Journal Article

Bispecific antibody derivatives with restricted binding functionalities that are activated by proteolytic processing

Silke Metz, Christian Panke, Alexander K. Haas, Jürgen Schanzer, Wilma Lau, Rebecca Croasdale, Eike Hoffmann, Britta Schneider, Johannes Auer, Christian Gassner, Birgit Bossenmaier, Pablo Umana, Claudio Sustmann and Ulrich Brinkmann

in Protein Engineering, Design and Selection

Volume 25, issue 10, pages 571-580
Published in print October 2012 | ISSN: 1741-0126
Published online September 2012 | e-ISSN: 1741-0134 | DOI:

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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 VH and one VL domain connected by a disulfide bond. The molecules are assembled by fusing a VH,Cys44 domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a VL,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 VH and VL. Tethering the VH and VL domains at the C-terminus of the CH3 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.

Keywords: antibody engineering; bispecific antibodies; disulfide stabilization; protease activation

Journal Article.  7008 words.  Illustrated.

Subjects: Proteins

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