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

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.

Pharmacokinetic, biodistribution, and biophysical profiles of TNF nanobodies conjugated to linear or branched poly(ethylene glycol).

Covalent attachment of poly(ethylene glycol) (PEG) to therapeutic proteins has been used to prolong in vivo exposure of therapeutic proteins. We have examined pharmacokinetic, biodistribution, and biophysical profiles of three different tumor necrosis factor alpha (TNF) Nanobody-40 kDa PEG conjugates: linear 1 × 40 KDa, branched 2 × 20 kDa, and 4 × 10 kDa conjugates. In accord with earlier reports, the superior PK profile was observed for the branched versus linear PEG conjugates, while all three conjugates had similar potency in a cell-based assay. Our results also indicate that (i) a superior PK profile of branched versus linear PEGs is likely to hold across species, (ii) for a given PEG size, the extent of PEG branching affects the PK profile, and (iii) tissue penetration may differ between linear and branched PEG conjugates in a tissue-specific manner. Biophysical analysis (R(g)/R(h) ratio) demonstrated that among the three protein-PEG conjugates the linear PEG conjugate had the most extended time-average conformation and the most exposed surface charges. We hypothesized that these biophysical characteristics of the linear PEG conjugate accounts for relatively less optimal masking of sites involved in elimination of the PEGylated Nanobodies (e.g., intracellular uptake and proteolysis), leading to lower in vivo exposure compared to the branched PEG conjugates. However, additional studies are needed to test this hypothesis.

Dimers and multimers of monoclonal IgG1 exhibit higher in vitro binding affinities to Fcgamma receptors.

The in vitro binding of monomeric, dimeric and multimeric forms of monoclonal IgG1 molecules, designated mAb1 and mAb2, to the extracellular domains of Fcgamma receptors RI, RIIA and RIIIB were investigated using a surface plasmon resonance (SPR) based biosensor technique. Stable noncovalent and covalent dimers of mAb1 and mAb2, respectively, were isolated from CHO cell expressed materials. The dissociation constants of monomeric mAb1 and mAb2 were determined to be 1 nM for the FcgammaRI-binding and 6-12 microM for the FcgammaRIIA- and FcgammaRIIIB-binding. Dimeric mAb1 and mAb2 exhibited increased affinities, by 2-3 fold for FcgammaRI and 200-800 fold for FcgammaRIIA and FcgammaRIIIB. Further increases in binding were observed when the antibodies formed large immune complexes with multivalent antigens, but not in a linear relation with size. The binding properties of monomeric mAb2 were identical with and without a bound monovalent antigen, indicating that antigen-binding alone does not induce measurable change in binding of antibodies to Fcgamma receptors. Dimerization is sufficient to show enhancement in the receptor binding. Given the wide distribution of the low-affinity Fcgamma receptors on immune effector cells, the increased affinities to aggregated IgG may lead to some biological consequences, depending on the subsequent signal transduction events. The SPR-based in vitro binding assay is useful in evaluating Fcgamma receptor binding of various species in antibody-based biotherapeutics.