ALBERT

Description: Abstract 2437 Poster Board II-414 Introduction: BK virus (BKV) is a polyomavirus that is ubiquitous in humans, infecting over 85% of normal individuals. After initial infection it persists in a latent state in the urothelium from whence it can reactivate causing disease in immunocompromised patients. BKV is a cause of haemorrhagic cystitis after allogeneic SCT and is emerging as one of the major causes of graft loss after renal transplant. Current treatment is limited to reduction of immunosuppression as possible. Aim: To develop a method for production of a T cell product with BKV specificity from normal donors for use in adoptive immunotherapy post hemopoietic stem cell transplantation. Methods: Peripheral blood mononuclear cells (PBMC) or monocyte derived dendritic cells (mo-DC) were pulsed with mixes of overlapping peptides covering the 5 BKV proteins (VP1, VP2, VP2 isoform 3, large T antigen (LTA) and small T antigen (sTA)). Mo-DC were produced by isolating monocytes by adherence to plastic and culturing for 7 days in GM-CSF and IL-4 containing media. On day 6 mo-DC were matured by the addition of TNF. T cells were stimulated on day 1 and 7 with peptide pulsed PBMC or mo-DC and cultured for 21 days with increasing doses of IL-2 from day 7. The cellular product was then analysed for phenotype, BKV specificity and functionality by examining cytokine production and cytotoxicity. Results: Cellular proliferation was seen in all of 10 normal donors with a mean increase of 6.4 fold in total cell number. All cellular products were 〉84% CD3 positive (Mean 96%, SEM 1.3) with CD4 and CD8 ratio varying significantly between individual donors (CD4 range 9.7 to 97.5%, mean 70.79; CD8 range 0.8 to 77.0%, mean 23.3). Cells were of memory phenotype, being CD28+ (mean 86.1%, SEM 6.5), CD45RO+ (mean 84.1%, SEM 5.9) and a variable proportion were of central memory phenotype (CD62L+ mean 21.6%, range 6.9 to 55.0). Cytokine responses to stimulation with BKV peptides could be elicited in 5 of 6 evaluable donors. Multiple cytokines were produced by the responding cells: IFN-γ (mean 29.9% of CD3 cells, range 4.5 to 78.8), TNF (mean 19.9%, range 2.7 to 63) and IL-2 (mean 12.8%, range 1.2 to 37.8). Cytokine responses were seen in both CD4 and CD8 cells and showed significant individual variation. VP1 and LTA specific cells dominated most cultures while a smaller percentage of the cells were specific for VP2, VP2 isoform 3 and sTA. CD8 specificity was mainly confined to a single protein whereas CD4 responses tended to be of lower magnitude but broader specificity. Cultures exhibited cytotoxic activity with the lysis of BKV antigen coated target cells in a pattern that correlated with the presence of CD8 positive cytokine producing cells (up to 78.9% specific lysis at effector to target ratio of 20:1). Discussion: The clinical utility of this product remains to be determined. Potential uses include prophylaxis and therapy of reactivation of BK virus after hemopoietic stem cell or renal transplantation. This method for large-scale expansion of BKV specific CTL could be utilised for analysis of BKV targeted immune responses and epitope identification. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Cytomegalovirus (CMV) reactivation post allogeneic haemopoietic stem cell transplant (HSCT) causes significant morbidity. Adoptive transfer of ex-vivo generated CMV specific T cells has the potential to restore immunity, prevent CMV reactivation and circumvent the need for pharmacotherapy. Donor derived CMV specific T cells were generated for prophylactic infusion into haemopoietic stem cell transplant recipients as part of a phase I/II clinical trial aiming to reduce the incidence of CMV reactivation. T cells were expanded by co-culturing with dendritic cells transfected with Ad5F35pp65, a recombinant adenovirus promoting the presentation of epitopes derived from the immunodominant CMV antigen pp65. The aim of this study was to determine if ex vivo expanded CMV specific T cells have the capacity to produce different cytokines and chemokines associated with protective immunity. Results: Ex vivo expanded Ad5F35pp65 stimulated cultures were primarily CD3+ T cells (median 92%) with a predominance of CD8+ (14–90%) over CD4+ (2.5–57%) cells. An assay measuring antigen specific production of interferon-γ (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor (TNF) and macrophage inflammatory protein 1β (MIP-1β) by intracellular cytokine flow cytometry was established to quantify the frequency of T cells with specificity towards CMV or adenovirus and to assess the quality of responses reflected by the simultaneous production of multiple cytokines. Ad5F35pp65 stimulated cultures were greatly enriched for CMV specific T cells producing cytokines in response to pp65 (mean 59%, 19.1–90%) compared to the starting PBMC population (0.5–1.5%). Responses directed towards the adenovirus hexon protein were also detected accounting for 0.65 to 9% of T cells. CMV specific CD8 T cells predominantly produced IFN-γ and MIP-1β followed by TNF with means of 61.3%, 59% and 44% respectively. The majority of CMV specific CD8+ T cells produced both IFN-γ and MIP-1β (80–96%) with a substantial proportion of these also producing TNF (72%, 44–88.5%). IL-2 producing CD8+ T cells were less frequent, ranging from 0.5–40%. However, IL-2 producers consistently exhibited the highest level of functionality with the production of all four cytokines. Furthermore, IFN-γ producing CD8+ T cells mobilized CD107, a marker of degranulation and cytotoxic activity. In the majority of cases, fewer CD4+ T cells exhibited specificity towards CMV pp65 (30%, 4–49.5%) however greater than 80% co-produced IFN-γ, MIP-1β and TNF. Furthermore, a high proportion of CD4+ T cells also produced IL-2 (53.4%). Adenovirus specific T cell responses were detected in all cultures but were mainly confined to the CD4+ population. Finally, we examined CMV specific responses in the starting donor PBMC population. Cytokine production profiles of CMV specific CD4 and CD8 T cells closely resembled those of ex vivo generated cultures suggesting the uniform expansion of CMV specific functional subsets. In conclusion, these results demonstrate that ex vivo expanded CMV specific T cells intended for adoptive transfer perform several functions associated with protective immunity in vivo, including the capacity to kill infected cells and the simultaneous production of multiple cytokines.

Description: Background: Cytomegalovirus (CMV) reactivation post-allogeneic haemopoietic stem cell transplant (HSCT) causes morbidity and mortality. Pharmacological therapy is limited by bone marrow suppression that can result in opportunistic infection. Adoptive transfer of ex-vivo generated CMV-specific T-cells has the potential to rapidly restore immunity, prevent CMV infection and circumvent the need for pharmacotherapy. Methods: We have recruited 18 patients into a trial of donor-derived CMV-specific T-cells generated using dendritic cells transduced with a fiber modified adenoviral vector encoding the immunodominant CMV matrix protein pp65. Thus far, 8 patients have received T-cells prophylactically starting at day 28 post-HSCT. Recipients were monitored for adverse reactions, CMV reactivation by polymerase chain reaction (PCR) and CMV-specific immune reconstitution. Results: 18 cultures have been completed with an 8.5–30 fold increase in total cell number (mean 2.1 × 108, range 0.8–5.1 × 108). Products consisted of T-cells (median 96.5%) displaying an effector memory phenotype (CD45RO+, CD62L−) and a predominance of CD8+ (14–90%) over CD4+ (2.5–57%) cells. All cultures exhibited negligible alloreactivity against recipient derived targets (0–3.6% specific lysis at E:T ratio of 20:1) but strong killing of CMV pp65 peptide pulsed targets (43–79% specific lysis at E:T 20:1). Killing was also observed against targets labeled with adenoviral antigens (2.7–12.5% lysis at E:T 20:1) demonstrating the bi-virus specificity of these cultures. In HLA-B7 and HLA-A2 donors, percentages of tetramer binding T-cells accounted for 3.2 to 40% of cells. Enrichment of cells recognizing known HLA-A24, B8 and B35 epitopes was not observed despite these cultures exhibiting strong cytotoxic activity against pp65 pulsed targets. Reasons for non-infusion include early death post-transplant (3 patients), failure of cultures to meet release criteria (2 patients), severe graft versus host disease (1 patient) and refusal (1 patient). 3 patients are currently awaiting infusion. Patients infused have been followed from 21 to 189 days post infusion. In 5 patients we have demonstrated rapid, polyepitope, CMV pp65-specific immune reconstitution using tetramer and ELISPOT analysis. CMV reactivation by PCR has occurred in 3 patients but none have progressed to CMV disease, suggesting the functional capacity of the cells to control viral reactivation. One patient received pharmacotherapy with valaciclovir while no other patients have required anti-viral antibiotics. There have been no infusion related adverse reactions. Graft versus host disease, non-CMV infections and other adverse events have not exceeded expected rates. Conclusions: Prophylactic adoptive transfer of CMV-specific T-cells is safe, hastens CMV-specific immune reconstitution and may reduce the need for anti-CMV pharmacotherapy in allogeneic HSCT recipients. Our data indicate the potential of specific cellular therapy to control opportunistic infections in severely immunosuppressed patients post-HSCT.

Description: Abstract 2435 Poster Board II-412 Introduction: Uncontrolled EBV replication can lead to life threatening post-transplant lymphoproliferative disorder (PTLD). The incidence of EBV reactivation and PTLD is proportional to the degree of cellular immunosuppression. Treatment with EBV specific cytotoxic T lymphocytes (CTL) effectively controls EBV replication but the generation of CTL using autologous EBV-transformed B cells as stimulators is cumbersome and not compliant with more strict regulatory requirements. Aim: To develop a rapid and reliable method for production of a clinical grade EBV specific T cell product using an adenoviral vector encoding genes for EBNA-1 and immunogenic LMP peptides. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood by Ficoll-paque centrifugation. Monocyte derived dendritic cells (mo-DC) were generated from PBMC by adherence to plastic and exposure to IL-4 and GM-CSF. On day 6 mo-DC were transfected with a clinical grade adenoviral vector encoding EBNA-1 and HLA Class I epitopes of LMP-1 and LMP-2a and matured with the addition of TNF. Transfected PBMC or mo-DC were used to stimulate T cells from the same donor and cultures were continued for 21 days from the first stimulation with a second stimulation on day 7 and increasing doses of IL-2 thereafter. The cellular product was analysed on day 21 for phenotype, antigen specificity and functional capacity by tetramer staining, cytokine production in response to antigen stimulation and cytotoxic activity against antigen coated targets. Results: Cellular proliferation was seen in all of 11 EBV seropositive normal donors with a mean fold increase in total cell number of 5.41 (SEM 1.3). In all donors, the cellular product was CD3 positive (mean 94.7%, range 81 to 99.4) with both CD4 (mean 65.7% of CD3, range 17.0 to 93.2) and CD8 cells present (mean 29.6%, range 4.4 to 74.4). Cells were of memory phenotype, being CD28+ (mean 91%, SEM 3.1), CD45RO+ (mean 89.4%, SEM 3.0) and a variable proportion were of central memory phenotype (CD62L+ mean 35.9%, range 10.6 to 73.3). EBV specificity was demonstrated by tetramer staining or intracellular cytokine detection in 7 of 10 donors. Tetramer analysis of 6 HLA-A2 positive donors showed up to 2498-fold expansion of LMP-2a tetramer specific CD8 cells compared to baseline (for epitope CLGGLLTMV, mean 469-fold increase, range 5.2 to 2498; for epitope FLYALALLL, mean 186-fold increase, range 2.1 to 601). Cytokine responses to stimulation with EBNA, LMP and adenoviral peptides were present in both CD4 and CD8 cells and showed significant individual variation. Cytotoxic activity was seen with up to 75% specific lysis of EBV antigen coated targets at an effector to target ratio of 20:1. Conclusion: The use of an adenoviral vector containing genes encoding EBNA-1 and LMP antigens allows the rapid generation of a clinical grade EBV-specific T cell product from the majority of EBV seropositive normal donors. This technique will permit the incorporation of adoptive immunotherapy for EBV into routine haemopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Description: Cytomegalovirus (CMV) and its therapy continue to contribute to morbidity and mortality in hemopoietic stem cell transplantation (HSCT). Many studies have demonstrated the feasibility of in vitro generation of CMV-specific T cells for adoptive immunotherapy of CMV. Few clinical trials have been performed showing the safety and efficacy of this approach in vivo. In this study, donor-derived, CMV-specific T cells were generated for 12 adult HSCT patients by stimulation with dendritic cells transduced with an adenoviral vector encoding the CMV-pp65 protein. Patients received a prophylactic infusion of T cells after day 28 after HSCT. There were no infusion related adverse events. CMV DNAemia was detected in 4 patients after infusion but was of low level. No patient required CMV-specific pharmacotherapy. Immune reconstitution to CMV was demonstrated by enzyme linked immunospot assay in all recipients with rapid increases in predominantly CMV-pp65 directed immunity in 5. Rates of graft-versus-host disease, infection, and death were not increased compared with expected. These results add to the growing evidence of the safety and efficacy of immunotherapy of CMV in HSCT, supporting its more widespread use. This study was registered at www.anzctr.org.au as #ACTRN12605000213640.