Nanobody formats and conjugates detecting, modulating, and targeting CXCR4

Cell surface receptors play a crucial role in orchestrating cellular responses to external signals such as the binding of extracellular ligands. The largest group of these receptors is that of G protein-coupled receptors (GPCRs). Within this group, chemokine receptors direct cell migration in response to ligands, which is vital for developmental processes and immune functions. The chemokine receptor CXCR4 is expressed at high levels in many cancers and plays a significant role in cancer progression. Another important group of transmembrane receptors is the receptor tyrosine kinases (RTKs). One of those is the epidermal growth factor receptor (EGFR) which promotes cell proliferation in physiological settings but also in the context of some cancers. Elevated expression levels of CXCR4 and EGFR result in the clustering of the respective receptor and increasing interactions with other receptors. Such interactions are thought to modify the receptor’s signaling output and hence might be implicated in the progression of several cancer types. The role of CXCR4 and EGFR in oncogenic settings makes them attractive targets for fundamental research as well as potential therapeutic applications. Several types of molecules have been developed targeting those receptors, including small molecules, peptides, antibodies, and antibody fragments. Nanobodies, also called VHHs or single-domain antibodies, are the variable domain of a heavy-chain-only antibody found in camelids. They have protruding binding sites enabling them to target buried epitopes such as the ligand-binding pocket of GPCRs. Moreover, nanobodies can be modified in several ways and for various applications, which is the topic of this thesis. After a general introduction (Chapter 1), this thesis first describes the use of fluorescently labeled nanobodies as probes in bioluminescence resonance energy transfer (BRET)-based studies (Chapter 2). The so-called NanoB2 workflow allows for monitoring the real-time binding of labeled nanobodies and unlabeled (competing) molecules to luciferase-tagged receptors. Next, the generation of new panels of CXCR4-targeting nanobodies with varying binding affinities and novel binding modes is described (Chapter 3). Interestingly, some of those nanobodies act as conformational sensors and detect small-molecule binding to luciferase-tagged CXCR4. The following three chapters make use of the novel high-affinity nanobodies in different applications. One of the new nanobodies, VUN410, was then further engineered for higher affinity (through avidity) by constructing various multivalent formats with up to ten nanobodies per molecule (Chapter 4). Among those, the bivalent nanobody-Fc construct seems best suited for avid binding and showed high potency in antagonizing ligand-induced signaling. In Chapter 5, the heteromerization of CXCR4 and EGFR is examined using functionalized nanobodies. Initially, CXCR4/EGFR heteromers and their modulability by endogenous ligands and nanobodies were detected using tagged receptors and BRET. Then, we have shown the existence of CXCR4/EGFR heteromers on cancer cells. As this detection required high sensitivity and specificity, a nanobody-based proximity ligation assay was established. This approach involves rolling circle DNA amplification upon close proximity binding of the target-specific nanobody-oligonucleotide conjugates. The applicability of bispecific, CXCR4- and either CD16- or CD3-targeted nanobodies as immune engagers for recruiting NK cells or T cells for antibody-dependent cellular cytotoxicity (ADCC) of CXCR4 expressing malignant cells was illustrated in Chapter 6. In these preliminary experiments, the enhanced ADCC observed for the nanobody-based engagers seemed unaffected by the CXCR4 oligomerization status. The thesis is concluded with a chapter in which the main approaches and findings of the previous chapters are discussed, as well as some future perspectives. Overall, this thesis describes the generation, reformatting, and functionalization of CXCR4-targeting nanobodies. Using nanobodies as tools, the receptor can be detected within the cell membrane, antagonized regarding ligand-induced signaling, and therapeutically targeted. This thesis illustrates the versatility of nanobodies in studying receptor biology and potential therapeutic applications.