In vitro assay development and optimization for the prediction of immune-related adverse events following immunotherapy

Abstract

Cancer immunotherapies activate the immune system to overcome tumor resistance and restore the antitumor immune response, however, excessive activation of the immune system may also affect normal tissues and organs and cause immune-related adverse events (irAEs) of varying severity. Current in vitro assays used for testing and predicting of irAEs induced by immunotherapeutics are associated with significant limitations, primarily due to unsatisfactory ability to accurately reproduce human physiological environment, indicating urgent need for more reliable and clinically relevant in vitro assays. While immune checkpoint inhibitors (ICIs) are becoming a more frequent treatment option in cancer therapy, case reports documenting ICI-induced cytokine release syndrome (CRS) are increasing in number. As part of this thesis project, a literature review on CRS as a rare irAE induced by ICIs was conducted. Although CRS represents one of the most serious irAEs, its pathophysiology remains unclear. Despite the fact that effective clinical management requires early recognition of the syndrome followed by prompt intervention, ICI-induced CRS does not have any defined diagnostic criteria and has not yet been included in the European Society of Medical Oncology (ESMO) clinical practice guidelines of management of the ICI-induced toxicities. Therefore, understanding of the molecular mechanisms and events behind CRS development triggered by different ICIs is crucial, as well as establishment of relevant test systems and clinical assays. Thus, the overall goal of the practical part of the thesis project was to test and develop in vitro assays that can be used to test and predict irAEs induced by immunotherapeutics. It was decided to focus on two types of assays, i.e., dendritic cell (DC) based assays, since DC-based models have shown great potential in immunogenicity assessment of immunotherapeutics, and on autologous cytokine release assays, since endothelial cell activation plays an important role in the development of ICI-induced CRS. More specifically, the aims of this work were, firstly, to test and compare two monocyte-derived dendritic cell (moDC) differentiation protocols in their ability to induce IL-2 production by moDCs, secondly, to test the IL-2 receptor expression by the moDCs with and without the immunotherapeutic drug Proleukin® (human recombinant IL-2) stimulation. Finally, to differentiate blood outgrowth endothelial cells (BOECs) from healthy donors’ peripheral blood mononuclear cells (PBMCs). The assessment was performed using flow cytometric analysis and enzyme-linked immunosorbent assay (ELISA). The current study could not identify any significant differences in IL-2 secretion by moDCs differentiated in the presence of IL-4 (+ GM-CSF) or IL-15 (+ GM-CSF). Furthermore, our study confirmed higher expression of IL-2R subunit CD132 by moDCs compared to the IL2R subunits CD122 and CD25. However, Proleukin® was found to have no effect on the expression of any of these nor on the activation of the moDCs. Based on this study, the effect of Proleukin® on IL-2 secretion by moDCs remains uncertain. Mastering the BOEC differentiation from PBMCs could be developed further in the future, by setting up an autologous coculture system of BOECs with PBMCs that is able to induce CRS, upon stimulation with different therapeutics.

Popular Abstract

Project title: Test system development for prediction of irAEs following immunotherapy
Immunotherapy is a type of cancer treatment that acts by activating the immune system, which helps to fight the disease. However, overactivation of the immune system may also affect normal tissues and organs and cause immune-related side effects (irAEs). Current laboratory tests aimed to identify eventual irAEs induced by immunotherapeutics are associated with limitations, mainly inaccuracy in mimicking human physiological processes. As a result, more reliable and clinically relevant laboratory tests are needed. Currently, so-called immune checkpoint inhibitors (ICIs) are the most widely used cancer immunotherapy. Recently, the number of cases documenting cytokine release syndrome (CRS) caused by ICIs, has been increasing. CRS is a severe immune reaction, characterized by overproduction of chemical messengers (cytokines) and overactivation of immune cells, and might lead to organ damage and even death. Even though CRS is one of the most dangerous irAEs, the exact mechanisms behind it are still unknown. Although it is crucial that the syndrome is recognized and dealt with promptly, ICI-induced CRS does not have any defined diagnostic criteria. As part of this project, a literature review on CRS as a rare irAE of ICIs was conducted and potential molecular mechanisms and events behind the CRS were analyzed. Apart from that, the overall goal of the study was to test and develop laboratory models that can be used for predicting irAEs following immunotherapies. One focus of the practical part of the project involved dendritic cell (DC) based test models. DCs comprise a type of immune cells that can present foreign molecules to other immune cells and, as a result, initiate and expand immune responses. This critical role of DCs makes them suitable in laboratory models for evaluating toxicities provoked by immunotherapeutics. IL-2 is a cytokine that can be produced by DCs and is critical for activating other types of immune cells. It affects cells by binding to the so-called IL-2 receptor, built up by the protein subunits CD25, CD122 and CD132. Sold under the name Proleukin®, IL-2 is a drug for activating the immune system. Firstly, we aimed to test two different protocols to develop DCs from monocytes (moDCs) and to compare eventual effects on IL-2 production by moDCs, and secondly, to investigate if Proleukin® had any effect on expression of the three IL-2 receptor subunits on the surface of moDCs. Our study could not identify any significant differences in IL-2 production by moDCs differentiated in accordance with the two protocols. Further, the study has confirmed higher expression of CD132 by moDCs compared to CD122 and CD25, however, Proleukin® had no effect on the expression of any of the three proteins nor on the activation of the moDCs. The other focus of the project was related to the literature review findings, namely the importance of inclusion of endothelial cells (ECs) in cytokine release models. With that in mind, a specific subtype of ECs, blood outgrowth ECs (BOECs), was developed from blood cells. Establishment of BOECs provides an opportunity to further develop a test system based on ECs and blood cells from the same donor that is able to induce CRS upon stimulation with different therapeutics. In conclusion, we have taken several different steps towards the development of laboratory test systems for prediction of irAEs following immunotherapy.

• author: Rosberg, Axel and Koval, Ekaterina
• supervisor: Christina Sakellariou; Malin Lindstedt
• organization: Department of Immunotechnology
• course: KIMM05 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• language: English
• id: 9100292
• date added to LUP: 2022-09-19 09:44:04
• date last changed: 2022-09-19 09:44:04