Detection and pharmacological disruption of CXCR4 oligomerization in lymphoid neoplasms

Lymphoid neoplasms cause significant morbidity and mortality. While first-line therapies often induce strong responses, many patients eventually relapse, with recurrence patterns varying by subtype. Despite advances such as CAR T-cells, bispecific antibodies and targeted drugs, many patients still face disease progression or treatment-related complications. A major driver of relapse is the persistence of malignant cells within protective niches in the bone marrow and lymph nodes. These microenvironments provide key survival and homing signals that enable tumor cells to evade treatment and progressively acquire resistance. These signals are mediated largely by members of the protein family of G protein-coupled receptors (GPCRs), with the chemokine receptor CXCR4 in a pivotal role. Disrupting CXCR4-mediated signaling is therefore a therapeutic strategy to weaken the advantage conferred by the protective microenvironment, rendering malignant cells more susceptible to therapeutic targeting. The GPCR protein family comprises the largest family of cell surface proteins in the human genome and are involved in a multitude of cellular processes by transducing extracellular signals across the plasma membrane. Long viewed as solitary actors, experimental evidence over the last two decades has indicated that a large variety of GPCRs are able to form dimers or higher-order clusters, a process referred to as oligomerization. GPCR oligomerization exerts distinct functional effects by modulating ligand affinity, G protein coupling and downstream pathway selection. CXCR4 oligomerizes both constitutively and upon binding of CXCL12. While CXCL12-induced oligomerization is essential for directed cell migration, the functional role of constitutive CXCR4 oligomers remains unknown as previous studies have primarily relied on heterologous over-expression models. This thesis seeks to address this gap by examining endogenous CXCR4 oligomerization in lymphoma cell lines and patient-derived samples, and by defining its functional consequences in the context of tumor-microenvironment interaction that contribute to disease persistence and therapy resistance. We developed nanobody-based biosensors that visualize and quantify endogenous CXCR4 oligomerization. We then dissected how disrupting these basal oligomers with small molecules and nanobodies alters downstream phospho-signaling, chemokinesis, drug resistance and spheroid growth. Finally, we translate these insights into therapeutic formats, bispecific nanobody engagers and high-affinity llama-derived monoclonal antibody formats, that simultaneously dismantle CXCR4 oligomers, antagonize CXCL12 binding and recruit immune effector cells. Collectively, this work converts CXCR4 oligomerization from a biophysical phenomenon into a targetable event that can be clinically exploited.