A general method for converting antibodies into fluorescent sensors of their antigens

About This Grant

ABSTRACT Fluorescently labeled antibodies have found extensive use as diagnostic tools due to their high affinities and specificities for their antigens as well as the high sensitivities associated with fluorescence-based assays. Despite this, most antibody-based diagnostic techniques require extensive experimental manipulations (e.g., multiple washing steps to remove unbound antibodies) and require costly and sophisticated equipment for detection. A new class of fluorescent antibodies that directly report on antigen binding through alterations in fluorescence properties that are readily detectable by eye could address this challenge. Not only would this eliminate the need for laborious experimental protocols but could also allow for rapid diagnostic analyses to be carried outside of laboratories. This possibility was explored almost 15 years ago when a coumarin-containing fluorescent non-canonical amino acid (fNCAA) was encoded within the binding site of a CD40L-binding antibody termed 5c8. The coumarin modified version of 5c8 exhibited an ~2-fold increase in fluorescence emission when bound to CD40L. More recently, structural studies of both wild type and coumarin-modified 5c8 revealed that, in both cases, a conformational change occurred far from the active site upon CD40L binding. This raised the intriguing possibility that moving the environmentally sensitive coumarin moiety from the combining site to this conformationally flexible region could elicit similar increases in fluorescence upon antigen binding without compromising affinity. In a recent set of preliminary studies, we have confirmed this hypothesis. If this approach is generalizable, fNCAAs could be used to convert essentially any antibody into a direct fluorescent sensor of its antigen without losses in affinity. Despite this promise, the generality of this approach has not been demonstrated; this is the focus of the proposed research. Here, we propose to build on our previous results in an effort to generate allosterically responsive fluorescent antibodies that directly report on the antigen binding event. Our efforts toward this goal will be carried out in two complementary aims: In Aim 1, we will seek to enhance the fluorescence properties of a previously identified, allosterically responsive fluorescent antibody by introducing tryptophan (Trp) residues in the vicinity of the fNCAA. Additionally, we will seek to identify new Fab variants with enhanced fluorescence properties through an extensive computational design campaign using the Rosetta software. In Aim 2, we will explore the generality of this approach by introducing coumarin-containing amino acids in other antibody scaffolds. As in Aim 1, these efforts will also be guided by a Rosetta-based design approach. Collectively, these efforts will pave the way for the development of a new paradigm of antibody-based diagnostics in which fluorescent antibodies not only directly sense their antigen targets but do so without the need of laborious experimental protocols or sophisticated instruments that facilitate detection.