Specific Antibodies to Difficult Targets: How to find the needles in the haystack
Peptides, often small and unstructured, are some of the most difficult targets in antibody discovery. Here, we recount how we tweaked our approach to mining our vast VHH libraries to isolate highly specific binders for difficult targets like these, even when other methods had failed.
VHH antibodies were discovered that would bind to the target peptide regardless of biotinylation chemistry (Bt1 / Bt2), but did not bind off-target surfaces.
Peptides as Difficult Targets
When a target antigen is too challenging to express, or where a highly specific epitope is desired, antibody discovery scientists can turn to peptides as target material. These peptides are small protein fragments typically composed of around 10 to 20 amino acids, and taken from a region of the biological target protein. However, targeting peptides with antibodies is notoriously challenging due to their small size and flexible structure.
The Peptide Challenge
Unlike larger proteins, peptides lack the well-defined structures that make conventional antibody binding straightforward. Their small and often unstructured (i.e., floppy) nature means that many antibody discovery platforms fail to find sufficiently specific or high-affinity binders. So when a client came to us with a VHH antibody discovery challenge against a highly specific peptide, everyone was excited to tackle such challenging targets.
A Client Story: Overcoming the Peptide Problem
The challenge was immense: a small, flexible peptide with only minor differences to other, non-target proteins. The client needed a highly specific antibody for a critical diagnostic application in oncology, but every approach they tried failed to deliver. They were out of options. As their last hope, they came to Isogenica. To make the task even more challenging, the target peptides were shipped blinded – we had no idea what we were working with. Despite these unknowns (and to our partner’s surprise!) our synthetic VHH libraries generated not one but three highly specific binders – a breakthrough where all other methods had failed. These binders were specific to the peptide of interest across different immobilisation chemistries, without binding to any of the “off-target” controls, including structural and charge-based analogs. This success led our partner to commission two new projects on similar peptides against further unknown targets. With the foundations laid in this project, subsequent work became more straightforward.
How did we overcome this challenge?
After initially limited success with our standard conditions for full-length protein antigens we conducted a deep-dive into what was happening, and what we could do to drive our selections towards the specificity needed. The luxury of a fully synthetic system is the control you can apply to selection conditions. Here, we employed several strategies:
- Deselecting against off-target peptides
- Specific elution with target peptides
- Adjusting blocking conditions
- Adjusting washing conditions
Of these, the most impactful was applying much more stringent blocking conditions, and using a new and highly complex blocking reagent.
Why did this work?
Through strategic depletion techniques, we further reduced the prevalence of ‘sticky’ antibodies, even more than our standard conditions, which tend to bind non-specifically. By doing so early in the selection process, we set the stage for more accurate and efficient identification of peptide-specific binders. The added challenge with peptide selections is that peptides are much smaller than typical antigens used for antibody discovery. This limited binding “real estate” means the overall number of specific antibodies in any library is lower compared to a larger protein with more available epitopes. Critically, this affects the ratio between specific and non-specific antibodies in subsequent rounds (see figure below). Identifying more stringent blocking conditions allowed us to reduce the number of non-specific binders, making it easier to enrich the VHH antibodies specific for the peptide.
Peptides are smaller than most antibody discovery targets, meaning there are fewer epitopes available for antibodies to bind to. In any selection, there will be a similar number of non-specific binders present in the first round. But in a peptide selection, the ratio of specific to non-specific binders can be skewed. Going into the second round it’s then much harder for the specific antibodies to find the peptide target, and “sticky” binders become more and more enriched. By identifying even more stringent blocking conditions, fewer non-specific phages were enriched, making it easier to enrich for those specific to a small target.
The AstraZeneca Success
In another example, AstraZeneca’s publication on VHH “intrabodies” highlights the power of Isogenica’s synthetic VHH in generating highly specific antibodies to difficult targets. In this case, the target was peptides derived from the cardiac target phospholamban – an intracellular protein required for regulating proper heart muscle contraction. Phospholamban is highly conserved between species, making it unsuitable for llama immunization, but no problem for a good synthetic antibody library.
‘Intrabodies’ – Isogenica’s VHH antibodies improve heart function when delivered intracellularly
Our VHH antibodies have been put to the test as a potential new therapy for heart failure in an early-stage study from AstraZeneca published in Nature Communications. In a step forward for intracellular biotherapeutics, this early stage investigational research also...
What We Learned
Our work has shown that blocking conditions for anti-peptide selections are even more critical than usual. Careful attention to these conditions is key to achieving successful outcomes in peptide targeting, and we have built an array of different blocking agents to address the needs of different projects. While we cannot disclose much about this project, it illustrates what our scientists can do, leveraging our huge VHH libraries when handling the most difficult targets.
A promising future
Isogenica’s biggest assets in the field of antibody discovery are the largest and most diverse synthetic VHH libraries available. But they can only be used to their biggest potential by our expert scientists. Our commitment to innovation and excellence ensures that we can provide solutions where others have not. Facing challenges with difficult targets? Let’s collaborate to turn your problems into groundbreaking solutions. Discover how Isogenica’s VHH libraries can be the key to your next success story. Read more about anti-peptide VHH discovery from our libraries in AstraZeneca’s published study, or reach out to us to discuss how we can support your VHH antibody discovery needs.
Sources:
1. Bever, C.S., et al., VHH antibodies: emerging reagents for the analysis of environmental chemicals. Anal Bioanal Chem, 2016. 408(22): p. 5985-6002.
2. De Genst, E., et al., Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure. Nat Commun, 2022. 13(1): p. 3018.
3. Pille, J., et al., Self-Assembling VHH-Elastin-Like Peptides for Photodynamic Nanomedicine. Biomacromolecules, 2017. 18(4): p. 1302-1310.
4. van Moorsel, M.V.A., et al., VhH anti-thrombomodulin clone 1 inhibits TAFI activation and enhances fibrinolysis in human whole blood under flow. J Thromb Haemost, 2022. 20(5): p. 1213-1222.