Abstract

Immunosuppressed patients are susceptible to virus reactivation or de novo infection. Adoptive immunotherapy, based on virus-specific T lymphocytes (VST), can prevent or treat viral diseases. However, donor availability, HLA-compatibility restrictions, high costs, and time required for the production of personalized medicines constitute considerable limitations to this treatment. Ex vivo rapid and large-scale expansion of VST, compliant with current good manufacturing practice (cGMP) standards, with an associated cell donor registry would overcome these limitations. This study aimed to characterize a VST product obtained through an expansion protocol transferable to cGMP standards. Antigenic stimulus consisted of cytomegalovirus (CMV) pp65 peptide pool-pulsed autologous dendritic cells (DCs) derived from monocytes. G-Rex technology, cytokines IL-2, IL-7, and IL-15, and anti-CD3 and anti-CD28 antibodies were used for culture. At day 14 of cell culture, the final product was characterized regarding T cell subsets, specificity, and functionality. The final product, comprised mainly CD4+ and CD8+ T lymphocytes (49.2 ± 24.7 and 42.3 ± 25.2, respectively). The culture conditions made it possible to achieve at least a 98.89-fold increase in pp65-specific CD3+ IFN-γ+ cells. These cells were specific, as pp65-specific cytotoxicity was demonstrated. Additionally, in complete HLA mismatch and without the presence of pp65, alloreactivity resulted in <5% cell lysis. In conclusion, a cGMP scalable process for the generation of a large number of doses of CMV-specific cytotoxic T cells was successfully performed.

Highlights

  • Immunodeficient patients are susceptible to infection by cytomegalovirus (CMV) and other viruses

  • The overall expansion factor of CD3+ cells for Peripheral blood mononuclear cells (PBMC) cocultured with pulsed dendritic cells (DCs) was an average of 49.3 ± 48.6 while for PBMCs cocultured with non-pulsed DC, it was an average of 9.6 ± 6.3

  • Expansion protocols for the creation of virus-specific T lymphocytes (VST) third-party banks for adoptive immunotherapy have been under development in different centers as an effective and feasible therapy for immunocompromised patients with herpesvirus infections [1, 5, 28,29,30,31]

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Summary

Introduction

Immunodeficient patients are susceptible to infection by cytomegalovirus (CMV) and other viruses. Herpesviruses, such as CMV, are often asymptomatic or mild in healthy individuals. In the context of immunocompromised patients, viral infections can be a severe cause of morbidity and mortality [1, 2]. Antiviral pharmacologic agents are effective against only some of these viruses; their use is costly, associated with significant toxicities and does not provide long-term protection [4, 5]. Adoptive immunotherapy based on virus-specific T lymphocytes (VST) is an attractive option, as T lymphocytes can confer long-term protection against the development of viral disease [6,7,8]

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