Alpaca-derived nanobodies as part of the antibody ‘toolbox’

Contributed by our own Will Fry, Ph.D. (Among his many accomplishments, Will has played a central role in building the custom Alpaca antibody program here at Antibodies Incorporated. Inquiries about this program can be placed on this page).

Rabbit and mouse antibodies are mainstay reagents found in all biological research labs, but some of you have likely heard the buzz around camelid antibodies. Camelids include camels, llamas, alpacas, and other lesser known species, though llamas and alpacas are the most commonly used camelids for antibody development since they are easier to handle.

Since antibodies are already developed in mice, rabbits, chickens, goats, guinea pigs, why do we need another animal host for making antibodies? Having antibodies from different hosts facilitates multiplex labeling by microscopy and also offers flexibility with respect to project cost and the amount of antibody produced.  However, for all of the above-mentioned animal hosts, the antibodies produced are structurally quite similar, with each antibody consisting of two identical heavy chains and two identical light chains and commonly referred to as a ‘conventional antibody’ (Figure 1).

Camelids however produce an entirely different type of antibody that is devoid of light chains and is commonly referred to as a ‘heavy chain only antibody’ (HCAb). These antibodies were first discovered serendipitously during a student-run project at the Belgian university Vrije Universiteit Brussel (VUB) in 1989. Since their initial discovery scientists have come to appreciate that, in addition to the absence of a light chain, these HCAbs possess special qualities that differentiate them from conventional antibodies.


Developing camelid antibodies

Shortly before the discovery of camelid HCAbs George P Smith developed the technique of phage display, which allows for the display of peptides and small proteins on the surface of bacteriophage viruses.  This technique couples phenotype (the protein displayed on the phage’s surface) with genotype (the DNA encoding the displayed protein which is artificially introduced into the phage). Using phage display, enormous antibody libraries on the order of a billion or more unique antibodies can be sifted through to identify antibodies with the most desirable properties, allowing scientists to isolate the veritable ‘needle in a haystack’.  When done with ‘conventional antibodies’, antibody phage display involves separately cloning out all of the variable light chains (VL) and variable heavy chains (VH) from the immune system to create a library of VLs and a 2nd library of VHs.  These separated VLs and VHs are then randomly mated together by molecular cloning to create an ‘ScFv’ (single chain variable fragment) library of ‘reconstituted’ antibodies. An issue with this is that these newly formed ScFv’s represent pairings between VLs and VHs that never existed in the original antibodies present in the host.

The camelid-derived equivalent of the ‘ScFv’ is called a Vhh or ‘nanobody’ and consists of the variable region of the camelid HCAb heavy chain domain. Unlike ScFv’s the Vhh is encoded by a single stretch of DNA in the host genome. Accordingly, when creating an antibody library from alpacas and other camelids, the library consists of intact antibodies that resemble the native antibodies originally present in the host.  Furthermore, because Vhh’s can be cloned in a single step, the creation of camelid (Vhh) antibody libraries is more straightforward.

What’s so special about camelid antibodies?

Beyond the technical advantages associated with their use in phage display, camelid antibodies also have unique properties that make them quite different from conventional IgGs.

Size: sometimes smaller is better.

The cloned Vhh ‘nanobody’ fragment is a fully functional antibody at a diminutive size of only 15kDa. Being small has advantages for certain applications including:

  • Crossing the blood brain barrier (BBB)- this is a hurdle for any therapeutic antibody since the BBB does not permit passage of high molecular weight drugs. However, several nanobodies have been found to cross the BBB and manipulation of the properties of these nanobodies is being explored as a way to optimize delivery.
  • Tissue clearing microcopy- various methods have been developed to allow whole tissues to be imaged by microscopy without having to section through the tissue and then reconstruct in 3D; these methods include iDISCO, CLARITY, among others. A limitation of these techniques is antibody penetration. Antibodies often label the exterior of the specimen but cannot thoroughly gain access to the interior.  The small size of nanobodies has recently been put to use in the development of ‘vDISCO’ where the ‘v’ is for Vhh. Using vDISCO the investigators were able to perform whole body labeling on mice and for the first time were able to visualize neuronal connectivity throughout the whole body of the animal (

Nanobodies can go where other antibodies cannot

The variable region of antibodies forms the ‘paratope’ or antigen-binding region of the antibody. This region consists of alternating ‘framework’ regions (FRs) and ‘complementarity-determining regions’ (CDRs). The variable region of camelids (Vhh) differs from that of conventional IgGs by having an elongated CDR3 that ranges from 12-24 amino acids relative to the comparatively shorter (9-12 amino acid) CDR3 of conventional IgGs (see Figure 2). The longer CDR3 of Vhh’s permits these antibodies to bind inside clefts and grooves that are generally inaccessible to conventional antibodies and make them great candidates for binding within enzyme active sites and other ‘hard to reach’ protein surfaces. This preference was well demonstrated in a study showing that the majority of camelid antibodies raised against the enzyme lysozyme bound within the active site and were inhibitory, whereas conventional IgGs raised in mouse against lysozyme preferentially bound outside of the enzyme active site  

Another place that nanobodies can go that other antibodies typically cannot is inside of living cells. When expressed inside of living cells these intracellular antibodies are referred to as intrabodies. Nanobodies are uniquely suited for use as intrabodies since, unlike conventional antibodies, they are generally resistant to the reducing environment of the cytosol and also fold efficiently upon expression.

The future of camelid antibodies

Efforts to develop nanobodies into therapeutics has been underway for some time now and just last year Cablivi, a nanobody targeting von Williebrand factor, was approved for the treatment of acquired thrombotic thrombocytopenic purpura. With many other nanobody-based therapeutics currently under development this is likely the first of many nanobody-based therapeutics.

The future also seems bright for the continued use of nanobodies as research tools with these reagents continuing to find new applications in cellular imaging and other applications.