Silva,Frederico Nuno Castanheira Aires daDionísio,Francisco André de Campos PereiraSilva,Juan José Ribeiro da2026-06-022026-06-022026http://hdl.handle.net/10400.5/118913Tese de Mestrado, Microbiologia Aplicada, 2026, Universidade de Lisboa, Faculdade de CiênciasBreast cancer remains one of the leading causes of cancer-related mortality worldwide, with HER2-positive subtypes representing particularly aggressive forms due to their heterogeneity and treatment resistance. Targeted therapies using monoclonal antibodies like Trastuzumab have improved patient outcomes; however, resistance and poor tumour penetration remain significant obstacles. Single-chain variable fragments (scFvs), which retain the antigen-binding regions of antibodies but are smaller in size, offer enhanced tumour penetration and are being explored as alternative therapeutic agents. This study aimed to engineer and characterise a novel recombinant immunotoxin using a microbiology based molecular approach. The therapeutic molecule consists of a fusion between an scFv derived from Trastuzumab (anti-HER2 monoclonal antibody) and the catalytic domain of diphtheria Toxin (DT) from Corynebacterium diphtheriae. The scFv-DT construct was cloned and expressed in Escherichia coli (BL21), enabling large-scale recombinant protein production. Purification was achieved through Immobilized Metal Affinity Chromatography (IMAC), followed by a refolding step via dialysis. In vitro characterisation using ELISA, flow cytometry and immunofluorescence confirmed preserved HER2-specific binding and reactivity in both human breast cancer and feline mammary carcinoma cells. Efficient cellular internalisation was also observed, an essential requirement for DT-mediated citotoxicity. However, cytotoxicity assessment using the WST-1 assay revealed no significant toxic effect, indicating that despite proper binding and internalization, the toxin was not being adequately activated inside the cells. To investigate this, a computational 3D model of the immunotoxin bound to HER2 was generated using AlphaFold and ChimeraX. suggesting possible conformational constraints. In conclusion, this study demonstrates the successful development of a HER2-targeted scFv-DT immunotoxin with strong binding and internalisation. Future studies should focus on linker optimization and genetic modifications to ensure proper intracellular activation of the toxin, paving the way for a more effective therapeutic strategy in both human and veterinary oncology.application/pdfporTrastuzumabimmunotoxinHER2diphtheria toxinmaster thesis