one form of the invention, the GLP-1 / linker / mTf fusion protein of the invention, eg, the protein corresponding to SEQ ID NO: 12, is at least one second It may be used in combination with therapeutic molecules to treat type II diabetes, obesity, and other diseases or conditions associated with abnormal blood glucose levels, where the second therapeutic molecule includes, for example, DPPIV inhibitors, neutral endopeptidase (NEP) 24.11 inhibitor or Glucophage ^{(R) (Glucophage (R)} ) ( metformin hydrochloride tablets) or Glucophage ^(R) XR (metformin hydrochloride sustained release tablets) is .

Glucophage ^(R) and Glucophage ^(R) XR are oral antihyperglycemic drugs for managing type II diabetes. Glucophage ^(R) XR is a sustained-release formulation of Glucophage. Accordingly, Glucophage ^(R) XR, since the drug is gradually released from the dosage form, may be administered once a day. Glucophage ^(R) promotes the reduction of glucose body production from the liver. Therefore, glucophage ^(R) is effective in controlling the blood glucose level of a patient. Glucophage ^(R) rarely causes hypoglycemia (hypoglycemia) because it does not produce any more insulin in the body.

Glucophage ^(R) also promotes the reduction of blood fat components, triglycerides and cholesterol, which are often high in people with type II diabetes. Metformin has been shown to help reduce appetite and reduce human weight by a few pounds when initiating drug intake.

  Metformin is approved for combination therapy with sulfonylurea or insulin, or monotherapy (by itself). Metformin is hypertensive in patients with insulin resistance (WO9112003-Upjohn), for clot lysis (in combination with t-PA-derivatives) (WO9108763, WO9108766, WO9108767 and WO9108765-Boehringer Mannheim) ), Ischemia and tissue oxygen deficiency (EP283369-Lipha), atherosclerosis (DE 1936274-Brunenglarber & Co., DE 2357875-Hurka), and US Pat. 205,087-ICI) has been suggested for use in the treatment of various cardiovascular diseases. In addition, it has been suggested that metformin is used in combination with prostaglandin-like cyclopentane derivatives as coronary dilators to lower blood pressure (US Pat. No. 4,182,772—Hoechst). ). In addition, metformin includes 2-hydroxy-3,3,3-trifluoropropionic acid derivatives (US Pat. No. 4,107,329-ICI), 1,2-diarylethylene derivatives (US Pat. No. 4,061,772). No.-Hoechst), substituted aryloxy-3,3,3-trifluoro-2-propionic acid, esters and salts (US Pat. No. 4,055,595-ICI), substituted hydroxyphenyl-piperidone (US Pat. , 024,267-Hoechst), and in combination with partially hydrogenated 1H-indeno- [1,2-B] -pyridine derivatives (US Pat. No. 3,980,656-Hoechst) Use is also suggested.

  Montanari et al. (Pharmacological Research, Vol. 25, No. 1, 1992) used metformin twice daily (bid) in an amount of 500 mg to give 850 mg metformin three times daily (t I.d.) describes increasing blood flow after ischemia in the same manner as when used. Sirtori et al. (J. Cardiovas. Pharm., 6: 914-923, 1984), in patients with peripheral vascular disease, metformin in an amount of 850 mg three times a day (tid) It is described to increase.

  The present invention provides for the treatment of a variety of diseases, including the combined use of a GLP-1 / linker / mTf fusion protein and one or more therapeutic agents (eg, metformin). In one embodiment, the GLP-1 / linker / mTf fusion protein and metformin can be used in combination to treat diseases and physical conditions associated with abnormal blood glucose levels, such as diabetes. Preferably, a GLP-1 / linker / mTf fusion protein and metformin can be used in combination to treat type II diabetes or obesity.

  The present invention provides for the treatment of type II diabetes and / or obesity using the GLP-1 / linker / mTf fusion protein of the present invention in combination with a thiazolidinedione such as a glitazone (rosiglitazone or pioglitazone). Thiazolidinedione reverses the insulin resistance seen in type II diabetes.

  Other therapeutic agents that can be used in combination with the GLP-1 / linker / mTf fusion protein of the present invention include, but are not limited to, DPPIV inhibitors, NEP inhibitors, sulfonylureas and sulfonylurea-like substances, peroxisome proliferator activity Receptor (PPAR) gamma modulator, PPAR alpha modulator, protein tyrosine phosphatase-1B inhibitor, insulin receptor tyrosine kinase activator, 11 beta-hydroxysteroid dehydrogenase inhibitor, glycogen phosphorylase inhibitor, glucokinase activator, Beta-3 adrenergic agonists, and glucagon receptor agonists.

Transgenic animals In one embodiment of the invention, the production of non-human transgenic animals comprising fusion constructs with a long serum half-life, high serum stability or high bioavailability of the invention is contemplated. Yes. In some embodiments, lactoferrin may be used as the Tf portion of the fusion protein to produce the fusion protein and secreted into milk.

  Numerous patents and publications have described the successful production of non-human transgenic animals, such as US Pat. No. 6,291,740 (issued September 18, 2001); US Pat. No. 6,281. , 408 (issued August 28, 2001); and US Pat. No. 6,271,436 (issued August 7, 2001), the contents of which are hereby incorporated by reference in their entirety. Are incorporated herein.

  The ability to alter the genetic makeup of animals, such as domestic mammals such as cows, pigs, goats, horses, cattle and sheep, allows for numerous commercial applications. These applications include animal production, weight gain, feed efficiency, carcass composition, milk production or content, disease resistance, high expression of exogenous proteins in a form that is easy to harvest (eg, expression in milk or blood) The production of animals with increased resistance to sex and infection by certain microorganisms, as well as the production of animals with enhanced growth rate or fertility. Animals that contain exogenous DNA sequences in their genome are referred to as transgenic animals.

  The most widely used method for the production of transgenic animals is microinjection of DNA into the pronucleus of fertilized embryos (Wall et al., J. Cell. Biochem. 49: 113 [1992]). Other methods for producing transgenic animals include infection of embryos using retroviruses or retroviral vectors. Infection of both pre-implantation 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 et al., Proc. Natl. Acad. Sci. USA 81: 7151 [1984]; Jahner et al., Proc. Natl. Acad Sci. USA 82: 6927 [1985]; (Proc. Natl. Acad Sci. USA 82: 6148-6152 [1985]; Stewart et al., EMBO J. 6: 383-388 [1987]).

  An alternative means of infecting embryos with retroviruses is the injection of virus or virus-producing cells into the blastocoel of mouse embryos (Jahner, D. et al., Nature 298: 623 [1982]). The introduction of transgenes into the germline of mice using retroviral infection into the uterus of mid-gestation mouse embryos has been reported (Jahner et al., Supra [1982]). Infection of bovine and sheep embryos with retroviruses or retroviral vectors to produce transgenic animals has been reported. These protocols include microinjection of retroviral particles or cell growth arrest (ie, mitomycin C treatment) that places retroviral particles into the periplasmic space of fertilized or early embryos (PCT International Publication WO 90/08832 [1990]; And Haskell and Bowen, Mol.Reprod.Dev., 40: 386 [1995] PCT International Application Publication No. WO 90/08832 describes the wild-type feline leukemia virus B into the clonal space of sheep embryos at the 2-8 cell stage. Fetuses derived from injected embryos have been shown to contain multiple insertion sites.

  US Pat. No. 6,291,740 (issued September 18, 2001) used retroviral vectors (eg, vectors derived from murine leukemia virus [MLV]) to transduce dividing cells, Describes the production of transgenic animals by introducing exogenous DNA into pre-mature and mature unfertilized oocytes (ie, pre-fertilized oocytes). The patent also describes methods and compositions driven by the cytomegalovirus promoter, as well as methods and compositions for murine breast cancer LTR expression of various recombinant proteins.

  US Pat. No. 6,281,408 (issued August 28, 2001) describes a method for producing transgenic animals using embryonic stem cells. Briefly, embryonic stem cells are used in the co-culture of mixed cells and morulas to produce transgenic animals. Prior to co-culture, exogenous genetic material is introduced into embryonic stem cells, for example, by electroporation, microinjection or retroviral delivery. ES cells transfected in this way are selected for gene integration via a selectable marker such as neomycin.

  US Pat. No. 6,271,436 (issued on Aug. 7, 2001) describes the production of transgenic animals using the following method, which isolates primordial germ cells and these To produce a cell line derived from primordial germ cells, transform both primordial germ cells and cultured cell lines, and use these transformed cells and cell lines to transform transgenic animals. Including production. Since the production efficiency of transgenic animals becomes extremely high, it becomes possible to utilize homologous recombination in the production of transgenic animal species other than rodents.

Gene Therapy In one embodiment of the present invention, the use of a modified transferrin fusion construct for gene therapy in which a modified transferrin protein or transferrin domain is linked to a therapeutic protein or peptide is contemplated. The modified transferrin fusion constructs with long serum half-life or high serum stability of the present invention are ideally suited for treatment by gene therapy.

  Describe the successful use of gene therapy to express soluble fusion proteins. Briefly, gene therapy by injecting an adenoviral vector containing a gene encoding a soluble fusion protein consisting of cytotoxic lymphocyte antigen 4 (CTLA4) and the Fc portion of human immunoglobulin G1 has recently been developed in recent years by Ijima et al. ( (June 10, 2001) Human Gene Therapy (USA) 12/9: 1063-77. In the gene therapy of this application, a mouse model of type II collagen-induced arthritis has been successfully treated by intra-articular injection of the vector.

  Gene therapy is also described in a number of US patents, for example, US Pat. No. 6,225,290 (issued May 1, 2001); US Pat. No. 6,187,305 (2001). Issued on Feb. 13); and US Pat. No. 6,140,111 (issued on Oct. 31, 2000).

  US Pat. No. 6,225,290 describes a method and construct that incorporates genetically engineered intestinal epithelial cells of a subject mammal so that a gene expressing a protein having a desired therapeutic effect can be activated. provide. Intestinal cell transformation is accomplished by administration of a formulation composed primarily of naked DNA, which can be administered orally. While oral or other gastrointestinal routes of administration provide a simple method of administration, the use of naked nucleic acids avoids the complications associated with the use of viral vectors to achieve gene therapy. The expressed protein is secreted directly into the gastrointestinal tract and / or the bloodstream, resulting in a therapeutic blood level of the protein, thereby treating patients in need of the protein. Transformed intestinal epithelial cells provide a short-term or long-term treatment for diseases associated with a deficiency of a particular protein, or diseases that can be treated with treatment by overexpression of the protein.

  US Pat. No. 6,187,305 provides a method of targeting genes or DNA in vertebrate cells, particularly mammalian cells. Briefly, DNA is introduced into primary or secondary cells of vertebrate origin by homologous recombination or DNA targeting and introduced into preselected sites of genomic DNA of primary or secondary cells. .

  US Pat. No. 6,140,111 (issued October 31, 2000) describes a retroviral gene therapy vector. The described retroviral vector contains an insertion site for the gene of interest, which can express high levels of protein derived from the gene of interest in a wide variety of transfected cell types. Also described are retroviral vectors lacking a selectable marker that allow them to be adapted for human gene therapy in the treatment of a wide variety of conditions without co-expressing marker products such as antibiotics. be able to. These retroviral vectors are particularly suitable for use with certain packaging cell lines. The ability of retroviral vectors to insert into the genome of mammalian cells makes them particularly promising candidates for use in genetic therapy for human and animal genetic diseases. Typically, genetic therapy involves (1) adding new genetic material to a patient's cells in vivo, or (2) removing patient cells from the body and adding new genetic material to the cells. , Reintroducing them into the body, ie in vitro gene therapy. For a discussion of how to perform gene therapy using retroviral vectors in various cells, see, for example, US Pat. No. 4,868,116 issued September 19, 1989, and December 25, 1990. 4,980,286 (epithelial cells), WO 89/07136 (hepatocytes) published on August 10, 1989, EP 378,576 (fibroblasts) published on July 25, 1990 And WO 89/05345 published June 15, 1989 and WO / 90/06997 (endothelial cells) published June 28, 1990 (the disclosures of which are herein incorporated by reference). ).

Kits comprising transferrin fusion proteins In a further embodiment, the invention provides kits comprising GLP-1 / mTf, or GLP-1 / linker / mTf fusion proteins, which kits may be used for therapeutic applications, for example. Alternatively, it may be used for purposes other than treatment. The kit includes a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. Such containers can be formed from a variety of materials such as glass or plastic. Such a container can contain a composition comprising a fusion protein that is effective for therapeutic uses as described above, or for non-therapeutic uses. The active substance in the composition is a therapeutic protein. A label on the container indicates that the composition is to be used for a specific therapeutic or non-therapeutic application, and further for use either in vivo or in vitro, such as those described above. The description about may be displayed.

  The kits of the present invention will typically include the containers described above and one or more other containers containing materials desirable from a commercial and user standpoint, such materials , Buffers, diluents, filters, needles, syringes, and package inserts including instructions for use.

  Even if there is no further explanation, those having ordinary skill in the art will be able to make the present invention using the above explanation and the following illustrative examples, and use them to claim. It is considered possible to implement the proposed method. For example, one skilled in the art should readily be able to measure the biological activity of the fusion protein constructs of the invention both in vitro and in vivo compared to the corresponding activity of the therapeutic moiety in their unfused state. is there. Similarly, a person skilled in the art can easily determine the serum half-life and serum stability of the construct according to the present invention. Therefore, the following examples specifically point out preferred embodiments of the present invention, but should not be construed as limiting the undisclosed remainder in any way.

Example 1: GLP-1 / transferrin fusion protein GLP-1 is a peptide that regulates insulin secretion. GLP-1 has anti-diabetic activity in human subjects suffering from diabetes, particularly type II diabetes. Like other peptides, GLP-1 has a short plasma half-life in humans. The present invention provides fusion proteins comprising GLP-1 fused to mTf having a long half-life, and pharmaceutical compositions of such fusion proteins, for the treatment of diseases associated with abnormal blood glucose levels.

  The invention also provides a fusion protein comprising a GLP-1 analog and mTf. In one embodiment of the invention, the GLP-1 analog comprises an additional His residue at the N-terminus. This His residue could be added to the N-terminus of GLP-1 or inserted after the His residue at the N-terminus of GLP-1. In other embodiments, the GLP-1 analog comprises an amino acid substitution at position 2. For example, Ala in GLP-1 (7-36) or GLP-1 (7-37) peptide is substituted with other amino acids.

  In this example, the process of producing a GLP-1 / mTf fusion protein is described. The same process can be used to produce transferrin fusion proteins containing GLP-1 peptide analogs.

  The amino acid sequences of GLP-1 (7-36) and GLP-1 (7-37) can be used to produce a GLP-1 / mTf fusion protein.

  For example, the peptide sequence of GLP-1 (7-36) may be back translated into DNA and codons optimized for yeast:

  The primers are specifically designed so that 5'XbaI and 3'KpnI sticky ends are formed after annealing and can be directly ligated to pREX0052 cleaved with XbaI / KpnI at just the 5 'end of the leader sequence and the N-terminus of mTf. Designed. Alternatively, they may be manipulated so that the other sticky end is ligated to another vector.

  After annealing and ligation, clones were sequenced to confirm correct insertion. This vector was named pREX0094. A cassette was excised from pREX0094 using NotI and subcloned into pSAC35, a yeast vector cleaved with NotI, to produce pREX0100.

  This plasmid was then electroporated into the host Saccharomyces cerevisiae yeast strain and transformants were selected for leucine prototrophy on minimal media plates. Expression was measured by growth in liquid minimal medium and supernatants were analyzed by SDS-PAGE, Western blot and ELISA.

Example 2: Addition of linker to improve activity Steric hindrance by mTf carrier may reduce the ability of GLP-1 to bind to its receptor, based on the hypothesis that GLP-1 mTf fusion The addition of a linker or spacer between GLP-1 and mTf as a method to improve activity was investigated. Thus, it was estimated that steric hindrance could be reduced and that activity was restored by inserting a linker to increase the distance between GLP-1 and mTf. Three different types of linkers were tested: a flexible linker, a short flexible linker, and a rigid linker.

The long flexible linkers tested were repeats of (SGGG) ₃ and linkers based on the GLP-2 sequence (“GLP-2 linker”). The linker (SGGG) ₃ repeat and similar sequences were used as linkers, for example in single chain Fv fragments with Vh and Vl linked together. The GLP-2 linker was tested because GLP-2 is in close proximity to GLP-1 in propeptides that naturally produce glucagon, GLP-1 and GLP-2 by processing. Since the intervening peptide sequence that is cleaved to give two GLP peptides is the N-terminal residue in GLP-2, it is removed and GLP-2 becomes inactive if it is cleaved from the linker by any means I did it. Furthermore, since GLP-2 is a natural sequence, it was speculated that it is unlikely to be immunogenic.

  The short flexible linkers tested were S, SS and SSG. This class of linkers was tested to determine the effect of flexibility at the junction of the two parts in the absence of GLP-1 spatially separated from mTf.

Rigid linkers, ie linkers that are not substantially helical, are designed based on the idea that GLP-1 clearly separates the two parts necessary to discover its receptor, although they are not mobile did. The use of the naturally occurring sequence between the N and C lobes of Tf, that is, the sequence naturally occurring in Cys331 to Cys339 (PEAPTDE) has resulted in the use of sequences found in high yields in the circulatory system, Doing so lowered the concern that they could be immunogenic. It was expected that the presence of a proline residue in this sequence would result in a less flexible peptide, similar to the proline residue at the second residue position of mTf. The linkers created were PE, PEA, PEAPTD, (PEAPTD) ₂ , (PEAPTD) 3, and (PEAPTD) 4.

  The hinge region from human IgG1 was investigated as an alternative, high generation amount of naturally occurring linker sequence. The IgG hinge is a region of a sequence that links the constant (Fc) and variable region (Vh) of the human IgG1 heavy chain. In the native protein, this hinge softens the IgG molecule upon antigen binding. This sequence is actually used to link the peptide or protein to the N-terminus of the Fc fusion. It has also been used to form flexible regions between two distinct protein domains in recombinant proteins (Doyle et al., 2003 Regulatory Peptides, 114, 153-158). A sequence commonly referred to as an IgG hinge is EPKSCDKTHTCPPCP (residues 224-238) (SEQ ID NO: 88), which has a cysteine that forms a disulfide bond with a light chain and a disulfide bond with the other heavy chain Fc region of an antibody It contains two cysteines that form.

This sequence was extended to 25 residues: V EPKSCDKTHTCPPCP APELLGGPS (SEQ ID NO: 124) to bring the linker to the desired length. This linker was modified by mutating the cysteine residue to serine to prevent disulfide bond formation of the free cysteine ( VEPKSSDKTHTSPPSP APELLGGPS (SEQ ID NO: 89)). This cysteine residue can be similarly substituted with alanine to produce VEPKSADKTHTAPAPAPELLGGPS (SEQ ID NO: 117). Furthermore, this cysteine residue and serine residue may be substituted with alanine to produce VEPKAADKTHTAPAPAPELLGGPA (SEQ ID NO: 125). Both SEQ ID NOs: 117 and 125 have reduced O-glycosylation as a result of removal of the serine residue. The hinge sequence described in this paragraph may be used with a single PEAPTD sequence, or with a PEAPTD multimer such as (PEAPTD) ₂ to further extend the linker.

  The C-terminal sequence of exendin-4 plays a major role in GLP-1 receptor binding affinity, and further adds a 9-amino acid C-terminal sequence: SSGAPPPS (SEQ ID NO: 15) to the GLP-1 peptide. Have increased their affinity for the GLP-1 receptor (Heuser et al., 2004 Int. J. Cancer 110: 386-394). This sequence was used as a linker with the IgG hinge.

A number of linkers were tested. The results are summarized below.

Construction of a long flexible linker (SGGG) ₃ (SEQ ID NO: 16) Primer such that when the linker construct pREX0216 [ΔL GLP-1 (7-36) (SGGG) ₃ mTf] is annealed together, an XbaI / KpnI overhang is generated. : Produced by designing P0281, P0282, P0283 and P028.

These were annealed by mixing 10 μL of each primer (20 pmol / μL) in a tube containing 10 μL of 10 × PCR buffer (without MgCl ₂ ). The reaction was performed at 68 ° C. for 5 minutes, 37 ° C. for 10 minutes, and 20 ° C. for 10 minutes. The annealed product was washed (Qiagen gel extraction kit, PCR protocol) and the product was cloned into pREX0095 digested with XbaI / KpnI to create pREX0216. The clone DNA was sequenced to determine if the insert was correct. The correct clone was obtained and the expression cassette was extracted by NotI / ScaI digestion and cloned into NotI digested and SAP-treated pSAC35, creating pREX0217. Clones were screened for inserts in the same orientation as the LEU2 gene using XbaI / SalI digestion. The pREX0217 DNA was transformed into a Saccharomyces cerevisiae strain (control strain) by electroporation using a BioRad Gene Pulser in a 2 mm cuvette (see WO05061718) on a BMM / S plate And then plated. A yeast stock of this construct was made: Y0160. Cultured to 52 ng / mL by flask shaking culture (from ELISA).

Fermentation of (SGGG) ₃ linker construct pREX0217 (F0079) produced only 0.218 μg / mL (by ELISA) (Table 1). However, the activity was greatly improved and the EC ₅₀ was 2.274 nM (cAMP060204-1) compared to about 0.6 nM for the GLP-1 peptide.

ΔL GLP-1 (7-36) GLP-2 linker mTf construct pREX0213 is made by designing linkers that can be annealed together: P0273, P0274, P0275, P0276, P0277, P0278, P0279 and P0280, They were cloned into pREX0095.

Basically, these primers were re-used with GLP-1 linked GLP-2 linker, each primer (20 pmol / μL) in a tube containing 10 μL of 10 × PCR buffer (without MgCl ₂ ). Annealed by mixing 10 μL. The reaction was performed at 68 ° C. for 5 minutes, 37 ° C. for 10 minutes, and 20 ° C. for 10 minutes. In order to complete the annealing, 5 μL of T4 DNA ligase was added to the reaction solution and incubated at room temperature for 2 hours. A PCR reaction was constructed using P0273 and P0280 as outer primers to amplify the annealed product. PCR reaction conditions are as follows: 94 ° C for 1 minute for 1 cycle, 94 ° C for 40 seconds, 55 ° C for 40 seconds, and 72 ° C for 1 minute for 25 cycles, followed by a final extension at 72 ° C. 7 minutes. The resulting PCR product was digested with XbaI / KpnI and cloned into pREX0095 digested with XbaI / KpnI to create pREX0213. The DNA between the two restriction sites of the clone was sequenced to check that the sequence was correct. Once the correct clone was obtained, the expression cassette was extracted by NotI / ScaI digestion and cloned into pSAC35 digested with NotI and treated with SAP to create pREX0214. Clones were screened for inserts in the same orientation as the LEU2 gene using XbaI / SalI digestion. PREX0214 DNA was transformed into a Saccharomyces cerevisiae control strain (WO05 / 061718) by electroporation using Bio-Rad Gene Pulser in a 2 mm cuvette and plated on BMM / S plates. Colonies were formed and grown in BMM / S shake flask culture (30 ° C., 200 rpm), and the production was determined to be 5 ng / mL (by anti-Tf-ELISA). A yeast stock (Y0172) of this construct was made.

The GLP-2 linker construct pREX0214 was fermented (F0080). Their production was 9 μg / mL (Table 1). Similar to the (SGGG) ₃ linker construct, the activity was greatly improved with an EC ₅₀ of 5.9.

Construction of short flexible linkers Short flexible linkers S, SS and SSG were made with various constructs.

Construction of short flexible linker variants was achieved by designing overlapping PCR primers.

  The linker was inserted between the GLP-1 mutant and mTf by one round of PCR using P0025 in combination with reverse mutagenesis primer and P0012 in combination with forward mutagenesis primer, Two rounds were performed and two one round products were combined with P0025 and P0012. The reaction conditions for one round were 15 cycles of 94 ° C. for 1 minute, 50 ° C. for 1 minute, and 72 ° C. for 1 minute, followed by a final extension at 72 ° C. for 10 minutes. Two rounds of reaction conditions consisted of 1 cycle at 94 ° C. for 1 cycle, 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 cycle for 25 cycles, followed by a final extension at 72 ° C. for 7 cycles. For minutes. The PCR product was then digested with AflII / BamHI and cloned into pREX0052 cut with AflII / BamHI. DNA between the restriction sites of the clone was sequenced to confirm correct insertion. The correct clone was selected and the expression cassette was extracted by NotI / PvuI digestion. The expression cassette was cloned into pSAC35 digested with NotI and treated with SAP. This clone was screened for inserts in the same orientation as the LEU2 gene using XbaI / SalI digestion. DNA was obtained by electroporation using Bio-Rad Gene Pulser in a 2 mm cuvette. It was transformed into S. cerevisiae strain A (WO05 / 061718). Stocks were made from this construct and analyzed for DNA sequence to confirm identity.

Similar to construct pREX0456, a single S linker produced 37 μg / mL from the fermentor (F0140). The productivity of the SS linker (pREX0507) and SSG linker (pREX0509) from the fermentor was 1200 μg / mL (F151 and 158), averaging 1346 μg / mL, respectively (F149, 152, 153 and 156) ( Table 3). Although the productivity was excellent, the activity was almost the same as that of the construct without a linker (pREX0505F150, 163, 164, 169, 170).

Construction of Rigid Linker The following shows a rigid linker construct prepared based on a sequence in which the N and C lobes of Tf are linked.

  In addition to the insertion of the PEAPTD-based linker, two further changes were made to the GLP-1 construct. An A8G mutation was incorporated for DPP IV resistance. To eliminate the possibility of kinking in the α-helix backbone of GLP-1 that might enhance the activity of GLP-1 analogs such as exendin-4, the G22E mutation was incorporated.

These variants were made by using mutagenic PCR primers in two rounds of PCR.

In one round of PCR, the forward mutagenesis primer was combined with P0012 and the reverse mutagenesis primer was combined with P0025. In two rounds of PCR, two products were combined with P0025 and P0012. The reaction conditions for one round were 15 cycles of 94 ° C for 1 minute, 50 ° C for 1 minute, and 72 ° C for 1 minute, followed by a final extension of 72 ° C for 10 minutes. Two rounds of reaction conditions consisted of one cycle at 94 ° C for 1 cycle, 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute for 25 cycles, followed by a final extension at 72 ° C for 7 cycles. For minutes. The PCR product was then digested with AflII / BamHI and cloned into pREX0052 cut with AflII / BamHI. DNA between the restriction sites of the clone was sequenced to confirm correct insertion. The correct clone was selected and the expression cassette was extracted by NotI / PvuI digestion. This expression cassette was cloned into pSAC35. This clone was screened for inserts in the same orientation as the LEU2 gene using XbaI / SalI digestion. DNA was obtained by electroporation using Bio-Rad Gene Pulser in a 2 mm cuvette. S. cerevisiae strain A was transformed (see WO05 / 061718). Stocks were made from this construct and analyzed for DNA sequence to confirm identity.

Both fermentations using PEA or PEAPTD based linker constructs showed excellent productivity of about 1 g / L (by ELISA) (Table 6). The PEA linker construct, pREX0518, has a potency of 375 nM, which is several times lower than that without the linker (pREX0505-2162 nM). The (PEAPTD) ₂ linker increased potency and productivity. The combination of A8G and (PEAPTD) ₂ yields the highest productivity and efficacy combination that is closest to the productivity and efficacy of GLP-1 peptide in cAMP analysis.

Mutants containing a single PEAPTD linker were made and fermented. Two construct variants were used to make a (PEAPTD) ₂ linker. One of them (pREX0585) was fermented to give a productivity of 1900 μg / mL (by ELISA) and a potency of 3 nM (Table 6).

Construction of IgG Hinge and Exendin-4 Linker The pREX0556 to pREX0559 were produced with the primers listed in Table 7 using a two-step PCR protocol.

  pREX0556 contains GLP-1 (7-37; A8G; G22E; K34A) fused to the N-terminus of transferrin with an IgG hinge linker (VEPKSSDKTHTSPPSPAPELLGGPS). In the first step, the 5 'segment from pREX0555 was amplified using primers P0723 and P0676 and the 3' segment from pREX0523 was amplified using P0677, P0724 and P0745. Since P0677 is a relatively long primer (87 bp), a shorter primer P0745 was included to improve amplification efficiency. The reaction conditions were 94 ° C. for 1 minute, 94 ° C. for 40 seconds, 55 ° C. for 40 seconds, and 72 ° C. for 1 minute for 15 cycles, followed by extension at 72 ° C. for 7 minutes. The 5 'and 3' segments were ligated by amplification using P0723 and P0724 using the above conditions for 18 cycles. The same PCR conditions were used for all four constructs. The product was digested with AflII / BamHI, cloned into the AflII / BamHI site of pREX0549 and sequenced. The expression cassette for the GLP-1 transferrin fusion was excised from pREX0549 by NotI / PvuI digestion, cloned into the NotI site of pSAC35, and analyzed for DNA sequence.

  pREX0557 contains exendin-4 C-terminus (CEx, SSGAPPPS) and GLP-1 (7-37; A8G; G22E; K34A) fused to the N-terminus of transferrin with an IgG hinge as a linker. In the first step, the 5 'segment was amplified from pREX0555 using primers P0723 and P0678 and the 3' segment was amplified from pREX0523 using P0679, P0724 and P0745. The 5 'and 3' segments were ligated by amplification with P0723 and P0724. The product was cloned into pCR2.1, DNA sequenced, then digested with AflII / BamHI and cloned into pREX0549. The expression cassette for the GLP-1 transferrin fusion was excised from pREX0549 by NotI / PvuI digestion, cloned into the NotI site of pSAC35, and analyzed for DNA sequence.

  pREX0558 contains GLP-1 (7-37; A8G; G22E; K34A) fused to the N-terminus of transferrin with PEAPTD and IgG hinge linker. In the first step, the 5 'segment was amplified from pREX0555 using primers P0723 and P0680, and the 3' segment was amplified from pREX0523 using P0681, P0724 and P0745. The 5 'and 3' segments were ligated by amplification at P0723 and P0724. The product was cloned into pCR2.1, DNA sequenced, digested with AflII / BamHI and cloned into pREX0549. The GLP-1 transferrin expression cassette was excised from pREX0549 by NotI / PvuI digestion, cloned into the NotI site of pSAC35, and subjected to DNA sequence analysis.

  pREX0559 contains GLP-1 (7-37; A8G; G22E; K34A) fused to the N-terminus of transferrin with a CEx linker. In the first step, the 5 'segment was amplified from pREX0555 using primers P0723 and P0719 and the 3' segment was amplified from pREX0523 using P0720 and P0724. The 5 'and 3' segments were ligated by amplification at P0723 and P0724. The product was digested with AflII / BamHI, cloned into pREX0549 and sequenced. The GLP-1 transferrin expression cassette was excised from pREX0549 by NotI / PvuI digestion, cloned into the NotI site of pSAC35, and subjected to DNA sequence analysis.

The construct was transformed into Saccharomyces cerevisiae strain A (see WO 05/061718) by electroporation using Bio-Rad Gene Pulser in a 2 mm cuvette and plated on BMM / S medium. Transformants were grown on BMM / S for 4 days and then applied on BMM / S plates containing anti-transferrin polyclonal serum (Calbiochem catalog number 616423). After 4 days, precipitated antibody / transferrin complex loops were visualized for all four constructs. The transformed clones were also streaked on a BMM / S plate to isolate a single colony. BMM / S shake flask cultures were inoculated from the patch, grown at 30 ° C. for 3 days and analyzed by ELISA to quantify secreted transferrin fusion protein. In all constructs, the addition of an IgG linker increased the productivity in shake flasks (Table 8) and the maximum productivity was observed with the PEAPTD IgG linker (pREX0558). This construct showed an approximately 7-fold increase in GLP-1 transferrin compared to the linker-free construct (pREX0505). When the CEx linker was added alone (pREX0559), the productivity did not increase much (Table 8).

Single cell stocks were made for constructs pREX0556-559 (Y0389, Y0384, Y0385 and Y0380, respectively) and DNA sequence analysis was performed to confirm identity. Two fermentations (F0187 and F0190) were performed on the IgG construct (pREX0556), and the concentration was measured by SDS-PAGE gel. The estimated yields were 679 and 719 μg / mL. In the fermentation F0191 of the PEAPTD IgG construct (pREX0558), a yield of 960 μg / mL was obtained by concentration measurement. The efficacy of the GLP-1 fusion was determined by cAMP analysis. F0187 and The EC ₅₀ values of F0190 (IgG) is 6.6nM and 8.6 nm, EC ₅₀ of F0191 (PEAPTD IgG) was 5.9 nM.

ELISA Data Table 9 below summarizes the ELISA data obtained using constructs containing GLP-1 and various linkers.

Conclusion 1. Long flexible linkers (SGGG) ₃ and GLP-2 were less productive, whereas GLP-2 linkers are extremely powerful.
2. The short flexible linkers, S, SS and SSG showed no improvement in potency, while the S linker decreased dramatically in productivity, while the SS and SSG linkers maintained productivity levels.
3. A linker that is not substantially helical has shown the most advantageous results. PE and PEA linkers showed no effect on either productivity or potency, but the PEAPTD linker, and in particular the double linker, (PEAPTD) ₂ , improved productivity and improved the natural GLP-1 peptide. Efficacy was brought within a range of about 5 to 10 times. Although the effects of 3 or 4 replicates were not tested for efficacy, productivity did not show the opposite result.
4). IgG linkers resulted in significant improvements in productivity, especially when combined with PEAPTD linkers. It has also been found that in vitro results in fusion potency similar to that of the native GLP-1 peptide, equivalent to the G22E mutation.
5. The construct pREX0585 nL GLP-1 (7-37; A8G; K34A) (PEAPTD) ₂ mTf showed the greatest improvement in potency, with the least difference from the native sequence.

Example 3: Pharmacokinetics of GLP-1 analog / linker / mTf fusion protein In this example, a GLP-1 analog / linker / mTf fusion protein was made and its pharmacokinetics was analyzed.
The present invention provides a fusion protein comprising a GLP-1 analog fused to mTf via a linker. In one embodiment, the GLP-1 (7-37) analog comprises amino acid substitutions at positions 8 and 34 (which correspond to amino acids 2 and 28 of SEQ ID NO: 6). For example, the GLP-1 (7-37) analog comprises a substitution of alanine with glycine at position 8 and a substitution of lysine with alanine at position 34. This prevents cleavage by dipeptidyl peptidase IV (substitution at the residue at position 8) and cleavage by the second enzyme (substitution at the residue at position 34).

GLP-1 (7-37; A8G, K34A) is fused to mTf via a linker such as (PEAPTD) ₂ to increase the productivity of the fusion protein. The amino terminus of GLP-1 (7-37; A8G, K34A) is fused to the nL leader sequence.

The complete nucleic acid and amino acid sequences of the fusion protein nL GLP-1 (7-37; A8G, K34A) (PEAPTD) ₂ mTf (see plasmids pREX0584 and pREX0585) are shown below (SEQ ID NOs: 65 and 66).

Pharmacokinetics of GLP-1 (A8G, K34A) -PEAPTDPEAPTD-mTf Two cynomolgus monkeys per group were injected subcutaneously or intravenously with 2250 μg / kg GLP-1-Tf fusion protein as measured by A ₂₈₀ . The fusion protein used was GLP-1 (A8G, K34A) -PEAPTDPEAPTD-mTf. Analysis of plasma samples uses a sandwich ELISA specific for GLP-1-Tf, using a monoclonal antibody against GLP-1 to capture the fusion protein and a polyclonal antibody against Tf to detect the bound protein. Was done. Plates were coated with rabbit anti-mouse Fab (1 μg / well), then washed and blocked with 1% BSA in PBS. Thereafter, mouse anti-GLP-1 MAb was added as a capture antibody. The plates were then washed and samples diluted with standard curves or 1% BSA in PBS were added to the plates. After incubation, the plate was washed, and biotinylated anti-human transferrin antibody was added as a detection antibody. While washing between each step, an antibody bound with HRP-streptavidin and a fluorescent substrate was detected.

  For bioassay, samples were incubated with CHO cells transfected with rat GLP-1 receptor. GLP-1 receptor (GLP-1R) is a membrane-bound G protein-coupled receptor, and when bound to a ligand, adenylyl cyclase is activated, resulting in a concentration-dependent increase in intracellular cAMP. CAMP produced by cells in response to GLP-1-Tf in plasma was measured by cAMP ELISA. The cAMP produced by the sample was compared with cAMP produced by known concentrations of GLP-1-Tf. PK analysis was performed using PK Summit software.

  The results are shown in FIG. The terminal half-life in these studies was calculated to be about 30 hours. This is comparable to the half-life of GLP-1 peptide being about 90 seconds in humans. In addition, a comparison of the area under the concentration curve for the subcutaneous or venous route shows high bioavailability via the subcutaneous route.

Example 4: Construction and use of BRX0585 for diabetes treatment and weight loss
Expression of BRX0585 in yeast An expression cassette was generated encoding GLP-1 (7-37) linked to the N-terminus of non-glycosylated human transferrin via the PEAPTDPEAPTD linker peptide (SEQ ID NO: 10). Of note, such as PEAPTD (SEQ ID NO: 13), IgG hinge linker (SEQ ID NO: 88, 89, and 117), IgG hinge linker and PEAPTD (SEQ ID NO: 118-123), and PEAPTDPEAPTPDPAPTD (SEQ ID NO: 14) Alternative linkers that are not substantially helical could also be used.

  This cassette was designed to replace the second amino acid of the GLP-1 moiety to prevent its effect on the GLP-1 moiety of DPP-IV. The transferrin sequence was modified so that two N-linked glycosylation sites were removed. This cassette was cloned in a high copy number plasmid (see FIG. 7; SEQ ID NO: 11) and expressed at high levels in Saccharomyces cerevisiae. Similarly, a transferrin sequence with the same modifications used to remove two N-linked glycosylation sites was cloned into a high copy number plasmid, It was expressed in cerevisiae. Using several chromatographic steps, the protein was purified until the purity was greater than 99.9% as measured by SDS-PAGE, SE-HPLC, and the N-terminus was sequenced. The resulting Tf and GLP-1 fusion protein corresponds to the sequence of SEQ ID NO: 12. In this example, the fusion peptide of SEQ ID NO: 12 is said to have the same meaning as BRX0585 and GLP-1-Tf.

GLP-1-Tf was obtained by stably transfecting Chinese hamster ovary (CHO) cells in which GLP-1 receptor had been activated in vitro with GLP-1 receptor (CHO / GLP-1R cells). (Montrose-Rafizadeh et al., 1997, J. Biol. Chem. 272: 21201-21206), cultured in Ham's F-12 medium (Cellgro) at 37 ° C. with 5% CO ₂ . CHO / GLP-1R cells were rinsed with Krebs-Ringer buffer (KRB, Sergro) and incubated at 37 ° C. for 1 hour to reduce endogenous intracellular cAMP levels. Subsequently, this was incubated briefly in KRB containing 4 mM IBMX (Sigma) to inhibit intracellular phosphodiesterase that degrades cAMP. Triplicate wells of the cells were treated with serial dilutions of GLP-1-Tf, GLP-1 (7-37) or Exendin-4 (the latter two from Bachem) at 37 ° C. for 60 minutes. The supernatant was then removed and the cells were lysed in 0.1N HCl. Cell lysates were analyzed using a competition-based chemiluminescent enzyme immunoassay (Assay Designs Inc.) to determine intracellular cAMP concentrations.

The GLP-1 receptor (GLP-1R) is a membrane-bound G protein-coupled receptor, and when bound to a ligand, adenylyl cyclase is activated and a concentration-dependent increase in intracellular cAMP occurs. GLP-1-Tf was found to activate adenylyl cyclase in CHO cells transfected with GLP-1R (CHO / GLP-1R). This results in a dose-dependent increase in intracellular cAMP levels and decreased activity, but the potency was similar compared to native GLP-1 and exendin-4 (EC ₅₀ : GLP-1 -2.28, 0.16 and 0.05 nM) in Tf, GLP-1 and exendin-4, respectively (Figure 8a).

The rat insulinoma cell line, RIN 1046-38 cells, was purchased from Samuel A. Obtained from Dr. Clark and cultured as described above (Wang et al., 2001, Endocrinology, 142: 1820-1827) in M199 medium (Sergro) at 37 ° C. with 5% CO ₂ . RIN1046-38 cells grown in 12-well plates and reaching 50-60% confluency were washed with glucose-free insulin secretion buffer (Biofluids) and the cells were washed with GLP-1 and GLP-1-Tf. Were incubated for 1 hour at 37 ° C. Also, exendin-9-39 (1 μmol / l: Bachem), an inhibitor of GLP-1R, was added to one set of experiments overnight. The supernatant was then collected and stored at −80 ° C. for determination of insulin content by ELISA (Crystal Chem). Cells were lysed and protein content was quantified using the Bradford method.

Islets were isolated from Sprague-Dawley rats as we described above (but with some modifications) (Wang et al., 1997, J. Clin. Invest. 99: 2883-2889). Briefly, the pancreas was digested with type XI collagenase (Sigma), and islets were separated by Ficoll-pack concentration gradient (Amersham Biosciences). The isolated islets are washed several times with Hanks balanced salt solution (Biosource) containing 0.2% bovine serum albumin (BSA, Sigma), manually picked up, 5% CO ₂ , 37 ° C. And cultured overnight in M199 medium supplemented with 5 mM glucose. Rat islets were then treated similarly to RIN cells as described above (30-40 per well).

  GLP-1-Tf was found to stimulate insulin secretion in a concentration-dependent manner in both rat insulinoma cells and isolated rat islets (FIGS. 8b and c).

Measurement of GLP-1-Tf in body fluids Male cynomolgus monkeys (Macaca fascicularis) (N = 4) received GLP-1-Tf (2.25 mg / kg) intravenous (IV) and subcutaneous (SC) boluses. Blood was collected at the times specified in FIG. 8d. Blood was centrifuged and serum was stored at −80 ° C. for GLP-1-Tf analysis. All animal experiments were performed with an approved protocol according to the Animal Care and Use Committee of the NIA.

  The concentration of GLP-1-Tf in the body fluid was measured using a sandwich ELISA. For this analysis, 200 ng of anti-GLP-1 monoclonal antibody (Antibody Shop) was immobilized on a microtiter plate coated with anti-mouse IgG. After washing, samples or standards were added to the plates and incubated overnight at 37 ° C. The plate was then washed and incubated with biotinylated chicken anti-human transferrin antibody (Fitzgerald Laboratories). Washing was repeated and the plates were incubated with horseradish-peroxidase (HRP) -streptavidin. Plates were washed again and bound HRP was measured using Pierce's Quanta Blu fluorogenic substrate. Measurements were made using a Spectramax Gemini fluorescent plate reader and Softmax Pro software. The buffer used in all steps was phosphate buffered saline (PBS) containing 1% BSA and 0.05% Tween 20.

After treatment with both routes of administration, the half-life (t _1/2 ) of GLP-1-Tf in serum is about 44 hours, comparing the subcutaneous and intravenous profiles to substantially 100 % Bioavailability was shown (FIG. 8d). Furthermore, by treating CHO / GLP-1R with serum from the monkey that received the injection, GLP-1-Tf measured by ELISA is biologically active, and in ELISA, It was found that the slope of intracellular cAMP decay accumulated in response to serum was similar to that of GLP-1-Tf (data not shown).

Measurement of glucose homeostasis and plasma insulin in non-diabetic and diabetic mice Male diabetic db / db mice lacking a functional leptin receptor (C57BLKS / J-Lepr ^db / Lepr ^db ) and their non-diabetic heterozygotes Litters were purchased at 4 weeks of age from Jackson laboratories (Bar Harbor). They were housed in two per cage and were allowed to eat freely for at least 5 days prior to any test. During the experiment, the same mice were placed together in cages.

  Diabetic db / db mice are homozygous for the mutated nonfunctional leptin receptor, so that they suffer from phagocytosis and have been eating 24 hours a day (during normal consumption during the dark hours). Eating (rather than eating), becoming obese, and having a high blood glucose level in the range of 300-400 mg / dl by 4-6 weeks of age (normal fasting blood glucose is 90-120 mg / dl). Their heterozygous litters (non-diabetic mice) are not overeating, are lean, and do not develop diabetes.

  After non-diabetic mice were fasted overnight, an intraperitoneal (IP) glucose tolerance test (IPGTT) (0.5 g / kg body weight) was performed. Tf and GLP-1-Tf were administered intraperitoneally 30 minutes before glucose injection. Blood glucose levels (Glucometer Elite, Bayer) were measured at 0, 30 and 60 minutes, and plasma insulin levels were measured at 0 and 30 minutes after IPGTT.

  In non-diabetic mice, IP administration of 1 and 10 mg / kg GLP-1-Tf enhanced insulin secretion. At 30 minutes, peak blood glucose decreased simultaneously with high insulin levels (Figure 9a).

  Tf and GLP-1-Tf were administered subcutaneously to non-diabetic and diabetic mice and blood glucose was frequently checked for a sustained effect for 48 hours. Plasma insulin levels were measured at 1 hour and 4 hours. In addition, diabetic mice administered with GLP-1-Tf or Tf subcutaneously at the start of fasting were fasted overnight for 12 hours, and then IPGTT (0.5 g / kg body weight) was also performed.

  In non-diabetic mice fed ad libitum, subcutaneous administration of GLP-1-Tf decreased blood glucose levels in a dose-dependent manner and increased insulin secretion (FIG. 9b). Within 3 hours of administration, the blood glucose level dropped to 70 mg / dl and gradually increased to the level before administration within 24 hours. When GLP-1-Tf was administered at higher doses (10 mg / kg), low blood glucose persisted for up to 36 hours.

  In diabetic mice fed freely, subcutaneous administration of GLP-1-Tf (both 1 and 10 mg / kg) reduced blood glucose levels to normal levels (from 358 ± 23 to 115 ± 18 mg / dl) The action began as early as 1 hour after injection (FIG. 10a). After administration of GLP-1-Tf at 1 and 10 mg / kg, increased plasma insulin levels persisted for at least 4 hours (FIG. 10b). At the maximum dose, the effect on blood glucose was sustained for at least 12 hours without causing hypoglycemia. At low doses of GLP-1-Tf (0.1 mg / kg), plasma insulin levels increase 1 hour after administration, which is accompanied by an acute, non-persistent hypoglycemic effect for 4 hours after administration. Occurs (Figures 10a and b).

  A diabetic mouse group was subcutaneously administered with Tf or GLP-1-Tf at 9 pm, fasted overnight, and IPGTT was performed the next morning. Fasted Tf-treated animals show lower fasting blood glucose than freely fed animals (238 ± 39 versus 358 ± 23 mg / dl; 0 hours for fasted animals in FIG. In addition, animals given 1 or 10 mg of GLP-1-Tf had lower fasting blood glucose than animals given Tf alone. (132 ± 27 mg / dl). After IPGTT, animals that received GLP-1-Tf had lower blood glucose levels and higher plasma insulin levels than animals treated with hTf (FIGS. 10c and d).

Male diabetic db / db mice (C57BLKS / J-Lepr ^db / Lepr ^db ) lacking food intake and blood glucose measurement functioning leptin receptor after administration of BRX0585 in non-diabetic and db / db mice , and their non-diabetic Diabetic heterozygous litters were purchased at 4 weeks of age from Jackson Laboratories (Bar Harbor).

  Mice (N = 5 / group) were adjusted by feeding once a day for 5 days (9-10 o'clock in the morning). On the morning of day 6, they were given intraperitoneal administration of Tf (10 mg / kg) or GLP-1-TF (0.1, 1 and 10 mg / kg), or exendin-4 (1 nmol / kg), followed by Feeded freely. Food intake and blood glucose levels were measured 2, 4, 7 and 24 hours after peptide administration.

  db / db mice (99 ± 9 mg / dl) maintained normal fasting blood glucose levels throughout the 5 days, and their levels were similar to non-diabetic animals (87 ± 2 mg / dl) (FIG. 11c). 0 hours, compared to 0 hours in Figure 11a). During the feeding time, both groups of animals ate about 0.5 grams of food pellets. Two animals were sacrificed on the morning of the sixth day and there was no food in the stomach or small intestine. GLP-1-Tf (0.1, 1 and 10 mg / kg), Tf (10 mg / kg) or exendin-4 (1 nmol / kg, diabetic mice only, before the rest of the animals were allowed to freely feed ) Was administered. Food intake was quantified and blood glucose levels (to measure postprandial glucose rise) were measured over the next 24 hour interval. In non-diabetic animals, GLP-1-Tf (10 mg / kg) reduced food intake by about half in 7 hours and GLP-1-Tf (1 mg / kg) in 2 hours and received only Tf Compared to animals, postprandial blood glucose levels decreased in a concentration-dependent manner at all three doses (FIGS. 11a and b), reflecting both decreased food intake and increased insulin secretion (FIG. 11). 10b). In the 7-24 hour time segment after injection, animals treated with GLP-1-Tf showed food intake similar to animals treated with Tf. Also, in db / db animals, when GLP-1-Tf (1 and 10 mg / kg) was used, food intake was reduced by about half in 2 hours, and again compared to Tf treatment, all three doses The blood glucose level after meal was lowered in a concentration-dependent manner. Exendin-4 prevented virtually any measurable food consumption for 7 hours and caused a reduction in food intake for the next 17 hours.

Analysis of β-cell proliferation and β-cell mass Male diabetic db / db mice lacking a functional leptin receptor (C57BLKS / J-Lepr ^db / Lepr ^db ) were placed 4 weeks from Jackson Laboratories (Bar Harbor). We purchased at age. They were housed at 2 per cage and fed ad libitum for at least 5 days prior to any study. During the experiment, the same mice were placed together in cages.

  db / db mice (6 per time point) received a single intraperitoneal injection of Tf (10 mg / kg) or GLP-1-Tf (10 mg / kg), and β- Sacrificed for measurement of cell proliferation, and sacrificed for measurement of islet mass after 3, 5, 8 and 10 days. Six hours before sacrifice, 60 mg / kg 5-bromo-2-deoxyuridine (BrdU, Sigma) was injected intraperitoneally. BrdU is a thymidine analog that is taken up during the S phase of cell mitosis. The pancreas was removed, fixed overnight in 4% buffered formalin, embedded in paraffin, and tissue sections (4 μm) were mounted on glass slides coated with poly-1-lysine. BrdU was detected with BrdU antibody (Sigma, 1: 500, further confirmed with Zymed antibody, 1: 250) and BrdU staining kit (Zymed Laboratories Inc.). Next, 5 to 600 islets were visualized from each condition, and the number of islets containing BrdU + cells and the percentage of BrdU + nuclei per total number of islet cells were quantified. Tissue sections were treated with guinea pig insulin antibody (Linco) for insulin staining and determination of β-cell mass. Antibody binding was visualized with 3,3-diaminobenzidine (DAB) and sections were counterstained with hematoxylin (Vectorstein ABC kit, Vector Laboratories, using goat anti-guinea pig IgG). The sections were examined using a video camera connected to a computer equipped with imaging software. Tissue images sectioned by a 2.5 × objective lens of a phase contrast optical microscope (Carl Zeiss) were obtained and digitized by a Sony Power HAD digital camera (per section) Average 20-30 images). All pancreatic and beta cell positive areas were quantified for each image using MetaMorph 4.6.3 software (Universal Imaging Inc., Westchester).

  Animals treated with GLP-1-Tf showed a clear increase in the number of BrdU + nuclei in the islets as early as 24 hours after GLP-1-Tf injection compared to treatment with Tf alone. On day 3 after active treatment, BrdU + nuclei show a 4.7-fold increase (p <0.01) (FIG. 12a) and all islets in GLP-1-Tf treated animals have one or more BrdU + In contrast, islets treated with hTf accounted for 35% (140 out of 400) of nuclei. In animals treated with GLP-1-Tf, islets were sometimes observed where BrdU + nuclei were observed in as many as 50% islet cell nuclei (Figure 12b). BrdU + nuclei were mainly present in β-cells containing insulin (FIG. 12b). After 3 days of treatment with GLP-1-Tf (10 mg / kg), the total mass of β cells increased 1.7-fold compared to Tf treatment, and on days 5, 8 and 10 β cells The total mass of was further increased by 2.2, 4.1 and 4.3 times, respectively (FIG. 12c). Using immunofluorescence and confocal microscopy, we have established GLP-1 by 24 hours in islets from fixed pancreas collected from Tf-treated and GLP-1-Tf-treated mice. The presence of -Tf could be detected (FIG. 14).

Measurement of Levels of hTf and GLP-1-Tf in Cerebrospinal Fluid (CSF) Male Sprague Dawley rats were purchased from Harlan Sprague Dawley, Indianapolis at 6 weeks of age. Rats were injected intraperitoneally with hTf and GLP-1-Tf. The animals were sacrificed after 2 and 24 hours to analyze the presence of proteins in plasma, CSF and brain. CSF was collected by inserting a 30-gauge needle with a syringe attached between the atlas and vertebrae and the CSF was gently aspirated. There was no blood contamination in the CSF used for analysis. In this experiment, rats were used to obtain a sufficient amount of CSF. The brain was removed, sliced, sonicated in SDS lysis buffer, and the homogenate was stored at −80 ° C. for subsequent processing in Western blot analysis.

  Plasma samples (0.1 μL) or CSF samples (5 μL) and brain homogenate were mixed with 10 μL of SDS-PAGE sample buffer and treated with 10% SDS-PAGE (Novex) according to the supplier's protocol. . The protein was transferred to a PVDF membrane (Bio-Rad) in 25 mM Tris + 192 mM glycine. The filter paper was equilibrated with 25 mM borate buffer (pH 8.0) +150 mM NaCl + 5% non-fat dry milk. Rabbit anti-Tf (Rockland Immunochemicals) was added to the blot and incubated overnight at room temperature. After washing thoroughly, HRP-conjugated goat anti-rabbit IgG (Pierce) was used as the secondary antibody. Antibody bands bound to Tf and GLP-1-Tf were detected by SuperSignal West Pico chemiluminescence detection kit (Pierce).

  Western blotting of plasma with anti-Tf showed an estimated 77 kDa size indicating the full length size, and no other fragments were observed, making concentration dependence clear (Figure 13a). Consistent with the ELISA, no immunoreactive Tf was observed in CSF in Western blot analysis. In addition, the present inventors also incubated CHO / GLP-1R cells with CSF 2 hours after collection from rats treated with GLP-1-Tf, and as a result, they can detect any increase in intracellular cAMP. In addition, according to Western blot analysis, homogenized brain sections from the above animals did not show Tf. It was therefore concluded that GLP-1-Tf does not cross the blood brain barrier.

C-Fos activation in the brain The expression pattern of c-Fos in the mouse brain (used as a marker for neuronal activation) is examined, and peripherally administered GLP-1-Tf activates the central nervous system. And the results were obtained when peripherally administered exendin-4 (1 nmol / kg = 4 μg / kg) administered intraperitoneally known to activate a given brain region The results were compared (Baggio et al., 2004, Gastroenterology. 127: 546-558).

Briefly, mice that received intraperitoneal injections were anesthetized with isoflurane 2 hours later, and mice that received intraventricular injections were anesthetized 1.5 and 24 hours later. Intracardiac perfusion with ice-cold 4% paraformaldehyde was performed immediately. At the end of the perfusion, the brains were immediately removed and maintained in ice-cold 4% paraformaldehyde for 72 hours and then transferred to a solution containing 4% paraformaldehyde and 25% sucrose until they floated. Each brain was cut into 30 μm sections using a sliding microtome equipped with a frozen platform. Brain sections from each animal were placed in 12-well plates and then processed for immunocytochemistry. First, free-floating sections from each animal were incubated in 0.3% H ₂ O ₂ for 30 minutes, and then washed with three 5-minute rinses in PBS once. The sections were then incubated for 45 minutes at room temperature in blocking serum (normal goat serum / PBS / 0.1% Triton). The sections were stained for 48 hours at 4 ° C. for c-Fos with an antibody directed against amino acid residues 4-17 in rabbit anti-human c-Fos (Calbiochem, 1: 30,000). The sections are then washed 6 times with PBS / 0.1% Triton and then with biotinylated goat anti-rabbit IgG (1: 600) in blocking serum (Vector Laboratories) at room temperature. Incubated for 1.5 hours. The sections were then visualized with DAB (Vector Stain ABC kit) and developed until the color reached the required intensity in the control sections. The reaction was stopped by immersing the slide in distilled water. After staining was complete, the sections were mounted on superfrost slides (Fisher Scientific), allowed to air dry for 1-2 days, and covered with a par mount. Sections were examined using an optical microscope to identify c-Fos positive cells.

  Peripheral administration of GLP-1-Tf (10 mg / kg dose) resulted in 2 hours post-administration, the final neuron (AP) (4/6 animals), solitary nucleus (NTS) (6/6 animals), and thalamus C-Fos was activated in the lower paraventricular nucleus (PVH) (6/6 animals) (FIG. 13b). GLP-1-Tf (1 mg / kg) activated NTS (6/6 animals) and PVH (6/6 animals), but AP (0/6 animals) was not activated (data not shown) ). Exendin-4 activated a region similar to GLP-1-Tf in all injected animals, but the AP response was considerably stronger than GLP-1-Tf (FIG. 13b).

  In other series of experiments, Tf and GLP-1-Tf were administered intraventricularly (ICV) and c-Fos activation was assessed 1.5 and 24 hours later. Ninety minutes after intracerebroventricular injection of GLP-1-Tf, c-Fos activation occurred in PVH and NTS, but not in AP (FIG. 13c). 24 hours after intracerebroventricular injection, c-Fos activation did not occur in any of the three brain regions (data not shown).

The results of all statistical methods are presented as mean ± SE. Student's t-test is based on the results of an F-test evaluating the equal variance of the two means. If the variances are statistically significantly different, the t test is based on unequal variances. An ANOVA test was used to calculate significant differences between two or more samples, followed by a post-hoc test using Scheffe test. A P value of less than 0.05 was considered statistically significant.

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

The alignment of the N and C domains of human (Hu) transferrin (Tf) is shown (SEQ ID NO: 3). Shown is an alignment of transferrin sequences from various species. Shown is an alignment of transferrin sequences from various species. Shown is an alignment of transferrin sequences from various species. Shown is an alignment of transferrin sequences from various species. The position of multiple insertion sites exposed on the surface of Tf for therapeutic proteins, polypeptides or peptides is indicated. FIG. 3 shows various variants of GLP-1, GLP-2 linkers and mTf used to construct various GLP-1 / GLP-2 / mTf fusion proteins. Shows the relative potency in cAMP analysis using rat GLP-1 receptor cloned into CHO cells. 2 shows a comparison of plasma concentrations of GLP-1 / mTf in monkeys as measured by ELISA and in vitro bioassay. It is a restriction map of pREX0585 (SEQ ID NO: 11) encoding GLP-1-Tf (SEQ ID NO: 12). a shows the percentage of maximal response of cAMP for various concentrations of GLP-1-Tf (SEQ ID NO: 12), GLP-1 and exendin-4. b indicates the percentage of their maximum secretion of insulin. c shows the percent of maximal response of insulin at various concentrations of GLP-1 and GLP-1-Tf. d indicates plasma GLP-1-Tf (SEQ ID NO: 12) over time. Shows blood glucose over time in non-diabetic mice treated with hTf, 1 mg / kg GLP-1-Tf or 10 mg / kg GLP-1-Tf by intraperitoneal (a) and subcutaneous (b) routes . a to d show blood glucose and plasma insulin secretion levels in db / db mice treated with hTf or GLP-1-Tf (SEQ ID NO: 12). Blood glucose (a and c) and food intake (b and d) in db / db and non-diabetic mice fed once daily for 5 days after treatment with hTf or GLP-1-Tf (SEQ ID NO: 12) Indicates. a-c shows BrdU + nuclei in pancreas of db / db mice treated with hTf or GLP-1-Tf (SEQ ID NO: 12). a is a Western blot showing fragments corresponding to Tf and GLP-1-Tf in plasma from rats treated with Tf or GLP-1-Tf (SEQ ID NO: 12). b to c are the last neuron, solitary nucleus and hypothalamic paraventricular nucleus of rats treated with hTf or GLP-1-Tf (SEQ ID NO: 12) by intraperitoneal injection (b) and intraventricular injection (c) It is an image of cFos activation in. Images are shown using immunofluorescence and confocal microscopy 24 hours after treatment with GLP-1-Tf in islets from fixed pancreas of mice treated with GLP-1-Tf or Tf.

Claims (59)

  A fusion protein comprising a GLP-1 peptide, a substantially non-helical polypeptide linker, and a modified transferrin (mTf) molecule that exhibits reduced glycosylation as compared to a native transferrin molecule. The fusion protein of claim 1, wherein the linker is selected from the group consisting of PEAPTD, (PEAPTD) ₂ , PEAPTD in combination with an IgG hinge linker, and (PEAPTD) _{2 in} combination with an IgG hinge linker.   The fusion protein of claim 1, wherein the linker substantially increases the productivity of fusion protein expression as compared to a fusion protein without a rigid linker or a fusion protein with a flexible polypeptide linker.   2. The fusion protein of claim 1, wherein the linker substantially increases the potency of the GLP-1 peptide compared to a fusion protein without a rigid linker or a fusion protein with a flexible polypeptide linker.   The fusion protein of claim 1, wherein the GLP-1 peptide exhibits a longer serum half-life compared to the unfused GLP-1 molecule.   2. The fusion protein of claim 1, wherein the GLP-1 peptide exhibits sustained biological activity compared to the unfused GLP-1 molecule.   2. The fusion protein of claim 1, wherein the GLP-1 peptide is present at the N-terminus of the fusion protein, the C-terminus of the fusion protein, or both the N- and C-termini of the fusion protein.   2. The fusion protein of claim 1 comprising at least two GLP-1 peptides.   The fusion protein of claim 1, wherein the GLP-1 peptide has been modified to substantially reduce cleavage by a protease.   The fusion protein of claim 1, wherein, prior to cleavage, the N-terminus of the fusion protein comprises a secretory signal sequence.   The fusion protein according to claim 10, wherein the signal sequence is a signal sequence derived from serum transferrin, lactoferrin, melanotransferrin, or a variant thereof.   The fusion protein according to claim 10, wherein the signal sequence is an HSA / MFα-1 hybrid leader sequence or a Tf signal sequence.   The fusion protein according to claim 11, wherein the signal sequence is a Tf signal sequence comprising amino acids 1 to 19 of SEQ ID NO: 2.   The fusion protein of claim 1, wherein GLP-1 (7-37) (SEQ ID NO: 6) has been modified.   15. The fusion protein of claim 14, wherein GLP-1 (7-37) is modified by mutating A8 to S, G or V (A2 of SEQ ID NO: 6 to S, G or V). .   15. The fusion protein of claim 14, wherein GLP-1 (7-37) is modified by mutating K34 to Q, A or N (K28 of SEQ ID NO: 6 to Q, A or N). .   The fusion protein according to claim 14, wherein GLP-1 (7-37) is modified so that V33 to G37 (V27 to G31 of SEQ ID NO: 6) are deleted.   The fusion protein according to claim 14, wherein GLP-1 is modified so that A30 to G37 (A24 to G31 of SEQ ID NO: 6) are deleted.   The fusion protein according to claim 14, wherein the modified GLP-1 is GLP-1 (7-37; A8G, K34A).   The fusion protein of claim 1, wherein the mTf molecule has a reduced affinity for the transferrin receptor (TfR).   21. The fusion protein of claim 20, wherein the mTf molecule does not substantially cross the blood brain barrier.   The fusion protein according to claim 1, wherein the mTf molecule is a modified lactoferrin or a modified melanotransferrin.   The fusion protein of claim 1, wherein the mTf protein has a reduced affinity for iron.   24. The fusion protein of claim 23, wherein the mTf protein does not bind iron.   The fusion protein of claim 1, wherein the mTf protein does not exhibit N-linked glycosylation.   The fusion protein of claim 1, wherein the mTf protein does not exhibit glycosylation.   2. The fusion protein of claim 1, wherein the mTf protein comprises at least one mutation that prevents glycosylation.   28. The fusion protein of claim 27, wherein the mutation is within or adjacent to an N-linked glycosylation site comprising the sequence NXS / T.   29. The fusion protein of claim 28, wherein the NXS / T site corresponds to amino acid N413 or N611 of SEQ ID NO: 3.   30. The fusion protein of claim 29, wherein the mutation is present in the N-linked glycosylation site at both N413 and N611 of SEQ ID NO: 3.   The fusion protein of claim 1, wherein the fusion protein comprises SEQ ID NO: 12.   A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 31.   A vector comprising the nucleic acid molecule of claim 32.   A host cell comprising the vector of claim 33.   A host cell comprising the nucleic acid molecule of claim 32.   35. A method for expressing a fusion protein comprising culturing the host cell of claim 34 under conditions such that the encoded fusion protein is expressed.   36. A method for expressing a fusion protein comprising culturing the host cell of claim 35 under conditions such that the encoded fusion protein is expressed.   35. The host cell of claim 34, wherein the cell is a prokaryotic cell or a eukaryotic cell.   36. The host cell according to claim 35, wherein the cell is a prokaryotic cell or a eukaryotic cell.   40. The host cell according to claim 38, wherein the cell is a yeast cell.   40. The host cell of claim 39, wherein the cell is a yeast cell.   A non-human transgenic animal comprising the nucleic acid molecule of claim 32.   43. A method for producing a fusion protein comprising isolating the fusion protein from the transgenic animal of claim 42.   A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 31 and a carrier.   35. A method of treating a subject comprising administering to the subject a therapeutically effective amount of the fusion protein of any one of claims 1-31.   46. The method of claim 45, wherein the subject has a high blood glucose level compared to a healthy subject.   48. The method of claim 46, wherein the elevated blood glucose level is associated with diabetes.   48. The method of claim 47, wherein the diabetes is type II diabetes.   32. A method of regulating blood glucose levels in a subject comprising administering to the subject a therapeutically effective amount of the fusion protein of any one of claims 1-31.   49. The method of claim 48, wherein the fusion protein is administered in combination with one or more substances selected from the group consisting of metformin, DPPIV inhibitors, NEP24.11 inhibitors and glitazones, or derivatives thereof. .   32. A method of reducing food intake in an animal comprising administering an effective amount of the fusion protein of any one of claims 1-31.   32. A method of inducing β-cell proliferation or β-cell mass increase in a patient, comprising administering an effective amount of the fusion protein of any one of claims 1-31.   A method for inducing insulin secretion in a patient in need of insulin secretion, comprising administering an effective amount of the fusion protein according to any one of claims 1-31.   A method for treating a patient with type I diabetes, comprising administering an effective amount of the fusion protein according to any one of claims 1 to 31.   32. A method of reducing gastric emptying in an animal comprising administering an effective amount of the fusion protein of any one of claims 1-31.   A method for inducing weight loss in an animal, comprising administering an effective amount of the fusion protein according to any one of claims 1 to 31.   A method for treating a patient with congestive heart failure comprising administering an effective amount of the fusion protein according to any one of claims 1 to 31.   A method for treating a patient with non-alcoholic, non-fatty liver disease, comprising administering an effective amount of the fusion protein according to any one of claims 1 to 31.   A fusion protein comprising a GLP-1 peptide, a polypeptide linker that is not substantially helical, and a transferrin molecule.

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