title: Engineering the expression of an anti-interleukin-13 antibody through rational design and mutagenesis.
abstract: High levels of protein expression are key to the successful development and manufacture of a therapeutic antibody. Here, we describe two related antibodies, Ab001 and Ab008, where Ab001 shows a markedly lower level of expression relative to Ab008 when stably expressed in Chinese hamster ovary cells. We use single-gene expression vectors and structural analysis to show that the reduced titer is associated with the VL CDR2 of Ab001. We adopted two approaches to improve the expression of Ab001. First, we used mutagenesis to change single amino-acid residues in the Ab001 VL back to the equivalent Ab008 residues but this resulted in limited improvements in expression. In contrast when we used an in silico structure-based design approach to generate a set of five individual single-point variants in a discrete region of the VL, all exhibited significantly improved expression relative to Ab001. The most successful of these, D53N, exhibited a 25-fold increase in stable transfectants relative to Ab001. The functional potency of these VL-modified antibodies was unaffected. We expect that this in silico engineering strategy can be used to improve the expression of other antibodies and proteins.
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Bojana
authors.initials: B
authors.lastname: Popovic
authors.affiliation: Department of Biopharmaceutical Development, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Gibson
authors.affiliation: Department of Biopharmaceutical Development, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Tarik
authors.initials: T
authors.lastname: Senussi
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Sara
authors.initials: S
authors.lastname: Carmen
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Sara
authors.initials: S
authors.lastname: Kidd
authors.affiliation: Department of Research Informatics, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Tim
authors.initials: T
authors.lastname: Slidel
authors.affiliation: Department of Respiratory, Inflammation and Autoimmunity, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Ian
authors.initials: I
authors.lastname: Strickland
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Xu
authors.initials: X
authors.lastname: Jianqing
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Spooner
authors.affiliation: Department of Biopharmaceutical Development, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Amanda
authors.initials: A
authors.lastname: Lewis
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Nathan
authors.initials: N
authors.lastname: Hudson
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Lorna
authors.initials: L
authors.lastname: Mackenzie
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Keen
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Ben
authors.initials: B
authors.lastname: Kemp
authors.affiliation: Department of Research Informatics, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Colin
authors.initials: C
authors.lastname: Hardman
authors.affiliation: Department of Biopharmaceutical Development, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Diane
authors.initials: D
authors.lastname: Hatton
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Trevor
authors.initials: T
authors.lastname: Wilkinson
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Tristan
authors.initials: T
authors.lastname: Vaughan
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: David
authors.initials: D
authors.lastname: Lowe
mesh_terms: D019943:Amino Acid Substitution; D006801:Humans; D018793:Interleukin-13 / Q000037:antagonists & inhibitors*; D016296:Mutagenesis; D020125:Mutation, Missense; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D018793:Interleukin-13; D057127:Single-Chain Antibodies
keywords: antibody dynamics; antibody engineering; antibody expression; antibody structure; structure-guided design