USING SORTASES TO INSTALL CLICK CHEMISTRY HANDLES FOR PROTEIN LIGATION

• US 20140249296A1
• Filed: 06/28/2012
• Published: 09/04/2014
• 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.
• Granted Patent: US 10,081,684 B2
• Time in Patent Office: Days
• US Class Current: 530/387.3
• 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 the target protein with a C-terminal sortase recognition sequence;

(b) contacting the target protein with a peptide or agent comprising 1-10 N-terminal glycine residues or an N-terminal alkylamine group and the click chemistry handle;

in the presence of a sortase enzyme under conditions suitable for the sortase to transamidate the target protein and the peptide comprising the click chemistry handle, thus conjugating the target protein to the click-chemistry handle.

2. The method of claim 1, wherein the target protein is fused to the sortase recognition sequence at the protein'"'"'s C-terminus.

5. The method of claim 1, wherein the sortase recognition motif is a sortase A recognition motif.

6. The method of claim 1, wherein the sortase recognition motif comprises the sequence LPXT.

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

8. The method of claim 1, wherein the peptide comprises a linker between the click chemistry group and the 1-10 glycine residues or the N-terminal alkylamine group, or between the click chemistry group and the sortase recognition sequence.

9. The method of claim 8, wherein the linker comprises an amino acid sequence of 1-100 amino acid residues.

10. The method of claim 1, wherein the click chemistry handle is selected from the group consisting of terminal alkyne, azide, strained alkyne, diene, dieneophile, alkoxyamine, carbonyl, phosphine, hydrazide, thiol, tetrazine, and alkene.

11. The method of claim 10, wherein the click chemistry handle is selected from the group consisting of cyclooctyne and azide.

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

4).

18. The method of claim 1, wherein

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

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

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

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

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

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

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

or

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

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

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

(b) contacting the target protein with a peptide comprising a sortase recognition motif, and the click chemistry handle;

in the presence of a sortase enzyme under conditions suitable for the sortase to transamidate the target protein and the peptide, thus conjugating the target protein to the click-chemistry handle.

4. The method of claim 3, wherein the target protein is fused to the 1-10 N-terminal glycine residues or an N-terminal alkylamine group at the protein'"'"'s N-terminus.

13. A method of post-translationally conjugating two proteins to form a chimeric protein, the method comprising:

contacting a first protein conjugated to a first click-chemistry handle with a second protein conjugated to a second click chemistry handle under conditions suitable for the first click chemistry handle to react with the second click chemistry handle, thus generating a chimeric protein comprising the two proteins linked via a covalent bond.

14-17. -17. (canceled)

19. A bispecific, chimeric antibody comprising

a first antibody or antigen-binding antibody fragment, comprising a sortase recognition sequence;

and

a second antibody or antigen-binding antibody fragment, comprising a sortase recognition sequence;

wherein the first and the second antibody or antibody fragment are conjugated together via click chemistry.

20. The chimeric antibody of claim 19, wherein the first and the second antibody or antibody fragment are conjugated together via a covalent bond at their C-termini (C—

C) or at their N-termini (N—

N).

21-28. -28. (canceled)

29. A protein, comprising a target protein with a sortase recognition motif, and a second molecule conjugated to the protein via click chemistry.

30. The protein of claim 29, wherein the sortase recognition motif comprises the sequence LPXT.

31-45. -45. (canceled)

46. A protein comprising

a sortase recognition motif;

and

a click chemistry handle conjugated to the sortase recognition motif.

47-55. -55. (canceled)

56. A kit comprising

(a) a first peptide comprising 1-10 glycine residues or a terminal alkylamine conjugated to a first click chemistry handle;

and

(b) a second peptide comprising a sortase recognition motif conjugated to a second click chemistry handle;

wherein the click chemistry handle of the first and the second peptide can react under suitable conditions; and

, optionally,

(c) a sortase enzyme.

57-62. -62. (canceled)