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Abstract Background Chimeric antigen receptor (CAR) T cell therapy is associated with high risk of adverse events. Glucocorticoids (GCs) are cornerstone in the management of high-grade cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Given the potentially deleterious effects of GCs on CAR T cells anti-tumor activity, increasing our understanding of GCs impact on CAR T cells is crucial. Methods Using several CAR T cells i.e., CD19, mesothelin (MSLN)-CD28 and MSLN-41BB CAR T cells (M28z and MBBz), we compared phenotypical, functional, changes and anti-tumor activity between i) transduced CD19 CAR T cells with untransduced T cells, ii) M28z with MBBz CAR T cells induced by Dexamethasone (Dx) or Methylprednisolone (MP) exposures. Results Higher levels of GC receptor were found in less differentiated CAR T cells. Overall, Dx and MP showed a similar impact on CAR T cells. Compared to untreated condition, GCs exposure increased the expression of PD-1 and TIM-3 and reduced the expression of LAG3 and function of T cells and CAR T cells. GC exposures induced more exhausted (LAG3 + PD1 + TIM3 +) and dysfunctional (CD107a-INFγ-TNF-IL2-) untransduced T cells in comparison to CD19 CAR T cells. GC exposure impaired more CD4 + than CD8 + CD19 CAR T cells. GC exposures increased more PD-1 expression associated with reduced proliferative capacity and function of M28z as compared to MBBz CAR T cells. CAR T cells anti-tumor activity was greatly affected by repeated GC exposure but partly recovered within 48h after GCs withdrawal. Conclusions In summary, GCs impacted phenotype and function of untransduced and CAR T cell with different magnitude. The nature of the CAR costimulatory domain influenced the magnitude of CAR T cell response to GCs.

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.

Successful translation of chimeric antigen receptor (CAR) T cell therapy for the treatment of solid tumors has proved to be troublesome, mainly due to the complex tumor microenvironment promoting T cell dysfunction and antigen heterogeneity. Mesothelin (MSLN) has emerged as an attractive target for CAR T cell therapy of several solid malignancies, including ovarian cancer. To improve clinical response rates with MSLN-CAR T cells, a better understanding of the mechanisms impacting CAR T cell functionality in vitro is crucial. Here, we demonstrated superior cytolytic capacity of CD28-costimulated MSLN-CAR T cells (M28z) relative to 4–1BB-costimulated MSLN-CAR T cells (MBBz). Furthermore, CD28-costimulated MSLN CAR T cells displayed enhanced cytolytic capacity against tumor spheroids with heterogeneous MSLN expression compared to MBBz CAR T cells. In this study, we identified CAR-mediated trogocytosis as a potential impeding factor for successful MSLN-CAR T cell therapy due to fratricide killing and contributing to tumor antigen heterogeneity. Moreover, we link antigen-dependent upregulation of LAG-3 with reduced CAR T cell functionality. Taken together, our study highlights the therapeutic potential and bottlenecks of MSLN-CAR T cells, providing a rationale for combinatorial 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.

Abstract Background Patients 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. Methods We 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. Results Anti-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

Background: Patients 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. Methods: Immune 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. Findings: Univariate 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. Interpretation: This 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.