Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules

Abstract

BackgroundGenetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells.MethodsTCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers.ResultsThe process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64–92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase.ConclusionLarge-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.

Prospective Evaluation of a Practical Guideline for Managing Positive Sterility Test Results in Cell Therapy Products

“…As pioneers in gene therapy manufacturing since the 1990s 22 , we introduced closed-system processing (from flask to bag-based cell culture) as a means to reduce product contamination 23 . These methods continue to be used and optimized for recent immunotherapies, including the manufacture of clinical grade CAR T-cells, TCR- transduced T-cells, mesenchymal stromal cells, and mutivirus specific T-cells 24–26 .…”

T lymphocytes as therapeutic arsenal for patients with hematological malignancies

“…TCR-transduced T cells are another therapeutic platform based on genetically modified cells to recognize malignant cells in the context of an HLArestricted and epitope-specific manner. MART1 and NY-ESO-1 TCRs are the most advances on the field and, despite the considerable progress in this highly personalizes cell therapy, several limitations [121], including manufacturing [122], have limited their widespread use.…”

Manufacturing chimeric antigen receptor T cells: issues and challenges

“…Unfortunately, the current yield of this fully automated and closed system ranges from only 1-5x10 9 cells, which is sufficient to treat patients enrolled on most CAR T-cell protocols for haematologic malignancies, but inadequate for many TCR T-cell protocols, which may require up to 100x10 9 cells. Although only partially closed, a large-scale manufacturing process using modular systems and semi-automated devices has been recently described, which resulted in highly functional clinical-grade TCR-transduced T cells 59 .…”

“…Highly proliferative T cells can lead to CRS, which may range from high fever and myalgias to unstable hypotension and respiratory failure. A key insight into CRS came with the observation that, in addition to the expected effector cytokines such as interferon (IFN)gamma, interleukin (IL)-6 can be quite elevated during the exponential phase of CAR T-cell therapy 59 . CRS is directly and possibly causally related to a complementary toxicity, which is macrophage activation syndrome 60 .…”