General structural comparison between conventional antibodies, heavy chain-only antibodies, single-chain Fv, and VHH. DOI: 10.3390/cancers16020371
Introduction to VHH Antibodies
VHH antibodies, also known as single-domain antibodies (sdAbs), are unique antibody fragments derived from the variable region of heavy-chain-only antibodies (HCAbs) naturally produced by camelids, including llamas, alpacas, and camels. Unlike conventional antibodies composed of two heavy and two light chains, HCAbs lack light chains entirely and do not possess the CH1 domain necessary for heavy-light chain pairing. Instead, each heavy chain contains a single antigen-binding variable domain (VHH), which is fully capable of binding antigens independently.
First discovered in 1989 by Professor Raymond Hamers-Casterman at the Vrije Universiteit Brussel in Trypanosoma-infected dromedary camels, these heavy-chain-only antibodies were shown to bind antigens efficiently without light chains. This discovery led to the identification of the VHH domain as a functionally independent unit with high affinity and specificity.1
VHH Antibody Structure and Features
VHH antibodies are small (~15 kDa), monomeric proteins composed of three complementarity-determining regions (CDRs) and four framework regions (FRs), forming a compact immunoglobulin fold. Their unique structural properties allow them to bind epitopes that are inaccessible to conventional antibodies.
Structural representation of framework 2 residues in human VH and camelid VHHs. DOI: 10.1080/19420862.2024.2341443
Structural features include:
Size and Solubility: The VHH domain measures approximately 2.5 × 4.0 nm. VHHs are highly soluble due to their hydrophilic FR2, which substitutes the hydrophobic residues typically found in conventional VH domains.
Framework Region Differences: FR2 in VHHs includes hydrophilic residues like F37, E44, R45, and G47, instead of the hydrophobic V37, G44, L45, W47 seen in conventional VH domains. This enables monomeric expression without aggregation.
CDR Architecture: VHHs exhibit an elongated CDR3, often stabilized by a disulfide bond with CDR1. This extended loop increases the paratope surface and enhances binding to cryptic or recessed epitopes. Structural studies show that CDR3 in VHHs contributes the majority of contact residues for antigen binding.
Paratope Shape: VHH paratopes are typically convex, in contrast to the flat or concave surfaces of conventional antibody paratopes. This configuration enables access to catalytic clefts and other concave antigenic sites.
Canonical Substitutions: A common FR1 substitution, L11S, is frequently observed in dromedary-derived VHHs and contributes to enhanced solubility and expression.2
Targeting immune checkpoints: VHHs blocking PD-1/PD-L1 and CTLA-4 are being developed for cancer immunotherapy.
Fc-fusion for effector function: VHH-Fc conjugates restore antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
Diagnostic Tools
VHH antibodies are particularly advantageous for diagnostics due to their robustness and modifiability.
Lateral Flow Immunoassays (LFIA): VHHs are used in point-of-care LFIA devices to detect pathogens like Trypanosoma spp. and norovirus, meeting the ASSURED criteria for diagnostics.
ELISA formats: Sandwich and competitive ELISAs using VHH pairs have been developed for detecting cancer biomarkers, food toxins, and pathogens.
Environmental and pharmaceutical QC: VHH-based LFIAs can detect contaminants such as aflatoxins and counterfeit drugs.
Research Reagents
Biosensors: VHHs serve as stable bioreceptors in electrochemical and optical biosensors. Example: a VHH-based biosensor for fibrinogen detection with a detection limit of 0.044 µg/mL.
In vivo imaging: Fluorescent or radioactively labeled VHHs provide high contrast, real-time tumor imaging with rapid clearance. Conjugates with IRDye800CW or ⁶⁸Ga have demonstrated fast tumor penetration and short half-lives ideal for imaging.
VHHs are modular, allowing the creation of bispecific or trispecific molecules that:
Engage multiple antigens (e.g., PD-L1 × 4-1BB)
Target tumor and immune cell receptors simultaneously
Combine effector and targeting functionalities
DOI: 10.3390/antib8010013
Example: Combining anti-PD-1/PD-L1 immunotherapy with anti-VEGF treatment has proven effective in cancer therapy, and a bispecific antibody targeting both pathways could further enhance treatment by concentrating VEGF inhibition within tumor tissue. While no such dual-targeting monotherapy is currently approved, early candidates like IMM2510 and PM8002 have shown promising results in clinical trials. The newly developed bispecific VHH antibody BB-203 demonstrates strong in vitro target-blocking and potent in vivo anti-tumor efficacy, along with favorable development characteristics, positioning it as a leading candidate in this class of therapies.3
Plasma Half-Life Enhancement via HSA Binding
Due to their low molecular weight, VHHs are subject to rapid renal clearance. Strategies to enhance half-life include:
Fusing HSA-binding VHH domains
PEGylation
Fc fusion
Example: Sonelokimab is a trivalent VHH construct (anti-IL-17A/F × anti-HSA) that extends serum half-life and is in Phase 3 trials for active psoriatic arthritis (PsA), a complex and debilitating inflammatory disease. Two of its VHH domains bind with high affinity to interleukin-17A (IL-17A) and interleukin-17F (IL-17F), enabling the inhibition of all biologically active IL-17 dimers: IL-17A/A, IL-17A/F, and IL-17F/F. These cytokines are known to play central roles in driving inflammation across multiple tissues. The third VHH domain binds to human serum albumin, which helps prolong the molecule’s half-life and enhances accumulation at sites of inflammation due to albumin’s natural distribution into edematous and inflamed tissues.4
T-Cell Engagers and Immunomodulation
VHHs can serve as:
CD3-directed T-cell engagers
Immune checkpoint inhibitors
Carriers for cytokine payloads (e.g., IL-2, IL-15)
Their size and ease of multimerization support customized immunomodulatory formats.
Clinical Trials Involving VHHs
VHH-based therapeutics span various disease categories and clinical phases.
Additional candidates are in trials for lupus, osteoarthritis, RSV, rotavirus, and campylobacter infections.
Role of Machine Learning in VHH Design
Structural Modeling
AlphaFold and NanoBodyBuilder2 can predict VHH 3D structure, especially for variable CDR3 loop regions critical to antigen binding.
Sequence Design & Affinity Maturation
AntiBERTy and AbLang use deep learning to infer residue-level effects on affinity and stability.
These models facilitate CDR randomization strategies in synthetic library design.
Optimization Tools
AntBO and LaMBO2 apply Bayesian optimization to guide experimental design, enabling in silico screening of large VHH variant libraries before lab validation.
Overview of VHH antibody discovery methods. DOI: 10.1080/19420862.2024.2341443
VHH Humanization and Developability
Humanization is crucial to mitigate immunogenicity for therapeutic VHH antibodies. This involves replacing camelid-specific framework residues with human equivalents, particularly in FR2 and FR1 regions.
Key challenges:
Loss of refolding efficiency upon framework substitution
Potential reduction in binding affinity
Reduced thermal stability and solubility
Tools such as AbNatiV assist in evaluating and optimizing human-likeness without compromising function. Moreover, synthetic libraries can be constructed using pre-humanized scaffolds with randomized CDRs, eliminating the need for animal immunization and simplifying regulatory pathways.
Summary: Why VHHs Are Transforming Antibody Research
Derived from heavy-chain-only camelid antibodies
Retain high binding affinity with just a single variable domain
Structural features (e.g., convex paratope, extended CDR3) enable access to hidden epitopes
Thermostable, soluble, and resistant to pH and proteases
Easily expressed in microbial systems and scalable
Useful across diagnostics, therapeutics, and research
Odongo, S., Radwanska, M., & Magez, S. (2023). NANOBODIES®: A Review of Generation, Diagnostics and Therapeutics. International Journal of Molecular Sciences, 24(6), 5994. https://doi.org/10.3390/ijms24065994
Alexander, E., & Leong, K. W. (2024). Discovery of nanobodies: a comprehensive review of their applications and potential over the past five years. Journal of Nanobiotechnology, 22(1). https://doi.org/10.1186/s12951-024-02900-y
Zhong, H., Li, D. H., Guo, D., Nguyen, P., & Yang, M. (2024). 505 A novel anti-PD-L1/anti-VEGF bispecific VHH antibody exhibits a synergistic anti-tumor effect compared to the monospecific VHH antibodies. Regular and Young Investigator Award Abstracts, A569–A569.https://doi.org/10.1136/jitc-2024-sitc2024.0505
