Development and psychometric evaluation of the Transdiagnostic Decision Tool:matched care for patients with a mental disorder in need of highly specialised care

BackgroundEarly identification of patients with mental health problems in need of highly specialised care could enhance the timely provision of appropriate care and improve the clinical and cost-effectiveness of treatment strategies. Recent research on the development and psychometric evaluation of diagnosis-specific decision-support algorithms suggested that the treatment allocation of patients to highly specialised mental healthcare settings may be guided by a core set of transdiagnostic patient factors.AimsTo develop and psychometrically evaluate a transdiagnostic decision tool to facilitate the uniform assessment of highly specialised mental healthcare need in heterogeneous patient groups. Method The Transdiagnostic Decision Tool was developed based on an analysis of transdiagnostic items of earlier developed diagnosis-specific decision tools. The Transdiagnostic Decision Tool was psychometrically evaluated in 505 patients with a somatic symptom disorder or post-traumatic stress disorder. Feasibility, interrater reliability, convergent validity and criterion validity were assessed. In order to evaluate convergent validity, the five-level EuroQol five-dimensional questionnaire (EQ-5D-5L) and the ICEpop CAPability measure for Adults (ICECAP-A) were administered.ResultsThe six-item clinician-administered Transdiagnostic Decision Tool demonstrated excellent feasibility and acceptable interrater reliability. Spearman's rank correlations between the Transdiagnostic Decision Tool and ICECAP-A (-0.335), EQ-5D-5L index (-0.386) and EQ-5D-visual analogue scale (-0.348) supported convergent validity. The area under the curve was 0.81 and a cut-off value of &gt;= 3 was found to represent the optimal cut-off value.ConclusionsThe Transdiagnostic Decision Tool demonstrated solid psychometric properties and showed promise as a measure for the early detection of patients in need of highly specialised mental healthcare.</p

The Role of Pseudomonas aeruginosa ExoY in an Acute Mouse Lung Infection Model

The effector protein Exotoxin Y (ExoY) produced by Pseudomonas aeruginosa is injected via the type III secretion system (T3SS) into host cells. ExoY acts as nucleotidyl cyclase promoting the intracellular accumulation of cyclic nucleotides. To what extent nucleotidyl cyclase activity contributes to the pathogenicity of ExoY and which mechanisms participate in the manifestation of lung infection is still unclear. Here, we used an acute airway infection model in mice to address the role of ExoY in lung infection. In infected lungs, a dose-dependent phenotype of infection with bacteria-expressing ExoY was mirrored by haemorrhage, formation of interstitial oedema in alveolar septa, and infiltration of the perivascular space with erythrocytes and neutrophilic granulocytes. Analyses of the infection process on the cellular and organismal level comparing infections with Pseudomonas aeruginosa mutants expressing either nucleotidyl cyclase-active or -inactive ExoY revealed differential cytokine secretion, increased prevalence of apoptosis, and a break of lung barrier integrity in mice infected with cyclase-active ExoY. Notably, of all measured cyclic nucleotides, only the increase of cyclic UMP in infected mouse lungs coincides temporally with the observed early pathologic changes. In summary, our results suggest that the nucleotidyl cyclase activity of ExoY can contribute to P. aeruginosa acute pathogenicity

Design and Characterization of an “All-in-One” Lentiviral Vector System Combining Constitutive Anti-GD2 CAR Expression and Inducible Cytokines

Genetically modified T cells expressing chimeric antigen receptors (CARs) so far have mostly failed in the treatment of solid tumors owing to a number of limitations, including an immunosuppressive tumor microenvironment and insufficient CAR T cell activation and persistence. Next-generation approaches using CAR T cells that secrete transgenic immunomodulatory cytokines upon CAR signaling, known as TRUCKs ("T cells redirected for universal cytokine-mediated killing"), are currently being explored. As TRUCKs were engineered by the transduction of T cells with two separate vectors, we developed a lentiviral modular "all-in-one" vector system that combines constitutive CAR expression and inducible nuclear factor of activated T cells (NFAT)-driven transgene expression for more efficient production of TRUCKs. Activation of the G(D2)-specific CAR via GD2(+) target cells induced NFAT promoter-driven cytokine release in primary human T cells, and indicated a tight linkage of CAR-specific activation and transgene expression that was further improved by a modified NFATsyn promoter. As proof-of-concept, we showed that T cells containing the "all-in-one" vector system secrete the immunomodulatory cytokines interleukin (IL)12 or IL18 upon co-cultivation with primary human GD2(+) tumor cells, resulting in enhanced effector cell properties and increased monocyte recruitment. This highlights the potential of our system to simplify application of TRUCK-modified T cells in solid tumor therapy

CAR-T cells and TRUCKs that recognize an EBNA-3C-derived epitope presented on HLA-B*35 control Epstein-Barr virus-associated lymphoproliferation

Background Immunosuppressive therapy or T-cell depletion in transplant patients can cause uncontrolled growth of Epstein-Barr virus (EBV)-infected B cells resulting in post-transplant lymphoproliferative disease (PTLD). Current treatment options do not distinguish between healthy and malignant B cells and are thereby often limited by severe side effects in the already immunocompromised patients. To specifically target EBV-infected B cells, we developed a novel peptide-selective chimeric antigen receptor (CAR) based on the monoclonal antibody Tu165 which recognizes an Epstein-Barr nuclear antigen (EBNA)-3C-derived peptide in HLA-B*35 context in a T-cell receptor (TCR)-like manner. In order to attract additional immune cells to proximity of PTLD cells, based on the Tu165 CAR, we moreover generated T cells redirected for universal cytokine-mediated killing (TRUCKs), which induce interleukin (IL)-12 release on target contact. Methods Tu165-based CAR-T cells (CAR-Ts) and TRUCKs with inducible IL-12 expression in an all-in-one construct were generated. Functionality of the engineered cells was assessed in co-cultures with EBNA-3C-peptide-loaded, HLA-B*35-expressing K562 cells and EBV-infected B cells as PTLD model. IL-12, secreted by TRUCKs on target contact, was further tested for its chemoattractive and activating potential towards monocytes and natural killer (NK) cells. Results After co-cultivation with EBV target cells, Tu165 CAR-Ts and TRUCKs showed an increased activation marker expression (CD137, CD25) and release of proinflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha). Moreover, Tu165 CAR-Ts and TRUCKs released apoptosis-inducing mediators (granzyme B and perforin) and were capable to specifically lyse EBV-positive target cells. Live cell imaging revealed a specific attraction of Tu165 CAR-Ts around EBNA-3C-peptide-loaded target cells. Of note, Tu165 TRUCKs with inducible IL-12 showed highly improved effector functions and additionally led to recruitment of monocyte and NK cell lines. Conclusions Our results demonstrate that Tu165 CAR-Ts recognize EBV peptide/HLA complexes in a TCR-like manner and thereby allow for recognizing an intracellular EBV target. Tu165 TRUCKs equipped with inducible IL-12 expression responded even more effectively and released IL-12 recruited additional immune cells which are generally missing in proximity of lymphoproliferation in immunocompromised PTLD patients. This suggests a new and promising strategy to specifically target EBV-infected cells while sparing and mobilizing healthy immune cells and thereby enable control of EBV-associated lymphoproliferation

GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD2 and Releasing Inducible IL-18

Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors. Thus, approaches using 4th advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs (“T cells redirected for universal cytokine-mediated killing”), are currently under investigation. Based on our previous development and validation of automated and closed processing for GMP-compliant manufacturing of CAR T cells, we here present the proof of feasibility for translation of this method to TRUCKs. We generated IL-18-secreting TRUCKs targeting the tumor antigen GD2 using the CliniMACS Prodigy® system using a recently described “all-in-one” lentiviral vector combining constitutive anti-GD2 CAR expression and inducible IL-18. Starting with 0.84 x 108 and 0.91 x 108 T cells after enrichment of CD4+ and CD8+ we reached 68.3-fold and 71.4-fold T cell expansion rates, respectively, in two independent runs. Transduction efficiencies of 77.7% and 55.1% was obtained, and yields of 4.5 x 109 and 3.6 x 109 engineered T cells from the two donors, respectively, within 12 days. Preclinical characterization demonstrated antigen-specific GD2-CAR mediated activation after co-cultivation with GD2-expressing target cells. The functional capacities of the clinical-scale manufactured TRUCKs were similar to TRUCKs generated in laboratory-scale and were not impeded by cryopreservation. IL-18 TRUCKs were activated in an antigen-specific manner by co-cultivation with GD2-expressing target cells indicated by an increased expression of activation markers (e.g. CD25, CD69) on both CD4+ and CD8+ T cells and an enhanced release of pro-inflammatory cytokines and cytolytic mediators (e.g. IL-2, granzyme B, IFN-γ, perforin, TNF-α). Manufactured TRUCKs showed a specific cytotoxicity towards GD2-expressing target cells indicated by lactate dehydrogenase (LDH) release, a decrease of target cell numbers, microscopic detection of cytotoxic clusters and detachment of target cells in real-time impedance measurements (xCELLigence). Following antigen-specific CAR activation of TRUCKs, CAR-triggered release IL-18 was induced, and the cytokine was biologically active, as demonstrated in migration assays revealing specific attraction of monocytes and NK cells by supernatants of TRUCKs co-cultured with GD2-expressing target cells. In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products

Ex Vivo Generation of CAR Macrophages from Hematopoietic Stem and Progenitor Cells for Use in Cancer Therapy

Chimeric antigen receptor (CAR) T-cell therapies have shown impressive results in patients with hematological malignancies; however, little success has been achieved in the treatment of solid tumors. Recently, macrophages (M phi s) were identified as an additional candidate for the CAR approach, and initial proof of concept studies using peripheral blood-derived monocytes showed antigen-redirected activation of CAR M phi s. However, some patients may not be suitable for monocyte-apheresis, and prior cancer treatment regimens may negatively affect immune cell number and functionality. To address this problem, we here introduce primary human hematopoietic stem and progenitor cells (HSPCs) as a cell source to generate functional CAR M phi s ex vivo. Our data showed successful CAR expression in cord blood (CB)-derived HSPCs, with considerable cell expansion during differentiation to CAR M phi s. HSPC-derived M phi s showed typical M phi morphology, phenotype, and basic anti-bacterial functionality. CAR M phi s targeting the carcinoembryonic antigen (CEA) and containing either a DAP12- or a CD3 zeta-derived signaling domain showed antigen redirected activation as they secreted pro-inflammatory cytokines specifically upon contact with CEA(+) target cells. In addition, CD3 zeta-expressing CAR M phi s exhibited significantly enhanced phagocytosis of CEA(+) HT1080 cells. Our data establish human HSPCs as a suitable cell source to generate functional CAR M phi s and further support the use of CAR M phi s in the context of solid tumor therapy