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. 15:1331345, p. 1–12. doi: 10.3389/fimmu.2024.1331345. Full text in Research Archive Show summary

  Chimeric antigen receptor (CAR) T cell technology has ushered in a new era of immunotherapy, enabling the targeting of a broad range of surface antigens, surpassing the limitations of traditional T cell epitopes. Despite the wide range of non-protein tumor-associated antigens, the advancement in crafting CAR T cells for these targets has been limited. Owing to an evolutionary defect in the CMP-Neu5Ac hydroxylase (CMAH) that abolishes the synthesis of CMP-Neu5Gc from CMP-Neu5Ac, Neu5Gc is generally absent in human tissues. Despite this, Neu5Gc-containing antigens, including the ganglioside GM3(Neu5Gc) have consistently been observed on tumor cells across a variety of human malignancies. This restricted expression makes GM3(Neu5Gc) an appealing and highly specific target for immunotherapy. In this study, we designed and evaluated 14F7-28z CAR T cells, with a targeting unit derived from the GM3(Neu5Gc)-specific murine antibody 14F7. These cells exhibited exceptional specificity, proficiently targeting GM3(Neu5Gc)-expressing murine tumor cells in syngeneic mouse models, ranging from B cell malignancies to epithelial tumors, without compromising safety. Notably, human tumor cells enhanced with murine Cmah were effectively targeted and eliminated by the 14F7 CAR T cells. Nonetheless, despite the detectable presence of GM3(Neu5Gc) in unmodified human tumor xenografts, the levels were insufficient to trigger a tumoricidal T-cell response with the current CAR T cell configuration. Overall, our findings highlight the potential of targeting the GM3(Neu5Gc) ganglioside using CAR T cells across a variety of cancers and set the stage for the optimization of 14F7-based therapies for future human clinical application.

• 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 Show summary

  Factor VII Activating protease (FSAP) has a protective effect in diverse disease conditions as inferred from studies in FSAP–/– mice and humans deficient in FSAP activity due to single-nucleotide polymorphism. The zymogen form of FSAP in plasma is activated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear whether this activation mechanism is specific and amenable to manipulation. Using a phage display approach, we have identified a Cys-constrained 11 amino acid peptide, NNKC9/41, that activates pro-FSAP in plasma. The synthetic linear peptide has a propensity to cyclize through the terminal Cys groups, of which the antiparallel cyclic dimer, but not the monocyclic peptide, is the active component. Other commonly found zymogens in the plasma, related to the hemostasis system, were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30–35) in an intrinsically disordered stretch in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41, and this was reversed by MA-FSAP-38C7, demonstrating the utility of this peptide. Peptide NNKC9/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP, elucidate its biological role, and provide a starting point for the pharmacological manipulation of FSAP activity.

• 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 Show summary

  Abstract TCR-like antibodies represent a unique type of engineered antibodies with specificity toward pHLA, a ligand normally restricted to the sensitive recognition by T cells. Here, we report a phage display-based sequential development path of such antibodies. The strategy goes from initial lead identification through in silico informed CDR engineering in combination with framework engineering for affinity and thermostability optimization, respectively. The strategy allowed the identification of HLA-DQ2.5 gluten peptide-specific TCR-like antibodies with low picomolar affinity. Our method outlines an efficient and general method for development of this promising class of antibodies, which should facilitate their utility including translation to human therapy.

• 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. Full text in Research Archive
• Frick, Rahel; Høydahl, Lene Støkken; Petersen, Jan; Pré, Fleur du; 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. 6:eabg4925(62), p. 1–16. doi: 10.1126/sciimmunol.abg4925. Full text in Research Archive Show summary

  Antibodies specific for peptides bound to human leukocyte antigen (HLA) molecules are valuable tools for studies of antigen presentation and may have therapeutic potential. Here, we generated human T cell receptor (TCR)–like antibodies toward the immunodominant signature gluten epitope DQ2.5-glia-α2 in celiac disease (CeD). Phage display selection combined with secondary targeted engineering was used to obtain highly specific antibodies with picomolar affinity. The crystal structure of a Fab fragment of the lead antibody 3.C11 in complex with HLA-DQ2.5:DQ2.5-glia-α2 revealed a binding geometry and interaction mode highly similar to prototypic TCRs specific for the same complex. Assessment of CeD biopsy material confirmed disease specificity and reinforced the notion that abundant plasma cells present antigen in the inflamed CeD gut. Furthermore, 3.C11 specifically inhibited activation and proliferation of gluten-specific CD4+ T cells in vitro and in HLA-DQ2.5 humanized mice, suggesting a potential for targeted intervention without compromising systemic immunity.

• 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 Show summary

  We describe a novel, easy and efficient combinatorial phage display peptide substrate‐mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti‐FLAG mAbs and its independency of any post‐translational modification. This library was tested with Factor‐VII activating protease (WT‐FSAP) and its single‐nucleotide polymorphism variant Marburg‐I (MI)‐FSAP. The WT‐FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI‐FSAP as a loss‐of‐function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT‐FSAP substrates than previously anticipated.

• 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 Show summary

  Abstract CD4+ T cells may induce potent antitumor immune responses through interaction with antigen-presenting cells within the tumor microenvironment. Using a murine model of multiple myeloma, we demonstrated that adoptive transfer of idiotype-specific CD4+ T cells may elicit curative responses against established multifocal myeloma in bone marrow. This finding indicates that the myeloma bone marrow niche contains antigen-presenting cells that may be rendered tumoricidal. Given the complexity of the bone marrow microenvironment, the mechanistic basis of such immunotherapeutic responses is not known. Through a functional characterization of antitumor CD4+ T-cell responses within the bone marrow microenvironment, we found that killing of myeloma cells is orchestrated by a population of bone marrow–resident CD11b+F4/80+MHC-IIHigh macrophages that have taken up and present secreted myeloma protein. The present results demonstrate the potential of resident macrophages as powerful mediators of tumor killing within the bone marrow and provide a basis for novel therapeutic strategies against multiple myeloma and other malignancies that affect the bone marrow.

• 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 (PNAS). 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. 2(2), p. 56–63. doi: 10.1093/abt/tbz007. Full text in Research Archive Show summary

  The successful use of chimeric antigen receptor (CAR) for hematological cancer treatment has influenced the direction taken in translational research toward an increasing focus on personalized targeted immunotherapy. Thus, a growing number of labs worldwide are now interested in testing their old antibody collections in this format to broaden the spectrum of utility and improve safety and efficacy. We herein present a straightforward protocol for the identification of an antibody from a hybridoma and the design of the single chain fragment that will be placed on the extracellular part of the CAR construct. We further show how to test the expression and the activity of the construct in primary T cells. We illustrate our demonstration with two new CARs targeted against the B cell receptor, more precisely the light chains κ and λ, that represent potential alternatives to the CD19 CAR used in the treatment of B-cell malignancies. Statement of Significance Chimeric antigen receptor molecules are becoming increasingly popular, and an easy-to-follow method to isolate and express them is herein presented.

• Høydahl, Lene Støkken; Frick, Rahel; Sandlie, Inger & Løset, Geir Åge (2019). Targeting the mhc ligandome by use of tcr-like antibodies. Antibodies. 8:32(2), p. 1–21. doi: 10.3390/antib8020032. Full text in Research Archive Show summary

  Monoclonal antibodies (mAbs) are valuable as research reagents, in diagnosis and in therapy. Their high specificity, the ease in production, favorable biophysical properties and the opportunity to engineer different properties make mAbs a versatile class of biologics. mAbs targeting peptide–major histocompatibility molecule (pMHC) complexes are often referred to as “TCR-like” mAbs, as pMHC complexes are generally recognized by T-cell receptors (TCRs). Presentation of self- and non-self-derived peptide fragments on MHC molecules and subsequent activation of T cells dictate immune responses in health and disease. This includes responses to infectious agents or cancer but also aberrant responses against harmless self-peptides in autoimmune diseases. The ability of TCR-like mAbs to target specific peptides presented on MHC allows for their use to study peptide presentation or for diagnosis and therapy. This extends the scope of conventional mAbs, which are generally limited to cell-surface or soluble antigens. Herein, we review the strategies used to generate TCR-like mAbs and provide a structural comparison with the analogous TCR in pMHC binding. We further discuss their applications as research tools and therapeutic reagents in preclinical models as well as challenges and limitations associated with their use.

• 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; Richter, Lisa Maria; Frick, Rahel; Snir, Omri; Gunnarsen, Kristin Støen & Landsverk, Ole J. B. [Show all 18 contributors for this article] (2018). 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 Show summary

  Background & Aims Development of celiac disease is believed to involve the transglutaminase-dependent response of CD4+ T cells toward deamidated gluten peptides in the intestinal mucosa of individuals with specific HLA-DQ haplotypes. We investigated the antigen presentation process during this mucosal immune response. Methods We generated monoclonal antibodies (mAbs) specific for the peptide–MHC (pMHC) complex of HLA-DQ2.5 and the immunodominant gluten epitope DQ2.5-glia-α1a using phage display. We used these mAbs to assess gluten peptide presentation and phenotypes of presenting cells by flow cytometry and enzyme-linked immune absorbent spot (ELISPOT) in freshly prepared single-cell suspensions from intestinal biopsies from 40 patients with celiac disease (35 untreated and 5 on a gluten-free diet) as well as 18 subjects with confirmed noninflamed gut mucosa (controls, 12 presumed healthy, 5 undergoing pancreatoduodenectomy, and 1 with potential celiac disease). Results Using the mAbs, we detected MHC complexes on cells from intestinal biopsies from patients with celiac disease who consume gluten, but not from patients on gluten-free diets. We found B cells and plasma cells to be the most abundant cells that present DQ2.5-glia-α1a in the inflamed mucosa. We identified a subset of plasma cells that expresses B-cell receptors (BCR) specific for gluten peptides or the autoantigen transglutaminase 2 (TG2). Expression of MHC class II (MHCII) was not restricted to these specific plasma cells in patients with celiac disease but was observed in an average 30% of gut plasma cells from patients and controls. Conclusions A population of plasma cells from intestinal biopsies of patients with celiac disease express MHCII; this is the most abundant cell type presenting the immunodominant gluten peptide DQ2.5-glia-α1a in the tissues from these patients. These results indicate that plasma cells in the gut can function as antigen-presenting cells and might promote and maintain intestinal inflammation in patients with celiac disease or other inflammatory disorders.

• 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. 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. 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.

• 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 Show summary

  Factor VII (FVII) activating protease (FSAP) is a circulating serine protease that is likely to be involved in a number of disease conditions such as stroke, atherosclerosis, liver fibrosis, thrombosis and cancer. To date, no systematic information is available about the substrate specificity of FSAP. Applying phage display and positional scanning substrate combinatorial library (PS-SCL) approaches we have characterised the specificity of FSAP towards small peptides. Results were evaluated in the context of known protein substrates as well as molecular modelling of the peptides in the active site of FSAP. The representative FSAP-cleaved sequence obtained from the phage display method was Val-Leu-Lys-Arg-Ser (P4-P1’). The sequence X-Lys/Arg-Nle-Lys/Arg (P4-P1) was derived from the PS-SCL method. These results show a predilection for cleavage at a cluster of basic amino acids on the nonprime side. Quenched fluorescent substrate (Ala-Lys-Nle-Arg-AMC) (amino methyl coumarin) and (Ala-Leu-Lys-Arg-AMC) had a higher selectivity for FSAP compared to other proteases from the hemostasis system. These substrates could be used to measure FSAP activity in a complex biological system such as plasma. In histonetreated plasma there was a specific activation of pro-FSAP as validated by the use of an FSAP inhibitory antibody, corn trypsin inhibitor to inhibit Factor XIIa and hirudin to inhibit thrombin, which may account for some of the haemostasis-related effects of histones. These results will aid the development of further selective FSAP activity probes as well as specific inhibitors that will help to increase the understanding of the functions of FSAP in vivo. This research has been published in Thrombosis and Haemostasis. © 2017 Schattauer

• 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. 2(17). doi: 10.1172/jci.insight.95193. Full text in Research Archive Show summary

  Selection of biased T cell receptor (TCR) repertoires across individuals is seen in both infectious diseases and autoimmunity, but the underlying molecular basis leading to these shared repertoires remains unclear. Celiac disease (CD) occurs primarily in HLA-DQ2.5+ individuals and is characterized by a CD4+ T cell response against gluten epitopes dominated by DQ2.5-glia-α1a and DQ2.5-glia-α2. The DQ2.5-glia-α2 response recruits a highly biased TCR repertoire composed of TRAV26-1 paired with TRBV7-2 harboring a semipublic CDR3β loop. We aimed to unravel the molecular basis for this signature. By variable gene segment exchange, directed mutagenesis, and cellular T cell activation studies, we found that TRBV7-3 can substitute for TRBV7-2, as both can contain the canonical CDR3β loop. Furthermore, we identified a pivotal germline-encoded MHC recognition motif centered on framework residue Y40 in TRAV26-1 engaging both DQB1*02 and the canonical CDR3β. This allowed prediction of expanded DQ2.5-glia-α2–reactive TCR repertoires, which were confirmed by single-cell sorting and TCR sequencing from CD patient samples. Our data refine our understanding of how HLA-dependent biased TCR repertoires are selected in the periphery due to germline-encoded residues. This research was originally published in JCI Insight. © 2018 American Society for Clinical Investigation

• Høydahl, Lene Støkken; Nilssen, Nicolay Rustad; Gunnarsen, Kristin Støen; Pré, Marie Fleur du; 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. 6. doi: 10.1038/srep39066. Full text in Research Archive Show summary

  Phage display screening readily allows for the identification of a multitude of antibody specificities, but to identify optimal lead candidates remains a challenge. Here, we direct the antibody-capsid fusion away from the signal sequence-dependent secretory SEC pathway in E. coli by utilizing the intrinsic signal sequence-independent property of pIX to obtain virion integration. This approach was combined with the use of an engineered helper phage known to improve antibody pIX display and retrieval. By direct comparison with pIII display, we demonstrate that antibody display using this pIX system translates into substantially improved retrieval of desired specificities with favorable biophysical properties in de novo selection. We show that the effect was due to less E. coli host toxicity during phage propagation conferred by the lack of a signal sequence. This pIX combinatorial display platform provides a generic alternative route for obtaining good binders with high stability and may thus find broad applicability.

• 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. Full text in Research Archive
• 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. doi: 10.3389/fonc.2014.00378. Full text in Research Archive Show summary

  Cancer immunotherapy has finally come of age, demonstrated by recent progress in strategies that engage the endogenous adaptive immune response in tumor killing. Occasionally, significant and durable tumor regression has been achieved. A giant leap forward was the demonstration that the pre-existing polyclonal T cell repertoire could be re-directed by use of cloned T cell receptors (TCRs), to obtain a defined tumor-specific pool of T cells. However, the procedure must be performed with caution to avoid deleterious cross-reactivity. Here, the use of engineered soluble TCRs may represent a safer, yet powerful, alternative. There is also a need for deeper understanding of the processes that underlie antigen presentation in disease and homeostasis, how tumor-specific peptides are generated, and how epitope spreading evolves during tumor development. Due to its plasticity, the pivotal interaction where a TCR engages a peptide/MHC (pMHC) also requires closer attention. For this purpose, phage display as a tool to evolve cloned TCRs represents an attractive avenue to generate suitable reagents allowing the study of defined pMHC presentation, TCR engagement, as well as for the discovery of novel therapeutic leads. Here, we highlight important aspects of the current status in this field.

• 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. Full text in Research Archive
• 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. 3. doi: 10.1038/srep01162. Full text in Research Archive Show summary

  We here report a novel phage display selection strategy enabling fast and easy selection of thermostabilized proteins. The approach is illustrated with stabilization of an aggregation-prone soluble single chain T cell receptor (scTCR) characteristic of the murine MOPC315 myeloma model. Random mutation scTCR phage libraries were prepared in E. coli over-expressing the periplasmic chaperone FkpA, and such over-expression during library preparation proved crucial for successful downstream selection. The thermostabilized scTCRmut variants selected were produced in high yields and isolated as monomers. Thus, the purified scTCRs could be studied with regard to specificity and equilibrium binding kinetics to pMHC using surface plasmon resonance (SPR). The results demonstrate a difference in affinity for pMHCs that display germ line or tumor-specific peptides which explains the tumor-specific reactivity of the TCR. This FkpA-assisted thermostabilization strategy extends the utility of recombinant TCRs and furthermore, may be of general use for efficient evolution of proteins.

• 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. Full text in Research Archive
• 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 Show summary

  Phage display has been instrumental in discovery of novel binding peptides and folded domains for the past two decades. We recently reported a novel pIX phagemid display system that is characterized by a strong preference for phagemid packaging combined with low display levels, two key features that support highly efficient affinity selection. However, high diversity in selected repertoires are intimately coupled to high display levels during initial selection rounds. To incorporate this additional feature into the pIX display system, we have developed a novel helper phage termed DeltaPhage that allows for high-valence display on pIX. This was obtained by inserting two amber mutations close to the pIX start codon, but after the pVII translational stop, conditionally inactivating the helper phage encoded pIX. Until now, the general notion has been that display on pIX is dependent on wild-type complementation, making high-valence display unachievable. However, we found that DeltaPhage does facilitate high-valence pIX display when used with a non-suppressor host. Here, we report a side-by-side comparison with pIII display, and we find that this novel helper phage complements existing pIX phagemid display systems to allow both low and high-valence display, making pIX display a complete and efficient alternative to existing pIII phagemid display systems.

• 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. 6(2). doi: 10.1371/journal.pone.0017433. Full text in Research Archive Show summary

  Background: Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display. Methodology/Principal Findings: Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient. Conclusions/Significance: Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before. © 2011 Wälchli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

• 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. Full text in Research Archive
• 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. 6(2). doi: 10.1371/journal.pone.0014702. Full text in Research Archive Show summary

  Background: Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII), or the minor coat protein III (pIII). Display on other coat proteins such as pVII allows for display of heterologous peptide sequences on the virions in addition to those displayed on pIII and pVIII. In addition, pVII display is an alternative to pIII or pVIII display. Methodology/Principal Findings: Here we demonstrate how standard pIII or pVIII display phagemids are complemented with a helper phage which supports production of virions that are tagged with octa FLAG, HIS6 or AviTag on pVII. The periplasmic signal sequence required for pIII and pVIII display, and which has been added to pVII in earlier studies, is omitted altogether. Conclusions/Significance: Tagging on pVII is an important and very useful add-on feature to standard pIII and pVII display. Any phagemid bearing a protein of interest on either pIII or pVIII can be tagged with any of the tags depending simply on choice of helper phage. We show in this paper how such tags may be utilized for immobilization and separation as well as purification and detection of monoclonal and polyclonal phage populations. © 2011 Wälchli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

• 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. 6(11). doi: 10.1371/journal.pone.0027930. Full text in Research Archive Show summary

  Although cloning and expression of T-cell Receptors (TcRs) has been performed for almost two decades, these procedures are still challenging. For example, the use of T-cell clones that have undergone limited expansion as starting material to limit the loss of interesting TcRs, must be weighed against the introduction of mutations by excess PCR cycles. The recent interest in using specific TcRs for cancer immunotherapy has, however, increased the demand for practical and robust methods to rapidly clone and express TcRs. Two main technologies for TcR cloning have emerged; the use of a set of primers specifically annealing to all known TcR variable domains, and 59-RACE amplification. We here present an improved 59-RACE protocol that represents a fast and reliable way to identify a TcR from 105 cells only, making TcR cloning feasible without a priori knowledge of the variable domain sequence. We further present a detailed procedure for the subcloning of TcRa and b chains into an expression system. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TcR transgene into different expression systems. The presented comprehensive method can be performed in any laboratory with standard equipment and with a limited amount of starting material. We finally exemplify the straightforwardness and reliability of our procedure by cloning and expressing several MART-1-specific TcRs and demonstrating their functionality. © 2011 Wälchli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

• 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. 10. doi: 10.1186/1472-6750-10-8. Full text in Research Archive Show summary

  Background Efficient expression systems exist for antibody (Ab) molecules, which allow for characterization of large numbers of individual Ab variants. In contrast, such expression systems have been lacking for soluble T cell receptors (TCRs). Attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. Here we present an optimized periplasmic bacterial expression system for soluble single chain (sc) TCRs. Results The effect of 1) over-expression of the periplasmic chaperon FkpA, 2) culture conditions and 3) molecular design was investigated. Elevated levels of FkpA allowed periplasmic soluble scTCR expression, presumably by preventing premature aggregation and inclusion body formation. Periplasmic expression enables disulphide bond formation, which is a prerequisite for the scTCR to reach its correct fold. It also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. Expression without IPTG induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. Shaker flask yield of mg levels of active purified material was obtained. The Vαβ domain orientation was far superior to the Vβα domain orientation regarding monomeric yield of functionally folded molecules. Conclusion The general expression regime presented here allows for rapid production of soluble scTCRs and is applicable for 1) high yield recovery sufficient for biophysical characterization and 2) high throughput screening of such molecules following molecular engineering.

• Lunde, Elin; Løset, Geir Åge; Bogen, Bjarne & Sandlie, Inger (2010). Stabilizing mutations increase secretion of functional soluble TCR-Ig fusion proteins. BMC Biotechnology. 10. doi: 10.1186/1472-6750-10-61. Full text in Research Archive Show summary

  Background Whereas T cell receptors (TCRs) detect peptide/major histocompatibility complexes (pMHCs) with exquisite specificity, there are challenges regarding their expression and use as soluble detection molecules due to molecular instability. We have investigated strategies for the production of TCR-immunoglobulin (Ig) fusion proteins. Two different TCRs that are characteristic of a mouse model for idiotype (Id) dependent immune regulation were engineered. They are structurally unrelated with different variable (V), diversity (D) and joining (J) segments, but each share one V gene segment, either Vα or Vβ, with the well characterized murine TCR, 2C. Results Several TCR-Ig formats were assessed. In one, the TCR V domains were fused to Ig constant (C) regions. In others, the complete extracellular part of the TCR was fused either to a complete Ig or an Ig Fc region. All molecules were initially poorly secreted from eukaryotic cells, but replacement of unfavourable amino acids in the V regions improved secretion, as did the introduction of a disulfide bridge between the TCR C domains and the removal of an unpaired cysteine. A screening strategy for selection of mutations that stabilize the actual fusion molecules was developed and used successfully. Molecules that included the complete heterodimeric TCR, with a stabilizing disulfide bridge, were correctly folded as they bound TCR-specific antibodies (Abs) and detected pMHC on cells after specific peptide loading. Conclusions We show that fully functional TCR-Ig fusion proteins can be made in good yields following stabilizing engineering of TCR V and C region genes. This is important since TCR-Ig fusions will be important probes for the presence of specific pMHCs in vitro and in vivo. In the absence of further affinity maturation, the reagents will be very useful for the detection of kinetic stability of complexes of peptide and MHC.

• 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. Full text in Research Archive 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive

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• Løset, Geir Åge; Mousavi, Seyed A. & Berg, Trond (2008). Sinusoidal liver cells and liver tolerance. Transworld Research Network. ISBN 9788178953649. 11 p. Full text in Research Archive

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• Pesci, Giacomo; Johannesen, Hedda; Løset, Geir Åge & Krengel, Ute (2020). A promising antibody to treat cancer. How does it work? Full text in Research Archive
• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Grzybek, Michal; Løset, Geir Åge & Krengel, Ute (2020). Molecular basis of specificity of a promising anti-tumour antibody. Full text in Research Archive
• 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. Full text in Research Archive
• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Heggelund, Julie Elisabeth; Løset, Geir Åge & Krengel, Ute (2019). 14F7 - A tumour specific antibody. Full text in Research Archive
• Johannesen, Hedda; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2019). An anti-tumour antibody that specifically recognises N-glycolyl GM3. Full text in Research Archive
• Johannesen, Hedda; hoås, Helene Mykland; Heggelund, Julie Elisabeth; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2018). 14F7, An anti-ganglioside scFv for cancer therapies. Full text in Research Archive
• Johannesen, Hedda; Mykland, Helene Hoås; Heggelund, Julie Elisabeth; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2018). An anti-ganglioside scFv for cancer therapies. Full text in Research Archive
• Johannesen, Hedda; Hoås, Helene Mykland; Heggelund, Julie Elisabeth; Bjerregaard-Andersen, Kaare; Løset, Geir Åge & Krengel, Ute (2018). 14F7, an antibody that recognises a tumour-specific ganglioside with high specificity. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• Johannesen, Hedda; Bousquet, Paula; Løset, Geir Åge & Krengel, Ute (2014). L`ASSASSINA PROTEIN THAT SPECIFICALLY KILLS TUMOUR CELLS. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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–+. Full text in Research Archive
• 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. Full text in Research Archive
• Løset, Geir Åge; Lunde, Elin & Sandlie, Inger (2007). Soluble T cell receptors. Not quite like antibodies. NBS-nytt. ISSN 0801-3535. p. 14–17. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• Pesci, Giacomo; Krengel, Ute; Johannesen, Hedda & Løset, Geir Åge (2020). Follow the Light - Tracking 14F7’s cellular journey with luminescence. Universitetet i Oslo. Full text in Research Archive 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. Full text in Research Archive
• 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. Full text in Research Archive Show summary

  Kreft er en sykdom som oppstår når en mutert celle vokser og deler seg, uavhengig av plassrestriksjoner eller tilgang på næring. Dette lager et indre press på andre organer i kroppen, hvis det ikke blir behandlet. I dag har vi ingen behandlingsform som dreper kreft uten at vi selv blir syke, derfor er det viktig å utarbeide nye kreftmedisiner for å fjerne kreftcellene mer effektivt I tillegg til effektiv celledeling, produserer kreftceller molekyler av andre sammensetninger og konsentrasjoner sammenlignet med friske celler. Denne kunnskapen utnyttes i immunterapi, hvor kroppens eget immunforsvar blir stimulert til å angripe kreftcellene. Et molekyl som kun eksisterer på celleoverflaten til kreft hos voksene mennesker, er gangliosidet NeuGc GM3. Dette gangliosidet blir gjenkjent av et antistoff med navnet 14F7, med kallenavnet Snikmorderen fordi det kun dreper kreftceller. Når 14F7 binder til gangliosidet, starter cellen å svulme, sprekke opp, og dø. Dette gjør 14F7 spesielt attraktiv for fremtidig kreftbehandling, siden den spesifikt dreper kreft celler. For å videreutvikle 14F7 og øke kunnskapen for hvordan antistoffer gjenkjenner karbohydratbaserte molekyler, må vi først kartlegges hvordan 14F7 interagerer med NeuGc GM3 gangliosidet. Strukturen til 14F7s fragment, antigen-bindende (Fab) ble kjent i 2004 ved hjelp av røntgenkrystallografi. På tross av iherdig innsats var det ikke mulig å reprodusere Fab krystallen, derfor finnes det i dag ikke et eksperimentelt svar på hvor og hvordan gangliosidet assosieres med 14F7. For å løse dette mysteriet prøves en ny vinkling, hvor 14F7 single-chain variable fragmenter (scFvs) blir uttrykt i periplasma til E. coli. scFv er det minste fragmentet som fortsatt inneholder det komplette bindingssete til gangliosidet. Siden scFv er mindre komplekst, sammenlignet med Fab og det komplette antistoffet, er det en lovende kandidat for å karakterisere interaksjonene mellom 14F7 og NeuGc GM3 gangliosidet ved bruk av røntgenkrystallografi. Fire forskjellige varianter av scFv 14F7 ble klonet og transformert inn i E. coli, hvor de ble isolert fra periplasma og renset med affinitetskromatografi. Bindingen til gangliosidet ble testet, siden konstruktene ikke gjenkjente gangliosidet ble scFv konstruktene optimalisert. Resultatet ble funksjonelle scFv proteiner med affinitet for NeuGc GM3. 14F7 scFv proteinene ble brukt til å utføre ELISA, ThermoFluor, SPR og krystalliseringsstudier. Vi klarte å gro krystaller, men fikk ikke brukbare diffraksjonsdata. Systemet trenger derfor å bli optimalisert videre. Denne masteroppgaven danner et grunnlag for videre arbeid både for krystalliseringsstudier, men også for hvordan ulike scFv design påvirker proteinets stabilitet og uttrykking i prokaryote celler.

• Løset, Geir Åge (2005). Antibody and T cell receptor engineering: Mechanistic studies and biotechnological applications. Universitetet i Oslo. Full text in Research Archive
• Løset, Geir Åge (2000). A comparison of the antigen binding ability of antibodies characteristic of the primary immune respons. Biologisk institutt. Full text in Research Archive

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