Abstract
Because monoclonal antibodies (mAbs) have exceptional specificity and favorable pharmacology, substantial efforts have been made to functionalize them, either with potent cytotoxins, biologics, radionuclides, or fluorescent groups for therapeutic benefit and/or use as theranostic agents. To exploit our recently discovered meditope–Fab interaction as an alternative means to efficiently functionalize mAbs, we used insights from the structure to enhance the affinity and lifetime of the interaction by four orders of magnitude. To further extend the lifetime of the complex, we created a mechanical bond by incorporating an azide on the meditope, threading the azide through the Fab, and using click chemistry to add a steric group. The mechanically interlocked, meditope–Fab complex retains antigen specificity and is capable of imaging tumors in mice. These studies indicate it is possible to “snap” functionality onto mAbs, opening the possibility of rapidly creating unique combinations of mAbs with an array of cytotoxins, biologics, and imaging agents.
Highlights
Because monoclonal antibodies have exceptional specificity and favorable pharmacology, substantial efforts have been made to functionalize them, either with potent cytotoxins, biologics, radionuclides, or fluorescent groups for therapeutic benefit and/or use as theranostic agents
We demonstrate that the mechanical bond permits the functionalization of a meditope site as meditope-enabled antibodies (memAbs), including the addition of fluorescent groups that permits the imaging of tumors in vivo
Based on the kinetic fits, this increase was largely due to a faster on-rate (Fig. 1b and Supplementary Table 1). The structure of this modified meditope bound to the Fab of the anti-human epidermal growth factor receptor 2 (HER2) memAb indicated that the diphenyl group straddles Leu[115] of the Fab heavy chain (Fig. 1c and Table 1), increasing the contact area by ~30 Å2 9
Summary
Because monoclonal antibodies (mAbs) have exceptional specificity and favorable pharmacology, substantial efforts have been made to functionalize them, either with potent cytotoxins, biologics, radionuclides, or fluorescent groups for therapeutic benefit and/or use as theranostic agents. We recently discovered a unique peptide binding site within a hole created by the light and heavy chains of the Fab domain of cetuximab[1], an anti-epidermal growth factor receptor monoclonal antibody (mAb) used clinically to treat head and neck and colorectal cancers (Fig. 1a). As with the other meditope/meditopeenabled Fab structures, the substitution did not substantially change the overall structure of the Fab (the root-mean-squaredeviation between the Fab domains of the Ile83Glu variant and the parental trastuzumab calculated over Cα is 0.28 Å) Combining these meditope (i.e., Leu[5] to diphenylalanine substitution and C-terminal extension) and meditope-enabled Fab (i.e., Ile83Glu substitution) modifications produced a dramatic increase in the affinity.
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