title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain.
abstract: We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.
authors.affiliation: a Antibody Discovery and Protein Engineering, MedImmune , Gaithersburg , MD , USA.
authors.forename: Nazzareno
authors.initials: N
authors.lastname: Dimasi
authors.affiliation: a Antibody Discovery and Protein Engineering, MedImmune , Gaithersburg , MD , USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Fleming
authors.affiliation: b Translational Sciences, IMED Oncology AstraZeneca , Waltham , MA , USA.
authors.forename: Kris F
authors.initials: KF
authors.lastname: Sachsenmeier
authors.affiliation: a Antibody Discovery and Protein Engineering, MedImmune , Gaithersburg , MD , USA.
authors.forename: Binyam
authors.initials: B
authors.lastname: Bezabeh
authors.affiliation: c Oncology Research, MedImmune , Gaithersburg , MD , USA.
authors.forename: Carl
authors.initials: C
authors.lastname: Hay
authors.affiliation: d Research Bioinformatics, MedImmune , Gaithersburg , MD , USA.
authors.forename: Jincheng
authors.initials: J
authors.lastname: Wu
authors.affiliation: c Oncology Research, MedImmune , Gaithersburg , MD , USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Sult
authors.affiliation: a Antibody Discovery and Protein Engineering, MedImmune , Gaithersburg , MD , USA.
authors.forename: Saravanan
authors.initials: S
authors.lastname: Rajan
authors.affiliation: a Antibody Discovery and Protein Engineering, MedImmune , Gaithersburg , MD , USA.
authors.forename: Li
authors.initials: L
authors.lastname: Zhuang
authors.affiliation: e Antibody Discovery and Protein Engineering, MedImmune , Cambridge , UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Cariuk
authors.affiliation: e Antibody Discovery and Protein Engineering, MedImmune , Cambridge , UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Buchanan
authors.affiliation: a Antibody Discovery and Protein Engineering, MedImmune , Gaithersburg , MD , USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Bowen
authors.affiliation: a Antibody Discovery and Protein Engineering, MedImmune , Gaithersburg , MD , USA.
authors.forename: Herren
authors.initials: H
authors.lastname: Wu
authors.affiliation: a Antibody Discovery and Protein Engineering, MedImmune , Gaithersburg , MD , USA.
authors.forename: Changshou
authors.initials: C
authors.lastname: Gao
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000302:isolation & purification; D066246:ErbB Receptors / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods*; D059748:Proteolysis; D017526:Receptor, IGF Type 1 / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D018033:Antibodies, Bispecific; D007074:Immunoglobulin G; D057127:Single-Chain Antibodies; C512478:EGFR protein, human; D066246:ErbB Receptors; D017526:Receptor, IGF Type 1
keywords: Antibody engineering; bispecific monovalent antibodies; dual targeting; single-chain antibody; tethered single-chain antibodies