Single-domain antibodies derived from camelids, also known as VHHs or nanobodies, are highly adaptable binding domains widely used in protein engineering.

They were pioneered by Raymond Hamers at Vrije Universiteit Brussel and developed commercially by Ablynx, then acquired by Sanofi [1,2]. Caplacizumab, a bivalent engineered single-domain antibody linked via a three-alanine linker, is approved for the treatment of thrombotic thrombocytopenic purpura (TTP) and thrombosis [3]. In addition, ozoralizumab — a trivalent VHH therapeutic licensed in Japan for rheumatoid arthritis — combines two anti-TNFα VHHs with an anti–human serum albumin VHH to prolong its half-life to approximately 18 days [4]. VHHs are fully modular and can be “bolted on” to other biomolecules to create various targeted conjugates (e.g. ADCs, ASOs or AOCs, targeted LNPs, and radiotherapeutics), often particularly relevant in oncology [5].

In addition to payload targeting, nanobodies have numerous key applications including:

1. half-life extension via albumin binding,
2. bi-/multi-specific engineering, and
3. intracellular applications, including targeted protein degradation (TPD).

This article explores these particular applications in more detail.

Half-life Extension with Albumin-Binding VHHs

Optimization of pharmacokinetics is a key challenge in the development of small biologic drugs. In traditional full-length mAbs, this happens naturally via the IgG Fc domain, extending the systemic half-life through recycling via the neonatal Fc receptor (FcRn). Albumin-binding VHHs achieve similar half-life extension by hitching a ride on serum albumin, the most abundant plasma protein, which also binds FcRn. However, albumin-binding VHH domains have two distinct advantages over IgG Fc [6]:

• No activation of macrophages, neutrophils, dendritic cells, NK cells, or complement;
• Smaller size for increased tissue penetration (~15 kDa vs. ~50 kDa for an Fc domain)

Many albumin-binding VHH domains have the added bonus of being cross-reactive across multiple species, including human, non-human primates (NHP), and rodents, which simplifies the requirements for and increases confidence in preclinical models used to test these nanobodies without the need for surrogate development [7].

Isogenica’s albumin-binding VHH ISOXTEND® modules, for instance, offer increased drug half-life and have cross-species reactivity. ISOXTEND® modules bind albumin with high affinity, to support prolonged systemic exposure. Their small size (~15 kDa) allows integration without disrupting therapeutic function [8].

Ozoralizumab illustrates this principle in practice, combining two anti-TNFα VHHs with an anti-albumin VHH to extend systemic half-life to ~18 days. [9].

Bi-/Multi-specific Engineering

A few bi-specific VHH antibodies are already under development, such as sonelokimab (SLK) — a bi-specific nanobody composed of three VHH domains that bind IL-17A, IL-17F and albumin [10]. VHH domains are particularly good candidates to generate bi-specific antibodies due to their modularity, small size, stability, solubility, high yields, and low aggregation. Expanded epitope binding libraries can be developed rapidly [11,12].

The reduced size of VHH domains increases formulation flexibility and can support higher concentrations when required. This enhances performance in applications where tissue penetration is important, such as solid tumors, fibrotic diseases, and mucosal disorders [13,14].

Traditional bi-specific antibodies, composed of full-length IgG scaffolds, require the asymmetric combination of two unique heavy chains and two unique light chains in order to generate an antibody that can bind two distinct antigens. This creates a mispairing risk because heavy and light chains can assemble incorrectly, requiring additional manufacturing steps. VHH domain antibodies overcome this mispairing challenge by creating symmetric bi-specific antibodies using established mAbs.

One of the key challenges with building bi-specifics is linker length and rigidity. Since both targets need to be engaged simultaneously, each target pair will have an optimum distance and flexibility that needs to be tested empirically with several different combinations. In this case study, we added anti-PD-L1 VHH antibodies to different, but symmetric, locations on the clinical anti-PD-1 mAb pembrolizumab. This study indeed showed differences in bi-specific activity depending on the location and orientation of the VHH – read the full study here: https://isogenica.com/supercharge-igg-therapeutics/

Intracellular Applications & Targeted Protein Degradation 

In contrast to conventional monoclonal antibodies (mAbs) — which cannot target intracellular targets in live cells — nanobodies can be delivered as intrabodies to modulate proteins inside cells. Due to their compact size (Table 1), nanobodies can fit within viral or plasmid vectors, even in genetic fusions with other biomolecules such as E3 ligase domains used for targeted protein degradation (TPD) [15].

Table 1: Molecular size of select nucleotide encoded domains.

In TPD, VHHs fused to E3 ligases create bioPROTACs that guide target proteins towards ubiquitin-mediated degradation. A striking example is the HuR–TRIM21 bioPROTAC, which breaks down HuR, countering tumor-promoting effects and halting growth in vivo. [16]. See the schematic below of a VHH–TRIM21 fusion degrader binding a target protein and directing it for degradation.

The stability and solubility of VHH domains which can allow them to fold effectively in the cytoplasm of a cell can be partially attributed to a conserved disulfide bond in the framework regions. Isogenica’s VHH scaffolds are engineered to contain only this single conserved disulfide bond for good expression and stability.

Discover more about Intracellular & TPD applications at https://isogenica.com/intrabodies/.

Ready to Build Smarter Therapeutics?

VHH domains provide a modular set of tools for improving biologics across various applications. Explore how Isogenica’s VHH platforms can mitigate early-stage risk and accelerate engineered biologic programmes.

You can also explore related pages on bi-specifics, ADCs, intracellular & TPD, and VHH libraries for deeper insights.

References

[1] Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hammers C, Bajyana Songa E, Bendahman N, Hammers R (1993). Naturally occurring antibodies devoid of light chains. Nature, 363(6428): 446–448. doi:10.1038/363446a0

[2] Sanofi S.A., Ablynx N.V. (2018). Sanofi completes its acquisition of Ablynx following the expiration of the squeeze-out procedure. GlobeNewswire. Available from: https://www.globenewswire.com/news-release/2018/06/19/1526178/0/en/SANOFI-COMPLETES-ITS-ACQUITISION-OF-ABLYNX-FOLLOWING-THE-EXPIRATION-OF-THE-SQUEEZE-OUT-PROCEDURE.html

[3] Sanofi S.A. (2018). Cablivi (caplacizumab) approved in Europe for adults with acquired thrombotic thrombocytopenic purpura (aTTP). PR Newswire. Available from: https://www.prnewswire.com/news-releases/cablivi-caplacizumab-approved-in-europe-for-adults-with-acquired-thrombotic-thrombocytopenic-purpura-attp-300705783.html

[4] Taisho Pharmaceutical Co., Ltd (2022). Notification of approval to manufacture and market Nanozora® 30 mg syringes for subcutaneous injection, a therapy for rheumatoid arthritis (Japan’s first NANOBODY® therapeutic). Available from: https://www.taisho.co.jp/en/company/news/20220926001109/

[5] De Pauw T, De Mey L, Debacker JM, Raes G, Van Ginderachter JA, De Groof TWM, Devoogdt N (2023). Current status and future expectations of nanobodies in oncology trials. Expert Opin Investig Drugs, 32(8):705–721. doi:10.1080/13543784.2023.2249814

[6] Harmsen MM, Ackerschott B, de Smit H (2024). Serum immunoglobulin or albumin binding single-domain antibodies that enable tailored half-life extension of biologics in multiple animal species. Front Immunol., 15: 1346328. doi:10.3389/fimmu.2024.1346328

[7] Shen Z, Xiang Y, Vergara S, Chen A, Xiao Z, Santiago U, et al. (2021). A resource of high-quality and versatile nanobodies for drug delivery. iScience, 24(9): 103014. doi:10.1016/j.isci.2021.103014

[8] Isogenica Ltd (2025). Tackling the challenge of short half-life for biological therapeutics. Available from: https://isogenica.com/half-life-extension/

[9] Takeuchi T (2023). Structural, nonclinical, and clinical features of ozoralizumab: a novel tumour necrosis factor inhibitor. Mod Rheumatol, 33(6): 1059–1067. doi:10.1093/mr/road038

[10] McInnes IB, Coates LC, Mease PJ, Ogdie A, Kavanaugh A, Eder L, et al. (2025). Sonelokimab, an IL-17A/IL-17F-inhibiting nanobody for active psoriatic arthritis: a randomized, placebo-controlled phase 2 trial. Nat Med. doi:10.1038/s41591-025-03971-6

[11] Mullin M, McClory J, Haynes W, Grace J, Robertson N, van Heeke G (2024). Applications and challenges in designing VHH-based bispecific antibodies: leveraging machine learning solutions. mAbs, 16(1): 2341443. doi:10.1080/19420862.2024.2341443

[12] Wang J, Kang G, Yuan H, Cao X, Huang H, de Marco A (2022). Research progress and applications of multivalent, multispecific and modified nanobodies for disease treatment. Front Immunol, 12: 838082. doi:10.3389/fimmu.2021.838082

[13] Li H, Zhou Q, Cao N, Hu C, Wang J, He Y, et al. (2025). Nanobodies and their derivatives: pioneering the future of cancer immunotherapy. Cell Commun Signal, 23(1): 271. doi:10.1186/s12964-025-02270-4

[14] Li Q, Humphries F, Girardin RC, Wallace A, Ejemel M, Amcheslavsky A, et al. (2022). Mucosal nanobody IgA as inhalable and affordable prophylactic and therapeutic treatment against SARS-CoV-2 and emerging variants. Front Immunol, 13: 995412. doi:10.3389/fimmu.2022.995412

[15] Silva-Pilipich N, Smerdou C, Vanrell L (2021). A small virus to deliver small antibodies: new targeted therapies based on AAV delivery of nanobodies. Microorganisms, 9(9):1956. doi:10.3390/microorganisms9091956

[16] Fletcher A, Clift D, de Vries E, Martinez Cuesta S, Malcolm T, Meghini F, et al. (2023). A TRIM21-based bioPROTAC highlights the therapeutic benefit of HuR degradation. Nat Commun, 14(1): 7093. doi:10.1038/s41467-023-42546-2