Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies

Abstract

We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This “crossover” retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible “CrossMab” formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab CH1-CL was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

“…The CrossMabs involve the KiH technology, and correct light chain pairing is enforced by a "crossed" light chain that does not associate with the heavy chain Fab region of the other arm of the bsAb. 20 When analyzed by MS, our deglycosylated CrossMab Fab , based on the same sequences as used in the quadroma and KiH antibodies, exhibits only the correct bsAb (compound aABb), besides some hole-hole and knobhole HC dimer (Fig. 4A).…”

“…In addition 2 Cys residues were introduced in the CH3 domains (S354C in the knob chain and Y349C in the hole chain) that form a stabilizing disulfide bridge. 20 Transfection (1:1:1:1 plasmid ratios) into HEK293-F cells (Invitrogen, 510029) was performed according to the cell supplier's instructions using Maxiprep (Qiagen, 12163) preparations of the antibody vectors, Opti-MEM I medium (Invitrogen, 31985) 293fectin (Invitrogen, 31985070), and an initial cell density of 1-2 £ 10 6 viable cells/mL in serumfree FreeStyle 293 expression medium (Invitrogen, 12338018). Antibody containing cell culture supernatants were harvested after 7 d of cultivation in shake flasks by centrifugation at 14,000 £ g for 30 min and filtered through a 0.22 mm sterile filter (Thermo Scientific, 566-0020).…”

“…17,18,19 Our solution, the CrossMabs, uses KiH for the HC heterodimerization and a domain exchange of HC and LC domains in one arm of the antibody to avoid LC mispairing. 20 Two antibodies of this type, both directed simultaneously against angiopoietin-2 (Ang2) and vascular endothelial growth factor A (VEGF-A) are currently being clinically investigated in oncology. 21 and ophthalmology.…”

EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies

“…An additional example of domain substitution involves the exchange of the variable heavy and light domains within an antigen-binding fragment (Fab), which has been used in the Crossmab bispecific format to control chain pairing. 15 Thus, we hypothesized that other Ig-fold domains may be used in place of CH1/CL as a strategy to prevent mispairing of light chains in a bispecific antibody.…”

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

“…Lastly, a crossover approach has been developed for the production of IgG-Bs antibodies. 23 This approach involved the molecular exchange of heavy chain and light chain domains within the antigen-binding fragment (Fab), which deviates from the canonical domain structure of IgG, and included mutations in the C H 3 domains to favor heterodimerization.…”