Barnase*Barstar-guided two-step targeting approach for drug delivery to tumor cells in vivo.

For precise ligation of a targeting and cytotoxic moiety, the use of Barnase-Barstar pair as a molecular glue is proposed for the first time. Targeting was mediated through the use of a scaffold protein DARPin_9-29 specific for the human epidermal receptor 2 (HER2) antigen that is highly expressed on some types of cancer and BarnaseBarstar native bacterial proteins interacted with each other with K 10 M. The approach proposed consists of prelabeling a target tumor with hybrid protein DARPin-Barnase prior to administration of cytotoxic component-loaded liposomes that have Barstar covalently attached to their surface. Based on in vivo bioimaging we have proven that DARPin-based BarnaseBarstar-mediated pretargeting possesses precise tumor-targeting capability as well as antitumor activity leading to apparent tumor-growth inhibition of primary tumors and distant metastases in experimental animals. The results obtained indicate that the new system combining DARPin and Barnase*Barstar can be useful both for the drug development and for monitoring the response to treatment in vivo in preclinical studies.