Development of HER2-targeted nanobodies for molecular optical imaging and therapy of breast cancer

Breast cancer is a complex disease and the most prevalent cancer in women worldwide. It has been estimated that 1 in 8 women and 1 in 1,000 men will develop breast cancer. Surgical-, chemical- and radiation based therapies are available to breast cancer patients. Early detection of cancer is crucial for its successful removal. Here a nanobody-based optical imaging approach for improved breast cancer imaging is presented. Nanobodies are derived from heavy chain only antibodies – a unique antibody species present in Camelidae. Nanobodies are the smallest, naturally derived antigen fragments, which even though 10x smaller than conventional antibodies, retain high affinity (in low nanomolar range) and specificity. In this thesis several protocols for obtaining nanobodies targeting HER2 receptor are presented. HER2 receptor is overexpressed in breast cancer and is associated with a more aggressive disease. Patients overexpressing HER2 receptor are subjected to a treatment with a monoclonal antibody - Herceptin® (trastuzumab). Nevertheless, less than 30% of patients respond to the initial treatment. Moreover, 70% of patients who initially responded will develop resistance. For this reason, finding alternative treatment options is of great importance. The first part of the thesis focuses on nanobody-based imaging. We developed HER2-specific nanobodies and conjugated them to a near infra red dye, IRDye800CW. We show that site-directed conjugation of the dye allows for preservation of nanobody affinity and specificity (random conjugation results in 1000 fold affinity drop). In in vivo studies we show that HER2-nanobodies-IRDye were able to clearly visualize HER2 positive breast cancer tumors shortly after injection. No fluorescence was observed in the HER2 negative tumors, proving the specificity of tested compounds. Furthermore, accumulation of nanobody-IRDye at the HER2 positive tumor resulted in obtaining the same contrast as with monoclonal antibody trastuzumab-IRDye much faster (4 hrs post injection versus 72 hrs post injection). We also showed that the contrast between normal tissue and tumor may be improved by injecting a combination of nanobodies recognizing different receptors. Injection of a combination of nanobodies differing in specificity and conjugated to two different fluorophores allows for determination of tumor molecular status. Determination of tumor molecular status is important for planning of patient’s treatment. Development of HER2-specific nanobodies binding to canine HER2 receptor is also presented in the thesis. This part was challenging as not all tools needed for nanobody development were available in this part of the project. We also show that HER2-specific nanobodies may be employed in electron microscopy based protocols for HER2 receptor visualization. Results obtained with nanobodies are comparable with stainings performed using monoclonal antibody trastuzumab. In the second part of the thesis two therapeutic approaches are presented. The first one is a nanobody based photodynamic therapy, where nanobodies are used to direct photosensitizer to the tumors site, whereas in the second approach nanobodies are used to target nanoparticles which encapsulate toxic payload, in this case an enzyme degrading RNA (namely RNase). In both cases we show that these nanobody-based drugs lead to death of HER2 positive cancer cells, whereas HER2 negative cells remain unaffected