Patent
The present invention, in some aspects, provides methods, reagents, compositions, and kits for the radiolabeling of proteins, for example, of proteins useful for positron emission tomography (PET) or single-photon emission computed tomography (SPECT) (e.g., for diagnostic and therapeutic applications), using sortase-mediated transpeptidation reactions. Some aspects of this invention provide methods for the conjugation of an agent, for example, a radioactive agent or molecule to diagnostic or therapeutic peptides or proteins. Compositions comprising sortagged, radiolabeled proteins as well as reagents for generating radiolalebed proteins are also provided. Kits comprising reagents useful for the generation of radiolabeled proteins are provided, as are precursor proteins that comprise a sortase recognition motif.
1. A method for labeling a protein having a sortase recognition motif comprising:
contacting the protein with a sortase substrate peptide in the presence of a sortase to transamidate the protein and the sortase substrate peptide, wherein the sortase substrate peptide comprises a click chemistry handle; and
contacting the protein with a radiolabeled agent, wherein the radiolabeled agent comprises a click chemistry handle which reacts with the click chemistry handle of the protein, thereby producing a radiolabeled protein,
wherein the radiolabeled agent comprises fluorodeoxyglucose ( 18 F-FDG), and wherein the click chemistry handle is linked to the fluorodeoxyglucose via an oxime linkage;
wherein the protein comprises a C-terminal sortase recognition motif LPXTG or NPXTX, wherein each instance of X independently represents any amino acid residue, and the sortase substrate peptide comprises a N-terminal sortase recognition motif oligoglycine or oligoalanine;
wherein the sortase substrate peptide is linked to the radiolabeled agent by an oxime linkage, a hydrazone linkage, a thiosemicarbazone linkage, a heterocyclylene linkage, an amide linkage, an ester linkage, an ether linkage, or a disulfide linkage.
2. The method of claim 1 , wherein the sortase substrate peptide comprises a tetrazine click chemistry handle, and the radiolabeled agent comprises a trans-cyclooctene click chemistry handle.
3. The method of claim 1 , wherein the sortase substrate peptide is linked to the radiolabeled agent by the oxime linkage, the hydrazone linkage, the thiosemicarbazone linkage, or the heterocyclylene linkage.
4. The method of claim 1 , wherein the C terminal sortase recognition motif is LPXTG.
5. The method of claim 1 , wherein the C-terminal sortase recognition motif is NPXTX.
6. The method of claim 1 , wherein the terminal sortase recognition motif is oligoglycine.
7. The method of claim 1 , wherein the N-terminal sortase recognition motif is oligoalanine.
8. The method of claim 1 , wherein the sortase is sortase A from Staphylococcus aureus (SrtA aureus ), sortase A from Streptococcus pyogenes (SrtA pyogenes ), sortase B from S. aureus (SrtB aureus ), sortase B from Bacillus anthracis (SrtB anthracis ), or sortase B from Listeria monocytogenes (SrtB monocytogenes ).
9. The method of claim 1 , wherein the protein is an antibody or an antibody fragment.
10. The method of claim 1 , wherein the protein is a therapeutic peptide.
11. The method of claim 4 , wherein the C-terminal sortase recognition motif is LPETG (SEQ ID NO:2).
12. The method of claim 5 , wherein the C-terminal sortase recognition motif is NPQTN (SEQ ID NO:4) or NPKTG (SEQ ID NO:5).
13. The method of claim 1 , wherein the oligoglycine or the oligoalanine comprises 1-10 N-terminal glycine residues or 1-10 N-terminal alanine residues, respectively.
14. The method of claim 1 , wherein the N-terminal sortase recognition motif comprises the sequence GGG.
15. The method of claim 1 , wherein the sortase substrate peptide comprises the sequence (G) n1 K, wherein n1 is an integer between 1 and 10, inclusive.
16. The method of claim 1 , wherein the protein is conjugated to the sortase substrate peptide in less than 20 minutes, less than 25 minutes, or less than 30 minutes.
17. The method of claim 1 , wherein at least 50% of the protein is labeled with the sortase substrate peptide.
18. The method of claim 16 , wherein the protein is conjugated to the sortase substrate peptide in less than 5 minutes.
19. The method of claim 16 , wherein the protein is conjugated to the sortase substrate peptide in less than 10 minutes.
20. The method of claim 16 , wherein the protein is conjugated to the sortase substrate peptide in less than 15 minutes.
21. The method of claim 1 , wherein the sortase substrate peptide comprises a trans-cyclooctene click chemistry handle, and the radiolabeled agent comprises a tetrazine click chemistry handle.
22. The method of claim 17 , wherein at least 60%, at least 75%, or at least 90%, of the protein is labeled with the sortase substrate peptide.