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Affinity targeting to the vitreous using bispecific protein conjugates

Ibeanu, Nkiruka Ozioma Amasi; (2023) Affinity targeting to the vitreous using bispecific protein conjugates. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Antibody-based medicines have revolutionised the treatment landscape of chronic retinal diseases, especially age-related macular degeneration. These medicines are administered by intravitreal injection as frequently as every two to four months. The advent of their use has prompted research into reducing the need for frequent intravitreal injections, which can be debilitating for patients. Affinity drug delivery offers a potential means of increasing retention of drugs in the vitreous chamber by interaction with hyaluronic acid or collagen II. Bispecific Fab-PEG-Fab molecules (bsFpFs) were prepared by site-specific conjugation capable of binding both to a component of the vitreous and to VEGF. Animal models are exclusively used to optimise intraocular medicines and formulations but are not appropriate for long-acting intraocular formulations due to the generation of anti-drug antibodies. The PK-Eye is a two-compartment aqueous outflow pharmaceutical model of the eye designed to estimate the outflow clearance of drugs as an alternative to animal models. Proof-of-concept experiments were utilized to prepare bispecific Fab conjugates by site-specific click conjugation, which involved the use of click-functionalised bis-alkylating reagents. Vitreous-anchoring bsFpFs (Fabbeva-PEG-Fabcoll and Fabbeva-PEG-FabHA) targeting collagen II and hyaluronic acid respectively were then prepared and evaluated for binding by dot blot studies. Three second-generation PK-Eye model setups were evaluated to better mimic the mass transfer properties of the eye which play a role in drug clearance. Bevacizumab was used to validate the models (ciliary inflow, posterior inflow and ciliary-hyaloid inflow models) with PBS as simulated vitreous fluid. The ciliary-hyaloid inflow model provided a means of evaluating the anchor concept with a simulated vitreous fluid composed of HA to which a synthesised bsFpF would bind for delayed clearance from the vitreous. The clearance t1/2s of IgGbeva (~5 days) and IgGHA (~14 days) were evaluated in the ciliary-hyaloid inflow model and HA SVF, with a longer t1/2 time for IgGHA due to vitreous anchoring. Although the clearance time of a bsFpF against HA (Fabbeva-PEG-FabHA) could not be determined due to low protein yield, the PK-Eye was effectively used as a simple, relatively inexpensive in vitro model to assess the anchor concept without the need for animals in early preclinical research.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Affinity targeting to the vitreous using bispecific protein conjugates
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology
URI: https://discovery.ucl.ac.uk/id/eprint/10180876
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