Crystal structures of mono- and bi-specific diabodies and reduction of their structural flexibility by introduction of disulfide bridges at the Fv interface

Abstract

Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Diabodies are engineered antibody fragments that are composed of two Fv domains connected by short peptide linkers. They are attractive candidates for mediators in assembling protein nano-structures because they can simultaneously bind to two different proteins and are rigid enough to be crystallized. However, comparison of previous crystal structures demonstrates that there is substantial structural diversity in the Fv interface region of diabodies and, therefore, reliable prediction of its structure is not trivial. Here, we present the crystal structures of ten mono- and bi-specific diabodies. We found that changing an arginine residue in the Fv interface to threonine greatly reduced the structural diversity of diabodies. We also found that one of the bispecific diabodies underwent an unexpected process of chain swapping yielding a non-functional monospecific diabody. In order to further reduce structural flexibility and prevent chain shuffling, we introduced disulfide bridges in the Fv interface regions. The disulfide-bridged diabodies have rigid and predictable structures and may have applications in crystallizing proteins, analyzing cryo-electron microscopic images and building protein nano-assemblies.

Diabody-Ig: a novel platform for the generation of multivalent and multispecific antibody molecules

“…Advantages include: 1) the C-termini of both Db-assembled V L domains are closely located to the N-termini of the different dimerization domains; 2) both antigen-binding sites are facing away from each other enabling unhindered access to both antigens; 3) Db exhibit (besides a V H -V L pairing within each Fv head) a strong V H -V H back-to-back interaction stabilizing a Fablike, closed conformation of the antigen-binding sites. 26 This particular structure of a diabody in the V H -V L orientation was further confirmed by other studies [30][31][32] and nicely reviewed by Kwon et al 33 In general, we assume that Db-Ig molecules should exhibit Ig-like flexibility, for example, the elbow angle, within each Db-Fab arm or the hinge angle between both Db-Fab arms, 34,35 due to the high structural similarity within the utilized dimerization domains (C H 1, C L , MHD2, and EHD2). In our examples, neither the inter-peptide linker within the Figure 3.…”

Redirected optimized cell killing (ROCK®): A highly versatile multispecific fit-for-purpose antibody platform for engaging innate immunity

“…There are models that postulate a very compact structure with the size of a Fab,- 88,89 with each specificity pointing to opposite directions. 90,91 Most likely, this structure may constitute a key element for an optimal innate immune cell engaging domain via CD16A. A similar rigid and compact structure with comparable architecture can also be postulated for the tandem diabody.…”

ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy

“…The structural modeling of LiTE and ATTACK antibodies and comparative analysis between their space sampling were performed thought comparative homology modeling with MODELLER. 43 In both molecules, the structure of a homo-specific diabody (pdb:5GS1) 44 was used a template for the anti-EFGR EgA1 V HH domain 45 [blast e-value of 3e-81 and a 54% of sequence identity], while the anti-CD3ε OKT3 scFv domain was modeled after the MFL-23 Recombinant Ab Fragment (pdb:1QOK) 46 (blast e-value of 1e-107 and 75% sequence identity). For ATTACK, the structure of human collagen XVIII trimerization domain (pdb:3HSH) 47 was used as template for the trimerization domain as described in previous works.…”