Defining the potential of mesothelin-directed CAR T cells for the treatment of ovarian cancer

Abstract

Chimeric antigen receptor (CAR) T cells have revolutionized the field of immunotherapy, by redirecting T cell specificity and effector functions. Co-stimulation has proven to be crucial for therapeutic effectiveness of CAR T cells and remarkable clinical response rates have been achieved with second generation CD19-directed CAR T cells containing either a CD28 or 4-1BB co-stimulatory domain for the treatment of B cell malignancies. Mesothelin (MSLN) has emerged as an attractive target for CAR T cell therapy in solid tumors, including ovarian cancer. Due to the complex tumor microenvironment niche of ovarian cancer, it is crucial to investigate the mechanisms impacting CAR T cell functionality to improve therapeutic effectiveness. The aim of this thesis was to evaluate the therapeutic potential of three different second generation MSLN-directed CAR T cells for the treatment of ovarian cancer.

Selection of the most advantageous co-stimulatory segment for functional MSLN-CAR T cell persistence in the relevant disease setting is of great importance for successful clinical application. In paper I and II, MSLN-directed CAR T cells containing either a CD28 (M28z) or 4-1BB (MBBz) co-stimulatory domain followed by the CD3ζ chain were compared in different models of ovarian cancer. M28z and MBBz CAR T cells elicited powerful anti-tumor responses in in vitro and preclinical in vivo models of ovarian cancer, although with different kinetics. Introduction of a CD28 co-stimulatory domain facilitated rapid activation of effector functions, while 4-1BB co-stimulation favored functional persistence of MSLN-CAR T cells.

In paper III, a new MSLN-CAR construct was included and evaluated with respect to the classical second generation CAR constructs M28z and MBBz. The novel construct (M1xx) had calibrated activation potential through mutations in the CD3ζ chain combined with CD28 co-stimulation. M1xx CAR T cells displayed superior tumor control as compared to M28z and MBBz CAR T cells in two different in vivo models. Treatment with M1xx CAR T cells resulted in tumor clearance and long-term remission in the orthotopic mice model. However, in a disseminated disease model M1xx CAR T cells treatment delayed tumor progression substantially but mice eventually succumbed to tumor burden.

In all papers, I attempted to elucidate the mechanisms affecting MSLN-CAR T cell functionality and several immune escape pathways were highlighted. Tumor cells were capable of evading immune control by downregulation of MSLN surface expression and upregulation of the PD-L1 and HLA-DR co-inhibitory ligands on the remaining MSLN+ tumor cells. Functional persistence of CAR T cells was limited due to exhaustion of MSLN-CAR T cells in vivo. Moreover, MSLN-CAR T cells displayed trogocytotic capacity thereby facilitating fratricide killing as well as tumor antigen escape.