Rational Design of Novel Antibody Decorated Nanoparticles Targeting Breast Cancer Cells

Abstract

Every year there are over 50,000 new cases of breast cancer in UK, which represents the most common UK cancer (CR-UK). Despite significant advances in anti-breast cancer treatments, there is still a need for improved therapeutics to overcome drug resistance. HER2 (Human Epidermal Growth Factor Receptor 2) is a transmembrane oncoprotein encoded by the HER2/neu gene and overexpressed in approximately 20 to 30% of invasive breast cancers. Tumours overexpressing HER2 are more aggressive and carry a poor prognosis; thus, the receptor is a priority therapeutic target. One targeting entity is Trastuzumab (Tz), a monoclonal antibody recognized as one of the most effective agents against HER2+ breast cancer and has also been attached to chemotherapeutics to form antibody-drug conjugate (ADC). These ADCs, such as Kadcyla®, require cell binding to HER2 and access to the cell interior by endocytosis to release the payload. HER2 is, however, commonly termed the "endocytosis deficient' member of the HER family of receptors, thus challenging attempts to design ADCs that need access to lysosomes for drug release and activity. Previous studies in the laboratory showed that HER2 endocytosis was significantly promoted with concomitant lysosomal delivery and degradation via Tz-mediated crosslinking, and this presented work lies under the hypothesis that nanoparticles (NPs) decorated with sufficient numbers of Tz could also cause HER2 cross-linking, endocytosis, and HER2 degradation. Later data showed that HER2 crosslinking induced a form of endocytosis termed macropinocytosis to drive cell entry. The work presented initially investigated macropinocytosis as a process in different cell types and ways to inhibit this process using inhibitors targeting the sodium proton exchanger (NHE1) as a regulator of intracellular pH and this endocytic process. EIPA (5-(N-Ethyl-N-isopropyl) amiloride) as a macropinocytosis inhibitor, surprisingly significantly increased the internalisation of HER2; a result not observed with other NHE1 inhibitors amiloride and the more selective NHE1 inhibitor cariporide. EIPA was also shown to increase the uptake of the fluid phase and macropinocytosis marker dextran but had no effect on endocytosis of transferrin via clathrin-coated vesicles. The results suggest that EIPA targets need further analysis as modulators of HER2 internalisation and targets for breast cancer therapy. Fluorescently labelled Tz- decorated Poly (lactic glycolic acid) NPs were then generated and found to be highly selective for HER2 expressing breast cancer cells over controls. Upon incubation with cells, the decorated NPs rapidly accumulated on the cell surface and also appeared as large intracellular structures suggestive of macropinosomes. Rhodamine and Doxorubicin encapsulated Tz-PLGA NPs were synthesised, showing their capacity to drive internalisation of the fluorophore and cytotoxic drug into vesicular structures, with the later formulation enhancing the cytotoxicity of the drug over its soluble counterpart.