Discovery of VHH Antibodies in Camelids

Antibodies are proteins produced by the immune system to recognize and bind specific foreign or abnormal molecules, called antigens. In humans and many other mammals, an IgG antibody is made of two heavy chains and two light chains. Together, these chains form antigen-binding regions that allow the antibody to recognize a specific target.

Camelids, a family of animals that includes camels, dromedaries, llamas, alpacas, and vicuñas, are unusual because they naturally produce not only conventional antibodies, but also a special class of antibodies called heavy-chain-only antibodies. These antibodies lack light chains and also lack the CH1 domain, a region normally involved in heavy-chain and light-chain pairing.

The antigen-binding region of a camelid heavy-chain-only antibody is a single variable domain called a VHH. VHH antibodies, also known as single-domain antibodies or, commercially, nanobodies. For lay readers, a VHH can be understood as the smallest naturally occurring antibody-derived unit that can still bind an antigen on its own. Unlike conventional antibody binding sites, which usually require both a heavy-chain and light-chain variable domain, a VHH functions as an independent antigen-binding domain.

This discovery showed that high-affinity and highly specific antigen recognition does not always require the paired heavy-chain and light-chain structure of conventional antibodies. Instead, a single compact domain from camelid antibodies can act as a stable and functional binding unit.

Introduction: What Are Camelid Antibodies?

The term camelid antibodies refers to antibodies produced by members of the camelid family, including llamas, alpacas, camels, and dromedaries. Camelids have a conventional antibody system similar to that of humans, but they also produce naturally occurring heavy-chain-only antibodies.

These heavy-chain-only antibodies are important because their antigen-binding site is formed by a single VHH domain. This gives VHHs several features that have made them attractive in antibody discovery and engineering:

• Small size: VHHs are much smaller than full-length IgG antibodies.

• Single-domain structure: They do not require light-chain pairing.

• Good solubility and stability: Many VHHs show favorable biophysical properties.

• Modular engineering potential: VHHs can be reformatted into multivalent, bispecific, or multispecific molecules.

A bispecific antibody is an engineered antibody that can bind two different targets. A multispecific antibody can bind more than two targets or epitopes. Because VHHs are small and do not require a paired light chain, they are especially useful as modular building blocks for these more complex antibody formats.

How Camelid Antibodies Differ from Human Antibodies

Human IgG antibodies are typically composed of two identical heavy chains and two identical light chains. Their antigen-binding site is formed by the pairing of a heavy-chain variable domain, called VH, and a light-chain variable domain, called VL.

Camelid heavy-chain-only antibodies have a different architecture. They are composed only of heavy chains and lack the light chains found in conventional antibodies. They also lack the CH1 domain. Instead of using a paired VH-VL binding site, they use a single VHH domain as the antigen-binding unit.

This difference is central to why VHHs became so important. In conventional antibodies, the VH domain normally depends on the VL domain for proper antigen recognition and structural support. In contrast, the camelid VHH domain is adapted to function without a light-chain partner.

The Breakthrough Discovery in the 1990s

The modern history of camelid VHH antibodies began with the discovery of naturally occurring heavy-chain-only antibodies in camelids. In the landmark 1993 report by Hamers-Casterman and colleagues, researchers described antibodies in camelids that were unexpectedly devoid of light chains.

This observation challenged the established understanding of antibody structure. At the time, antibodies were generally understood as molecules requiring both heavy and light chains to form functional antigen-binding sites. Camelid heavy-chain-only antibodies showed that this was not always the case.

The key structural implication was that the antigen-binding region of these antibodies was contained in a single heavy-chain variable domain: the VHH. This domain could recognize antigen independently, without a light-chain variable domain.

This finding generated scientific curiosity because it raised several important questions:

• How could a single variable domain remain soluble without its usual light-chain partner?

• How could it generate enough binding diversity to recognize many antigens?

• Could this compact binding unit be isolated and engineered for research or therapeutic use?

Subsequent studies helped answer these questions and established VHHs as a new class of antibody-derived binding molecules.

Early Research and Structural Insights into VHH Domains

To understand why VHHs work as independent antigen-binding domains, it is useful to define a few antibody structure terms.

Complementarity-determining regions, or CDRs, are the loops of an antibody that contribute directly to antigen binding. In a VHH, there are three CDRs: CDR1, CDR2, and CDR3. Among these, CDR3 is often especially important because it can make extensive contact with the antigen.

Framework regions are the more conserved structural regions that support the CDR loops. They help maintain the antibody domain’s overall fold.

VHHs contain structural features that help them function without a light chain. One important feature is the presence of characteristic amino acid residues in framework region 2, often called VHH hallmark residues. In conventional VH domains, some of these positions are normally buried at the interface with the light-chain variable domain. In VHHs, these positions are exposed to solvent and are adapted to maintain solubility and stability without VL pairing.

Another important feature is the structure of the CDR3 loop. VHH CDR3 regions are often longer and more variable than those of conventional human VH domains. This can allow VHHs to reach recessed or difficult-to-access epitopes.

An epitope is the specific part of an antigen recognized by an antibody. Some epitopes are flat and exposed, while others are hidden in grooves, clefts, or enzyme active sites. Because of their compact size and extended CDR3 loops, VHHs can sometimes access epitopes that may be less accessible to conventional antibodies.

Some VHHs also contain non-canonical disulfide bonds. A disulfide bond is a stabilizing chemical link between two cysteine amino acids. In VHHs, additional disulfide bonds can help stabilize long CDR3 loops and support antigen binding.

Together, these features explain how VHHs combine small size with functional antigen recognition.

From Discovery to Biotech Applications of Camelid Antibodies

The discovery of camelid heavy-chain-only antibodies led to the rise of VHHs as research tools, diagnostic reagents, and therapeutic building blocks.

Because VHHs are small, stable, and genetically simple, they can be expressed and engineered in formats that are difficult or less efficient with conventional antibodies. They can be linked together in tandem, fused to Fc domains, attached to albumin-binding domains for half-life extension, or incorporated into multispecific antibody formats.

A key advantage of VHHs is that they do not require light-chain pairing. In conventional bispecific antibody development, incorrect heavy-chain and light-chain pairing can create manufacturing and purification challenges. VHHs avoid this specific issue because the binding domain is contained in a single chain.

VHHs have been explored in multiple biotechnology and therapeutic areas, including:

• Therapeutics: VHHs can block disease-associated proteins, redirect immune cells, or serve as components of engineered biologics.

• Diagnostics: VHHs can be used to detect biomarkers, pathogens, or disease-associated proteins.

• Bioimaging: Their small size can support tissue penetration and rapid target engagement.

• Targeted delivery: VHHs can guide payloads to specific cells or tissues.

• Cell therapy: VHHs can serve as antigen-recognition domains in engineered immune cell receptors.

One of the best-known VHH-based therapeutic examples is caplacizumab, a bivalent VHH-based molecule targeting von Willebrand factor. Other VHH-containing therapeutic formats include VHH-Fc fusions, trivalent VHH constructs, and chimeric antigen receptor T cell therapies that use VHHs as targeting domains.

Continued Research in Camelids Like Alpacas

Camelids remain important models for VHH generation. Llamas, alpacas, camels, and dromedaries can be immunized with a target antigen to produce antigen-specific heavy-chain-only antibody responses.

Immunization means exposing the animal’s immune system to an antigen so that B cells generate antibodies against it. B cells are immune cells that produce antibodies. During an immune response, B cells can undergo affinity maturation, a natural process that improves antibody binding strength over time.

A typical VHH discovery workflow from immunized camelids involves several steps:

1. The camelid is immunized with the target antigen.

2. Blood is collected after the immune response develops.

3. Peripheral blood mononuclear cells, or PBMCs, are isolated from the blood.

4. RNA is extracted from these immune cells.

5. Complementary DNA, or cDNA, is synthesized.

6. VHH sequences are amplified by PCR.

7. A VHH library is constructed and screened for antigen binders.

PBMCs are immune cells found in blood, including B cells and T cells. They are useful in antibody discovery because B cells contain the genetic information encoding antibody sequences.

PCR, or polymerase chain reaction, is a laboratory method used to amplify specific DNA sequences. In VHH discovery, PCR can be used to amplify the genes encoding VHH domains.

After VHH sequences are amplified, they are often inserted into a display library. One widely used method is phage display. Phage display is a screening method in which antibody fragments are displayed on the surface of bacteriophages, which are viruses that infect bacteria. Researchers expose the phage library to the target antigen and enrich phages displaying VHHs that bind the target. This process is often called panning.

Camelid Antibodies in Modern Antibody Engineering

The value of camelid VHH antibodies extends beyond their discovery format. Their single-domain architecture makes them especially useful in advanced antibody engineering.

VHHs can be used to build:

• Bivalent molecules, where two binding domains recognize the same target.

• Biparatopic molecules, where two binding domains recognize different epitopes on the same target.

• Bispecific molecules, where binding domains recognize two different targets.

• Multispecific molecules, where several binding domains are combined.

• Fc-fusion proteins, where VHHs are linked to an antibody Fc region to improve half-life.

• Albumin-binding formats, where one VHH binds serum albumin to extend circulation time.

• CAR-T recognition domains, where VHHs help engineered T cells recognize cancer-associated antigens.

Summary: How Camelid Antibodies Changed Antibody Science

The discovery of camelid heavy-chain-only antibodies opened a new direction in antibody discovery. VHHs are stable, soluble, and modular. Their structure allows them to access certain epitopes, tolerate diverse engineering formats, and support the development of diagnostics, research tools, and therapeutic candidates.

Camelid antibodies continue to influence modern antibody science through immunized VHH libraries, synthetic and semi-synthetic libraries, multispecific antibody engineering, cell therapy design, and emerging computational discovery approaches.

For researchers developing next-generation antibody candidates, VHHs provide a flexible route from antigen recognition to engineered biologic design. Biointron supports VHH discovery and camelid antibody engineering workflows to help accelerate the identification, screening, and production of high-quality VHH candidates for research and therapeutic development.

References:

1. Odongo, S., Radwanska, M., & Magez, S. (2023). Nanobodies: A Review of Generation, Diagnostics and Therapeutics. International Journal of Molecular Sciences, 24(6). https://www.mdpi.com/1422-0067/24/6/5994

2. Evers, A., Guarnera, E., Pekar, L., & Zielonka, S. (2025). From discovery to the clinic: structural insights, engineering options, clinical, and 'next wave' applications of camelid-derived single-domain antibodies. mAbs, 17(1), 2583210. https://doi.org/10.1080/19420862.2025.2583210