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Abstract
Differences in conformational dynamics between two full-length monoclonal antibodies have been probed in detail using Fast Photochemical Oxidation of Proteins (FPOP) followed by proteolysis and LC-ESI-MS/MS analyses. FPOP uses hydroxyl radical labeling to probe the surface-accessible regions of proteins and has the advantage that the resulting covalent modifications are irreversible, thus permitting optimal downstream analysis. Despite the two monoclonal antibodies (mAbs) differing by only three amino acids in the heavy chain complementarity determining regions (CDRs), one mAb, MEDI1912-WFL, has been shown to undergo reversible self-association at high concentrations and exhibited poor pharmacokinetic properties in vivo, properties which are markedly improved in the variant, MEDI1912-STT. Identifying the differences in oxidative labeling between the two antibodies at residue level revealed long-range effects which provide a key insight into their conformational differences. Specifically, the amino acid mutations in the CDR region of the heavy chain resulted in significantly different labeling patterns at the interfaces of the CL-CH1 and CH1-CH2 domains, with the nonaggregating variant undergoing up to four times more labeling in this region than the aggregation prone variant, thus suggesting a change in the structure and orientation of the CL-CH1 interface. The wealth of FPOP and LC-MS data obtained enabled the study of the LC elution properties of FPOP-oxidized peptides. Some oxidized amino acids, specifically histidine and lysine, were noted to have unique effects on the retention time of the peptide, offering the promise of using such an analysis as an aid to MS/MS in assigning oxidation sites.
Highlights
Long-range conformational interactions between the various domains of monoclonal antibodies, the variable (V) and constant (C) domains, has been a hotly debated topic since the 1970s.1−3 numerous studies have suggested a functional allosteric link between antigen binding in the V domains of the antigenbinding fragment (Fab) and C domain function in the fragment crystallizable (Fc) region, the prevailing hypothesis is that the Fab and Fc domains are functionally independent,[4,5] supported by the perceived difficulty of transmitting allosteric conformational changes via the highly flexible hinge connecting the two regions.[6]
Henderson et al demonstrated that a double mutation of amino acid sidechains in the elbow and framework regions of the V domain resulted in major increases in flexibility of the elbow joint and in the more distant complementarydetermining regions (CDRs) loops of both the H and L chains.[16]
The most obvious differences in identified modification sites were directly proximal to the W30S and F31T mutations in the heavy chain CDRs of the monoclonal antibodies (mAbs)
Summary
Long-range conformational interactions between the various domains of monoclonal antibodies (mAbs), the variable (V) and constant (C) domains, has been a hotly debated topic since the 1970s.1−3 numerous studies have suggested a functional allosteric link between antigen binding in the V domains of the antigenbinding fragment (Fab) and C domain function in the fragment crystallizable (Fc) region (reviewed in ref 2), the prevailing hypothesis is that the Fab and Fc domains are functionally independent,[4,5] supported by the perceived difficulty of transmitting allosteric conformational changes via the highly flexible hinge connecting the two regions.[6]. It is wellestablished that class switches in the C domain of Fab arms can affect antigen binding affinity[7−10] and instigate changes in cooperative unfolding.[11] Further, class switches in either the C or V domains can change the elbow angle of the Fab arm.[7,12] Together these effects suggest that the conformation of the antigen binding site can, to some extent, be modulated by changes in the C domain, via long-range conformational changes mediated via the elbow angle.[9] Other studies suggested that the reverse communication is possible, whereby antigen binding may change the conformation of the C domains.[13] a systematic comparison of antigenbound and antigen-free mAb structures in the Protein Data Bank (pdb) revealed conformational changes in the relative orientation of V and C domains in the Fab for both heavy (H) and light (L) chains, as well as significant movement of the CH1−1 loop, an essential component of the CH1−CL interface,[14] suggesting that long-range conformational changes within the Fab arm can be instigated from either direction Subtle changes, such as single amino acid substitutions, have been observed to cause long-range conformational changes in Fabs. FPOP offers many advantages over alternative structural MS techniques, such as hydrogen−deuterium exchange (HDX), where single residue resolution is typically difficult to achieve,[26] and conditions required to minimize back exchange of the reversible deuterium label limit the available analysis time and choice of proteases for digestion.[17]
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