Tools for ADC drug discovery – screening VHHs with pre-engineered C-terminal cysteine handles
In the fast-paced world of biotechnology, innovation often stems from taking smart, calculated risks. At Isogenica, we are used to running unusual antibody drug discovery projects. But sometimes, even we have our doubts. One such moment arose when several Antibody-Drug Conjugate (ADC) and radiopharmaceutical companies approached us with a seemingly simple request: Could we add a C-terminal cysteine to our VHHs?
Figure 1. Representation of a VHH antibody structure with an additional Cysteine at the C-terminus
The Cysteine Question: A Risk Worth Taking?
The VHH scaffold we use at Isogenica is known for its simplicity. It features only one disulfide bond (Cys-Cys), which makes it highly efficient for expression in our high-throughput E. coli system. The streamlined nature of this scaffold reduces the chances of misfolding, so introducing a third cysteine into this carefully balanced system felt risky. Potential concerns were low yields, potential misfolding, and aggregation, challenges that could outweigh any potential benefits.
However, when one of our partners emphasized the importance of this modification for their downstream bioconjugation processes, we discussed the potential risks with them and decided to take the leap. It was a moment of hesitation – could this minor tweak derail the entire process? But we chose to push forward together, knowing that innovation often demands calculated risks.
The Experiment: A Leap of Faith
To test the hypothesis, we modified our in-house vector to incorporate an additional cysteine at the C-terminal of our VHHs using In-Fusion Site-Directed Mutagenesis. These modified VHHs were then cloned into this new vector and expressed in E. coli.
The main objective was to be able to screen our VHH selection outputs in high-throughput primary screening assays with the C-terminal cysteine already present. This was important to make sure our lead panel would work properly with this modification for the partner’s final application, which involved using a cysteine handle for conjugation.
Protein production and purification
To test if this was likely to work, we took a panel of well-characterized leads from a previous program and cloned them again into the new vector, then tested their expression and purification yields in our standard system – cytoplasmic E. coli expression and two-step bead-based purification.
We purified cysteine-tagged VHHs using our usual magnetic bead-based IMAC/IEX protocol. To our surprise, the cysteine-tagged VHHs not only produced similar protein yields, but some even exceeded the non-tagged versions (Table 1). Overall, the sequence of each VHH clone had a much greater impact on yield, as is usually seen in most antibody discovery campaigns. With these encouraging purification results, the next question was how this modification affected the VHH binding properties and overall stability.
Table 1. Protein concentration (µM) after purification.
Spontaneous dimers – double trouble?
It’s well known that free cysteine residues can spontaneously dimerize, so the first thing to check was if this was happening. These purified proteins were analyzed by non-reducing SDS-PAGE (Figure 2) and size exclusion chromatography (SEC) (Figure 3) to determine whether the VHHs were forming dimers and the percentage of dimer population compared to monomers.
From the SDS-PAGE and SEC results, we observed that cysteine-tagged VHHs did form dimers, as indicated by higher band in Figure 2 and the earlier peak in Figure 3, which represents a slightly larger molecule (the dimer) alongside the monomer. This dimerization is expected because free cysteines naturally tend to bond together under the right conditions, forming disulfide pairs.
Even with this tendency, most of the sample stayed monomeric, likely due to a combination of factors. The buffer conditions may not have been ideal for promoting efficient disulfide bond formation. Additionally, the polar FLAG-tags on the VHHs could have caused repulsion when brought close together by the disulfide bond, preventing full dimerization.
Figure 3. Size Exclusion Chromatography (SEC) traces showing dimer peaks only in the presence of the C-terminal cysteine.
Antigen Binding
Knowing that dimers were present in the samples presented two further challenges for accurately measuring the affinity of each VHH.
- Concentration – additional disulfide bonding can affect molarity calculations, making accurate quantitation of the sample more challenging.
- Avidity – this property is the sum of all affinity interactions between two molecules. For many antibody fragments, bivalency means much higher avidity, sometimes binding 100x more strongly than a monomer. By creating spontaneous dimers, it was very likely that the dimeric molecules in the sample would have a very different binding profile to the monomers.
Impact on Avidity
Because we were developing this vector with the aim of using the C-terminal cysteine format for hit screening, it was important to understand the effect of the C-terminal tag on the binding properties of individual clones. Would we identify the wrong molecules as hits?
For these known control clones, affinities were measured using ELISA and SPR methods on versions expressed from the standard vector and the C-terminal tag vector. For most VHHs tested, a shift in the ELISA binding curve was shown. For some clones, this shift was variable clone-to-clone, possibly due to the small dimer population or kinetic characteristics that reduce the impact of avidity.
To dive more deeply, a kinetic analysis using SPR demonstrated that for clone A4, the binding affinity (KD) improved moderately, from 8.4 nM to 1.8 nM—a 4-fold decrease—due to the presence of a C-terminal cysteine. On the other hand, clone C4 exhibited a much more significant change, with the KD decreasing >40-fold, from 316 nM to 7.4 nM (Figure 4). We hypothesised that this difference was likely due to the off-rate, which is the rate at which the drug leaves the binding site. Clone A4 has a slow off-rate, making it less dependent on avidity, while clone C4, with a faster off-rate, benefits more from dimerization.
Figure 4. Surface plasmoson resonance (SPR) traces demonstrating the differences in avidity gains for clones with different off-rates.
While the dimerization of VHHs via a C-terminal cysteine occurred at a low percentage (<25%) in these conditions, the effect on avidity is significant in certain clones. This meant that screening in this format could give individual clones with faster off-rates an advantage when determining which clones progress further as ‘hits’ in VHH discovery. However, in high-avidity applications such as targeting a virus or other small particle, this can actually be very attractive. In these applications the epitope is particularly important, so being able to keep lots of diversity in the hit panel is helpful, while the kinetics of monomer binding are much less relevant.
Implications and Future Prospects
This success has opened new possibilities for Isogenica and our partners. Adding a C-terminal cysteine without compromising VHH functionality allows us to move quickly into formatted testing for applications such as:
- Antibody drug conjugates (ADC)
- Fluorescent labelling
- Nanoparticle targeting
- Oligonucleotide labelling
- Radionuclide labelling
Furthermore, because we are able to perform primary screens in this vector, we can choose hits based on the final application. For example, “dirty” targets that are expressed on healthy tissues at lower levels are particularly attractive for avidity-based targeting (Figure 5).
Figure 5. Differential binding of low-affinity bi-valent antibody to healthy and cancer cells based on tumor antigen expresión levels
Conclusion: Embracing Innovation
What started as a cautious “maybe” became a successful “yes.” This experience highlights the importance of venturing into the unknown, which can lead to groundbreaking advancements. We were transparent with our partner about the perceived risks, drawing from our extensive experience. Together, we took the leap, and now they’re reaping the rewards, while we expand our ADC drug discovery toolbox.
Are you an innovator? At Isogenica, we embrace high-risk, high-reward projects. If you’re interested in exploring how our VHH technologies can enhance your projects, let’s discuss potential collaborations.
Here at Isogenica, we share our experience in VHH antibody discovery and engineering to help move innovative projects forward. Contact our experts today to find a solution to your challenge.