HK1135112B - Exendin fusion proteins - Google Patents

Description

Exendin fusion proteins

Technical Field

The present invention relates to fusion proteins comprising exendin-4 (exendin-4) and transferrin and their use for the treatment of diseases associated with elevated glucose serum levels and for weight loss, such as type II diabetes. The fusion proteins of the invention may also be used to treat other diseases known to benefit from treatment with exendin-4 and other GLP-1 receptor agonists, such as type I diabetes, congestive heart failure, myocardial infarction, irritable bowel syndrome, neurological diseases such as Alzheimer's disease and Huntington's disease, and non-alcoholic, non-fatty liver disease.

Background

Diabetes refers to a disease process that derives from multiple etiologic factors and is characterized by elevated plasma glucose levels or hyperglycemia during fasting or after administration of glucose during an oral glucose tolerance test. Generally, there are two recognized forms of diabetes. In type I diabetes, or Insulin Dependent Diabetes Mellitus (IDDM), patients produce little or no insulin, which is a hormone that regulates glucose utilization. For type II diabetes, or non-insulin dependent diabetes mellitus (NIDDM), patients typically have the same or even higher plasma insulin levels as compared to non-diabetic subjects. However, these patients develop resistance to the stimulatory effects of insulin on glucose and lipid metabolism in the major insulin-sensitive tissues, muscle, liver and adipose tissue. Plasma insulin levels, while elevated, are not sufficient to overcome significant insulin resistance, resulting in hyperglycemia.

Persistent or uncontrolled hyperglycemia is associated with increased and early (prematurity) morbidity and mortality. In general, abnormal glucose homeostasis is directly and indirectly associated with alterations in lipid, lipoprotein and apolipoprotein metabolism and other metabolic and hemodynamic disease. For example, patients with type II diabetes have a significantly increased risk of macrovascular and microvascular complications, including coronary heart disease, stroke, peripheral vascular disease, hypertension, renal disease and neurological disease.

Obesity and overweight are generally defined by the Body Mass Index (BMI) which is related to total body fat and is a measure of the risk of certain diseases. BMI is calculated by dividing body weight in kilograms by the square of height in meters (kg/m)²). Generally, overweight is defined as 25-29.9kg/m²The BMI of (1), obesity is defined as 30kg/m²Or higher BMI. See, for example, national Heart, Lung, and Blood Institute, Clinical Guidelines on The identification, Evaluation, and Treatment of upside and Obesistance in additives, The Evaluation Report, Washington, DC: U.S. department of Health and Human Services, NIH publication No.98-4083 (1998).

Overweight or obese individuals have an increased risk of contracting ailments, such as hypertension, dyslipidemia, type II (non-insulin dependent) diabetes, insulin resistance, glucose intolerance, hyperinsulinemia, coronary heart disease, angina pectoris, congestive heart failure, stroke, gallstones, cholecystitis, cholelithiasis, gout, osteoarthritis, obstructive sleep apnea and respiratory problems, gallbladder disease, certain forms of cancer (e.g., endometrial, breast, prostate, and colon cancer) and psychological disorders (e.g., depression, eating disorders, distorted body image (and low self-esteem). The negative health consequences of obesity make it the second leading cause of preventable death in the united states and manifest significant economic and psychosocial effects in society. See McGinnis M, Foege WH., "Actual mice of death in the United States," JAMA 270: 2207-12, 1993.

Obesity is now recognized as a chronic disease that requires treatment to reduce its associated health risks. While weight loss is an important therapeutic outcome, one of the primary goals of obesity control is to improve cardiovascular and metabolic values to reduce obesity-related morbidity and mortality. It has been shown that a 5-10% reduction in body weight can substantially improve metabolic values such as blood glucose, blood pressure and lipid concentrations. Thus, it is believed that a 5-10% reduction in body weight may reduce morbidity and mortality. Currently available prescription drugs for the management of obesity typically reduce body weight by reducing dietary fat absorption, such as orlistat, or by causing energy deficit by reducing food intake and/or increasing energy expenditure, such as sibutramine.

Current treatments for type II diabetes include administration of exogenous insulin, oral medication and dietary therapy and exercise regimens. In 2005, exenatide (exenatide) (exendin-4;) FDA approved as an adjuvant therapy for type II diabetic patients taking metformin and/or sulfonylureas, but not achieving adequate glycemic (glycemic) control. Exenatide is exendin-4, a potent GLP-1 receptor agonist, an endogenous product of the salivary gland of exendin. Like GLP-1, Exendin-4 is an incretin. It is insulinotropic, inhibits feeding and gastric emptying, and is trophic for rodent beta cells (tropic) (Parks et al, Metabolism.50: 583-. Furthermore, due to the presence of a glycine in position 2 at its N-terminal end, exendin-4 is not a substrate for DPPIV like GLP-1. The disadvantage of using exenatide is that it has to be injected twice a day, because of its t_1/2Only for 2-4 hours (Kolterman et al, J.Clin.Endocrinol.Metab.88: 3082-3089, 2003 and Fineman et al, Diabetes Care.26: 2370-2377, 2003).

Thus, there is a need for longer lasting, degradation resistant GLP-1 receptor agonist molecules that can be used as therapeutic agents to provide glycemic control as well as to reduce body weight. The development of long-acting incretin mimetics provides the ability to enhance control of elevated insulin by sustained enhancement of glucose-dependent insulin secretion and provides the convenience of less frequent dosing. The present invention fulfills this need by providing exendin-4 molecules fused to modified transferrin that extend the in vivo circulating half-life of exendin-4 while maintaining biological activity. Thus, the use of the fusion proteins of the invention can reduce the high incidence of nausea and vomiting commonly associated with incretin use.

Summary of The Invention

The present invention provides fusion proteins comprising an exendin-4 fused to a transferrin (Tf) molecule by a peptide linker (linker), preferably by a non-helical polypeptide linker.

Preferably, the linker is selected from PEAPTD (SEQ ID NO: 6), (PEAPTD)₂(SEQ ID NO: 5), PEAPTD bound to IgG hinge linker (SEQ ID NO: 6), and PEAPTD bound to IgG hinge linker₂(SEQ ID NO: 6). More preferably, the linker is (PEAPTD)₂(SEQ ID NO：5)。

The Tf moiety of a fusion protein of the invention may be derived from any mammalian Tf, preferably, from human Tf. More preferably, Tf is a Tf (mtf) modified to exhibit reduced glycosylation as compared to the native transferrin molecule, and even more preferably, Tf has the amino acid sequence as shown in SEQ id no: 17. In other preferred embodiments, Tf is modified to reduce iron binding and/or binding to a Tf receptor.

In another preferred embodiment, the N-terminus of the fusion protein further comprises a secretory signal sequence, preferably the signal sequence is from serum transferrin, lactoferrin, melanotransferrin (melantotrasferrin), or a variant thereof, more preferably a Human Serum Albumin (HSA)/MF α -1 hybrid leader sequence, a modified HSA/MF α -1 hybrid leader sequence, or a Tf signal sequence, and still more preferably the signal sequence is a human Tf signal sequence (nL), such as SEQ ID NO: 18, respectively.

In a preferred embodiment, the present invention provides a composition comprising Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) a fusion protein of mTf fusion protein, wherein said fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 23 or SEQ ID NO: 25, wherein the latter further comprises an nL leader sequence at the N-terminus. In other preferred embodiments, the exendin-4 is exendin-4 (1-39) and has the amino acid sequence as set forth in SEQ ID NO: 4, and/or an exendin-4 molecule is fused at the N-terminus of the fusion protein, the C-terminus of the fusion protein or both the N-terminus and the C-terminus of the fusion protein.

The invention also provides nucleic acid molecules encoding the above fusion proteins, and corresponding vectors comprising the nucleic acid molecules, and host cells comprising the nucleic acid molecules and vectors.

The invention also features pharmaceutical compositions comprising any of the above fusion proteins and a pharmaceutically acceptable carrier.

In a preferred embodiment, the pharmaceutical composition comprises SEQ ID NO: exendin-4 (1-39) (PEAPTD) shown as 23₂(SEQ ID NO: 5) mTf fusion proteins, and in some embodiments, comprise the amino acid sequence of SEQ ID NO: exendin-4 (1-39) (PEAPTD) shown as 23₂(SEQ ID NO: 5) compositions of mTf fusion proteins are suitable for administration in a dosage range of about 0.5mg to about 50mg or about 1mg to about 100 mg.

In another preferred embodiment, the composition is suitable for administration by inhalation.

The invention also features a method of treating I I type diabetes or reducing blood glucose in a human patient in need thereof comprising administering to the patient a therapeutically effective amount of a fusion protein comprising an exendin-4 fused to a Tf via a polypeptide linker, preferably a non-helical linker.

Preferably, the method comprises administering a pharmaceutical composition comprising an amino acid sequence as set forth in SEQ ID NO: 23 to a pharmaceutically acceptable salt thereofExendin-4 (1-39) (PEAPTD) of sequence₂(SEQ ID NO: 5) mTf fusion proteins, and in some embodiments, as set forth in SEQ ID NO: 23 at a dose of about 0.5mg to 50mg, at a frequency of about once per week, once every two weeks, or once per month. In another embodiment, an exendin-4 fused to a Tf, via a polypeptide linker, preferably a non-helical linker, and more preferably, as set forth in SEQ ID NO: 23, is administered less frequently than exenatide and achieves therapeutic efficacy at comparable therapeutic doses.

The invention also features a method of treating obesity or reducing body weight in a human patient in need thereof comprising administering a therapeutically effective amount of a fusion protein comprising an exendin-4 fused to a Tf via a polypeptide linker, preferably a non-helical linker. Preferably, the fusion protein comprises a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 23 (1-39) (PEAPTD)₂(SEQ ID NO: 5) mTf fusion proteins, and, in some embodiments, as set forth in SEQ ID NO: 23 at a dose of about 1mg to about 100mg, at a frequency of about once per week, once every two weeks, or once per month. In another embodiment, an exendin-4 fused to a Tf via a polypeptide linker, preferably, as set forth in SEQ ID NO: 23, administered at a frequency that is too low as compared to exenatide for therapeutic efficacy.

The invention also provides the use of an exendin-4/Tf fusion protein, or a pharmaceutical composition, comprising an exendin-4 (1-39) (PEAPTD), for the manufacture of a medicament for the treatment of type II diabetes or for the reduction of blood glucose in a human patient in need thereof, preferably wherein the medicament is suitable for administration at a dose of about 0.5mg to 50mg, or for the manufacture of a medicament for the treatment of obesity or weight loss in a human patient in need thereof, preferably wherein the medicament is suitable for administration at a dose of about 1mg to about 100mg₂(SEQ ID NO: 5) a Tf fusion protein, and more preferably, wherein the fusion protein is as set forth in SEQ ID NO: shown at 23.

"Exendin-4" refers to a peptide as set forth in SEQ ID NO: 4(1-39), and an exendin-4 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to yield, for example, an exendin-4 fragment of exendin-4 (1-31) or exendin-4 (1-30), and a peptide having at least 90%, and preferably at least 95% identity to exendin-4 (1-39) or one of the other above-mentioned exendin-4 fragments.

As used herein, two or more DNA coding sequences are said to be "linked" or "fused" when the DNA coding sequences are translated as a fusion polypeptide as a result of in-frame fusion between the DNA coding sequences. The term "linked" or "fused" may also be used to refer to a peptide that is fused by alternative methods, e.g., chemical methods. The term "fusion" with respect to transferrin (Tf) fusions includes, but is not limited to, attachment of at least one therapeutic protein, therapeutic polypeptide or therapeutic peptide to the N-terminus of Tf, to the C-terminus of Tf, and/or insertion between any two amino acids within Tf.

"pharmaceutically acceptable" refers to a substance or composition that must be compatible chemically and/or toxicologically with the other ingredients included in the formulation and/or the mammal to be treated therewith.

A "therapeutically effective amount" refers to an amount of an exendin-4/Tf fusion protein of the invention that reduces blood glucose, caloric intake, reduces body weight and/or reduces body fat as compared to an appropriate control value determined prior to treatment or in a vehicle-treated group.

The terms "treatment", "treated" or "therapy" include both prophylactic treatment (i.e., treatment that prevents disease) and palliative treatment.

Brief description of the drawings

FIG. 1 shows comparative GLP-1(7-37, A8G, K34A) (PEAPTD)₂(SEQ ID NO: 5) mTf fusion protein (GLP-1/Tf) (FIG. 1A) andexendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) map of in vitro collagenase resistance (MMP-1) for the mTf fusion protein (Exendin-4/Tf) (FIG. 1B).

FIG. 2 shows Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) graph of dose effect of mTf fusion protein (Exendin-4/Tf) on blood glucose in diabetic (db/db) mice and demonstrates the comparative effect of the Exendin-4 control. Each point represents the average glucose measurement (n-3).

FIG. 3 shows daily administration of Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) graph of dose effect of mTf fusion protein (Exendin-4/Tf) on body weight and shows comparative effect of Exendin-4 and mTf control.

FIG. 4 is a comparison of Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) relative potency of mTf fusion proteins (Exendin-4/Tf) and Exendin-4. It was determined by cell-based cAMP assay. Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) EC50 for the mTf fusion protein was 31.3pM and EC50 for Exendin-4 was 6.6 pM.

Detailed Description

Exendin-4/Tf fusion proteins

An exendin-4/Tf fusion protein of the invention comprises an exendin-4 fused to a Tf peptide via a polypeptide linker. Preferably, the peptide is encoded with full-length exendin-4 (1-39) (SEQ ID No.: 4), or a sequence selected from SEQ ID NO: 4 to yield, for example, an exendin-4 fragment of exendin-4 (1-31) or exendin-4 (1-30).

Preferably, a non-helical polypeptide linker moiety is used to link exendin-4 to Tf.

A preferred linker is PEAPTDPEAPTD (SEQ ID NO: 5). The other linkers may be selected from PEAPTD (SEQ ID NO.: 6), PEAPTD (SEQ ID NO.: 6) bound to the I gG hinge linker (SEQ ID NO: 7-16), and PEAPTDPEAPTD (SEQ ID NO: 5) bound to the IgG hinge linker (SEQ ID NO: 7-16). The fusion proteins of the invention comprising a substantially non-helical linker moiety may exhibit increased expression yields compared to similar fusion proteins that do not have a substantially non-helical linker. Further, an exendin-4/Tf fusion protein comprising a substantially non-helical linker may exhibit increased expression yield compared to a similar fusion protein having a helical polypeptide linker.

Preferred Exendin-4/Tf fusion proteins comprise a linker (PEAPTD)₂(SEQ ID NO: 5) and SEQ ID NO: mTf-linked Exendin-4 (1-39) (SEQ ID NO: 4) provided in 17. For the preparation, Exendin-4 (1-39) (PEAPTD) is preferred₂(SEQ ID NO: 5) the mTf fusion protein also contains the human transferrin secretion signal or leader sequence (nL) (SEQ ID NO: 18). Encoding a preferred Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) the nucleic acid sequences of the individual components of the mTf fusion protein are as follows: nL leader sequence (SEQ ID NO: 19), Exendin-4 (1-39) (SEQ ID NO: 20), (PEAPTD)₂(SEQ ID NO: 21), and mTf (SEQ ID NO: 22). Intact Exendin-4 (1-39) (PEAPTD) without nL leader sequence₂(SEQ ID NO: 5) the amino acid sequence of the mTf fusion protein is SEQ ID NO: 23; the corresponding nucleic acid sequence is SEQ ID NO: 24. complete Exendin-4 (1-39) (PEAPTD) with nL leader sequence at the N-terminus₂(SEQ ID NO: 5) the amino acid sequence of the fusion protein of mTf is SEQ ID NO: 25; the corresponding nucleic acid sequence is SEQ ID NO: 26.

although the preferred mTf is as described above, any transferrin can be used to make the exendin-4/Tf fusion proteins of the invention. As an example, wild-type human Tf is a 679 amino acid protein of about 75kDa (not counting glycosylation) with two major domains or lobes (lobes), N (about 330 amino acids) and C (about 340 amino acids) that appear to be derived from a gene repeat. See GenBank accession nos. NM _001063, XM _002793, M12530, XM _039845, XM _039847, and S95936 (all of which are incorporated herein by reference in their entirety), and SEQ ID NO: 2 and 3(SEQ ID NO: 2 contains the additional 19 amino acid sequences of the nL human transferrin leader sequence). The two domains differ over time but retain a large degree of identity/similarity.

The N and C leaflets are each further divided into two subdomains, N1 and N2, C1 and C2. Tf functions to transport iron to the cells of the body. This process is mediated by the Tf receptor (TfR), which is expressed on all cells, especially actively growing cells. TfR recognizes the iron-binding form of Tf (each receptor binds to two of its molecules), causing endocytosis, whereby the TfR/Tf complex is transported to the endosome. The local drop in pH in the endosome results in the release of bound iron and circulation of the TfR/Tf complex to the cell surface and the release of Tf (apoTf, a form known as unbound iron). Receptor binding occurs through the C domain of Tf. Because the unglycosylated iron-binding Tf binds to the receptor, the two glycosylation sites in the C domain do not appear to be involved in receptor binding.

Each Tf molecule can transport two iron ions (Fe)³⁺). It complexes in the space between the N1 and N2, C1 and C2 subdomains, resulting in conformational changes in the molecule.

For SEQ ID NO: 3, the iron binding site comprises at least the amino acids Asp63 (Asp 82 of SEQ ID NO: 2, SEQ ID NO: 2 including the natural Tf signal sequence), Asp392 (Asp 411 of SEQ ID NO: 2), Tyr95 (Tyr 114 of SEQ ID NO: 2), Tyr426 (Tyr 445 of SEQ ID NO: 2), Tyr188 (Tyr 207 of SEQ ID NO: 2), Tyr514 or 517 (Tyr 533 or Tyr536 of SEQ ID NO: 2), His249 (His 268 of SEQ ID NO: 2), and His585 (His 604 of SEQ ID NO: 2). The hinge region comprises at least SEQ ID NO: 3, and C domain amino acid residues 425, 427, 581, 582 and/or 652, 658. SEQ ID NO: 3 comprises at least the amino acids Thr120 (Thr 139 of SEQ ID NO: 2), Thr 452 (Thr 471 of SEQ ID NO: 2), Arg124 (Arg 143 of SEQ ID NO: 2), Arg456 (Arg 475 of SEQ ID NO: 2), Ala126 (Ala 145 of SEQ ID NO: 2), Ala 458 (Ala 477 of SEQ ID NO: 2), Gly 127 (Gly 146 of SEQ ID NO: 2), and Gly 459 (Gly 478 of SEQ ID NO: 2).

Preferably, the modified exendin-4/Tf fusion protein is of human origin, although any animal Tf molecule may be used to prepare the fusion proteins of the invention, including human Tf variants, bovine, porcine, ovine, canine, rabbit, rat, mouse, hamster, echnida, platypus, chicken, frog, anthriscus, monkey, and other bovine, canine and avian species. All of these Tf sequences are readily available in GenBank and other published databases. Human Tf nucleic acid sequences are available (see SEQ ID NO: 1 and accession numbers above) and can be used to make gene fusions between domains of Tf or Tf and selected therapeutic molecules. Fusions can also be made from related molecules, such as lactoferrin (lactoferrin) GenBank Acc: NM — 002343) or murine melanin transferrin (GenBank acc. NM — 013900).

Melanin transferrin is a glycosylated protein found at high levels in malignant melanoma cells and was originally designated as human melanoma antigen p97(Brown et al, 1982, Nature, 296: 171-. It has high sequence homology with human serum transferrin, human lactoferrin, and chicken transferrin (Brown et al, Nature, 296: 171-126173, 1982; Rose et al, Proc. Natl. Acad. Sci. USA, 83: 1261-1265, 1986). However, unlike these receptors, the melanotransferrin cellular receptor has not been identified. Melanin transferrin binds iron reversibly and it exists in two forms, one of which is anchored to the cell membrane by glycosylphosphatidylinositol and the other of which is soluble and actively secreted (Baker et al, FEBSLett, 298, 1992: 215-.

Lactoferrin (Lf), a natural defense against iron binding proteins, has been found to have antibacterial, antifungal, antiviral, antitumor and anti-inflammatory activity. This protein is present in exocrine secretions that are normally exposed to normal flora: emulsion, tears, nasal discharge, saliva,Bronchial mucus, gastrointestinal fluids, cervical vaginal mucus, and semen. In addition, Lf is a major component of secondary specific particles of circulating polymorphonuclear neutrophils (PMNs). Apolipoprotein release occurs under degranulation of PMNs in septic regions. The primary function of Lf is to scavenge free iron in the body fluids and inflamed areas to inhibit free radical mediated damage and reduce the effectiveness of metals to attack microorganisms and tumor cells. In testing adults¹²⁵In studies of the I Lf turnover rate, Lf was shown to be rapidly taken up by the liver and spleen, and radioactivity persists in the liver and spleen for several weeks (Bennett et al, Clin. Sci. (Lond.). 57: 453-.

The transferrin moiety of the exendin-4/Tf fusion proteins of the invention includes transferrin splice variants. In one embodiment, the transferrin splice variant can be a human transferrin splice variant. In particular, the human transferrin splice variant can be that of Genbank accession AAA 61140.

The transferrin moiety of the exendin-4/Tf fusion proteins of the invention includes lactoferrin splice variants. In one embodiment, the human serum lactoferrin splice variant can be a novel splice variant of neutrophil lactoferrin. In particular, the sequence of the neutrophil lactoferrin splice variant can be as shown in Genbank accession No. AAA 59479. The neutrophil lactoferrin splice variant may also comprise the amino acid sequence EDCIALKGEADA (SEQ ID NO: 27) which includes a novel region of splice variation.

Alternatively, the transferrin moiety of the exendin-4/Tf fusion proteins of the invention comprises a melanotransferrin variant.

Modified Tf fusions may be made with any Tf protein, fragment, domain or engineered domain. For example, a fusion protein can be made with a full length Tf sequence with or without the native Tf signal sequence. Tf fusion proteins can also be made with a single Tf domain, such as the N or C domain alone or a modified form of Tf comprising a 2N or 2C domain (see US patent US 2006/0130158). Fusions of therapeutic proteins with a single C domain can be made, where the C domain is altered to reduce, inhibit, or prevent glycosylation. Alternatively, a single N domain may be useful as Tf glycosylation site residues in both the C domain and the N domain. Preferably, the Tf fusion protein has a single N domain expressed at high levels.

As used herein, a C-terminal domain or leaflet modified to function as an N-like domain is modified to exhibit a glycosylation pattern or iron-binding properties substantially similar to a native or wild-type N domain or leaflet. Preferably, the C domain or leaflet is modified so that it is not glycosylated and does not bind iron by substituting relevant C domain regions or amino acids to those present in the corresponding regions or sites of the native or wild type N domain.

As used herein, a Tf moiety comprising "two N domains or leaflets" includes a modified Tf molecule that replaces a native C domain or leaflet with a native or wild-type N domain or leaflet or a modified N domain or leaflet, or that comprises a C domain that is modified to function substantially like a wild-type or modified N domain.

Analysis of the two domains by overlap (overlap) of the two domains (Swiss PDB Viewer 3.7b2, Iterative Magic Fit) and by direct amino acid alignment (ClustalW multiplex) showed that the two domains differed over time. Amino acid alignment shows that the two domains have 42% identity and 59% similarity. However, about 80% of the N domain matches the C domain in terms of structural equivalence. The C domain also has some additional disulfide bonds compared to the N domain.

In one embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises at least two N-terminal leaflets of transferrin. In a further embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises at least two N-terminal leaflets of a transferrin derived from human serum transferrin.

The transferrin moiety of the exendin-4/Tf fusion protein may also comprise: at least two N-terminal leaflets of transferrin having a mutation at least one amino acid residue selected from the group consisting of SEQ ID NO: 3 Asp63, Gly65, Tyr95, Tyr188, and His 249; in SEQ ID NO: 3, a recombinant human serum transferrin N-terminal leaflet mutant having a mutation at Lys206 or His 207; or at least two C-terminal leaflets of transferrin. In a further embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises at least two C-terminal leaflets of transferrin derived from human serum transferrin.

In a further embodiment, the C-terminal leaflet variant further comprises SEQ ID NO: 3, Asn413 and Asn611, which does not allow glycosylation.

In another embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises at least two C-terminal leaflets having a mutant transferrin at least one amino acid residue selected from the group consisting of SEQ ID NO: 3 Asp392, Tyr426, Tyr514, Tyr517 and His585, wherein the mutant retains the ability to bind metal. In an alternative embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises at least two C-terminal leaflets of a transferrin protein having a mutation at least one amino acid residue selected from the group consisting of SEQ ID NO: 3 Tyr426, Tyr514, Tyr517 and His585, wherein the mutant has a reduced ability to bind metal. In another embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises at least two C-terminal leaflets of a transferrin protein having a mutation at least one amino acid residue selected from the group consisting of SEQ ID NO: 3, Asp392, Tyr426, Tyr517 and His585, wherein the mutant does not retain the ability to bind metals and functions substantially like an N domain.

When the C domain of Tf is part of a fusion protein, to prevent glycosylation or high mannose glycosylation (hypermannosylation) and to extend the serum half-life of the fusion protein and/or therapeutic protein (to make asialo-Tf, or in some examples, monosialo-Tf or disialo-Tf) for expression in a yeast system, two N-linked glycosylation sites can be mutated, corresponding to SEQ ID NO: 3, N413 and N611. In addition to the Tf amino acids corresponding to N413 and N611, adjacent residues within the N-X-S/T glycosylation site can be mutated to prevent or substantially reduce glycosylation. See us patent 5,986,067. It has been reported that the N domain of Tf expressed in Pichia pastoris (Pichia pastoris) undergoes O-linked glycosylation at S32 with a single hexose, which may also be mutated or modified to prevent such glycosylation.

Thus, the exendin-4/Tf fusion proteins may also include modified transferrin molecules, wherein the transferrin exhibits reduced glycosylation, including but not limited to asialo-, monosialo-and bis-sialic acid forms of Tf. In another embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises a recombinant transferrin mutant that is mutated to prevent glycosylation. The transferrin moiety of the exendin-4/Tf fusion protein can also comprise recombinant transferrin mutants, which are fully glycosylated. In a further embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises a recombinant human serum transferrin mutant mutated to prevent N-linked glycosylation, wherein the sequence of SEQ ID NO: 3 at least one of Asn413 and Asn611 has been mutated to an amino acid which does not allow glycosylation. In another embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises a recombinant human serum transferrin mutant that is mutated to prevent or substantially reduce glycosylation, wherein, for example, adjacent residues within the N-X-S/T glycosylation site are mutated, e.g., the mutation of S/T residues. In addition, glycosylation can be reduced or prevented by mutating serine or threonine residues. Further, changing X to proline is known to inhibit glycosylation.

As discussed in more detail below, modified Tf fusion proteins of the invention may also be engineered to not bind iron and/or to bind the Tf receptor. In other embodiments of the invention, the ability to retain iron binding and Tf to bind iron may be used to deliver therapeutic proteins or peptides into cells across epithelial or endothelial cell membranes. These embodiments that bind to iron and/or Tf receptors are typically engineered to reduce or prevent glycosylation, thereby extending the serum half-life of the therapeutic protein. When loaded with iron, the N domain alone is unable to bind TfR, while the iron-binding C domain can bind TfR, but does not have the same affinity as the intact molecule.

Alternatively, the transferrin moiety of the exendin-4/Tf fusion protein may comprise 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 moiety of the exendin-4/Tf fusion protein comprises a recombinant transferrin mutant having a mutation, wherein the mutant has a weaker binding affinity for metal ions than wild-type serum transferrin. In an alternative embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises a recombinant transferrin mutant having a mutation, wherein the mutant has a stronger binding affinity for a metal ion than a wild-type serum transferrin.

In another embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises a recombinant transferrin mutant having a mutation, wherein the mutant does not retain the ability to bind a transferrin receptor. For example, the exendin-4 and Tf fusion proteins of the present invention can bind to the cell surface GLP-1 receptor but not to the Tf receptor. Such fusion proteins may be therapeutically active on the cell surface (i.e., not entering the cell).

Alternatively, the transferrin moiety of an exendin-4/Tf fusion protein may comprise: a recombinant transferrin mutant having a mutation, wherein the mutant has a weaker binding affinity for transferrin receptor than wild type serum transferrin; a recombinant transferrin mutant having a mutation, wherein the mutant has a stronger binding affinity for transferrin receptor than wild type serum transferrin; a recombinant transferrin mutant having a mutation, wherein the mutant does not retain the ability to bind carbonate ions; a recombinant transferrin mutant having a mutation, wherein the mutant has a weaker binding affinity for carbonate ions than wild type serum transferrin; or a recombinant transferrin mutant having a mutation, wherein the mutant has a stronger binding affinity for carbonate ions than wild type serum transferrin.

In another embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises a recombinant human serum transferrin mutant that has a modified amino acid sequence selected from the group consisting of SEQ ID NOs: 3 has a mutation in an amino acid residue of Asp63, Gly65, Tyr95, Tyr188, His249, Asp392, Tyr426, Tyr514, Tyr517 and His585, wherein the mutant retains the ability to bind metal ions. In an alternative embodiment, the recombinant human serum transferrin mutant has a mutation in at least one amino acid sequence selected from the group consisting of SEQ ID NO: 3 has a mutation in an amino acid residue of Asp63, Gly65, Tyr95, Tyr188, His249, Asp392, Tyr426, Tyr514, Tyr517 and His585, wherein the mutant has an attenuated ability to bind metal ions. In another embodiment, the recombinant human serum transferrin mutant has a mutation in at least one amino acid sequence selected from the group consisting of SEQ ID NO: 3 has a mutation in an amino acid residue of Asp63, Gly65, Tyr95, Tyr188, His249, Asp392, Tyr426, Tyr517 and His585, wherein the mutant does not retain the ability to bind metal ions.

In another embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 3, a recombinant human serum transferrin mutant having a mutation at Lys206 or His207, wherein the mutant has a stronger binding affinity for metal ions than wild type human serum transferrin (see us patent 5,986,067). In an alternative embodiment, the transferrin moiety of the exendin-4/Tf fusion protein is comprised in SEQ ID NO: 3, a recombinant human serum transferrin mutant having a mutation at Lys206 or His207, wherein the mutant has a weaker binding affinity for metal ions than wild type human serum transferrin. In a further embodiment, the transferrin moiety of the exendin-4/Tf fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 3, a recombinant human serum transferrin mutant having a mutation at Lys206 or His207, wherein the mutant does not bind metal ions.

Any available technique may be used to prepare the exendin-4/Tf fusion proteins of the present invention, including but not limited to common Molecular techniques, such as those described in Molecular Cloning, Sambrook et al: a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989. When nucleotide substitutions are made using techniques well known in the art for effecting site-specific mutagenesis, the alteration of the encoded amino acid is preferably of a subclass (minor nature), i.e., conservative amino acid substitutions, however other, non-conservative substitutions are also contemplated, particularly when making a modified transferrin portion of a Tf fusion protein, e.g., a modified Tf protein exhibiting reduced glycosylation, reduced iron binding, and the like. Especially contemplated are amino acid substitutions, small deletions or insertions, typically of 1 to about 30 amino acids; insertions between transferrin domains; a small amino-or carboxy-terminal extension, such as a methionine residue at the amino-terminus, or a small linker peptide of less than 50, 40, 30, 20 or 10 residues between or linking transferrin and exendin-4 or a small extension that facilitates purification, such as a polyhistidine tract (trace), an epitope or a binding domain.

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

Non-conservative substitutions include substitutions of amino acids in one set with amino acids in another set. For example, a non-conservative substitution may include the substitution of a hydrophobic amino acid with a polar amino acid. For a general description of nucleotide substitutions see, e.g., Ford et al, prot. exp. pur.2: 95-107, 1991. Non-conservative substitutions, deletions and insertions are particularly useful for making Tf fusion proteins of the invention that exhibit no or reduced binding to iron, no or reduced binding to the Tf receptor, and/or no or reduced glycosylation.

By mutating (including deleting, substituting or inserting) a nucleotide sequence corresponding to SEQ ID NO: 3, Asp63, Tyr95, Tyr188, His249, and/or C-domain residue Asp392, Tyr426, Tyr514, and/or His585, may reduce or disrupt iron binding and/or receptor binding. Also by mutating seq id NO: amino acids Lys206, His207 or Arg632 of 3 affect iron binding. By mutating (including deleting, substituting or inserting) a nucleotide sequence corresponding to SEQ ID NO: 3, Thr120, Arg124, Ala126, Gly 127 and/or Thr 452, Arg456, Ala 458 and/or Gly 459, may reduce or disrupt carbonate binding. Reduction or disruption of carbonate binding can negatively affect iron and/or receptor binding.

Binding to the Tf receptor may be reduced or abolished by mutation (including deletion, substitution or insertion) of an amino acid residue corresponding to one or more of the above-described Tf N domain residues for iron binding.

As described above, glycosylation can be reduced or prevented by mutating (including deleting, substituting or inserting) amino acid residues corresponding to one or more of the Tf C domain residues near the N-X-S/T site corresponding to C domain residues N413 and/or N611 (see U.S. Pat. No. 5,986,067). For example, N413 and/or N611 may be mutated to a Glu residue.

In cases where a Tf fusion protein of the invention is not modified to prevent glycosylation, iron binding, carbonate binding and/or receptor binding, glycosylation, iron and/or carbonate ions may be stripped or cleaved from the fusion protein. For example, available deglycosylases (deglycosylases) can be used to cleave glycosylated residues from fusion proteins, particularly sugar residues linked to Tf moieties, glycosylase deficient yeast can be used to prevent glycosylation and/or recombinant cells can be cultured in the presence of agents that prevent glycosylation (e.g., tunicamycin).

By treating the fusion protein with deglycosylation enzyme, the sugars on the fusion protein can be enzymatically reduced or completely removed. Deglycosylation enzymes are known in the art. Examples of deglycosylation enzymes include, but are not limited to, galactosidase, PNGase a, PNGase F, glucosidase, mannosidase, fucosidase, and Endo H deglycosylation enzyme.

However, in certain instances, it may be preferred for oral delivery that the Tf moiety of the fusion protein be sufficiently glycosylated.

Other mutations to Tf may be made to alter the three-dimensional structure of Tf, such as modification of the hinge region to prevent conformational changes required for iron binding and Tf receptor recognition. For example, mutations can be made in or near N domain amino acid residues 94-96, 245-247 and/or 316-318 and C domain amino acid residues 425-427, 581-582 and/or 652-658. In addition, mutations can be made in or near the flanking regions of these sites to alter the structure and function of Tf.

An exendin-4/Tf fusion protein can function as a carrier protein to extend the half-life or bioavailability of a therapeutic protein, and in some instances, to deliver the therapeutic protein into a cell and/or across the Blood Brain Barrier (BBB). In an alternative embodiment, the fusion protein comprises a modified transferrin molecule, wherein transferrin does not retain the ability to cross the BBB.

In another embodiment, an exendin-4/Tf fusion protein comprises a modified transferrin molecule, wherein the transferrin molecule retains the ability to bind to a transferrin receptor and transport therapeutic peptides into a cell. In an alternative embodiment, the exendin-4/Tf fusion protein comprises a modified transferrin molecule, wherein the transferrin molecule does not retain the ability to bind to a transferrin receptor and transport therapeutic peptides into cells.

In a further embodiment, the exendin-4/Tf fusion protein comprises a modified transferrin molecule, wherein the transferrin molecule retains the ability to bind to a transferrin receptor and transport therapeutic peptides into cells and retains the ability to cross the BBB. In an alternative embodiment, the exendin-4/Tf fusion protein comprises a modified transferrin molecule, wherein the transferrin molecule retains the ability to cross the BBB but does not retain the ability to bind to transferrin receptor and transport therapeutic peptides into cells.

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 may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by natural processes, such as post-translational processing, or by chemical modification techniques known in the art. Such modifications are described in detail in basic texts and in more detailed monographs, as well as in a large body of research literature.

Modifications may be present anywhere in the polypeptide, including the peptide backbone, the amino acid side chains and the amino or carboxyl termini. It is understood that the same kind of modification may be present to the same or different extent at several sites in a given polypeptide. A given polypeptide may also contain many types of modifications. The polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translational natural processing or may be prepared by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, tetradecylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, sulfation, transfer of RNA mediated addition of amino acids to Proteins (e.g., arginylation), and ubiquitination (see, e.g., protein-structures and Molecular Properties, 2nd Ed., T.E.Creighton, W.H.Freeman and Company, New York, 1993; Post-transformation of protein Modification of Proteins, B.C.Johnson, Ed, K, N.S., academycpress, New York, pgs.1-12, 1983; and Seifter et al meth.enzymol.182: 626-646, 1990).

Nucleic acid molecules encoding exendin-4/Tf

The invention also provides nucleic acid molecules encoding exendin-4/Tf fusion proteins. Preferred nucleic acid molecules encode the nucleic acid sequence of SEQ ID NO: 23, which is by (PEAPTD)₂(SEQ ID NO: 5) amino acid sequence of Exendin-4 (1-39) linked to mTf. Exemplary nucleic acid sequences are set forth in SEQ ID NO: as shown at 24. More preferably, the nucleic acid sequence of the invention encodes SEQ ID NO: 25 which is exendin-4 (1-39) (PEAPTD) plus an additional N-terminal 19 amino acids representing the human transferrin secretion signal or leader sequence₂(SEQ ID NO: 5) amino acid sequence of mTf. Exemplary codes for SEQ ID NO: 25 is as set forth in SEQ ID NO: shown at 26.

The sequence encoding the exendin-4/Tf fusion protein may also include a C-terminal stop codon (e.g., tga, taa, tag) and may be readily obtained by a variety of methods including, but not limited to, chemical synthesis, gene mutation of wild-type exendin-4 and transferrin polynucleotide sequences obtained from cDNA or genomic library screening, expression library screening, and/or Polymerase Chain Reaction (PCR) amplification of the cDNA. Nucleic acid molecules encoding exendin-4/Tf fusion proteins can be prepared by site-directed mutagenesis, PCR amplification or other suitable methods wherein the primers have the desired point mutation. The recombinant DNA methods and mutagenesis methods described herein are generally Sambrook et al, Molecular Cloning: a Laboratory Manual, Cold spring Harbor Laboratory Press, 1989, and Current Protocols in molecular Biology, Ausubel et al, Green Publishers Inc. and Wileyand Sons, 1994.

Nucleic acid polynucleotides encoding the amino acid sequence of an exendin-4/Tf fusion protein can be identified by expression cloning using detection of positive clones based on the properties of the expressed protein. Typically, nucleic acid libraries are screened for binding of antibodies or other binding partners (e.g., receptors or ligands) to cloned proteins expressed and displayed on the surface of host cells. The antibody or binding partner is modified with a detectable label to identify those cells expressing the desired clone.

Recombinant expression techniques can be performed in accordance with the description set forth below to prepare exendin-4/Tf fusion protein-encoding polynucleotides and express the encoded polypeptides. For example, a large number of desired nucleotide sequences can be readily prepared by one skilled in the art by inserting a nucleic acid sequence encoding the amino acid sequence of an exendin-4/Tf fusion protein into a suitable vector. This sequence can then be used to generate detection probes or amplification primers. Alternatively, a polynucleotide encoding an exendin-4/Tf fusion protein amino acid sequence may be inserted into an expression vector. The encoded exendin-4/Tf fusion protein can be prepared in large quantities by introducing the expression vector into a suitable host.

Another method for obtaining a suitable nucleic acid sequence is the Polymerase Chain Reaction (PCR). In this method, cDNA is prepared from poly (A) + RNA or total RNA using reverse transcriptase. Two primers (usually complementary to two separate regions of the cDNA encoding the amino acid sequence of the exendin-4/Tf fusion protein) are then added to the cDNA along with a polymerase, such as Taq polymerase, which amplifies the region of the cDNA between the two primers.

The DNA fragment encoding the amino terminus of the polypeptide may have an ATG encoding a methionine residue. Methionine may or may not be present on the mature form of an exendin-4/Tf fusion protein, depending on whether the polypeptide produced in the host cell is designed to be secreted from the cell. The codon encoding isoleucine may also be used as the start site. Other methods known to those skilled in the art may also be used. In some embodiments, the nucleic acid variant comprises an altered codon to optimize expression of the exendin-4/Tf fusion protein in a given host cell. The specific codon changes depend on the exendin-4/Tf fusion protein and the host cell chosen for expression. Such codon optimization may be performed by various methods, for example, by selecting codons that are preferably used in highly expressed genes of a given host cell. A Computer algorithm incorporating a codon frequency table for codon bias of highly expressed bacterial genes (e.g., "Eco _ high. cod") provided by the University of Wisconsin Package Version 9.0(Genetics Computer Group, Madison, Wis.) may be used. Other useful codon frequency tables include "Celegans _ high.cod," Celegans _ low.cod, "Drosophila _ high.cod," Human _ high.cod, "Maize _ high.cod," and "Yeast _ high.cod.

Carrier

The nucleic acid molecule encoding the amino acid sequence of the exendin-4/Tf fusion protein is inserted into a suitable expression vector using standard ligation techniques. The vector is typically selected to be functional in the particular host cell being used (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene may occur). The nucleic acid molecule encoding the exendin-4/Tf fusion protein amino acid sequence may be amplified/expressed in prokaryotic, yeast, insect (baculovirus system) and/or eukaryotic host cells. For a review of expression vectors, see meth.enz, vol.185, d.v. goeddel, Academic Press, 1990.

In general, expression vectors used in any host cell contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences" in some embodiments, typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence comprising donor and acceptor splice sites, a sequence encoding a leader sequence for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

Optionally, the vector may comprise a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5 'or 3' end of the exendin-4/Tf fusion protein coding sequence; the oligonucleotide sequence encodes a polyHis (e.g., 6His), or other "tag" such as FLAG, HA (influenza virus hemagglutinin), or myc against which commercially available antibodies are present. Upon expression of the polypeptide, the tag is typically fused to the polypeptide and can be used as a means for affinity purification of the exendin-4/Tf fusion protein from the host cell. Affinity purification can be accomplished, for example, by column chromatography using an anti-tag antibody as an affinity matrix. Optionally, the tag can then be removed from the purified exendin-4/Tf fusion protein by various methods, e.g., using some peptidases for cleavage, e.g., digestion of the 3' end of the FLAG tag sequence upstream of one of the amino acid sequences with enterokinase.

The flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), heterozygous (i.e., a combination of flanking sequences from more than one source), or synthetic, or the flanking sequences may be native sequences that normally function to modulate exendin-4 expression. The source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, so long as the flanking sequences play a role in and are activatable by the host cell machinery.

Useful flanking sequences may be obtained by any method known in the art. Typically, flanking sequences useful herein have been previously identified by localization and/or by restriction endonuclease digestion, and thus may be isolated from a suitable tissue source with an appropriate restriction endonuclease. In some cases, the entire nucleotide sequence of the flanking sequences may be known. Here, the flanking sequences may be synthesized using the methods described herein for nucleic acid synthesis or cloning.

Wherein all or only a portion of the flanking sequences are known, which may be obtained by PCR and/or by screening a genomic library with suitable oligonucleotides and/or fragments of the flanking sequences from the same or another species. Where the flanking sequences are unknown, the DNA segment comprising the flanking sequences may be isolated from a larger quantity of DNA which may comprise, for example, the coding sequence or even another gene or genes. The separation can be accomplished by: digested with restriction endonucleases to produce suitable DNA fragments, then purified using agarose gel,column chromatography (Qiagen, Chatsworth, Calif.), or other methods known to those skilled in the art. It will be apparent to those skilled in the art that the selection of suitable enzymes will serve this purpose.

The origin of replication is typically part of those prokaryotic expression vectors that are commercially available, and this origin helps the vector to expand in the host cell. The vector was amplified to a copy number, which in some instances was important for optimal expression of the exendin-4/Tf fusion protein. If the vector selected does not contain a replication origin, the replication origin can be chemically synthesized on the basis of known sequences and ligated into the vector. For example, the origin of replication of plasmid pBR 322(New engllandbiolabs, Beverly, MA) is suitable for most gram-negative bacteria and different origins of replication (e.g., SV40, polyoma virus, adenovirus, Vesicular Stomatitis Virus (VSV), or papilloma virus such as HPV or BPV) are suitable for cloning vectors in mammalian cells. In general, for mammalian expression vectors, the origin of replication component is not required (e.g., the SV40 origin is often used simply because it contains an early promoter).

Transcription termination sequences are typically located at the 3' end of the polypeptide coding region and serve to terminate transcription. In general, the transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly-T sequence. When the sequence is readily cloned from a library or even purchased commercially as part of a vector, the sequence can also be readily synthesized using nucleic acid synthesis methods such as those described herein.

The selectable marker gene element encodes a protein that is essential for the survival and growth of host cells cultured in a selective medium. Representative selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins to prokaryotic host cells, e.g., ampicillin, tetracycline, or kanamycin; (b) complement the auxotrophy of the cell; or (c) supplying key nutrients not available from complex media. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. The neomycin resistance gene may also be used in the selection of prokaryotic and eukaryotic host cells.

Other selection genes may be used to amplify the gene to be expressed. Amplification is a process in which a large number of genes required for the production of proteins necessary for growth are repeated in sequence within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. Mammalian cell transformants are placed under selective pressure, wherein only the transformants are uniquely adapted to survive by virtue of the selectable gene present in the vector. The selection pressure is exploited by culturing transformed cells under conditions in which the concentration of the selection agent in the culture medium is continuously varied, resulting in the amplification of both the selection gene and the DNA encoding the exendin-4/Tf fusion protein. As a result, an increased number of Exendin-4/Tf fusion proteins are synthesized from the amplified DNA.

Ribosome binding sites are usually necessary for translation initiation of mRNA and are characterized by Shine-Dalgarno sequences (prokaryotes) or Kozak sequences (eukaryotes). This element is usually located 3 'to the promoter and 5' to the coding sequence of the exendin-4/Tf fusion protein to be expressed. Shine-Dalgarno sequences are diverse but are typically polypurine (i.e., have a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using the methods described herein and used in prokaryotic vectors.

The terms "secretory signal sequence" or "secretory leader sequence" are used interchangeably and are described, for example, in U.S. patents 6,291,212 and 5,547,871. The secretory signal sequence or secretory leader sequence encodes a secretory peptide. Secretory peptides are amino acid sequences used to direct secretion of a mature polypeptide or protein from a cell. Secretory peptides are generally characterized by a hydrophobic amino acid core and are typically found (but not exclusively) at the amino terminus of newly synthesized proteins. Often, during secretion, the secretory peptide is cleaved from the mature protein. The secretory peptide may contain a processing site that allows cleavage of the signal peptide from 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 by, for example, in vitro mutagenesis.

Secretory peptides may be used to direct secretion of the fusion proteins of the invention. One such secretory peptide that may be used in conjunction with other secretory peptides is the alpha pairing factor leader sequence. Secretory signal sequences or secretory leader sequences are necessary for a complex series of post-translational processing steps leading to protein secretion. If the complete signal sequence is present, the expressed protein enters the lumen of the rough endoplasmic reticulum and is then transported via the Golgi apparatus to the secretory vesicles and finally to the outside of the cell. Typically, the signal sequence follows the start codon and encodes a signal peptide at the amino terminus of the protein to be secreted. In most cases, the signal sequence is cleaved by a specific protease (called signal peptidase). Preferred signal sequences enhance the efficiency of processing and export of recombinant proteins expressed using viral, mammalian or yeast expression vectors.

In one embodiment, the native Tf signal sequence may be used for expression and secretion of the fusion proteins of the invention. Since transferrin molecules are present in many types of secretions, such as blood, tears, and milk, many different transferrin signal peptides exist. For example, the transferrin signal peptide can be from serum transferrin, lactoferrin, or melanotransferrin. The native transferrin signal peptide can also be from a variety of species, such as insects, mammals, fish, frogs, ducks, chickens, or other species. Preferably, the signal peptide is derived from a mammalian transferrin molecule. More preferably, the signal peptide is derived from human serum transferrin. The signal peptide sequence from various mammalian transferrin molecules is described in U.S. patent publication No. 2006/0205037.

Preferably, the transferrin-derived signal sequence is used to secrete a heterologous protein, e.g., any protein of interest heterologous to the Tf signal sequence can be expressed and secreted using the Tf signal. In particular, the Tf signal sequence may be used for secretion of proteins from recombinant yeast. Preferably, the signal peptide is derived from human serum transferrin (SEQ ID NO: 18; encoded by SEQ ID NO: 19). Other preferred signal peptides include HSA/MF α -1(SEQ ID NO: 40; encoded by SEQ ID NO: 41), and modified HSA/MF α -1(SEQ ID NO: 42; encoded by SEQ ID NO: 43).

To ensure efficient removal of the signal peptide sequence, in some cases it may be preferable to include a short propeptide sequence between the signal sequence and the mature protein, where the C-terminal portion of the propeptide comprises a recognition site for a protease (e.g., the yeast kex2p protease). Preferably, the propeptide sequence is from about 2 to 12 amino acids in length, more preferably from about 4 to 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).

Expression and cloning vectors typically comprise a promoter that is recognized by the host organism and operably linked to a molecule encoding an exendin-4/Tf fusion protein. Promoters are non-transcribed sequences, located upstream (i.e., 5' to) the start codon of a structural gene (typically within about 100 to 1000 bp), which control transcription of the structural gene. Promoters are routinely divided into two classes: inducible promoters and constitutive promoters. In response to some change in culture conditions, such as the presence or absence of nutrients or a change in temperature, an inducible promoter causes an increase in the level of transcription of the DNA under its control. On the other hand, constitutive promoters cause sustained gene product production; that is, little or no control of gene expression. A large number of promoters are known which are recognized by a variety of potential host cells. A suitable promoter is operably linked to DNA encoding an exendin-4/Tf fusion protein by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector. Native exendin-4 or transferrin promoter sequences can be used to direct the amplification and/or expression of an exendin-4/Tf fusion protein nucleic acid molecule. However, a heterologous promoter is preferred if it allows for a greater amount of transcription and higher yield of the expressed protein compared to the native promoter, and if it is compatible with the system of the host cell chosen for use.

Suitable promoters for use in yeast hosts are also known in the art and are discussed further below. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use in mammalian host cells are known and include, but are not limited to, those obtained from the viral genome, such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis b virus and most preferably simian virus 40(SV 40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat shock promoters and actin promoters.

Promoters suitable for use in prokaryotic hosts include the beta-lactamase and lactose promoter systems; coli T7 inducible RNA polymerase; alkaline phosphatase; a tryptophan (trp) promoter system; and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their sequences have been disclosed so as to enable one skilled in the art to ligate them to the desired DNA sequence by using the required linkers or linkers (to provide any useful restriction sites).

Other promoters that are useful for controlling expression of an exendin-4/Tf fusion protein include, but are not limited to: SV40 early promoter region (Bemoist and Chambon, Nature 290: 304-10, 1981); a CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Rous sarcomavir) (Yamamoto et al, Cell 22: 787-97, 1980); the herpes thymidine kinase (hepes thymidines kinase) promoter (Wagner et al, Proc. Natl. Acad. Sci. U.S.A.78: 1444-45, 1981); regulatory sequences of the metallothionein (metallothionine) gene (Brinster et al, Nature 296: 39-42, 1982); prokaryotic expression vectors such as the beta-lactamase promoter (Villa-Kamaroff et al, Proc. Natl. Acad. Sci. U.S.A.75: 3727-31, 1978); or the tac promoter (DeBoer et al, Proc. Natl. Acad. Sci. U.S.A., 80: 21-25, 1983).

Enhancer sequences can be inserted into vectors to increase transcription of DNA encoding exendin-4/Tf fusion proteins in higher eukaryotic cells. Enhancers are cis-acting elements of DNA, usually about 10-300bp in length, that act on a promoter to increase transcription. Enhancers are relatively independent of orientation and position. They have been found at the 5 'and 3' ends of the transcription unit. Some enhancer sequences obtained from mammalian genes are known (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically an enhancer from a virus is used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and the adenovirus enhancer are exemplary enhancing elements for activating eukaryotic promoters. While the enhancer may be spliced into the vector at the 5 ' or 3 ' end position of the exendin-4/Tf fusion protein encoding nucleic acid molecule, it is usually located at a site 5 ' to the promoter.

Expression vectors can be constructed from starting vectors, such as commercially available vectors. Such vectors may or may not contain all of the desired flanking sequences. When one or more of the flanking sequences described herein are not present in a vector, they may be obtained separately and ligated into the vector. Methods for obtaining the individual flanking sequences are well known to those skilled in the art.

Suitable yeast vectors for use in the present invention are described, for example, in U.S. Pat. No. 6,291,212 and include YRp7(Struhl et al, Proc. Natl. Acad. Sci. USA 76: 1035-. Useful yeast plasmid vectors also include pRS403-406, pRS413-416 and Pichia vectors from Stratagene Cloning Systems (La Jolla, Calif.). Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integrative plasmids (YIp) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA 3. Plasmids pRS 413-41.6 are yeast centromere plasmids (YCp).

Such vectors typically include a selectable marker, which can be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable selection of transformants. Preferred selectable markers are those that complement the auxotrophy of the host cell, provide antibiotic resistance or enable the cell to utilize a particular carbon source, and include LEU2(Broach et al supra), URA3(Botstein et al, Gene 8: 17, 1979), HIS3(Struhl et al, supra) or POT1(Kawasaki and Bell, European patent EP 171, 142). Other suitable selectable markers include the CAT gene, which confers chloramphenicol resistance to yeast cells. Preferred promoters for use in yeast include promoters from the yeast glycolytic genes (Hitzeman et al, J biol. chem.225: 12073-12080, 1980; Alber and Kawasaki, J. mol. appl. Genet.1: 419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or the alcohol dehydrogenase genes (Young et al, genetic Engineering of Microorganisms for Chemicals, Hollander et al, p.355, Plenum, N.Y., 1982; Amerer, meth. enzymol.101: 192-201, 1983). In this regard, a particularly preferred promoter is TPI1 promoterMover (Kawasaki, U.S. Pat. No. 4,599,311) and ADH2-4^C(see U.S. Pat. No. 3, 6,291,212 promoter (Russell et al, Nature 304: 652-Asa 654, 1983.) expression units may also include a transcription terminator A preferred transcription terminator is TPI1 terminator (Alber and Kawasaki, supra.) other preferred vectors and preferred components such as promoters and terminators of yeast expression systems are disclosed in European patents EP 0258067, EP 0286424, EP0317254, EP 0387319, EP 0386222, EP 0424117, EP0431880, EP 1002095EP, EP 0828759, EP 0764209, EP 0749478, and EP 0889949, PCT publications WO 00/44772 and WO 94/04687, and U.S. Pat. Nos. 5,739,007, 5,637,504, 5,302,697, 5,260,202, 5,667,986, 5,728,386,386, 5,783,423, 5,965,133, 6,6150,924, 5,379,714, and 5,377,714.

In addition to yeast, the fusion proteins of the invention may also be expressed in filamentous fungi, such as Aspergillus (Aspergillus) strains. Examples of useful promoters include promoters from the glycolytic genes of Aspergillus nidulans (Aspergillus nidulans), such as the adh3 promoter (McKnight et al, EMBO J.4: 2093-2099, 1985) and the tpiA promoter. An example of a suitable terminator is the adh3 terminator (McKnight et al, supra). Expression units utilizing such components can be cloned into, for example, a vector capable of inserting into the chromosomal DNA of Aspergillus.

Other vectors are vectors compatible with bacterial, insect, and mammalian host cells. Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1(Invitrogen, Carlsbad, CA), pBSII (Stratagene), pET15(Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2(Clontech, PaloAlto, CA), pETL (BlueBacll, Invitrogen), pDSR-alpha (PCT publication No. WO 90/14363), and pFastBacDual (Gibco-BRL, Grand Island, NY).

Additional suitable vectors include, but are not limited to, cosmids, plasmids, or modified viruses, but it is understood that the vector system must be compatible with the host cell of choice. Such vectors include, but are not limited to, plasmids, e.g.Plasmid derivatives (high copy number ColE 1-based phagemids, Stratagene), PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g.,TAa kit of parts for the detection of a drug,plasmid derivatives, Invitrogen), and mammalian, yeast or viral vectors, such as baculovirus expression systems (pBacPAK plasmid derivatives, Clontech).

Also included in the expression vector is a polyadenylation signal located downstream of the coding sequence of interest. Polyadenylation signals include early or late polyadenylation signals from SV40 (Kaufman and Sharp, supra), polyadenylation signals from the 5E1B region of adenovirus, and human growth hormone gene terminator (DeNoto et al, Nucl. acid Res.9: 3719-3730, 1981). A particularly preferred polyadenylation signal is V_HGene terminators (see U.S. patent 6,291,212). The expression vector may include a non-coding viral leader sequence, such as an adenovirus 2 tripartite leader sequence, located between the promoter and the RNA splice sites. Preferred vectors may also include enhancer sequences such as the SV40 enhancer (see U.S. Pat. No. 6,291,212) and the mouse enhancer (Gillies, Cell 33: 717-728, 1983). The expression vector may also include sequences encoding an adenoviral VA RNA.

After construction of the vector and insertion of the nucleic acid molecule encoding the exendin-4/Tf fusion protein into the appropriate site of the vector, the complete vector can be inserted into a suitable host cell for amplification and/or polypeptide expression. Transformation of an expression vector for an exendin-4/Tf fusion protein into a selected host cell can be accomplished by well-known methods, including methods such as transfection, infection, electroporation, microinjection,lipofection, the DEAE-dextran method, or other known techniques. The method selected will be, in part, a function of the type of host cell used. These and other suitable methods are well known to those skilled in the art and are described, for example, in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989. Cloned DNA sequences comprising the fusion proteins of the invention can be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al, Cell 14: 725, 1978; Corsaro and Pearson, physical Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973.). Other techniques for introducing cloned DNA sequences into mammalian cells may also be used, such as electroporation (Neumann et al, EMBO J.1: 841-845, 1982), or lipofection. To identify cells that have integrated the cloned DNA, a selectable marker is typically introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers for use in mammalian cells in culture include genes that confer resistance to drugs (e.g., neomycin, hygromycin and methotrexate). The selectable marker may be an amplifiable selectable marker. Preferred amplifiable selectable markers are the DHFR gene. Particularly preferred amplifiable markers are DHFR^r(see U.S. Pat. No. 6,291,212) cDNA (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80: 2495-. Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass.) and selection of selectable markers is within the level of ordinary skill in the art.

Host cell

The invention also includes cells, preferably yeast cells, transformed to express an exendin-4/Tf fusion protein of the invention. In addition to the transformed host cells themselves, the present invention also includes cultures of these cells in nutrient media, preferably monoclonal (clonally homologous) cultures, or cultures derived from monoclonal cultures. If the polypeptide is secreted, the medium will contain the polypeptide, and the cells, or no cells (if the cells have been removed by filtration or centrifugation).

A particularly useful host cell for the production of the Exendin-4/Tf fusion proteins of the present invention is Pichia methylotrophica (Steinlein et al, protein express. Purif.6: 619-. Pichia pastoris has been developed as an important host for the production of foreign proteins since its alcohol oxidase promoter was isolated and cloned; its transformation was first reported in 1985. Pichia pastoris can utilize methanol as a carbon source in the absence of glucose. The pichia expression system may use the methanol-induced alcohol oxidase (AOX1) promoter, which controls the gene encoding the expression of alcohol oxidase, which catalyzes the first step of methanol metabolism. This promoter has been characterized and incorporated into a range of pichia expression vectors. Since the proteins produced in pichia are usually correctly folded and secreted into the culture medium, fermentation of genetically engineered pichia provides an excellent alternative to the e. Many proteins have been produced using this system, including tetanus toxin fragments, Bordetella pertussis adhesin (Bordetella pertussis pertactin), human serum albumin and lysozyme.

A strain of Saccharomyces cerevisiae is another preferred host. In a preferred embodiment, a yeast cell is used, or more particularly, a s.cerevisiae host cell comprising a genetic defect in a gene necessary for asparagine-linked glycosylation of glycoproteins. Saccharomyces cerevisiae host cells with such defects can be prepared using standard techniques of mutation and selection, although many available yeast strains have been modified to prevent or reduce glycosylation or high mannose glycosylation. Ballou et al (J.biol.chem.255: 5986-5991, 1980) have described the isolation of defective mannoprotein biosynthetic mutants in genes which influence asparagine-linked glycosylation. Gentzsch and Tanner (Glycobiology 7: 481-486, 1997) have described a family of at least six genes (PMT1-6) encoding enzymes responsible for the first step of O-glycosylation of proteins in yeast. Mutants deficient in one or more of these genes exhibit reduced O-linked glycosylation and/or altered specificity of O-glycosylation.

In one embodiment, the host is a strain of Saccharomyces cerevisiae described in PCT publication No. WO 05/061718. For example, the host may comprise pSAC 35-based plasmids carrying one copy of PDI1 gene or any other chaperone gene, respectively, in a strain with a host version (host version) with a knock-out of PDI1 or other chaperone. Such constructs confer enhanced stability.

For optimal production of the heterologous protein, it is also preferred that the host strain carries a mutation, for example the Saccharomyces cerevisiae pep4 mutation (Jones, Genetics 85: 23-33, 1977), which results in a reduction of the proteolytic activity. Host strains containing mutations in other protease coding regions are particularly useful for producing large quantities of the exendin-4/Tf fusion proteins of the present invention.

When cultured under appropriate conditions, the host cell synthesizes an exendin-4/Tf fusion protein, which can then be collected from the culture medium (if the host cell secretes it into the culture medium) or produced directly from the host cell (if it is not secreted). The choice of an appropriate host cell depends on a variety of factors, such as the desired expression level, the polypeptide modifications desired or necessary for activity (e.g., glycosylation or phosphorylation) and the ease of folding into a biologically active molecule.

Other host cells may be prokaryotic host cells (e.g., E.coli) or eukaryotic host cells (e.g., insect or vertebrate cells). Many suitable host cells are known in the art and many are available from the American Type Culture Collection (ATCC), Manassas, Va. Examples include, but are not limited to, mammalian cells, such as Chinese hamster ovary Cells (CHO), CHO DHFR (-) cells (Urlaub et al, Proc. Natl. Acad. Sci. U.S.A.97: 4216-20, 1980), Human Embryonic Kidney (HEK)293 or 293T cells, or 3T3 cells. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening, product production, and purification are known in the art. Other suitable mammalian cell lines are monkey COS-1 and COS-7 cell lines, and CV-1 cell lines. Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell lines obtained from in vitro culture of primary tissues, and primary explants are also suitable. The candidate cells may be genotypically deficient in the selection gene, or may contain a dominant acting selection gene. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3 cell lines from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines. Each of these cell lines is known and available to those skilled in the art of protein expression.

Similarly useful as suitable host cells are bacterial cells. For example, various strains of E.coli (e.g., HB101, DH5 α, DH10, and MC1061) are well known host cells in the biotechnology arts. Bacillus subtilis (B.subtilis), Pseudomonas spp (Pseudomonas spp.), other Bacillus spp (Bacillus spp.) and Streptomyces spp (Streptomyces spp) may also be used.

In addition, when desired, insect cell systems can be used to express exendin-4/Tf fusion proteins. Such systems are described, for example, in Kitts et al, Biotechniques 14: 810-17, 1993; lucklow, curr. opin.biotechnol.4: 564-72, 1993; and Lucklow et al, j.virol, 67: 4566-79, 1993. Preferred insect cells are Sf-9 and Hi5 (Invitrogen).

Production of Exendin-4/Tf fusion cells

The host cells of the invention comprising the DNA construct are cultured in a suitable growth medium. As used herein, the term "suitable growth medium" means a medium that comprises nutrients necessary for cell growth. Nutrients necessary for cell growth may include carbon sources, nitrogen sources, essential amino acids, vitamins, minerals, and growth factors. The growth medium typically selects for cells comprising the DNA construct by, for example, drug selection or lack of essential nutrients (which are supplemented by a selectable marker on or co-transfected with the DNA construct). Yeast cells, for example, are preferably cultured in chemically defined media containing a carbon source (e.g., sucrose), a non-amino acid nitrogen source, inorganic salts, vitamins, and essential amino acid supplements. The pH of the medium is preferably maintained at a pH of greater than 2 and less than 8, preferably at a pH of 5.5-6.5. Methods for maintaining a stable pH include buffering and constant pH control. Preferred buffers include succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, Mo.). Yeast cells deficient in the genes necessary for asparagine-linked glycosylation are preferably cultured in a medium comprising an osmotic pressure stabilizer. A preferred osmotic pressure stabilizer is sorbitol added to the medium at a concentration of between 0.1M and 1.5M, preferably 0.5M or 1.0M.

Suitable media for culturing E.coli cells include, for example, Luria Broth (LB) and/or Terrific Broth (TB). Suitable media for culturing eukaryotic cells include Roswell Park mental Institute Medium 1640(RPMI 1640), Minimum Essential Medium (MEM) and/or Dulbecco's Modified EagleMedium (DMEM), all of which can supplement the serum and/or growth factors necessary for the particular cell line being cultured. Suitable media for insect cell culture are Grace's medium supplemented with yeast powder (yeastolate), whey protein hydrolysate, and/or fetal bovine serum (if necessary).

Typically, antibiotics or other compounds for selective culture of transfected or transformed cells are added to the culture medium as supplements. The compound used may be determined by the selectable marker element present in the plasmid used to transform the host cell. For example, when the selectable marker element is kanamycin resistance, the compound added to the medium is kanamycin. Other compounds useful for selective culture include ampicillin, tetracycline and neomycin.

Baculovirus/insect cell expression systems may also be used to produce the modified Tf fusion proteins of the invention. BacPAK^TMBaculovirus expression System (BD Biosciences (Clontech)) in insect hostsThe recombinant protein is expressed at high levels in the cell. The target gene was inserted into a transfer vector that was co-transfected into an insect host cell with linearized BacPAK6 viral DNA. BacPAK6DNA lacks essential parts of the baculovirus genome. When the DNA is recombined with the vector, the essential elements are repaired and the target gene is transferred to the baculovirus genome. After recombination, some viral plaques were selected and purified and tested for recombination phenotype. The new isolated recombinant virus can then be amplified and used to infect insect cell cultures to produce large quantities of the desired protein.

The exendin-4/Tf fusion proteins of the present invention can also be produced in transgenic plants and animals. For example, sheep and goats may produce therapeutic proteins in their emulsions. Or the tobacco plants may contain proteins in their leaves. The production of transgenic plant and animal proteins involves the addition of a novel gene encoding a fusion protein to the genome of an organism. Not only can a transgenic organism produce a novel protein, but it can also transmit this ability to its progeny.

The amount of exendin-4/Tf fusion protein produced by a host cell can be assessed using standard methods known in the art. Such methods include, but are not limited to, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, High Performance Liquid Chromatography (HPLC) separation, immunoprecipitation, and/or activity assays, such as DNA binding gel shift assays.

If an exendin-4/Tf fusion protein has been designed to be secreted from a host cell line, the majority of the polypeptide can be found in the cell culture medium. However, if the polypeptide is not secreted from the host cell, the polypeptide is present in the cytoplasm and/or nucleus (for eukaryotic host cells) or in the cytosol (for gram-negative bacterial host cells).

For exendin-4/Tf fusion proteins located in the cytoplasm and/or nucleus (for eukaryotic host cells) or cytosol (for gram-negative bacterial host cells), intracellular material (including inclusion bodies of gram-negative bacteria) can be extracted from the host cell using any standard technique known to those skilled in the art. For example, the host cells can be lysed to release the periplasmic/cytoplasmic contents by french press, homogenization, and/or sonication followed by centrifugation.

If the exendin-4/Tf fusion protein forms inclusion bodies in the cytosol, the inclusion bodies are usually bound to the medial and/or lateral cell membrane and can therefore be found mainly in the pelleted material after centrifugation. The precipitated material may then be treated with a chaotropic agent, such as a detergent, guanidine derivative, urea, or urea derivative, at extreme pH or in the presence of a reducing agent (e.g., dithiothreitol at basic pH or tricarboxyethylphosphine at acidic pH) to release, split, and solubilize the inclusion bodies. Solubilized exendin-4/Tf fusion proteins are then analyzed by gel electrophoresis, immunoprecipitation, and the like. If it is desired to isolate the polypeptide, standard procedures can be used, for example as described herein and in Marston et al, meth.Enz.182: 264-75, 1990 to achieve separation.

If inclusion bodies are not formed in significant amounts after expression of the exendin-4/Tf fusion protein, the polypeptide can be found predominantly in the supernatant after centrifugation of the cell homogenate. The polypeptide may be further isolated from the supernatant using methods such as those described herein.

Many other methods for producing polypeptides are known in the art and these methods can be used to produce exendin-4/Tf fusion proteins. See, e.g., Roberts et al, proc.natl.acad.sci.u.s.a.94: 12297-303, 1997, which describe the production of fusion proteins between mRNA and the peptide encoded thereby. See also Roberts, curr, opin, chem, biol.3: 268-73, 1999.

Methods for producing peptides or polypeptides are also described in U.S. Pat. nos. 5,763,192, 5,814,476, 5,723,323, and 5,817,483. The method includes generating random genes or fragments thereof, and then introducing these genes into a host cell that produces one or more proteins encoded by the random genes. The host cells are then screened to identify those clones that produce peptides or polypeptides having the desired activity. Other methods for recombinant peptide expression are disclosed in U.S. Pat. Nos. 6,103,495, 6,210,925, 6,627,438, and 6,737,250. This method uses E.coli and the common secretory pathway of E.coli. Peptide fusion to signal sequence; thus, the peptide is targeted for secretion.

Another method for producing a peptide or polypeptide is described in PCT publication No. WO 99/15650. The disclosed method, referred to as random activation of gene expression for discovery of genes, includes activation of endogenous gene expression or overexpression of genes by in situ recombination methods. For example, expression of an endogenous gene is activated or increased by integrating regulatory sequences into a target cell capable of activating gene expression by non-homologous or aberrant recombination. First the target DNA is subjected to radiation and the gene promoter is inserted. The promoter is finally located in the cleft (break) in front of the gene, initiating gene transcription. This results in the expression of the desired peptide or polypeptide.

Isolation/purification of Exendin-4/Tf fusion proteins

The secreted, biologically active, exendin-4/Tf fusion protein can be isolated from the culture medium in which the host cell is cultured under conditions that allow secretion of the biologically active fusion protein. Cell material is removed from the culture medium and the biologically active fusion protein is isolated using isolation techniques known in the art. Suitable separation techniques include precipitation and separation by a variety of chromatographic methods including gel filtration, ion exchange chromatography and affinity chromatography.

Particularly preferred purification methods are affinity chromatography on iron-binding or metal-chelating columns or immunoaffinity chromatography using transferrin or antigens of therapeutic proteins against the polypeptide fusion. The antigen is preferably immobilized or bound to a solid support or matrix. In one embodiment, the substrate is CNBr activated Sepharose (Pharmacia LKB Technologies, inc., Piscataway, n.j.). By this method, the medium is bound to the antigen/substrate under conditions that allow binding to occur. The complex is washed to remove unbound material and the exendin-4/Tf fusion protein is released or eluted by using conditions that are unfavorable for complex formation. Particularly useful elution methods include changing the pH, wherein the immobilized antigen has a high affinity for an exendin-4/Tf fusion protein at a first pH and a reduced affinity at a second pH (higher or lower); varying the concentration of certain chaotropic agents; or by using detergents.

Purification of exendin-4/Tf fusion proteins from solution can be achieved by a variety of techniques. If a polypeptide comprising a tag (e.g.6 histidine 9 or other small peptide, such as FLAG (Eastman Kodak Co., New Haven, CT) or myc (Invitrogen)) has been synthesized at its carboxy-or amino-terminus, purification can be carried out in a one-step process by passing the solution through an affinity column, where the column matrix has a high affinity for the tag.

For example, polyhistidine binds nickel with high affinity and specificity. Thus, a nickel affinity column (e.g., a nickel affinity column)Nickel column) can be used for purification. See Current Protocols in molecular Biology, 10.11.8 (supra).

In addition, an exendin-4/Tf fusion protein can be purified by using a monoclonal antibody capable of specifically recognizing and binding to an exendin-4/Tf fusion protein.

When the exendin-4/Tf fusion protein is preferably partially or completely purified so that it is partially or substantially contaminant free, standard methods known to those skilled in the art may be used. Such methods include, but are not limited to, electrophoretic separation followed by electroelution, various types of chromatography (affinity, immunoaffinity, molecular sieve, and ion exchange), HPLC, and preparative isoelectric focusing ("Isoprotie" instrument/technique, Hoefer Scientific, San Francisco, Calif.). In some cases, two or more purification techniques may be combined to achieve increased purity.

Pharmaceutical composition

The exendin-4/Tf fusion proteins of the present invention may generally be administered in the form of a pharmaceutical composition. The pharmaceutical compositions may, for example, be in a suitable form for oral administration (e.g. tablets, capsules, pills, powders, solutions, suspensions), for parenteral injection (e.g. sterile solutions, suspensions or emulsions), for intranasal administration (e.g. aerosol drops (aerosol drops) or the like), for rectal administration (e.g. suppositories) or for transdermal administration (e.g. patches). The pharmaceutical compositions may be in unit dosage form suitable for single administration of precise dosages. Pharmaceutical compositions will comprise an exendin-4 Tf fusion protein of the invention as an active ingredient and may comprise conventional pharmaceutical carriers. In addition, it may include other pharmaceutical agents, adjuvants, and the like.

Methods for preparing various pharmaceutical compositions of biologically active peptides are known in the art of pharmacy. See, for example, U.S. patent 2005/0009748 (for oral administration); and us patent 2004/0157777, 2005/0002927 and 2005/0215475 (for transmucosal administration, e.g., intranasal or buccal administration). See also Remington: the Practice of pharmacy, Lippincott Williams and Wilkins, Baltimore, MD, 20^th ed.，2000。

Traditionally, peptide and protein drugs are administered by injection because of the low bioavailability when administered orally. These drugs are prone to chemical and conformational instability and are often degraded by the acidic environment in the stomach and by enzymes in the gastrointestinal tract. In response to these delivery problems, several techniques for oral delivery have been developed, such as encapsulation into nanoparticles consisting of polymers with hydrophobic backbones and hydrophilic branches as drug carriers, encapsulation into microparticles, liposomes inserted into emulsions, and conjugation with other molecules. All of these techniques can be used with the fusion molecules of the present invention.

Examples of nanoparticles include mucoadhesive nanoparticles coated with chitosan and carbopol (Takeuchi et al, adv. drug Deliv. Rev.47: 39-54, 2001) and nanoparticles comprising a charged binding polyester, poly (2-sulfobutyl-vinyl alcohol) and a polylactic acid-polyglycolic acid copolymer (Jung et al, Eur. J. Pharm. Biopharm. 50: 147-160, 2000). Nanoparticles comprising a surface polymer having poly-N-isopropylacrylamide domains and cationic poly-vinylamine groups showed improved absorption of salmon calcitonin when orally administered to rabbits.

Drug delivery particles composed of alginate and pectin, reinforced with polylysine, are relatively resistant to acids and bases and can be used as drug carriers. These particles combine the advantages of bioadhesion, enhanced absorption and sustained release (Liu et al, J.Pharm.Pharmacol.51: 141-149, 1999).

In addition, it has been shown that conjugation to N-and C-terminal lipoamino acid groups and lipopolysaccharide groups of peptides, such as synthetic somatostatin, produces amphoteric surfactants, resulting in compositions that retain biological activity (Toth et al, J.Med.chem.42 (19): 4010-.

Examples of other peptide delivery techniques include carbopol coated mucoadhesive emulsions comprising the peptide of interest and nitroso-N-acetyl-D, L-penicillamine and carbopol or taurocholate and carbopol. These techniques have been shown to be effective when orally administered to rabbits to reduce blood calcium concentrations (Ogiso et al, biol. pharm. Bull. 24: 656-661, 2001). Phosphatidylethanol derived from phosphatidylcholine is used to prepare liposomes containing phosphatidylethanol as a carrier for insulin. These liposomes have been shown to be functional when administered orally to rabbits (Kisel et al, int.J.Pharm.216: 105-114, 2001).

Insulin has been formulated into poly (vinyl alcohol) -gel spheres comprising insulin and a protease inhibitor (e.g., aprotinin or bacitracin). The glucose-lowering properties of these gel beads have been demonstrated in rabbits, where insulin is released in large amounts in the lower intestine (Kimura et al, biol. pharm. Bull. 19: 897-900, 1996.

Oral delivery of insulin has also been studied using nanoparticles prepared from poly (alkylcyanoacrylates) dispersed with a surfactant in the oil phase (Damge et al, J.Pharm.Sci.86: 1403-1409, 1997) and using calcium alginate beads coated with chitosan (Onal et al, artist.Cells blood plasma Substitt.Immobil.Biotechnol.30: 229-237, 2002).

In other methods, the N-and C-termini of the peptide are linked to polyethylene glycol and then to an allyl chain to form conjugates with enhanced resistance to enzymatic degradation and enhanced diffusivity through the gastrointestinal wall (GIwell) (r)www.nobexcorp.com)。

Is a cationic lipid mixture that interacts non-covalently with peptides to produce a protective coating or layer. The peptide-lipid complex may be fused to the plasma membrane of the cell, whereby the peptide is internalized into the cell.

In processes using liposomes as starting material, helical particles have been developed as pharmaceutical vehicles. The peptide is added to a suspension of liposomes containing predominantly negatively charged lipids. The addition of calcium causes the liposomes to disintegrate and fuse into a large sheet (sheet) consisting of lipid bilayers, which then spontaneously roll up or pack into a helix (U.S. Pat. No. 5,840,707).

In addition, the invention includes pulmonary delivery of exendin-4/Tf fusion protein formulations. Pulmonary delivery is particularly promising for delivery of macromolecules, which are difficult to deliver by other routes of administration. Such pulmonary delivery is effective for both systemic delivery and local delivery to treat pulmonary disease because the drug delivered to the lung is readily absorbed directly into the blood circulation through the alveolar region.

The present invention provides compositions suitable for forming pharmaceutical dispersions for oral inhalation (pulmonary delivery) to treat various conditions or diseases. The fusion protein formulations can be delivered by different routes, such as liquid nebulizers, aerosol-based metered dose inhalers (MDI's), and dry powder dispensing devices. Pharmaceutically acceptable carriers including surfactants or surfactants and bulking (bulking) carriers are typically added in the process of formulating compositions for pulmonary delivery to provide stability, dispersibility, consistency and/or bulking (bulking) properties to enhance uniform pulmonary delivery of the composition to a subject.

The surfactant or surfactants facilitate the absorption of the peptide through the mucosa or mucosal lining. Useful surfactants or surfactants include fatty acids and salts thereof, bile salts, phospholipids, or alkyl sugars. Examples of the fatty acid and its salt include octanoic acid (C)₈) Decanoic acid (C)₁₀) Lauric acid (C)₁₂) And myristic acid (C)₁₄) Sodium, potassium and lysine salts. Examples of bile salts include cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, lithocholic acid, ursodeoxycholic acid.

Examples of the phospholipid include single-chain phospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine, or double-chain phospholipids such as diacylphosphatidylcholine, diacylphosphatidylglycerol, diacylphosphatidylethanolamine, diacylphosphatidylinositol, and diacylphosphatidylserine. Examples of the alkyl sugar include alkyl glucoside or alkyl maltoside, such as decyl glucoside and dodecyl maltoside.

Pharmaceutical excipients used as carriers include stabilizers such as Human Serum Albumin (HSA), bulking agents such as sugars, amino acids and polypeptides; a pH adjusting agent or buffer, and a salt such as sodium chloride. These carriers may be crystalline or amorphous or may be a mixture of both.

Examples of the sugar used as the swelling agent include monosaccharides such as galactose, D-mannose, and sorbose, disaccharides such as lactose and trehalose; cyclodextrins, such as 2-hydroxypropyl- β -cyclodextrin, and polysaccharides, such as raffinose, maltodextrin, and extrans, sugar alcohols, such as mannitol and xylitol. Examples of polysaccharides useful as bulking agents include aspartame. Amino acids include alanine and glycine, preferably glycine.

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

Suitable pH adjusting agents or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, and sodium ascorbate; sodium citrate is preferred.

The GLP-1 receptor agonist fusion composition for pulmonary delivery may be packaged as a unit dose, wherein the therapeutically effective amount of the composition is present in a unit dose container, such as a blister pack or gelatin capsule. Blister packs or gelatin capsules are generally prepared by methods generally known in the packaging art.

Us patent 6,524,557 discloses pharmaceutical aerosol formulations comprising (a) an HFA propellant; (b) a pharmaceutically active polypeptide dispersed in a propellant; and (C) a surfactant which is C₈-C₁₆A fatty acid or salt thereof, a bile salt, a phospholipid, or an alkyl sugar, said surfactant enhancing systemic absorption of the polypeptide in the lower respiratory tract. The invention also provides methods of making such formulations and the use of such formulations in treating patients.

One method for pulmonary delivery of dry powder drugs utilizes a handheld device having a hand pump for providing 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 is made available for inhalation by the patient.

Dry powder dispersion devices are described in several patents. Us patent 3,921,637 describes a manual pump with a needle for piercing individual capsules of powdered medicament. The use of multiple reservoir trays or drug strips is described in european patent EP 0467172; PCT publication Nos. WO 91/02558 and WO 93/09832; and U.S. Pat. nos. 4,627,432, 4,811,731, 5,035,237, 5,048,514, 4,446,862, 5,048,514, and 4,446,862.

Aerosolization of protein therapeutics is disclosed in european patent EP 0289336. Therapeutic aerosol formulations are disclosed in PCT publication No. WO 90/09781.

Method of treatment

The exendin-4/Tf fusion proteins of the present invention can be used in combination with other pharmaceutical agents for the treatment of the conditions or diseases described herein. Thus, methods of treatment comprising administering the compounds of the invention in combination with other pharmaceutical agents are also provided by the invention.

In a method aspect of the invention, an exendin-4/Tf fusion protein of the invention, alone or in combination with one or more pharmaceutical agents, is administered peripherally to a patient, separately or together, in any conventional manner known in the art for peripheral administration. Thus, the exendin-4/Tf fusion protein or combination may be administered to a patient parenterally (e.g., intravenously, intraperitoneally, intramuscularly, or subcutaneously), intranasally, orally, sublingually, buccally, by inhalation (e.g., by aerosol), rectally (e.g., by suppository), or transdermally. Parenteral, but not oral administration (e.g., injection) is the preferred method of administration, and subcutaneous administration is the preferred method of parenteral administration. Pulmonary delivery by inhalation is also a preferred method of administration.

Compositions suitable for parenteral injection typically include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers or diluents (including solvents and vehicles) include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, including triglycerides of vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate.

These compositions for parenteral injection may also contain excipients such as preservatives, wetting agents, solubilizing agents, emulsifying agents and dispersing agents. Various antibacterial and antifungal agents may be used to prevent microbial contamination of the composition, for example, parabens (parabens), chlorobutanol, phenol and sorbic acid. It may also be desirable to include isotonic agents, for example, sugars and sodium chloride. Prolonged absorption of injectable pharmaceutical compositions can be brought about by the use of agents capable of delaying absorption, for example, aluminum monostearate and gelatin.

The exendin-4/Tf fusion proteins of the present invention may be administered to a patient at doses that vary depending on a number of factors, including the mode of administration, the age and weight of the subject, the severity of the disease, the condition or disorder being treated, and the pharmaceutical activity of the exendin-4/Tf fusion protein to be administered. The determination of dosage ranges and optimal dosages for a particular patient are well within the skill of the ordinary artisan.

For parenteral injection for treatment to reduce blood glucose, the peptide of SEQ ID NO: exendin-4 (1-39) (PEAPTD) shown as 23₂(SEQ ID NO: 5) the mTf fusion protein can be administered to a human subject at a dosage level ranging from about 0.5-50mg per dose, more preferably 0.5-20mg per dose, and dosed about once a week, once every two weeks, or once a month.

For parenteral injection for treatment to reduce body weight, the dosage range may be higher than the dosage range for reducing blood glucose. Thus, for parenteral administration for treatment to reduce body weight, the peptide of SEQ ID NO: exendin-4 (1-39) (PEAPTD) shown as 23₂(SEQ ID NO: 5) the mTf fusion protein can be administered to a human subject at a dosage level ranging from about 1-100mg per dose, and dosed about once a week, once every two weeks, or once a month.

The invention also provides an exendin-4/Tf fusion protein for use in the treatment of type II diabetes or reducing blood glucose in a human patient. Further provided are exendin-4/Tf fusion proteins of the invention for use in the treatment of obesity or reduction of food intake in a human patient. A further aspect of the invention provides the use of an exendin-4/Tf fusion protein of the invention in the manufacture of a medicament for the treatment of type II diabetes or for lowering blood glucose in a human patient. A further aspect provides the use of an exendin-4/Tf fusion protein of the invention in the manufacture of a medicament for the treatment of obesity or for reducing food intake. Features of the method aspect of the invention may be applied to these aspects.

Embodiments of the present invention are illustrated by the following examples. It should be understood, however, that the embodiments of the invention are not limited to the particular details of these examples, as other variations thereof will be apparent to those skilled in the art, or may be apparent from the instant disclosure and the appended claims. All references cited herein are incorporated by reference in their entirety.

Examples

Example 1: construction of Exendin-4/Tf fusion proteins

Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) mTf fusion protein

The Exendin-4 (1-39) DNA sequence (SEQ ID NO: 20) was inserted between the secretory signal sequence (nL) (SEQ ID NO: 19) and the mTf sequence (SEQ ID NO: 22) of pREX0549 using site-overlap extension (SOE) PCR. Two primers, P0702(SEQ ID NO: 28) and P0703(SEQ ID NO: 29), were designed to insert the sequence using pREX0549 as a template.

The DNA sequence (SEQ ID No.: 30) was obtained by retranslating the exendin-4 amino acid sequence using codons optimized for yeast expression. Two PCR products were initially generated using primers P0177(SEQ ID NO: 31) and P0702 with an AflII site at 5 'or primers P0014(SEQ ID NO: 32) and P0703 with a BamHI site at 3'. The products from these reactions were gel purified and added to a second round of PCR using only the outer primers P0177 and P0014.

The product from this second reaction was gel purified and digested with restriction enzymes AflII and BamHI as performed on plasmid pREX 0549. The appropriate products from these reactions were ligated together to give pREX0561, which was DNA sequenced between the AflII and BamHI sites to determine the correct insertion of the exendin-4 sequence. The expression cassette was recovered from pREX0561 by digestion with restriction enzyme NotI and ligated into NotI-digested bovine intestinal alkaline phosphatase-treated pSAC35 to give pREX 0589.

SOE PCR was performed using pREX0561 as a template and primers P1810(SEQ ID NO: 33) and P1811(SEQ ID NO: 34) to introduce a linker peptide sequence (PEAPTD) between the C-terminus of the exendin-4 sequence encoding the C-terminus and the N-terminus of the mTf sequence encoded by the same method as described above₂(SEQ ID NO.：21)。

The final product from this PCR was gel purified and digested with restriction enzymes AflII and BamHI as performed on plasmid pREX 0549. The appropriate products from these reactions were ligated together to give pREX0935, which was DNA sequenced between the AflII and BamHI sites to determine the code (PEAPTD)₂(SEQ ID NO: 5) correct insertion of the sequence. The expression cassette was recovered from pREX0935 by digestion with restriction enzyme NotI and ligated into NotI-digested, alkaline phosphatase-treated pSAC35 to give pREX 0936. Exendin-4 (1-39) (PEAPTD) without nL leader sequence₂(SEQ ID NO: 5) the amino acid sequence of the mTf fusion protein is provided herein as SEQ ID NO: 23. encoding the amino acid sequence of SEQ ID NO: 23 is provided herein as SEQ ID NO: 24. exendin-4 (1-39) (PEAPTD) with nL leader sequence₂(SEQ ID NO: 5) the amino acid sequence of the mTf fusion protein is provided herein as SEQ ID NO: 25. encoding the amino acid sequence of SEQ ID NO: 25 is provided herein as SEQ ID NO: 26.

other Exendin-4/Tf constructs

Exendin-4 has an additional 9 amino acids at the C-terminus compared to GLP-1. In the case of free peptides, these additional residues are believed to confer increased affinity for the GLP-1 receptor and greater protease resistance. However, it may also be responsible for the immunogenicity of the peptide to some extent. Two additional constructs were prepared in essentially the same manner as described above to prepare a construct having only a sequence homologous to GLP-1, i.e. Exendin-4 (1-31) or Exendin-4 (1-30), by deleting the DNA sequence encoding residues 32-39 or 31-39, respectively. For Exendin-4 (1-31), primers P0904(SEQ ID NO: 35) and P0941(SEQ ID NO: 36) were used and the appropriate products were ligated (pREX0629/pREX 0658). For Exendin-4 (1-30), primers P0942(SEQ ID NO: 37) and P0943(SEQ ID NO: 38) were used and the appropriate products were ligated (pREX0630/pREX 0659).

Exendin-4 (1-39) sequences and optional linkers, e.g. (GGGGS)₃(SEQ ID NO: 39), PEAPTD (pREX1005) (SEQ ID NO: 6), or IgG hinge (pREX0938) (SEQ ID NO: 7-16).

Additional Exendin-4/Tf constructs with other Signal sequences (affecting relative Productivity)

The constructs were generated to express exendin-4/mTf (SEQ ID NO: 23; encoded by SEQ ID NO: 24) linked to the signal sequence HSA/MF α -1(pREX 1354) (SEQ ID NO: 40; encoded by SEQ ID NO: 41) and modified HSA/MF α -1(pREX 1345) (SEQ ID NO: 42; encoded by SEQ ID NO: 43). Comparison of the productivity of yeast strains expressing exendin-4/mTf with three different signal sequences showed that the relative productivity of transferrin signal sequence (nL)/HSA/MF α -1/modified HSA/MF α -1 was 1/1.75/1.32.

Example 2: determination of the potency of Exendin-4/Tf fusion proteins

The potency was calculated from the measured cAMP response generated as a result of GLP-1 receptor mediated ligand binding in CHO cells transfected with rabbit GLP-1 receptor (CHO-GLP-1R) after incubation with the sample. 96-well tissue culture plates were seeded with CHO-GLP-1R cells and cultured overnight. The next day, cells were washed with Krebs-Ringer buffer (KRB) and incubated with 3-isobutyl-1-methylxanthine (IBMX) which is a phosphodiesterase inhibitor2mM) KRB to inhibit intracellular enzymes that process cAMP. Serial dilutions of test compound and control were prepared in KRB/IBMX and wells of (inoculate) cells were inoculated with sample and control (in triplicate). After incubation, with a competition-based fluorescence immunoassay (campfluoro Assay Kit, Molecular Devices corp., Sunnyvale, CA) assayed individual sample lysates to measure increases in intracellular cAMP levels. The amount of cAMP accumulation in the cell following GLP-1 receptor mediated ligand binding is used to determine biological activity and relative potency.

The data in Table 1 indicate that the exendin-4/Tf fusion proteins activate the GLP-1 receptor more than GLP-1(7-37, A8G, K34A) (PEAPTD)₂(SEQ ID NO: 5) the mTf fusion protein is more potent. The mTf portion in each fusion protein has the amino acid sequence of SEQ ID NO: 17.

Table 1: potency of GLP-1/mTf and Exendin-4/Tf fusion proteins

Example 3: exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) MMP resistance of mTf fusion proteins

Testing Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) resistance of the mTf fusion protein (SEQ ID NO: 23) to in vitro inactivation by matrix metalloproteinase I (MMP-1, collagenase). Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) mTf fusion protein and GLP-1(7-37, A8G, K34A) (PEAPTD)₂(SEQ ID NO: 5) samples of mTf fusion protein were incubated with recombinant MMP-1 at 37 ℃ for 48 hours and then tested for activity. FIG. 1 shows that an exendin-4/Tf fusion protein (FIG. 1B) is resistant to inactivation of MMP-1 compared to a GLP-1/mTf fusion protein (FIG. 1A). Albeit with an amino group in the active part of the moleculeThe sequences were very similar, but again this difference occurred in the degradation.

Example 4: exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) Effect of mTf fusion proteins on blood glucose in diabetic mice

With different doses of Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) mTf fusion protein (SEQ ID NO: 23), GLP-1(7-37, A8G, K34A) (PEAPTD)₂The mTf fusion protein, or exendin-4 (Bachem, King of Prussia, PA), was injected into diabetic (db/db) mice and blood glucose concentration was monitored by analyzing blood samples with a glucometer. As shown in FIG. 2, exendin-4 (1-39) (PEAPTD) at doses as low as 1.3nmole/kg₂(SEQ ID NO: 5) the mTf fusion protein significantly reduced blood glucose in these animals over a3 hour period following subcutaneous injection. Glucose levels were almost normalized in all treatment groups and the levels lasted 24 hours. The glucose concentration was gradually increased to the pre-treatment level over a period of 48-72 hours after treatment, depending on the dose administered. Exendin-4 peptides lower blood glucose than Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) the mTf fusion protein is abundant, regardless of dose, and the glucose-lowering effect of Exendin-4 is completely lost after about 12 hours. The maximal reduction in blood glucose achievable with exendin-4 was about 37%, which is consistent with literature reports; with Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) the reduction observed for the mTf fusion protein was about 70%. Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) the effect of mTf fusion protein on blood glucose was also significantly greater than the equivalent dose of GLP-1(7-37, A8G, K34A) (PEAPTD)₂(SEQ ID NO: 5) the mTf fusion protein is stronger and has a longer duration.

Example 5: exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) Effect of mTf fusion protein on Rabbit weight

With different doses of Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) mTf fusion protein (SEQ ID NO: 23), and Exendin-4 subcutaneous SpragueDawley rabbit. mTf or saline was used as a control. Rabbits were weighed daily (during dosing period before dosing). The animals were in close proximity to food and water at all times.

As shown in FIG. 3, Exendin-4 (1-39) (PEAPTD) was administered at doses of 10 and 100nmole/kg₂(SEQ ID NO: 5) mTf fusion protein treated animals lost weight after the first injection and continued to lose weight throughout the dosing period. By day 5, animals treated with the 100nmole/kg dose had an average 75 gram body weight (17%) reduction compared to controls and the weight reduction was correlated with a reduction in food and water intake. Daily drug administration was stopped on day 5 because of the significant and dramatic weight loss observed. All animals gained weight at a similar rate after the 5 day dosing period. However, with Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) the mTf fusion protein treated group, especially the high dose group, was still lighter than the control 20 days after the last dose.

Example 6: exendin-4 (1-39) (PEAPTD) for glycemic control in type II diabetic patients and for weight loss₂(SEQ ID NO: 5) predicted dose of mTf fusion protein

Based on published data, a therapeutic single dose of 10 μ g of exenatide () Produces a Cmax of 200pg/mL in humans. In terms of blood sugar lowering effect, exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) the difference in molecular size between the mTf fusion proteins (SEQ ID NO: 23) (4.2kDa vs. 80.5kDa) indicates an approximately 3.8ng/mL of Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) blood levels of mTf fusion protein are equivalent to therapeutic levels of exenatide. Exendin-4 (1-39) (PEAPTD) based on in vitro testing in CHO cells expressing the human GLP-1 receptor, in addition to molecular size₂(SEQ ID NO: 5) the mTf fusion protein is approximately 5-fold less potent than Exendin-4 (FIG. 4). Thus, to achieve similar therapeutic activity of 10 μ g of exenatide,about 20ng/ml of Exendin-4 (1-39) (PEAPTD) is required₂(SEQ ID NO: 5) circulating concentration of mTf fusion protein.

Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) mTf fusion protein and GLP-1(7-37, A8G, K34A) (PEAPTD)₂(SEQ ID NO: 5) the mTf fusion proteins are similar in molecular size and structure. Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) the molecular weight of the mTf fusion protein is 80.5kDa and GLP-1(7-37, A8G, K34A) (PEAPTD)₂(SEQ ID NO: 5) the molecular weight of the mTf fusion protein is 79.6 kDa. Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) mTf fusion protein vs GLP-1(7-37, A8G, K34A) (PEAPTD)₂(SEQ ID NO: 5) mTf fusion proteins are about 4-8 times more effective. Exendin-4 (1-39) (PEAPTD) administered to macaques at 1mg/kg intravenously and subcutaneously₂(SEQ ID NO: 5) the mean pharmacokinetic parameters of the mTf fusion proteins are listed in Table 2. Subcutaneous administration of GLP-1(7-37, A8G, K34A) (PEAPTD) at 0.6mg/kg to macaques₂(SEQ ID NO: 5) the mean pharmacokinetic parameters of the mTf fusion proteins are listed in Table 3.

Table 2: exendin-4 (1-39) (PEAPTD) administered to macaques at 1mg/kg intravenously and subcutaneously₂(SEQ ID NO: 5) summary of mean pharmacokinetic parameters of mTf fusion proteins

Parameter(s)	Intravenous administration of drugs	Under the skin
			Cmax(ng/mL)	33,981±14,826(4)	5,236±1,038(4)
Tmax(h)	0.542(4)	9.02(4)
			AUC(0-t)(h·ng/mL)	567,364±68,102(4)	278,067±24,367(4)
AUC(inf)(h·ng/mL)	572,314±68,660(4)	280,279±29,261(3)
			λz(h-1)	0.0313±0.0137(4)	0.0252±0.0084(3)
T1/2(h)	25.5±10.3(4)	29.3±08.2(3)
			CL(mL/min/kg)	1.77±0.24(4)	-
Vz(mL/kg)	65.9±31.0(4)	-
			F(％)	-	49.0

TABLE 3 GLP-1(7-37, A8G, K34A) (PEAPTD) after subcutaneous administration to macaques at 0.6mg/kg₂(SEQ ID NO: 5) summary of mean pharmacokinetic parameters of mTf fusion proteins

Parameter(s)	Male sex	Female
			Cmax(ng/mL)	2,922±1,530(3)	3,173±1,767(3)
Tmax(h)	12.0(3)	24.2(3)
			AUC(0-t)(h·ng/mL)	168,087±58,749(3)	165,474±32,756(3)
AUC(inf)(h·ng/mL)	171,963±61,998(3)	186,065±140(2)
			λz(h-1)	0.0216±0.0042(3)	0.0250±0.0002(2)
t(h)	32.9±6.78(3)	27.7±0.23(2)
			CL(mL/min/kg)	-	-
Vz(mL/kg)	-	-
			F(％)	-	-

In a separate experiment, GLP-1(7-37, A8G, K34A) (PEAPTD)₂(SEQ ID NO: 5) bioavailability of mTf fusion proteins was shown to be about 50% in cynomolgus monkeys.

Exendin-4 (1-39) (PEAPTD) for intravenous and subcutaneous administration₂(SEQID NO: 5) half-life (t) of clearance of mTf fusion protein_1/2) Range of Tmax, and bioavailability (F (%)) vs GLP-1(7-37, A8G, K34A) (PEAPTD)₂(SEQ ID NO: 5) monkey pharmacokinetic parameters for the mTf fusion proteins were similar.

GLP-1(7-37, A8G, K34A) (PEAPTD) was used previously₂Human experience with (SEQ ID NO: 5) mTf shows that the pharmacokinetics are linear from a dose of 30. mu.g/kg to 900. mu.g/kg, with a mean Tmax at 48 hours and a mean t_1/2About 50 hours. Cmax is 758 + -435 ng/mL at a dose of 300 μ g/kg (or 30mg/100kg patient) and C at a dose of 900 μ g/kg (90mg/100kg patient)max was 1,609. + -. 805 ng/mL. However, neither the fusion protein showed a robust effect (robust effect) on blood glucose levels in diabetic subjects at these doses, or at the 1800 μ g/kg dose.

Due to the similarity in size and structure, and the similar pre-clinical pharmacokinetic profile between the two compounds in monkeys, Exendin-4 (1-39) (PEAPTD) is expected₂(SEQ ID NO: 5) pharmacokinetic characteristics of mTf fusion proteins in humans with GLP-1(7-37, A8G, K34A) (PEAPTD)₂(SEQ ID NO: 5) mTf fusion proteins are similar.

Based on the combination with GLP-1(7-37, A8G, K34A) (PEAPTD)₂(SEQ ID NO: 5) similarity of mTf to GLP-1(7-37, A8G, K34A) (PEAPTD)₂(SEQ ID NO: 5) Exendin-4 (1-39) (PEAPTD) in mTf comparison₂(SEQ ID NO: 5) the in vitro potency of the mTf fusion protein was relatively 4-8 fold higher, compared to exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) the in vitro potency of mTf fusion proteins was relatively low 5-fold, surprisingly 2mg dose of Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) mTf fusion proteins can have glucose lowering effects. Further, a 10mg dose per subject is required, once weekly, to achieve a stable Cmin of 20 ng/mL. Exendin-4 (1-39) (PEAPTD) for use in the treatment of blood glucose lowering₂(SEQ ID NO: 5) an effective dose of mTf fusion protein (SEQ ID NO: 23) ranges from 0.5 to 50mg per dose administered once a week. Such doses may also be administered once every two weeks or once every month.

Exendin-4 (1-39) (PEAPTD) of 10nmole/kg or more in addition to the effect on blood glucose₂(SEQ ID NO: 5) the mTf fusion protein is associated with a reduction in animal body weight in mice and rabbits. Mixing 10 or 100nmole/kg Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) A single dose of mTf fusion protein resulted in an average reduction of 6% and 14% in body weight, respectively, 24 hours after administration to mice, compared to an average reduction of 1% in control or in 1nmole/kg of Exendin-4 treated animals. Administered daily at a dose of 100nmol/kgExendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) the mTf fusion protein also resulted in a 16% reduction in rabbit body weight. The weight loss can be attributed to the administration of Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) reduction in food intake observed in animals with mTf fusion proteins, a known pharmacokinetic effect of GLP-1 receptor activation. Available data indicate that the dose required for weight loss is about 2-3 times higher than the dose required for glucose lowering. Exendin-4 (1-39) (PEAPTD) for weight reduction₂(SEQ ID NO: 5) an effective dose of mTf fusion protein (SEQ ID NO: 23) ranges from 1.0 to 100mg per dose administered once a week. Such doses may also be administered once every two weeks or once every month.

Example 7: exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) delivery of mTf fusion proteins by inhalation

An aerosol was generated with an Aerotech II compressed air jet nebulizer (CIS-usnc, Bedford, MA) and introduced through a 1.58cm diameter stainless steel aerosol delivery line into a 24 port flow through rodent exposure system (IN-TOX, ABQ, NM). The exhaust flow rate outside the chamber was 11.5L/min. Atomizer pressure was maintained at-30 psi.

To test the nebulizing effect of the fusion proteins, 10mg/mL Exendin-4 (1-39) (PEAPTD) in 10mM histidine pH 7.4, 100mM NaCl₂(SEQ ID NO: 5) mTf fusion protein (SEQ ID NO: 23) solution nebulization and then 5mL of concentrated liquid from the aerosol was collected in a biosampler for more than 8 minutes and tested for integrity and activity. The nebulization process was on Exendin-4 (1-39) (PEAPTD) as judged by SDS-PAGE and SEC-HPLC₂(SEQ ID NO: 5) No detectable side effects were observed with the structure of the mTf fusion protein; there was no significant decomposition or aggregate formation. The recovered material also showed biological activity.

For in vivo testing, diabetic mice (db/db) were placed in the inhalation chamber and allowed to breath aerosolized exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) mTf fusion proteins for varying lengths of time. At the end of the inhalation exposure period, the mice were monitored for blood glucose during the next 72 hours. The time of inhalation in the cavity was chosen so that the animals received exposures equivalent to subcutaneous administration of 0.3, 1 and 3mg/kg doses. As a control, these doses were administered Subcutaneously (SC) to compare the in vivo activity of administration by the inhaled route. Receiving Exendin-4 (1-39) (PEAPTD) by inhalation₂(SEQ ID NO: 5) blood glucose levels in animals with mTf fusion proteins showed a significant decrease after exposure to the drug. Exendin-4 (1-39) (PEAPTD)₂(SEQ ID NO: 5) evaluation of circulating levels of mTf fusion protein indicated that the bioavailability of this non-optimized system and formulation was about 10%.

Claims (21)

1. A fusion protein comprising an exendin-4 (SEQ ID NO: 4) fused to a transferrin (Tf) via a polypeptide linker (peasttd) 2(SEQ ID NO: 5), wherein the Tf is modified to exhibit reduced glycosylation as compared to a native transferrin molecule, and the Tf has the amino acid sequence as set forth in SEQ ID NO: 17.

2. The fusion protein of claim 1, wherein the exendin-4 molecule is fused at the N-terminus of the fusion protein, the C-terminus of the fusion protein or both the N-terminus and the C-terminus of the fusion protein.

3. The fusion protein of claim 1, wherein the N-terminus of the fusion protein further comprises a secretion signal sequence.

4. The fusion protein of claim 3, wherein the signal sequence is a signal sequence from serum transferrin, lactoferrin, melanotransferrin, or a variant thereof.

5. The fusion protein of claim 3, wherein the signal sequence is a Human Serum Albumin (HSA)/MF α -1 hybrid leader sequence, a modified HSA/MF α -1 hybrid leader sequence, or a Tf signal sequence.

6. The fusion protein of claim 3, wherein the signal sequence is as set forth in SEQ ID NO: 18, human Tf signal sequence shown in fig. 18.

7. A nucleic acid encoding the fusion protein of any one of claims 1-6.

8. A vector comprising the nucleic acid of claim 7.

9. A host cell comprising the vector of claim 8.

10. A fusion protein comprising an exendin-4 fused to a modified transferrin (mTf), wherein said fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 23, or a pharmaceutically acceptable salt thereof.

11. Encoding the amino acid sequence of SEQ ID NO: 23.

12. The nucleic acid of claim 11, wherein said nucleic acid comprises the sequence set forth as SEQ ID NO: 24, or a sequence shown in fig. 24.

13. A vector comprising the nucleic acid of claim 11 or 12.

14. A host cell comprising the vector of claim 13.

15. A pharmaceutical composition comprising the fusion protein of any one of claims 1-6 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising the fusion protein of claim 10 and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, wherein the composition is suitable for administration at a dose ranging from 0.5mg to 50 mg.

18. The pharmaceutical composition of claim 17, wherein the composition is suitable for administration at a dose of 1mg to 100 mg.

19. The pharmaceutical composition of claim 15 or 16, wherein the composition is suitable for administration by inhalation.

20. Use of a therapeutically effective amount of a fusion protein according to any one of claims 1-6 and 10 in the manufacture of a medicament for treating type II diabetes or reducing blood glucose in a human patient in need thereof.

21. Use of a therapeutically effective amount of a fusion protein according to any one of claims 1-6 and 10 in the manufacture of a medicament for treating obesity or reducing body weight in a human patient in need thereof.