US 11,912,984 B2
Preparation of libraries of protein variants expressed in eukaryotic cells and use for selecting binding molecules
John McCafferty, Babraham (GB); Michael Dyson, Cambridge (GB); and Kothai Parthiban, Cambridge (GB)
Assigned to Iontas Limited, Cambridge (GB)
Filed by IONTAS LIMITED, Cambridge (GB)
Filed on Feb. 11, 2020, as Appl. No. 16/787,960.
Application 16/787,960 is a continuation of application No. 15/308,570, granted, now 11,286,477, previously published as PCT/GB2015/051287, filed on May 1, 2015.
Claims priority of application No. 1407852 (GB), filed on May 2, 2014.
Prior Publication US 2020/0165596 A1, May 28, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); G01N 33/567 (2006.01); C12N 15/63 (2006.01); C12N 15/87 (2006.01); C12N 15/10 (2006.01); C12N 15/90 (2006.01); C07K 16/00 (2006.01); A61K 48/00 (2006.01)
CPC C12N 15/102 (2013.01) [C07K 16/005 (2013.01); C12N 15/1037 (2013.01); C12N 15/907 (2013.01); C07K 2317/24 (2013.01); C07K 2317/622 (2013.01); C07K 2317/64 (2013.01); C07K 2319/03 (2013.01); C07K 2319/035 (2013.01); C07K 2319/036 (2013.01); C12N 2310/20 (2017.05); C12N 2800/30 (2013.01); C12N 2800/80 (2013.01)] 15 Claims
 
1. A method of producing a library of at least 105 mammalian cell clones containing DNA encoding a diverse repertoire of antibody molecules which differ in one or more complementarity-determining regions (CDRs), wherein the antibody molecules are displayed on surfaces of the mammalian cell clones, wherein the antibody molecules comprise or are linked to membrane anchors, and wherein the library contains donor DNA integrated at a fixed locus or at duplicate fixed loci, the method comprising
providing donor DNA molecules encoding antibody fragments comprising one or more CDRs, and mammalian cells with a genome size of greater than 2×107 base pairs,
introducing the donor DNA molecules into the mammalian cells and providing a site-specific nuclease within the mammalian cells, wherein the nuclease cleaves a recognition sequence in cellular DNA to create an integration site at which the donor DNA becomes integrated into the cellular DNA, integration occurring through DNA repair mechanisms endogenous to the mammalian cells, thereby creating recombinant cells containing donor DNA integrated at a fixed locus or at duplicate fixed loci in the cellular DNA, and
culturing the recombinant cells to produce clones,
thereby providing a library of at least 105 mammalian cell clones containing the donor DNA encoding the repertoire of antibody molecules, wherein the antibody molecules are displayed on the surface of their expressing cells.