Periodic Reporting for period 1 - CINK (Advancing cancer immunotherapy using natural killer cells for hematological and metastatic cancers) Periodo di rendicontazione: 2018-02-15 al 2020-02-14 Sintesi del contesto e degli obiettivi generali del progetto Cancer immunotherapy has become a powerful weapon for the treatment of cancer in the last decades. Yet the design of an effective immunotherapy is complicated by various factors such as tumor-specific scape mechanisms that renders an immunosuppressive environment, immune-modulating effects of conventional treatments and treatment-related toxicities, all of which results in failure of sustained anti-tumor responses. Therefore, new and more effective approaches are still in much need. NK cells have proven to have a critical role in the immunoediting process by which the immune system controls the progression of cancer cells as well direct cytotoxic functions. Indeed, multiple immunotherapeutic efforts that individually targets the anti-tumor potential of these immune cells have been done with a relative success, but progress still needs to be done. Unfortunately, the development of exhaustion during prolong stimulation as well as tumor NK cell-specific scape mechanisms that reduces NK cell survival and/or functionality, have limited the use of NK cell-based immunotherapy in cancer treatment. Here we are exploring the mechanisms by which NK cell activation is regulated in order to develop strategies that result in sustained and stronger anti-tumor responses while the manifestation of exhaustion is minimized. By combining immune stimulation signals, such as IL-2, IL-12 and/or IL-15 co-stimulation, with inhibition of immunosuppression with immune checkpoint blockade therapy (for example PD1/PDL1, CTLA4 or NKG2A) we expect to improve NK cell efficacy and reduce tumor progression. This project, thus, pretends to unveil the mechanisms regulates NK cell activation to better utilized the potential of these immune cells to prevent metastasis and cancer relapse as well as set up the bases required for its clinical translation. Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti During the funded period, we have primarily worked in developing strategies that leads to better natural killer (NK) cell survival and function with limited exhaustion outcome upon activation in order to improve current NK cell-based immunotherapies in cancer treatment. Despite the tremendous advances in immunotherapy of cancer, tumor scape and consequent cancer relapse is still a major problem and the search for a more efficacious and durable therapy is still on. Intrinsic or extrinsic mechanisms involved in the induction of exhaustion, in both NK and T cells, are an important limiting factor in the efficacy of cancer immunotherapy and thus new approaches are still needed. We have observed in mouse models that continues treatment with recombinant IL-15 and IL-15Rα resulted in a phenotype that was characteristic of exhaustion with downregulation of the activating transcription factor eomesodermin and reduced functional and proliferation capacities. In contrast, acute IL-15/IL-15Rα treatment achieved a better activation and functional effects. However, the overall expansion of NK cells after acute treatment was limited. Thus, we next evaluated the impact of hydrodynamic delivery of plasmid-IL-15 on NK cells, which has previously been used to induce NK cell activation and improve NK cell reconstitution. We observed that this approach resulted in a strong NK cell expansion, a significant increase of NK cell activation associated with improved natural cell cytotoxicity, and no exhaustion signals. Because this method has previously been associated with important toxicity side effects and a rather limited effect in overall tumor survival, we decided to modify our original approach and evaluate the efficacy of activated NK cell intratumoral delivery as a mean to limit treatment-related toxicities while causing a systemic anti-tumor effect that can potentially prevent metastasis and cancer relapse. In both colon cancer and metastatic melanoma mouse models, we observed that intratumoral delivery of IL-15 activated NK cells resulted in an improvement of tumor survival and a delay of tumor growth. Alternative, IL-12 mRNA transfection of NK cells is currently being tested to minimize treatment-related toxicities associated with the hydrodynamic delivery approach and accomplish a sustained IL-15 expression that maintains NK cell functionality without causing exhaustion. IL-12, a cytokine that has been proposed to be involved in the crosstalk between NK and DC and in NK cell activation, is also a very attractive candidate for intervention in NK cell efficacy. Other immunotherapeutic approaches are currently being tested to enhance even further NK cell activation and function with promising results. Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) Cancer immunotherapy that takes advantage of the immune system to eliminate tumor cells has become an indispensable weapon for cancer treatment in the last decades. Despite the tremendous recent progress in this field, cancer treatment has not reached that desirable goal of frequently becoming a chronic disease. Unfortunately, tumor heterogeneity and tumor immune escape mechanisms as well as intrinsic immune regulatory mechanisms account for the many reasons that cancer relapse still occurs. NK-cell immunobiology and the regulation of its activation has proven far more complex than was initially thought through the existence of self-regulatory mechanisms that influence NK cell activation or function leading to an exhaustion phenotype. Additionally, NK cell-based immunotherapy has also proven to have important limitations and fail to accomplish long-term efficacy due to NK cell specific tumor evasion mechanisms such as recruitment of immunosuppressor cells, downregulation of activating receptors, and/or extrinsic induction of exhaustion, among other mechanisms. Therefore careful analysis of the pathways involved in NK cell activation that might limit NK cell function are critical to understand how NK cells are regulated and to be able to exploit this immune cell as an immunotherapeutic weapon. Here, we are exploring different approaches to induce a stronger and sustained NK cell activation and function to uncover a novel intrinsic and complex regulatory mechanism that controls cytotoxicity against tumors and limits NK cell-based immunotherapy efficacy. This analysis is helping us to develop a new, and needed strategy to expand ex vivo activated NK cells and improve overall NK cell function utilizing cutting edge technology such as available new mAbs, siRNA, synthetic drugs and/or selection by immunomagnetic or FACS cell sorting, that makes possible to engineer NK cells to reach our expectations. Thus far, forcing the production of IL-15 has given promising results, and has proven efficacious in expanding and activating NK cells with strong anti-tumor responses. Therapeutic efficacy will be evaluated next in clinically meaningful mouse and xenograft models with relevant mouse and human tumor models respectively to demonstrate the versatility and the clinical translation capabilities of our proposed therapeutic strategies. Therapeutic efficacy will be also evaluate in combination with current immune checkpoint blockade therapies to accomplish an even better outcome. These strategies share the goal of achieving more efficacious immunotherapy approaches, especially for the treatment of hematological and metastatic cancers, to prevent cancer relapse and limit treatment-related toxicities, prolonging tumor-free survival rate and improving quality of life of cancer patients. Indirect effect of PD-/PD-L1 pathway on NK cell exhaustion
