1074. The Use of Genetically Modified Antigen Presenting Cells to Generate Donor-Derived Virus-Specific Cytotoxic T Lymphocytes Reactive Against CMV and Adenoviral Antigens for Clinical Use
Authors: Gary D. Myers, Ann Leen, Helen Huls, Elizabeth Buza, Jeff Chang, Robert A. Krance, George Carrum, Malcolm K. Brenner, Cliona M. Rooney, Helen E. Heslop, Catherine M. Bollard
Journal: Molecular Therapy (2005)

CMV and Adenovirus 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 PBMC from CMV seropositive normal donors that should restore cellular immunity to both CMV and adenovirus simultaneously using gene transfer to modify antigen-presenting cells. We have generated a clinical grade recombinant adenovirus type 5 vector pseudotyped with a type 35 fiber (Ad5f35pp65) carrying a transgene for the immunodominant CMV antigen, pp65. We have developed a protocol utilizing an initial round of stimulation with autologous mononuclear cells transduced with Ad5f35pp65, 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, four CTL cultures contained a mean of 96% (range 93–99%) CD8+ve and a mean of 4% (range 1.3–7%) CD4+ve cells. All CTL lines showed significant cytotoxicity in chromium release assays against CMV targets and 3/4 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 3 patients in this phase I CMV prophylaxis study. All patients received 1 infusion of virus-specific CTL at a dose of 110e7/m2 from day 30 post transplant. We observed up to a 28-fold increase in CMV pentamer positive CD8+ve T cells post CTL. At last follow-up (2–10 weeks) all patients remain CMV negative. One patient was culture positive for adenovirus in her stool pre CTL therapy. Within 2 weeks of CTL infusion we observed a 2-log reduction of adenovirus copies/gram stool at which time the patient's symptoms (fever, loose stools) resolved. In summary, we have developed a protocol for the efficient generation using genetically modified monocytes and LCL to expand CTL ex vivo with specificity for CMV and adenovirus. CMV-pentamer specific CD8+ve T cells increase in the peripheral blood post CTL infusion despite infusing relatively low numbers of cells. 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 infections post transplant.