Nanobodies – small, but oh my

Re-evolutionizing the antique immunology toolbox of llamas, sharks & co.

Writer: Annika Schulz
Editor: Elizabeth Jovena Sulistyo
Artist: Stephanie Chang

Towards the end of the 20th century, an astonishing finding was made in camelid and shark blood serum. Sharks are known to have evolved the earliest form of adaptive immunity, whereby immunological memory can be generated and pathogens targeted with high specificity. In these animals, a previously unknown miniature form of antibody was discovered to form part of their adaptive immune system. Similarly, these small antibodies have also been identified in quite different vertebrates like camelids, which includes camels, llamas and alpacas.

Structurally, conventional antibodies are Y-shaped multimeric proteins consisting of a homodimeric heavy-chain and two light-chains that attach at the small arms of the Y-shape. However, in serum from nurse sharks or llamas, you will also find antibodies possessing just the heavy-chains made up of a single variable domain and two (camelids) to five (sharks) constant domains. While not much is known about how these seemingly incomplete structures emerged in distantly related species, their discovery has certainly provided a stepping stone for a wide range of biomedical research endeavours. 

So what does a heavy-chain-only antibody possibly have to offer? The extensive repertoire of antigen-binding sites generated by these smaller antibodies in camels suggests they are not the result of faulty design. Instead, by isolating the single variable domain from heavy-chain-only antibodies, minute structures known as nanobodies were created. Being the smallest known antibody fragment capable of antigen binding, these nanobodies have their own methods of creating binding specificity and diversity. 

A variable domain commonly consists of three complementary determining regions (CDRs). Of these, the CDR3 loop region is thought to play the most important role in antigen binding. That said, nanobodies possess an extended CDR3 loop, which allows them to reach unique target epitopes inaccessible to their shorter counterparts in antibodies. Nanobodies also don’t limit their antigen-binding capacity to the loop regions, thereby even offering a greater diversity of antigen-binding sites than classical antibodies. These phenomenal characteristics, in addition to high-yield production in E. coli biofactories, open up a range of applications in cutting-edge research across the board. 

In the lab…

One of the most significant achievements to date that involved the use of nanobodies was the determination of the human β2 adrenergic receptor (ADRB2) structure. In 2012, Brian Kobilka received the Nobel Prize in Chemistry for his contribution. Using nanobodies, his lab was able to lock ADRB2 in its active state to obtain a crystal structure. Other fragments from regular antibodies had previously failed to stabilise the receptor but with the significantly smaller nanobodies, they obtained sufficient data to solve it. Today ADRB2 is a well-defined member of the 7-transmembrane receptor class, which forms the target of a major proportion of currently approved drugs

In diagnostics…

A significant challenge in the field of cancer imaging is the poor penetration exhibited by classical antibodies used to deliver visualisation agents. Nanobodies overcome this problem by offering higher levels of tissue penetration while staying in the body for a minimal period of time, since they are rapidly filtered out by the kidneys due to their small size (notably, this can be modified for therapeutic purposes). This also makes them a good probe for tumour type detection. For instance, a phase II clinical trial will be completed this year investigating the efficacy of nanobody-based detection of HER2 expression in breast cancer. What is more, nanobodies have excellent properties for rapid diagnostic assays used to identify infectious agents. They are very heat-stable compared to antibodies, which has major implications for diagnostic testing in settings lacking appropriate refrigeration facilities. 

In patients…

Over past decades, the use of monoclonal antibodies as drugs has seen a dramatic increase. Nanobodies offer these therapies novel prospects with enhanced efficacy and administrative advantages. For instance, nanobodies with two different antigen-binding sites fused together have been shown to inhibit solid tumour growth. However, given the lack of the heavy-chain constant domain, nanobodies don’t elicit the same immunological effects as classical antibodies.

That said, nanobodies can be employed advantageously to modulate inflammation in other ways. For example, rheumatoid arthritis and inflammatory bowel disease management includes anti-cytokine antibody therapies. These are delivered by intravenous infusions but with the more robust, heat- and acid-stable nanobodies, these treatments could potentially be delivered by inhalation or orally. A novel antiviral therapy for rotavirus based on the orally administered nanobody ARP1 was found to be effective in controlling viral diarrhoea in infants. Further developments envision delivering ARP1 in the form of immunity-boosting functional foods like rice.

What’s next?

In the lab, nanobodies will soon lend a helping hand to CRISPR technologies to enable the control of gene expression and epigenetic memory without causing DNA breakage. Recently, multivalent nanobodies have also been put forward for the development of desperately needed therapies for COVID-19. SARS-CoV-2 neutralising nanobodies work by not only interfering with viral receptor binding, but may also induce conformational changes that prematurely activate fusion machinery. Multivalent nanobodies provide fantastic prospects for reducing mutational escape of the virus and could even be administered by inhalation.

How odd to think this all started with the discovery of seemingly flawed antibody isotypes in camelids and sharks…

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