Ferritin-based anticancers

In this PhD thesis, human heavy chain ferritin (hereafter HuHf) and some of its variants were successfully produced for a dual purpose: the development of HuHf-based bioconjugates for the targeted delivery of metal-based anticancer drugs and the development of platforms for ferritin-based anticancer vaccines. HuHf was conjugated with various metal-based compounds, in particular the two gold drugs auranofin (AF) and aurothiomalate (AuTM), and the candidate drug gold carbene Au(NHC). By integrating ESI-MS results obtained on the wild-type protein and site-specific mutants with MD modeling and cryo-EM structural determination, the binding sites and binding modes of these metal compounds were firmly established, highlighting a number of peculiarities in the coordination chemistry of protein-bound gold(I) species. For comparison, the binding of HuHf to Hg2+ and Pt2+ compounds was also characterized. The high affinity of gold(I) compounds for HuHf is also responsible for their stability with respect to other biological competitors. The use of HuHf as a nanocarrier for anticancer metallodrugs is based on its ability to interact and be internalized via the TfR1 receptor. We have shown here that TfR1 is expressed at significantly higher levels in several cancer cell lines (A2780, U87MG, MCF-7, HCT-116) than in non-tumor lines (HEK and MRC-5) and that the extent of HuHf uptake (free or metal-conjugated) is indeed proportional to TfR1 levels. Focusing on A2780 ovarian cancer cells, we demonstrated that HuHf conjugates with the two drugs (namely HuHf@AF and HuHf@AuTM) are more cytotoxic than the free drugs. Importantly, under the same toxicity conditions, 1H NMR analyses on A2780 ovarian cancer cells showed the same effect on the metabolomic profile regardless of whether the treatment was based on the free or HuHf conjugated compounds. Finally, we demonstrated that HuHf can efficiently enter dendritic cells. A prototype antigen-exposing ferritin (HuHf-OVA) was produced. Together, these two results provide proof-of-concept for the development of HuHf-based cancer vaccines.