An Antibody with a Variable‐Region Coiled‐Coil “Knob” Domain

Abstract

The X-ray crystal structure of a bovine antibody (BLV1H12) revealed a unique structure in its ultralong heavy chain complementarity determining region 3 (CDR3H) that folds into a solvent-exposed b-strand "stalk" fused to a disulfide crosslinked "knob" domain. We have substituted an antiparallel heterodimeric coiled-coil motif for the b-strand stalk in this antibody. The resulting antibody (Ab-coil) expresses in mammalian cells and has a stability similar to that of the parent bovine antibody. MS analysis of H-D exchange supports the coiled-coil structure of the substituted peptides. Substitution of the knob-domain of Ab-coil with bovine granulocyte colonystimulating factor (bGCSF) results in a stably expressed chimeric antibody, which proliferates mouse NFS-60 cells with a potency comparable to that of bGCSF. This work demonstrates the utility of this novel coiled-coil CDR3 motif as a means for generating stable, potent antibody fusion proteins with useful pharmacological properties.

A Novel Her2/VEGFR2/CD3 trispecific antibody with an optimal structural design showed improved T-cell-redirecting antitumor efficacy

“…In addition, to spatially separate the nanobody inserts and Fab backbone, an antiparallel coiled-coil stalk was used as a rigid linker to connect the nanobody and CD3 Fab to increase the stability of the structures. A similar linker strategy was previously used successfully to fuse EPO, GCSF and Exendin-4 into CDR or non-CDR loops of human or bovine antibodies [28,[53][54][55][56]. Different from TriTACs [57] and TriTE [20], which showed impaired functionalities due to steric hindrance, our Fab-based bsAbs and tsAbs using the special linker design and the site-specific recombination strategy potentially retained the affinity of each antibody domain.…”

Conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies

“…Cyclotides such as Kalata B1 are being used as scaffolds for the design of small binding proteins (43)(44)(45)(46), and their structural homology with the knob domains on the bovine antibodies suggests that libraries of bovine antibodies with extensive natural variation in the loop regions and disulfide pairings could be used to search for small knob domains with specific affinities or functions. Antibody engineering inspired by the bovine antibody template has already taken off at a rapid pace, with knob domains replaced with other proteins, stalk regions replaced by stable structures such as a coiled-coiled domain, or stalk/knob structures transplanted into the CDRs of human antibodies (47)(48)(49)(50)(51)(52)(53)(54). Antibodies with novel activities have also been created through insertion of large protein domains into the CDR H3 of conventional antibodies by functional selection using libraries of permissive junctions (55).…”

Influence of Domain Interactions on Conformational Mobility of the Progesterone Receptor Detected by Hydrogen/Deuterium Exchange Mass Spectrometry

“…HDX-MS has emerged as a powerful technique to characterize conformational flexibility associated with protein-protein or protein-ligand interaction in solution state (Chalmers et al, 2006; Englander, 2006; Konermann et al, 2011; Landgraf et al, 2013; Zhang et al, 2013). HDX-MS has advantages for large molecular proteins such as SRs since it is not limited by protein size and can localize changes in conformational flexibility to specific sequences.…”