US 12,466,872 B2
Method for the generation of an FCRN expressing cell by targeted integration of multiple expression cassettes in a defined organization
Tilman Schlothauer, Penzberg (DE); Stefan Seeber, Sindelsdorf (DE); and Jasmin Maria Wehrstein, Ulm (DE)
Assigned to Hoffmann-La Roche Inc., Little Falls, NJ (US)
Filed by Hoffmann-La Roche Inc., Little Falls, NJ (US)
Filed on Sep. 30, 2021, as Appl. No. 17/490,979.
Application 17/490,979 is a continuation of application No. PCT/EP2020/058452, filed on Mar. 26, 2020.
Claims priority of application No. 19166030 (EP), filed on Mar. 29, 2019.
Prior Publication US 2022/0119482 A1, Apr. 21, 2022
Int. Cl. C07K 14/735 (2006.01)
CPC C07K 14/70535 (2013.01) 9 Claims
 
1. A method for producing C-terminally biotinylated FcRn comprising the steps of
a) cultivating a mammalian cell comprising a deoxyribonucleic acid encoding FcRn and biotin-[acetyl-CoA-carboxylase] ligase (BirA) in a biotin containing medium, and
b) recovering C-terminally biotinylated FcRn from the cell or the cultivation medium, wherein the deoxyribonucleic acid encoding FcRn and BirA is stably integrated into the genome of the mammalian cell and comprises in 5′- to 3′-direction
a first expression cassette encoding class I major histocompatibility complex-like protein (α-FcRn) comprising an Avi-tag at the C-terminus,
a second expression cassette encoding β2-microglobulin (β2m),
a third expression cassette encoding α-FcRn comprising an Avi-tag at the C-terminus,
a fourth expression cassette encoding β2m, and
a fifth expression cassette encoding biotin-[acetyl-CoA-carboxylase] ligase.