Week 3, June 2025: Advances in Single-Domain Antibodies

Single-domain antibodies (sdAbs), also known as VHH antibodies or nanobodies, are the smallest functional fragments of antibodies, consisting of a single variable domain derived from heavy-chain-only antibodies found in camelids or engineered from human immunoglobulin frameworks. Despite their small size (about 12–15 kDa), they retain full antigen-binding ability. Their compact structure, high solubility, stability, and ease of genetic manipulation have positioned sdAbs as powerful tools in diagnostics, therapeutics, and biotechnology. These properties give them applications across intracellular targeting, penetration of dense tissues, and formulation for noninvasive delivery routes.

In a recent preprint, researchers developed a modular synthetic nanobody phage display vector based on the clinically validated 2Rs15d scaffold, allowing precise manipulation of binding and framework regions. This platform overcomes key limitations of camelid immunization, enabling library generation for non-immunogenic or toxic antigens and facilitating pharmacokinetic tailoring. The resulting library, with over 1.7 × 10⁶ unique CDR2 variants per microliter, yielded Cal3, which is a nanobody cross-reactive with human and mouse calreticulin and suitable for PET imaging when radiolabeled with ⁶⁴Cu-NOTA. These findings establish synthetic libraries as a scalable foundation for generating clinically relevant single-domain antibodies, supporting downstream efforts in solubility and delivery engineering.

Further downstream, a recent study focused on improving the solubility of single-domain antibodies for therapeutic development. They examined two sdAbs with human VH-like hydrophobic hallmark residues that exhibited low solubility despite being derived from an alpaca immune library. By introducing VHH-like mutations (V37Y, G44E, L45R, W47L) into framework region 2 (FR2), they successfully increased the solubility of one sdAb without buffer additives. For the second sdAb, additional modification, specifically a W99A substitution informed by spatial aggregation propensity calculations, was required to achieve monomeric behavior. Both engineered sdAbs retained functional integrity, demonstrating that solubility can be rationally improved even in the absence of structural information. 

Besides solubility, researchers are advancing the pharmacokinetics of single-domain antibodies. A recent study introduced a universal, site-selective conjugation strategy for variable domains of heavy-chain-only antibodies (VHHs) using thiol-maleimide chemistry. By engineering unpaired cysteine residues into C-terminal extensions of VHHs, they enabled efficient conjugation to maleimide-functionalized polysaccharides such as dextran-maleimide (Dex-Mal) and hyaluronic acid-maleimide (HA-Mal). Conjugation efficiency varied across six VHHs due to structural heterogeneity, but was significantly improved by inserting a glycine-serine (G4S) linker or multiple cysteine residues. This platform supports robust and customizable bioconjugation without compromising antigen-binding activity, facilitating the development of sdAb-based diagnostics and therapeutics. Such conjugation methods are particularly advantageous when integrated with delivery strategies targeting mucosal or systemic compartments. 

On the treatment delivery aspect, recent work has demonstrated the potential for topical or oral delivery. 

A recent paper describes human single-domain antibodies (UdAbs) for noninvasive, localized, topical treatment of mucosal inflammatory diseases. In a murine study, the anti-IL-33 UdAb A12 was delivered topically and via inhalation to target inflammation at the ocular surface and in the lungs. Despite limited interference with IL-33 receptor binding, A12 effectively inhibited IL-33 signaling, reduced dry eye severity, and ameliorated allergic asthma symptoms. Compared to the IgG control itepekimab, A12 achieved significantly higher corneal concentrations with minimal systemic penetration. These findings highlight the potential of UdAbs for targeted modulation of mucosal inflammation through localized delivery routes, broadening the therapeutic landscape for sdAb-based biologics. 

Meanwhile, oral biologic therapy has been a long-standing challenge due to protease degradation and poor gastrointestinal stability of conventional antibodies. Researchers engineered an anti-interleukin-23 receptor (IL-23R) VHH with both high affinity and protease resistance, achieving picomolar binding through contributions from both CDR and framework residues. The optimized VHH remained stable for over 8 hours in intestinal fluid and 24 hours in fecal samples. Oral administration in mouse and cynomolgus monkey models led to deep, sustained inhibition of IL-23R signaling, validating functional delivery to the gut. These findings establish orally delivered VHHs as a promising therapeutic platform for chronic inflammatory diseases such as inflammatory bowel disease.

Last month, a study reported on a panel of single-domain antibodies targeting the conserved S2 subunit of the SARS-CoV-2 spike protein, offering broad and potent neutralization of both SARS-CoV-1 and SARS-CoV-2. One VHH in particular bound a highly conserved quaternary epitope within the Heptad Repeat 2 (HR2) region, locking the spike protein in its prefusion conformation. When fused to a human IgG1 Fc and administered systemically at low doses, this VHH prevented SARS-CoV-2 infection in two animal models. Escape mutations were rare and rendered the virus poorly infectious. This work highlights the therapeutic potential of sdAbs as next-generation antivirals and underscores their unique ability to target structurally conserved epitopes inaccessible to conventional antibodies.