Evaluation and optimisation of new engineered human human apoferritins: protein nanocages for targeted drug delivery and intracellular cargo release

The targeted delivery of drugs which maximises their therapeutic efficacy whilst minimising the side effects has been a significant goal since Paul Ehrlich coined the term 'magic bullet' in 1907. The advent of monoclonal antibody technology in 1975 provided a protein that could target specific cells and was heralded as an example of a 'magic bullet'. Antibodies can be used as therapeutic agents on their own as demonstrated with the breast cancer treatment Herceptin or more recently, as drug carriers and therapeutics (Antibody Drug Conjugates) to target less selective drugs to tumour cells as demonstrated by Kadcyla. Whilst antibody-based systems are used clinically, they do have significant disadvantages which include only being able to deliver a small number of drugs per antibody and being expensive to produce because this requires mammalian cells. We wish to develop a new targeted drug delivery system based on the human protein, apoferritin. This protein is made up of 24 subunits and self-assembles above pH 2.0 to form a hollow sphere (nanocage) 12 nm in diameter. We can trap up to 500 drug molecules in a single nanocage (compared with 3-8 per antibody, attached to their external surface). Apoferritin is naturally taken up into cells using a membrane receptor called TfR1. It does this as encapsulates iron ions and delivers these to the cytoplasm of the cell for them to grow. Whilst some cancers express elevated amounts of TfR1 as they grow faster and this allows them to be targeted by natural apoferritin, many cancers express other surface proteins (biomarkers). These can be targeted by antibodies or other proteins including a recently developed much smaller protein called an affibody. Affibodies that selectively bind epidermal growth factor receptors (EGFR, HER2, HER3) that are found at much higher levels in some cancers have been identified. By combining the targeting ability of affibodies with the drug encapsulation and membrane crossing ability of apoferritin using synthetic biology, we can generate new drug delivery systems that delivery much higher amounts of drugs per protein including ones that are sensitive to being metabolised in the blood if not protected by encapsulation. In this project we will make a library of affibody-apoferritin fusion proteins that can be mixed together in different ratios to optimise the targeted drug delivery properties against a range of common cancer cell types. In a preliminary study, we have shown that apoferritin encapsulation of the brain cancer drug temozolomide makes it effective against cells that have developed resistance to the action of the drug if delivered on its own. If this is seen with other drugs, it offers the opportunity to extend the period a drug is effective. As part of the study we will examine two classes of compounds that have good activity against cancer cells in vivo but because they are not taken up by cells efficiently, they cannot be used as therapeutics. The Mission award will allow us to comprehensively evaluate the affibody-apoferritin system to determine if it can become the 'next generation' targeted drug delivery system or 'trojan horse' following on from antibodies, for a wider variety of different drug types. Unlike immunoglobulin G antibodies, the affibody-apoferritin subunits can be produced in bacteria or other non-mammalian cells. This means that they can be produced at low cost and on a larger scale and much more sustainably than antibodies as will be required if they are to be readily available worldwide.

The mission award will be used to comprehensively evaluate the drug delivery properties of a range of apoferritin (AFt) based protein nanocages (capable of encapsulating 500 drug molecules) that have been modified to incorporate affibodies (Afbs) that target human epidermal growth factor receptors (HERX). The project will focus on fully exploiting the pH dependent self-assembly properties of AFt to bring together defined mixtures of engineered AFt and Afb-AFt subunits to form a medium size library of nanocages that display different ratios of the affibodies. The library of cages will be screened individually using a combination of FACS and automated microscopy to identify those that are most efficiently taken up by a range of 5 cell lines that express the targeted receptors at varying levels. Single molecule fluorescence microscopy methods will be used to determine the uptake mechanism of the Afb-AFt cages that are internalised in the largest quantities in each cell line and also the fate of fluorescent drug cargoes. The top performing Afb-AFts will then be radiolabelled and their distribution in both healthy and tumour-bearing mice determined. The uptake studies will dissect out the relative importance of Afb-HERX and AFt-transferrin receptor 1 (TfR1) mediated endocytosis to internalisation as well as the influence the AFt TfR1 binding has on in vivo distribution of the nanocages. Complementary engineering of the AFt cages to prevent cargo leakage and improve cargo release in the early endosome will be undertaken to further enhance the drug delivery properties. The encapsulation and biological activity of two new classes of therapeutic cargo will be examined to exemplify the versatility of the AFt cage and its ability to internalise challenging molecules. The Afb-AFt system can be expressed in E. coli as it does not require any disulfide bond formation which makes it an attractive, more sustainable and low cost alternative to an antibody-based drug delivery system.