Compositions and methods for producing bioactive fusion proteins

• US 20090118181A1
• Filed: 05/22/2008
• Published: 05/07/2009
• Est. Priority Date: 05/22/2007
• Status: Active Grant

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Walker, Kenneth W., Gegg, Colin V. JR.
• Granted Patent: US 8,420,779 B2
• Time in Patent Office: Days
• US Class Current: 514/1.100
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: C07K 14/43504 from invertebrates
• CPC Class Codes: C07K 14/43522 from scorpions
• CPC Class Codes: C07K 14/524 Thrombopoietin, i.e. C-MPL ligand
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• 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

Abstract

Disclosed is a composition of matter involving a recombinant fusion protein comprising a a pharmacologically active protein partner, and a small pharmacologically inactive protein domain partner of human origin, such as but not limited to, a 10^th fibronectin III domain, a SH3 domain, a SH2 domain, a CH2 domain of IgG1, a PDZ domain, a thrombospondin repeat domain, an ubiquitin domain, a leucine-rich repeat domain, a villin headpiece HP35 domain, a villin headpiece HP76 domain, or a fragment or modification of any of these. Also disclosed are nucleic acids (e.g., DNA constructs) encoding the fusion protein, expression vectors and recombinant host cells for expression of the fusion protein, and pharmaceutical compositions containing the recombinant fusion protein and a pharmaceutically acceptable carrier, and method of producing a pharmacologically active recombinant fusion protein.

Claims

1. A composition of matter comprising a recombinant fusion protein, wherein said fusion protein comprises:

(a) a pharmacologically inactive protein domain of human origin, wherein said pharmacologically inactive protein domain

(i) has a mass of about 3 kDa to about 20 kDa, and

(ii) characteristically forms protein aggregates of less than about 10 percent of total mass of protein when suspended without other proteins in a pharmaceutically acceptable formulation buffer of interest; and

(b) at least one pharmacologically active protein about 5 to about 80 amino acid residues in length.

2. The composition of matter of claim 1 of the formula

(F¹)_a—

X

and multimers thereof, wherein;

F¹ is a half-life extending moiety, and a is 0 or 1;

X² is D-(L)_c-(P⁵)_d—

(X³)_e, (X⁴)_f—

(P⁵)_d-(L)_c-D, or (X⁴)_f—

(P⁵)_d-(L)_c-D-(L)_g-(P⁶)_h—

(X³)_i, wherein c and g are each independently 0 or 1, d and h are 1, and e, f, and i are each independently 0, 1, 2, 3, or 4;

X³ is -(L)_j-(P⁷), j is 0 or 1;

X⁴ is (P⁸)-(L)_k-, k is 0 or 1;

D is the pharmacologically inactive protein domain of human origin;

P⁵, P⁶, P⁷ and P⁸ are each independently pharmacologically active proteins; and

L is in each instance a peptidyl linker.

3. The composition of matter of claim 1 or claim 2, wherein the pharmacologically inactive protein domain is selected from a 10^th fibronectin III domain, a SH3 domain, a SH2 domain, a CH2 domain of IgG1, a PDZ domain, a thrombospondin repeat domain, an ubiquitin domain, a leucine-rich repeat domain, a villin headpiece HP35 domain, and a villin headpiece HP76 domain, or a fragment or modification of any of these that is soluble and maintains its native, or near-native, secondary or tertiary structure in a biologically compatible aqueous buffer at physiological pH and temperature.

4. The composition of matter of claim 1 or claim 2, wherein the pharmacologically inactive protein domain comprises an amino acid sequence selected from SEQ ID NOS:

1 through 8, 13, 50, 61, 86 through 90, 94, and 102 through 110.

5. The composition of matter of claim 1 or claim 2, wherein the pharmacologically active protein is a toxin peptide, a CGRP peptide antagonist, a bradykinin B1 receptor peptide antagonist, a PTH agonist peptide, a PTH antagonist peptide, an ang-2 binding peptide, a myostatin binding peptide, an EPO-mimetic peptide, a TPO-mimetic peptide, a NGF binding peptide, a BAFF antagonist peptide, a GLP-1 or peptide mimetic thereof, or a GLP-2 or peptide mimetic thereof.

6. The composition of matter of claim 5, wherein the toxin peptide is selected from ShK, a ShK peptide analog, OSK1 and an OSK1 peptide analog.

7. The composition of matter of claim 2, wherein a is 1, and F¹ is a polyethylene glycol, a copolymer of ethylene glycol, a polypropylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid, a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycosylated chain, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group;

or a combination of any of these members.

8. The composition of matter of claim 2, wherein a is 1, and F¹ is a polyethylene glycol.

9. The composition of matter of claim 2, wherein a is 1, and F¹ is selected from peptide ligands or small molecule ligands that have binding affinity for a long half-life plasma protein.

10. A pharmaceutical composition, comprising the composition of matter of claim 1 or claim 2, and a pharmaceutically acceptable carrier.

11. A nucleic acid comprising a polynucleotide sequence encoding a recombinant fusion protein comprising:

(a) a pharmacologically inactive protein domain of human origin, wherein said pharmacologically inactive protein domain

(i) has a mass from about 3 kDa to about 20 kDa, and

(ii) characteristically forms protein aggregates of less than about 10 percent of the total mass of protein when suspended without other proteins in a pharmaceutically acceptable formulation buffer of interest; and

(b) a pharmacologically active protein of about 5 to about 80 amino acid residues in length.

12. The nucleic acid of claim 11, wherein the pharmacologically inactive protein domain is selected from a 10^th fibronectin III domain, a SH3 domain, a SH2 domain, a CH2 domain of IgG1, a PDZ domain, a thrombospondin repeat domain, an ubiquitin domain, a leucine-rich repeat domain, a villin headpiece HP35 domain, and a villin headpiece HP76 domain, or a fragment or modification of any of these that is soluble and maintains its native, or near-native, secondary or tertiary structure in a biologically compatible aqueous buffer at physiological pH and temperature.

13. The nucleic acid of claim 11, wherein the encoded pharmacologically inactive protein domain comprises an amino acid sequence selected from SEQ ID NOS:

1 through 8, 13, 50, 61, 86 through 90, 94, and 102 through 110.

14. The nucleic acid of claim 11, wherein the nucleic acid is a DNA.

15. An expression vector comprising the nucleic acid of claim 14.

16. The expression vector of claim 14, further comprising a coding sequence encoding a secretory signal peptide operably linked to the polynucleotide sequence encoding the recombinant fusion protein.

17. A recombinant host cell comprising the expression vector of claim 15.

18. The recombinant host cell of claim 17, wherein the host cell is a prokaryotic host cell.

19. The recombinant host cell of claim 18, wherein the prokaryotic host cell is an Escherichia coli.

20. A method of producing a pharmacologically active recombinant fusion protein, comprising:

(a) placing the recombinant host cell of claim 17 in a growth medium, such that the recombinant fusion protein is expressed; and

(b) isolating the fusion protein from the cell or growth medium.