Hedda Johannesen

• Bjerregaard-Andersen, Kaare; Johannesen, Hedda; Abdel-Rahman, Noha Mansour Hassan; Heggelund, Julie Elisabeth; Hoås, Helene Mykland; Abraha, Fana; Bousquet, Paula; Høydahl, Lene Støkken; Burschowsky, Daniel; Rojas, Gertrudis; Oscarson, Stefan; Løset, Geir Åge & Krengel, Ute (2018). Crystal structure of an L chain optimised 14F7 anti-ganglioside Fv suggests a unique tumour- specificity through an unusual H-chain CDR3 architecture. Scientific Reports.  ISSN 2045-2322.  8:10836, s 1- 11 . doi: 10.1038/s41598-018-28918-5 Fulltekst i vitenarkiv. Vis sammendrag

  Targeted cancer immunotherapy offers increased efficacy concomitantly with reduced side effects. One antibody with promising clinical potential is 14F7, which specifically recognises the NeuGc GM3 ganglioside. This antigen is found in the plasma membrane of a range of tumours, but is essentially absent from healthy human cells. 14F7 can discriminate NeuGc GM3 from the very similar NeuAc GM3, a common component of cell membranes. The molecular basis for this unique specificity is poorly understood. Here we designed and expressed 14F7-derived single-chain Fvs (scFvs), which retained the specificity of the parent antibody. Detailed expression and purification protocols are described as well as the synthesis of the NeuGc GM3 trisaccharide. The most successful scFv construct, which comprises an alternative variable light chain (VLA), allowed structure determination to 2.2 Å resolution. The structure gives insights into the conformation of the important CDR H3 loop and the suspected antigen binding site. Furthermore, the presence of VLA instead of the original VL elucidates how this subdomain indirectly stabilises the CDR H3 loop. The current work may serve as a guideline for the efficient production of scFvs for structure determination.

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• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Grzybek, Michal; Løset, Geir Åge & Krengel, Ute (2020). Molecular basis of specificity of a promising anti-tumour antibody.
• Pesci, Giacomo; Krengel, Ute; Johannesen, Hedda & Løset, Geir Åge (2020). Follow the Light - Tracking 14F7’s cellular journey with luminescence. Vis sammendrag

  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.

• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Grzybek, Michal; Ünal, Coskun; Løset, Geir Åge & Krengel, Ute (2019). Crystal structure of an anti-tumour antibody in complex with its tumour-specific antigen.
• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Heggelund, Julie Elisabeth; Løset, Geir Åge & Krengel, Ute (2019). 14F7 - A tumour specific antibody.
• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2019). An anti-tumour antibody that specifically recognises N-glycolyl GM3.
• Solbakke, Kristoffer Gudesen; Krengel, Ute; Johannesen, Hedda; Løset, Geir Åge & Bjerregaard-Andersen, Kaare (2019). 4G9: An antibody that potentially imitates a cancer-related carbohydrate.
• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Heggelund, Julie Elisabeth; Løset, Geir Åge & Krengel, Ute (2018). NeuGc GM3 - a cancer-specific ganglioside that is recognised by the antibody 14F7.
• Johannesen, Hedda; Hoås Mykland, Helene; Heggelund, Julie Elisabeth; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2018). An anti-ganglioside scFv for cancer therapies.
• Johannesen, Hedda; Mykland hoås, Helene; Heggelund, Julie Elisabeth; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2018). 14F7, An anti-ganglioside scFv for cancer therapies.
• Johannesen, Hedda; Mykland Hoås, Helene; Heggelund, Julie Elisabeth; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2018). 14F7, an antibody that recognises a tumour-specific ganglioside with high specificity.
• Johannesen, Hedda; Hammerstad, Marta; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III.
• Hammerstad, Marta; Hersleth, Hans-Petter; Lofstad, Marie; Johannesen, Hedda; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2016). Structural Insight into the Function of Ribonucleotide Reductase.
• Johannesen, Hedda & Foss, Øystein (2016). Grensesprengende kjemi – et heftig møte mellom magi, myter og vitenskap.
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A Structural and Functional Investigation of Ribonucleotide Reductase Class III In Bacillus Cereus.
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A structural and functional investigation of Ribonucleotide reductase Class III in Bacillus cereus. NBS-nytt.  ISSN 0801-3535.  s 65- 65
• Johannesen, Hedda; Bousquet, Paula; Løset, Geir Åge & Krengel, Ute (2014). L`ASSASSINA PROTEIN THAT SPECIFICALLY KILLS TUMOUR CELLS.
• Johannesen, Hedda; Krengel, Ute; Løset, Geir Åge & Bousquet, Paula (2014). The Assassin - Recombinant single-chain Fv production of the anti-tumour antibody 14F7.
• Johannesen, Hedda (2013). A PERFECTLY DESIGNED TRAP: A MOLECULE THAT SPECIFICALLY KILLS TUMOUR CELLS.
• Johannesen, Hedda (2013). Snikmorderen - Vinner av Kjemi Grand Prix. Vis sammendrag

  Proteinet 14F7 dreper kreftcelle. For 8 millioner år siden skjedde det en mutasjon som gjorde at vi mennesker ikke lenger klarte å lage et spesifikt karbohydrat, men når en vanlig celle blir til en kreftcelle dukker dette sukkeret opp igjen i cellen. 14F7 binder seg til dette karbohydratet og lager et hull i cellen slik at den dør. Jeg ønsker å finne ut av hvordan 14F7 binder til karbohydratet. Hvis jeg kan oppdage hvordan 14F7 og sukkermolekylet interagerer kan jeg jobbe med å forbedre bindingen slik at 14F7 blir enda mer effektiv.

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