Cytotoxic T Cells Specific for Adenovirus, BK Virus, Cytomegalovirus, Epstein Barr Virus and Varicella Zoster Virus Produced for Clinical Use In Immune Reconstitution Post Allogeneic Stem Cell Transplantation

Adoptive immunotherapy using donor-derived cellular effectors can restore immunity and prevent virus related disease after haemopoietic stem cell transplantation (HSCT) as shown by trials targeting EBV, CMV and adenovirus. It is therefore desirable to expand the repertoire of T cells for adoptive immunotherapy to include other viruses that cause disease in this patient group. We have developed a method for the production of a T cell product specific for multiple viruses that does not rely on EBV transformed cells for stimulation. In this study we assessed the efficacy and reproducibility of the method for generating antigen specific T cells with broad specificity.

Monocyte-derived dendritic cells (mo-DC) were used to present antigens from 5 viruses to T cells of normal donors. Presentation of CMV, EBV and human adenovirus (HadV) antigens was achieved by transfection with recombinant adenovirus vectors encoding the entire CMV pp65 gene (Ad5F35pp65) or epitopes derived from EBV proteins EBNA1, LMP1 and LMP2a (AdE1-LMPpoly). Additionally, mo-DC were infected with a clinically approved cell free vaccine preparation of the live attenuated Varicella Zoster virus (VZV) OKA strain or incubated with overlapping peptide mixes covering the BK virus (BKV) genes LTA, STA, VP1, VP2 and VP3.

Activated antigen-specific T cells were preferentially expanded with two dendritic cell stimulations on day one and seven with the addition of increasing doses of IL-2 from day 7 to day 21. Methods were initially established for stimulation with individual (VZV and BKV) or dual viruses (EBV/Hadv and CMV/Hadv). In subsequent cultures, responding cells were combined at various time-points to optimise the total number of cells in the final product. Cultures were assessed by cell count, viability and phenotype. Specificity for antigens was demonstrated with multimer staining where available (EBV and CMV) and by intracellular cytokine detection (IL-2, IFN-g and TNF) after restimulation with antigens. The quality of antigen specific T cells was assessed by analysis of the production of multiple cytokines.

The final multi-virus cell products from 7 cultures increased in cell number from baseline (mean increase in total cell number was 8-fold), were CD3 positive (mean 92.1%), with variation between individuals in CD4:CD8 ratio. Cells were CD45RO⁺ (mean 87.7%) and a variable number expressed CD62L⁺ (mean 27.12%, range 12.1 to 41.0%). The total percentage of T cells in the final product that produced cytokines in response to restimulation with the various antigens ranged from 2.6 to 52% (mean 14.63). This was composed of both CD4 (mean 12.9%, range 5.5 to 44.5%) and CD8 cells (mean 20.3%, range 0.9 to 61.5%). Fold increase in MHC multimer positive cells compared to baseline was as follows: CMVpp65 HLA-A2 NLV 1742-fold, CMVpp65 HLA-B7 TPR 1689-fold, EBV LMP2a HLA-A2 CLG 124-fold, EBV LMP2a HLA-A2 FLY 180-fold. The quality of T cell responses in combined cultures was commensurate with the responses of cultures of cells specific for individual viruses. T cells responding to VZV, CMV and BKV antigens consistently co-produced up to 3 cytokines, IFN-γ, TNF and IL-2 indicative of the multi-functional quality of the expanded T cell product.

To our knowledge, this is the first report of a methodology for production of T cells specific for 5 viruses for use in adoptive immunotherapy in HSCT. We have been able to generate cells with multiple TH1 cytokine functions in all of 7 cultures from 5 normal donors. There are several advantages to our methodology: it can be tailored to individual serostatus, it is not HLA-dependent, it is rapid and it potentially enhances the protection available to immunocompromised transplant recipients. The use of antigen and reagent sources that are consistent with Good Manufacturing Practice will allow for rapid clinical translation. Safety and efficacy data from upcoming clinical trial of this product are awaited.

No relevant conflicts of interest to declare.