President TriBiotica Beverly, Massachusetts, United States
Matthew Lawler (TriBiotica)| James Kurnick (Massachusetts General Hospital)| Leah Fagundes (TriBiotica)| Milto Junior (TriBiotica)| Lenora Rose (TriBiotica, TriBiotica)| Ian Dunn (TriBiotica)
Click chemistry has found wide utility in basic biological research, molecular diagnostics, and drug discovery. Given this important and expanding role in molecular biology research, methods for facile detection of click reaction products have emerged as a key to the development of novel assays and therapeutics. While these reactions have previously been detected by appending molecular tags or fluorogenic elements to click reagents, an antibody directed against a minimal click reaction product structure allows for much more versatile and selective detection of click reaction sites in the absence of additional molecular tags. We describe the isolation, specificity, and uses of antibodies specific for unique structures created when azide groups react with dibenzocyclooctyne (DBCO) to assemble varied structures for diagnostic and therapeutic applications.
In vitro phage display was used to select a panel of scFv molecules (from Creative Biolabs Inc) specifically recognizing and binding small peptides covalently joined together by azide-DBCO click chemistry. Subsequently, the best scFv binder was converted into a full human IgG1 antibody (IgG1-51). Importantly, neither smaller precursor in isolation is recognized by these novel binding molecules, which specifically bind the assembled product. Specificity studies showed that for recognition, IgG1-51 preferred peptide sequences flanking the azide-DBCO reaction product, but the identity of the specific residues involved was not crucial. Notably, IgG1-51 could strongly recognize the product of the original DBCO-peptide precursor when reacted with azidosugars positioned on cell surfaces by metabolic labeling, and some DBCO-peptides elicited even stronger cell-surface responses than the original peptide.
Significant applications of IgG1-51 derive from the principle of ‘haplomers’, which we define as structures that are inert until brought into close proximity by molecular templating, after which haplomer assembly into biologically and immunologically functional products occurs. In situ assembly of functional haplomers, on or within a pathological cell target, can focus specific diagnostic and therapeutic applications while potentially greatly reducing deleterious background toxicities. An important extension to this technology is thus the deployment of haplomers incorporating the original azide- and DBCO-peptides whose reaction products are recognizable by IgG1-51. Additional antibody reagents against alternative click products (under development) will further extend the range of applications for these molecular tools. Antibodies that recognize cell surface markers that are known to exhibit abnormally high densities on certain tumor cells can be separately conjugated with specific azide- and DBCO-peptides. When two such antibodies bind to surface markers in molecular proximity, their azide- and DBCO-peptide tags can click-react, enabling subsequent recognition by IgG1-51. This in turn provides a means for selectively delivering therapeutic or diagnostic payloads to corresponding target cells.