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

Abstract The compromise between affinity and specificity in TCR-dependent targeting of HLA-restricted tumor-associated antigens presents a significant challenge in developing efficacious immunotherapies. As such, T cell engagers which circumvent these limitations are of particular interest. We have established a process to generate bispecific Designed Ankyrin Repeat Proteins (DARPins) that simultaneously target HLA-I molecules in complex with tumor-associated peptides and CD3ε. High-affinity HLA-A*0201/NY-ESO1157-165-specific DARPins were isolated after only four rounds of in-vitro selection from naïve DARPin libraries. Combining HLA-A*0201/NY-ESO1157-165-specific DARPins with a CD3ε-specific DARPin created potent T cell engagers which elicited CD8+ T cell activation towards tumor targets with high peptide specificity, as confirmed by X-scanning mutagenesis and functional killing assays. The cryo-EM structure of a ternary DARPin/HLA-A*0201/NY-ESO1157-165 complex revealed a rigid and concave DARPin surface that binds to the entire length of the peptide-binding cleft, contacting both α-helices and the peptide. The present results unveil promising immuno-oncotherapeutic approaches with the possibility of rapidly developing DARPins with high affinity and specificity to HLA/peptide targets that can be readily combined with a new generation of anti-CD3ε-specific DARPins. Competing Interest Statement NV, SM, SF, MP, NP, DV, SB, AC, TH and MW are employed by Molecular Partners AG and hold options or shares in the company. VL was employed by Molecular Partners AG at the time of this research and holds options/shares in the company. All the other authors do not have any conflict of interest to disclose.