Inhibiting Lysine Demethylase 1A Improves L1CAM-Specific CAR T Cell Therapy by Unleashing Antigen-Independent Killing via the FAS-FASL Axis

Simple Summary: Solid tumor cells can lose or heterogeneously express antigens to become resistant to chimeric antigen receptor (CAR) T cell therapy. Here, we explore whether epigenetic manipulation to unleash antigen-independent killing mechanisms can overcome this hurdle. KDM1A is overexpressed in many cancers and removes lysine methylation on histones that keeps the DNA firmly packed to selectively activate or repress gene activity, depending on the specific lysine target. KDM1A also regulates the expression of nonhistone proteins. We inhibited KDM1A in the childhood tumor, neuroblastoma, to increase FAS expression on tumor cells. The FAS receptor can be triggered to induce cell death when bound by the FAS ligand on CAR and other activated T cells present in the tumor environment, even if the tumor cells lack the target antigen. FAS upregulation via KDM1A inhibition sensitized neuroblastoma cells to FAS-FASL-mediated killing and augmented CAR T cell therapy against antigen-poor or even antigen-negative neuroblastoma. Abstract: Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising treatment strategy, however, therapeutic success against solid tumors such as neuroblastoma remains modest. Recurrence of antigen-poor tumor variants often ultimately results in treatment failure. Using antigen-independent killing mechanisms such as the FAS receptor (FAS)-FAS ligand (FASL) axis through epigenetic manipulation may be a way to counteract the escape achieved by antigen downregulation. Analysis of public RNA-sequencing data from primary neuroblastomas revealed that a particular epigenetic modifier, the histone lysine demethylase 1A (KDM1A), correlated negatively with FAS expression. KDM1A is known to interact with TP53 to repress TP53-mediated transcriptional activation of genes, including FAS. We showed that pharmacologically blocking KDM1A activity in neuroblastoma cells with the small molecule inhibitor, SP-2509, increased FAS cell-surface expression in a strictly TP53-dependent manner. FAS upregulation sensitized neuroblastoma cells to FAS-FASL-dependent killing and augmented L1CAM-directed CAR T cell therapy against antigen-poor or even antigen-negative tumor cells in vitro. The improved therapeutic response was abrogated when the FAS-FASL interaction was abolished with an antagonistic FAS antibody. Our results show that KDM1A inhibition unleashes an antigen-independent killing mechanism via the FAS-FASL axis to make tumor cell variants that partially or totally suppress antigen expression susceptible to CAR T cell therapy

Aberrant signaling of immune cells in Sjögren's syndrome patient subgroups upon interferon stimulation

Background: Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease, characterized by mononuclear cell infiltrates in the salivary and lacrimal glands, leading to glandular atrophy and dryness. Patient heterogeneity and lack of knowledge regarding its pathogenesis makes pSS a difficult disease to manage. Methods: An exploratory analysis using mass cytometry was conducted of MAPK/ERK and JAK/STAT signaling pathways in peripheral blood mononuclear cells (PBMC) from 16 female medication free pSS patients (8 anti-Sjögren’s syndrome-related antigen A negative/SSA- and 8 SSA+) and 8 female age-matched healthy donors after stimulation with interferons (IFNs). Results: We found significant differences in the frequencies of memory B cells, CD8+ T central and effector memory cells and terminally differentiated CD4+ T cells among the healthy donors and patient subgroups. In addition, we observed an upregulation of HLA-DR and CD38 in many cell subsets in the patients. Upon IFNα2b stimulation, slightly increased signaling through pSTAT1 Y701 was observed in most cell types in pSS patients compared to controls, while phosphorylation of STAT3 Y705 and STAT5 Y694 were slightly reduced. IFNγ stimulation resulted in significantly increased pSTAT1 Y701 induction in conventional dendritic cells (cDCs) and classical and non-classical monocytes in the patients. Most of the observed differences were more prominent in the SSA+ subgroup, indicating greater disease severity in them. Conclusions: Augmented activation status of certain cell types along with potentiated pSTAT1 Y701 signaling and reduced pSTAT3 Y705 and pSTAT5 Y694 induction may predispose pSS patients, especially the SSA+ subgroup, to upregulated expression of IFN-induced genes and production of autoantibodies. These patients may benefit from therapies targeting these pathways.publishedVersio

Integrating multiple dimensions of ecological stability into a vulnerability framework

Ecological stability encompasses multiple dimensions of functional and compositional responses to environmental change. Though no single stability dimension used in isolation can fully reflect the overall response to environmental change, a common vulnerability assessment that integrates simultaneously across multiple stability components is highly desirable for ecological risk assessment. We develop both functional and compositional counterparts of a novel, integrative metric of overall ecological vulnerability (OEV). We test the framework with data from a modularized experiment replicated in five lakes over two seasons, examining functional and compositional responses to both pulse and press disturbances across three trophic groups. OEV is measured as the area under the curve integrated over the entire observation period, with the curve delimiting the difference between the disturbance treatment and undisturbed parallel controls, expressed either as the log response ratio of biomass (functional OEV) or community dissimilarity index (compositional OEV). Both, functional and compositional OEV correlated negatively with functional and compositional 'resistance', 'temporal stability' and 'final/extent of recovery' following both pulse and press disturbances, though less so with 'resilience' following a pulse disturbance. We also found a positive correlation between functional and compositional OEV, which reveals the potential to also evaluate the intricate linkage between biodiversity and functional change. Our findings demonstrate that OEV comprises a robust framework to: (a) capture simultaneously multiple functional and compositional stability components, and (b) quantify the functional consequences of biodiversity change. Our results provide the basis for an overarching framework for quantifying the overall vulnerability of ecosystems to environmental change, opening new possibilities for ecological risk assessment and management. Synthesis. Ecological stability comprises multiple dimensions that together encapsulate how ecosystems respond to environmental change. Considering these multiple aspects of stability simultaneously often poses a problem in environmental assessments, which frequently require overarching indicators of risk or vulnerability. While an analysis of multiple dimensions allows for deeper exploration of mechanisms, here we develop and test a new univariate indicator that integrates stability aspects under a broad range of disturbance regimes. Using a modularized experiment in Swedish lakes, we show that this integrative measure captures multiple stability dimensions reflecting compositional and functional vulnerability and their relationships between them

Combination of GD2-directed bispecific trifunctional antibody therapy with Pd-1 immune checkpoint blockade induces anti-neuroblastoma immunity in a syngeneic mouse model

Introduction: Despite advances in treating high-risk neuroblastoma, 50-60% of patients still suffer relapse, necessitating new treatment options. Bispecific trifunctional antibodies (trAbs) are a promising new class of immunotherapy. TrAbs are heterodimeric IgG-like molecules that bind CD3 and a tumor-associated antigen simultaneously, whereby inducing a TCR-independent anti-cancer T cell response. Moreover, via their functional Fc region they recruit and activate cells of the innate immune system like antigen-presenting cells potentially enhancing induction of adaptive tumor-specific immune responses. Methods: We used the SUREK trAb, which is bispecific for GD2 and murine Cd3. Tumor-blind trAb and the monoclonal ch14.18 antibody were used as controls. A co-culture model of murine dendritic cells (DCs), T cells and a neuroblastoma cell line was established to evaluate the cytotoxic effect and the T cell effector function in vitro. Expression of immune checkpoint molecules on tumor-infiltrating T cells and the induction of an anti-neuroblastoma immune response using a combination of whole cell vaccination and trAb therapy was investigated in a syngeneic immunocompetent neuroblastoma mouse model (NXS2 in A/J background). Finally, vaccinated mice were assessed for the presence of neuroblastoma-directed antibodies. We show that SUREK trAb-mediated effective killing of NXS2 cells in vitro was strictly dependent on the combined presence of DCs and T cells. Results: Using a syngeneic neuroblastoma mouse model, we showed that vaccination with irradiated tumor cells combined with SUREK trAb treatment significantly prolonged survival of tumor challenged mice and partially prevent tumor outgrowth compared to tumor vaccination alone. Treatment led to upregulation of programmed cell death protein 1 (Pd-1) on tumor infiltrating T cells and combination with anti-Pd-1 checkpoint inhibition enhanced the NXS2-directed humoral immune response. Conclusion: Here, we provide first preclinical evidence that a tumor vaccination combined with SUREK trAb therapy induces an endogenous anti-neuroblastoma immune response reducing tumor recurrence. Furthermore, a combination with anti-Pd-1 immune checkpoint blockade might even further improve this promising immunotherapeutic concept in order to prevent relapse in high-risk neuroblastoma patients

GD2-directed bispecific trifunctional antibody outperforms dinutuximab beta in a murine model for aggressive metastasized neuroblastoma

Background: Neuroblastoma is the most common extracranial solid tumor of childhood. Patients with high-risk disease undergo extremely aggressive therapy and nonetheless have cure rates below 50%. Treatment with the ch14.18 monoclonal antibody (dinutuximab beta), directed against the GD2 disialoganglioside, improved 5-year event-free survival in high-risk patients when administered in postconsolidation therapy and was recently implemented in standard therapy. Relapse still occurred in 57% of these patients, necessitating new therapeutic options. Bispecific trifunctional antibodies (trAbs) are IgG-like molecules directed against T cells and cancer surface antigens, redirecting T cells (via their CD3 specificity) and accessory immune cells (via their functioning Fc-fragment) toward tumor cells. We sought proof-of-concept for GD2/CD3-directed trAb efficacy against neuroblastoma. Methods: We used two GD2-specific trAbs differing only in their CD3-binding specificity: EKTOMUN (GD2/human CD3) and SUREK (GD2/mouse Cd3). This allowed trAb evaluation in human and murine experimental settings. Tumor-blind trAb and the ch14.18 antibody were used as controls. A coculture model of human peripheral blood mononuclear cells (PBMCs) and neuroblastoma cell lines was established to evaluate trAb antitumor efficacy by assessing expression of T-cell surface markers for activation, proinflammatory cytokine release and cytotoxicity assays. Characteristics of tumor-infiltrating T cells and response of neuroblastoma metastases to SUREK treatment were investigated in a syngeneic immunocompetent neuroblastoma mouse model mimicking minimal residual disease. Results: We show that EKTOMUN treatment caused effector cell activation and release of proinflammatory cytokines in coculture with neuroblastoma cell lines. Furthermore, EKTOMUN mediated GD2-dependent cytotoxic effects in human neuroblastoma cell lines in coculture with PBMCs, irrespective of the level of target antigen expression. This effect was dependent on the presence of accessory immune cells. Treatment with SUREK reduced the intratumor Cd4/Cd8 ratio and activated tumor infiltrating T cells in vivo. In a minimal residual disease model for neuroblastoma, we demonstrated that single-agent treatment with SUREK strongly reduced or eliminated neuroblastoma metastases in vivo. SUREK as well as EKTOMUN demonstrated superior tumor control compared with the anti-GD2 antibody, ch14.18. Conclusions: Here we provide proof-of-concept for EKTOMUN preclinical efficacy against neuroblastoma, presenting this bispecific trAb as a promising new agent to fight neuroblastoma