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 antigendependent manner while MSLN þ tumor cells expressed the corresponding ligands (PD-L1 and HLA-DR), demonstrating that coin-hibitory 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 þ 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.

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BackgroundPatients with advanced malignancies, e.g. lung cancer, ovarian cancer or melanoma, frequently present with brain metastases. Clinical presentation and disease progression of cancer is in part shaped by the interaction of the immune system with malignant cells. Antigen-targeted immune responses have been implicated in the prolonged survival of patients with cancer. This includes the tumor-associated antigen (TAA) mature mesothelin, a 40 kDa cell surface-bound antigen that is overexpressed in several malignancies including lung ovarian and pancreatic cancer. We examined in an observational, prospective study the survival of patients with brain metastases in association with clinical parameters and cellular immune responses to molecularly defined TAAs or viral (control) target antigens.MethodsImmune cells in peripheral blood obtained from thirty-six patients with brain metastases were tested for cytokine production in response to a broad panel of defined viral and TAA target antigens, including full-length mesothelin. Incubation of immune cells with antigenic targets was carried out in i) medium alone, (ii) in a cytokine cocktail of interleukin (IL)-2/IL-15/IL-21, or (iii) IL-2/IL-7. Supernatants were tested for interferon gamma (IFN-γ) production, after which univariate and multivariate analyses (Cox stepwise regression model) were performed to identify independent clinical and immunological factors associated with patient survival. Patients were followed-up for at least 500 days after surgery or until death.FindingsUnivariate analysis identified age, gender, radiotherapy and mutational load as clinical parameters affecting survival of patients with brain metastases. Cox multivariate analysis showed that radiotherapy (P = 0·004), age (P = 0·029) and IFN-γ responses to mature mesothelin, conditioned by IL-2/IL-7 (P = 0·045) were independent predictors of the survival of patients from surgery up to follow-up or death.InterpretationThis is the first evidence that immune responses to mesothelin serve as a marker of increased overall survival in patients with brain metastases, regardless of the primary tumor origin. Analyses of immunological markers could potentially serve as prognostic markers in patients with brain metastases and help to select patients in need for adjunct, immunological, treatment strategies.

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.

BackgroundPatients with brain tumor or pancreatic cancer exhibit the poorest prognosis, while immune fitness and cellular immune exhaustion impacts their survival immensely. This work identifies differences in the immune reactivity to the common human pathogens cytomegalovirus (CMV) and Epstein–Barr virus (EBV) between patients with brain tumor in comparison to those with pancreatic cancer and healthy individuals.MethodsWe characterized the humoral and cellular immune responses of patients with brain tumor or pancreatic cancer to cytomegalovirus structural protein pp65 (CMV-pp65) as well as Epstein–Barr nuclear antigen-1 (EBNA-1) by whole-blood assay and ELISA.ResultsAnti-CMV-pp65 plasma immunoglobulin gamma (IgG) titers were significantly lower in patients with brain tumor compared to healthy donors and patients with pancreatic cancer. Among the responding patients with GBM, those with a weak anti-CMV IgG response also had a decreased median overall survival (p = 0.017, 667 vs 419 days) while patients with brain tumor showed a generally suppressed anti-CMV immune-reactivity. Patients with brain tumor exhibited a significantly lower interferon gamma (IFNγ) response to EBNA-1 and CMV-pp65 compared to patients with pancreatic cancer or healthy donors. This antigen-specific response was further amplified in patients with brain tumor upon conditioning of whole blood with IL-2/IL-15/IL-21. Exclusively in this setting, among the responding patients with GBM, those exhibiting a EBV-specific cellular immune response above the median also displayed an increased median overall survival pattern compared to weak responders (753 vs 370 days, p < 0.001).ConclusionsThis report provides (i) a fast and easy assay using common viral antigens and cytokine stimulation to screen for immune fitness/exhaustion of patients with brain tumor in comparison to pancreatic cancer and healthy individuals and (ii) EBV/CMV-induced IFNγ production as a potential marker of survival in patients with brain tumor.Electronic supplementary materialThe online version of this article (10.1186/s12967-018-1557-9) contains supplementary material, which is available to authorized users.