What is a nanobody?

Introduction to Nanobodies and Their Structure

In 1993, Professor Hamers-Casterman, an immunologist at the Université libre de Bruxelles in Belgium, and his colleagues discovered a novel antibody in the serum of camels (Camelidae; later studies confirmed this also included dromedaries and alpacas) that differed in structure from traditional antibodies. This antibody consisted solely of two heavy chains and was named the heavy-chain antibody (HCAb). The variable region of the heavy chain in HCAb is referred to as VHH (variable domain of the heavy chain of heavy-chain antibody). When produced via recombinant expression in vitro, the VHH molecule has a molecular weight of only 15 kDa—approximately one-tenth that of a conventional antibody and half that of an antigen-binding fragment (scFv or VH-VL)—hence earning the name nanobody (Nb).

Due to the absence of a light chain, nanobodies possess only three antigen-recognition regions (complementarity-determining regions, CDRs) belonging to the heavy chain. To compensate for the missing biological activity of the light chain CDRs, nanobodies have an extended amino acid sequence in their CDR3 region (16–18 amino acid residues, compared to 8–15 in conventional antibodies), enhancing the diversity and specificity of their CDRs. Additionally, the CDR3 region of a nanobody can form a large, exposed convex loop that extends like a "finger" into the crevices or clefts of an antigen, enabling it to reach epitopes inaccessible to traditional antibodies. A cysteine residue within the CDR3 loop can also form a disulfide bond with a cysteine at position 45 in either CDR1 or framework region 2 (FR2), conferring structural stability to the nanobody. This significantly reduces the energy required for antigen binding, making it easier to obtain nanobodies with high affinity.

Advantages of Nanobodies

1、Nanobodies Exhibit Stable Physicochemical Properties

Nanobodies possess remarkable stability in terms of biophysical and biochemical properties, including solubility, thermostability, and resistance to proteolysis. The folding of their CDR3 loop and the framework-2 (FR2) region contain numerous hydrophilic amino acids, enabling high solubility in aqueous solutions and reducing aggregation tendencies. Nanobodies also demonstrate excellent conformational and thermal stability, retaining full binding capacity even after one week at 37°C. Beyond thermostability, they remain stable under conditions involving proteases, chaotropic agents, and extreme pH levels. These stability features allow nanobodies to be administered via various routes, including intravenous injection, oral delivery, and inhalation formulations—routes that are often challenging for traditional antibody drugs.

When applied in clinical diagnostics, the high stability of nanobodies under extreme conditions (e.g., high organic solvent concentrations, extreme pH, or temperature variations) provides significant advantages for detecting small molecules or proteins.

2、Nanobodies Are More Feasible for Large-Scale Production

Traditional antibodies, due to their long amino acid sequences and complex protein structures, require sophisticated equipment for production and can only be manufactured in eukaryotic systems. Additionally, achieving therapeutic effects with clinical antibody drugs typically necessitates high doses, further increasing production demands. These factors result in the need for large quantities of mammalian cells and lengthy purification processes, making traditional antibody production extremely costly.In contrast, nanobodies can be efficiently expressed in microbial systems (e.g., bacteria, yeast, or fungi) and rapidly screened from display libraries. This gives nanobodies a significant advantage in terms of lower production costs.

3、Nanobodies exhibit low immunogenicity.

The structure of nanobodies is highly similar to the variable heavy chain (VH) region of human antibodies, consisting of three hypervariable regions (HVRs) flanked by four framework regions (FRs). Additionally, their genetic homology reaches 90%. This indicates that nanobodies have low immunogenicity. To date, there have been no reports of adverse reactions caused by the immunogenicity of nanobodies.

4、Nanobodies have a small molecular weight, strong tissue penetration ability, and rapid blood clearance.

The passive diffusion rate of drugs within tissues is inversely proportional to their molecular size. Compared to conventional antibodies (150 kDa), monovalent nanobodies (15 kDa) exhibit superior tissue penetration, enabling them to reach diseased tissues (such as solid tumors) that are inaccessible to traditional antibodies, thereby exerting therapeutic effects. Additionally, nanobodies can effectively cross the blood-brain barrier, offering a novel approach for brain-targeted drug delivery. Due to their small size, nanobodies are rapidly cleared by the kidneys, resulting in a short half-life, which minimizes toxicity and makes them advantageous for clinical imaging applications.

However, the short half-life also limits their use as therapeutic agents. This can be addressed by strategies such as PEGylation, albumin binding or fusion, or Fc fusion to extend their half-life.

5、Nanobodies can recognize epitopes inaccessible to conventional antibodies

Due to their small size and unique CDR3 structure, nanobodies can interact with novel epitopes that traditional antibodies cannot reach, enabling them to bind and neutralize targets that are difficult for conventional antibodies to hit.

Because of their low molecular weight and weak inter-monomer adhesion, nanobodies can be easily linked and expressed as multimers through genetic engineering. If designed as clinical therapeutics, this feature allows the development of multivalent or bispecific antibodies, enhancing their antigen-binding capability. Additionally, this property can be utilized to extend the half-life of nanobodies by forming multimers or conjugating them with an anti-serum albumin nanobody.

6、Nanobodies have the ability to form multimers

Due to their small molecular weight and weak inter-monomer adhesion, nanobodies can be easily linked and expressed as multimers through genetic engineering. When designed as clinical therapeutics, this feature enables the development of multivalent or bispecific antibodies, enhancing their antigen-binding capability. Additionally, leveraging this property, the half-life of nanobodies can be extended by forming multimers or conjugating them with an anti-serum albumin nanobody.

Screening and Production of Nanobodies

1、Construction of Nanobody Gene Libraries

Antigen-specific nanobodies can be generated from immune libraries, naïve libraries, or synthetic libraries, among which immune library construction is a commonly used technique for nanobody development.

Purified target antigens are used to immunize alpacas. After successful immunization, lymphocytes containing mature B cells are extracted from the alpacas' blood. RNA is isolated from the lymphocytes and reverse-transcribed into a cDNA library. Using specially designed primers, PCR amplification is performed on the cDNA library to obtain specific gene fragments. These gene fragments are then ligated into phage vectors and transfected into E. coli, ultimately completing the construction of the nanobody gene library.

2. Screening of Specific Antibodies

The target antigen or antigen-coated material is directly or indirectly immobilized on a solid carrier. The phage display library is then added to allow interaction with the antigen. After incubation, positive phages bind to the antigen, while unbound or non-specifically bound phages are removed by washing the solid carrier surface. Subsequently, a strong acid or alkali is used to disrupt the binding between the antigen and the positive phages, releasing the phages into solution. Typically, 2-3 rounds of screening are sufficient to obtain nanobody clones that meet development requirements.

3、Production and Preparation of Nanobodies

After obtaining antibody clones that meet the required standards, large-scale production and preparation of the antibodies are necessary. The production of nanobodies involves the transcription of DNA into mRNA, followed by translation into proteins. Due to the complex structure of antibodies, the expression system requires sophisticated cellular machinery to ensure proper expression, folding, and processing. Currently, the industry has a range of well-established expression systems for antibody production, each with its own advantages and disadvantages. Optimal functional expression and purification of antibody designs are critical factors determining their successful application. Therefore, selecting the appropriate expression system is a key step in maximizing benefits.

Mammalian cell lines are the preferred expression system for therapeutic antibodies due to their low immunogenicity and their ability to provide optimal folding, secretion, and post-translational modifications. However, they also have drawbacks such as high production costs and technical challenges. The E. coli expression system is the most popular due to its convenience in production and operation, fast production cycle, and well-established platform. Nevertheless, E. coli cannot express recombinant antibodies with complex structures or rich disulfide bonds due to limitations in oxidative environment and folding capacity. Yeast is another widely used microbial expression host, combining the advantages of prokaryotic systems (short cycle, low cost, simple operation) and eukaryotic systems (proper folding, modification, and secretion), making it the most favored host for expressing full-length IgG or other antibody formats.Other systems can also be used to express recombinant antibodies, such as filamentous fungi, algae, protozoa, insect cells, transgenic plants, and animals. Among these, research on transgenic plants and animals is still in its early stages and requires further in-depth investigation.

For nanobodies, due to their simple structure and good water solubility, cost-effective bacterial and yeast expression systems can be utilized for large-scale production. For example, the Belgian company Ablynx has established a well-developed Pichia pastoris expression and purification process. There are few reports on nanobody production in mammalian cell lines, as the high production costs limit the development of this system. Additionally, numerous studies have reported nanobody production in plants and animals. Similar to traditional antibody production systems, these two approaches offer advantages such as low cost, minimal contamination, and high safety, demonstrating potential application value.

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