Medical biotechnology : Nanomedicine

Objectives and competences

Targeted drug delivery using nanoparticles, in vivo imaging with high affinity binding domains, high-resolution clinical treatment using radiolabeled and fluorescent antibodies, ultrasensitive biosensors based on selective binders: medical technologies rely on the availability of molecules characterized by elevated affinity and high selectivity. At the same time, in vitro synthesis allows the preparation of binder libraries of extremely large diversity. The course will analyse how to design, build, screen, characterize, and functionalize such binders to produce reagents suitable for the different applications.
Course will be active every two years.

Prerequisites

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Content

• Recombinant Ab technology and alternative scaffolds
• Libraries and panning: experimental design using purified antigens and cells, Yeast and bacterial display
• Binder sequencing (deep sequencing), characterization, subcloning, production
• NP structures, NP bioactivation strategies, NP applications in imaging and therapy, NP toxicology
• Parameters influencing in vivo biodistribution
• Mechanisms of action and cell uptake
• Abs for imaging (PET, IR, …) and fluorescent Abs in surgery
• Radiotherapy
• In vitro diagnostics
• Imunopurifikacija
• Antibody coupling and biosensor surface activation, AFM biosensors
• Chemical detection

Intended learning outcomes

Students will learn the principles of different approaches to identify antigen specific binders of different formats using phage display technology and alternative display methods. The characterization, biological validation, expression alternatives, and production options available for recombinant antibodies will be exposed. Successively, the course will describe different nanoparticles and the strategies used to functionalize them with binders able to optimize their targeting in vivo for application in imaging and therapy. NPs possible side-effects will be discussed.
A further lesson block will deal with the relationships between antibody mass, avidity, biodistribution, cell uptake, and endosomal transport. These parameters influence the binder activity and their suitability for the different applications in vivo. Mass and avidity are also crucial for in vitro diagnostic, affinity purification, and the building of effective biosensors.

Readings

  • Bradbury, A.R.M.; Sidhu, S.; Dübel, S.; McCafferty, J. Beyond the natural antibodies: the power of in vitro display technologies. Nat. Biotechnol., 2011, 29, 245-254.

  • Monegal, A.; Ami, D.; Martinelli, C.; Huang, H.; Aliprandi, M.; Capasso, P.; Francavilla, C.; Ossolengo, G.; de Marco, A. Immunological applications of single domain llama recombinant antibodies isolated from a naïve library. Prot. Engineer. Des. Sel., 2009, 22, 273-280.

  • Vaneycken, I.; D’huyvetter, M.; Hernot, S.; De Vos, J.; Xavier, C.; Devoogdt, N.; Caveliers, V.; Lahoutte, T. Immuno-imaging using nanobodies. Curr. Op. Biotechnol., 2011, 22, 1-5.

  • Jain, R.K. Transport of molecules in the tumor interstitium: a review. Cancer Res., 1987, 47, 3039-3051.

  • Vosjan, M.J.; Vercammen, J.; Kolkman, J.A.; Stigter-van Walsum, M.; Revets, H.; van Dongen, G.A. Nanobodies targeting the hepatocyte growth factor: potential new drugs for molecular cancer therapy. Mol. Cancer Ther., 2012, 11, 1017-1025.

  • Oliveira, S.; Schiffelers, R.M.; van der Veeken, J.; van der Meel, R.; Vongpromek, R.; van Bergen En Henegouwen, P.M.; Storm, G.; Roovers, R.C. Downregulation of EGFR by a novel multivalent nanobody-liposome platform. J. Control. Release, 2010, 145, 165-175.

  • Shen, B.Q.: Xu, K.; Liu. L.; Raab. H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K.L.; Tien, J.; Yu, S.F.; Mai, E.; Li, D.; Tibbitts, J.; Baudys, J.; Saad, O.M.; Scales, S.J.; McDonald, P.J.; Hass, P.E.; Eigenbrot, C.; Nguyen, T.; Solis, W.A.; Fuji, R.N.; Flagella, K.M.; Patel, D.; Spencer, S.D.; Khawli, L.A.; Ebens, A.; Wong, W.L.; Vandlen, R.; Kaur, S.; Sliwkowski, M.X.; Scheller, R.H.; Polakis, P.; Junutula, J.R. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol., 2012, 30, 184-189.

  • de Marco, A. 2014 Methodologies for the isolation of alternative binders with improved clinical potentiality over conventional antibodies. Crit. Rev. Biotech., 33(1):40-8

  • de Marco A. 2013 Co-expression and co-purification of antigen-antibody complexes in bacterial cytoplasm and periplasm. Methods Mol Biol. 1129:125-35.

  • Zou T, Dembele F, Beugnet A, Sengmanivong L, Trepout S, Marco S, de Marco A, Li M-H (2015) Nanobody-functionalized PEG-b-PCL polymersomes and their targeting study. J Biotechnology, 214:147-155

  • Crépin R, Gentien D, Duché A, Rapinat A, Reyes C, Némati F, Massonnet G, Decaudin D, Djender S, Moutel S, Desrumeaux K, Cassoux N, Piperno-Neumann S, Amigorena S, Perez F, Roman Roman S, de Marco A (2017) Nanobodies against surface biomarkers enable the analysis of tumor genetic heterogeneity in uveal melanoma Patient Derived Xenografts. Pigment Cell Melanoma Res, 30:317-327

  • Ambrosetti E, Paoletti P, Bosco A, Parisse P, Scaini D, Tagliabue E, de Marco A, Casalis L (2017) Quantification of circulating cancer biomarkers via sensitive topographic measurements on single binder nanoarrays. ACS Omega, 2:2618-2629

Assessment

During the course students will prepare individual projects in a written form that will be defended orally in an open discussion with professor and students (30%).

Lecturer's references

Ario de Marco is associate professor in the field of biotechnology at the University of Nova Gorica.

Selected Bibliography:
1. LEVI-SCHAFFER, Francesca, DE MARCO, Ario. COVID-19 and the revival of passive immunization : antibody therapy for inhibiting SARS-CoV-2 and preventing host cell infection : IUPHAR review: 31. British Journal of Pharmacology. 2021, 1-35. DOI: 10.1111/bph.15359.
2. OLOKETUYI, Sandra, ANNOVI, Giulia, DE MARCO, Ario. Peroxidase zymograms obtained by agarose native gel electrophoresis have unmet resolution and completeness. International journal of biological macromolecules. 2020, 156:869-873. DOI: 10.1016/j.ijbiomac.2020.04.058.
3. POPOVIC, Milica, MAZZEGA, Elisa, TOFFOLETTO, Barbara, DE MARCO, Ario. Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions. Microbial cell factories. 2018, 17:1-13. DOI: 10.1186/s12934-017-0856-9.
4. ROMANI, Chiara, DE MARCO, Ario, et al. Evaluation of a novel human IgG1 anti-claudin3 antibody that specifically recognizes its aberrantly localized antigen in ovarian cancer cells and that is suitable for selective drug delivery. Oncotarget. 2015, 6(33):34617-34628. DOI: 10.18632/oncotarget.5315.
5. MASSA, Paul E., PANICCIA, Aida, MONEGAL, Ana, DE MARCO, Ario, RESCIGNO, Maria. Salmonella engineered to express CD20-targeting antibodies and a drug-converting enzyme can eradicate human lymphomas. Blood. 2013, 122(5):705-714. DOI: 10.1182/blood-2012-12-474098.