Jun 10, 2026
With the continuous advancement of genetic engineering technologies, the development of antibody fragments has become increasingly feasible. In certain specific application scenarios, portions of native immunoglobulins can be omitted to generate novel molecular architectures. Among these, camelid heavy-chain antibody variable domains (VHH) and single-chain variable fragments (scFv) have garnered considerable attention. Although scFv has been widely applied, VHH is progressively emerging as a promising alternative owing to its superior physicochemical properties, enhanced solubility and stability, smaller molecular size, and lower production costs. This review provides a comprehensive comparative analysis of these two antibody formats from the perspectives of structural properties and therapeutic applications.
Immunoglobulin G (IgG) molecules consist of two functionally distinct regions: the antigen-binding fragment (Fab) and the crystallizable fragment (Fc). The binding specificity of the entire Ig molecule is exclusively determined by the Fab region, particularly the Fv domain, which is composed of the heavy-chain variable region (VH) and the light-chain variable region (VL). This modular organization of immunoglobulins facilitates convenient genetic modification and engineering. To date, various antibody derivatives—including Fab, scFv, diabodies, triabodies, and single-domain antibodies—have been developed and are progressively replacing full-length Ig molecules in research, clinical diagnostics, and therapeutic applications. Among these, scFv and VHH are particularly prominent.
In conventional Ig, the Fab fragment is responsible for antigen-binding specificity, with the Fv domain representing its minimal structural unit. The Fv comprises VH and VL domains, each stabilized by an intra-chain disulfide bond, and these two domains interact through a hydrophobic interface. Additionally, an inter-chain disulfide bond between CH1 and CL domains further stabilizes the Fab structure. The scFv is an engineered derivative of the Fv domain, in which the VH and VL sequences are linked by a flexible peptide linker to form a single antigen-binding entity. Both VH and VL contain three hypervariable regions, termed complementarity-determining regions (CDRs), which are interspersed by four framework regions (FRs). The FRs provide a stable scaffold that maintains the three-dimensional conformation of the VH and VL domains. Compared with CDRs, the FR sequences are considerably more conserved in amino acid usage. In conventional Ig, the heavy-chain CDR3 (CDR3H) is highly variable and contributes approximately 29% of the antigen-binding force, whereas in VHH, CDR3H accounts for over 60% of the binding energy.
Given the predominant contribution of VH to antigen recognition, researchers have long attempted to generate functional antibodies comprising only heavy chains. However, early studies demonstrated that isolated VH domains exhibited poor secretion, low affinity, and inadequate solubility. Fortunately, the discovery of VHH—the variable domain of camelid heavy-chain-only antibodies—overcame these obstacles. Compared with VH, VHH possesses distinct amino acid substitutions in the VH-VL interaction interface, particularly within FR2, where highly hydrophobic residues in VH are replaced by highly hydrophilic residues in VHH. Substituting the hydrophobic residues in VH with their hydrophilic VHH counterparts significantly improves solubility and expression levels. Moreover, the CDR3H in VHH is, on average, longer than that in VH. This extended CDR3H folds into a structure that shields the hydrophobic patch at the VH-VL interface, thereby enhancing solubility. The elongation and enhanced diversity of VHH CDR3H also compensate for the loss of diversity and binding affinity resulting from the absence of VL. In certain VHH subfamilies, additional disulfide bonds further stabilize the extended CDR3 loops, contributing to overall structural stability.
The structural differences between VHH and scFv result in markedly distinct physicochemical properties. In terms of size, scFv (approximately 30 kDa) is roughly twice the molecular weight of VHH (approximately 15 kDa). The smaller dimensions of VHH facilitate genetic manipulation, and its three binding loops enable superior access to cryptic epitopes. Notably, the elongated CDR3H of VHH forms a finger-like structure that can penetrate cavities and grooves present in multi-pass transmembrane proteins and enzymes. Both formats have molecular weights below the renal filtration threshold (~65 kDa), resulting in short serum half-lives. Nevertheless, their small sizes confer excellent tissue penetration properties—particularly for VHH—which establishes a solid structural foundation for diverse pharmaceutical applications. Both VHH and scFv can be modified via PEGylation, Fc fusion, albumin fusion, or anti-albumin antibody fusion to increase molecular weight, prolong half-life, and expand application scenarios.
VHH exhibits superior solubility compared with scFv, attributable to the substitution of highly hydrophobic residues in FR2 of VH with hydrophilic residues in VHH. In scFv, four residues in FR2 (V37, G44, L45, and W47) form a hydrophobic interface that facilitates VH-VL association; however, this hydrophobic region also reduces solubility and promotes aggregation. In contrast, VHH possesses polar or smaller amino acid substitutions at corresponding positions (F37 or Y37, E44, R45, and G47), rendering the molecule more hydrophilic and thus significantly more soluble than scFv. These non-polar-to-polar amino acid transitions also confer enhanced thermodynamic stability, increased resistance to chemical denaturation and enzymatic degradation, and greater stability under extreme pH and ionic strength conditions. Furthermore, the presence of additional disulfide bonds in VHH contributes to its higher conformational stability, albeit at the cost of reduced conformational flexibility.
Both scFv and VHH can be derived from immunized or naïve antibody libraries through various display technologies and single B-cell approaches. For display-based methods, peripheral blood mononuclear cells (PBMCs) are isolated from animals, total RNA is extracted, and cDNA is synthesized via reverse transcription. The antibody fragments are then amplified by PCR. For scFv, VH and VL fragments are separately amplified and subsequently linked via a flexible linker in either VH-VL or VL-VH orientation. The scFv fragment is then cloned into display vectors to achieve genotype-phenotype coupling, followed by antigen-binding selection and enrichment of specific binders. For VHH, a two-round PCR strategy is typically required: the first-round amplification yields both conventional VH-hinge-CH1-CH2 fragments (~900 bp) and VHH-hinge-CH2 fragments (~600 bp); the 600 bp product is then used as template for a second-round PCR with VHH-specific primers to obtain the full VHH gene. Subsequent display and screening procedures are analogous to those for scFv. Notably, during scFv construction, the fusion of VH and VL segments is prone to mispairing and frameshift mutations, necessitating codon optimization of the linker sequence.
Regarding recombinant expression, VHH is considerably easier to produce than scFv. Although scFv can be expressed in the bacterial cytoplasm or periplasm—where chaperone proteins and disulfide isomerases in the periplasm facilitate proper folding—yields are typically low. Moreover, unpaired cysteine residues in scFv may form non-specific disulfide bonds with periplasmic proteins, leading to precipitation. In the reducing environment of the cytoplasm, disulfide bond formation is hindered, exposing the hydrophobic VH-VL interface and resulting in aggregation and inclusion body formation. Refolding from inclusion bodies is time-consuming and often yields low-activity products, limiting scFv applications. Although hydrophobic-to-hydrophilic substitutions in the VH-VL interface can mitigate aggregation, such modifications often compromise antigen-binding affinity. In contrast, VHH can be efficiently expressed in both the cytoplasm and periplasm without pronounced aggregation issues, enabling simpler and more cost-effective production. Furthermore, while scFv expression in yeast remains challenging, VHH can be readily expressed in Saccharomyces cerevisiae.
scFv is commonly derived from rodents, particularly mice and rats, with murine VL and VH sequences exhibiting only 53% and 51% sequence identity, respectively, to their human counterparts. In contrast, VHH shares 75–90% sequence homology with the human VH3 gene family, suggesting lower immunogenicity in clinical applications. Consequently, humanization of VHH is more straightforward. Even after humanization, murine-derived scFv may still elicit anti-idiotypic responses, as elimination of key residues in the variable region could compromise antigen binding.
Although scFv and VHH exhibit comparable affinities, they display distinct epitope preferences. VHH preferentially binds to concave epitopes, such as grooves and cavities on ion channels, viral glycoproteins, or immunological synapses, owing to its elongated CDR3H loops that form convex structures capable of accessing recessed epitopes. Conversely, scFv more readily recognizes planar or linear epitopes. Notably, VHH also demonstrates good affinity for flat epitopes, indicating its ability to form diverse interface complexes. Additionally, VHH exhibits lower non-specific background binding compared with scFv.
Owing to their small dimensions, both scFv and VHH are highly suitable for a wide spectrum of applications, ranging from diagnostics and therapeutics to fundamental research.
Targeted therapeutic agents have substantially expanded treatment options for numerous diseases, particularly in oncology. As high-affinity antibody fragments, both scFv and VHH play pivotal roles in targeted therapies and have been developed in various formats for the treatment of diverse pathologies.
Both scFv and VHH can function as neutralizing agents that directly bind and inactivate exogenous particles, including toxins, viruses, cancer antigens, or disease-related cytokines and growth factors. These antibody fragments are administered as naked antibodies in monovalent or multivalent forms to block target functions, or they are fused to Fc regions to extend serum half-life and elicit immune effector functions.
Nanobodies offer five distinctive advantages in this context: (1) their smaller size enables superior tissue diffusion and extends neutralization capacity beyond the vascular compartment; (2) enhanced flexibility and binding propensity allow bivalent or trivalent VHH constructs to achieve improved neutralization efficacy; (3) lower immunogenicity and a more straightforward humanization process; (4) high stability facilitating cost-effective production; and (5) structural features enabling access to otherwise inaccessible epitopes. These attributes collectively position VHH as a highly versatile platform for neutralizing applications.
Toxin neutralization represents a critical but often overlooked health concern worldwide. Plasma anti-venom serum (PAS) provides passive immunotherapy with rapid and effective antibody responses. Toxin neutralizers require rapid diffusion and clearance capabilities to efficiently recognize and neutralize toxins with high tissue penetrance, followed by timely renal clearance. Therefore, antibody fragments such as VHH and scFv are particularly well-suited for toxin inactivation. While scFv has been successfully applied in vitro, VHH has demonstrated superior performance in neutralizing various lethal toxins in numerous studies.
Antiviral neutralization: VHH-based neutralizers have been developed against diverse animal and human viruses, including HIV-1, human respiratory syncytial virus (hRSV), and H5N1 influenza. Upon binding to viral envelope glycoproteins, VHH prevents virus–coreceptor interactions on host cells. Multivalent constructs (bivalent and trivalent) recognize similar or distinct epitopes on envelope glycoproteins with enhanced efficiency, thereby broadening neutralization breadth, as demonstrated for anti-HIV and anti-hRSV trivalent nanobodies. scFv has also been employed as viral neutralizers against HIV, influenza, porcine epidemic diarrhea virus (PEDV), and HPV. However, VHH possesses a distinct advantage, as its smaller size enables more effective matching and occupancy of viral envelope protein cavities.
Cancer immunotherapy: Antibody fragments can inhibit tumor immune evasion by directly binding to tumor cells or blocking critical components involved in tumor growth and invasion. Given the essential role of growth factors in angiogenesis, particularly within the tumor microenvironment, blocking these factors or their cognate receptors can promote tumor regression. Both scFv and VHH have been utilized to bind and neutralize vascular endothelial growth factor receptor 2 (VEGFR-2).
Cytokine/chemokine neutralization: Suppression of cytokines and chemokines represents a potential therapeutic strategy for both cancer and autoimmune diseases. VHH and scFv have been developed to block various inflammatory and immunomodulatory cytokines for the treatment of rheumatoid arthritis, chronic inflammation, autoimmune inflammatory diseases, cancer, and stroke. Cytokine–nanobody complexes conjugated to serum albumin exhibit prolonged half-life while maintaining sufficient neutralizing activity. Moreover, the high permeability of nanobodies and their ability to cross the blood–brain barrier enable attenuation of neuroinflammatory responses following cerebral ischemia.
Antibody fragments can also serve as drug delivery vehicles to transport payloads to target organs or diseased tissues, thereby reducing non-specific cytotoxicity and off-target damage while prolonging systemic half-life and enhancing therapeutic efficacy. The most well-known examples include antibody fragments conjugated to cytotoxic molecules for targeted cell killing. Both scFv and VHH have been employed in drug conjugation. Immunotoxins and small-molecule drugs can be conjugated to both formats; however, VHH exhibits superior tissue penetration and physicochemical properties, rendering it more advantageous in ADC development. Emerging conjugation strategies also encompass antibody–oligonucleotide conjugates, in which siRNA is delivered to target organs for the treatment of congenital genetic disorders.
Both scFv and VHH represent excellent antigen-targeting moieties for genetically engineered cell therapies, including CAR-T and CAR-NK modalities. The core component of chimeric antigen receptors (CARs)—the extracellular antigen-binding domain—is typically composed of either scFv or VHH. Owing to its smaller size, ease of humanization, and superior physicochemical stability, VHH is increasingly favored in cell therapy applications.
The inherent specificity of antigen–antibody recognition ensures that antibodies will continue to serve as highly effective therapeutic molecules. With ongoing advances in gene cloning technologies, iterative improvements in antibody discovery platforms, and continuous exploration of novel structural formats, the application landscapes of both scFv and VHH are expected to expand further. Cell therapy, antibody–drug conjugates, bispecific antibodies, trispecific antibodies, and multispecific antibody formats are poised to make significant contributions across a broad range of disease areas.
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