The Clinical Use of Donor-Derived Virus-Specific Cytotoxic T Lymphocytes Reactive Against Cytomegalovirus (CMV), Adenovirus and Epstein Barr Virus (EBV).
Authors: Myers Doug , Leen Ann M. , Sili Ulahan , Huls Mary H. , Buza Elizabeth , Leung Kathy , Carrum George , Krance Robert A. , Brenner Malcolm K. , Rooney Cliona M. , Heslop Helen E. , Bollard Catherine M. 
Published: April 2020

Abstract
CMV, Adenovirus and EBV are major viral pathogens causing morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation. Previous studies have shown that prophylactic adoptive immunotherapy with donor-derived Cytotoxic T Lymphocytes (CTL) directed against EBV and CMV can effectively prevent the clinical manifestations of these viruses. We have extended these studies by generating CTL from normal donor PBMC that can restore cellular immunity to CMV, EBV and adenovirus simultaneously. We have developed a protocol utilizing an initial round of stimulation with autologous mononuclear cells transduced with a recombinant adenovirus type 5 vector pseudotyped with a type 35 fiber carrying a transgene for the immunodominant CMV antigen, pp65 (Ad5f35pp65). This is followed by 2 rounds of weekly stimulation with autologous EBV-lymphoblastoid cell lines (LCL) transduced with the same vector using MOIs of 10 and 100 respectively. After 3 rounds of stimulation, 9 CTL cultures contained a mean of 83% (range 8.4–98.99%) CD8+ve and a mean of 19.6% (range 2.2–91.6%) CD4+ve cells. In chromium release and/or IFNg ELISPOT assays, all CTL lines showed specific activity against CMV and EBV targets. 8/9 lines also showed specific lysis against adenovirus targets. Further, using MHC-peptide multimers we have been able to demonstrate the simultaneous presence of CD8+ve cells recognizing peptide epitopes from CMV pp65 (range 2.32–21%) and Adenovirus hexon (1.07–8.08%) in the CTL cultures. So far we have treated 6 patients in this phase I CMV prophylaxis study, 3 on dose level 1 (1x10e7/m2) and 3 on dose level 2 (5x10e7/m2). Patients received 1 infusion of virus-specific CTL from 54–120 days post transplant. We observed up to a 28-fold increase in CMV pentamer positive CD8+ve cells post CTL. At last follow-up (7–35 weeks post CTL infusion) all patients are CMV and EBV negative. 2 patients were transiently positive for CMV by PCR 4–9 weeks post CTL but both were negative 7 days later without anti-viral therapy, with a corresponding rise in CMV-specific CTL detected in the peripheral blood. 2 patients were culture positive for adenovirus in their stool pre CTL therapy. One of these patients was infected with adenovirus species from subgroups A, C and D. In both patients, we observed a 2-log reduction of adenovirus copies/g stool within 2–3 weeks post CTL infusion at which time their symptoms (fever, loose stools) resolved. In summary, we have developed a protocol for the efficient generation of multi-virus specific CTL: infusion of small numbers of these cells increased virus-specific CD8+ve T cells in the peripheral blood post CTL infusion. Further, reduction in adenovirus load in stool suggests efficacy of adenovirus specific CTL in vivo. However, expansion of virus-specific CTL in vivo may require presence of antigen. We will therefore complete this prophylaxis study and then proceed to using virus-specific CTL for the treatment of CMV and adenovirus disease post transplant.