Evidence for intermolecular domain exchange in the Fab domains of dimer and oligomers of an IgG1 monoclonal antibody

Abstract

Recombinant protein therapeutics have become increasingly useful in combating human diseases, such as cancer and those of genetic origin. One quality concern for protein therapeutics is the content and the structure of the aggregated proteins in the product, due to the potential immunogenicity of these aggregates. Collective efforts have led to a better understanding of some types of protein aggregates, and have revealed the diversity in the structure and cause of protein aggregation. In this work we used a broad range of analytical techniques to characterize the quinary structure (complexes in which each composing unit maintains native quaternary structure) of the stable non-covalent dimer and oligomers of a monoclonal IgG1λ antibody. The results supported a mechanism of intermolecular domain exchange involving the Fab domains of 2 or more IgG molecules. This mechanism can account for the native-like higher order (secondary, tertiary and disulfide bonding) structure, the stability of the non-covalent multimers, and the previously observed partial loss of the antigen-binding sites without changing the antigen-binding affinity and kinetics of the remaining sites (Luo et al., 2009, mAbs 1:491). Furthermore, the previously observed increase in the apparent affinity to various Fcγ receptors (ibid), which may potentially promote immunogenicity, was also explained by the quinary structure proposed here. Several lines of evidence indicated that the formation of multimers by the mechanism of intermolecular domain exchange took place mostly during expression, not in the purified materials. The findings in this work will advance our knowledge of the mechanisms for aggregation in therapeutic monoclonal antibodies.

Structural basis of polyethylene glycol recognition by antibody

“…Dimer formations without antigen have also been observed in therapeutic monoclonal antibodies [30]. They can be a result of domain exchange or other covalent and noncovalent interactions between the antibodies, probably induced by stress [30,31]. In contrast, PEG binding to the CDR region of Fab is more common.…”

Heterologous recombinant expression of non-originator NISTmAb

“…SEC is a seemingly simple, yet critical and informative assay regarding product quality because increased high molecular weight (HMW) species content has been associated with the potential for immunogenicity 15,16 . A SEC method was previously qualified during RM 8671 value assignment, which yielded a typical mAb profile consisting of a dominant monomer peak, low abundance LMW and HMW species, and a void volume peak arising from the L-histidine buffer (elution time ≈ 6.25 minutes) 17 .…”

Structural and thermodynamic insights into antibody light chain tetramer formation through 3D domain swapping

“…Higher secondary structure stability may prevent aggregation of the light chain that could lead to systemic amyloidosis. On the other hand, 3D-DS can occur in various manners in antibodies [24][25][26] . The native antibody light chain has a cysteine residue in the constant region that can causes dimerization of antibodies through a disulfide bond.…”

“…For instance, the human antibody 2G12, an HIV-1 neutralizer, can undergo 3D-DS by exchanging the heavy chain variable region between two antigen-binding (Fab) regions 24 . 3D-DS of exchanging the light chain variable region between two Fab regions has also been suggested 25 . The single domain VH from camelid heavy chain (VHH) can dimerize by engaging in 3D-DS for the C-terminal β-strand, where the hinge loop region converts to a β-strand in the dimer 26 .…”