Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS Prodigy

Abstract

Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

Functionality and Cell Senescence of CD4/ CD8-Selected CD20 CAR T Cells Manufactured Using the Automated CliniMACS Prodigy® Platform

“…The CliniMACS Prodigy® system is currently the most widely used automated platform for the manufacturing of CAR T cells [9], achieving successful results as shown previously [6-8, 10, 11]. The ability to conduct all manufacturing steps needed for gene modification of designated target cells, i.e., the CD4/CD8 cell immunomagnetic selection, T cell activation, transduction, expansion, and formulation of the final product in a sterile single-use tubing set (TS520) using GMP-grade reagents in a closed system, enables safe manufacturing of clinical-grade cell therapeutics according to GMP regulations.…”

B Cell-Based Cancer Immunotherapy

“…Manufacturing of B cells under GMP-conditions comprises no obstacles anymore after today’s experience with CAR trials [86] and B cell-adoptive transfer [81], and suitable tumor antigens for pulsing have been discovered in many solid tumor entities [87]. The whole isolation and activation process of CD40B cells in general would also be suitable for an automated manufacturing process, e.g., in the CliniMACS Prodigy (Miltenyi Biotec) [88]. The most straightforward approach would thus include isolation of B cells from TILs by CD19+ microbeads, activation and expansion with the CD40L, loading with antigen after control of the activation status, and reinjection into the patient (Fig.…”

“…The current success of chimeric antigen-receptor T cells will lead to a more widespread establishment of the infrastructure required for the clinical application of cellular therapies. In addition, technological advances such as small, automated, closed system cell manufacturing platforms that enable the decentralized “point-of-care” generation of cellular therapies will further ease the clinical testing of cellular immunotherapies such as CD40B-cell cancer vaccines [88]. Therefore, it can be expected that the near future will see the first clinical trials of B cell-based cancer vaccines.…”

Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion

“…The proportion of the most abundantT-cell subtype, theTSCM-like population comprising roughly half of the cells, was not substantially influenced by the amount of IL-2 supplementation. Also, 100 IU/mL IL-2, considered here as the current standard often used inT-cell production [36], generated on average a 2.5-fold smaller proportion of TCM and 5.5-fold more TEff compared with T cells expanded without supplemental IL-2 (P = 0.04 and P = 0.01, respectively; Figure 2). …”