Silent in one assay, potent in another: VHHs unlock a new way to block the cancer resistance target CDK2

Joint case study profiles VHHs against CDK2/CycE1 in functional biochemical assays and finds binders acting through two distinct mechanisms.

CAMBRIDGE, UK and MILAN, ITALY, 27 October 2026 — A VHH antibody that had no effect on CDK2 kinase activity turned out to block the protein interaction that activates it, in a joint case study from Isogenica Ltd. and Axxam S.p.A. The study profiled VHHs against CDK2/CycE1, a cell-cycle complex linked to resistance to standard-of-care therapy in ovarian, breast and endometrial cancers. It found binders that inhibit the target in two ways: by blocking kinase activity, or by preventing the complex from forming.

CDK2/CycE1 regulates the G1 to S transition of the cell cycle. Selective small-molecule CDK2 inhibitors have entered clinical trials, but tumours can escape them by switching to the CDK4/6 pathway or by increasing CDK2 expression, which raises the dose needed and, with it, the risk of toxicity. According to the study, no VHHs against CDK2 or the complex had previously been reported.

Isogenica ran VHH selections against pre-activated CDK2 using their large, diverse synthetic libraries, discovering 192 VHH antibodies that bound the CDK2 antigen. Protein purification methods were introduced to Axxam’s HTS automation, providing each VHH as a pure protein for testing in functional assays.

In the first assay, which measured CDK2 enzyme inhibition, four VHHs showed two-digit nanomolar potency. Three of these also stopped the complex forming in a second assay. But the two assays did not always agree. One potent VHH had no effect on complex formation, and one that looked inactive in the first assay blocked the complex, suggesting it binds where CycE1 docks rather than at the enzyme’s active site.

A third assay, using a different readout, confirmed the inhibition at lower potency, demonstrating the advantage of using multiple assays to deepen understanding through parallel datasets, especially when looking for binders to novel epitopes or with unique functionality.

“Binding tells you a VHH has found its target. It doesn’t tell you what the VHH does there,” said Lurdes Rodrigues Duarte PhD, Director of R&D at Isogenica. “Working with Axxam, we could profile a large, diverse set of binders in functional assays early. That showed us mechanisms a binding screen would have missed, including a molecule that demonstrated an unexpected way of operating.”

“This study demonstrates how combining high-throughput VHH production with functional screening can transform the way we identify and characterize promising antibody candidates,” said Jean-François Rolland, Senior Vice President, Early Drug Discovery at Axxam. “By developing an automated platform capable of producing and purifying hundreds of VHHs in just a few hours, we can move rapidly from binder identification to functional characterization. This approach enables us to explore different mechanisms of action and identify promising candidates that might otherwise be overlooked.”

The study is the first output of the partnership Isogenica and Axxam announced in September 2024, combining Isogenica’s VHH discovery with Axxam’s functional assays and, now, its automated VHH production. Following this proof-of concept study, significantly larger VHH panels can be explored directly following selections, enabling function-first discovery for rare functional binders such as agonists.

The full case study is available at https://axxam.com/application-of-vhh-in-drug-discovery/