Determinants of the assembly and function of antibody variable domains

Abstract

The antibody Fv module which binds antigen consists of the variable domains VL and VH. These exhibit a conserved ß-sheet structure and comprise highly variable loops (CDRs). Little is known about the contributions of the framework residues and CDRs to their association. We exchanged conserved interface residues as well as CDR loops and tested the effects on two Fvs interacting with moderate affinities (KDs of ~2.5 µM and ~6 µM). While for the rather instable domains, almost all mutations had a negative effect, the more stable domains tolerated a number of mutations of conserved interface residues. Of particular importance for Fv association are VLP44 and VHL45. In general, the exchange of conserved residues in the VL/VH interface did not have uniform effects on domain stability. Furthermore, the effects on association and antigen binding do not strictly correlate. In addition to the interface, the CDRs modulate the variable domain framework to a significant extent as shown by swap experiments. Our study reveals a complex interplay of domain stability, association and antigen binding including an unexpected strong mutual influence of the domain framework and the CDRs on stability/association on the one side and antigen binding on the other side.

Bispecific antibodies targeting dual tumor-associated antigens in cancer therapy

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Specific Residue Interactions Regulate the Binding of Dengue Antigens to Broadly Neutralizing EDE Antibodies

“…Overall, the binding of the antigen to EDE1 C8 Fab corresponded to a reduced mobility of the flexible CDR loops locking these loops in a distinct conformational state, and strong reduction of the mobility of these loops has also been observed in the past in cases of strong antigen–Fab binding …”

Molecular mechanism of amyloidogenic mutations in hypervariable regions of antibody light chains

“…CDR loops are not only involved in antigen recognition, they have also been shown to play important structural roles in antibody domain architecture and V H /V L domain association. Various experimental and computational studies on V L domains demonstrated that CDRs can have a strong influence on the folding pathway, stability, and conformation of the protein ( 69 , 70 , 71 , 72 ). The involvement of a CDR mutation in LC amyloidogenicity has been shown for a proline residue in the CDR3 loop of an amyloidogenic V L domain.…”