Conventional proteomic approach using different modifications of two-dimensional electrophoresis combined with mass spectrometry analysis (2D-PAGE & MS) in looking for gastric adenocarcinoma (GC) biomarkers was in 2007/08 upgraded by nanobody-based proteomic technology, and besides to still running search for GC biomarkers, reseach was focused to the investigation of molecular basis of glioma and glioblastoma (GBM) and search of proteomic biomarkers specific to GBM stem cells (GSCs). In fact, the two inventions, which were based on previous research, represented the basic methodological platform of ongoing research: (A) an innovative method of the production of both VHH and VH nanobodies simultaneously (doi: 10.1016/j.jim.2009.08.016), and (B) discovery of an algorithm, Global Sequence Signature (GSS), able to take into account multiple functional and/or local sequence properties to detect scattered evolutionary constraints into protein sequences. This study has unravelled a robust interaction network on antibody structure surrounding the CDR3 loop. In addition to the general applicability of the GSS algorithm (https://doi.org/10.1016/j.bbagen.2013.02.014), which can bring together functional and sequence data to locate hot spots of constrained evolution, the relationship between CDR3 and scaffold is taken into account in protein engineering when designing antibody libraries.
Nanobodies (Nbs) are single-domain antigen-binding fragments of Camelid heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small (4.0 x 2.5 nm; 12-15 kDa), soluble and stable particles, they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments.
Glioblastoma (GBM) is the most frequent and most malignant primary brain tumor in adults, with the incidence of 3–5 cases per 100,000 EU inhabitants. Its localization to the brain, invasive behavior and extremely poor prognosis make it one the most lethal forms of cancer. Despite improvement of surgical removal, chemotherapy and radiotherapy, the median survival of patients with multimodal treatment approaches is approximately 15 months, with only 3-5% of patients surviving longer than 36 months. The presence of disseminating tumor stem-like cells (GSCs) that support tumor self-renewal and are particularly resistant to chemo- and radio-therapy, is the major factor in preventing complete surgical resection and causing tumor recurrence. If GCSs are responsible for tumor formation, maintenance and recurrence, then new treatments should aim to specifically target and destroy these cells. Given the complex genetic and epigenetic heterogeneity of GBM, it is unlikely that the expression of a single marker will define cancer stem cells in every tumor. Hence, a combination of markers will probably best define glioma stem cells, thus making a way to the development of better tailored brain cancers tretament(s). Consequently the discovery of new, more specific GSC markers remains one of priorities in the research on how to effectively battle this type of cancer.
Following immunization of Southamerican llamas with GBM/GSC protein extracts we recently identified, with the nanobody-based reverse proteomics methodology, several putative human glioblastoma biomarkers which are now in the process of in vitro and in vivo functional assessment and studied as candidates for the design of the nanobody-based diagnostic approaches and targeting strategies. In this context engineered transport vesicles such as GPMVs, archeosomes end exosomes are considered.
⇒ Link to a current core research project: TRANS-GLIOMA
Past and Present Research Projects:
- INTERREG EC Project 2017: New Therapies of Glioblastoma by using a Transborder Translational Research Platform (Acronym: TRANS-GLIOMA): University of Ljubljana (MCMB, Faculty of Medicine), Lead Partner; R. Komel, Project Leader/Coordinator; 1/7/2017 – 30/11/2020: https://www.ita-slo.eu/en/transglioma-0; https://www.facebook.com/TransGlioma/.
- Z3-2649: Identification of novel glioblastoma bio-markers for non-invasive liquid biopsy; basic post-doctoral research project, N. Šamec, 1/9/2020 – 31/8/2022: Z3-2649 (B)
- 3M200315 CELSA: Generation of nanobodies against immunomodulating checkpoint receptors in glioblastoma tumor cells; CELSA research project, I. Jovčevska, 1/9/2020 – 31/8/2022: CELSA-2020; CELSA.
- Z3-1869: Development of anti-FREM2 nanobody and its use for targeting glioblastoma cells; basic post-doctoral research project, I. Jovčevska, 1/7/2019 – 30/06/2021: Z3-1869
- National research program P1-0390 Functional Genomics and Biotechnology for Health; R. Komel, 1/1/2015 – 10/7/2017 / D. Rozman, 10/7/2015 – 31/12/2020 (previously P1-0104; 2009-2014, R. Komel); A/ Molecular and Cellular Understanding of Selected Complex Multifactorial Disorders: WP1a – Molecular roots of carcinogenesis (R. Komel). B/ Search for Molecular Targets, New Drugs and Delivery Systems Design: WP 3′ – Development of new therapeutic approaches and delivery systems of nano-particles (R. Komel): http://www.sicris.si/search/prg.aspx?lang=slv&id=6087; http://www.sicris.si/search/prg.aspx?lang=slv&id=9665.
- INTERREG EC Project 2011, Ref. No. 42: Identification of novel biomarkers to brain tumors – glioma, for diagnosis and as new therapeutic targets (Acronym: GLIOMA); https://www.keep.eu/keep/project-ext/21554/GLIOMA?ss=14b907cacd0802f2d5c154c4f9762589&espon; T. Lah Turnšek & R. Komel, 1/11/2011 ― 30/04/2015.
- J1―9740 Development of protein microarray for research on gastric cancer proteome; basic national research project; R. Komel, 1/1/2007―31/12/2009; http://www.sicris.si/search/prj.aspx?lang=slv&id=5337; DOI:10.3390/microarrays5030019.
- PICS-3294 Project »Characterisation of proteome markers in cancer cell lines and tissues using surface plasmon resonance imagery on chip« [Joint Research Project of the International Agreements of Slovenian Government (Ministry for High Education, Science and Technology) with French Government (CNRS, France)]; R. Komel & D. Pompon, 2004 ― 2006; https://www.cnrs.fr/derci/spip.php?article881&lang=en.
Clinical Partners:
- University Clinical Centre Ljubljana (UKC), Neurosurgery Clinic (Boštjan Matos, M.D.; Andrej Vranič, M.D.)
- Institute of Oncology Ljubljana (OI), Pathology Dept. (Uroš Smrdel, M.D.; As. Prof. Lorna Zadravec Zaletel, M.D.; Marija Skoblar Vidmar, M.D.)
Collaborating Research Institutes:
- National Institute of Biology (NIB), Dept. for Genetic Toxicology and Cancer Biology, Ljubljana (Prof. Tamara Lah Turnšek, PhD)
- Faculty of Medicine UL, Institute of Pathology (Dr. Jože Pižem, M.D.; Jernej Mlakar, M.D.)
- Faculty of Medicine UL, Institute of Biophysics (Špela Zemljič Jokhander, PhD)
- Faculty of Medicine UL, Institute for Forensic Medicine (Prof. Jože Balažic, M.D.)
- Biosistemika d.o.o., Ljubljana (Matija Valinger)
- Euroservis S.r.l., Trieste, Italy (Mojca Zajc)
International Collaboration:
- Free University Brussels (VUB), Cellular and Molecular Immunology, Brussels, Belgium (Prof. Serge Muyldermans, PhD)
- International Centre for Genetic Engineering and Biotechnology (ICGEB), Protein Networks, Trieste, Italy (Mike Myers, PhD)
- Azienda Sanitaria Universitaria Integrata di Udine (ASUIUD), Udine, Italy
- Elettra Sincrotrone Trieste S.C.p.A., Trieste, Italy
- Azienda Unita Locale Socio- Sanitaria N. 12 Veneziana, Venezia, Italy
- Formerly also: Institut National de Recherche Scientifique (INSERM), Gif-sur-Yvette, France (Prof. Denis Pompon, PhD)
Researchers involved:
| Researcher | Position |
| Radovan Komel | Group and Project Leader |
| Mirjana Liović | Senior Researcher |
| Ivana Jovčevska | PostDoc Researcher |
| Neja Šamec | PostDoc Researcher |
| Alja Zottel | Junior Researcher / PhD student |
| Former Group Members | |
| Ana Kump | Technical Assistant |
| Lucija Raspor Dall’Olio | PostDoc Researcher |
| Marko Vidak | PhD student |
| Nina Kočevar | PhD student & PostDoc |
| Damjana Kastelic | PhD student & PostDoc |
Education:
| Student | Thesis | Title | Year | Supervisors |
| Alja Zottel | PhD | Evaluation of nanobodies against the selected protein biomarkers of glioblastoma and attempt of their delivery with exosomes. | 2021 | Radovan Komel |
| Neja Zupanec | PhD | Implementation of nanobodies for the design of glioblastoma targeting therapy. | 2018 | Radovan Komel |
| Marko Vidak | PhD | Cell communication network analysis of glioblastoma stem cell marker candidates β-actin, CD9, FTL, S100A9 and TRIM28. | 2018 | Radovan Komel |
| Ivana Jovčevska | PhD | Llama heavy-chain antibody-derived nanobodies as tools to identify protein markers for human glioma. | 2015 |
Radovan Komel S. Muyldermans |
| Filip Mihalič | MSc | Determination of cytotoxic effects of nanobodies specific to TRIM28, β-actin and factor 206, on three cell lines. | 2015 |
Radovan Komel Mirjana Liović |
| Maša Mirković | Dipl | Analysis of selected proteins in patients with gastric adenocarcinoma with Western blotting and immunodetection. | 2014 |
Radovan Komel Nina Kočevar |
| Nina Kočevar | PhD | Searching for protein biomarkers in gastric adenocarcinoma. | 2011 | Radovan Komel |
| Damjana Kastelic | PhD | Identification of markers and development of protein biochips for characterization of proteome and typing of malignant tissues. | 2009 |
Radovan Komel Denis Pompon |
| Marko Šnajder | Dipl | Two-dimensional electrophoresis in the study of gastric adenocarcinoma. | 2008 | Radovan Komel |
| Nina Kočevar | Dipl | Proteome analysis of gastric cancer with two-dimensional gel electrophoresis: optimization of the procedure and preliminary experiments. | 2007 |
Radovan Komel D. Žgur Bertok |
Selected publications:
- Zottel A., Jovčevska I., Šamec N. and Komel R. (2021): Cytoskeletal proteins as glioblastoma biomarkers and targets for therapy: a systematic review (Review). Critical Reviews in Oncology/Hematology xx: 1-29; https://xxx, doi: xxx.
- Zottel A. , Šamec N., Kump A., Raspor Dall’olio L., Pužar Dominkuš P., Romih R., Hudoklin S., Mlakar J., Nikitin D., Sorokin M., Budzin A. A., Jovchevska I., Komel R. (2020): Analysis of miR-9-5p, miR-124-3p, miR-21-5p, miR-138-5p, and miR-1-3p in glioblastoma cell lines and extracellular vesicles. International Journal of Molecular Sciences 21(22): 1-22; https://www.mdpi.com/1422-0067/21/22/8491/htm, doi: 10.3390/ijms21228491.
- Zottel A., Jovčevska I., Šamec N., Mlakar J., Šribar J., Križaj I., Zadravec Zaletel L., Komel R. (2020): Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma (stem) cell migration. Therapeutic Advances in Medical Oncology 12:1-29; https://journals.sagepub.com/doi/pdf/10.1177/1758835920915302, doi: 10.1177/ 1758835920915302.
- Zottel A., Šamec N., Videtič Paska A., Jovchevska I. (2020): Coding of glioblastoma progression and therapy resistance through long noncoding RNAs (Review). Cancers 12: 1-23; https://www.mdpi.com/2072-6694/12/7/1842, doi: 10.3390/cancers12071842.
- Šamec N., Zottel A., Videtič Paska A., Jovčevska I. (2020): Nanomedicine and Immunotherapy: A Step Further towards Precision Medicine for Glioblastoma. Molecules 25(3), pii: E490; https://www.mdpi.com/1420-3049/25/3/490/pdf, doi: 10.3390/molecules25030490.
- Jovčevska I. and Muyldermans S. (2020): The therapeutic potential of nanobodies. BioDrugs 34(1): 11-26; https://www.bjbms.org/ojs/index.php/bjbms/article/view/3717, doi: 0.1007/s40259-019-00392-z.
- Jovčevska I. (2020): Next generation sequencing and machine learning technologies are painting the epigenetic portrait of glioblastoma (Review). Frontiers in Oncology 10: 1-14; https://www.frontiersin.org/articles/10.3389/fonc.2020.00798/full, doi: 10.3389/fonc.2020.00798.
- Jovčevska I., Zottel A., Šamec N., Mlakar J., Sorokin M., Nikitin D., Buzdin A., Komel R. (2019): High FREM2 gene and protein expression levels are associated with favorable prognosis of IDH-wild type glioblastomas. Cancers 11: 1-18; doi: 10.3390/cancers11081060.
- Zottel A., Videtič Paska A., Jovčevska I. (2019): Nanotechnology meets oncology: nanomaterials in brain cancer research, diagnosis and therapy. Materials 12(10): pii: E1588; doi:10.3390/ma12101588 .
- Jovčevska I. (2019): The genetic secrets of long term glioblastoma survivors. Bosn. J. Basic Med. Sci. 19(2): 116-124; doi:10.17305/bjbms.2018.3717.
- Vidak M., Liović M., Jovčevska I. et al. (2018): Meta-analysis of transcriptome data from public databases and subsequent experimental validation exposed SPRY1 and FREM2 as new glioblastoma stem cell marker candidates. International Journal of Molecular Sciences 19(5): pii: E1369; doi: 10.3390/ijms19051369.
- Šamec N., Jovčevska I., Zottel A. et al. (2018): Glioblastoma-specific anti-TufM nanobody for in vitro immunoimaging and cancer stem cell targeting. Oncotarget 9(25): 17282-17299; doi: 10.18632/oncotarget.24629.
- Zemljič Jokhadar Š. et al. (2018): GPMVs in variable physiological conditions: could they be used for therapy delivery? BMC Biophysics 11: 1-12.
- Jovčevska I. (2018): Sequencing the next generation of glioblastomas. Crit. Rev. Clin. Lab. Sci. 55(4): 264-282; doi: 10.1080/10408363.2018.1462759.
- Jovchevska I., Zupanec N., Urlep Ž., et al. (2017): Differentially expressed proteins in glioblastoma multiforme identified with a Nanobody-based anti-proteome approach and confirmed by OncoFinder as possible tumor class predictive biomarker candidates. Oncotarget 8(27): 44141-44158; doi: 10.18632/oncotarget.17390.
- Kunz P. et al. (2017): Exploiting sequence and stability information for directing nanobody stability engineering. Biochim. Biophys. Acta General Subjects 1861(9): 2196–2205; doi: 10.1016/j.bbagen.2017.06.014.
- Vidak M., Rozman D., Komel R. (2015): Effects of flavonoids from food and dietary supplements on glial and glioblastoma multiforme cells. Molecules 20(10): 19406-19432; doi: 10.3390/molecules201019406.
- Hudler P., Kocevar N., Komel R. (2014): Proteomic approaches in biomarker discovery: new perspectives in cancer diagnostics. Sci. W. J. 2014:260348. doi: 10.1155/2014/260348.
- Zavec A. B. et al. (2014): Archaeosomes can efficiently deliver different types of cargo into epithelial cells grown in vitro. J. Biotechnol. 192 Pt A:130-5. doi: 10.1016/j.jbiotec.2014.09.015.
- Jovčevska I. et al. (2014): TRIM28 and β-actin identified via nanobody-based reverse proteomics approach as possible human glioblastoma biomarkers. PloS One 9(11): e113688; doi: 10.1371/journal.pone.0113688.
- Kočevar N. et al. (2013): The progress of proteomic approaches in searching for cancer biomarkers. New Biotechnology 30(3): 319-326; doi: 10.1016/j.nbt.2012.11.011.
- Kastelic D. et al. (2013): The global sequence signature algorithm unveils a structural network surrounding heavy chain CDR3 Loop in Camelidae variable domains. BBA General Subjects 1830(6): 3373-3381; doi:1 0.1016/j.bbagen.2013.02.014.
- Jovčevska I., Kočevar N., Komel R. (2013): Glioma and glioblastoma – how much do we (not) know? Mol. Clin. Oncol. 1(6):935-941; doi: 10.3892/mco.2013.172.
- Schmidt R. et al. (2013): Optimised ‘on demand’ protein arraying from DNA by cell free expression with the ‘DNA to Protein Array’ (DAPA) technology. Proteomics 88: 141-148; doi: 10.1016/j.jprot.2013.02.002.
- Kočevar N. et al. (2012): Proteomic analysis of gastric cancer and immunoblot validation of potential biomarkers. World J. Gastroenterol. 18(11): 1216-1228; doi 10.3748/wjg.v18.i11.1216.
- Hudler P. et al. (2010): Proteomic strategies in tumor metastasis research. Clin. Exp. Metastasis 27(6): 441-451; doi: 10.1007/s10585-010-9339-7.
- Kastelic D. et al. (2008): A single-step procedure of recombinant library construction for the selection of efficiently produced llama VH binders directed against cancer markers. J. Immunol. Meth. 350(1-2): 54-62; doi: 10.1016/j.jim.2009.08.016.