Development of mirror-image monobodies targeting the oncogenic BCR::ABL1 kinase

Abstract

Mirror-image proteins, which are composed of d-amino acids, are an attractive therapeutic modality, as they exhibit high metabolic stability and lack immunogenicity. Development of mirror-image binding proteins is achieved through chemical synthesis of the d-target protein, phage display library selection of an l-binder and chemical synthesis of the (mirror-image) d-binder that consequently binds the physiological l-target. Monobodies are among the most well-established synthetic (l-)binding proteins and their small size (~90 residues) and lack of endogenous cysteine residues make them particularly accessible to chemical synthesis. Here we developed monobodies with nanomolar binding affinities against the d-SH2 domain of the leukemic tyrosine kinase BCR::ABL1. Two crystal structures of heterochiral monobody-SH2 complexes revealed targeting of the pY binding pocket by an unconventional binding mode. We then prepared stable and potent d-monobodies by either ligating two chemically synthesized d-peptides or by self-assembly without ligation. Their proper folding and stability were determined and high affinity binding to the l-target was shown. d-monobodies were protease-resistant, showed long-term plasma stability, inhibited BCR::ABL1 kinase activity and bound BCR::ABL1 in cells. Hence, we demonstrate that functional d-monobodies can be developed readily, enabling their use as future d-protein therapeutics to target a broad spectrum of protein-protein interactions.