Epstein Barr virus (EBV) is a human herpes virus with a prevalence of over 90% in adults [1]. Acute infection is generally asymptomatic, but can present clinically as infectious mononucleosis (glandular fever) and infection persists latently throughout life in B lymphocytes. Intermittent viral reactivation drives proliferation of infected cells, which can transform to malignant lymphoblasts in the absence of immune surveillance. In immunocompetent individuals, EBV-specific T cells control these reactivations [2]. However, iatrogenic immunosuppression of patients following haematopoietic stem cell transplant (HSCT) or solid organ transplant (SOT) can result in potentially fatal EBV-driven post-transplant lymphoproliferative disorder (PTLD), where EBV-transformed B cells develop into an aggressive B cell lymphoma [3].

Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cell lines has proven to be an effective clinical treatment for patients with rituximab-resistant or refractory PTLD [4]. The Scottish National Blood Transfusion Service (SNBTS) EBV-specific T cell bank has delivered third-party partially HLA-matched EBV-specific T cells for PTLD patients for over 15 years, allowing a rapid therapeutic intervention following diagnosis, with an initial 52% complete response rate in a phase 2 multicentre trial [5]. As of December 2020, over 100 SOT and HSCT patients have been treated with the more recent second-generation SNBTS T cell bank, issued under a Medicines and Healthcare Regulatory Agency (MHRA) Manufacturing Specials licence for therapeutic use, and a recent follow-up study of 64 patients with refractory disease treated between 2011 and 2017 indicates that overall survival at 3 years post treatment was more than 40%, and higher (62%) in SOT patients [6]. This corresponds well with other clinical studies using adoptive therapy with EBV-specific T cells, where overall response rates were 63-68% [7-9].

Clinical manufacture of allogeneic EBV-specific T cells for therapy requires a generation protocol that conforms to current good Manufacturing Process (cGMP) regulations. Manufacture of the current SNBTS second-generation SNBTS T cell bank was undertaken using a conventional process for generating EBV-specific T cells, using mononuclear cells (MNC) co-cultured over 6-8 weeks with autologous EBV-transformed lymphoblastoid cell lines (LCL). The LCL act as antigen-presenting cells (APC) to induce proliferation of T cells recognizing EBV viral proteins such as BZLF1 and BMLF1 involved in early lytic cycle transactivation, and viral latency proteins such as EBNA1, EBNA2, EBNA3, LMP1 and LMP2 [10,11]. Multiple rounds of stimulation with irradiated autologous LCL are required to induce EBV-specific T cell expansion to a suitable therapeutic dose [12]. The introduction of rapid expansion protocols in development of new T cell therapies [13] indicates a need for both robust, rapid methods for EBV-specific T cell manufacture, and improved analytic methods to assess the quality of the material throughout.

Replacement of standard flask culture with gas-permeable rapid expansion devices (G-Rex flasks, Wilson Wolf) is now established for T cell culture, and T cell lines are generally initiated in culture with a ratio of 40 MNC : 1 LCL for the first stimulation round. Subsequent stimulation rounds are generally at a 1 T cell : 5 LCL ratio in rapid expansion protocols [14], though variations in this approach are evident between studies [15-18]. As part of this study, we determined the optimal number of stimulation rounds and optimized our previous manufacture protocol by transfer to EU GMP-compliant culture reagents (medium and cytokine) and G-Rex flask culture throughout. We confirmed appropriate T cell: LCL stimulation ratio parameters for culture and in each case utilised an extensive multi-parameter flow cytometric analysis approach to dissect the composition of the cultures.

The critical quality attributes of the SNBTS therapeutic EBV-specific T cells products are stringent and include viability, T cell lineage, and absence of other lymphocytes; for this process optimization study we introduced a broader flow cytometric analysis panel which allowed characterization of the T cell compartment to a much higher level of discrimination. The new panels were able to determine T cell development, differentiation and activation status, and provide markers of efficacy through intracellular cytokine analysis. We utilized these panels to determine the efficacy of the modified protocol in comparison with the current process, and assess quality from starting mononuclear cells through to final T cell product. Visualizing this data is complex, and we applied t-Stochastic Neighbour Embedding (t-SNE) algorithms to incorporate multiple parameters into a single image, allowing visual comparison of the T cell phenotype. This approach provides clear evidence for an improved process for manufacture of virus-specific T cells to a standard suitable for clinical use.