We were pleased to see that a PHERAstar FS was put to good use is a recent Nature Communications paper. The paper describes the discovery, characterization and therapeutic potential of nanobodies directed to the metabotropic glutamate G protein-coupled receptors (GPCR’s). The work is a collaborative effort among French scientists including our friends at Cisbio and reinforces the versatility of HTRF and how HTRF assays can be used to investigate a variety of different aspects of cell signaling. Nanobody interaction with receptors, agonist binding, receptor conformational changes and IP-1 production were all monitored with HTRF assays!
Cartoons illustrating the principles of the HTRF nanobody binding assay (left) and mGlu2 conformational sensor assay Figure 1a and 2a, respectively, in https://www.nature.com/articles/s41467-017-01489-1
So what exactly is a nanobody? The short answer is it is a smaller antibody.
Most of you are familiar with conventional mammalian IgG antibodies, the hetero-tetramer proteins, composed of heavy-chains and light chains, which have been widely used for biotechnology applications and as biological agents in therapeutic treatments. You may not be aware that other forms of antibodies occur naturally, such as heavy chain only antibodies in camelids. Camelids include dromedary and Bactrian camels, llamas and alpacas. Indeed, the nanobodies used in the Nature Communications paper were derived from llamas. And you thought llamas were only good for providing wool for making your favorite sweater!
Sera from camelids contain both conventional and heavy chain only versions of antibodies. While it is unclear why the distribution of heavy chain only antibodies is restricted their attributes have been studied and used as the basis to create some of the single domain antibody fragments that are now available. These nanobodies are championed by the biotechnology community for a variety of virtues.
The first attribute is their size. Nanobodies are on average 10 times smaller than a conventional antibody. The small size of nanobodies means they can access epitopes that were previously inaccessible due to protein folding. Using nanobodies to conserved cryptic epitopes in GPCR’s and ion channels is an area of particular interest. These membrane spanning proteins have precious little area that is exposed and traditional antibodies have yielded few products that have therapeutic utility. It seems logical that the smaller size of nanobodies will be useful where previous efforts may have failed. Sometimes small is better, like asking someone with small hands to help you get the last olive out of the bottom of the jar.
Despite their small size have a high binding efficiency and specificity AND are very resilient. Since the single binding domain has evolved to work alone in camels it turns out that the binding domain continues to work well even when it is isolated from the heavy chain. This seems to be less true for binding domains isolated from the typical mammalian form of antibodies.
Nanobodies are encoded by a single gene. Thus they are readily produced by a number of hosts; mammalian cells, as well as, yeast and bacteria. Regardless of host, production has been scaled up so that high concentration formulations can be created. The ability to get high concentration yields make nanobodies suitable for a variety of administration routes. Indeed, injection, nebulization (for inhalation) and oral treatments have already been prepared and of course humanized versions will be vital when used as a therapeutic.
The single gene nature of nanobodies make them easy to implement as recombinant proteins for a variety of biotechnology tasks such as immunoprecipitation and live cell imaging. The recombinant capabilities are also a feature that can be exploited for therapeutic purposes. Multiple nanobody building blocks can be linked together to bind multiple targets or multiple locations on the same target to improve therapeutic efficacy. They can also be targeted to specific cells and linked to drugs for higher specificity of action. Furthermore this recombinant production makes them very consistent as they are clonal in nature. So their binding and functional capabilities will not change over time.
For its advocates, the sky is the limit for the utility of nanobodies!
One application of nanobodies would be to have them bind to a protein of interest and change that proteins function. In the Nature Communications paper 3 different nanobodies were characterized and had very distinct pharmacological effects: one bound indiscriminately while the other two were found to be positive allosteric modulators (PAMs). These two could be further separated by the fact that one has intrinsic agonist activity making it ago-PAM.
What is a PAM? Maybe we should break that down. P is for positive so it is something that has an enhancing effect on the agonist receptor relationship. A is for allosteric which contains allo from Greek meaning other so the effect is indirect, not at the agonist binding site. M is for modulator meaning that it is usually acting in concert with the agonist. The usual mode of action for an allosteric modulator is to act at a site away from the agonist binding site and induce a conformational change that alters the binding or functional effect of the agonist. PAM’s are sometimes also called allosteric enhancers. There are situations where the PAM is able to activate the receptor in the absence of agonist and increase both the potency and efficacy of the agonist.
Although this study focused on effects that would be seen in the central nervous system they do point out that these metabolic glutamate receptors are also present in other parts of the body. So the potential of the characterized nanobodies in a cancer treatment will await future studies.