Nanobodies targeting human and viral chemokine receptors

G protein-coupled receptors (GPCRs) control many physiological processes by converting input from the extracellular environment (e.g., light, neurotransmitters, hormones, or other ligands) into intracellular signal transduction. Due to their crucial role in various disease states and their tractability (e.g., predominant plasma membrane localization), GPCRs represent a major therapeutic class. CXCR4 is a member of the chemokine receptors, a family of GPCRs with a central role in the development and homeostasis of the immune system. Additionally, CXCR4 is overexpressed in many cancers, in which the receptor is implicated in oncogenic processes like proliferation and metastasis. It became clear that some GPCRs not only exist as monomeric entities but also form interactions among themselves or with other receptors, a process called oligomerization. In artificial systems, CXCR4 switches from mainly monomeric at low expression levels to predominantly dimeric or higher-order oligomeric at high expression levels. The human cytomegalovirus (HCMV) is a widespread pathogen that has infected a large fraction of the population. In immunocompetent individuals, this herpesvirus establishes latent and asymptomatic infections. However, in organ-transplant or immunocompromised settings, HCMV may reactivate with severe consequences. HCMV infections are associated with several malignancies, including glioblastoma. In this most aggressive type of brain cancer, HCMV is believed to be oncomodulatory. HCMV encodes four viral GPCRs, human chemokine receptor homologs, of which US28 is the best-studied one. This receptor is vital for the establishment and maintenance of latency as well as the oncomodulatory effects. Yet, there are outstanding questions regarding US28 conformations and mechanisms underlying the oncomodulatory effects. Nanobodies or VHHs are the single variable fragments of heavy chain-only antibodies found in camelids. The long CDR3 and protruding paratope make nanobodies ideal for targeting discontinuous cryptic epitopes, such as the binding pockets of GPCRs. This thesis describes the development of nanobodies to study the mechanisms underlying oncogenic signaling by CXCR4 and US28. The first study introduces fluorescently labeled nanobodies as probes for BRET-based binding studies with Nanoluciferase-tagged GPCRs. In the second study, we report on VUN103, a nanobody that targets the intracellular site of US28. A comparison of VUN103 with a previously published US28-targeting nanobody, Nb7, demonstrates the existence of different active conformations. While VUN103 recognizes the apo constitutively active state of US28, Nb7 recognizes the ligand-bound active state. By interfering with G protein binding, VUN103 could fully inhibit US28 signaling and associated oncomodulatory effects in glioblastoma cells. The third study sheds light on how US28 rewires and relocalizes cellular signaling networks in glioblastoma cells, thereby contributing to HCMV-mediated oncomodulatory effects. US28 activates the sphingosine kinase-1 (SK1)/sphingosine-1-phosphate receptor 1 (S1P1) signaling axis, apparently from intracellular sites, thereby engaging in malignant feed-forward loops that exert proliferative and anti-apoptotic effects. The fourth study describes the characterization of a series of CXCR4-targeting nanobodies to study the role of receptor oligomerization in downstream signal transduction. Using nanobodies that modulate CXCR4 oligomerization, we corroborate the role of receptor oligomerization in activating the oncogenic JAK-STAT pathway. In the fifth study, we detected endogenous CXCR4 oligomers in B-cell malignancies using nanobodies. Identifying non-competitive CXCR4-targeting nanobodies and small molecules allowed the demonstration of pharmacological disruption of endogenous CXCR4 clusters. Eventually, malignant signaling and phenotypes could be associated with CXCR4 clusters, highlighting the potential of ligands that can selectively interfere with this process. Overall, the research described in this thesis provides insights into the mechanisms underlying the oncogenic signaling of human and viral chemokine receptors. The existence and functional relevance of GPCR oligomers and multiple active conformations, as well as malignant intracellular signaling complexes, are key findings. Furthermore, this thesis highlights the versatility of nanobodies to study and modulate various aspects of GPCR interactions and signaling.