Cancer is one of the leading causes of premature death in the world and its incidence and mortality are rapidly growing worldwide. Despite the biological heterogeneity of cancer, most patients are still treated with generic therapies, which often result in negative aftermath on the patients’ health. Adverse effects can be reduced by treatments targeting molecules which are exclusively expressed by cancer cells. One such molecule is the ganglioside N-glycolyl GM3 neuraminic acid (NeuGc GM3), which is present on the surface of several human cancer cell types and is not found in healthy tissue. This tumor-specific ganglioside is targeted with high specificity by the monoclonal antibody 14F7, which was produced in Cuba two decades ago. Unlike most anti-tumor antibodies, 14F7 has the outstanding ability to induce cancer cell death without recruiting any component of the immune system. To date, the cell killing mechanism of 14F7 is still poorly understood. In order to fully exploit its potential in cancer therapy, the fate of 14F7 after binding to the target has to be unraveled. The aim of this Master’s project was to gain insights into the internalization abilities of 14F7. After binding to the ganglioside, does the antibody travel inside the cell? Here, a 14F7 single-chain variable fragment (scFv) was covalently connected to a luciferase called NanoLuc. Due to its bright luminescence and pH-dependent activity, the NanoLuc has been identified as a suitable reporter for internalization studies. The vector coding for the fusion protein 14F7 scFv-NanoLuc was cloned through a PCR- based method, named Gibson assembly. A production protocol based on control of the glucose feed in Escherichia coli was established. The protein was effectively purified with immobilized metal affinity chromatography followed by size-exclusion chromatography. The binding affinity of the 14F7 scFv domain to NeuGc GM3 was confirmed by ELISA. The NanoLuc revealed a very high brightness and a robust pH- dependent activity. A recently developed assay to quantify the amount of a cell surface receptor, named Topanga assay, allowed quantification of NeuGc GM3 on several cell lines. Using the Topanga assay as a starting point, a novel assay to obtain insights into the internalization kinetics was developed. Interestingly, high amounts of NeuGc GM3 on the cell surface corresponded to faster internalization. This work paves the way for the application of scFv-C1-Nluc in bioluminescence microscopy in live cells.