Applications for nanobodies in therapy and cell research

Bates, Jack Anthony (2020): Applications for nanobodies in therapy and cell research. Dissertation, LMU München: Fakultät für Biologie

Abstract

The incidental discovery of special heavy chain only antibodies (HCAbs) in the early 90s has gone on to become an important landmark in numerous fields of antibody application. HCAbs are lightweight camelid derived antibodies that lack both the light chains and CH1 domains of standard structure antibodies. Unlike classic antibodies, HCAbs therefore bind to antigens using only monomeric heavy chain variable domains (VHHs). When these VHHs, also called nanobodies, are expressed in isolation, they form single domain antibodies (sdAbs) with some extraordinary properties. Their minute size enables them to bind to otherwise inaccessible epitopes and to diffuse excellently through tissues, they are often exceptionally stable and minimally immunogenic and since nanobodies remain soluble when expressed in the cytoplasm, they can also be used to target intracellular antigens and guide fused effectors modules. By exploiting such attributes nanobody technology has now evolved to fill a massive range of scientific, pharmaceutical and diagnostic functions. In this work we have made use of these exciting molecules to generate new methods with applications in therapy and cell research. To assist in the study of the protein function, we have constructed highly tunable nanobody targeted tools, which enable the rapid modulation of target protein levels upon the application of light or small molecules. These DiPD (drug induced protein degradation) and LiPD (light induced protein degradation) systems can be programmed to target unmodified cellular proteins within diverse organisms and furthermore, can be combined to degrade multiple proteins simultaneously. We anticipate that these systems will be extremely useful to those studying long half-life, essential and redundant proteins. To therapeutically target disease machinery present inside of cells, we have designed and developed highly optimized nanobody chimeras capable of potently and selectively killing cells that harbor these antigens. We have demonstrated the function of these target responsive apoptotic proteins (TRAPs) using the capsid proteins of HIV-1 and Hepatitis B virus and a pipeline for the production of these molecules has been described. We suggest that almost any cytosolic or nuclear protein of permissive concentration could be targeted using this system and that TRAP-like molecules could therefore be effective against a number of diseases. Lastly, since the successful delivery of nanobodies to the cytosol still represents a major obstacle to their therapeutic implementation, we have investigated and optimized cellular protein delivery using Mesoporous Silica Nanoparticles (MSNs). We developed a background free sensor to track protein delivered into in vitro cell populations and showed that MSNs can facilitate impressive rates of protein transfection which are comparable to those of numerous commercial products. This study demonstrates the great potential of MSN mediated protein delivery and also provides a highly tractable protein delivery sensor which would be well suited to study a range of delivery methods.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 21. Februar 2020
• 1. Bericht­erstatter:in: Leonhardt, Heinrich