VHHs, also known as nanobodies or single-domain antibodies, are compact antibody fragments capable of recognizing their targets with high specificity. Their small size, structural stability, efficient tissue penetration, and ability to access epitopes that are difficult to reach with conventional antibodies make them increasingly attractive formats for targeted therapeutic development.
Initially considered mainly as research or diagnostic tools, VHHs are now clinically validated therapeutic formats. Several drugs or therapeutic modalities using VHHs or single-domain antibody domains have reached the market, while the number of preclinical and clinical projects continues to increase in oncology, inflammatory diseases, hematology, molecular imaging, targeted radiotherapy, and cell therapy.
This growth creates a rising need for platforms capable of producing complex, homogeneous, and modified VHHs with a high level of molecular control. Chemical synthesis of VHHs directly addresses this need.
Several examples demonstrate that VHHs are no longer only experimental formats:
A humanized bivalent Nanobody targeting von Willebrand factor, used in acquired thrombotic thrombocytopenic purpura. Its approval marked an important milestone, demonstrating that a VHH-based format can reach the market as a biological drug.
A trivalent anti-TNFα Nanobody approved in Japan for rheumatoid arthritis. Its structure combines two anti-TNFα domains and one human serum albumin-binding domain to extend plasma half-life. This format illustrates the value of VHHs for the rational design of multivalent proteins with optimized pharmacokinetics.
An anti-PD-L1 single-domain antibody fused to an Fc domain, approved in China for unresectable or metastatic MSI-H/dMMR solid tumors. Its subcutaneous administration highlights the potential of compact antibody domains for developing new immunotherapy formats and administration routes.
An anti-BCMA CAR-T therapy used in multiple myeloma, incorporates two single-domain antibody domains in its chimeric receptor. This demonstrates that VHHs can serve as targeting building blocks in complex therapeutic architectures beyond conventional soluble VHH formats.
The use of VHHs is expanding rapidly, driven by several factors:
In oncology, VHHs are being investigated for the targeting of immune checkpoints, tumor receptors, surface biomarkers, stromal targets, and epitopes that are difficult to access with conventional antibodies. Their small size may facilitate tumor penetration and rapid targeting, which is particularly relevant for imaging, targeted radiotherapy, and drug conjugate applications.
This trend reinforces the value of chemical synthesis: the more complex therapeutic formats become, the more critical it is to control sequence design, conjugation site, drug-to-antibody ratio, and final product homogeneity.
Recombinant production remains a major approach for generating VHHs. However, some therapeutic formats require a level of control that is difficult to achieve with conventional bioproduction: incorporation of non-natural amino acids, site-specific functionalization, homogeneous conjugation, half-life optimization, creation of multivalent formats, or controlled addition of therapeutic payloads.
Chemical synthesis enables the construction of VHHs from peptide fragments, which are then assembled to obtain a defined, modifiable, and reproducible protein. It offers a strategic alternative when bioproduction becomes limiting due to molecular complexity, batch heterogeneity, or modification constraints.
Chemical synthesis enables direct incorporation of non-natural or functionalized amino acids into the VHH sequence.
VHHs are naturally robust, but chemical synthesis optimizes their structure rationally.
VHHs are attractive candidates for targeted conjugates with precise control over attachment sites.
Chemical synthesis enables radiolabeling at precise positions for imaging and therapy.
Chemical synthesis enables complex architectures beyond monovalent VHHs.
Chemical synthesis produces defined molecules with controlled sequence and modifications.
Chemical synthesis reduces dependency on cellular systems andavoids time-consuming steps associated with bioproduction such as cell line development, clone selection, expression optimization, and biological impurity management.
This modular approach accelerates the transition from design to GMP-compatible production, which is particularly valuable when several therapeutic variants must be compared.
GMP production of recombinant proteins requires lengthy and costly development phases. For chemically accessible VHHs, chemical synthesis can reduce overall development and GMP production costs through:
Some VHHs are difficult to produce by recombinant expression, particularly when they show low yield, poor solubility, a tendency to aggregate, toxicity to the host cell, or purification challenges.
In these situations, chemical synthesis can represent an alternative to conventional biological production. It enables the direct production of the VHH sequence, followed by controlled folding, purification, and analytical characterization steps.
Low production yield can limit the validation, characterization, or development of a VHH. Before modifying the sequence, changing the expression system, or abandoning the candidate, it may be relevant to evaluate production by chemical synthesis.
This approach is particularly useful for obtaining defined quantities of VHH for feasibility studies, structure–activity relationship studies, conjugation testing, or functional validation.
Biological systems are often limited when modified amino acids need to be introduced into a VHH. Chemical synthesis enables the direct incorporation of specific modifications into the sequence, with a high level of control.
It can be used to introduce non-canonical amino acids, D-amino acids, isotopic labels, PEGylated, glycosylated, or phosphorylated residues, as well as functionalized amino acids designed for downstream conjugation.
The conjugation of a VHH to an active molecule, a fluorophore, biotin, or another functional group can generate heterogeneous products when performed in an uncontrolled manner. Random conjugation may affect the affinity of the VHH for its target, its stability, or the reproducibility of the final product.
Chemical synthesis enables the introduction of a defined conjugation site at a precise position within the VHH. This approach facilitates the production of more homogeneous VHH conjugates, with improved control over the attachment site and the number of molecules coupled per VHH.
For VHH–drug conjugates, controlling the number of active molecules attached to the VHH is a critical parameter. Non-specific conjugation can lead to a mixture of products with variable drug-to-VHH ratios.
By incorporating a specific conjugation handle during the design phase, chemical synthesis enables better control over the final architecture of the conjugate. This approach helps generate VHH–drug conjugates that are more homogeneous, easier to characterize, and more reproducible.
Yes. Chemical synthesis can enable the controlled incorporation of a chelator, a radiolabeling handle, or a functional group compatible with radioactive labeling.
This strategy is particularly relevant for the development of VHHs intended for PET imaging, SPECT imaging, targeted radiotherapy, or theranostic applications.
Multivalent or multisite VHH formats can be complex to produce using conventional biological methods, particularly when several modules need to be assembled with a precise architecture.
Chemical synthesis and chemoselective ligation strategies make it possible to assemble multiple VHHs or functional modules in a controlled manner. This approach can be used to design bivalent, bispecific, biparatopic, branched, or multifunctional VHH constructs.
Yes, provided that an appropriate folding and analytical control strategy is implemented. VHHs generally contain cysteine residues that are important for their structure. Producing the sequence alone is therefore not sufficient: the molecule must also be correctly folded, with the appropriate disulfide bonds.
Chemical synthesis must therefore be combined with folding, purification, and characterization steps to confirm the quality of the final VHH.
Recombinant production remains suitable for many standard VHHs. Chemical synthesis becomes particularly relevant when the VHH is difficult to express, when the production yield is low, when purification is complex, or when a precise modification is required.
It is also indicated for VHHs containing non-canonical amino acids, site-specifically conjugated VHHs, radiolabeled VHHs, multivalent VHHs, or formats requiring a strictly controlled molecular architecture.
GMP-compatible production can be considered when the synthesis, purification, folding, and characterization strategy is sufficiently robust and reproducible.
This approach can be relevant for complex modified VHH candidates, early clinical batches, or formats requiring a high level of molecular control, either as a complement or an alternative to conventional bioproduction.
Chemical synthesis of VHHs opens a complementary pathway to conventional bioproduction for the development of next-generation therapeutic proteins. It enables the production of homogeneous, modified, and functionalized VHHs with a high level of molecular control.
The arrival of the first medicines based on VHHs or single-domain antibodies, together with the increasing number of preclinical and clinical projects, confirms the growing interest in these formats for next-generation therapies.
For applications requiring non-natural amino acids, optimized stability, precise conjugation, radiolabeling, multivalence, or rapid transition to GMP, chemical synthesis represents a strategic advantage.
By combining rational design, automated synthesis, chemical assembly, controlled folding, and analytical characterization, this approach supports the development of next-generation therapeutic VHHs, from initial concept to GMP-compatible production.
