The problem of recognising 'friend' or 'foe' is essential in all security systems. For millennia, over the course of evolution, living organisms have formed and improved systems of protection from alien 'outsiders' to a given subject in order to maintain internal consistency and individuality. The most progressive of these systems is undoubtedly the mammalian immune system.
Antibodies are the 'special agents' that recognise foreign structures in the immune system. They are produced by immune system cells when they encounter viruses, bacteria, and other potentially dangerous substances generally referred to as 'antigens', short for 'antibody-generator'. When an antigen is recognised, the immune system is activated and tries to neutralise the malicious agent. Classical antibodies, immunoglobulins, are glycoproteins built from two heavy (H) and two light (L) chains. In turn, H- and L-chains contain variable (V) and constant (C) domains (Fig. 1). The antibody variable domains recognise antigens, while the constant domains interact with their own immune system cells1.
The ability to produce antibodies against virtually any antigens and their extreme specificity make antibodies a unique tool for both basic research and the diagnosis and treatment of various diseases. Immunotherapy is currently one of the most successful and fastest growing branches of biomedicine. However, there are significant limitations in the use of antibodies - they are rather large protein molecules with a mass of about 150 kDa and are approximately about 14.2×8.5 ×3.8nm 2 in size (for comparison, the mass of the main protein of chicken eggs is only around 45 kDa). This can complicate and even impede the penetration and distribution of antibodies in the tissue. In addition, antibodies can provoke an immune response in the recipient, that is, they are potentially immunogenic2-5. It is also worth noting that their production process is time-consuming and complex.3,4,6,7
In this regard, the discovery in 1993 by Belgian scientists of special, non-canonical antibodies with a simplified structure was truly significant.7 Antibodies were found in the blood of Camelids (such as camels and llamas) that had shortened heavy chains and were completely devoid of light chains. These camelid antibodies were named HcAb (Heavy chain Antibody).7,8 Later, based on their only antigen-recognising variable domain (VHH - Variable domain of the Heavy chain of the Heavy chain Ab), miniscule proteins were created that were able to efficiently recognise and specifically bind the antigen — single-domain antibodies. They are about 10 times smaller than classical antibodies and are about 2.5×4nm in size. 2-9 Due to their size, single-domain antibodies are called 'nanoantibodies' or simply, as they are now widely known, 'nanobodies', which is a registered trademark of the Belgian company Ablynx.9,10
Schematic depiction of classical human monoclonal antibody (human mAb conventional antibody), Camelid hcAb antibody, and nanoantibody (Nb). Their approximate molecular weight in kDa is also indicated (kD). H-heavy chains, L-light, C-constant domains, V-variable domains. Adapted from Bannas et al., 2017.5
Their small size, as well as other structural features, gave the nanobodies a number of significant advantages over classical antibodies. Firstly, they can easily penetrate hard-to-reach organs and tissues and are rapidly excreted from the body through the kidneys. Secondly, they are more stable over a wide range of temperatures (they can withstand heating to 60-80°C) and a more acidic environment.2-7. They also have a higher solubility and a lower tendency towards aggregation due to the replacement of some hydrophobic amino acids with hydrophilic ones.6,7,9 An important and practically significant feature of the nanobody's structure is that it can form long finger-like protruding structures that can penetrate the hidden places of antigens inaccessible to larger antibodies3,5-7,9. Due to their similarity to equivalent human proteins, nanobodies also have a low immunogenicity.2-4,7,9,10
However, one of the most important advantages of using nanobodies is their ready availability: because they are the product of only one gene, they are relatively inexpensive and easy to produce in large quantities3,6,7,9,10. It is also easy to perform various genetic engineering manipulations with nanobodies, for instance, 'sewing' various markers, medicines, or nanoparticles onto them, or combining two or more nanobodies.3,5,6,7,9 Thus, it has become clear that nanobodies are a powerful tool which has an enormous potential and can be used in both scientific research and for biomedical purposes.
Variants of therapeutic constructions created using nanobodies. (A) Conjugate of two identical nanobodies (dimer), a bispecific complex (a conjugate of nanobodies to different antigens), a conjugate of nanobodies with albumin for a longer-lasting effect (half-life extended). (B) Conjugate of nanobodies with tags and toxins (immunotoxin). (C) Radioactive (radionuclide) and fluorescent (chromobody) nanobodies. (D) Conjugate of nanobodies and nanoparticles for targeted drug delivery. Adapted from Bannas et al., 2017.5
A lot of research has been devoted to the use of nanobodies for cancer diagnosis and therapy. Due to their small size, nanobodies can easily penetrate tumours and be distributed within them.2-7,10 It is possible to synthesise nanobodies which block the molecules that accelerate the development of cancer cells and the growth of blood vessels in a tumour. This has a destructive effect on cancer cells, preventing the tumour’s growth and metastasis. It is also possible to connect nanobodies which 'recognise' cancer cells to special markers which make the tumour 'visible' and/or more vulnerable to immune system cells, or to a toxin, anti-cancer drug or nanoparticle carrying such a drug2,3,5,6. The interaction of nanobodies with biomarkers of human pathological conditions can be used to monitor and quantify when diagnosing diseases.6,11 Nanobodies can also be combined with radioactive and fluorescent markers. Due to their selective effect on tumours and rapid excretion, the patient's radiation dose is significantly reduced, and the diagnostic effectiveness of the markers also increases.2,3,5,6 Thus, it is possible to transfer large doses of radiation to tumours without any damage to healthy tissue,3,6 and to visualise tumours in real time before surgery.6
Nanobodies have great potential in the battle against viral and bacterial infections,.3,6,7,12,13. as well as in neutralising poisons and toxins.3,6,7,14.. The efficacy of nanobodies has been shown against viruses with high variability, such as HIV, influenza, and hepatitis C.3,6.. Nanobodies can also be used to fight the Helicobacter pylori bacterium, which causes the development of gastritis and duadenitis..3,6.
To date, a large number of therapeutic nanobody molecules have been developed. Among them, pride of place goes to nanobodies against chronic inflammatory and autoimmune diseases such as rheumatoid arthritis (Vobarilizumab, Ozoralizumab), psoriasis (ALX-0761) and systemic lupus erythematosus (ALX-0061). All are currently undergoing various stages of clinical trials3,6. Some nanobodies (Caplacizumab) are able to bind to the activated form of von Willebrand factor and thus have an antithrombotic effect. A drug based on them was clinically tested and was approved by the US Food and Drug Administration in February 2019 for the treatment of thrombotic thrombocytopenic purpura.15.
An extremely interesting field of research is the use of nanobodies as intracellular antibodies or 'intrabodies'. In this case, with the help of genetic engineering, the gene of a specific nanobody is delivered into the cell, and the cell itself begins to synthesise it. Thus, nanobodies can affect intracellular antigens that are not accessible from the outside, for example in patients with neurodegenerative diseases or infected with Salmonella.3,7,16 This type of gene therapy has been actively studied in recent times, but the potential for its use in treating humans is currently seen as problematic.3,16.
Russian scientists are also successfully engaged in the study of nanobodies. In the Molecular Biotechnology Laboratory at the Russian Academy of Sciences' Institute of Gene Biology, Moscow, new nanobodies have been synthesised, the procedure for their preparation improved, and gene therapy methods used to synthesise nanobodies inside cells.6,9,11-13
2. Hu Y., Liu C., Muyldermans S. Nanobody-Based Delivery Systems for Diagnosis and Targeted Tumor Therapy. Front Immunol. 2017, 8: 1442.
3. Steeland S., Vandenbroucke R.E., Libert C. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today. 2016, 21 (7): 1076–113.
4. Arezumand R., Alibakhshi A., Ranjbari J., Ramazani A., Muyldermans S. Nanobodies As Novel Agents for Targeting Angiogenesis in Solid Cancers. Front. Immunol. 2017, 8: 1746
5. Bannas P., Hambach J., Koch-Nolte F. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Front. Immunol. 2017 8: 1603.
6. Gorshkova E.N., Vasilenko E.A., Tillib S.V., Astrakhantseva I.V. Single-domain antibodies and bio-engineering drugs on their basis: new opportunities for diagnostics and therapy. Medical immunology. 2016, Vol. 18, No. 6, pp. 505-520.
7. Li C., Tang Z., Hu Z., Wang Y., Yang X., Mo F., Lu X. Natural Single-Domain Antibody-Nanobody: A Novel Concept in the Antibody Field. J Biomed Nanotechnol. 2018, 14 (1): 1-19.
8. Hamers-Casterman C., Atarhouch T., Muyldermans S., Robinson G., Hamers C., Bajyana Songa E., Bendahman N., Hamers R. Naturally occurring antibodies devoid of light chains. Nature. 1993, 363, 446-448.
9. Tillib S.V. “Camel Nanoantibody” is an Efficient Tool for Research, Diagnostics and Therapy. Molecular biology. 2011. Vol. 45, No. 1. pp. 77-85.
10. Allegra A., Innao V., Gerace D., Vaddinelli D., Allegra A.G., Musolino C. Nanobodies and Cancer: Current Status and New Perspectives. Cancer Invest. 2018, 36 (4): 221-237.
11. Tillib S.V., Ivanova T.I., Lysyuk E.Yu., Larin S.S., Kibardin A.V., Korobko E.V., Vikhreva P.N., Gnuchev N.V., Georgiev G .P., Korobko I.V. Nanoantibodies for detection and blocking of bioactivity of human vascular endothelial growth factor a165 Biochemistry (Moscow). 2012. Vol. 77, No. 6. pp. 659-665.
12. Tillib S., Ivanova T.I., Vasilev L.A., Rutovskaya M.V., Saakyan S.A., Gribova I.Y., Tutykhina I.L., Sedova E.S., Lysenko A.A., Shmarov M.M., Logunov D.Y., Naroditsky B.S., Gintsburg A.L. Formatted single-domain antibodies can protect mice against infection with influenza virus (H5N2). Antiviral Research. 2013, 97, 245–254.
13. Burmistrova D.A., Tillib S.V., Shcheblyakov D., Dolzhikova I.V., Shcherbinin D.N., Zubkova O.V., Ivanova T.I., Tukhvatulin A.I., Shmarov M.M., Logunov D.Y., Naroditsky B.S., Gintsburg A.L. Genetic passive immunization with adenoviral vector expressing chimeric nanobody-Fc molecules as therapy for genital infection caused by Mycoplasma hominis. PLoS One. 2016. Vol. 11, p. e0150958.
14. Alirahimi E., Kazemi-Lomedasht F., Shahbazzadeh D., Habibi-Anbouhi M., Hosseininejad Chafi M., Sotoudeh N., Ghaderi H., Muyldermans S., Behdani M. Nanobodies as novel therapeutic agents in envenomation. Biochimica et Biophysica Acta (BBA) - General Subjects. 2018, 1862 (12): 2955-2965.
15. FDA approved caplacizumab-yhdp
16. Messer A., Joshi S.N. Intrabodies as neuroprotective therapeutics. Neurotherapeutics. 2013, 10 (3): pp.447-58.