Engineering Carriers for Therapeutic Protein Delivery to Intracellular Targets
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Dhankher, Anshul
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Abstract
Over the past several decades, advances in recombinant DNA technology and protein engineering have led to the use of protein therapeutics in the treatment of many diseases. Despite their many benefits, protein therapeutics cannot cross the cell membrane and are restricted to extracellular targets in the bloodstream or on the surface of cells. At the same time, many diseases could be treated by delivery of these proteins to certain intracellular targets which have long been considered undruggable by traditional small molecule therapies. To address this delivery challenge, we previously developed the Hex carrier that could bind antibodies for intracellular delivery. We showed delivery of functional, bioactive antibodies to intracellular targets with this carrier. In this work, we further developed the Hex carrier for broader applications to deliver other binding proteins while also evaluating the structure of Hex carrier assemblies and developing a mechanistic understanding of its delivery into cells. By using a suite of characterization techniques, we identified the most stable configuration of Hex-antibody complexes and controlled their dynamic rearrangement to promote formation of stable, uniform protein assemblies. Although characterization of Hex-antibody complexes’ stability in serum showed that antibodies would not remain bound to the Hex carrier in vivo limiting the translational potential of this strategy, fundamental insights into the role of different building blocks on the delivery function of Hex suggested a broader use for delivering other proteins aside from just antibodies. Therefore, we created fusion proteins of the Hex carrier and the DARPin protein scaffold to deliver anti-KRAS binding DARPins into cancer cells. Mutations in KRAS are found in a number of cancers and represent a key undruggable target in cancer biology. We found that delivery of anti-KRAS DARPins reduced proliferation of colon and lung cancer cells, showing that Hex carriers could deliver functional binding proteins into cells for inhibition of undruggable targets in cancer. This work establishes the DARPin Hex proteins as a potential anti-KRAS cancer therapy and shows that Hex carriers are a generalizable platform for intracellular delivery of therapeutic proteins.
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2021-08-25
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Dissertation