Camelid single-domain antibodies (also known as nanobodies or VHHs) are derived from the Camelidae family of mammals such the llamas, camels, and alpacas. Unlike other antibodies, camelid antibodies lack a light chain and are composed of two identical heavy chains.

The unique structure of these heavy chain antibodies (HCAbs) drew the interest of various researchers for their application in various scientific and therapeutic fields – such as in vivo cellular imaging and antibody therapeutics in cancer therapy.

Discovery of Camelid Antibodies

Camelid antibodies were accidentally discovered in the late 1980s by a group of students who were working in the laboratory of Professor Raymond Hamers in Brussels, Belgium. The total and fractionated immunoglobulin-G (IgG) molecules isolated from the serum of a dromedary camel were analyzed with the intention of developing a serodiagnostic kit that could help in detecting trypanosome infection in water buffaloes and camels.

During the analysis, the students realized that the antibodies present in the camel did not have the usual structure and contained an HCAb antigen-binding domain that was subsequently named as the VHH domain. This VHH is found on a single stretch of amino acids and can recognize antigens that are as small as 12 to 15 kilodaltons (kDa).

The discovery of camelid antibodies led to the widespread use of these antibodies in biotechnological research, primarily due to the relative ease with which these antibodies could be handled. A number of preclinical and clinical studies are underway, such as the effect of camelid antibodies as imaging reagents or for therapy studies against platelet aggregation, respiratory syncytial virus (RSV) infection, venom toxins, rheumatoid arthritis, but also as radiolabelled nanobodies.

Properties of Camelid antibodies

Camelid antibodies are very specific and tend to display strict monomeric behavior, along with high thermostability and good solubility. Furthermore, their relatively small size allows for easy genetic engineering, thereby lowering overall production cost.

These VHHs have a good penetration rate into tissues and low immunogenicity – therefore, they are used to target antigenic epitopes that are difficult for large molecules (such as conventional monoclonal antibodies) to access. This ability of camelid antibodies to recognize cryptic epitopes renders them ideal as enzyme inhibitors, or for infection diagnosis.

The relatively short life of camelid antibodies is helpful in tumor imaging of diseased tissues that require rapid clearance. The shelf life can be increased to improve pharmacokinetic behavior by using options such as fusing of VHHs to an anti-serum albumin moiety or serum albumin.

Production of Camelid Antibodies

Completely functional antibodies are efficiently produced in mammalian cells only, and appropriate glycosylation of these antibodies is extremely important for exerting the required therapeutic activity. However, microbial production systems such as Escherichia coli (E. coli), filamentous fungi, or yeasts are used for large scale and economic production of antibodies.

VHH can generally be produced well in microorganisms but the production level depends on the VHH sequence patterns. Sagt and his colleagues have reported an increase in the production of VHH in yeast due to the presence of a potential N-linked glycosylation site.

Moreover, studies on baker’s yeast have demonstrated that the production of VHH in yeast can be increased up to five fold by addition of ethanol and supplementing the growth medium with ethylenediaminetetraacetic acid (EDTA), sorbitol, or casaminoacids.

In yet another research study, there was a reduction in the production of VHH due to the presence of unpaired C-terminal cysteines. DNA shuffling also enhances VHH production by random molecular evolution.

By using several expression formats, monovalent VHHs can be genetically fused into two or more VHHs to improve functional affinity. Zhang et al developed pentameric recombinant antibodies, also known as pentabodies, by binding VHH to the B-subunits of an E. coli toxin. This binding was observed to be so strong that it resulted in self-assembling of the VHH into a homopentamer.

Therapeutic Applications and Future Research

Until recently, therapeutic applications of VHH antibodies were limited, as they could not recruit essential antibody effector functions (such as antibody-dependent cellular cytotoxicity  and complement-dependent cytolysis) that are required for correct glycosylation of the second heavy-chain constant (CH2) domain.

Thus, the production of functional antibodies could be achieved only in higher eukaryotic cells. However, recent research has made it possible to develop functional antibodies in (P. pastoris). The aforementioned microorganism is a methylotrophic yeast that is known for its ability to produce recombinant proteins in gram amounts in one liter of culture.

In conclusion, camelid VHHs are suitable for a variety of therapeutic applications, e.g. in sleeping sickness, infant diarrhea caused by rotavirus, foot-and-mouth disease, sepsis, rheumatoid arthritis, brain disorders, neurodegenerative diseases, etc. VHHs are especially effective as oral immunotherapy in diarrhea due to their ability to withstand extreme pH values.

Recent research endeavors are now focusing on developing nanobodies that do not require effector functions and that have minimal side effects compared to conventional whole antibodies.

Further Reading

• All Camelid Content
• Camelid Antibodies for the Treatment of Allergies
• Camelid Antibodies – Advantages and Limitations
• Structure of Camelid Antibodies
• Nanobody Development

Written by

Deepthi Sathyajith

Deepthi spent much of her early career working as a post-doctoral researcher in the field of pharmacognosy. She began her career in pharmacovigilance, where she worked on many global projects with some of the world's leading pharmaceutical companies. Deepthi is now a consultant scientific writer for a large pharmaceutical company and occasionally works with News-Medical, applying her expertise to a wide range of life sciences subjects.