Monitoring the Structural Changes of Conjugated Antibodies by High-Resolution Electron Microscopy and Individual-Particle Electron Tomography

Abstract

Peptides have promise as potent and selective drug candidates but have not succeeded primarily because of poor pharmacokinetics. The fusion of these peptides to our scaffold antibody has produced molecules, CovX-Bodies, which protect the peptide from renal elimination and/or enzymatic degradation. However, it is possible that the fusion process may in itself affect the intrinsic properties of the antibody scaffold (eg. effector function). Changes in function may relate to changes in structure. However, the structural study of highly dynamic and structurally heterogenous antibodies by X-ray crystallization and NMR is extremely difficult. These studies were designed to examine structural changes that may have occurred during the conjugation process.Here, we studied the structural changes of antibodies with the high-resolution electron microscopy and individual-particle electron tomography (IPET, for details, see the abstract presented by Lei Zhang and Gang Ren). We found 1) the average angle between the Fab regions was 55 degrees +-15 in unconjugated antibodies, but 40 degrees +-10 in unconjugated antibodies and 2) the average sizes of the Fc domain were similar regardless of conjugation. However, 3) the Fc domain shape differed significantly depending on conjugation. The Fc domains of conjugated antibodies were significantly elongated (>30%). Using IPET, we reconstructed a dozen of three-dimensional density maps of individual conjugated and unconjugated antibodies. Comparing the maps, it showed that all three domains of conjugated antibodies were elongated after fusing with the drug. This example demonstrates that this method could be a useful tool for monitoring the structural changes of antibodies.