Technology - Bioengineering Platform for Delivery of Peptides into Cells

Bioengineering Platform for Delivery of Peptides into Cells

A platform bioengineering approach, which combines orthogonal protein crosslinking with supercharging protein surfaces, to design and develop cell-penetrating monobodies.

Background:

Intracellular targets, including a number of high-profile oncology-related ones, remain "undruggable". While small molecules are generally able to cross biological membranes more effectively than monoclonal antibodies (mAbs), they are often ineffective at modulating such intracellular protein-protein interactions (PPIs), especially those wherein there is a poorly defined binding pocket. On the flip side, mAbs offer superior specificity and affinity for their targets and appear to be more suitable for addressing PPIs when compared to small molecules but are generally unable to effectively penetrate cells and reach targets in the cytosol or nucleus. Efforts to address these shortcomings have led to the exploration and development of both nanobodies and monobodies, the latter of which are a class of antibody mimetics, or synthetic binding proteins, that are small in size, possess high specificity and affinity, lack disulfide bonds and can be genetically encoded for "synthesis" in even the reducing environments of the cytoplasm and nucleus. Despite these favorable attributes and although they have been demonstrated to be potent and selective inhibitors of key oncoproteins, monobodies still lack the ability to efficiently cross the plasma membrane when delivered exogenously.

Technology Overview:

This University at Buffalo invention provides compositions and methods related to the bioengineering of monobodies. More specifically, a novel, genetically encoded, electrophilic amino acid may be site-specifically introduced into a protein (e.g. monobody) of interest, which then undergoes spontaneous, intramolecular, proximity-driven orthogonal crosslinking with a nearby nucleophilic residue, resulting in a more stable product. When compared against several alternative approaches to the genetic incorporation of electrophilic amino acids into glutathione-S-transferase (GST), a model that has been used to assess the electrophilicity of noncanonical amino acids, the compositions of this invention appear to provide a superior crosslinking approach. A supercharged monobody prepared using this approach displayed not only improved cellular uptake when compared to a non-crosslinked monobody but also enhanced proteolytic resistance against cathespin B, an enzyme responsible for the degradation of protein cargoes in endosomes.