EBV-specific T Cells for Therapeutic Use Demonstrate Differences in Phenotype, Transcriptional Profile and Function Depending Upon Manufacturing Process

Background & Aim Adoptive immunotherapy with Epstein-Barr virus (EBV)-specific T cells (VST) has proven effective for patients with refractory EBV-related malignancies. Two principal manufacturing approaches have been utilized to generate sufficient allogeneic T cells for therapy; sequential stimulation of donor PBMC with autologous EBV-positive lymphoblastic cell lines (LCLs), or in vitro expansion of interferon-gamma selected EBV-VST identified using EBV peptides. The Scottish National Blood Transfusion Service has a LCL-based EBV-VST bank which has been used to treat refractory PTLD patients for the last ten years on a Specials basis. This bank is being replaced by a next-generation bank based on EBV peptide selection and expansion in GMP-compliant closed-process manufacture. We have investigated the therapeutic material from both manufacturing processes and compared the phenotypic and functional characteristics of both to determine comparability. Methods, Results & Conclusion VST from both manufacturing processes were assessed for surface and intracellular markers by flow cytometry, which indicated that there were no significant differences in most parameters including CD3 content, CD4:CD8 ratio, and differentiation status. Peptide-generated products did demonstrate a significantly increased central memory compartment and mean IFNg expression. Further transcriptional analysis was undertaken using a custom TaqMAN low density array for relevant chemokine receptors, and indicated that EBV peptide-isolated VST had significantly increased levels of CCR7, CXCR4 and CXCR5, which correlate strongly with enhanced expansion and retention of central memory T cells. Transcriptional and phenotypic results were correlated with functional cytokine and migration assay to confirm responses. LCL-generated VST products showed significantly increased levels of CD38, indicating an accumulation of terminally differentiated functional T cells with reduced proliferative potential. The results from this study indicate that both EBV-VST manufacturing methods generate effective functional cells, but peptide-based VST demonstrate enhanced central memory cell retention which may predicate towards improved persistence in PTLD recipients. Adoptive immunotherapy with Epstein-Barr virus (EBV)-specific T cells (VST) has proven effective for patients with refractory EBV-related malignancies. Two principal manufacturing approaches have been utilized to generate sufficient allogeneic T cells for therapy; sequential stimulation of donor PBMC with autologous EBV-positive lymphoblastic cell lines (LCLs), or in vitro expansion of interferon-gamma selected EBV-VST identified using EBV peptides. The Scottish National Blood Transfusion Service has a LCL-based EBV-VST bank which has been used to treat refractory PTLD patients for the last ten years on a Specials basis. This bank is being replaced by a next-generation bank based on EBV peptide selection and expansion in GMP-compliant closed-process manufacture. We have investigated the therapeutic material from both manufacturing processes and compared the phenotypic and functional characteristics of both to determine comparability. VST from both manufacturing processes were assessed for surface and intracellular markers by flow cytometry, which indicated that there were no significant differences in most parameters including CD3 content, CD4:CD8 ratio, and differentiation status. Peptide-generated products did demonstrate a significantly increased central memory compartment and mean IFNg expression. Further transcriptional analysis was undertaken using a custom TaqMAN low density array for relevant chemokine receptors, and indicated that EBV peptide-isolated VST had significantly increased levels of CCR7, CXCR4 and CXCR5, which correlate strongly with enhanced expansion and retention of central memory T cells. Transcriptional and phenotypic results were correlated with functional cytokine and migration assay to confirm responses. LCL-generated VST products showed significantly increased levels of CD38, indicating an accumulation of terminally differentiated functional T cells with reduced proliferative potential. The results from this study indicate that both EBV-VST manufacturing methods generate effective functional cells, but peptide-based VST demonstrate enhanced central memory cell retention which may predicate towards improved persistence in PTLD recipients.