Combining bacterial display and protein language models to engineer a CD69-binding affibody for molecular imaging of immune activation

Recent years have seen remarkable clinical success with therapies that harness the patient's immune system, with checkpoint inhibitors in oncology as a prominent example. Non-invasive monitoring of immune activation in vivo has the potential to accelerate both basic immunology research and clinical drug development, offering a valuable means to track responses to emerging immunotherapies. CD69 is a rapidly induced activation marker on lymphocytes and other leukocytes, making it an attractive imaging target. For such applications, affibody molecules offer distinct advantages as radio imaging tracers due to their small size, high affinity, and rapid pharmacokinetics, resulting in excellent imaging contrast. Here, we combined directed evolution with computational design to optimise a CD69-binding affibody molecule. An alanine scan of the parental binder informed the construction of a diversification library, which was displayed on Escherichia coli and subjected to iterative MACS and FACS selections with stringent off-rate competition. The top selection hit was subsequently refined through site-directed mutagenesis, including variants suggested by a general protein language model. The resulting lead, Z1525, bound human CD69 with single-digit nanomolar affinity and showed improved thermal stability while retaining solubility and refolding capacity, consistent with suitability for radiolabelling and in vivo targeting. Most importantly, it displayed selective binding to CD69 on stimulated Jurkat cells, with negligible binding to resting cells. These results establish E. coli display with off-rate-driven selection as an efficient strategy for affibody affinity maturation and demonstrate that protein language models can effectively guide improvements in folding stability. Z1525 represents a promising radio imaging tracer candidate for monitoring immune activation in vivo and warrants further preclinical development.