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

Patent metadata

Field	Value
Publication number	US-10344311-B2
Application number	US-201815862312-A
Country	US
Kind code	B2
Filing date	Jan 4, 2018
Priority date	Feb 5, 2015
Publication date	Jul 9, 2019
Grant date	Jul 9, 2019

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 of producing a protein comprising a formylglycine residue, the method comprising:
culturing a cell comprising:
a formylglycine-generating enzyme (FGE); and
a protein comprising an FGE recognition site,
in a cell culture medium comprising Cu 2+ under cell culture conditions in which the 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.

2. The method according to claim 1 , wherein the Cu 2+ is present in the cell culture medium at a concentration of from 1 nM to 10 mM.

3. The method according to claim 2 , wherein the Cu 2+ is present in the cell culture medium at a concentration of from 1 μM to 1 mM.

4. The method according to claim 1 , wherein the FGE is endogenous to the cell.

5. The method according to claim 1 , wherein the cell is genetically modified to express an FGE.

6. The method according to claim 1 , wherein the protein containing an FGE recognition site is endogenous to the cell.

7. The method according to claim 1 , wherein the cell is genetically modified to express the protein containing an FGE recognition site.

8. The method according to claim 1 , wherein the cell is a eukaryotic cell.

9. The method according to claim 8 , wherein the eukaryotic cell is a mammalian cell.

10. The method according to claim 9 , wherein the mammalian cell is selected from the group consisting of: a CHO cell, a HEK cell, a BHK cell, a COS cell, a Vero cell, a Hela cell, an NIH 3T3 cell, a Huh-7 cell, a PC12 cell, a RAT1 cell, a mouse L cell, an HLHepG2 cell, an NSO cell, a C127 cell, a hybridoma cell, a PerC6 cell, a CAP cell, and a Sp-2/0 cell.

11. The method according to claim 9 , wherein the mammalian cell is a human cell.

12. The method according to claim 8 , wherein the eukaryotic cell is a yeast cell.

13. The method according to claim 8 , wherein the eukaryotic cell is an insect cell.

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

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

16. The method according to claim 15 , 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.

17. The method according to claim 15 , wherein the antibody specifically binds to a tumor-associated antigen or a tumor-specific antigen.

18. The method according to claim 17 , 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.

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

20. The method according to claim 19 , wherein the ligand is a growth factor or a hormone.

21. The method according to claim 1 , further comprising conjugating an agent to the protein comprising the formylglycine residue via an aldehyde moiety of the formylglycine residue.

22. The method according to claim 21 , wherein the agent is a therapeutic agent.

23. The method according to claim 22 , 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.

24. The method according to claim 21 , wherein the agent is an imaging agent.

Assignees

• Scherer Technologies Llc R P

Inventors

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

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61P31/12

  Antivirals · CPC title

• A61P31/10

  Antimycotics · CPC title

• A61P31/04

  Antibacterial agents · CPC title

• C12N9/0051

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