SITE-SPECIFIC MODIFICATION OF PROTEINS THROUGH CHEMICAL MODIFICATION ENABLING PROTEIN CONJUGATES, PROTEIN DIMER FORMATION, AND STAPLED PEPTIDES

• US 20110263832A1
• Filed: 02/18/2011
• Published: 10/27/2011
• Est. Priority Date: 02/18/2010
• Status: Active Grant

• Current Assignee: Advanced Proteome Therapeutics Inc. (Advanced Proteome Therapeutics Corp.)
• Original Assignee: Advanced Proteome Therapeutics Inc. (Advanced Proteome Therapeutics Corp.)
• Inventors: Krantz, Alexander, Yu, Peng
• Granted Patent: US 8,927,485 B2
• Time in Patent Office: Days
• US Class Current: 530/391.1
• CPC Class Codes: A61K 47/55 the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds

Abstract

The present invention generally provides methods for the site-specific modification of peptides, polypeptides, and proteins, e.g., granulocyte macrophage colony-stimulating factor, human superoxide dismutase, annexin, leptin, antibodies and the like, cytokines and chemokines, at their N-termini and at sites at which unnatural aminoacids have been introduced along the protein framework. The modifications described herein can be used for the synthesis and application of the adducts in radio-labeling, molecular imaging and protein therapeutic applications, and the treatment of disorders such as rheumatoid arthritis, lupus erythematosus, psoriasis, multiple sclerosis, type-1 diabetes, Crohn'"'"'s disease, and systemic sclerosis, Alzheimer disease, cancer, liver disease (e.g., alcoholic liver disease), and cachexia.

Claims

1. A method of synthesizing a compound having a structure according to Formula (V),

2. The method of claim 1, wherein Q is W(O)_p1(CH₂)_q(O)_p2-L.

3. The method of claim 1, wherein Bio is selected from a pharmaceutical, an imaging moiety, or an antibody.

4. The method of claim 1, wherein Q is O.

5. A method of covalently attaching proteins to a protein or a biologically active agent comprising

(a) combining a first protein with a second reagent selected from biologically active agent or a protein;

(b) forming a covalent attachment between said first protein and said biologically active agent or second protein, wherein said covalent attachment is formed via a reversible reaction; and

(c) subsequently forming a second covalent attachment between said first protein and said biologically active agent or second protein, wherein said covalent attachment is formed via an irreversible reaction.

6. The method of claim 5, wherein

said reversible reaction of step (b) comprises a reaction between a reagent comprising a linker group comprising bisaminoxy, bis-hydrazine, or bis-semicarbazide functionality, or any combination thereof, and

(i) said first protein comprising a ketone or aldehyde group, and/or

(ii) said second reagent comprising a ketone or aldehyde group;

or wherein

said reversible reaction of step (b) comprises a reaction between a reagent comprising a linker group comprising two carbonyl groups selected from aldo- or keto- groups, and

(i) said first protein comprising a bisaminoxy, bis-hydrazine, or bis-semicarbazide functionality, and/or

(ii) said second reagent comprising a bisaminoxy, bis-hydrazine, or bis-semicarbazide functionality, or any combination thereof.

7. The method of claim 6, comprising

said first protein having a structure according to the formula

8. The method of claim 5, wherein said irreversible reaction of step (c) is cycloaddition reaction that comprises an alkynyl component and an azido component, or a diene component and a dienophile component.

9. A compound having the following structure,

10. A method of site specifically producing a multimeric protein comprising combining a multifunctional linker and one or more proteins, wherein said multifunctional linker can form a covalent attachment to said protein.

11. The method of claim 10, wherein said protein is a four helix bundle protein or comprises a zipper binding motif.

12. The method of claim 10, wherein said multimer is a dimer.

13. The method of claim 12, wherein said dimer is an immunoconjugate further comprising an intact antibody fragment, a single-chain variable fragment (scFv), a diabody, a minibody, or a scFv-Fc fragment.

14. The method of claim 10, wherein said protein comprises an amino acid residue having an aldo- or keto-functional group amino acid residue, and wherein said amino acid residue is the N-terminal amino acid or is a surface available residue.

15. The method of claim 10, wherein said multifunctional linker is a bifunctional linker comprising a functional group that is aminooxy, hydrazine, or semicarbazide.

16. The method of claim 15, wherein said bifunctional linker is a bis-aminooxyalkane NH₂O(CH₂)_nONH₂ where n is an integer between 1-20.

17. The method of claim 10, wherein said protein comprises a glycine or alanine residue at the N-terminus.

18. The method of claim 17, wherein said multi-functional linker is a bis-anhydride, bis-imidate, bis-carbonyl imidazole, or a heterobifunctional reagent.

19. The method of claim 10, wherein said protein comprises a thiol function group at the N-terminus.

20. The method of claim 10, wherein said proteins further comprise functional groups that can form a covalent linkage to a second protein via an irreversible reaction.

21. A method of labeling a protein comprising a thiol function group, said method comprising

(a) combining said protein with 3-bromopyruvate;

(b) treating said product with a reagent comprising a linker group comprising bisaminoxy, bis-hydrazine, or bis-semicarbazide functionality, and further comprising a biologically active agent.

22. A method of forming a covalent bond between two functional groups in a protein comprising treating said protein with a bifunctional linker.

23. The method of claim 22, wherein said functional groups are two carbonyl groups selected, independently, from an aldo- and a keto-group.

24. The method of claim 23, wherein one of said carbonyl groups is derived from an amino acid selected from the group consisting of

25. The method of claim 23, wherein said bifunctional linker comprises a functional group that is aminooxy, hydrazine, or semicarbazide.

26. The method of claim 25, wherein said bifunctional linker is selected from:

a bis-aminooxyalkane NH₂O(CH₂)_nONH₂, wherein n is an integer between 1-20, or

a bis-semicarbazide-alkane NH₂NRCO(CH₂)_nOCNRNH₂, wherein n is an integer between 1-20.

27. The method of claim 25, wherein said protein comprises two amino acid residues comprising aminooxy groups.

28. The method of claim 27, wherein said bifunctional linker has a structure according to RC(═

O)—

(CH₂)_n—

C(═

O)R, wherein n is an integer from 1-20, and each R is, independently, optionally substituted C_1-20 alkyl, optionally substituted C_2-20 alkynyl, optionally substituted C_6-14 aryl, or optionally substituted 5-14 membered heteroaryl.

29. The method of claim 25, wherein said protein comprises two amino acid residues comprising thiol groups.

30. The method of claim 29, wherein said bifunctional linker has a structure according to XCH₂C(═

O)—

(CH₂)_n—

C(═

O)CH₂X, wherein X is a halogen.