Combinatorial strategy to generate antibody-cytokine fusions with "activity-on-demand" for cancer therapy

Immunotherapy has emerged as the fourth cornerstone of the cancer treatment arsenal alongside radiotherapy, surgery, and chemotherapy, with impressive therapeutic outcomes in a fraction of patients. Cytokines are a class of immunomodulatory proteins with the potential to harness the activity of the immune system and recombinant preparations of certain cytokine payloads have gained marketing authorization for cancer therapy. However, due to their potency and toxicity, recombinant cytokines are typically administered at sub-optimal doses, preventing the use of potentially curative regimens. The antibody-based delivery of cytokine payloads to the tumor site represents an avenue to expand the therapeutic window of cytokine biopharmaceuticals. Our group has focused on the design of antibody-cytokine fusions (immunocytokines) directed against stromal components of the tumor using the F8 and L19 antibodies recognizing splice variants of fibronectin. Immunocytokines have proven to preferentially localize at the site of disease for long periods of time, and fusions featuring tumor necrosis factor (TNF), interleukin 12 (IL12), and interleukin 2 (IL2) have shown substantially improved anti-cancer efficacy. However, they typically retain full cytokine activity and may cause toxicities at early time points after intravenous administration, comparable to the naked payload at equivalent doses. There is, therefore, an urgent need to develop immunocytokines with “activity-on-demand”, virtually silent systemically, and only active upon accumulation at the tumor site. This thesis describes the preclinical validation of a technology named “Intra-Cork”, which relies on the administration of pathway-selective small molecule inhibitors able to mask cytokine signaling systemically after intravenous administration. The small molecule inhibitor functions as a cork on a bottle, releasing the cytokine signaling “on-demand”. The technology allows to preserve the long residence time of the cytokine and its pro-inflammatory activity within the tumor mass, as small molecule inhibitors are rapidly cleared from circulation. Janus kinase (JAK) inhibitors may be ideally suited for this purpose, as JAK proteins are key signaling mediators for several payloads, including IL12 and IL2. In the first part of this thesis, the Intra-Cork technology has been validated on L19-IL12. The JAK2 inhibitor Ruxolitinib expanded the therapeutic index of L19-IL12 in a preclinical model of cancer. Indeed, it massively reduced systemic pro-inflammatory cytokine levels and completely protected the liver from necrosis, preserving the potent anti-cancer activity of L19-IL12 and its immunological remodeling at the tumor site. The second part of this thesis describes the validation of the Intra-Cork technology on F8-IL2 using the approved JAK1-selective inhibitor Upadacitinib. One of the major life-threatening toxicities of IL2-based products is vascular leak syndrome (VLS), leading to generalized edema and organ failure, which could be fully recapitulated in our in vivo model. Upadacitinib spared healthy organs from VLS manifestations and allowed the administration of curative doses of F8-IL2. Overall, the results obtained indicate that the Intra-Cork strategy may help to overcome dose-limiting toxicities associated with antibody-cytokine fusions. Future clinical trials will shed light on the potential of this state-of-the-art technology.