Cancer is the second-most common cause of death for humans and engendered by malignant human cells dividing uncontrollably due to the failure of cell growth control mechanisms. A major bottleneck for the development of new diagnostics and therapeutics is the identification of specific tumor biomarkers and the generation of suitable tools to target these biomarkers. Tumor-associated carbohydrate antigens (TACAs) and aberrant surface thiol groups are promising classes of tumor biomarkers. However, the number of available tools to study them is very limited. I established an approach to generate heavy-chain antibodies targeting TACAs by immunization of an alpaca, thereby expanding the toolbox against TACAs. Conjugates of TACAs and carrier proteins were produced and injected into an alpaca, inducing the formation of glycan-specific conventional and heavy-chain antibodies in the animal. These heavy-chain antibodies formed the basis for the generation of nanobodies binding to the tumor-associated glycan Globo-H. By serendipity, I discovered a new tool for targeting thiol groups on cancer cells, as nanobody CB2 specifically binds to several subtypes of B cell lymphoma and breast cancer via thiol-thiol interactions. I demonstrated that CB2 binding is linked to increased thiol levels on lymphoma cells and requires the presence of an unusual cysteine in the antigen-binding region of the nanobody. CB2 was endowed with additional properties through functionalization, using a combination of chemical and enzymatic conjugation methods. On one hand, I showed that CB2 modified with a rhamnosylated glycopeptide can recruit antibodies to lymphoma cells, resulting in complement activation and cancer cell death. On the other hand, using fluorophore- coupled CB2, I proved that thiol-mediated CB2 binding is followed by internalization into lymphoma cells. Finally, I demonstrated that CB2 internalization can be exploited for the delivery of cytotoxic agents to cancer cells, again leading to cancer cell death. In summary, the results of my work illustrate the potential of combining thiol-binding, internalizing nanobodies with further functionalization, thereby laying the foundation for numerous applications in cancer diagnostics and therapeutics.
