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Holden, Isobel R (2025) Development and Encapsulation of Affibody-Apoferritin Biologics for the Targeted Treatment of Breast Cancer. PhD thesis, University of Nottingham.

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Abstract

Targeted cancer therapies produce more favourable clinical outcomes for cancer patients compared to unselective small-molecule cytotoxic chemotherapeutics. Currently, the most widely available targeted anti-cancer therapies are antibody-drug conjugates (ADC) with an example being tratuzumab-DM1 (Kadcyla®) which is a first line treatment for human epidermal growth factor 2 (HER2, ErbB-2) positive breast cancer. To develop a new improved biological anti-cancer therapy and drug delivery system, a fusion protein has been constructed, composed of a small 5 kDa affibody targeted towards an ErbB receptor: EGFR (ErbB-1), HER2 (ErbB-2) or HER3 (ErbB-3) and attached via a flexible glycine-serine linker at the N-terminus of a subunit of human heavy chain apoferritin. This creates a hollow spherical affibody-apoferritin nanocage (8 nm internal diameter) with 24 targeting affibodies displayed in an ordered pattern on the cage surface (14 -15 nm external diameter).

These protein nanocages display high levels of selective cytotoxic activity, producing IC50 values of: 13 pM for ZEGFR-apoferritin against MDA-MB-468 (a high EGFR expression breast cancer); 190 pM for ZHER2-apoferritin against SKBR3 (a high HER2 expression breast cancer) and 18 pM for ZHER3-apoferritin against T47D (a high HER3 expression breast cancer). In this research, the affibody-apoferritin cages were also used for encapsulation via reassembly; resulting in apoferritin cages with multiple different types of targeting affibodies displayed on the surface and small molecular dyes or anti-cancer therapeutics encapsulated. To aid this functionality, a new method of encapsulation was developed, ‘nanotitration’ (NTC), which qualitatively improves the number of small molecules that can be encapsulated. This encapsulation method also increased cell inhibition as measured by MTT and luciferase assays. The affibody-apoferritin fusion protein has been shown to outperform current anti-HER2 agent, trastuzumab in vitro and has even greater efficacy against EGFR and HER3 positive cell lines, overall evidencing affibody-apoferritins to be selective and highly potent experimental anti-tumour agents.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Thomas, Neil R. Bradshaw, Tracey D.
Keywords:	breast cancer, anti-cancer therapies, affibody-apoferritin, nanocages
Subjects:	Q Science > QP Physiology > QP501 Animal biochemistry R Medicine > RC Internal medicine > RC 254 Neoplasms. Tumors. Oncology (including Cancer) R Medicine > RS Pharmacy and materia medica
Faculties/Schools:	UK Campuses > Faculty of Science > School of Pharmacy
Item ID:	78748
Depositing User:	Holden, Isobel
Date Deposited:	30 Jul 2025 04:40
Last Modified:	30 Jul 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/78748

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