Single-domain antibodies (sdAbs), often referred to as VHHs or nanobodies, are derived from the heavy-chain-only antibodies found in camelids such as llamas and alpacas. They are the smallest naturally occurring antibody fragments, about 15 kDa in size, and consist of a single variable domain.
Key structural and functional features include:
Monomeric design that folds independently.
High solubility and stability, even under extreme pH or temperature.
Extended CDR3 loop, which enables access to cryptic or recessed epitopes.
Single-chain variable fragments (scFvs) are engineered molecules created by fusing the variable heavy (VH) and variable light (VL) domains of an antibody with a short peptide linker. They are larger than VHHs, typically 25–30 kDa, and mimic the antigen-binding site of a full IgG.
Characteristics include:
Dual-domain structure requiring correct VH–VL pairing for function.
Prone to misfolding or aggregation, especially in non-native environments.
Shorter CDR3 loops compared to VHHs, limiting access to deep epitopes.
Despite these challenges, scFvs have been widely applied in therapeutic formats such as CAR-T cells, antibody–drug conjugates (ADCs), and diagnostics.
Beyond their general size and folding differences, specific sequence substitutions within the VHH framework confer distinct physicochemical advantages. Camelid VHHs preserve the IgV β-sandwich but replace the canonical VH framework-2 hydrophobes (Val37, Gly44, Leu45, Trp47) used for VH–VL packing with hydrophilic residues such as Phe37 or Tyr37, Glu44, Arg45, and Gly47. These substitutions, together with a CDR3 that partially caps the former VL-facing surface, raise surface hydrophilicity and mitigate solvent-exposed hydrophobic patches—key contributors to the exceptional solubility and low aggregation propensity of VHHs compared with scFvs.
This sdAb comparison highlights how structural simplicity makes VHHs more stable and versatile, while scFvs remain important in established therapeutic systems. These structural adaptations extend to the antigen-binding loops themselves, where VHHs have evolved compensatory mechanisms. Loss of the VL partner is counterbalanced by elongation of CDR1 and CDR3, providing a total antigen-contact surface (~600–800 Ų) comparable to six conventional CDR loops. To mitigate entropic penalties from long, flexible loops, camelid VHHs often introduce an additional disulfide tether connecting CDR3 to CDR1, CDR2, or FR2, enhancing conformational rigidity and preserving affinity for recessed epitopes.
When comparing single-domain antibody vs scFv, several functional differences emerge:
Stability: VHHs are highly resistant to heat, pH extremes, and denaturants, while scFvs show moderate stability and may lose function under stress.
Binding affinity: Both formats can achieve nanomolar affinities, but VHHs are especially suited to cryptic or recessed epitopes due to their long CDR3 loop.
Humanization: VHHs, being camelid in origin, often require more framework adjustments to minimize immunogenicity. scFvs, which are usually derived from human IgGs, may be closer to therapeutic compatibility.
In addition to stability and affinity, the geometry of the paratope dictates preferred epitope classes. While both fragments can achieve high affinities, the convex CDR3 topology of VHHs enables access to concave or recessed surfaces, such as ion channel vestibules or viral glycoprotein canyons, whereas scFvs tend to favor planar or linear epitopes defined by cooperative VH–VL surfaces. Notably, VHHs also exhibit reduced nonspecific background binding, improving their performance in complex biological matrices.
Both VHHs and scFvs are valuable tools in antibody engineering, but their applications differ due to their structural properties:
Multimerization potential: The small size and simple structure of VHHs make them especially amenable to formats such as bispecific, trispecific, or tandem constructs. scFvs can also be multimerized and are widely used in formats like BiTEs, though they are more prone to folding and stability issues in these contexts.
CAR-T therapy: scFvs remain the predominant recognition domains in current CAR-T therapies. VHH-based CARs are under active investigation and show promise, offering potential advantages such as reduced aggregation and enhanced stability, but most are still in preclinical or early clinical stages.
Intracellular targeting: VHHs are particularly suited for “intrabody” applications because their compact, soluble nature allows efficient folding within the reducing environment of the cytoplasm. scFvs, by contrast, often misfold or aggregate under these conditions.
An important engineering consideration in scFv design involves linker composition and domain orientation. scFv functionality depends strongly on the 10-25 amino acid peptide linker that joins VH and VL domains. Linkers rich in Gly-Ser confer flexibility, while Glu-Lys stretches enhance solubility. Suboptimal linker length or composition can disrupt domain pairing, leading to misfolding, aggregation, or loss of antigen binding despite intact CDRs.
Each antibody fragment type has limitations that must be considered. From a production standpoint, the two formats behave very differently across expression systems. VHHs express efficiently in bacterial cytoplasm or yeast due to their single-domain nature and limited need for disulfide-assisted folding. scFvs often require periplasmic export in E. coli for correct disulfide formation, lowering yield, or accumulate as inclusion bodies requiring denaturation–refolding. In yeast, scFvs’ hydrophobic interfaces can overload folding chaperones, while VHHs fold properly and achieve higher yields, translating to lower manufacturing complexity and cost.
VHHs: They have a short serum half-life, which usually requires extension strategies such as Fc or albumin-binding fusions. They also depend on camelid origin for discovery, though synthetic libraries are increasingly available. This rapid clearance, while advantageous for imaging or diagnostic purposes, can limit therapeutic exposure. Unmodified VHHs fall below the renal filtration threshold (~65 kDa), leading to rapid glomerular elimination. To extend circulation time, strategies such as Fc fusion, albumin binding, or PEGylation are employed, and each balances half-life extension with retention of tissue penetration and activity.
scFvs: Their major drawbacks include folding instability, risk of VH–VL domain mismatch, and aggregation. These scFv limitations can affect reproducibility, especially in large-scale production. Humanization requirements also differ significantly between the two fragment types. Murine-derived scFvs share only about 50-55% sequence identity with human frameworks and often trigger anti-idiotypic responses, whereas camelid VHH frameworks share 75-90% identity with human VH3 family genes. Consequently, VHHs generally exhibit lower immunogenicity risk and simpler humanization, though specific CDR sequences still require optimization to minimize immunogenic epitopes.
The choice between VHH vs scFv depends on the intended use:
VHHs are ideal for applications requiring deep tissue penetration, robust stability, and access to hidden epitopes. They are also well suited for imaging, biosensors, and modular constructs.
scFvs are preferred when compatibility with existing IgG-based scaffolds, effector functions, or established therapeutic platforms such as CAR-T is needed.
Imaging applications further highlight these pharmacokinetic contrasts. When labeled for PET or SPECT, VHHs show rapid tissue distribution and high tumor-to-background ratios owing to their small size and fast clearance, while scFvs display slower kinetics and higher background retention. However, VHHs’ renal clearance can produce strong kidney and bladder signals, so target site proximity must be considered in imaging design.
Ultimately, both formats are complementary tools in antibody engineering. Evaluating the antibody fragment differences helps researchers and developers align structural advantages with therapeutic goals.
The comparison of VHH vs scFv reveals that while both are valuable antibody fragments, their differences in size, stability, and folding requirements drive distinct applications. VHHs are compact, stable, and suited for innovative designs, whereas scFvs remain indispensable in traditional therapeutic systems like CAR-T cells.
Understanding these differences ensures better use of each format in research and clinical development. Both continue to play important roles in antibody engineering, with ongoing innovations expanding their potential in biotechnology.
Their discovery and library generation workflows also differ mechanistically. VHH libraries are typically constructed from immunized or naïve camelid B cells via straightforward RT-PCR and phage or yeast display, producing single-domain binders without VH–VL pairing ambiguity. In contrast, scFv libraries require combinatorial pairing of VH and VL genes through techniques such as SOE-PCR, which introduces mispaired clones and reduces the proportion of functional, developable fragments—an upstream factor influencing overall discovery efficiency.
References:
Yasaman Asaadi, Fatemeh Fazlollahi Jouneghani, Janani, S., & Fatemeh Rahbarizadeh. (2021). A comprehensive comparison between camelid nanobodies and single chain variable fragments. Biomarker Research, 9(1). https://doi.org/10.1186/s40364-021-00332-6
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