Activated formylglycine-generating enzymes and methods of producing and using the same

Patent metadata

Field	Value
Publication number	US-12018308-B2
Application number	US-202318132279-A
Country	US
Kind code	B2
Filing date	Apr 7, 2023
Priority date	Feb 5, 2015
Publication date	Jun 25, 2024
Grant date	Jun 25, 2024

Abstract

The present disclosure provides activated formylglycine-generating enzymes (FGE), methods of producing activated FGE, and their use in methods of producing a protein comprising a formylglycine (FGly) residue. The methods of producing activated FGE, as well as methods of use of activated FGE in producing FGly-containing proteins, include both cell-based and cell-free methods. Compositions and kits that find use, e.g., in practicing the methods of the present disclosure are also provided.

First claim

What is claimed is:

1. A method comprising:
expressing a formylglycine-generating enzyme (FGE) and a protein comprising an FGE recognition site in a cell-free reaction mixture comprising Cu 2+ to activate the FGE, under conditions in which the activated FGE converts a cysteine residue or a serine residue of the FGE recognition site to a formylglycine residue, to produce a protein comprising a formylglycine residue; and
conjugating an agent to the protein comprising the formylglycine residue via an aldehyde moiety of the formylglycine residue.

2. The method according to claim 1 , wherein the agent is a therapeutic agent.

3. The method according to claim 2 , wherein the therapeutic agent is selected from the group consisting of: a cytotoxic agent, an antiproliferative agent, an antineoplastic agent, an antibiotic agent, an antifungal agent, and an antiviral agent.

4. The method according to claim 1 , wherein the agent is an imaging agent.

5. The method according to claim 4 , wherein the imaging agent is selected from the group consisting of: a fluorescent dye, a near-infrared (NIR) imaging agent, and a single-photon emission computed tomography (SPECT)/CT imaging agent, a nuclear magnetic resonance (NMR) imaging agent, a magnetic resonance imaging (MRI) agent, a positron-emission tomography (PET) agent, an x-ray imaging agent, a computed tomography (CT) imaging agent, a K-edge imaging agent, an ultrasound imaging agent, a photoacoustic imaging agent, an acoustic optical imaging agent, microwave imaging agent, a nuclear imaging agent, and combinations thereof.

6. The method according to claim 1 , wherein the activated FGE and the protein comprising the FGE recognition site are combined in a reaction mixture comprising a reducing agent.

7. The method according to claim 6 , wherein the reducing agent promotes conversion of the cysteine residue or the serine residue of the FGE recognition site to the formylglycine residue.

8. The method according to claim 7 , wherein the reducing agent is 2-mercaptoethanol.

9. The method according to claim 1 , wherein the Cu 2+ is provided by a source of Cu 2+ selected from copper sulfate, copper citrate, copper tartrate, Fehling's reagent, and Benedict's reagent.

10. The method according to claim 9 , wherein the source of Cu 2+ is copper sulfate.

11. The method according to claim 1 , wherein the FGE is an N-terminally truncated FGE.

12. The method according to claim 11 , wherein the FGE is a N-terminally truncated human FGE.

13. The method according to claim 1 , wherein the protein is an antibody, an antibody fragment, a ligand, an enzyme, or an antigen.

14. The method according to claim 1 , wherein the protein is an antibody or antibody fragment.

15. The method according to claim 14 , wherein the antibody or antibody fragment is selected from the group consisting of: an IgG or fragment thereof, a Fab, a F(ab′)2, a Fab′, an Fv, an ScFv, a bispecific antibody or fragment thereof, a diabody or fragment thereof, a chimeric antibody or fragment thereof, a monoclonal antibody or fragment thereof, a humanized antibody or fragment thereof, and a fully human antibody or fragment thereof.

16. The method according to claim 14 , wherein the antibody specifically binds to a tumor-associated antigen or a tumor-specific antigen.

17. The method according to claim 16 , wherein the tumor associated antigen or tumor-specific antigen is selected from the group consisting of: HER2, CD19, CD22, CD30, CD33, CD56, CD66/CEACAM5, CD70, CD74, CD79b, CD138, Nectin-4, Mesothelin, Transmembrane glycoprotein NMB (GPNMB), Prostate-Specific Membrane Antigen (PSMA), SLC44A4, CA6, and CA-IX.

18. The method according to claim 1 , wherein the protein is a ligand.

19. The method according to claim 18 , wherein the ligand is a growth factor.

20. The method according to claim 18 , wherein the ligand is a hormone.

Assignees

• Scherer Technologies Llc R P

Inventors

• Rabuka David
• Dehart Gregory W
• Holder Patrick
• Baker Jeanne

Classifications

• A61K47/68

  the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment · CPC title

• C12Y108/99

  with other acceptors (1.8.99) · CPC title

• C12N9/0051

  acting on a sulfur group of donors (1.8) · CPC title

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title