Q&A on Nanobody Expression and Purification Techniques

1. What is the mechanism of action of nanobodies?

Answer: Traditional antibody drugs primarily function through two mechanisms: target neutralization and Fc-mediated effects (including ADCC, CDC, and ADCP). The former is achieved through antigen binding by the antibody's variable region, while the latter relies on the Fc fragment's interaction with complement or receptors. Since nanobodies lack an Fc fragment, their mechanism of action is limited to target neutralization.

2. What are the structural features of nanobodies?

Answer: The VH domain of nanobodies contains four hydrophilic amino acid residues in FR2 (F42 or Y42, E49, R50, and G52). Compared to conventional antibodies, nanobodies have a longer CDR3 region, which can form a convex structure. This enhances their ability to recognize hidden tumor antigen epitopes.

3. Why do nanobodies exhibit higher water solubility than conventional antibodies?

Answer: In conventional antibodies, the FR2 region of the VH domain contains four highly conserved hydrophobic amino acid residues (V42, G49, L50, and W52). In contrast, VHH nanobodies replace these residues with hydrophilic ones (F42/Y42, E49, R50, and G52), thereby significantly enhancing their water solubility.

4. What are the advantages of nanobodies compared to conventional antibodies?

Answer: Compared to conventional antibodies, nanobodies exhibit superior physicochemical properties:① Small size and molecular weight, enabling excellent tissue penetration;② Enhanced stability, allowing adaptation to various administration routes;③ Low immunogenicity, with human-like VH domain structure showing up to 90% homology;④ Unique CDR3 structure that facilitates superior epitope recognition;⑤ Efficient large-scale production with lower manufacturing costs.

5. Why pursue the development of nanobodies?

Answer: Compared to monoclonal antibodies, nanobodies offer distinct advantages including smaller molecular weight, superior solubility, enhanced stability, and stronger tissue penetration capabilities.

6. Can nanobodies recognize epitopes that conventional antibodies cannot?

Answer: Yes. The smaller size and unique CDR3 structure of nanobodies enable interactions with novel epitopes inaccessible to conventional antibodies. This allows nanobodies to bind and neutralize targets that are difficult for traditional antibodies to engage.

7.Can nanobodies treat bacterial infections?

Answer: Due to their high specificity, nanobodies can combat bacterial infections by precisely binding to surface antigens of pathogenic bacteria and blocking bacterial adhesion to host cells, as well as interfering with bacterial motility and virulence factors. This makes them a promising new approach for developing antibacterial therapies against bacterial infectious diseases.

8.What are the functions of nanobodies?

Answer: Due to their excellent properties, nanobodies have been widely applied in research fields such as molecular imaging and diagnostics, drug delivery, tumor immunotherapy, and cell therapy. Particularly in the field of tumor treatment, various therapeutic agents have been developed, including bispecific nanobodies, nanobody-drug conjugates (NDCs), CAR-T therapy, CAR-NK therapy, and targeted radionuclide therapy.