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Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are still being understood. For example, we currently lack a comprehensive understanding of the mechanisms of neurotoxicity observed in patients receiving T cell therapies, including recent reports of encephalitis caused by human herpesvirus 6 (HHV-6) reactivation1. Here, via petabase-scale viral RNA data mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in human CD4+ T cells in standard in vitro cultures. Using single-cell sequencing, we identify a rare population of HHV-6 ‘super-expressors’ (~1 in 300-10,000 cells) that possess high viral transcriptional activity in chimeric antigen receptor (CAR) T cell culture before spreading to infect other cells in vitro. Through the analysis of single-cell sequencing data from patients receiving cell therapy products that are FDA-approved2 or used in clinical studies3,4, we identify the presence of CAR+, HHV-6 super-expressor T cells in vivo. Together, our study implicates cell therapy products as a source of lytic HHV-6 reported in clinical trials1,5–7 and has broad implications for the design, production, and monitoring of cell therapies.

In recent years, high temperature short time (HTST) treatment technology has been increasingly adopted for medium treatment to mitigate the potential risk of viral contamination in mammalian cell culture GMP manufacturing facilities. Mouse minute virus (MMV), also called minute virus of mice (MVM), implicated in multiple viral contamination events is commonly used as a relevant model virus to assess the effectiveness of HTST treatment of cell culture media. However, results from different studies vary broadly in inactivation kinetics as well as log reduction factors (LRFs) achieved under given treatment conditions. To determine whether the reported discrepancies stemmed from differences in MMV strains, laboratory-scale HTST devices, medium matrices, and/or experimental designs, we have taken a collaborative approach to systematically assess the effectiveness of HTST treatment for MMV inactivation. This effort was conceptualized based on a media treatment gap analysis conducted by the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) under the MIT Center for Biomedical Innovation (CBI). Specifically, two different MMV strains were used to evaluate the effectiveness of HTST at various treatment conditions with regard to exposure temperature and hold time duration by two independent laboratories within two different companies. To minimize experimental variations, the two sites used the same batches of MMV stocks, the same commercially purchased medium, and the same model of thermocyclers as the laboratory-scale HTST device. The two independent laboratories yielded similar MMV inactivation kinetics and comparable LRF. No significant differences were observed between the two MMV strains evaluated, suggesting that the variations from prior studies were likely due to differences in equipment, medium matrices, or other factors. The data presented here indicate that MMV inactivation by HTST treatment obeys first-order kinetics and can be mathematically modeled using an Arrhenius equation. The model-based extrapolation provides a quantitative estimate of MMV inactivation by the current industry standard HTST condition (102°C for a hold time of 10 s) used for medium treatment. Finally, based on the data from the current study and the industry experience, it is recommended that any alternative virus barrier technologies adopted for medium treatment should provide a clearance of at least 3.0 LRF based on a worst-case model virus to effectively mitigate potential risks of viral contamination.

Allogeneic chimeric antigen-receptor T-cell process flows comprise large-scale primary cultures incorporating traditional biologics processing steps such as cell expansion, with novel processing steps such as electroporation. In addition, these process flows begin with donor-derived peripheral blood mononuclear cells and generate single batches of product, which may be used to treat hundreds of patients. The corresponding unit operation-process safety considerations are individually diverse, and in combination are currently unique and rapidly evolving. The use of available regulations and the principles of quality by design are useful in designing appropriate control strategies into the process during development to ensure process safety.

Recombinant protein therapeutics, vaccines, and plasma products have a long record of safety. However, the use of cell culture to produce recombinant proteins is still susceptible to contamination with viruses. These contaminations cost millions of dollars to recover from, can lead to patients not receiving therapies, and are very rare, which makes learning from past events difficult. A consortium of biotech companies, together with the Massachusetts Institute of Technology, has convened to collect data on these events. This industry-wide study provides insights into the most common viral contaminants, the source of those contaminants, the cell lines affected, corrective actions, as well as the impact of such events. These results have implications for the safe and effective production of not just current products, but also emerging cell and gene therapies which have shown much therapeutic promise.