Antibodies are traditionally defined as molecules with two heavy chains and two light chains. However, there was an important change in the traditional understanding of antibodies in 1989. This research conducted by Professor Raymond Hamers of the Vrije University Brussel (VUB) resulted in the unexpected discovery of heavy chain-only antibodies (HCAbs) which lack a light chain (Figure 1). This discovery happened via student-led research which formulated a sero-diagnostic assay in order to diagnose trypanosome infection in camels and water buffalos (Muyldermans, 2013).

The discovery of Camelid heavy-chain antibodies has prompted widespread interest in utilizing these antibody domains in a variety of applications such as research, diagnostics and therapeutics (Muyldermans, 2013). These camelid heavy-chain antibodies are also known as VHHs/sdAbs/nanobodies (Figure 1). The formation of camelid VHHs for medicinal purposes occurred in three separate stages. The first decade (1993–2003) could potentially be considered as the exploration period (Arbabi-Ghahroudi, 2017). During 1996 and 2001, numerous patents were granted to research institutions in Belgium and Netherlands with an emphasis on potential commercial uses. Ablynx was established in 2001 with the primary goal of advancing nanobody-based medications and examining their therapeutic potential (Arbabi-Ghahroudi, 2017).

During the period of 2003–2013, a significant increase in publications surpassing 1,000 by 2013 was observed which suggests a substantial increase in attention and research focus on VHHs (Arbabi-Ghahroudi, 2017). There has been an evident increase in publications throughout the current developmental period starting from 2014 to the present and numerous VHHs have progressed into clinical trials or are getting ready for market release (Arbabi-Ghahroudi, 2017). Two decades of continuous work by Ablynx led to the formulation of the first nanobody medication known as caplacizumab (Cablivi) (Scully et al., 2019). The approval was obtained from the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) in 2018 and 2019, respectively (Bergstrand et al., 2022). This novel drug cures a rare blood clotting disorder called the acquired thrombotic thrombocytopenic purpura (TPP) (Scully et al., 2019). Multiple variables are accountable for the long duration that passed between the discovery of camelid single-domain antibodies (sdAbs) and their release into the market. One of the major variables is the novel nature of this approach.

Camelidae species are immunized against specific targets or antigens which result in the production of heavy chain antibodies (HCAb) and conventional antibody repertoires in vivo. Phage-display libraries provide a reliable representation of the various in vivo-matured heavy chain repertoires since they are generated by cloning amplified VHH repertoires with barely any alteration (Arbabi-Ghahroudi, 2017).

The remarkable specificity and affinity of VHHs are similar to those of conventional antibodies. Also, they exhibit excellent solubility, stability at different temperatures and possess monomeric behavior (Ikeuchi et al., 2021). VHHs are extremely tiny, measuring around 2.5 nm in diameter and 4 nm in length with a molecular weight of about 15 kDa (Hoey et al., 2019). They are easier to genetically engineer and can easily be produced for a relatively low price (Hoey et al., 2019). Moreover, they exhibit low immunogenicity and have improved tissue penetration properties (Khodabakhsh et al., 2018).

The remarkable thermostability of nanobodies is demonstrated by their capacity to retain 80% of their activity even after exposure to 37°C for a week (Paul et al., 2023). Furthermore, they exhibit resistance to proteases, denaturing agents and high pH levels (Paul et al., 2023). Despite their extremely short development time, research suggests that nanobodies can be generated in large quantities employing a microbiological system (de Marco, 2020). Nanobodies offer a promising alternative to conventional antibodies in disease diagnosis and treatment due to their unique advantages.

Recent advances in silico approaches have greatly contributed to the design and optimization of nanobody based therapeutics for asthma. Using computational tools such as molecular dynamics simulations and homology modeling (Cheng et al., 2019), researchers have focused on designing single-domain antibodies with enhanced stability, solubility, and specificity (Cheng et al., 2019).

One study utilized a camelization approach to create three specific mutated single-domain antibodies targeting a key pro-inflammatory cytokine implicated in allergic asthma. Using a monoclonal antibody structure as a template, these mutations significantly improved solubility and stability. Simulations revealed stable, long-lasting interactions mediated primarily by complementary-determining regions (CDRs). The engineered single-domain antibodies demonstrated improved binding affinity, stability, and solubility compared to their wild-type counterparts, highlighting their therapeutic potential (Araújo et al., 2023).

In recent preclinical studies, several promising nanobody-based therapies have been developed for the treatment of asthma and related allergic conditions, focusing on different therapeutic targets. For instance, Ma, L. et al. developed a trivalent bispecific nanobody targeting IL-5 and albumin to improve efficacy and address limitations of current IL-5 therapies (Ma et al., 2022). This nanobody showed superior efficacy over existing IL-5 therapies like mepolizumab, being 58 times more effective in inhibiting TF-1 cell proliferation. It also demonstrated excellent pharmacokinetics and sustained eosinophil suppression in primates. These results suggest the nanobody’s potential as a next-generation therapeutic for severe eosinophilic asthma, offering improved efficacy and longer-lasting effects (Ma et al., 2022). Similarly, Li, Shijie et al. engineered nanobodies suitable for inhalation administration that target IL-5, a cytokine critical for eosinophil proliferation and activation. Among the candidates, AIL-A96-Fc was identified as a highly effective nanobody that blocked the IL-5/IL-5Rα interaction and demonstrated cross-species activity with both human and cynomolgus IL-5. AIL-A96-Fc exhibited significant blocking effects, underscoring its potential as an inhaled therapeutic for eosinophilic asthma (Shijie et al., 2024).

Additionally, Qiu, W. et al. produced a bispecific antibody targeting both IL-4Rα and IL-5, utilizing humanized VHHs derived from alpacas (Qiu et al., 2020). They further investigated the epitope interactions of these VHHs with IL-4Rα and IL-5. Structural and biochemical analyses demonstrated that the nanobodies effectively inhibited the interactions between IL-4, IL-5, IL-13, and their respective receptors. Compared to dupilumab, which targets only IL-4Rα and has limited efficacy in severe disease, this bispecific antibody simultaneously attenuates the activity of three cytokines (IL-4, IL-5, and IL-13), offering enhanced therapeutic potential (Qiu et al., 2020).

Furthermore, Zhu, M. et al. designed an inhalable nanobody (Nb) targeting the IL-4Rα chain for asthma treatment, capitalizing on the inherent stability and efficacy advantages of nanobodies. By utilizing three immunized Nb libraries, they created the bivalent Nb, LQ036, which exhibited high affinity and specificity for human IL-4Rα. Preclinical tests in humanized mice demonstrated that LQ036 effectively inhibited key asthma-related biomarkers, including IgE and CCL17, reduced airway inflammation, and showed favourable pharmacokinetics and safety profiles. These findings underscore the potential of LQ036 as an effective inhalable biologic for the treatment of asthma (Zhu et al., 2024).

Meanwhile, Gevenois, P. J. Y. et al. developed nanobodies targeting IL-13, a key cytokine in allergy, inflammation, and fibrosis. While the initial nanobodies showed good affinity, they were ineffective at inhibiting IL-13 biological activity in vitro. To enhance efficacy, multimeric constructs were created, resulting in a significant increase in both affinity and biological activity, suggesting that multimeric nanobodies could be a promising approach for more effective IL-13 targeting (Gevenois et al., 2021).

In a similar manner, Rinaldi, M. et al. constructed ALX-0962, a bispecific nanobody targeting IgE and human serum albumin to extend plasma half-life (Rinaldi et al., 2013). Unlike Omalizumab, ALX-0962 demonstrated dual functionality, effectively neutralizing soluble IgE with higher potency while displacing preformed IgE-FcεRI complexes on basophils. This dual action significantly reduced basophil degranulation at nanomolar concentrations. These findings highlight ALX-0962s potential to provide a faster onset of clinical improvement in asthma treatment (Rinaldi et al., 2013).

In addition, Bauernfeind, C. et al. developed high-affinity Bet v 1-specific nanobody trimers to outcompete IgE binding and prevent allergic reactions. The engineered trimers showed enhanced cross-reactivity, slower dissociation rates, and better inhibition of IgE-allergen interactions compared to monomers. They effectively reduced IgE binding to Bet v 1 and related allergens while suppressing allergen-induced basophil degranulation. These results highlight the potential of nanobody trimers as a promising therapeutic strategy to prevent allergic reactions caused by Bet v 1 and its cross-reactive allergens (Bauernfeind et al., 2024).

Likewise, a study produced an anti-IgE nanobody derived from the Indian dromedarius camel to reduce hypersensitivity in allergic asthma. Using an ovalbumin-induced mouse model, the nanobody significantly suppressed IgE production and alleviated symptoms of airway inflammation, including bronchoconstriction and airway hyperresponsiveness. The results suggest that this camelid-derived nanobody could be a promising therapeutic strategy for allergic inflammation (Paul et al., 2023).

SAR443765, developed by Sanofi, is the first and only nanobody to date to reach a Phase 1 clinical trial for asthma treatment, marking a significant advancement in biologics targeting type 2 airway inflammation (Deiteren et al., 2023). This bifunctional NANOBODY^®, designed to block both TSLP and IL-13, demonstrated promising safety and efficacy results in the trial (NCT05366764). In 36 mild-to-moderate asthma patients with elevated FeNO, a single subcutaneous dose significantly reduced FeNO at week 4, outperforming the effects of monovalent biologics targeting either pathway. Reductions in blood biomarkers, such as IL-5 and IgE, aligned with these findings, and numerical improvements in prebronchodilator FEV1 were observed. The treatment was well-tolerated, with only mild to moderate Treatment-emerging adverse events such as nasopharyngitis and injection site reactions. These results highlight SAR443765s potential as a groundbreaking therapeutic for asthma (Deiteren et al., 2023).

The advancement of SAR443765 into clinical trials marks a significant milestone, demonstrating the transformative potential of nanobodies as promising therapeutic agents for asthma. This success highlights the urgent need for further research and development to translate more preclinical breakthroughs into clinical applications, paving the way for nanobodies to revolutionize asthma treatment and address critical unmet medical needs.