Structural landscape of engineered multivalent antibody fragments and their application as crystallization scaffolds

Abstract

Multivalent recombinant antibody fragments, "multibodies", are produced by fusing antibody VH and VL domains and provide the ability to bind multiple antigens simultaneously. The oligomeric state of a multibody is believed to be determined by the length of the linker region between the V-domains, with longer linkers resulting in diabodies (60 kDa) and shorter linkers leading to the formation of triabodies (90 kDa), tetrabodies (120 kDa), and larger oligomers. In this work, we investigate this design space by engineering multibodies from the sequences of human mAbs CR57 and Imdevimab, and resolve their crystal structures at 2.25 [A] and 2.55 [A] resolution, respectively. Our results show that despite minimizing the length of the hinge region between the V-domains, these constructs form diabodies. This indicates that linker length is not the sole determinant of a multibodys oligomeric state, and additional factors such as the mAb origin species and light chain type must also be taken into account when designing multibodies. Moreover, we confirmed that the native paratope of the antibody is well- maintained in the diabody format, and conducted a proof-of-concept trial comparing the crystallization propensity of a diabody versus a Fab in antibody-antigen complex crystallization. Our results show that a diabody can promote crystallization more effectively than a Fab, demonstrating the potential of diabodies as crystallization scaffolds for antibody- antigen complexes.