Small Antibodies from Not So Small Organisms: Harnessing the Unique Immune System of Llamas, Camels, Sharks, and Lampreys

Antibodies are proteins produced by our immune systems in response to antigens, bits and pieces of foreign and harmful substances, including pathogens such as viruses and bacteria. The primary function of antibodies is to protect us by specifically binding antigens to neutralize them or target them for destruction.

Conventional antibodies have a complex structure with a characteristic Y-shape. They consist of 4 components: 2 long pieces called heavy chains (HC) and 2 shorter pieces called light chains (LC). Each chain contains a variable and a constant region. Each HC is linked to a LC through disulphide bonds and the combination of the  variable domains forms the antigen-specific binding sites (also called paratopes). The stem of the Y is called the constant region, Fc, which binds Fc receptors on immune cells to trigger specialized activities such as elimination of pathogens.

Since the Nobel Prize winning advent of hybridoma technology by Köhler and Milstein in 1975, there has been a continuous surge of antibody production in the scientific field. The power of conventional antibodies has been harnessed for many immunological techniques in research as well as therapeutic drug development.

Interestingly, in 1989, a group of researchers in Brussels, by chance, decided to analyze camel blood and discovered antibodies with unusual structure – they were 10-fold smaller than conventional antibodies. Further analysis revealed that although these small antibodies had a complex repertoire and were able to bind with high specificity to antigens, they consisted of only two HC with no LC, hence named heavy-chain antibodies (HCAb). Subsequently, similar small antibodies have also been identified in llamas and cartilaginous fish such as sharks. Whereas camelid HCAb variable regions are named variable heavy homodimers (VHH), shark HCAb have been named immunoglobin  new antigen receptors (IgNAR) with the variable region designated as VNAR.

Similar to conventional antibody production, the first step to generate antigen-specific HCAb is animal immunization. Several months later, HCAb can be isolated from blood samples and sequenced to obtain the genetic instructions for the small antibodies. Unlike conventional antibodies, the HCAb genes can then be provided to bacteria to easily produce large quantities of these antibodies without sacrificing or continuously bleeding animals.

HCAb are more soluble and stable at high temperatures. Their small size allows for binding to cryptic binding sites on antigens inaccessible to conventional antibodies, easy penetration into cells and tissues, and low toxicity due to rapid clearance from tissues, rendering them ideal candidates for therapeutic diagnostics and drugs. These characteristics inspired the engineering of nanobodies – engineered single domain antibodies consisting of only the paratope of HCAb. Due to their stability, nanobodies can be delivered intravenously, subcutaneously, through inhalation, or even ingested. Nanobodies are not only chemically stable, but can be fused to other proteins for binding to multiple targets. For example, tumour-specific nanobodies or HCAb can be fused to radioactive or fluorescent molecules to visualize tumours or deliver drugs.

Many nanobody therapeutics are currently being developed. In 2019, a VHH nanobody was approved for treatment of a rare clotting disease, thrombotic thrombocytopenic purpura. Another drug has completed Phase II clinical trials for rheumatoid arthritis. More recently, llama nanobodies are being investigated for treatment of SARS-CoV-2. Due to the respiratory nature of this virus, this is a particularly attractive option as nanobodies can be inhaled. Preliminary results in laboratory experiments have shown promising results in preventing viral entry into cells via binding of spike proteins on SARS-CoV-2. Interestingly, due to their rapid clearance from tissues, this characteristic can also prevent effective drug delivery. To circumvent this issue, many of the drugs being tested are a fusion of two nanobodies which effectively increases the stickiness of the drug to their targets.

Besides therapeutic purposes, small antibodies can be used to manipulate other proteins. For example, nanobodies have been used in crystallography to stabilize proteins that have historically been difficult to crystallize due to their dynamic nature. Kobilka and colleagues utilized a llama nanobody to uncover the structure of the β2 adrenergic receptor, leading to a Nobel Prize in 2012. Other research applications of nanobodies include tagging specific proteins for destruction or manipulating protein function. Since IgNAR are even smaller than camelid HCAbs, VNAR fusion nanobodies are being investigated for therapeutics that require crossing the blood-brain barrier. One such nanobody drug from Ossianix targets a receptor on cells that control access through the blood-brain barrier to open the passage for delivery of drugs to the brain. Another VNAR drug, AD-214, is now in Phase I clinical trials for the treatment of idiopathic pulmonary fibrosis.

In addition to HCAb, a unique antibody-like molecule has been found in jawless vertebrates. First identified in 2004 by Cooper and colleagues, variable lymphocyte receptors (VLR) are expressed in lampreys and hagfish immune cells in response to pathogens. Two unique VLR types (VLRA, VLRB) exist in the hagfish genome, whereas three types exist in the lamprey genome (VLRA, VLRB, and VLRC). The three types share similar structural properties; however, the structure of VLR differs vastly from both conventional antibodies and HCAb. Forming a C-shape, a VLR is made up of repeating modules, where the concave portion forms the paratope that displays high specificities to antigens. Interestingly, a similar defense system can be found in plants, suggesting evolutionary conservation. Due to their unique structure, antigen-specific VLR discovery may open avenues in antigen recognition that is currently not possible by conventional antibodies. VLR are also being tested in drug delivery to the brain for treatment of neurological disorders such as brain tumours, Alzheimer’s, multiple sclerosis, and traumatic injuries.

Alternatives such as HCAb and VLR are enabling novel strategies for developing research tools and therapeutic molecules that can complement or even surpass the limits of conventional antibodies. Intriguingly, it is currently unknown why or how these molecules evolved in nature. Future work to further understand their origin, development, and unique functions in immunity will further advance nanobody and VLR engineering to applications even beyond our current repertoire of research.


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Ling Ling Tai

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