An IL-10 variant with higher affinity for a receptor subunit remains effective at low concentrations.

The role of Epigenetics in Epithelial Mesenchymal Transition (EMT) has recently emerged. Two epigenetic enzymes with paradoxical roles have previously been associated to EMT, EZH2 (Enhancer of Zeste 2 Polycomb Repressive Complex 2 (PRC2) Subunit), a lysine methyltranserase able to add the H3K27me3 mark, and the histone demethylase KDM6B (Lysine Demethylase 6B), which can remove the H3K27me3 mark. Nevertheless, it still remains unclear how these enzymes, with apparent opposite activities, could both promote EMT. In this study, we evaluated the function of these two enzymes using an EMT-inducible model, the lung cancer A549 cell line. ChIP-seq coupled with transcriptomic analysis showed that EZH2 and KDM6B were able to target and modulate the expression of different genes during EMT. Based on this analysis, we described INHBB, WTN5B, and ADAMTS6 as new EMT markers regulated by epigenetic modifications and directly implicated in EMT induction.

Chimeric Antigen Receptor T (CAR T) cell therapy targeting the CD19 antigen represents a significant advancement in the treatment of various subtypes of CD19-expressing hematologic malignancies. Nevertheless, the current commercial CAR T manufacturing process is intricate, time-consuming, and costly, involving a supply chain that initiates at the treating center with mononuclear cell apheresis, followed by cryopreservation and shipping to and from a centralized third-party manufacturing facility. This process can introduce treatment delays due to logistical requirements. We hypothesized that point-of-care (POC) manufacturing of CAR T cells could reduce vein to vein time which is one of the major challenges with commercial CAR T processes. This study presents the outcomes of large-scale, clinical-grade, in-house POC CAR T manufacturing, including assessments of transduction efficiency, vector copy number, phenotype, and effector function of CAR T cells. Utilizing the CliniMACS Prodigy system, we processed mononuclear cells from fresh leukapheresis harvests of healthy donors. The cells underwent automated processing and activation with TransAct, a polymeric nanomatrix activation reagent containing CD3/CD28-specific antibodies. Following activation, the cells were transduced and expanded in the Centricult-unit under stabilized conditions, with automated feeding and media exchange. For transduction, we employed the Miltenyi Lentigen third-generation lentiviral vector, which delivers a second-generation CD19 CAR construct with a 4-1BB costimulatory domain and TNFRSF19 transmembrane domain. Our POC manufacturing data demonstrated the successful generation of large-scale CAR T cells, sufficient to prepare multiple doses within a shortened production period of 9 days, followed by cryopreservation for quality control assessment and batch certification for patient infusion. The transduction efficiencies, phenotype, and function of the Lentigen CD19 CAR T cells were comparable to previously published procedures, with overall T-cell yields sufficient for therapeutic application. CAR expression was evaluated by flow cytometry and vector copy number. In-process testing ensured the quality and suitability of CAR T cells in terms of sterility and cell number for the desired dose preparations. The final product was also subjected for the assessment of any replicative competent lentivirus. CAR-mediated target cell killing was assessed via co-culture assays, and cytokine release was measured by ELISA. To evaluate the shelf life of the cryopreserved product, stability and viability were checked at approximately 6 and 12 months. Our results indicate that in-house POC manufacturing at King Faisal Specialist Hospital & Research Centre (KFSH&RC) enables regulatory-compliant production of CAR T cells, eliminating the need for third-party shipping logistics.

Nous avons produit des CART-cells et des LT-mock ont été produits ex-vivo. L'efficacité de transduction est mesuré par l'expression CD3+/CD19+ (82% en moyenne, n=S), en cytométrie en flux. L'expression protéique de CAR a été vérifiée par western blot et en cytométrie en flux attestant de son expression membranaire. Nous avons testé la fonctionnalité de la cassette suicide iCaspase9/ AP1903 sur CART-cells après 48h de traitement à I' AP1903. Nous avons montré que plus de 90% des CART-cells entrent en apoptose (Marquage 7-AAD en cytométrie en flux). Nous avons ensuite réalisé des tests fonctionnels in-vitro des CART-cells contre des lignées LMC exprimant naturellement IL-lRAP ou contre des lignées transfectées pour exprimer la forme membranaire d'IL-lRAP. Après co­culture des CART-cells en présence des cellules cibles IL-lRAP+ nous avons montré qu'ils prolifèrent (test CFSE), qu'ils sécrètent de l'interféron y et des cytokines inflammatoires. Enfin, par marquage 7-AAD, nous avons démontré la cytotoxicité des CART-cells contre les cellules ILl-RAP+. Finalement, nous avons montré l'efficacité des CART-cells in vivo, dans un modèle de xénogreffe tumorales dans des souris immunodéficiences NSG greffées par la lignée K562-IL1RAP+ / Luciférase +. On constate une diminution de la masse tumorale 4 jours après injection des CART-cells, jusqu'à disparition totale. Cette preuve de concept laisse entrevoir des perspectives thérapeutiques alternatives dans le traitement de la LMC ou LAM, qui devront être optimiser dans des modèles murins (séquence et nombres d'injection, nombres de cellules, associations avec ITKS ou autres chimiothérapies) afin de conduire un essai clinique de phase I, pour démontrer la faisabilité et la sécurité de l'approche pour envisager une démonstration d'efficacité chez l'homme. La sécurisation par la cassette suicide permet d'envisager l'utilisation des CART-cells en situation autologue ou allogénique (Donor Lymphocytes infusion, DU)