VHH antibodies for payload targeting
Learn how to harness the power of these small format VHH antibodies (often called nanobodies) for targeting payloads such as small molecule drugs, radionuclides, oligonucleotides, LNPs and more.
Scientific & Therapeutic Context
About ADC applications
Since the first antibody drug conjugates (ADCs) in 2000, researchers have been pushing the limits of which payloads antibodies can be used to target. Thanks to the unique ability of antibodies to bind specifically to their target, antibody conjugates can be used successfully to limit off-target effects. Many companies are now pursuing programs on numerous conjugate technologies such as:
– Antibody Drug Conjugates (ADC) – small molecules drugs, often used to deliver cytotoxic molecules to cancer cells.
– Antibody Radioconjugates (ARC) – radioactive isotopes, used in diagnostic or therapeutic settings.
– Antibody Oliognuclide Conjugates (AOC) – DNA or RNA encoding a genetic therapy. This can be naked or modified genetic material, or even viral vectors and lipid nanoparticles (LNPs).
Why consider VHH antibodies for payload targeting?
Small, simple, and robust – VHH antibodies make ideal building blocks for advanced therapies. However, advantageous properties should be balanced with desirable half-life and drug-antibody ratio (DAR) to identify the right solution for your application.
Advantages of VHH antibodies for payload targeting
With all the binding affinity of a full-length monoclonal in just 10% of the size, VHHs (sometimes known as nanobodies) have unique properties that are advantageous for developing antibody drug conjugates:
– Small size – their compact nature allows VHHs to penetrate tissues quickly, for deeper target engagement. Many groups are even using VHHs to deliver therapies across the blood-brain barrier.
– High solubility – naturally monomeric, VHH single domains are more soluble than the VH domains found in traditional IgGs, for improved drug developability.
– Simple and robust – Isogenica’s VHH libraries have a fixed scaffold with just one internal disulphide bond. Together with the innate robustness of the molecules, they fold well across a wide pH range allowing buffer transitions needed for payload conjugation.
– Low immunogenicity – With 4 VHH-based therapies already approved, patient studies show low immunogenicity risks for VHHs generally. To mitigate this further, Isogenica offers pre-humanised solutions to reduce requirements for lead engineering.
– Unique CDR3 domains – VHHs have longer CDR3s than traditional IgGs, this means they can penetrate deep into binding pockets to hit difficult targets.
– Ease of manufacture – Due to their simplicity, VHHs can be manufactured readily in microbial systems such as bacteria or yeast. This can be a big advantage when using non-canonical amino acid handles for payload conjugation.
Antibody-drug conjugation with VHHs
Knowing where your drug payload is conjugated on your targeting antibody is critical for making a consistent and reliable ADC drug. Even on small antibodies like VHHs, there are several options for site-specific drug conjugation, but they need to be built in.
C-terminal conjugation handles
The most straightforward is to use a C-terminal handle. With a C-terminal handle present on your VHH antibody, a wide variety of payloads can be conjugated specifically and effectively, including:
– small molecule drugs for ADCs
– radionuclides for radiopharmaceutical or radiodiagnostic applications
– oligonucleotides for DNA or RNA delivery as antibody oligonucleotide conjugates (AOCs)
– viral particles for gene therapy delivery
– LNPs for delivery of genetic and other payloads
The C-terminal of the VHH is a low-risk choice for payload conjugation because it extends away in the opposite direction from the binding region of the VHH antibody. This limits any interference of the DC payload in antibody binding. The two most popular choices of tag with our partners are:
1. C-terminal cysteine (Cys) for thiol conjugation to maleimide-linked payloads including chelators, flurophores, polymers, small molecules and many more.
2. C-terminal sortase for enzymatic conjugation to payloads with a polyglycine tag.
Potential handles for site-specific antibody drug conjugation on VHH antibodies.
Non-canonical amino acid handles
Additionally, synthetic “non-canonical” amino acids can be encoded into VHHs to create bio-orthogonal handles within the VHH molecule itself at appropriate sites in the frameworks. Due to unique reactive groups present on non-canonical amino acids, incredibly specific and efficient chemical reactions can be used to link the desired payload to the amino acid side chain, as described by Axup et al in their PNAS article from 2012.
Thanks to the simplicity of VHHs, they can be produced in most biological systems, including microbial expression platforms like E. coli which are easily engineered to incorporate non-canonical amino acids.
Are VHH antibodies right for your conjugation project?
Alongside the advantages, the small size of VHH single domain antibodies can have some limitations which should be considered:
Drug-antibody ratio (DAR): Unlike using traditional mAbs for ADC, your conjugation handles for VHHs do not come included in its genetic sequence. This allows complete precision for conjugation, but does require some pre-planning. While low DAR solutions are readily available, achieving high DAR may require tailored solutions such as branched linkers.
Serum half-life: Left unmodified, VHH antibodies are cleared quickly from the bloodstream. While ideal for radiodiagnostics, this needs modifying for many other applications. The best approach will depend on the biophysical properties of the payload, which may be half-life-extending by itself. You can learn more about different options, including Isogenica’s ISOXTEND® platform, here: https://isogenica.com/half-life-extension/
Partnering for ADC and bioconjugation projects
Isogenica has developed vector solutions which allow high-throughput screening to take place with VHH antibodies already in possession of a C-terminal cysteine or sortase tag. This approach ensures that selected lead antibodies can tolerate the desired conjugation handle in the right position.
In our recent blog we share the story of how our C-terminal cysteine vector was developed, including little-to-no impact on purification yield in our high-throughput system, and the effect of spontaneous Cys-Cys dimer formation.
Our further partnerships include research collaborations in radiodiagnostics, proprietary linker technologies, and multiple ADC discovery programs.
Not sure if VHH are right for your application?
Explore the science behind our antibody discovery platforms
Access our latest white papers and application notes to see how Isogenica’s synthetic VHH technologies are accelerating innovation in CAR-T, bispecifics, and immuno-oncology.
White Paper “Data-Driven Validation of Synthetic VHHs”
This white paper provides a data-driven validation of Isogenica’s synthetic VHH libraries, powered by Colibra® technology. Designed for biotech and pharmaceutical scientists, it demonstrates how these libraries enhance and accelerate drug discovery, particularly in oncology and immunotherapy. DownloadExtending half-lives of VHH antibodies
Because VHHs are small, they can be cleared quickly from the bloodstream. This can be a useful feature for some applications, but often a longer plasma half-life is desirable. DOWNLOADAdvantages of VHH in bi-specifics
To learn more about the application of VHHs in bi-specifics, we have condensed our expertise into a downloadable Application Note. DOWNLOADOptimizing CAR-T and T-cell antibody engagers: a role for VHH single domain antibodies
This whitepaper summarises the clinical and research landscape for CAR-T and T-cell engaging antibody therapies and show how single domain VHH antibodies can be applied to optimise the next generation of these important new therapeutic modalities. DOWNLOADIsogenica’s PD-L1 VHH as Functional Antagonists
PD-1 is an immune checkpoint protein expressed on the surface of multiple types of immune cells, including antigen-stimulated T-cells and tumour specific T-cells1. Interaction between PD-1 and its ligands (PD-L1 or PD-L2), is responsible for the regulation of T-cell activation, apoptosis, proliferation and cytokine production. DOWNLOADAnti-LRP5/6 VHH inhibits WNT pathway and prevents tumour growth
VHH are the variable domain of heavy chain only antibodies. They are small in size (~15 kD) and biophysically robust. With tunable half-lives, these antibodies are ideal for targeting inaccessible epitopes, achieving enhanced tissue penetration, multi-target binding and formatting for payload delivery… DOWNLOAD