A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.
Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2×105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture – total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by >90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.
Viral infections account for substantial morbidity and mortality in patients who are immunocompromised by their disease or its treatment. After HSCT, for example, infections caused by persistent herpesviruses such as EBV and CMV, as well as by respiratory viruses such as Respiratory Syncytial Virus (RSV), are well known, while the importance of infections caused by Adv, BK virus, and human herpesvirus (HHV)-6 have more recently been appreciated. While pharmacological agents are standard therapy for some infections, they have substantial toxicities, generate resistant variants, and are frequently ineffective. In contrast, virus-specific T cells derived from stem cell donors have proven safe and effective for the prevention and treatment of viral infection or disease in the hemopoietic stem cell transplant (HSCT) setting2,5,6,18-21. However, the broader implementation of T cell immunotherapy is ultimately limited by the cost, complexity and time required for CTL production.
Our novel and rapid approach to generate multivirus CTL, described in the current manuscript, should substantially increase the feasibility of cytotoxic T cell therapy for viral diseases, enabling the strategy to become a standard of care for the immunocompromised host. The use of plasmid nucleofected DCs as APCs enables antigen presentation on both MHC class I and II without competition from viral vectors or indeed from multiple viral antigens being expressed within a single cell since different DC populations are utilized for each plasmid3. The use of IL-4/7 increases T cell survival and proliferation, which correspondingly helps increase the frequency and repertoire of responding antigen-specific T cells16,17. Finally, culture in the G-Rex dramatically reduces T cell apoptosis during culture. Gas exchange (O2 in and CO2 out) occurs across a gas permeable silicon membrane at the base of the flask, preventing hypoxia while allowing a greater depth of medium above the cells, providing more nutrients and diluting waste products. This platform can also be extended to additional viruses as when protective antigens are identified.
The authors have nothing to disclose.
This work is supported by a Production Assistance for Cellular Therapies (contract NIH-NHLBI (HB-10-03) HHSN26820100000C) (C.M.R.), a Specialized Centers for Cell-based Therapy Grant NIH-NHLBI 1 U54 HL081007 (C.M.R.), an ASBMT Young Investigator Award (U.G. and J.V.), a Leukemia and Lymphoma Society Special Fellow in Clinical Research Award (U.G.), and an Amy Strelzer Manasevit Scholar Award (A.M.L).
Name of the reagent | Company | Catalogue number |
---|---|---|
CellGenix | CellGenix | 2005 |
IL4 | R+D Systems | 204-IL/CF |
IL7 | Peprotech | 200-15 |
Hyclone RPMI 1640 | Thermo Scientific | SH30096.01 |
Human AB serum | Valley Biomedical Inc. | HP1022 |
Nucleofector | Amaxa/Lonza | AAF-1001B & AAF-1001X |
Nucleofection Kit | Amaxa/Lonza | V4XP-3012 |
Plasmids | NTC | n/a |
GM-CSF | R&D | 215-GM/CF |
IL1 | R&D | 201-LB-025 |
IL6 | R&D | 206-IL-CF |
TNFα | R&D | 210-TA-010 |
PGE2 | Sigma | P6532-1MG |
G-Rex | Wilson Wolf Manufacturing | AY11-00027 |