geiralo

• Heinzelbecker, Julia; Fauskanger, Marte; Jonson, Ida; Krengel, Ute; Løset, Geir Åge & Munthe, Ludvig Andre [Show all 7 contributors for this article] (2024). Chimeric antigen receptor T cells targeting the GM3(Neu5Gc) ganglioside. Frontiers in Immunology. ISSN 1664-3224. 15. doi: 10.3389/fimmu.2024.1331345.
• Berge-Seidl, Sebastian; Nielsen, Nis Valentin; Alfonso, Armando A. Rodriguez; Etscheid, Michael; Kandanur, Sai Priya Sarma & Haug, Bengt Erik [Show all 15 contributors for this article] (2022). Identification of a phage display-derived peptide interacting with the N-terminal region of factor VII activating protease (FSAP) enables characterization of zymogen activation. ACS Chemical Biology. ISSN 1554-8929. 17(9), p. 2631–2642. doi: 10.1021/acschembio.2c00538. Full text in Research Archive
• Frick, Rahel; Høydahl, Lene Støkken; Hodnebrug, Ina; Vik, Erik Sebastian; Dalhus, Bjørn & Sollid, Ludvig Magne [Show all 9 contributors for this article] (2022). Affinity maturation of TCR-like antibodies using phage display guided by structural modeling. Protein Engineering Design & Selection. ISSN 1741-0126. 35. doi: 10.1093/protein/gzac005. Full text in Research Archive
• Minton, Annabel R.; Smith, Lindsay D.; Bryant, Dean J.; Strefford, Jonathan C.; Forconi, Francesco & Stevenson, Freda K. [Show all 12 contributors for this article] (2022). B-cell receptor dependent phagocytosis and presentation of particulate antigen by chronic lymphocytic leukemia cells. Exploration of Targeted Anti-tumor Therapy. ISSN 2692-3114. 3(1), p. 37–49. doi: 10.37349/etat.2022.00070.
• Frick, Rahel; Høydahl, Lene Støkken; Petersen, Jan; du Pré, Fleur; Kumari, Shraddha & Berntsen, Grete [Show all 20 contributors for this article] (2021). A high-affinity human TCR-like antibody detects celiac disease gluten peptide-MHC complexes and inhibits T cell activation. Science immunology. ISSN 2470-9468. 6:eabg4925(62), p. 1–16. doi: 10.1126/sciimmunol.abg4925. Full text in Research Archive
• Kara, Emrah; Nielsen, Nis Valentin; Eggertsdottir, Bergrun; Thiede, Bernd; Kanse, Sandip & Løset, Geir Åge (2020). Design and characterization of a new pVII combinatorial phage display peptide library for protease substrate mining using Factor VII activating protease (FSAP) as model. ChemBioChem. ISSN 1439-4227. 21(13), p. 1875–1884. doi: 10.1002/cbic.201900705. Full text in Research Archive
• Haabeth, Ole Audun; Hennig, Kjartan; Fauskanger, Marte; Løset, Geir Åge; Bogen, Bjarne & Tveita, Anders Aune (2020). CD4+ T-cell killing of multiple myeloma cells is mediated by resident bone marrow macrophages. Blood Advances. ISSN 2473-9529. 4(12), p. 2595–2605. doi: 10.1182/bloodadvances.2020001434. Full text in Research Archive
• Huszthy, Peter Csaba; Gopalakrishnan, Ramakrishna Prabhu; Jacobsen, Johanne Tracey; Haabeth, Ole Audun; Løset, Geir Åge & Braathen, Ranveig [Show all 10 contributors for this article] (2019). B cell receptor ligation induces display of V-region peptides on MHC class II molecules to T cells. Proceedings of the National Academy of Sciences of the United States of America. ISSN 0027-8424. 116(51), p. 25850–25859. doi: 10.1073/pnas.1902836116. Full text in Research Archive Show summary

  The B cell receptors (BCRs) for antigen express variable (V) regions that are enormously diverse, thus serving as markers on individual B cells. V region-derived idiotypic (Id) peptides can be displayed as pId:MHCII complexes on B cells for recognition by CD4+ T cells. It is not known if naive B cells spontaneously display pId:MHCII in vivo or if BCR ligation is required for expression, thereby enabling collaboration between Id+ B cells and Id-specific T cells. Here, using a mouse model, we show that naive B cells do not express readily detectable levels of pId:MHCII. However, BCR ligation by Ag dramatically increases physical display of pId:MHCII, leading to activation of Id-specific CD4+ T cells, extrafollicular T-B cell collaboration and some germinal center formation, and production of Id+ IgG. Besides having implications for immune regulation, the results may explain how persistent activation of self-reactive B cells induces the development of autoimmune diseases and B cell lymphomas.

• Köksal, Hakan; Baken, Elizabeth; Warren, David; Løset, Geir Åge; Inderberg, Else Marit & Wälchli, Sébastien (2019). Chimeric antigen receptor preparation from hybridoma to T-cell expression. Antibody Therapeutics. ISSN 2516-4236. 2(2), p. 56–63. doi: 10.1093/abt/tbz007. Full text in Research Archive
• Frick, Rahel; Gunnarsen, Kristin Støen; Dahal-Koirala, Shiva; Risnes, Louise Fremgaard; Sollid, Ludvig Magne & Sandlie, Inger [Show all 8 contributors for this article] (2019). A TRAV26-1-encoded recognition motif focuses the biased T cell response in celiac disease. European Journal of Immunology. ISSN 0014-2980. 50, p. 142–145. doi: 10.1002/eji.201948235. Full text in Research Archive
• Høydahl, Lene Støkken; Frick, Rahel; Sandlie, Inger & Løset, Geir Åge (2019). Targeting the mhc ligandome by use of tcr-like antibodies. Antibodies. ISSN 2073-4468. 8:32(2), p. 1–21. doi: 10.3390/antib8020032. Full text in Research Archive
• Høydahl, Lene Støkken; Richter, Lisa Maria; Frick, Rahel; Snir, Omri; Gunnarsen, Kristin Støen & Landsverk, Ole J. B. [Show all 18 contributors for this article] (2019). Plasma Cells Are the Most Abundant Gluten Peptide MHC-expressing Cells in Inflamed Intestinal Tissues From Patients With Celiac Disease. Gastroenterology. ISSN 0016-5085. 156(5), p. 1428–1439.e10. doi: 10.1053/j.gastro.2018.12.013. Full text in Research Archive
• Bjerregaard-Andersen, Kaare; Johannesen, Hedda; Abdel-Rahman, Noha Mansour Hassan; Heggelund, Julie Elisabeth; Hoås, Helene Mykland & Abraha, Fana [Show all 13 contributors for this article] (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, p. 1–11. doi: 10.1038/s41598-018-28918-5. Full text in Research Archive Show summary

  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.

• Gunnarsen, Kristin Støen; Høydahl, Lene Støkken; Neumann, Ralf Stefan; Bjerregaard-Andersen, Kaare; Nilssen, Nicolay Rustad & Sollid, Ludvig Magne [Show all 8 contributors for this article] (2018). Soluble T-cell receptor design influences functional yield in an E. coli chaperone-assisted expression system. PLOS ONE. ISSN 1932-6203. 13:e0195868(4), p. 1–15. doi: 10.1371/journal.pone.0195868. Full text in Research Archive Show summary

  There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles. A celiac disease patient-derived TCR with specificity for gluten was used, and we achieved detectable expression for all formats and variants. We found that expression in RosettaBlueTM without IPTG induction resulted in the highest periplasmic yields. Moreover, after large-scale expression and protein purification, only the scTCR format was obtained in high yields. Importantly, stability engineering of the scTCR was a prerequisite for obtaining reliable biophysical characterization of the TCR-pMHC interaction. The scTCR format is readily compatible with high-throughput screening approaches that may enable both development of reagents allowing for defined peptide MHC (pMHC) characterization and discovery of potential novel therapeutic leads.

• Gunnarsen, Kristin Støen; Høydahl, Lene Støkken; Risnes, Louise Fremgaard; Dahal-Koirala, Shiva; Neumann, Ralf Stefan & Bergseng, Elin [Show all 15 contributors for this article] (2017). A TCRα framework–centered codon shapes a biased T cell repertoire through direct MHC and CDR3β interactions. JCI Insight. ISSN 2379-3708. 2(17). doi: 10.1172/jci.insight.95193. Full text in Research Archive
• Kara, Emrah; Manna, Dipankar; Løset, Geir Åge; Schneider, E L; Craik, C S & Kanse, Sandip (2017). Analysis of the substrate specificity of Factor VII activating protease (FSAP) and design of specific and sensitive peptide substrates. Thrombosis and Haemostasis. ISSN 0340-6245. doi: 10.1160/TH17-02-0081. Full text in Research Archive
• Høydahl, Lene Støkken; Nilssen, Nicolay Rustad; Gunnarsen, Kristin Støen; du Pré, Marie Fleur; Iversen, Rasmus & Roos, Norbert [Show all 11 contributors for this article] (2016). Multivalent pIX phage display selects for distinct and improved antibody properties. Scientific Reports. ISSN 2045-2322. 6. doi: 10.1038/srep39066. Full text in Research Archive
• Grødeland, Gunnveig; Fredriksen, Agnete; Løset, Geir Åge; Vikse, Elisabeth Lea; Fugger, Lars & Bogen, Bjarne (2016). Antigen targeting to human HLA Class II molecules increases efficacy of DNA vaccination. Journal of Immunology. ISSN 0022-1767. 197(9), p. 3575–3585. doi: 10.4049/jimmunol.1600893.
• Løset, Geir Åge; Berntzen, Gøril; Frigstad, Terje; Pollmann, Sylvie; Gunnarsen, Kristin Støen & Sandlie, Inger (2015). Phage display engineered T cell receptors as tools for the study of tumor peptide–MHC interactions. Frontiers in Oncology. ISSN 2234-943X. doi: 10.3389/fonc.2014.00378. Full text in Research Archive
• Frigstad, Terje; Løset, Geir Åge; Sandlie, Inger & Bogen, Bjarne (2013). A Public T cell Receptor Recognized by a MonoclonalAntibody Specific for the D-J Junction of the b-chain. Scandinavian Journal of Immunology. ISSN 0300-9475. p. 345–351. doi: 10.1111/sji.12098.
• Gunnarsen, Kristin Støen; Kristinsson, Solveig Gunn; Justesen, Sune; Frigstad, Terje; Buus, Søren & Bogen, Bjarne [Show all 8 contributors for this article] (2013). Chaperone-assisted thermostability engineering of a soluble T cell receptor using phage display. Scientific Reports. ISSN 2045-2322. 3. doi: 10.1038/srep01162. Full text in Research Archive
• Løset, Geir Åge & Sandlie, Inger (2012). Next generation phage display by use of pVII and pIX as display scaffolds. Methods. ISSN 1046-2023. 58(1), p. 40–46. doi: 10.1016/j.ymeth.2012.07.005.
• Nilssen, Nicolay Rustad; Frigstad, Terje; Pollmann, Sylvie; Roos, Norbert; Bogen, Bjarne & Sandlie, Inger [Show all 7 contributors for this article] (2012). DeltaPhage-a novel helper phage for high-valence pIX phagemid display. Nucleic Acids Research (NAR). ISSN 0305-1048. 40(16). doi: 10.1093/nar/gks341. Full text in Research Archive
• Wälchli, Sebastien; Løset, Geir Åge; Kumari, Shraddha; Johansen, Jorunn; Yang, Weiwen & Sandlie, Inger [Show all 7 contributors for this article] (2011). A Practical Approach to T-Cell Receptor Cloning and Expression. PLOS ONE. ISSN 1932-6203. 6(11). doi: 10.1371/journal.pone.0027930. Full text in Research Archive
• Qiao, Shuo Wang; Raki, Melinda; Gunnarsen, Kristin Støen; Løset, Geir Åge; Lundin, Knut E A & Sandlie, Inger [Show all 7 contributors for this article] (2011). Posttranslational Modification of Gluten Shapes TCR Usage in Celiac Disease. Journal of Immunology. ISSN 0022-1767. 187(6), p. 3064–3071. doi: 10.4049/jimmunol.1101526.
• Løset, Geir Åge; Bogen, Bjarne & Sandlie, Inger (2011). Expanding the Versatility of Phage Display I: Efficient Display of Peptide-Tags on Protein VII of the Filamentous Phage. PLOS ONE. ISSN 1932-6203. 6(2). doi: 10.1371/journal.pone.0014702. Full text in Research Archive
• Løset, Geir Åge; Roos, Norbert; Bogen, Bjarne & Sandlie, Inger (2011). Expanding the Versatility of Phage Display II: Improved Affinity Selection of Folded Domains on Protein VII and IX of the Filamentous Phage. PLOS ONE. ISSN 1932-6203. 6(2). doi: 10.1371/journal.pone.0017433. Full text in Research Archive
• Lunde, Elin; Løset, Geir Åge; Bogen, Bjarne & Sandlie, Inger (2010). Stabilizing mutations increase secretion of functional soluble TCR-Ig fusion proteins. BMC Biotechnology. ISSN 1472-6750. 10. doi: 10.1186/1472-6750-10-61. Full text in Research Archive
• Gunnarsen, Kristin Støen; Lunde, Elin; Kristiansen, Per Eugen; Bogen, Bjarne; Sandlie, Inger & Løset, Geir Åge (2010). Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA. BMC Biotechnology. ISSN 1472-6750. 10. doi: 10.1186/1472-6750-10-8. Full text in Research Archive
• Løset, Geir Åge; Kristinsson, Solveig Gunn & Sandlie, Inger (2008). Reliable titration of filamentous bacteriophages independent of pIII fusion moiety and genome size by using trypsin to restore wild-type pill phenotype. BioTechniques. ISSN 0736-6205. 44(4), p. 551–554. doi: 10.2144/000112724. Show summary

  Phage display holds a key position in the use of combinatorial library approaches for the purpose of protein engineering and discovery. However, modifying the pIII protein of the phage can severely and negatively influence the infectiousness of the phage particle. This concern is particularly relevant when large pIII fusions in combination with multivalent display systems are at use. We here describe the use of trypsin to restore wt pIII phenotype as a small modification to the standard titration protocol. The results show that the trypsin treatment has a very large, but heterogeneous effect on the phage infection efficiency depending on the pIII fusion domain and the valence of display.

• Løset, Geir Åge; Lunde, Elin; Bogen, Bjarne; Brekke, OH & Sandlie, Inger (2007). Functional phage display of two murine alpha/beta T-cell receptors is strongly dependent on fusion format, mode and periplasmic folding assistance. Protein Engineering Design & Selection. ISSN 1741-0126. 20, p. 461–472. doi: 10.1093/protein/gzm044.
• Løset, Geir Åge; Løbersli, Inger; Kavlie, Anita; Stacy, John; Borgen, Tine & Kausmally, Louise [Show all 9 contributors for this article] (2005). Construction, evaluation and refinement of a large human antibody phage library based on the IgD and IgM variable gene repetoire. Journal of Immunological Methods (JIM). ISSN 0022-1759. 299(01.feb), p. 47–62.
• Reiersen, Herald; Løbersli, Inger; Løset, Geir Åge; Hvattum, Else; Simonsen, Bjørg & Stacy, John Eric [Show all 11 contributors for this article] (2005). Covalent antibody display--an in vitro antibody-DNA library selection system. Nucleic Acids Research (NAR). ISSN 0305-1048. 33(1). doi: 10.1093/nar/gni010.
• Løset, Geir Åge; Løbersli, Inger; Kavlie, Anita; Stacy, John Eric; Borgen, Tine & Kausmally, Louise [Show all 10 contributors for this article] (2005). Construction, evaluation and refinement of a large human antibody phage library based on the IgD and IgM variable gene repertoire. Journal of Immunological Methods (JIM). ISSN 0022-1759. 299(1-2), p. 47–62. doi: 10.1016/j.jim.2005.01.014.
• Løset, Geir Åge; Roux, K.H.; Zhu, P.; Michaelsen, Terje Einar & Sandlie, Inger (2004). Differential segmental flexibility and reach dictate the antigen binding mode of chimeric IgD and IgM: Implications for the function of the B cell receptor. Journal of Immunology. ISSN 0022-1767. 172, p. 2925–2934.
• Brekke, Ole Henrik & Løset, Geir Åge (2003). New technologies in therapeutic antibody development. Current opinion in pharmacology (Print). ISSN 1471-4892. 3(5), p. 544–550. doi: 10.1016/j.coph.2003.05.002.
• Thommesen, John Einar; Michaelsen, Terje Einar; Løset, Geir Åge; Sandlie, Inger & Brekke, Ole Henrik (2000). Lysine 322 in the human IgG3 C(H)2 domain is crucial for antibody dependent complement activation. Molecular Immunology. ISSN 0161-5890. 37(16), p. 995–1004.

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• Løset, Geir Åge; Mousavi, Seyed A. & Berg, Trond (2008). Sinusoidal liver cells and liver tolerance. Transworld Research Network. ISBN 8178953641. 11 p.

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• Pesci, Giacomo; Johannesen, Hedda; Løset, Geir Åge & Krengel, Ute (2020). A promising antibody to treat cancer. How does it work?
• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Grzybek, Michal; Løset, Geir Åge & Krengel, Ute (2020). Molecular basis of specificity of a promising anti-tumour antibody .
• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2019). An anti-tumour antibody that specifically recognises N-glycolyl GM3.
• 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; Heggelund, Julie Elisabeth; Løset, Geir Åge & Krengel, Ute (2018). NeuGc GM3 - a cancer-specific ganglioside that is recognised by the antibody 14F7.
• 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; Hoås Mykland, Helene; Heggelund, Julie Elisabeth; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2018). An anti-ganglioside scFv for cancer therapies.
• Kara, Emrah; Manna, Dipankar; Løset, Geir Åge; Schneider, Eric L.; Craik, Charles S. & Kanse, Sandip (2017). Analysis of the Substrate Specificity of Factor VII Activating Protease (FSAP) and Design of a Specific and Sensitive Peptide Substrate. Res Pract Thromb Haemost. p. 560–560. doi: 10.1002/rth2.12012.
• Mandecki, W.; Goldman, E.; Sandlie, Inger & Løset, Geir Åge (2015). Phage Display and Selection of Protein Ligands. In Goldman, Emanuel & Green, Laurence (Ed.), Practical Handbook of Microbiology, Third Edition. CRC Press. ISSN 9781466587397. p. 101–114.
• Johannesen, Hedda; Bousquet, Paula; Løset, Geir Åge & Krengel, Ute (2014). L`ASSASSINA PROTEIN THAT SPECIFICALLY KILLS TUMOUR CELLS.
• Høydahl, Lene Støkken; Neumann, Ralf Stefan; Qiao, Shuo Wang; Sollid, Ludvig Magne; Sandlie, Inger & Løset, Geir Åge (2011). Development of a High Affinity TCR Reagent by Phage Display to Study Antigen Presentation in Celiac Disease. Scandinavian Journal of Immunology. ISSN 0300-9475. 73(4), p. 372–372.
• Qiao, Shuo Wang; Raki, Melinda; Gunnarsen, Kristin Støen; Løset, Geir Åge; Sandlie, Inger & Sollid, Ludvig Magne (2011). Post-Translational Modification of Gluten Shapes the T Cell Response in Coeliac Disease by Direct Interaction with the T Cell Receptor. Scandinavian Journal of Immunology. ISSN 0300-9475. 73(4), p. 356–356.
• Gunnarsen, Kristin Støen; Kristinsson, Solveig Gunn; Bogen, Bjarne; Sandlie, Inger & Løset, Geir Åge (2011). Thermostability Engineering of a Soluble Single Chain T Cell Receptor (scTCR) Using Phage Display. Scandinavian Journal of Immunology. ISSN 0300-9475. 73(4), p. 352–352.
• Qiao, Shuo Wang; Raki, Melinda; Løset, Geir Åge; Sandlie, Inger & Sollid, Ludvig Magne (2010). TCR Vbeta Bias and Semi-Public T Cell Responses In DQ2-Gliadin-Alpha-II Specific T Cells From Coeliac Disease Patients. Scandinavian Journal of Immunology. ISSN 0300-9475. 71(6), p. 511–+.
• Gunnarsen, Kristin Støen; Kristinsson, Solveig Gunn; Bogen, Bjarne; Sandlie, Inger & Løset, Geir Åge (2009). Thermostability engineering of a soluble single chain T cell receptor using phage display.
• Løset, Geir Åge; Lunde, Elin & Sandlie, Inger (2007). Soluble T cell receptors. Not quite like antibodies. NBS-nytt. ISSN 0801-3535. p. 14–17.
• Flobakk, Morten; Løset, Geir Åge; Rosenhave, Ellen Maryann; Sandlie, Inger; Aalen, Reidunn B. & Blomhoff, Heidi Kiil [Show all 7 contributors for this article] (2004). PBL på grunnkurset i cellebiologi og genetikk på Institutt for molekylær Biovitenskap. NBS-nytt. ISSN 0801-3535. p. 20–26.
• Kristiansen, Helene Sørum; Krengel, Ute; Løset, Geir Åge & Progida, Cinzia (2022). Let it bind, and light you may find - Investigation of 14F7's cellular journey and effects. Universitetet i Oslo.
• Pesci, Giacomo; Krengel, Ute; Johannesen, Hedda & Løset, Geir Åge (2020). Follow the Light - Tracking 14F7’s cellular journey with luminescence. Universitetet i Oslo. Show summary

  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.

• Solbakke, Kristoffer Gudesen; Krengel, Ute; Johannesen, Hedda; Løset, Geir Åge & Bjerregaard-Andersen, Kaare (2019). 4G9: An antibody that potentially imitates a cancer-related carbohydrate. Universitetet i Oslo.
• Johannesen, Hedda; Krengel, Ute; Løset, Geir Åge & Bousquet, Paula (2014). The Assassin - Recombinant single-chain Fv production of the anti-tumour antibody 14F7. Universitetet i Oslo.
• Løset, Geir Åge (2005). Antibody and T cell receptor engineering: Mechanistic studies and biotechnological applications. Unipub forlag. ISSN 1501-7710.
• Løset, Geir Åge (2000). A comparison of the antigen binding ability of antibodies characteristic of the primary immune respons. Biologisk institutt.

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