Mapping the immune landscape in solid tumors : implications for immunotherapy

Our cells are programmed with various safety mechanisms to avoid transformation into tumor cells. In case these fail, we have a guarding immune system ready to recognize and eliminate these cells. Despite these safety measurements, cancer is one of the leading causes of death worldwide. The tumor cells find ways to escape the immune system. Paradoxically, components of the immune system can contribute to the progression of tumors by the use of various immunosuppressive pathways. However, the immune system can also be harnessed, and the anti-tumor functions restored to regain control of the tumor development. This has been highlighted in the past decade, with the introduction of novel immunotherapeutic approaches, such as checkpoint blockade, to target the naturally occurring brakes called co-inhibitory receptors. The work presented in this thesis consists of four papers which contribute with knowledge on infiltrating immune cells in prostate cancer (Paper I) and ovarian cancer (Paper II-IV). In the work of Paper I-IV, we have looked into tumor-infiltrating lymphocytes and mapped the presence, composition, expression pattern and functionality of various T cell subsets in these two solid tumor types. The work was performed by retrieving material from cancer patients undergoing surgery, isolating immune cells and performing phenotypic descriptions by flow cytometry. We also have assessed the soluble environment in which the immune infiltrates reside and assessed T cell functionality by looking into cytokine secretion, cytotoxicity and/or proliferation by various readouts. In Paper I, we performed phenotyping of immune infiltrates in peripheral blood and prostates with malignant, benign or healthy histology. In Paper II, we assessed the immunophenotype in peripheral blood, ascites and metastasized tumor tissue of advanced ovarian cancer patients. The results in Paper I and II showed lymphocyte infiltration to be common in both tumor types, in particular of CD8+ effector memory T cells. PD-1, which enables inhibition of effector functions by binding to its ligands, was the most abundantly expressed co-inhibitory receptor in both tumor types. However, in Paper I, PD-1 expression was also common in healthy prostates indicating a role in the homeostasis of the prostate environment. In Paper II, we correlated our findings to patient outcome and identified eight immune-related risk factors (both cellular and soluble) in ascites and/or tumor associated with overall patient survival. In Paper III, we investigated the functionality of infiltrating T cells isolated from ovarian cancer patients. We wanted to explore if functionality, in terms of cytokine responsiveness, could be enhanced using immunotherapeutic PD-1-targeting conventional monoclonal antibodies (mAbs) nivolumab/pembrolizumab and novel scaffold proteins called DARPin® proteins. The results showed improved secretion of several important effector cytokines using the PD-1 targeting reagents. A bivalent PD-1 targeting DARPin® protein showed comparable results to the clinically approved mAbs which warrants further investigation. However, despite boosted cytokine responsiveness, our results indicated that tumor-derived T cells are still highly dysfunctional, presenting challenges in restoring anti-tumor responses. In Paper IV, we investigated the features of the unconventional subset γδ T cells in ovarian cancer. Our aim was to investigate their features and contribution in this cancer type. We profiled their T cell receptor (TCR) characteristics, their phenotype and functional response to various stimuli. We found the ascites-derived and tumor-derived γδ T cell repertoires to be distinct from one another. We suggested the ascites γδ T cells to be driven by adaptive TCR-driven pathways due to the observed clonal focusing in this compartment, while tumor γδ T cells displayed a high diversity and likely respond through innate pathways. In summary, we found the γδ T cells to be beneficial for the patients by anti-tumor functions including cytotoxicity and production of important effector cytokines. Importantly, we identified their functionality to be associated to outcome, where higher functionality was linked to increased patient survival. We observed a negative impact of CD39 on γδ functionality, which warrants further investigation to understand how γδ T cell functionality can be boosted. Future optimization of immunotherapeutic approaches requires basic understanding of immune infiltrates in tumors. By learning more about these tumor-infiltrating immune cells, what they express and how their functionality can be affected, new strategies can be outlined based on this knowledge. I hope that by reading this thesis, you will obtain insight into this exciting research field and how the presented work has contributed.

Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins

PD-1/PD-L1 blockade has revolutionized the field of immunooncology. Despite the relative success, the response rate to anti-PD-1 therapy requires further improvements. Our aim was to explore the enhancement of T-cell function by using novel PD-1-blocking proteins and compare with clinically approved monoclonal antibodies (mAbs). We isolated T-cells from the ascites and tumor of 17 patients with advanced epithelial ovarian cancer (EOC) and analyzed the effects using the mAbs nivolumab and pembrolizumab and two novel engineered ankyrin repeat proteins (DARPin® proteins). PD-1 blockade with either mAb or DARPin® molecule significantly increased the release of IFN-γ, granzyme B, IL-2, and TNF-α, demonstrating successful reinvigoration. The monovalent DARPin® protein was less effective compared to its bivalent equivalent, demonstrating that bivalency brings an additional benefit to PD-1 blockade. Overall, we found a higher fold increase of lymphokine secretion in response to the PD-1 blockade by tumor-derived T-cells; however, the absolute amounts were significantly lower compared to the release from ascites-derived T-cells. Our results demonstrate that PD-1 blockade can only partially reinvigorate functionally suppressed T-cells from EOC patients. This warrants further investigation preferably in combination with other therapeutics. The study provides an early pilot proof-of-concept for the potential use of DARPin® proteins as eligible alternative scaffold proteins to block PD-1