Aldehyde-Tagged Protein-Based Drug Carriers and Methods of Use

• US 20100210543A1
• Filed: 02/16/2010
• Published: 08/19/2010
• Est. Priority Date: 02/17/2009
• Status: Active Grant

• Current Assignee: Redwood Bioscience, Inc (Catalent, Inc.)
• Original Assignee: Redwood Bioscience, Inc (Catalent, Inc.)
• Inventors: Rabuka, David, Breidenbach, Mark Alan
• Granted Patent: US 9,238,878 B2
• Time in Patent Office: Days
• US Class Current: 514/1.100
• CPC Class Codes: A61K 38/23 Calcitonins
• CPC Class Codes: A61K 38/26 Glucagons
• CPC Class Codes: A61K 47/643 Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 301/06 Sulfuric ester hydrolases (3.1.6)
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof

Abstract

The disclosure provides aldehyde-tagged protein carriers that can be covalently and site-specifically bound to drug to provide a drug-containing scaffold. The invention also encompasses methods of production of such drug-containing scaffolds and intermediates, as well as methods of use.

Claims

1. A carrier protein-drug conjugate comprising:

a carrier protein and a covalently bound drug, wherein the carrier protein comprises a modified sulfatase motif of the formula;

X₁(FGly′

)X₂Z₂X₃Z₃

where FGly′

is of the formula;

2. The carrier protein-drug conjugate of claim 1, where in the carrier protein-drug conjugate comprises two or more modified sulfatase motifs.

3. The carrier protein-drug conjugate of claim 2, where in the carrier protein-drug conjugate comprises three or more modified sulfatase motifs.

4. The carrier protein-drug conjugate of claim 1, where in the modified sulfatase motifs are positioned in the carrier protein-drug conjugate at least one of an N-terminus of the carrier protein, a C-terminus of the carrier protein, and a solvent-accessible loop of the carrier protein.

5. The carrier protein-drug conjugate of claim 2, wherein the modified sulfatase motifs of provided as concatamer and are separated by a flexible linker.

6. The carrier protein-drug conjugate of claim 1, wherein the carrier protein is albumin.

7. The carrier protein-drug conjugate of claim 1, wherein the covalently bound drug is a peptide drug.

8. The carrier protein-drug conjugate of claim 7, herein the peptide drug is glucagon-like peptide 1 (GLP-1) or a biologically active variant thereof.

9. The carrier protein-drug conjugate of claim 7, herein the peptide drug is calcitonin or a biologically active variant thereof.

10. The carrier protein-drug conjugate of claim 1, wherein the covalently bound drug is a small molecule drug.

11. The carrier protein-drug conjugate composition of claim 1, wherein Z₃ is arginine (R).

12. The carrier protein-drug conjugate of claim 1, wherein X₁, when present, X₂, and X₃ are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.

13. The carrier protein-drug conjugate of claim 1, wherein the X₁, when present, is L, M, V, S or T.

14. The carrier protein-drug conjugate of claim 1, wherein X₂ and X₃ are each independently S, T, A, V, G, or C.

30. A formulation comprising:

a carrier protein-drug conjugate according to claim 1; and

a pharmaceutically acceptable excipient.

31. A method of treating a subject having or at risk of having condition amenable to treatment with glucagon-like peptide 1 (GLP-1), the method comprising:

administering to a subject in of treatment a carrier protein-drug conjugate according to claim 1, wherein the covalently bound drug is glucagon-like peptide 1 (GLP-1) or a biologically active variant thereof;

wherein said administering is effective to treat the condition in the subject.

32. A method of treating a subject having or at risk of having condition amenable to treatment with calcitonin, the method comprising:

administering to a subject in of treatment a carrier protein-drug conjugate according to claim 1, wherein the covalently bound drug is calcitonin or a biologically active variant thereof;

wherein said administering is effective to treat the condition in the subject.

33. A recombinant nucleic acid comprising a nucleic acid encoding an aldehyde-tagged carrier protein according to claim 10, wherein Z₁ is a cysteine residue or a serine residue.

15. An aldehyde-tagged carrier protein comprising an amino acid sequence comprising:

X₁Z₁X₂Z₂X₃Z₃ 

 

(I)

where

Z₁ is a cysteine, a serine, or a 2-formylglycine residue;

Z₂ is a proline or alanine residue;

X₁ is present or absent and, when present, is any amino acid, with the proviso that when the heterologous sulfatase motif is at an N-terminus of the aldehyde tagged polypeptide, X₁ is present; and

X₂ and X₃ are each independently any amino acid; and

Z₃ is a basic amino acid;

wherein the carrier protein presents the covalently bound drug on a solvent-accessible surface when in a folded state.

16. The aldehyde-tagged carrier protein of claim 15, where in the carrier protein comprises two or more modified sulfatase motifs.

17. The aldehyde-tagged carrier protein of claim 16, where in the carrier protein-drug conjugate comprises three or more modified sulfatase motifs.

18. The aldehyde-tagged carrier protein of claim 15, where in the modified sulfatase motifs are positioned in the carrier protein-drug conjugate at least one of an N-terminus of the carrier protein, a C-terminus of the carrier protein, and a solvent-accessible loop of the carrier protein.

19. The aldehyde-tagged carrier protein of claim 15, wherein the carrier protein is albumin.

20. The aldehyde-tagged carrier protein of claim 15, wherein Z₃ is arginine (R).

21. The aldehyde-tagged carrier protein of claim 15, wherein X₁, when present, X₂, and X₃ are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.

22. The aldehyde-tagged carrier protein of claim 15, wherein the X₁, when present, is L, M, V, S or T.

23. The aldehyde-tagged carrier protein of claim 15, wherein X₂ and X₃ are each independently S, T, A, V, G, or C.

24. A library of modified carrier proteins, the library comprising:

a population of aldehyde-tagged carrier proteins according to claim 15, or nucleic acid constructs encoding the aldehyde-tagged carrier proteins, where Z₁ is a cysteine or serine residue, wherein the population comprises members having differently aldehyde-tagged carrier proteins.

25. The library of claim 24, wherein the population comprises aldehyde-tagged carrier proteins comprising two or more aldehyde tags.

26. The library of claim 24, wherein the population comprises aldehyde-tagged carrier proteins comprising at least one aldehyde tag at one or more of the N-terminus, the C-terminus, or an interior loop and the carrier protein.

27. The library of claim 24, wherein the library is provided in a population of recombinant cells genetically modified to express the nucleic acid constructs.

28. A method of producing a carrier protein-drug conjugate, the method comprising combining in a reaction mixture

an aldehyde-tagged carrier protein of claim 15, wherein Z₁ is a 2-formyl-glycine residue and

a drug for conjugation to the carrier protein, wherein the drug comprises a aminooxy or hydrazide reactive group,

wherein the drug is provided in the reaction mixture in an amount sufficient to provide for a desired ratio of drug to carrier protein, said combining being under conditions suitable to promote reaction between an aldehyde of the carrier protein and reactive group of the drug to generate a carrier protein-drug conjugate; and isolating the carrier protein-drug conjugate from the reaction mixture.

29. The method of claim 28, wherein the carrier protein is folded prior to said combining.