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Cytokine Receptor Signaling Revisited: Implementing novel concepts for cytokine-based therapies

Final Report Summary - CYRE (Cytokine Receptor Signaling Revisited: Implementing novel concepts for cytokine-based therapies)

Protein interactions are key for cytokine receptor function: from (1) the extracellular recognition of a cytokine ligand (2) that induces receptor clustering to (3) the subsequent activation of the signalling machinery. The CYRE program aimed at applying novel insights and technologies to all three steps.
(1) With AcTakines (Activity-on-Target cytokines), proof-of-concept for a novel class of cell-specific immunocytokines was demonstrated. They are composed of a mutant cytokine, with strongly reduced binding affinity for its receptor complex, and a targeting moiety that binds a cell-specific surface marker. This way, the systemic toxicity associated with clinical use of pleiotropic, pro-inflammatory cytokines is avoided. Indeed, AcTakines remain inactive “en route” through the body, and only unveil their activity by avidity effects as a result of cell-specific targeting. We demonstrated this principle for structurally diverse cytokines such as type I and II interferons (IFN), tumor necrosis factor (TNF) and interleukin-1 (IL-1). In vitro, AcTakine effects (i.e. the ratio of specific activities on targeted over untargeted cell types) of up to 1000-fold are typically obtained. In murine tumor (and auto-immune disease) models, very promising results were obtained with therapeutic indices far above what can be observed for classic (immuno)cytokines. Complete growth stasis was achieved for B16 melanoma and 4T1 breast carcinoma tumors using type I IFN-based AcTaferons directly targeted to the tumor cells or to the immune compartment, e.g. to the cDC1 dendritic cells. Combination with doxorubicin led to complete tumor eradication and cured animals were immune against a tumor rechallenge . In case of TNF-based AcTafactors targeted to the tumor (neo)-vasculature, complete eradication was demonstrated of very large, established B16 tumors in combination treatments with either type II-AcTaferon, IL-1-based AcTaleukin or with CAR-T cells. In all cases, no or minimal in vivo toxicity was observed.
(2) We studied non-canonical receptor clustering in case of leptin, a cytokine that acts as a metabolic switch, regulating body weight and relating the body’s energy stores to high energy consuming processes like reproduction and immune responses. Using biochemical and genetic approaches, we could show that leptin’s metabolic and immuno-regulatory functions could be uncoupled at the leptin receptor (LR) level. More specifically, the immunoglobulin-like domain (IGD) in the LR ectodomain was essential for the homomeric LR clustering that underlies metabolic signaling, but dispensable for immune signaling. Interestingly, we subsequently found that the LR can cluster with the epidermal growth factor receptor (EGFR) in an IGD-independent way. Based on these insights, we developed a selective leptin antagonist that only interfered with LR signaling on immune cell types.
(3) We developed a series of technologies to study protein-protein interactions (PPI) in intact human cells: MAPPIT, KISS and Virotrap. Furthermore, a semi-automated, high-throughput tool was put in place to enable proteome-wide human PPI screening. With this platform at hand, we performed large-scale interactomics screens focused on intracellular trafficking. A high-confidence protein interaction network (PIN) was built encompassing key proteins involved in 4 different steps: plasma membrane to early endosome sorting, recycling, retrograde transport and the degradation pathway. Functional studies were performed on selected proteins that participate in these processes. On top of this “steady-state” PIN, a “dynactomics” approach, whereby cellular perturbations are applied to map the adaptability of the PIN, is underway. Likewise, screens to evaluate the effect of protein perturbations on network behaviour, so-called “edgetics” screens, are being performed as well. In line with this endeavour, a high-throughput screening strategy is in place to map PPI interfaces. In parallel, a next-gen sequencing approach is being put in place to perform massive parallel PPI surface mapping, and its application on cancer driver proteins is a next goal.