title: Bispecific antibody derivatives with restricted binding functionalities that are activated by proteolytic processing.
abstract: We have designed bispecific antibodies that bind one target (anti-Her3) in a bivalent IgG-like manner and contain one additional binding entity (anti-cMet) composed of one V(H) and one V(L) domain connected by a disulfide bond. The molecules are assembled by fusing a V(H,Cys44) domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a V(L,Cys100) to another H-chain. To ensure heterodimerization during expression in mammalian cells, we introduced complementary knobs-into-holes mutations into the different H-chains. The IgG-shaped trivalent molecules carry as third binding entity one disulfide-stabilized Fv (dsFv) without a linker between V(H) and V(L). Tethering the V(H) and V(L) domains at the C-terminus of the C(H)3 domain decreases the on-rates of the dsFv to target antigens without affecting off-rates. Steric hindrance resolves upon removal of one side of the double connection by proteolysis: this improves flexibility and accessibility of the dsFv and fully restores antigen access and affinity. This technology has multiple applications: (i) in cases where single-chain linkers are not desired, dsFvs without linkers can be generated by addition of furin site(s) in the connector that are processed during expression within mammalian cells; (ii) highly active (toxic) entities which affect expression can be produced as inactive dsFvs and subsequently be activated (e.g. via PreScission cleavage) during purification; (iii) entities can be generated which are targeted by the unrestricted binding entity and can be activated by proteases in target tissues. For example, Her3-binding molecules containing linkers with recognition sequences for matrix metalloproteases or urokinase, whose inactivated cMet binding site is activated by proteolytic processing.
authors.affiliation: Roche Pharma Research & Early Development, Large Molecule Research, Nonnenwald 2, D-82372 Penzberg, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Metz
authors.forename: Christian
authors.initials: C
authors.lastname: Panke
authors.forename: Alexander K
authors.initials: AK
authors.lastname: Haas
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schanzer
authors.forename: Wilma
authors.initials: W
authors.lastname: Lau
authors.forename: Rebecca
authors.initials: R
authors.lastname: Croasdale
authors.forename: Eike
authors.initials: E
authors.lastname: Hoffmann
authors.forename: Britta
authors.initials: B
authors.lastname: Schneider
authors.forename: Johannes
authors.initials: J
authors.lastname: Auer
authors.forename: Christian
authors.initials: C
authors.lastname: Gassner
authors.forename: Birgit
authors.initials: B
authors.lastname: Bossenmaier
authors.forename: Pablo
authors.initials: P
authors.lastname: Umana
authors.forename: Claudio
authors.initials: C
authors.lastname: Sustmann
authors.forename: Ulrich
authors.initials: U
authors.lastname: Brinkmann
mesh_terms: D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001666:Binding Sites, Antibody; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000276:immunology; D020780:Matrix Metalloproteinase 9 / Q000276:immunology; D010447:Peptide Hydrolases / Q000378:metabolism; D015202:Protein Engineering; D059748:Proteolysis; D020893:Receptor, ErbB-3 / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000276:immunology
chemical_list: D018033:Antibodies, Bispecific; D004220:Disulfides; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D020893:Receptor, ErbB-3; D010447:Peptide Hydrolases; D014568:Urokinase-Type Plasminogen Activator; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9