EXTENDED RECOMBINANT POLYPEPTIDES AND COMPOSITIONS COMPRISING SAME

• US 20100239554A1
• Filed: 02/03/2010
• Published: 09/23/2010
• Est. Priority Date: 02/03/2009
• Status: Active Grant

• Current Assignee: Amunix Pharmaceuticals, Inc. (Sanofi )
• Original Assignee: Amunix, Inc.
• Inventors: Wang, Chia-wel, Silverman, Joshua, Schellenberger, Volker, To, Wayne, Spink, Benjamin, Stemmer, Willem P., Cleland, Jeffrey L., Geething, Nathan C.
• Granted Patent: US 8,673,860 B2
• Time in Patent Office: Days
• US Class Current: 424/94.300
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/14 Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 5/06 of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• CPC Class Codes: A61P 5/10 of the posterior pituitary hormones, e.g. oxytocin, ADH
• CPC Class Codes: A61P 5/22 for decreasing, blocking or antagonising the activity of calcitonin
• CPC Class Codes: A61P 5/48 : of the pancreatic hormones
• CPC Class Codes: A61P 5/50 : for increasing or potentiating the activity of insulin
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/04 : Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/06 : Antianaemics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/12 : Antihypertensives
• CPC Class Codes: A61P 9/14 : Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• CPC Class Codes: C07K 14/001 : by chemical synthesis
• CPC Class Codes: C07K 14/47 : from mammals
• CPC Class Codes: C07K 14/545 : IL-1
• CPC Class Codes: C07K 14/605 : Glucagons
• CPC Class Codes: C07K 14/61 : Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C07K 14/745 : Blood coagulation or fibrinolysis factors
• CPC Class Codes: C07K 2319/31 : fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/35 : containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
• CPC Class Codes: C12N 9/6437 : Coagulation factor VIIa (3.4.21.21)
• CPC Class Codes: C12N 9/644 : Coagulation factor IXa (3.4.21.22)

Abstract

The present invention relates to compositions comprising biologically active proteins linked to extended recombinant polypeptide (XTEN), isolated nucleic acids encoding the compositions and vectors and host cells containing the same, and methods of using such compositions in treatment of glucose-related diseases, metabolic diseases, coagulation disorders, and growth hormone-related disorders and conditions.

Claims

1. An isolated extended recombinant polypeptide (XTEN) comprising greater than about 400 to about 3000 amino acid residues, wherein the XTEN is characterized in that:

(a) the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues constitutes more than about 80% of the total amino acid sequence of the XTEN;

(b) the XTEN sequence is substantially non-repetitive;

(c) the XTEN sequence lacks a predicted T-cell epitope when analyzed by TEPITOPE algorithm, wherein the TEPITOPE algorithm prediction for epitopes within the XTEN sequence is based on a score of −

9 or greater.

(d) the XTEN sequence has greater than 90% random coil formation as determined by GOR algorithm; and

(e) the XTEN sequence has less than 2% alpha helices and 2% beta-sheets as determined by Chou-Fasman algorithm.

2. The isolated XTEN of claim 1, wherein the XTEN is further characterized in that:

(a) the sum of asparagine and glutamine residues is less than 10% of the total amino acid sequence of the XTEN; and

/or

(b) the sum of methionine and tryptophan residues is less than 2% of the total amino acid sequence of the XTEN.

3. The isolated XTEN of claim 1, wherein the XTEN is further characterized in that:

(a) no one type of amino acid constitutes more than 30% of the XTEN sequence;

(b) the XTEN comprises a sequence in which no three contiguous amino acids are identical unless the amino acid is serine, in which case no more than three contiguous amino acids are serine residues; and

/or

(c) the XTEN sequence has a subsequence score of less than 10.

4. The isolated XTEN of claim 1, wherein the XTEN is further characterized in that:

(a) at least about 80% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the sequence motifs has about 9 to about 14 amino acid residues and wherein the sequence of any two contiguous amino acid residues does not occur more than twice in each of the sequence motifs;

(b) the sequence motifs consist of four to six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and

(c) the XTEN enhances pharmacokinetic properties of a biologically active protein (BP) when linked to the BP as a fusion protein wherein the pharmacokinetic properties are ascertained by measuring the blood concentration of the fusion protein after administration of a therapeutically effective dose to a subject in comparison to the corresponding BP not linked to XTEN and administered to a subject at a comparable dose.

5. The isolated XTEN of claim 4, wherein the enhanced pharmacokinetic property is selected from an increase in terminal half-life of at least three-fold and blood concentrations that remain within the therapeutic window for the fusion protein for a period at least about three-fold longer compared to the corresponding BP not linked to XTEN.

6. The isolated XTEN of claim 4, wherein the sequence motifs are selected from one or more sequences of Table 1.

7. An isolated fusion protein, comprising the XTEN of claim 1 linked to a BP selected from the group consisting of glucose regulating peptide, metabolic protein, coagulation protein, and growth hormone.

8. The isolated fusion protein of claim 7, wherein the BP is glucose regulating peptide.

9. The isolated fusion protein of claim 8, wherein the glucose regulating peptide is exendin-4.

10. The isolated fusion protein of claim 7, wherein the BP is metabolic protein.

11. The isolated fusion protein of claim 10, wherein the BP is IL-1ra.

12. The isolated fusion protein of claim 7, wherein the BP is a coagulation factor.

13. The isolated fusion protein of claim 12, wherein the BP is factor IX.

14. The isolated fusion protein of claim 12, wherein the BP is factor VII.

15. The isolated fusion protein of claim 7, wherein the BP is growth hormone.

16. An isolated fusion protein, comprising the XTEN of claim 1 linked to a BP exhibiting at least 90% sequence identity to a sequence selected from Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8.

17. The isolated fusion protein of claim 7, further comprising a second XTEN sequence wherein the cumulative total of amino acid residues in the XTEN sequences is greater than about 400 to about 3000 residues.

18. The isolated fusion protein of claim 7, further comprising a spacer sequence between the BP and XTEN, wherein the spacer sequence comprises between 1 to about 50 amino acid residues that optionally comprises a cleavage sequence.

19. The isolated fusion protein of claim 18, wherein the cleavage sequence is susceptible to cleavage by a protease selected from FXIa, FXIIa, kallikrein, FVIIa, FIXa, FXa, thrombin, elastase-2, granzyme B, MMP-12, MMP-13, MMP-17 or MMP-20, TEV, enterokinase, rhinovirus 3C protease, and sortase A.

20. The isolated fusion protein of claim 7, wherein the fusion protein exhibits binding for a target receptor or ligand of the corresponding native BP in the range of about 0.1%-30% of the binding capability of the corresponding BP that is not linked to the fusion protein.

23. An isolated nucleic acid comprising a polynucleotide sequence selected from (a) a polynucleotide encoding the XTEN of claim 1 or the fusion protein of claim 7 or 21, or (b) the complement of the polynucleotide of (a).

24. An expression vector comprising the polynucleotide sequence of claim 23.

25. The expression vector of claim 24, further comprising a recombinant regulatory sequence operably linked to the polynucleotide sequence.

26. The expression vector of claim 24, wherein the polynucleotide sequence is fused in frame to a polynucleotide encoding a secretion signal sequence.

27. The expression vector of claim 26, wherein the secretion signal sequence is a prokaryotic signal sequence.

28. A host cell, comprising the expression vector of claim 24, wherein the host cell is selected from a prokaryotic cell or a eukaryotic cell.

29. The host cell of claim 28, wherein the prokaryotic host cell is E. coli.

30. The host cell of claim 28, wherein the eukaryotic host cell is CHO.

21. An isolated fusion protein comprising a sequence that has at least 90% sequence identity to a sequence selected from Table 40, Table 41, Table 42, Table 43, and Table 44.

22. A pharmaceutical composition comprising the fusion protein of claim 7 or 21 and at least one pharmaceutically acceptable carrier.

31. A kit, comprising packaging material, at least a first container comprising the pharmaceutical composition of claim 22, a label identifying the pharmaceutical composition and storage and handling conditions, and a sheet of instructions for the reconstitution and/or administration of the pharmaceutical compositions to a subject.

32. A method of treating a disease, disorder or condition, comprising administering a therapeutically effective dose of the pharmaceutical composition of claim 22 to a subject in need thereof.

33. The method of claim 32, wherein the disease, disorder or condition is selected from type 1 diabetes, type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin production, insulin resistance, syndrome X, excessive appetite, insufficient satiety, glucagonomas, dyslipidemia, retinal neurodegenerative processes, Factor VII deficiency, Factor X deficiency, Factor XII deficiency, hemophilia A, hemophilia B, Von Willebrand'"'"'s disease, hypertension, acute coronary syndrome, rheumatoid arthritis, reperfusion injury following ischemia, growth-hormone deficiency, Turner'"'"'s Syndrome, Prader-Willi Syndrome, idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn'"'"'s disease, ulcerative colitis, and muscular dystrophy.

34. The method of claim 32, wherein the pharmaceutical composition is administered subcutaneously, intramuscularly, or intravenously.

35. The method of claim 32, wherein the therapeutically effective dose results in maintaining blood concentrations of the fusion protein within a therapeutic window for the fusion protein at least three-fold longer compared to the corresponding BP not linked to the fusion protein and administered at a comparable dose to a subject.

36. The method of claim 32, wherein multiple consecutive doses of the pharmaceutical composition are administered using a therapeutically effective dose regimen.

37. The method of claim 36, wherein the therapeutically effective dose regimen results in a gain in time of at least four-fold between at least two consecutive C_max peaks and/or C_min troughs for blood levels of the fusion protein compared to the corresponding BP not linked to the fusion protein and administered using a comparable dose regimen to a subject.

38. The method of claim 36, wherein the therapeutically effective dose regimen results in an improvement in at least one measured parameter in a subject using less frequent dosing or a lower total dosage in moles of the fusion protein of the pharmaceutical composition administered to the subject compared to the corresponding BP not linked to the fusion protein and administered to a subject

39. The method of claim 38, wherein the one measured parameter is selected from fasting glucose level, response to oral glucose tolerance test, peak change of postprandial glucose from baseline, HbA_lc, caloric intake, satiety, rate of gastric emptying, insulin secretion, peripheral insulin sensitivity, response to insulin challenge, beta cell mass, body weight, prothrombin time, bleeding time, thrombin-antithrombin III complex (TAT), D-dimer, incidence of bleeding episodes, erythrocyte sedimentation rate (ESR), C-reactive protein, bone density, muscle mass, blood pressure, plasma triglycerides, HDL, cholesterol, LDL cholesterol, incidence of angina, and cardiac output.