Development of DARPin T cell engagers for specific targeting of tumor-associated HLA/peptide complexes.

Venetz-Arenas N, Schulte T, Müller S, Wallden K, Fischer S, Resink T, Kadri N, Paladino M, Pina N, Radom F, Villemagne D, Bruckmaier S, Cornelius A, Hospodarsch T, Alici E, Ljunggren HG, Chambers BJ, Han X, Sun R, Carroni M, Levitsky V, Sandalova T, Walser M, Achour A

iScience 28 (12) 113926 [2025-12-19; online 2025-11-03]

The balance between affinity and specificity in T cell receptor (TCR)-dependent targeting of HLA-restricted tumor-associated antigens presents a significant challenge for immunotherapy development. T cell engagers that circumvent these limitations are therefore of particular interest. We established a process to generate bispecific designed ankyrin repeat proteins (DARPins) that simultaneously target HLA-I/peptide complexes and CD3e. These high-affinity T cell engagers elicited CD8+ T cell activation against tumor targets with strong peptide specificity, as confirmed by X-scanning mutagenesis and functional killing assays. A cryo-EM structure of the ternary DARPin/HLA-A∗0201/NY-ESO1157-165 complex revealed a rigid, concave DARPin surface spanning the full length of the peptide-binding cleft, contacting both α-helices and the peptide. The present findings reveal promising immuno-oncotherapeutic approaches and demonstrate the feasibility of rapidly developing DARPins with high affinity and specificity for HLA/peptide targets, which can be readily combined with a new generation of anti-CD3e-specific DARPins.

PubMed 41321628

DOI 10.1016/j.isci.2025.113926

Crossref 10.1016/j.isci.2025.113926

pmc: PMC12664392
pii: S2589-0042(25)02187-X