Single Domain Antibody

Monoclonal and polyclonal antibodies (Abs) are well-corroborated tools adopted in basic biomedical research and innovative key components of numerous therapeutic platforms today on the market ¹. The vast majority of the Abs are obtained from easily accessible laboratory animals, such as mice, hamsters and rabbits, and consist of two heavy and two light chains that give to the whole molecule two identical antigen-binding sites.

Although extensively used, these Abs have an intrinsic and insurmountable Achille’s heel: a molecular weight of 150 kDa on average that significantly weaken their in vivo therapeutic applicability. Indeed, as large size molecules, they cannot access solid and compact tissues neither cross the blood brain barrier nor access cryptic antigen binding site².

To address these issues, and given the increasing demand on Abs based therapeutic products, it is therefore of paramount importance to find new formats with equal binding specificity of conventional Abs but with improved in vivo performance.

Back in the early 90s, pioneering studies shed ligth on the un-conventional structures of camelids and sharks derived Abs ^3,4. Of interest and importantly, camelids heavy chain only Abs (HCAbs) and shark immunogobulin new antigen receptors (IgNARs), own a peculiar structure that completely lacks of light chains resulting per se significantly smaller than that of conventional Abs. Nowadays, after decades of intense reseach, these classes of particular Abs are at the cutting-edge for Abs based research.

Single domain Ab the new frontier in Abs based therapy
Single domain Ab (sdAb) is the variable region isolated from HCAbs and IgNARs, called VHH and vNAR respectively, with preserved complete functional antigen-binding capacity. With a size of just 15 kDa, one 10th of convetional Ab, sdAb represents the smallest antigen binding fragment discovered so far ⁵. Given its small dimension, sdAb offers a series of adavanteges that cannot be overlooked:

•    Improved tissue penetration
•    Ability to trespass the blood-brain barrier
•    Unique recognition of hiden epitope
•    Optimal affinity and specificity
•    Highly stable over a wide range of pHs and temperatures
•    Highly soluble
•    Rapid clereance
•    Easily re-formatted for different downstream applications
•    Cost-effective
•    Possibility of large-scale production.

According to the features listed above, sdAbs result highly suitable for in vivo application and consequently their therapeutic potential has not gone unnoticed. Today, without doubt this new format of Ab is an ideal building block for the generation of innovative revolutionising therapy and high performing diagnostic tools across a number of therapeutic area, with the field of oncology, infectious diseases and rare disease as major beneficiaries.

SdAbs boost the biotechnology market and inspire new therapeutic solution
Explosive interest is increasing around sdAbs as their performance is for the majority recognized as superior to conventional Abs. As such, the great biomedical potential of sdAb has triggered a quick action from venture capitalist and the biomedical industry as well. In line with several market analysis reports, the sdAb market size is estimated USD 132.2 million in 2021 and is rapidly projected to reach USD ca. 700 million in 2028 (CAGR ca. 26,9%). Of note and importantly, in 2018 FDA approved the first ever VHH, a Sanofi’s single domain Abs, named Caplacizumab, for the treatment of the acquired thrombotic thrombocytopenic purpura - a rare emathological disorder characterised by the formation of circulating small blood clots. With this first approval, a sudden and massive growth of the entire sdAb market is expected ⁶. Indeed, a handful of other camelid VHH candidates, mainly discovered by Ablynx’s platform, are today under clinical evaluation. The FDA’s approval of one of those candidates will exponentially expand the size of sdAb market wich is optimistically predicted ca. USD 9 billions by 2030. In a long term projection, as more candidates will find clinical approval, sdAbs are expected to conquer the entire Abs market.

Moreover, the flourishing R&D activities around the sdAb field positively impacts the demand and sales of these products in the global market. Indeed, big and medium size biotechnology companies are focusing their financial effort into R&D and commercialisation of sdAb- based therapeutic tools, and offering complete solution for the discovery, production and engineering of sdAb. Equally relevant is the action of small start-ups and academic laboratories that are pivoting their effort toward generating the next generation Ab-based therapeutic platforms.

In this respect is worth of mentioning targeted protein degradation (TPD) a rapidly growing field of medicinal chemistry that is revolutionising the drug discovery process⁷. Briefly, TPD allows for the degradation of specific disease causing proteins by using the cell’s natural degradation machinary systems in conjugation with targeting motifs. Recent development of this innovative technology demonstrated that TPD greatly benefits from the use of sdAb to specifically degrade well-known therapeutic target such as the immune checkpoint Programmed death-ligand 1 (PD-L1) ⁸. Given the impressive interest growing around this promising therapeutic strategy, it is herein easily expectable a progressive leavering of the flexibility of this technology. No surprising, venture capitalists are closely looking into this opportunity and have already pivoted substantial financial effort to accelerate the development and commercialisation of TPD sdAb based platform and other sdAb technologies.

From camelids to sharks and the necessity for alternative animal sources of sdAb
Camelids, including camels, lamas and alpcas are so far the most commonly used animal sources for sdAb discovery and production. However, several challenges are faced with respect to the farming cost, as experienced staff is required for the the handling of these large size animals. Additionally, as naïve animal are needed for each sdAb discovery, a permanent housing facility must host each animal at the end of the immunisation project. However, an increasing number of research farms is ready to offer a series of highly customisable services ranging from animal immunisation untill complementary DNA (cDNA) synthesis. Thanks to these commercial services, the in-house animal facility is dispensabile and even small academic laboratories have started to explore the exciting world of sdAb.

As aforementioned, the vNAR of IgNAR from several species of shark, including nurse shark and wobbegong shark, is a valid alternative to camelids derived products. Over the years, the reliability of shark vNARs as immunodiagnostic and therapeutic tool has been confirmed in a countless number of preclinical studies. From a purely commercial stand point, as VHHs still remain the first choice for sdAb based product development, the market size of vNARs is still limited thus vNAR discovery services and products are significantly less costly than camelid counterpart.

Unfortunately, the large size of shark species together with the impossibility of captivity, the slow maturity, the aggressive temper and the unpredictable behaviour are today the major issue hampering the success of vNAR derived products.

To address these issues, scientists from City University of Hong Kong firstly demonstrated that a commonly used home acquarium carpet fish, named whitespotted bamboo shark (Chiloscyllium plagiosum), could be successfully adopted for sdAb production ⁹. Indeed, the small size (the adult size is less than 1 meter in length), the affordable cost, the docile behaviour and the possibility for large-scale husbandry, make the whitespotted bamboo shark an ideal source of sdAb. Accordingly, this animal promises a saving of 50% cost and production time when compared to both camelids and nurse shark sdAb production pipeline respectively.

Behind the development of this novel and exciting animal model there are years of intensive research: in vitro and in vivo validation, analysis of the genome, transcriptome, proteomics, and the study of the adaptive humoral immunity of bamboo shark.

As a proof of concept, the authors discovered the GFP-specific vNARs, which performance, picomolar binding affinity and ability to recognise unique conformational epitopes, resulted comparable to that of VHH products.

These encouranging data have recently led to the establishment of Jotbody (HK) Limited, a start-up with dual operating sites located in Hong Kong and Shenzhen. Jotbody, along with camelids derived sdAb products, continues to explore the potential of whitespotted bamboo shark vNARs with the discovery and validation of new sdAbs against different targets including PD-L1 and SARS-CoV-2 spike protein among others.

Without doubt, there is still a long way to go before the performace of vNARs will be globally recognised as equal as VHHs. In this respect, future studies should also explore alternative models such as the whitespotted bamboo shark, this will certainly help to expand the biomedical use of vNARs and will positively impact the future trajectory of sdAbs research.

Conclusion and remarks
The discovery of the unique structure of camelids and sharks derived Abs is a fundamental breakthrough in immunology. As highlighted in this short communication, the derived sdAbs own the unique biological and structural features needed to overcome the technical limitations of convetional Abs. Thanks to the intensive research, the sdAb field is continuously evolving with new isolation and formatting approaches, alternative animal models and innovative therapeutic platforms.

Additional spike of attention is expected from industry and investors as more sdAbs will find clinical approval. In the meanwhile, academic institutions and start up companies are already preparing the way for the next generation of sdAb based platforms.

A new era of Abs based therapy has just began, and is moving quikly from lab bench to clinical setting where is likely to leave an indelible mark on human healthcare.

References

1.     https://www.antibodysociety.org.
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4.     Greenberg AS, Avila D, Hughes M, Hughes A, McKinney EC, Flajnik MF. A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 1995;374(6518):168-73. doi: 10.1038/374168a0
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