Nanobodies (Nbs) are small, stable, and highly specific antigen-binding proteins derived from the variable domains of heavy chain-only antibodies (HCAbs) found in camelids. These nanobodies have unique features, including their small size, high stability, and accessibility to cryptic epitopes. Because of these properties, nanobodies have emerged as powerful tools for various applications in both plants and animals. This article provides an overview of nanobodies.
Heavy chain-only antibodies (HCAbs), lacking light chains, are naturally found in the serum of camelids such as llamas, camels, alpacas, guanacos, and dromedaries. The recombinant variable domains of the heavy chain of HCAbs, known as nanobodies (Nbs) or single-domain antibodies (sdAbs), have gained considerable attention due to their unique properties (Muyldermans 2013). Nanobodies, with a size of only 4 nm in length and 2.5 nm in diameter, are the smallest antigen-binding proteins known to date. They are approximately one-tenth the size of conventional mammalian antibodies (Chakravarty et al. 2014; Jovcevska and Muyldermans 2020). Despite their small size, nanobodies exhibit high specificity and affinity for their epitopes. Nanobodies can bind to their targets with dissociation constants reaching the nanomolar or picomolar range, comparable to conventional antibodies (Ingram et al. 2018; Liu et al. 2018).
Nanobodies offer several advantages over conventional monoclonal antibodies (mAbs). Firstly, nanobodies are highly soluble and stable even under harsh conditions. Their sequence homology to the variable domains of human immunoglobulins (Igs) ensures the retention of four conserved framework regions (FR1-4), maintaining the core structure, and three complementary determining regions (CDR1-3), responsible for antigen recognition (Muyldermans et al. 2009). Nanobodies have improved solubility and stability compared to VH domains of IgGs due to amino acid mutations that enhance hydrophilicity in hydrophobic areas responsible for VH/VL interaction within FR (Schumacher et al. 2018). Nanobodies can be concentrated up to 1-10 mg/mL in standard phosphate or Tris buffers (Muyldermans 2013). Unlike conventional mAbs that are susceptible to extreme environmental conditions due to their multimeric structure, monomeric nanobodies exhibit excellent tolerance to high temperatures (60-80 °C, even 90 °C) and a wide pH range (pH 3.0-9.0). The ability of nanobodies to efficiently refold after denaturation without compromising their antigen-binding capacity contributes to their exceptional stability (De Vos et al. 2013; Harmsen and De Haard 2007). In addition, many camel VHHs possess an extra interloop disulfide bond, which enhances stability and resistance to degradation by pepsin or chymotrypsin. These characteristics make nanobodies potential candidates for oral administration (Hussack et al. 2011; Ingram et al. 2018). Secondly, nanobodies are amenable to genetic engineering due to their monomeric structure. They can be easily fused with different proteins to confer specificity, enabling multifunctional molecular applications. Unlike most mAbs, which are predominantly produced in mammalian cells, nanobodies can be efficiently produced in bacteria and yeast because they lack the Fc fragment with N-linked oligosaccharide. Large-scale and cost-effective microbial production systems have been established for nanobodies (Liu and Huang 2018).
The generation of nanobodies commonly involves screening libraries constructed from immunized or non-immunized camelids using phage display technology. Immune libraries provide nanobodies with high affinity but limited diversity due to the affinity maturation process during animal immunization. In contrast, naïve libraries offer nanobodies with high diversity but limited specificity and affinity. Synthetic libraries, developed in recent years, are artificially constructed by randomizing the CDRs using selected nanobody scaffolds. Synthetic libraries are suitable for screening nanobodies against any antigen, including toxic or low-immunogenic antigens that are challenging to obtain through conventional animal immunization strategies. Unlike immune libraries, which require the construction of a new library for each target antigen, synthetic libraries allow screening for nanobodies against different antigens (Liu et al. 2018). Although nanobodies isolated from synthetic libraries may exhibit lower affinity due to the absence of in vivo antibody maturation, this drawback can be mitigated by increasing the size and diversity of the synthetic libraries and utilizing optimized screening methods (Zimmermann et al. 2020).
Related Services provided by Alpha Lifetech:
References
1 Chakravarty, R., Goel, S., Cai, W. 2014. Nanobody: The "Magic Bullet" for Molecular Imaging? Theranostics. 4(4): 386-398.
2 De Genst, E., Silence, K., Decanniere, K., Conrath, K., Loris, R., Kinne, J., Muyldermans, S., Wyns, L. 2006. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci U S A. 103(12): 4586-4591.
3 De Vos, J., Devoogdt, N., Lahoutte, T., Muyldermans, S. 2013. Camelid Single-Domain Antibodies: A Versatile Tool for in Vivo Imaging of Extracellular and Intracellular Antigen. Mol. Ther. 21(2): 1-18.
4 Desmyter, A., Spinelli, S., Boutton, C., Saunders, M., Blachetot, C., De Genst, E., Cambillau, C., Muyldermans, S. 1996. Functional Aberrations of Single Domain Antibodies. Science. 263(5154): 1362-1365.
5 Hussack, G., Hirama, T., Ding, W., MacKenzie, R., Tanha, J. 2011. Engineered single-domain antibodies with high protease resistance and thermal stability. PLoS One. 6(10): 1-7.
.jpg){kind=logo}
