Camelid Antibodies and Nanobodies

Advantages of Camelid Antibodies and Nanobodies

The V_HH domains of camelid heavy chain-only antibodies can be effectively engineered, and are designated as single-domain antibodies (sdAbs), or nanobodies, in reference to their small nanometre size range (Figure 2). In addition to many sought-after properties, nanobodies are capable of targeting epitopes of antigens that are inaccessible for conventional antibodies due to their characteristically lengthier CDR3 loops (Table 1).

Table 1. Important Properties of Camelid Antibodies. (*Beneficial for certain applications that favour rapid clearance, but not for treatment of viral diseases. In this case, camelid antibodies would require to be readministered multiple times compared to canonical antibodies⁴.)

Scientists working on antibody therapeutics and diagnostics are also drawn to use nanobodies because of the relative ease at which they can be produced and engineered. Unlike many other antibody fragments such as Fab or single-chain Fv (scFv), recombinant production of nanobodies does not require the association of V_H and V_L domains, thus avoiding mispairing issues. In addition, the single-domain nature of nanobodies greatly facilitates their genetic engineering into formats suited for a variety of purposes.

Camelid Antibodies and Nanobodies for Therapeutic and Research Applications

Thanks to their advantageous biophysical features, nanobodies have opened new research perspectives in the development of innovative human therapeutics. In February 2019, the FDA approved the first V_HH-based nanobody drug, caplacizumab, for the treatment of acquired thrombotic thrombocytopenic purpura (TTP). Caplacizumab was constructed by linking two humanized V_HH domains that were initially identified from llamas. Nowadays, hundreds of nanobody therapeutics are under development against various diseases including hematology, infectious diseases, inflammation, and COVID-19, with many being studied in clinical trials. 

Benefits of using nanobodies as therapeutics include the following:

• Small but powerful: Target epitopes with high affinity at locations that are inaccessible for traditional antibodies
• Better stability and solubility: Resistant to high acidity and high temperature; less prone to aggregation
• Higher tissue penetration: enter tissues and tumors more rapidly and deeply than  traditional antibody drugs allowing for increased efficacy
• Ease of engineering & manufacturing: Flexible to be tailored into different formats facilitating lead optimization; relatively low production cost 

For many of the above reasons, nanobodies are also pursued for diagnostic purposes, and for manufacturing reagents to aid in research and development.

How are Camelid Antibodies and Nanobodies Developed?

Prior to producing nanobodies, camelids (typically, llamas) are first immunized with a target antigen. Then, nanobodies are commonly discovered through a nucleotide sequencing approach called next generation sequencing (NGS). NGS is performed on the nucleic material extracted from the llamas’ circulating peripheral blood lymphocytes. Then the heavy chain genes are cloned into a phagemid vector to generate a phage display library, which will be used to perform affinity maturation in vitro. To bypass cell collection and potential contamination, another method can be used to access the circulating nanobodies directly. This method is called next generation protein sequencing (NGPS).

Discovering Camelid Antibodies and Nanobodies through NGPS

NGPS is a robust and complementary technology that can be used to power camelid biologics pipeline from discovery through engineering, development, validation, and manufacturing (Figure 3).

Figure 3. Rapid Novor’s next generation protein sequencing platform for the discovery and validation of camelid antibodies and nanobodies

REpAb^® is Rapid Novor’s antibody discovery platform that is rooted in mass spectrometry and supported by big data-powered machine learning. REpAb^® can be easily integrated into already existing antibody discovery pipelines utilizing nucleic data, or be directly employed on affinity-purified bleeds from inoculated animals. REpAb^® captures the circulating antibody pool already primed against the target and does not require the culling of the production animal. Full sequences of functional antibodies have been directly derived from rabbit, goat, and now alpaca using our REpAb^® technology. For more information about our REpAb^® antibody discovery platform, please refer to our article: What is Polyclonal Antibody Sequencing?

Enabling Camelid Antibody and Nanobody Engineering and Validation with NGPS

When developing therapeutic antibody fragments and nanobodies, the selection and screening process that usually relies on hybridoma and phage display can be tedious and time-consuming due to the large number of choices for fragment combinations. These processes also do not include relevant information on post-translational modifications (PTMs). By obtaining information on PTM type and location, NGPS can predict potential enhancement or detrimental effects of physicochemical properties, and function of antibodies. As such, NGPS is a powerful tool that can complement and optimize pipelines relying on hybridoma and/or phage display.

NGPS at Rapid Novor includes services such as REpAb^® and REmAb^®, Rapid Novor’s established de novo protein sequencing platform; these can be applied to rational design and generation of proof-of-concept nanobody candidates based on more comprehensive structural information afforded by amino acid sequences. Furthermore, our MATCHmAb™ peptide mapping service offers a quick alternative to traditional peptide mapping of verifying the protein sequence of isolated antibodies using our proven protein sequencing technology to help researchers avoid cross-reactivity and reproducibility problems down the road.