Isabelle Magalhaes

Tuned activation of MSLN-CAR T cells induces superior antitumor responses in ovarian cancer models

Background Limited persistence of functional CAR T cells in the immunosuppressive solid tumor microenvironment remains a major hurdle in the successful translation of CAR T cell therapy to treat solid tumors. Fine-tuning of CAR T cell activation by mutating CD3ζ chain immunoreceptor tyrosine-based activation motifs (ITAMs) in CD19-CAR T cells (containing the CD28 costimulatory domain) has proven to extend functional CAR T cell persistence in preclinical models of B cell malignancies. Methods In this study, two conventional second-generation MSLN-CAR T cell constructs encoding for either a CD28 co-stimulatory (M28z) or 4-1BB costimulatory (MBBz) domain and a novel mesothelin (MSLN)-directed CAR T cell construct encoding for the CD28 costimulatory domain and CD3ζ chain containing a single ITAM (M1xx) were evaluated using in vitro and in vivo preclinical models of ovarian cancer. Two ovarian cancer cell lines and two orthotopic models of ovarian cancer in NSG mice were used: SKOV-3 cells inoculated through microsurgery in the ovary and to mimic a disseminated model of advanced ovarian cancer, OVCAR-4 cells injected intraperitoneally. MSLN-CAR T cell treatment efficacy was evaluated by survival analysis and the characterization and quantification of the different MSLN-CAR T cells were performed by flow cytometry, quantitative PCR and gene expression analysis. Results M1xx CAR T cells elicited superior antitumor potency and persistence, as compared with the conventional second generation M28z and MBBz CAR T cells. Ex vivo M28z and MBBz CAR T cells displayed a more exhausted phenotype than M1xx CAR T cells as determined by co-expression of PD-1, LAG-3 and TIM-3. Furthermore, M1xx CAR T cells showed superior ex vivo IFNy, TNF and GzB production and were characterized by a self-renewal gene signature. Conclusions Altogether, our study demonstrates the enhanced therapeutic potential of MSLN-CAR T cells expressing a mutated CD3ζ chain containing a single ITAM for the treatment of ovarian cancer. CAR T cells armored with calibrated activation potential may improve the clinical responses in solid tumors.

Mesothelin Expression in Patients with High-Grade Serous Ovarian Cancer Does Not Predict Clinical Outcome But Correlates with CD11c+ Expression in Tumor

Mesothelin (MSLN) is overexpressed in several tumors including ovarian cancer and is the target of current trials. There is limited and conflicting data on MSLN prognostic impact in ovarian cancer. We performed a retrospective study on patients with high-grade serous ovarian cancer, analyzing MSLN expression by immunohistochemistry and examining the correlation of its expression to overall and progression-free survival. Correlations of expression of MSLN, CD8, and macrophage markers in different tumor compartments were also investigated. Positive MSLN expression was detected in 55.1% of primary tumors and 51.5% of the metastases. MSLN expression was not correlated with survival. We observed a significant positive correlation (r = 0.34, p = 0.01) between MSLN expression in the metastatic site and CD11c expression in total tumor area and perivascular area in the primary tumor. Our results show that MSLN expression does not correlate with clinical outcome. The impact of the correlation between MSLN and CD11c

Mesothelin-Specific CAR T Cells Target Ovarian Cancer

Abstract New therapeutic options for patients with ovarian cancer are urgently needed. Therefore, we evaluated the efficacy of two second-generation mesothelin (MSLN)-directed CAR T cells in orthotopic mouse models of ovarian cancer. Treatment with CAR T cells expressing an MSLN CAR construct including the CD28 domain (M28z) significantly prolonged survival, but no persistent tumor control was observed. Despite lower response rates, MSLN-4–1BB (MBBz) CAR T cells induced long-term remission in some SKOV3–bearing mice. Tumor-infiltrating M28z and MBBz CAR T cells upregulated PD-1 and LAG3 in an antigen-dependent manner while MSLN+ tumor cells expressed the corresponding ligands (PD-L1 and HLA-DR), demonstrating that coinhibitory pathways impede CAR T-cell persistence in the ovarian tumor microenvironment. Furthermore, profiling plasma soluble factors identified a cluster of M28z- and MBBz-treated mice characterized by elevated T-cell secreted factors that had increased survival, higher CD8+ T-cell tumor infiltration, less exhausted CAR T-cell phenotypes, and increased HLA-DR expression by tumor cells. Altogether, our study demonstrates the therapeutic potential of MSLN-CAR T cells to treat ovarian cancer. Significance: These findings demonstrate that MSLN-directed CAR T cells can provide antitumor immunity against ovarian cancer.