Automated Enrichment, Transduction, and Expansion of Clinical-Scale CD62L⁺ T Cells for Manufacturing of Gene Therapy Medicinal Products

Abstract

Multiple clinical studies have demonstrated that adaptive immunotherapy using redirected T cells against advanced cancer has led to promising results with improved patient survival. The continuously increasing interest in those advanced gene therapy medicinal products (GTMPs) leads to a manufacturing challenge regarding automation, process robustness, and cell storage. Therefore, this study addresses the proof of principle in clinical-scale selection, stimulation, transduction, and expansion of T cells using the automated closed CliniMACS® Prodigy system. Naïve and central memory T cells from apheresis products were first immunomagnetically enriched using anti-CD62L magnetic beads and further processed freshly (n = 3) or split for cryopreservation and processed after thawing (n = 1). Starting with 0.5 × 108 purified CD3+ T cells, three mock runs and one run including transduction with green fluorescent protein (GFP)-containing vector resulted in a median final cell product of 16 × 108 T cells (32-fold expansion) up to harvesting after 2 weeks. Expression of CD62L was downregulated on T cells after thawing, which led to the decision to purify CD62L+CD3+ T cells freshly with cryopreservation thereafter. Most important in the split product, a very similar expansion curve was reached comparing the overall freshly CD62L selected cells with those after thawing, which could be demonstrated in the T cell subpopulations as well by showing a nearly identical conversion of the CD4/CD8 ratio. In the GFP run, the transduction efficacy was 83%. In-process control also demonstrated sufficient glucose levels during automated feeding and medium removal. The robustness of the process and the constant quality of the final product in a closed and automated system give rise to improve harmonized manufacturing protocols for engineered T cells in future gene therapy studies.

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

“…Both results are in line with previously published data from similar manufacturing processing with the CliniMACS Prodigy® system [8, 11]. This reflects an improvement of manufacturing process based on CD62L-selected T cells where a wider range of 82–99% CD3+ T cells was reported [7]. Additionally, the transduction efficiency of a median of 23% of the overall T cells in the present study was sufficient to produce relevant amounts of target cells in the clinical product.…”

“…The expansion rate of CD8+ T cells (median 79-fold) was significantly higher than the expansion rate of CD4+ T cells (median 56-fold, p = 0.023). As a result, the ratio of CD4/CD8 decreased during the cultivation period (from 2.4 to 1.7), but still at no time point was it < 1 in contrast to previous data [7]. Interestingly, the transduction efficiency was significantly higher ( p < 0.05) for CD4+ compared to CD8+ T cells, in a median of 30 versus 17%, respectively.…”

“…Some contaminating other cell types, mainly NKT, NK cells, some B cells, and monocytes, were reduced during the following cultivation in the T cell-adapted culture medium. As only a small amount of the CD4/CD8 enriched target cell fraction is used for the lentiviral vector-transduction and cell expansion, the remaining enriched fraction can be cryopreserved and used later for additional manufacturing processes if necessary, as already shown [7]. The T cell activating reagent used (MACS GMP T cell TransAct), with biodegradable colloidal nanomatrix particles and covalently bound CD3/CD28, combines the advantages of efficient T cell stimulation with no need for additional removal steps.…”

“…1). The following modifications to the manufacturing process previously described by Priesner et al [7] were carried out. An enriched CD4+ and CD8+ cell fraction was used as the starting material instead of CD62L+ selected cells.…”

Gene Modified T Cell Therapies for Hematological Malignancies

“…2). Recently, aspects of CAR-T cell manufacturing have been automated (eg, the CliniMACS Prodigy has been shown to support lentiviral T cell modification with minimal hands-on time), 72,73 and phase I testing of cells manufactured in such a system is underway in the allogeneic transplant setting in London (clinicaltrials.gov, NCT02893189). Effector function and cell persistence are critical for effective immunotherapy.…”