Single-domain antibodies, or nanobodies, have recently been thrust into the spotlight as a potential treatment for COVID-19. In fact, a COVID-19 nanobody-based treatment developed by the Beroni Group, an Australian biotechnology company, is currently in preclinical testing. Even though nanobodies are only beginning to realize their therapeutic potential, they have been studied for decades.

In 1989, two graduate students at Vrije Universiteit Brussel serendipitously discovered a unique property of camelids (camels, llamas, and alpacas among others). While testing frozen camel blood serum, the students found that camelids not only produce conventional antibodies, but also a unique secondary set of single-chain antibodies (scAbs) composed of two identical heavy-chain polypeptides, each of which incorporates two contiguous constant domains, a hinge region, and a variable domain. (The constant domains of each heavy chain run parallel; beyond the hinge region, the variable domains diverge like the arms of the letter “Y.”) Each of the variable domains of the scAb serves as an antigen-binding module.

This exciting discovery was just the start. Subsequent work revealed that only a tiny fragment of the scAb, a single variable domain, is required to recognize an antigen. This fragment weighs in at only 12–15 kDa, hence the name “nanobody.”

In contrast, human antibodies are composed of two identical heavy-chain and two identical light-chain polypeptides. These proteins are large, with a molecular weight of approximately 150 kDa. Unlike camelid scAbs, the antigen-binding site of human antibodies spans both the heavy and light chains (or rather the variable domains of these chains), meaning all the chains are needed to detect an antigen.

Nanobody advantages

As human antibodies are large, they often have difficulty accessing small binding spaces on viruses, certain cells, and targets deep within tumor tissue. However, the small nanobodies can navigate tight spaces and may be an attractive alternative to human antibodies for therapeutic developers. Additionally, the binding domain of nanobodies is long, producing a “finger like” structure that enhances the ability of nanobodies to reach their targets.

One huge advantage of nanobodies, compared with conventional human antibodies, is their easy manufacturability. The relatively simple process starts with immunization of a camelid with the desired antigen. The camelid’s immune system produces a scAb that recognizes the antigen. A blood sample is subsequently taken from the camelid (left otherwise unharmed), and the mRNA for the scAb is extracted from the sample.

The genes for the variable antigen-binding domain, that is, the nanobody, are then amplified from the mRNA. Large quantities of the final nanobody can then be produced inside microorganisms, typically Escherichia coli, at a low cost.

Newer methods are taking animals completely out of the equation by testing antigens against a preproduced library of nanobodies. Twist Bioscience, though its Twist BioPharma division, offers several types of nanobody libraries within a llama scAb framework or partially humanized scAb framework. Billions of nanobody sequences can be tested at once, making antibody discovery and development extremely fast and relatively inexpensive.

Nanobody applications

Originally, nanobodies were used only or primarily for research purposes. However, exploration into nanobodies’ use as a therapeutic has dramatically increased over the last decade. In February 2019, a significant advance was made when the first nanobody therapeutic was approved by the FDA.

The drug, called Cablivi, was developed by Ablynx for the treatment of acquired thrombotic thrombocytopenic purpura. Cablivi acts as an anti-von Williebrand factor and prevents platelets from aggregating around organs.

Nanobodies for varied conditions are in clinical trials. For example, nanobodies are being evaluated as treatments for psoriasis, rheumatoid arthritis, and viral infections.

In addition to constituting solo therapies, nanobodies may contribute to combination therapies. Of interest are the clinical trials exploring the combination of nanobodies and chimeric antigen receptor (CAR) T-cell therapies for cancer.

CAR T cells are genetically engineered to recognize and target antigens on the surface of tumors. Thus far, CAR T-cell therapies have been very promising treatments for blood cancers that are unresponsive to more conventional treatments. However, CAR T-cell therapies have not yet been successful against solid tumors.

To take on solid tumors, CAR T cells may need to home in on alternative targets. The usual targets include cancer-specific antigens, which are proving hard to find, and cancer-associated antigens, which are easier to find but are harder to engage safely, since they also appear on healthy cells.

These targets pose yet another yet another difficulty. They are usually targeted by CAR T cells that incorporate an antigen-recognition domain derived from a human monoclonal antibody. However, human antibodies can cause immunogenicity leading to side effects and a reduction in CAR T-cell efficacy.

What alternative targets might be suitable? Possibilities abound in the extracellular matrix, a web of proteins that shields solid tumors and harbors immunosuppressive molecules. The idea of hitting targets in the extracellular matrix appealed to scientists at Boston Children’s Hospital. Ultimately, these scientists decided to engineer CAR T cells with antigen-recognition domains derived from nanobodies.

Using mouse models of cancer, the scientists demonstrated that nanobody-based CAR T cells are only weakly immunogenic and capable of recognizing specific antigens in the tumor microenvironment. To construct these CAR T cells, the scientists used the Gibson Assembly method, a technique that allows multiple DNA fragments to be combined and cloned.

CAR T cell–nanobody constructs are capable of damaging tumor-nourishing blood vessels and tumor-protecting elements of the extracellular matrix. The damage to the tumor microenvironment significantly slows growth and allows other treatments, such as chemotherapy, access to the inside of the tumor.

Nanobody development issues

It took 30 years after nanobodies were discovered in 1989 for a nanobody therapeutic to reach the market. The first 10 years were focused on research into the structure, composition, and properties of nanobodies. Just after the 10-year mark, in 2001, Vrije Universiteit Brussel attempted to commercialize nanobodies with multiple patents issued in its name. These patents were subsequently passed onto the Vlaams Interuniversitair Instituut voor Biotechnologie (VIB) and then onto a VIB-founded company, Ablynx, in 2002.

It is likely that intellectual property limitations on the composition of matter of nanobodies contributed to the long delay between the discovery of nanobodies and the first approval of a nanobody-based drug. However, the main patent claims on this biomolecule expired in 2014 in Europe and 2017 in America, allowing Ablynx to significantly expand its collaboration with some of the largest pharmaceutical companies in the world, including Merck & Co., Boehringer Ingelheim, and Sanofi.

These collaborations have resulted in a flurry of clinical trials being registered involving nanobodies and the long-awaited approval of Cablivi. Additionally, diminishing intellectual property barriers associated with nanobodies’ composition of matter has allowed even more companies to show an interest in the further commercialization of these super-molecules.

As with all therapies, nanobodies do have drawbacks. Their small size results in rapid clearance through the kidneys, reducing their half-lives. Therefore, to ensure a high enough volume of nanobodies are present in the blood to achieve the desired effect, frequent dosing is required, which may induce kidney toxicity. There is also a small risk that patients could have an immune response to therapeutic nanobodies as they are a biologic material.

Fortunately, these problems can be overcome. Research has shown that fusing nanobodies to serum albumin, an abundant transport protein found in blood, significantly increases the half-lives of nanobodies, allowing them to remain in the blood for longer and in larger quantities. The immunogenicity of nanobodies can be reduced via humanization, a process that modifies some of the nanobody protein sequences to increase their similarity to human antibodies, reducing the risk of a negative immune reaction.

While there have been delays to the commercialization of nanobodies as therapeutics, now that multiple companies are able to invest in these wonderful and unique molecules, it is anticipated that there will soon be an explosion of nanobodies being used as therapeutics for a multitude of diseases, from viral infections to cancer. Camelid nanobodies have not only proven their worth but could also change the landscape of antibody therapy and hail in a new generation of therapeutics.

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