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Hackett, Gavin S. (2012) Intracellular delivery of therapeutic antibodies. PhD thesis, University of Nottingham.

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Abstract

Therapeutic antibodies are highly versatile macromolecules that can be engineered to bind and inhibit a target with high specificity. Unfortunately, the cell membrane is impenetrable to antibody reagents, thus limiting their use almost entirely to extracellular targets. Expanding the application of therapeutic antibodies to intracellular targets is an exciting concept that could have a huge impact on how intracellular protein-protein interactions involved in diseases can be modulated. The modification of therapeutic antibodies with Cell Penetrating Peptides (CPPs) can enable cellular penetration, however, no general approach to modifying antibodies with CPPs has been developed that allows for systematic optimisation of both the in vivo and cell penetrating properties.

In this study, neutralising single-chain variable fragment antibodies (scFvs) have been isolated from naïve scFv libraries using antibody phage display that are specific to the model intracellular targets Bcl-2 and Bcl-xL. Lead scFvs showed potent inhibition of these proteins in an in vitro assay with IC50 values of <10 nM being calculated, which is superior to the small molecule Bcl-2/xL inhibitor ABT-737. The lead anti-Bcl-xL scFv was conjugated to the CPPs octa-arginine, HIV Tat49-57 or Antp52-58. These peptides were synthesised to possess either an N-isobutyryl cysteinyl or N-maleimidopropionyl moiety and a C-terminal lysine residue, allowing for site-specific conjugation to an unpaired cysteine residue introduced to the scFv construct and regioselective introduction of 5-carboxyfluorescein to the CPP, respectively. Live-cell confocal microscopy showed that the scFv-octa-arginine conjugate possessed superior cell entry capabilities compared to the scFv-Tat49-57 and scFv-Antp52-58 conjugates. Further studies using a panel of cancer cell lines are required to determine if the anti-Bcl-xL scFv-octa-arginine conjugate can induce apoptosis through inhibition of cellular Bcl-xL.

Additonally, a novel approach to controlling the cell penetrating properties of the CPP octa-arginine has been developed. It was demonstrated that carbamate protection of octa-arginine’s guanidine functionality effectively inhibited its cell entry capabilities. Moreover, esterase-labile acyloxymethyl carbonyl (AM) protecting groups were utilised to protect the guanidine functionality of octa-arginine, inhibiting its cell entry capabilities. In a HPLC based assay it was demonstrated that the AM protected octa-arginine was deprotected by pig liver esterase, suggesting that in vivo deprotection could be achieved by serum esterases. The controlled unmasking of octa-arginine is predicted to increase its circulation time and reduce non-specific tissue uptake in vivo, potentially making this CPP more suitable for in vivo applications.

The methodologies utilised for the preparation of scFv-CPP conjugates and developed for the controlled unmasking of octa-arginine in this study will allow for optimisation of the cell penetrating and in vivo properties of this promising class of macromolecular therapeutic, thus providing a gateway to unlocking the immense potential of therapeutic intracellular antibodies.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Fischer, P.M. Chan, W.C. Jackson, R.
Subjects:	Q Science > QP Physiology
Faculties/Schools:	UK Campuses > Faculty of Science > School of Pharmacy
Item ID:	12611
Depositing User:	EP, Services
Date Deposited:	01 Oct 2012 13:01
Last Modified:	16 Dec 2017 21:15
URI:	https://eprints.nottingham.ac.uk/id/eprint/12611

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