Chimeric antigen receptor T cells (CAR T cells) with T stem (TSCM) cell-like phenotypic characteristics promote sustained antitumor effects. We performed an unbiased and automated high-throughput screen of a kinase-focused compound set to identify kinase inhibitors (KIs) that preserve human TSCM cell-like CAR T cells. We identified three KIs, UNC10225387B, UNC10225263A and UNC10112761A, that combined in vitro increased the frequency of CD45RA+CCR7+TCF1hi TSCM cell-like CAR T cells from both healthy donors and patients with cancer. KI-treated CAR T cells showed enhanced antitumor effects both in vitro and in vivo in mouse tumor models. The KI cocktail maintains TSCM cell-like phenotype preferentially in CAR T cells originating from naive T cells and causes transcriptomic changes without arresting T cell activation or modulating the chromatin organization. Specific kinases, ITK, ADCK3, MAP3K4 and CDK13, targeted by the KI cocktail in a dose-dependent manner are directly associated with the preservation of TSCM cell-like CAR T cells. Knockdown of these kinases individually or in combination enriches for TSCM cell-like CAR T cells, but only CAR T cells generated in the presence of the KI cocktail show robust expansion and differentiation on stimulation with tumor cells. Overall, transient pharmacological inhibition of strategically targeted kinases maintains stem-like features in CAR T cells and improves their antitumor activity.
© 2025. The Author(s).

Sarcomas are rare, mesenchymal tumors, representing about 10-15% of all childhood cancers. GD2 is a suitable target for chimeric antigen receptor (CAR) T-cell therapy due to its overexpression in several solid tumors. In this preclinical study, we investigated the potential use of iCasp9.2A.GD2.CAR-CD28.4-1BBζ (CAR.GD2) T-cells as a treatment option for patients who have GD2-positive sarcomas and we sought to identify factors shaping hostile tumor microenvironment in this setting. GD2 expression was evaluated by flow-cytometry on primary tumor biopsies of pediatric sarcoma patients. GD2 expression in sarcoma cells was also evaluated in response to an enhancer of zeste homolog 2 (EZH2) inhibitor (Tazemetostat). The antitumor activity of CAR.GD2 T-cells was evaluated both in vitro and in vivo preclinical models of orthotopic and/or metastatic soft-tissue and bone sarcomas. GD2 expression was detected in 55% of the primary tumors. Notably, the Osteosarcoma and Alveolar Rhabdomyosarcomas subtypes exhibited the highest GD2 expression levels, while Ewing sarcoma showed the lowest. CAR.GD2 T-cells show a significant tumor control both in vitro and in vivo models of GD2-expressing tumors. Pretreatment with an EZH2 inhibitor (Tazemetostat) upregulating GD2 expression, sensitizes GD2dim sarcoma cells to CAR.GD2 T-cells cytotoxic activity. Moreover, in mouse models of disseminated Rhabdomyosarcomas and orthotopic Osteosarcoma, CAR.GD2 T-cells showed both a vigorous anti-tumor activity and long-term persistence as compared to un-transduced T-cells. The presence of immunosuppressive murine myeloid-derived suppressor (MDSC) cells significantly reduces long-term anti-tumour activity of infused CAR.GD2 T-cells. Tumor-derived G-CSF was found to be one of the key factors driving expansion of immunosuppressive murine and human MDSC, thus indirectly limiting the efficacy of CAR.GD2 T-cells. Our preclinical data strongly suggest that CAR.GD2 T-cells hold promise as a potential therapeutic option for the treatment of patients with GD2-positive sarcomas. Strategies to tackle hostile immunosuppressive MDSC are desirable to optimize CAR.GD2 T-cell activity.
© 2024. The Author(s).

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

Different eosinophil subpopulations have been identified in asthma and other eosinophilic disorders. However, there is a paucity of data on eosinophil subpopulations in patients with chronic obstructive pulmonary disease (COPD). The aim of this study was to compare eosinophil phenotypes in blood and induced sputum in patients with COPD, asthma and controls. Stable patients with mild-to-moderate COPD (n = 15) and asthma (n = 14) with documented blood eosinophilia ≥100 cells/µL in the year prior to the study and the control group (n = 11) were included to the study. The blood and sputum eosinophil phenotypes were analyzed by flow cytometry. IL-5, IL-13, CCL5 and eotaxin-3 levels were measured in the induced sputum. The marker expression on blood eosinophils was similar among control, asthma and COPD groups. The expressions of CD125, CD193, CD14 and CD62L were higher on blood than on sputum eosinophils in all three groups. We found increased levels of CD193+ and CD66b+ sputum eosinophils from COPD patients, and an elevated level of CD11b+ sputum eosinophils in asthma compared to COPD patients. The results of our study suggest that the profile of marker expression on COPD sputum eosinophils differed from other groups, suggesting a distinct phenotype of eosinophils of COPD patients than in asthma or healthy subjects.

Despite the success of CAR-T cell cancer immunotherapy, challenges in efficacy and safety remain. Investigators have begun to enhance CAR-T cells with the expression of accessory molecules to address these challenges. Current systems rely on constitutive transgene expression or multiple viral vectors, resulting in unregulated response and product heterogeneity. Here, we develop a genetic platform that combines autonomous antigen-induced production of an accessory molecule with constitutive CAR expression in a single lentiviral vector called Uni-Vect. The broad therapeutic application of Uni-Vect is demonstrated in vivo by activation-dependent expression of (1) an immunostimulatory cytokine that improves efficacy, (2) an antibody that ameliorates cytokine-release syndrome, and (3) transcription factors that modulate T cell biology. Uni-Vect is also implemented as a platform to characterize immune receptors. Overall, we demonstrate that Uni-Vect provides a foundation for a more clinically actionable next-generation cellular immunotherapy.
Copyright © 2022 Elsevier Inc. All rights reserved.