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

Mirror-image proteins, 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 d-target proteins, phage display library selection of l-binders and chemical synthesis of (mirror-image) d-binders that consequently bind the physiological l-targets. Monobodies are 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 develop 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 reveal targeting of the pY binding pocket by an unconventional binding mode. We then prepare potent d-monobodies by either ligating two chemically synthesized d-peptides or by self-assembly without ligation. Their proper folding and stability are determined and high-affinity binding to the l-target is shown. d-monobodies are protease-resistant, show long-term plasma stability, inhibit BCR::ABL1 kinase activity and bind BCR::ABL1 in cell lysates and permeabilized cells. Hence, we demonstrate that functional d-monobodies can be developed readily. Our work represents an important step towards possible future therapeutic use of d-monobodies when combined with emerging methods to enable cytoplasmic delivery of monobodies.