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US 11,530,277 B2Granted
Compositions and methods for making antibodies based on use of an expression-enhancing locus
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsRobert Babb, Darya Burakov, Gang Chen, James P. Fandl
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Abstract
This invention relates to site-specific integration and expression of recombinant proteins in eukaryotic cells. In particular, the invention includes compositions and methods for improved expression of antibodies including bispecifc antibodies in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, by employing an expression-enhancing locus.
Claims41
A cell comprising an exogenous nucleic acid sequence integrated at a specific site within an enhanced expression locus,
- wherein the exogenous nucleic acid sequence comprises a first exogenous nucleic acid comprising a nucleotide sequence encoding a first light chain fragment (LCF), a second exogenous nucleic acid comprising a nucleotide sequence encoding a first heavy chain fragment (HCF), and a third exogenous nucleic acid comprising a nucleotide sequence encoding a second HCF;
- wherein the nucleotide sequence encoding the first LCF is operably linked to a first promoter, the nucleotide sequence encoding the first HCF is operably linked to a second promoter, and the nucleotide sequence encoding the second HCF is operably linked to a third promoter;
- wherein the first and second HCFs, and the first LCF, are fragments of a bispecific antigen-binding protein a bispecific antibody.
A set of vectors, comprising
- a first vector comprising from 5′ to 3′:
- a first recombinase recognition site (RRS), a first nucleic acid comprising a nucleotide sequence encoding a first LCF, and a third RRS;
- a second vector comprising from 5′ to 3′, said third RRS, a second nucleic acid comprising a nucleotide sequence encoding a first HCF, a second RRS;
- wherein either the first or the second nucleic acid further comprises a nucleotide sequence encoding a second HCF;
- wherein the first and second HCFs, and the first LCF, are fragments of a bispecific antigen-binding protein;
- and
- wherein the nucleotide sequence encoding the first LCF is operably linked to a first promoter, the nucleotide sequence encoding the first HCF is operably linked to a second promoter, and the nucleotide sequence encoding the second HCF is operably linked to a third promoter.
4 more elements
A system comprising a cell and a set of vectors,
- wherein the cell comprises, integrated within an enhanced expression locus of its genome from 5′ to 3′:
- a first recombinase recognition site (RRS), a first exogenous nucleic acid, a third RRS, a second exogenous nucleic acid, and a second RRS, wherein the three RRSs are different from one another;
- wherein the set of vectors comprises
- a first vector comprising from 5′ to 3′, the first RRS, a first nucleic acid comprising a nucleotide sequence encoding a first light chain fragment (LCF), and the third RRS;
- a second vector comprising the third RRS, a second nucleic acid comprising a nucleotide sequence encoding a first heavy chain fragment (HCF), and the second RRS;
- and
- wherein either the first nucleic acid or the second nucleic acid further comprises a nucleotide sequence encoding a second HCF;
- wherein the nucleotide sequence encoding the first LCF is operably linked to a first promoter, the nucleotide sequence encoding the first HCF is operably linked to a second promoter, and the nucleotide sequence encoding the second HCF is operably linked to a third promoter;
- and
- wherein upon introduction of the vectors into the cell, the first and second nucleic acids in the vectors integrate into the enhanced expression locus through recombination mediated by the first, second and third RRSs.
7 more elements
Dependencies are inferred from the claim text.