Cell lines

Patent metadata

Field	Value
Publication number	US-10253345-B2
Application number	US-201715611116-A
Country	US
Kind code	B2
Filing date	Jun 1, 2017
Priority date	Sep 24, 2012
Publication date	Apr 9, 2019
Grant date	Apr 9, 2019

Abstract

There is provided inter alia a process for stabilizing a eukaryotic cell line which expresses PylRS and tRNAPyl and which is suitable for incorporation of a gene encoding a target protein containing one or more non-natural amino acids encoded by a nonsense codon which comprises culturing said cell line under conditions in which the adverse effect of tRNAPyl expression on cell viability and/or cell growth is reduced or eliminated.

First claim

The invention claimed is:

1. A process for preparing a target protein containing one or more non-natural amino adds encoded by an amber codon which comprises:
(A) introducing into a eukaryotic cell line which expresses PyiRS and tRNAPyl and is capable of incorporating a gene encoding a target protein comprising one or more non-natural amino acids encoded by an amber codon a gene encoding the target protein such that the target protein is stably expressed in the cell line,
(B) culturing the eukaryotic cell line in the presence of a decoy amino add which is a substrate for PylRS but which is incapable of being incorporated into an extending protein chain, wherein the decoy amino acid is selected from:
(a) a compound of formula VIIA:

wherein K is CO or SO 2 ; and
Q=H, C 1-6 alkyl, aryl, heteroaryl, OC 1-6 alkyl, OCH 2 aryl, OCH 2 heteroaryl, C 2-6 alkenyl or OC 2-6 alkenyl; and
(b) a compound of formula VIIB:

wherein G=H;
a=4 or 5; and
R═C 1-6 alkyl, C 2-6 alkenyl, —CH 2 aryl, C 2-6 alkynyl, C 1-6 haloalkyl or C 1-6 azidoalkyl; and
(C) expressing the target protein in the presence of one or more non-natural amino adds and in the absence of the decoy amino add.

2. The process according to claim 1 wherein the eukaryotic cell line is a mammalian cell line selected from CHO, HEK293, PER6, COS-1, COS-7, HeLa, VERO, mouse hybridoma and mouse myeloma cell lines.

3. The process according to claim 1 which further comprises the step of chemically modifying the resultant target protein using a first functional group incorporated into the target protein via the non-natural amino acid.

4. The process according to claim 3 wherein the first functional group on the non-natural amino acid is reacted with a second functional group on a reagent to covalently bind the reagent to the target protein.

5. The process according to claim 3 wherein the reagent is covalently bound to the target protein via a triazole linkage.

6. The process according to claim 3 wherein the non-natural amino acid contains an azide group and is reacted with an alkyne group present on the reagent; the non-natural amino acid contains the alkyne group and is reacted with the azide group present on the reagent; the non-natural amino acid contains the azide group and is reacted with an alkene group on the reagent; or wherein the non-natural amino acid contains the alkene group and is reacted with the azide group on the reagent.

7. The process according to claim 4 wherein the functional group on the reagent is attached to the target protein via a linker.

8. The process according to claim 4 wherein the reagent is selected from a protein, a toxin, a cytotoxic drug, or a PEG group.

9. The process according to claim 3 wherein the target protein comprises an antibody.

10. The process according to claim 9 wherein the antibody comprises a monoclonal antibody.

11. The process according to claim 9 wherein the antibody comprises an IgG antibody.

12. The process according to claim 9 wherein the antibody is a full-length antibody or an antibody fragment selected from Fab, Fab2 and single chain antibody fragments (scFvs).

13. The process according to claim 5 , wherein the decoy amino acid is selected from:

Assignees

• Medimmune Ltd

Inventors

• Grabstein Kenneth H
• Van Brunt Michael
• Marelli Marcello
• Brady William
• Johnson Jeffrey C

Classifications

• C07K16/244

  Interleukins [IL] · CPC title

• C07K17/08

  the carrier being a synthetic polymer · CPC title

• C07C271/20

  to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups · CPC title

• C07C271/22

  to carbon atoms of hydrocarbon radicals substituted by carboxyl groups · CPC title

• C12Y601/01

  Ligases forming aminoacyl-tRNA and related compounds (6.1.1) · CPC title