A Platform for Engineering Cyclotide Therapeutics
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porth-oporth-phd-be-2025-thesis.pdf
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Author(s)
Porth, Owen T.
Advisor(s)
Wittrup, K. Dane
Date Issued
May 2025
Publisher
Massachusetts Institute of Technology
Abstract
Cyclotides are tiny, disulfide-rich cyclic peptides that exhibit exceptional stability to extremes of pH, heat, and protease concentration. Their solvent-exposed loops are amenable to extensive mutation, similar to antibody complementarity determining regions. Unlike antibodies, their small size and hyperstability makes them exciting frameworks for the engineering of orally delivered therapeutics. Such binders could be used as protein-protein interaction inhibitors for gastrointestinal indications. Seeing this therapeutic opportunity, our goal was to create a versatile yeast surface display library for the isolation of cyclotide binders and use this platform to develop an orally available inhibitor of TNF. In this thesis, we describe a set of tools and methods comprising a nascent cyclotide platform, along with recommendations for their successful implementation and ideas for their improvement.
We created a yeast surface display library from linearized versions of MCoTI-I and kB1 cyclotides randomized in loop 1 and 6 for MCoTI-I and loop 6 in kB1. We screened these naïve libraries with and without terminal constraints that were imposed by a bovine ultralong (BUL) single chain variable fragment. The MCoTI-I libraries outperformed the kB1 libraries and the BUL-imposed terminal constraints proved essential to the isolation of an MCoTI-I-derived binder to murine TNF (mTNF). The efficacy of the libraries was stymied by the rapid accumulation of nonspecific binders and the low conformational diversity of the wildtype loop length libraries. We developed methods to better deplete the library of nonspecificity and were able to generate a double-digit nanomolar binder to murine SHP2.
We affinity matured the mTNF-binding clones, yielding a significant improvement in binding signal. We found that these binders were resilient to framework mutations, were relatively resistant to protease, and competed with murine TNF receptor 1 (mTNFR1) to bind mTNF. We developed methods to produce linear and cyclic versions of the binders. We solved the structure of the affinity matured, framework-stabilized mTNF binder – gaining insight into an unusual proline isomerization phenomenon that affects their binding. Biophysical characterization revealed that the binders benefited from a massive avidity effect when binding the trimer mTNF on yeast. Epitope mapping revealed that the binders share overlapping epitopes with Humira and mTNFR1/2. Surprisingly, the peptides have no inhibitory effect in HEK-Blue-TNF assay and their BUL-antibody versions actually enhance TNF signal. This suggested an unusual binding mechanism more similar to small molecule inhibitor of TNF than Humira. We modified the peptides to bind human TNF and devised new affinity maturation methods to drastically increase their affinity and efficacy as inhibitors.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
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