Large Scale In-House Point-of-Care Manufacturing of Clinical Grade Autologous Anti-CD19 CAR T Cells in Saudi Arabia
Authors: Warda Walid , El Fakih Riad , Alrasheed Nouf , Alkhaldi Moumenah , Hamideddin Noura , Alqudairy Hanan , Alsuliman Abdullah , Almozyan Sheema , Alaiya Ayodele , Doubi Alaa Osama , Alothaimeen Haya S , Alahmari Ali D , Alfraih Feras A , Aljurf Mahmoud , Alzahrani Hazzaa , Ahmed Syed Osman , Syed Farhatullah 
Published: June 2024

ABSTRACTChimeric 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.