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Difference Between VHH and Conventional Antibodies
Biointron2025-05-21Read time: 10 mins
DOI: 10.1111/imr.13385
Overview of Antibodies
Antibodies function as key components of the adaptive immune response. They neutralize pathogens, facilitate phagocytosis via opsonization, and activate immune cells. Conventional antibodies, specifically immunoglobulin G (IgG), are heterotetrameric proteins composed of two heavy chains and two light chains. Each chain contains a variable domain responsible for antigen recognition and a constant domain that defines the antibody's effector functions. The antigen-binding site is formed by the interaction of three complementarity-determining regions (CDRs) from each variable domain (VH and VL). These six loops (3 in VH and 3 in VL) work synergistically to determine specificity and affinity.
In clinical settings, monoclonal antibodies (mAbs) such as rituximab and adalimumab are widely used for targeted therapies, ranging from autoimmune disorders to various cancers. Their mechanism often relies on Fc-mediated immune responses, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Meanwhile, diagnostic antibodies are employed in immunoassays such as ELISA and Western blotting.
What Are VHH Antibodies?
VHH antibodies, also referred to as nanobodies or single-domain antibodies (sdAbs), are derived from heavy-chain-only antibodies (HCAbs) naturally present in Camelidae species (e.g., camels, llamas, and alpacas). Unlike conventional IgG, HCAbs lack light chains and the CH1 domain of the heavy chain. The variable domain of these HCAbs, termed VHH, functions independently as a stable, antigen-binding unit.
VHHs have several advantageous properties:
Small molecular weight (~15 kDa)
High solubility and stability under extreme pH and temperature
Ability to bind concave and enzymatic cleft epitopes
Efficient tissue penetration due to their small size
VHH domains consist of three CDRs (CDR1-3) flanked by four framework regions (FR1-4), forming a β-barrel structure of nine anti-parallel β-strands. Notably, CDR3 in VHHs is longer than in conventional VH domains, which enables unique paratope conformations such as upright, half-roll, and roll structures. These long loops allow VHHs to access otherwise inaccessible epitopes, such as enzyme active sites or receptor clefts. Structural enhancements like additional disulfide bonds between CDRs also contribute to their conformational stability. Camelid VHHs contain hallmark framework residues that facilitate high solubility, making them ideal candidates for research, diagnostics, and therapeutics.
Structural features of nanobody MU551, a prototype llama VHH. DOI: 10.1111/imr.13385
Evolutionary Origin of VHH Antibodies
Heavy-chain-only antibodies (HCAbs) are a relatively recent evolutionary development, unique to the camelid lineage (camels, llamas, alpacas), emerging roughly 45 million years ago. A key mutation in a common ancestor inactivated the splicing site of the CH1 exon in one IgG gene, preventing light chain pairing and leading to the formation of soluble, functional heavy-chain-only antibodies. Over time, further adaptive mutations accumulated, such as hydrophilic substitutions in framework 2 (FR2) and cysteine insertions near CDR loops, thus enhancing stability, solubility, and antigen affinity. This evolutionary pressure likely selected for improved resistance to pathogens in harsh environments.
DOI: 10.1038/s41586-021-03676-z
VHH vs Conventional Antibodies
The differences between VHHs and conventional antibodies stem from their structural simplification. The lack of light chains and a single-domain configuration allow VHHs to be produced in microbial systems, which are more cost-effective and scalable than mammalian expression systems required for full IgG. Furthermore, the superior tissue penetration of VHHs makes them suitable for applications in solid tumors and central nervous system (CNS) targets. However, the absence of an Fc domain in VHHs means they lack immune effector functions such as ADCC or CDC, limiting their standalone efficacy in some therapeutic contexts. This trade-off necessitates engineering approaches, such as fusion with Fc domains or albumin-binding modules, to enhance their pharmacological profile.
Structural Comparison: VHH vs IgG Antibodies
Conventional IgG antibodies are Y-shaped molecules composed of two heavy and two light chains, each with variable and constant domains. The antigen-binding site is formed by the interaction of three CDRs from both the VH and VL domains. These domains fold into a conserved immunoglobulin β-barrel structure composed of nine β-strands arranged into two antiparallel β-sheets.
In contrast, VHHs are single-domain molecules that structurally resemble the VH domain of an antibody but have evolved to function independently in the absence of a light chain partner. These domains also adopt the classical immunoglobulin fold, yet exhibit unique sequence and structural features that confer improved stability and solubility.
Key differences:
Framework 2 Adaptations: VHH domains possess hallmark framework 2 residues at positions 37 (F/Y), 44 (E), 45 (R), and 47 (G), which form a hydrophilic surface patch. This adaptation enhances solubility in aqueous environments and compensates for the absence of the hydrophobic VH–VL interface found in conventional antibodies.
CDR3 Architecture: VHH CDR3 loops are generally longer and more conformationally flexible than those in conventional VH domains. This increased length enables VHHs to bind concave or enzymatic cleft epitopes that are typically inaccessible to conventional antibodies, which prefer flatter, surface-accessible targets.
Disulfide Bond Variability: Additional disulfide bonds often form between CDR1 and CDR3 or between CDR2 and CDR3 in camelid-derived VHHs. These covalent linkages further stabilize the long CDR3 loop conformations and enhance thermal and conformational resilience.
CDR3 Conformations and Framework Involvement: Structural studies have shown that the long CDR3 loops in VHHs can adopt upright, half-roll, or roll conformations. In upright loop structures, residues in framework 2 actively participate in forming the antigen-binding paratope, a feature rarely observed in conventional VH domains.
Folding and Expression Efficiency: These hallmark residues in VHHs provide intrinsic hydrophilicity, compensating for the loss of stabilizing interactions with a variable light chain. In conventional VH domains, these same positions are involved in hydrophobic interactions with VL domains, often leading to aggregation or poor solubility when expressed alone. In VHHs, the long CDR3 can also fold back over the hydrophobic patch in framework 2, further stabilizing the domain.
Sequence Homology with Human VH Domains: Camelid VHHs exhibit over 80% sequence identity with human VH3–23, reducing the risk of immunogenicity and making them favorable candidates for therapeutic humanization.
These structural adaptations not only enable VHHs to function independently as antigen-binding domains but also support higher expression yields in heterologous systems like E. coli. In contrast, conventional VH domains typically require complex folding aids or refolding steps after expression. VHHs, therefore, offer a more tractable scaffold for biotechnological and therapeutic applications.
DOI: 10.1186/s40364-021-00332-6
Functional and Biophysical Differences
Stability: VHHs exhibit thermal melting points often exceeding 60°C and can refold after denaturation, maintaining binding activity.
Solubility: Hydrophilic residues and long CDR3s help reduce aggregation and increase expression yield.
Tissue Penetration: Due to their small size, VHHs diffuse more effectively into dense tissues and tumors.
Pharmacokinetics: VHHs are rapidly cleared by the kidneys; half-life extension strategies include fusion to human serum albumin (HSA) or the Fc domain.
Despite high thermal resilience, efforts have been made to further enhance VHH stability through targeted mutagenesis of framework residues and optimization of CDR loop sequences. Some VHHs demonstrate remarkable conformational refolding even after exposure to extreme heat or acidic pH, making them viable for oral and inhalation-based drug delivery. The rapid renal clearance of monovalent VHHs, while a limitation in sustained therapeutic exposure, is advantageous in imaging applications where fast background clearance improves signal-to-noise ratios. Strategies like PEGylation, Fc-fusion, or inclusion of albumin-binding domains have successfully extended the half-life of therapeutic VHHs to match or exceed that of some conventional antibodies.
VHHs can be expressed efficiently in E. coli and yeast. However, their lack of glycosylation may affect function in some cases, necessitating mammalian expression for specific applications. Unlike full IgG, VHHs' small size and solubility reduce formulation constraints and support alternative delivery routes such as intranasal, oral, or inhaled administration—advantageous for infectious disease and CNS-targeting therapies.
Engineering and Application Differences
VHHs are excellent scaffolds for engineering:
Multivalency and Bispecificity: VHHs are easily linked to create multivalent or bispecific constructs.
Production: VHHs can be efficiently produced in microbial hosts (E. coli, yeast), while IgGs typically require mammalian systems.
IgG: Long-term systemic therapy with Fc effector functions
The ease of VHH expression and engineering enables the rapid generation of synthetic libraries using phage display or yeast display platforms. These libraries can be tailored using sequence databases like the Observed Antibody Space (OAS) or INDI to design VHHs with optimal developability profiles. Furthermore, machine learning tools now enable prediction of aggregation-prone motifs and immunogenic regions, streamlining candidate selection. VHHs are particularly suited for fusion with functional moieties such as radionuclides, enzymes, or cytokines, making them powerful tools for both diagnostic imaging and therapeutic payload delivery. In contrast, full-length antibodies require more complex engineering to incorporate such functionalities without compromising stability.
Recent advances in nanobody discovery include the use of synthetic VHH libraries and transgenic animal models like the LamaMouse and NanoMouse. These platforms allow high-throughput generation of nanobody-based heavy chain antibodies against diverse human antigens. Techniques like cDNA immunization enable the generation of nanobodies that recognize native conformations of membrane proteins expressed in vivo, broadening the utility of VHHs in both therapeutic and diagnostic development.
Numerous preclinical studies have demonstrated VHH efficacy in modalities including radioimmunotherapy, photodynamic therapy, and CAR-T therapy. The use of VHHs in CAR constructs, such as in FDA-approved Carvykti for multiple myeloma, highlights their utility in cellular immunotherapy. Additionally, the ability of VHHs to access cryptic or recessed epitopes expands the landscape of druggable targets, particularly in oncology and infectious diseases. Envafolimab, a PD-L1-targeting VHH-Fc fusion, is under clinical investigation for its ease of subcutaneous delivery and promising immune checkpoint blockade activity, further validating the clinical potential of engineered VHHs.
Rituximab mechanism of action. DOI: 10.3390/vaccines11121756
Limitations and Considerations
VHH limitations:
Short serum half-life (minutes to hours)
Lack Fc-mediated immune functions
Require humanization to reduce immunogenicity
Conventional mAbs limitations:
Poor tissue penetration
High production cost due to mammalian cell requirements
Both formats may trigger immune responses, though humanized VHHs show promising profiles. Pre-existing anti-drug reactivity (PEAR) against VHHs' C-terminal tags is a concern and may be mitigated by specific amino acid substitutions (e.g., terminal prolines or alanine).
While human VHH analogs share >80% sequence identity with human VH3 domains, potential immunogenicity remains a concern. Studies have shown that framework residues and terminal tags may elicit anti-drug antibodies (ADAs) in certain patients. The phenomenon of PEAR complicates immunogenicity assessments, masking true responses to therapeutic agents. Engineering strategies, such as adding proline or alanine residues at the C-terminus, have been shown to mitigate PEAR and improve the safety profile. Nonetheless, detailed assessments using humanized mouse models and long-term clinical trials are essential for accurate immunogenicity profiling. In contrast, conventional antibodies may also require glycoengineering and Fc optimization to minimize off-target effects and enhance efficacy.
Cong, Y., Devoogdt, N., Philippe Lambin, Dubois, L. J., & Ala Yaromina. (2024). Promising Diagnostic and Therapeutic Approaches Based on VHHs for Cancer Management. Cancers, 16(2), 371–371. https://doi.org/10.3390/cancers16020371
Koch-Nolte, F. (2024). Nanobody-based heavy chain antibodies and chimeric antibodies. Immunological Reviews, 328(1), 466-472. https://doi.org/10.1111/imr.13385
