Abstract
Biologics are a class of therapeutic molecules which can be characterised by their specificity and potency of action. Administration of biologics is limited to injection due to negligible intestinal absorption. This method of administration is typically less accepted by patients and expensive to manufacture and administer. Several barriers limit systemic absorption of biologics, but the intestinal epithelium is the most formidable and challenging to overcome. Current approaches to this challenge employ 'absorption enhancers' that non-selectively disrupt and increase intestinal permeability, but safety concerns around the chronic use of these methods (e.g., many surfactants) have hindered clinical translation. Therefore, there is an unmet need in safe and effective technologies for the oral delivery of biologics.Key to safe and effective oral delivery of biologics are materials that 'smuggle' drugs selectively across the intestinal mucosa without barrier disruption. Ideally, these would be conjugated to the drug or drug delivery systems. This project has focussed on developing novel transcytosis-enabling materials that meet these criteria. In this case these are peptide-based, taking inspiration from IgG which readily crosses the intestinal mucosa by transcytosis via the neonatal Fc receptor (FcRn). This method of receptor-targeted drug delivery responds to and addresses safety concerns surrounding the chronic use of permeation enhancers. Preparation of transcytosis-enabling compounds has been informed by the receptor and ligand binding epitopes of IgG and HSA to FcRn.
Proxima Concepts' discovery platforms (Mozaic™ and Almanac™) have been used to prepare transcytosis-enabling peptide constructs (Lexicon™). Using Mozaic, the structure of prototype transcytosis-enabling peptides is informed by a screening library of micellar nanoparticles which bear different combinations of amino acids on their surfaces. In parallel, Almanac (algorithms derived from data of structural analyses of protein-protein interactions) has been used to infer the structure of ligand-binding peptides based on receptor binding domain sequences. As an extension of Almanac, molecular dynamics simulations of peptide and protein binding interactions has been used to inform and validate prototype peptide designs.
The cytotoxicity of Mozaic micelles and transcytosis-enabling Lexicon peptides was quantified using MTS and LDH toxicity assays, from which suitable concentrations for use in tissue culture experiments were determined. LQS Mozaic micelles showed transport over time across the Caco-2 epithelial model, through the detection of a fluorescent rhodamine label in the basolateral media, in partial agreement with previous results in a mouse model.
The Almanac method was used to design Lexicon peptides which were determined to have a rupture force ~1200 pN greater than control peptides, using constant velocity pulling steered molecular dynamics (CVP SMD). Once synthesised, these peptides were shown to bind to FcRn using the Lumit FcRn binding assay from Promega, and their trans-epithelial transport was quantified using the Caco-2 intestinal cell model.
This project confirmed the effectiveness of the Almanac method for designing peptides which bind to a selected target site. The designed peptides did not show significant improvement in FcRn binding or transport across Caco-2 monolayers over similar control peptides, but their transport in the intestinal model was inhibited by the native ligand, indicating successful targeting of the FcRn transcytosis pathway. This project has also established a methodology for the design and testing, in silico and in vitro, of peptides which bind selectively to any target protein of choice. With further testing and refinement, the Lexicon peptide is a promising candidate for an orally-administered and specifically-targeted biologic delivery platform.
Date of Award | 1 Jan 2024 |
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Original language | English |
Awarding Institution |
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Supervisor | Maya Thanou (Supervisor) & Driton Vllasaliu (Supervisor) |