Using sortases to install click chemistry handles for protein ligation

• US 10,081,684 B2
• Filed: 06/28/2012
• Issued: 09/25/2018
• Est. Priority Date: 06/28/2011
• Status: Active Grant

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: Ploegh, Hidde L., Witte, Martin D., Yoder, Nicholas C.
• Primary Examiner(s): Ha, Julie
• Assistant Examiner(s): Hellman, Kristina M
• Application Number:
  US14/127,736
• Publication Number:
  US 20140249296A1
• Time in Patent Office: 2,280 Days
• Field of Search: None
• CPC Class Codes: C07K 1/107 by chemical modification of precursor peptides
• CPC Class Codes: C07K 1/1072 by covalent attachment of residues or functional groups
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/569 Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: C07K 2317/62 comprising only variable region components
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea

Abstract

Methods and reagents for the installation of click chemistry handles on target proteins are provided, as well as modified proteins comprising click chemistry handles. Further, chimeric proteins, for example, bi-specific antibodies, that comprise two proteins conjugated via click chemistry, as well as methods for their generation and use are disclosed herein.

Claims

1. A method of installing a click chemistry handle to the C-terminus of a target protein, the method comprising the steps of:

(a) providing a first target protein with a C-terminal sortase recognition sequence, and a second target protein with a C-terminal sortase recognition sequence;

(b) contacting the first target protein with a peptide or agent in the presence of a sortase, wherein the peptide or agent comprises 1-10 N-terminal glycine residues or an N-terminal alkylamine group, and wherein the peptide or agent comprises a first click chemistry handle, thereby producing a first target protein conjugated to the first click chemistry handle, wherein the first click chemistry handle is selected from the group consisting of terminal alkynes, azides, strained alkynes, dienes, dieneophiles, alkoxyamines, phosphines, hydrazides, thiols, tetrazines, and alkenes;

(c) contacting the second target protein with a peptide or agent in the presence of a sortase, wherein the peptide or agent comprises 1-10 N-terminal glycine residues or an N-terminal alkylamine group, and wherein the peptide or agent comprises a second click chemistry handle, thereby producing a second target protein conjugated to the second click chemistry handle, wherein the second click chemistry handle is selected from the group consisting of terminal alkynes, azides, strained alkynes, dienes, dieneophiles, alkoxyamines, phosphines, hydrazides, thiols, tetrazines, and alkenes; and

(d) contacting the first target protein conjugated to the first click chemistry handle of (b) with the second target protein conjugated to the second click chemistry handle of (c), thereby generating a chimeric protein comprising the first target protein and the second target protein.

2. The method of claim 1, wherein the sortase enzyme is sortase A.

3. The method of claim 1, wherein the sortase recognition sequence is a sortase A recognition sequence.

4. The method of claim 1, wherein the sortase recognition sequence comprises the sequence LPXT (SEQ ID NO:

144), wherein X is any amino acid.

5. The method of claim 1, wherein the 1-10 N-terminal glycine residues are three N-terminal glycine residues.

6. The method of claim 1, wherein the peptide or agent comprises a linker between the first and/or second click chemistry handle and the 1-10 glycine residues or the N-terminal alkylamine group.

7. The method of claim 6, wherein the linker comprises an amino acid sequence of 1-100 amino acid residues.

8. The method of claim 1, wherein the first or second click chemistry handle is selected from the group consisting of cyclooctyne and azide.

9. The method of claim 1, wherein the sortase recognition sequence is LPETG (SEQ ID NO:

4).

10. The method of claim 1, wherein the first target protein and/or the second target protein comprises an antigen-binding domain.

11. The method of claim 10, wherein the antigen binding domain comprises a camelid antibody, a VHH domain, a single-domain antibody, an scFv, a nanobody, or an antigen-binding fragment thereof.

12. The method of claim 1, wherein

(i) the first click chemistry handle is a terminal alkyne, and the second click chemistry handle is an azide;

(ii) the first click chemistry handle is a strained alkyne, and the second click chemistry handle is an azide;

(iii) the first click chemistry handle is a diene, and the second click chemistry handle is a dieneophile;

(iv) the first click chemistry handle is a phosphine, and the second click chemistry handle is an azide;

(v) the first click chemistry handle is a thiol, and the second click chemistry handle is an alkene;

or

(vi) the first click chemistry handle is a cyclooctyne, and the second click chemistry handle is an azide.

13. A method of installing a click chemistry handle to the N-terminus of a target protein, the method comprising

(a) providing a first target protein with 1-10 N-terminal glycine residues or an N-terminal alkylamine group;

(b) contacting the first target protein with a peptide in the presence of a sortase enzyme, wherein the peptide comprises a sortase recognition sequence and a first click chemistry handle, thereby producing a first target protein conjugated to the first click chemistry handle, wherein the first click chemistry handle is selected from the group consisting of terminal alkynes, azides, strained alkynes, dienes, dieneophiles, alkoxyamines, carbonyls, phosphines, hydrazides, thiols, tetrazines, and alkenes; and

(c) contacting the first target protein conjugated to the first click chemistry handle of (b) with a second protein conjugated to a second click chemistry handle, thereby generating a chimeric protein comprising the first target protein and the second protein, wherein the second click chemistry handle is selected from the group consisting of terminal alkynes, azides, strained alkynes, dienes, dieneophiles, alkoxyamines, carbonyls, phosphines, hydrazides, thiols, tetrazines, and alkenes.

14. The method of claim 13, wherein the sortase enzyme is sortase A.

15. The method of claim 13, wherein the peptide comprises a linker between the first click chemistry handle and the sortase recognition sequence.

16. The method of claim 15, wherein the linker comprises an amino acid sequence of 1-100 amino acid residues.

17. The method of claim 13, wherein the sortase recognition sequence is a sortase A recognition sequence.

18. The method of claim 13, wherein the sortase recognition sequence comprises the sequence LPXT (SEQ ID NO:

144), wherein X is any amino acid.

19. The method of claim 13, wherein the 1-10 N-terminal glycine residues are three N-terminal glycine residues.

20. The method of claim 13, wherein the first click chemistry handle is selected from the group consisting of cyclooctyne and azide.

21. The method of claim 13, wherein the sortase recognition sequence is LPETG (SEQ ID NO:

4).

22. The method of claim 13, wherein the first target protein and/or the second protein comprises an antigen-binding domain.

23. The method of claim 22, wherein the antigen binding domain comprises a camelid antibody, a VHH domain, a single-domain antibody, an scFv, a nanobody, or an antigen-binding fragment thereof.

24. The method of claim 13, wherein

(i) the first click chemistry handle is a terminal alkyne, and the second click chemistry handle is an azide;

(ii) the first click chemistry handle is a strained alkyne, and the second click chemistry handle is an azide;

(iii) the first click chemistry handle is a diene, and the second click chemistry handle is a dieneophile;

(iv) the first click chemistry handle is an alkoxamine, and the second click chemistry handle is a carbonyl;

(v) the first click chemistry handle is a phosphine, and the second click chemistry handle is an azide;

(vi) the first click chemistry handle is a hydrazide, and the second click chemistry handle is a carbonyl;

(vii) the first click chemistry handle is a thiol, and the second click chemistry handle is an alkene;

or

(viii) the first click chemistry handle is a cyclooctyne, and the second click chemistry handle is an azide.