Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Camelidae—camels, alpacas, llamas—are unique among mammals in producing functional antibodies composed solely of two identical heavy chains (HCs). These camelid antibodies, often called heavy chain antibodies (HCAbs), are a biological curiosity, and the special properties endowed by their unique structure make them of increasing interest for basic research and therapeutic applications alike.
Much of this interest stems from the fact that the HCAb antigen-binding domain, called a VHH (or VHH) domain, is a found on a single stretch of amino acids. The VHH retains its antigen-recognition capabilities even as a diminutive 12- to 15-kDa fragment. This allows it access to small spaces, while enabling relatively facile in vitro manipulation of the single (approximately) 360-base-pair stretch of DNA that encodes it, as well.
Antibodies and nanobodies
Conventional antibodies are comprised of two identical HCs and two identical light chains (LCs). Each heterologous tetrameric antibody contains two identical monospecific antigen-binding domains, each made up of the variable region of one LC and one HC—picture the tips of the archetypal Y-shaped antibody. Because both chains are required to detect an antigen, it is their combination that is selected (rather than each chain separately) in vivo in the bone marrow as well as during in vitro antibody production.
HCAbs, on the other hand, are homodimers, with each chain independently recognizing its cognate antigen. Thus the selection process requires only a single pairing. This vastly simplifies the in vitro phage-display process, during which clones are selected for antigen affinity. “If you do this with conventional antibodies, you always have to find the right light and heavy chain which go together the best,” explains Tina Romer, head of R&D for ChromoTek, a camelid antibody developer.
Producing HCAbs, too, can be much simpler, requiring only a vector encoding a single amino acid chain. They can be successfully and reproducibly expressed in a variety of systems, including Chinese hamster ovary (CHO), bacteria, yeast and plant cell cultures. The chains can be cloned in frame with tags such as a polyhistidine or FLAG tag, allowing for simplified purification, as well.
Camelid antibodies also can be recombinantly expressed as strictly antigen-binding fragments that lack other functional domains. The smallest such fragment of a traditional IgG, the approximately 25-kDa scFv (single chain variable fragment), is made by joining the variable regions of the HC and LC by a linker sequence.
Natural and recombinant VHHs—which need no extra linker—are the camelid equivalent HCAb fragments. They go by several names, including single domain antibodies (sdAbs), but are probably best known as Nanobodies® (Nbs), a registered trademark of biopharmaceutical company Ablynx.
What’s the use?
Das Prayaga, president and CEO of Antibody Research Corporation, sees Nbs as an emerging market and believes their appeal comes from their comparatively small size and concomitant stability.
“They can actually bind to epitopes that are in really small areas in a lot of folded proteins that are inaccessible to larger antibodies,” points out Sonya Paske, vice president of operations at Capralogics. This is partly because of the VHH’s unique architecture: It is composed of three relatively long, flexible loops rather than the six shorter loops of a traditional antibody or scFv. This enables Nbs to associate with concave antigenic surfaces such as enzyme active sites and ion-channel cavities [1].
Nbs are robust, with the ability to withstand both elevated temperatures and a wide pH range, extending both their in vivo and in vitro utility—for example, making them attractive for diagnostic biosensors, such as ELISA kits that do not need refrigeration. Molecules selected through panning have been found that can withstand the presence of detergents and other denaturants, such as urea. Panned Nbs also have been found that can cross the blood-brain barrier [2].
Another advantage, says Maxine Chen, marketing liaison for discovery biology at GenScript, is that Nbs can “penetrate tissues relatively easily and have better bioavailability than traditional antibodies.”
This all has made Nbs attractive for therapeutics. Ablynx and its partners, for example, have at least four Nbs in clinical trials, and others in discovery and pre-clinical stages, for indications including hematology, neurology, inflammation, pulmonary disease and oncology.
Like traditional antibodies and their fragments, camelid antibodies and Nbs can be conjugated to solid surfaces or fluorescently tagged for use in imaging. Endogenously expressed GFP- and RFP-tagged Nbs, for example, have enabled researchers to make real-time movies of DNA replication and to view the cytoskeleton with single-molecule localization using super-resolution microscopy.
Various properties, such as their size and ability to withstand harsh conditions and insinuate into crevices, have also allowed Nbs to be used “as a crystallization chaperone for structural biology, for really difficult-to-crystalize structures, such as the 5HT3 serotonin receptor,” says Chen.
Where to?
There are few conjugated and unconjugated camelid antibodies commercially available—most of these are polyclonal—and even fewer Nbs. And needless to say, most organizations in the developed world don’t maintain their own herds of camels, llamas and alpacas. But fortunately for the researcher interested in camelid antibodies against their own particular targets of interest, there are firms that can be contracted to provide custom services that range from husbandry and immunization “through phage display, cloning, characterization” and large-scale VHH antibody production, notes David Barraclough, vice president for marketing at Abcore.
Generation of polyclonal serum takes about two months and costs roughly $2,000. Taking that serum from engineering and screening all the way through to a fully optimized monoclonal Nbs can run $40,000 to $50,000 or more and take upwards of four to six months, depending on the target and the engineering required. Prayaga suggests—if you have the option—testing your theory with the full-length antibody to find out if recognition and affinity are good enough. “Then you chop it down and use Nbs.”
References
[1] Muyldermans, S, “Nanobodies: Natural single-domain antibodies,” Annu Rev Biochem, 82:775–97, 2013. [PubMed ID: 23495938]
[2] Abulrob, A, et al., “The blood-brain barrier transmigrating single domain antibody: Mechanisms of transport and antigenic epitopes in human brain endothelial cells,” J Neurochem, 95:1201–1214, 2005. [PubMed ID: 16271053]
Image: iStockPhoto