VHH antibodies for ADC applications

Learn how to harness the power of these small format VHH antibodies for antibody-drug conjugate (ADC) applications.
Icon depicting a VHH-drug-conjugate binding to a cell surface target.

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

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, and the effect of spontaneous Cys-Cys dimer formation.
ADC drug discovery

Our further partnerships include research collaborations to demonstrate the efficacy of VHHs conjugated to proprietary cytotoxic payloads on a variety of different cell types.

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