VHH antibodies for ADC applications
Why consider VHH antibodies for ADC drug targeting?
Advantages of VHH antibodies for ADC applications
Small, simple, and robust – VHH antibodies make ideal building blocks for advanced therapies.
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:
– 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 ADC payload in antibody binding. The two most popular choices of tag with our partners are:
1. C-terminal cysteine (Cys) for thiol conjugation to e.g. maleimide-linked payloads
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.
Partnering for ADC and bioconjugation projects
Our further partnerships include research collaborations to demonstrate the efficacy of VHHs conjugated to proprietary cytotoxic payloads on a variety of different cell types.
Explore the science behind our antibody discovery platforms
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