Lymphotoxin beta-activated LTBR/NIK/RELB axis drives proliferation in cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the intrahepatic (iCCA) or extrahepatic (eCCA) bile ducts with poor prognosis and limited treatment options. Prior evidence highlighted a significant contribution of the non-canonical NF-κB signalling pathway in initiation and aggressiveness of different tumour types. Lymphotoxin-ß (LTß) stimulates the NF-κB-inducing kinase (NIK), resulting in the activation of the transcription factor RelB. However, the functional contribution of the non-canonical NF-κB signalling pathway via the LTß/NIK/RelB axis in CCA carcinogenesis and progression has not been established. METHODS: Human CCA-derived cell lines and organoids were examined to determine the expression of NF-κB pathway components upon activation or inhibition. Proliferation and cell death were analysed using real-time impedance measurement and flow cytometry. Immunoblot, qRT-PCR, RNA sequencing and in situ hybridization were employed to analyse gene and protein expression. Four in vivo models of iCCA were used to probe the activation and regulation of the non-canonical NF-κB pathway. RESULTS: Exposure to LTα1/ß2 activates the LTß/NIK/RelB axis and promotes proliferation in CCA. Inhibition of NIK with the small molecule inhibitor B022 efficiently suppresses RelB expression in patient-derived CCA organoids and nuclear co-translocation of RelB and p52 stimulated by LTα1/ß2 in CCA cell lines. In murine CCA, RelB expression is significantly increased and LTß is the predominant ligand of the non-canonical NF-κB signalling pathway. CONCLUSIONS: Our study confirms that the non-canonical NF-κB axis LTß/NIK/RelB drives cholangiocarcinogenesis and represents a candidate therapeutic target.

Evaluation of all-cause mortality and cardiovascular safety in patients receiving chimeric antigen receptor T cell therapy: a prospective cohort study.

Background: Assessment of cardiovascular risk is critical for patients with cancer. Previous retrospective studies suggest potential cardiotoxicity of CAR T cell therapies. We aimed to prospectively assess cardiotoxicity and the predictive value of cardiac biomarkers and classical risk factors (age, cardiac function, diabetes, arterial hypertension, smoking) for cardiac events and all-cause mortality (ACM). Methods: In this prospective cohort study, all patients treated with CAR T cell constructs (axi-cel, tisa-cel, brexu-cel, ide-cel, or the 3rd generation CAR HD-CAR-1) from Oct 1, 2018, to Sept 30, 2022 at the University Hospital Heidelberg were included. Surveillance included cardiac assessment with biomarkers (high-sensitive Troponin T (hs-cTnT), N-terminal brain natriuretic peptide (NT-proBNP)), 12-lead-ECG, and 2D echocardiography. ACM was defined as the primary study endpoint, while cardiotoxicity, defined by clinical syndromes of heart failure or decline in ejection fraction, served as a secondary endpoint. Findings: Overall, 137 patients (median age 60, range 20-83, IQR 16), were included in the study. 46 patients died during the follow up period (median 0.75 years, range 0.02-4.33, IQR 0.89) 57 month, with a median survival of 0.57 years (range 0.03-2.38 years, IQR 0.79). A septal wall thickness above 11 mm (HR 2.48, 95%-CI = 1.10-5.67, p = 0.029) was associated with an increased risk of ACM, with a trend seen for reduced left ventricular ejection fraction prior to therapy (LVEF <40%; HR 9.17, 95%-CI = 1.30-183.11, p = 0.051). Secondary endpoint was reached by 93 patients while no baseline parameter was able to predict an elevated risk. However, hs-cTnT change from baseline of 50% or more during the first 14 days after CAR infusion predicted ACM (HR 3.81, 95%-CI = 1.58-9.45; p = 0.003). None of the baseline characteristics was able to predict the incidence of cardiac events. Interpretation: Reduced pre-lymphodepletion ejection fraction and early post-infusion biomarker kinetics may be associated with increased ACM and cardiotoxicity events. These findings may help to identify patients who could benefit from intensified cardio-oncological surveillance. Funding: The German Center for Cardiovascular Research, German Research Foundation, and the Federal Ministry of Education and Research.

Comparative analysis of Bcl-2 family protein overexpression in CAR T cells alone and in combination with BH3 mimetics.

Approximately 50% of patients with hematologic malignancies relapse after chimeric antigen receptor (CAR) T cell treatment; mechanisms of failure include loss of CAR T persistence and tumor resistance to apoptosis. We hypothesized that both of these challenges could potentially be overcome by overexpressing one or more of the Bcl-2 family proteins in CAR T cells to reduce their susceptibility to apoptosis, both alone and in the presence of BH3 mimetics, which can be used to activate apoptotic machinery in malignant cells. We comprehensively investigated overexpression of different Bcl-2 family proteins in CAR T cells with different signaling domains as well as in different tumor types. We found that Bcl-xL and Bcl-2 overexpression in CAR T cells bearing a 4-1BB costimulatory domain resulted in increased expansion and antitumor activity, reduced exhaustion, and decreased apoptotic priming. In addition, CAR T cells expressing either Bcl-xL or a venetoclax-resistant Bcl-2 variant led to enhanced antitumor efficacy and survival in murine xenograft models of lymphoma and leukemia in the presence or absence of the BH3 mimetic venetoclax, a clinically approved BH3 mimetic. In this setting, Bcl-xL overexpression had stronger effects than overexpression of Bcl-2 or the Bcl-2(G101V) variant. These findings suggest that CAR T cells could be optimally engineered by overexpressing Bcl-xL to enhance their persistence while opening a therapeutic window for combination with BH3 mimetics to prime tumors for apoptosis.

Optimization of a flow cytometry test for routine monitoring of B cell maturation antigen targeted CAR in peripheral blood.

Chimeric antigen receptor (CAR) modified T cell therapies targeting BCMA have displayed impressive activity in the treatment of multiple myeloma. There are currently two FDA licensed products, ciltacabtagene autoleucel and idecabtagene vicleucel, for treating relapsed and refractory disease. Although correlative analyses performed by product manufacturers have been reported in clinical trials, there are limited options for reliable BCMA CAR T detection assays for physicians and researchers looking to explore it as a biomarker for clinical outcome. Given the known association of CAR T cell expansion kinetics with toxicity and response, being able to quantify BCMA CAR T cells routinely and accurately in the blood of patients can serve as a valuable asset. Here, we optimized an accurate and sensitive flow cytometry test using a PE-conjugated soluble BCMA protein, with a lower limit of quantitation of 0.19% of CD3+ T cells, suitable for use as a routine assay for monitoring the frequency of BCMA CAR T cells in the blood of patients receiving either ciltacabtagene autoleucel or idecabtagene vicleucel.

Mesothelin CAR T Cells Secreting Anti-FAP/Anti-CD3 Molecules Efficiently Target Pancreatic Adenocarcinoma and its Stroma.

PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.