Renee Simms

Donor-derived CMV specific T-cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation

We investigated the use of adoptively transferred donor-derived cytomegalovirus (CMV) specific cytotoxic T lymphocytes (CTL) as immune reconstitution postallogeneic transplant in a phase 2 study. Fifty patients were infused with a single dose of 2 × 10(7)cells/m(2) after day 28 post-transplant. Twenty-six patients reactivated CMV posttransplant (only 5 post-CTL infusion) and 9 required therapy with ganciclovir or foscarnet (only 1 post-CTL infusion). There was 1 case of fatal CMV disease, attributable to high levels of antithymocyte globulin at the time of T cell infusion. We compared the patients in the phase 2 study with a group of contemporaneous controls also treated at the trial centers. There was no increase in acute or chronic graft-versus-host disease attributable to CTL infusion; overall and progression-free survival were similar in both groups. There was a reduction in the percentage of patients who required CMV directed antiviral therapy (17% vs 36%, P = .01) and in the total number of treatment days in the cohort receiving CTL (3.4 days vs 8.9 days, P = .03) without a reduction in CMV reactivation rates. We postulate that adoptively transferred cells are able to expand in response to viral antigen, limit viral replication, and prevent progression to tissue infection. This study was registered on the Australian Clinical Trial Registry as #ACTRN12605000213640 and #ACTRN12607000224426.

BK virus-specific T cells for use in cellular therapy show specificity to multiple antigens and polyfunctional cytokine responses.

Background. BK virus (BKV) infection causes hemorrhagic cystitis posthemopoietic stem-cell transplant and graft loss in renal transplant recipients. Reactivation occurs in up to 60% of patients in both groups. Treatment-related cellular immunosuppression is a major contributor to the development of BKV-related disease, but the targets of the immune response are not well characterized. Immunotherapy by adoptive transfer of cellular effectors has been shown to be effective in controlling and preventing some virus-related diseases in transplant recipients, particularly Epstein-Barr virus and cytomegalovirus. Infusion of BKV-specific T cells may potentially reconstitute functional BKV immunity and reduce clinical complications of BKV infection. Methods. BKV-specific T cells for clinical use in adoptive immunotherapy were generated using monocyte-derived dendritic cells pulsed with overlapping peptide mixes spanning the five BKV proteins VP1, VP2, VP3, large T antigen, and small T antigen. Phenotypic and functional characteristics of the cells were investigated as well as their antigen specificity. Results. Expanded CD4+ and CD8+ cells responded to restimulation with BKV peptides principally from VP1, large T, or small T antigens; produced multiple cytokines; and showed cytotoxic activity against antigen-coated targets. Conclusions. Possible clinical uses for BKV-specific T cells generated using this method include immune reconstitution posthemopoietic stem-cell transplantation or prophylaxis and treatment of immune deficiency in renal transplant recipients, fulfilling the need for effective therapy for BKV-related hemorrhagic cystitis and renal dysfunction.

Clinical-grade varicella zoster virus-specific T cells produced for adoptive immunotherapy in hemopoietic stem cell transplant recipients

BACKGROUND AIMS Varicella zoster virus (VZV) causes life-long latent infection in healthy individuals, which reactivates in 10-68% of stem cell transplant patients. Reconstituting immunity through adoptive transfer of T cells specific for VZV may aid in the prophylaxis and treatment of VZV infections. The potential for generating T cells specific for VZV using a clinically approved VZV vaccine strain was investigated. METHODS The Varivax® vaccine was used to stimulate peripheral blood mononuclear cells from healthy donors. Only reagents approved for clinical manufacture were used. Monocyte-derived dendritic cells pulsed with Varivax (R) were used to stimulate autologous mononuclear cells at a responder to stimulator ratio of 10:1. On day 7, a second stimulation was performed; 20 U/mL interleukin (IL)-2 were added from day 7 and 50 U/mL IL-2 from day 14 onwards. Cell phenotype and functionality were assessed after 21 days of culture. RESULTS A mean increase of 11-fold in cell number was observed (n= 18). Cultures were mainly T cells (mean CD3 (+) 89.7%, CD4 (+) 54.2%, CD8 (+) 28.7%) with effector and central memory phenotypes. Cells produced one or more T helper (Th)1 cytokine (interferon-γ, tumor necrosis factor-α and IL-2), and CD4 (+) (but not CD8 (+) ) cells expressed the cytoxicity marker CD107 when restimulated with VZV antigens. CONCLUSIONS We have demonstrated a clinically applicable method that yields high numbers of highly reactive T cells specific for VZV. We propose that reconstructing host immunity through adoptive transfer of VZV-specific T cells will reduce the frequency of clinical VZV infection in the period of severe immune suppression that follows allogeneic stem cell transplantation.

In vitro generation of influenza-specific polyfunctional CD4+ T cells suitable for adoptive immunotherapy.

BACKGROUND AIMS Influenza viruses cause potentially fatal respiratory infections in stem cell transplant patients. Specific T cells provide long-lived host adaptive immunity to influenza viruses, and the potential for generating such cells for clinical use was investigated. METHODS The inactivated influenza vaccine (Fluvax) approved for human use was used as the antigen source. Monocyte-derived dendritic cells pulsed with Fluvax were used to stimulate autologous peripheral blood mononuclear cells (PBMC) on days 0 and 7. Cells were expanded with interleukin (IL)-2 from day 7 onwards. Cell numbers and phenotype were assessed on day 21. The presence of influenza virus-specific cells was assessed by cytokine production and proliferative responses following restimulation with influenza antigens. RESULTS Over 21 days of culture, a mean fold increase of 26.3 in cell number was observed (n = 7). Cultures were predominantly effector and central memory CD4+ cells, and expressed a phenotype characteristic of activated antigen-specific cells capable of B-cell helper function. Cytotoxic CD4+ and CD8+ cells specific for influenza and a high percentage of CD4+ cells specific for each of three influenza viruses targeted by Fluvax (H1N1, H3N2 and Brisbane viruses) were generated. In addition, T cells expanded when restimulated with antigens derived from influenza viruses. CONCLUSIONS We have demonstrated a clinically usable method for producing influenza virus-specific T cells that yield high numbers of highly reactive CD4+ cells suitable for adoptive immunotherapy. We propose that reconstructing host immunity through adoptive transfer of influenza virus-specific T cells will reduce the frequency of influenza-related deaths in the period of severe immune suppression that follows stem cell transplantation.

Long-term control of recurrent or refractory viral infections after allogeneic HSCT with third-party virus-specific T cells

Donor-derived adoptive T-cell therapy is a safe and effective treatment of viral infection posttransplant, but it is limited by donor serostatus and availability and by its personalized nature. Off-the-shelf, third-party virus-specific T cells (VSTs) appear promising, but the long-term safety and durability of responses have yet to be established. We conducted a prospective study of 30 allogeneic hemopoietic stem cell transplant (HSCT) patients with persistent or recurrent cytomegalovirus (CMV) (n = 28), Epstein-Barr virus (n = 1), or adenovirus (n = 1) after standard therapy. Patients were treated with infusions of partially HLA-matched, third-party, ex vivo-expanded VSTs (total = 50 infusions) at a median of 75 days post-HSCT (range, 37 to 349 days). Safety, viral dynamics, and immune recovery were monitored for 12 months. Infusions were safe and well tolerated. Acute graft versus host disease occurred in 2 patients, despite a median HLA match between VSTs and the recipient of 2 of 6 antigens. At 12 months, the cumulative incidence of overall response was 93%. Virological control was durable in the majority of patients; the reintroduction of antiviral therapy after the final infusion occurred in 5 patients. CMV-specific T-cell immunity rose significantly and coincided with a rise in CD8+ terminal effector cells. PD-1 expression was elevated on CD8+ lymphocytes before the administration of third-party T cells and remained elevated at the time of viral control. Third-party VSTs show prolonged benefit, with virological control achieved in association with the recovery of CD8+ effector T cells possibly facilitated by VST infusion. This trial was registered at www.clinicaltrials.gov as #NCT02779439 and www.anzctr.org.au as #ACTRN12613000603718.

Adjuvant Peptide Pulsed Dendritic Cell Vaccination in Addition to T Cell Adoptive Immunotherapy for Cytomegalovirus Infection in Allogeneic Hematopoietic Stem Cell Transplantation Recipients

Adoptive cellular immunotherapy has been shown to be effective in the management of cytomegalovirus (CMV) reactivation and disease. Whether adjuvant dendritic cell (DC) vaccination will provide additional benefit in prophylaxis or treatment of CMV in hematoietic cell transplantation (HSCT) recipients is unknown. In this study, we administered prophylactic CMV-peptide specific T cell infusions, followed by 2 doses of intradermal CMV peptide-pulsed DC vaccine, to 4 HSCT recipients. There were no immediate adverse events associated with T cell infusion or DC vaccinations. One of the 4 patients developed grade III acute gut graft-versus-host disease. Immune reconstitution against CMV was detected in all 4 patients. Patients receiving CMV peptide-specific T cells and DC vaccination had peak immune reconstitution at least 10 days after the second DC vaccination. In summary, combining DC vaccine with T cell infusion appears feasible, although further study is required to ascertain its safety and efficacy in augmenting the effects of infusing donor-derived CMV-specific T cells.

Establishment and Operation of a Third-Party Virus-Specific T Cell Bank within an Allogeneic Stem Cell Transplant Program

Hematopoietic stem cell transplantation (HSCT) donor-generated virus-specific T cells (VSTs) can provide effective treatment for viral infection post-HSCT but are not readily accessible to all patients. Off-the-shelf cryopreserved VSTs suitable for treatment of multiple patients are an attractive alternative. We generated a bank of 17 cytomegalovirus (CMV)-, 14 Epstein-Barr virus (EBV)-, and 15 adenovirus (AdV)-specific T cell products from 30 third-party donors. Donors were selected for expression of 6 core HLA antigens expressed at high frequency in the local transplant population. T cells were generated by co-culturing venous blood or mobilized hematopoietic stem cell (HSC)-derived mononuclear cells with monocyte-derived dendritic cells pulsed with overlapping peptides covering CMV pp65, AdV5 hexon, or EBV BZLF1/LMP2A/EBNA1 proteins. Addition of a CD14+ selection step instead of plate adherence to isolate monocytes before culture initiation significantly improved expansion in cultures from HSC material. Phenotyping showed the CD8+ subset to have significantly higher numbers of terminal effector T cells (CD45RA+62L-) and lower numbers of effector memory T cells (CD45RA-62L-) when compared with the CD4+ subset. Increased expression of the immunoinhibitory markers PD-1 and TIM-3 was noted on CD4+ but not CD8+ cells when compared with the control group. VST showed antiviral activity restricted through a variety of common HLAs, and modelling suggested a suitably HLA-matched product would be available for >90% of HSCT patients. Only a small number of carefully selected third-party donors are required to generate a VST bank of broad coverage, indicating the feasibility of local banking integrated into existing allogeneic HSCT programs.

449. Multipathogen-Specific T Cells for Immune Reconstitution – A Decade of Manufacturing Development and Clinical Use

Since 2003 we have treated 101 patients with antigen specific T cells for treatment or prevention of opportunistic infections in the context of haemopoietic progenitor cell (HPC) and solid organ transplant. Over this time, we have modified laboratory procedures to progressively incorporate T cell therapy into standard transplant practice. We have increased the number of pathogen targets, utilised HPC products and streamlined culture techniques. We are now able to produce a multipathogen product within 3 weeks of HPC harvest in a manner consistent with good manufacturing practice and within the scope of systems established for HPC processing. Our approach could be used by most accredited transplant laboratories.We have developed robust, simple and inexpensive methods that use marrow or G-CSF mobilised HPC products as starting material for T cell expansion. This has major practical importance in minimising repeated donor screening, blood draws and apheresis. Our current procedure uses monocyte derived dendritic cells (mo-DC) isolated using a modified CD14 selection method that requires minimal quantities of CD14 selection reagent. Maturation of mo-DCs utilises GM-CSF, IL-4, PG-E2, IL-6 and IL-1β. Multiple antigens are presented to T cells by mo-DCs including peptide mixes for cytomegalovirus (CMV), Epstein Barr virus (EBV), BK virus and adenovirus, vaccines (Varicella Zoster and Influenza virus) and mycelial lysate (Aspergillus). Activated antigen specific T cells are expanded using IL-2. We use no T cell selection/depletion procedures, feeder layers, cytokine, tetramer capture or clinical grade sorting methodologies. The overall cost of manufacture of each product, including reagents, staffing and ongoing infrastructure costs is approximately AU

Cytomegalovirus-Specific Cytotoxic T Lymphocytes Can Be Efficiently Expanded from Granulocyte Colony-Stimulating Factor–Mobilized Hemopoietic Progenitor Cell Products Ex Vivo and Safely Transferred to Stem Cell Transplantation Recipients to Facilitate Immune Reconstitution

2400 per dose.The phenotypic composition of multipathogen products is mainly CD3 (mean 97%, range 90-97), with variable CD4:8 ratio, minimal contaminating B cells (mean 0.1%), monocytes (mean 0.3%) and NK cells (mean 3%). We have demonstrated antigen specificity using intracellular cytokine staining, MHC multimer analysis (CMV and EBV), interferon-γ Elispot and chromium (Cr51) release cytotoxicity assay. Products do not show alloreactivity by Cr51 release assay (Mean 0.8% specific lysis at E:T ratio of 20:1, range 0-5.3).Clinical results of the first 50 patients treated with CMV specific T cells have previously been reported and showed infusion to be safe and to reduce the need for CMV-directed antiviral therapy. Clinical outcomes of the remaining patients treated with multi-pathogen T cells specific for up to 8 pathogens will be reported when data is mature.Future areas of development will involve extension of the range of infectious pathogens targeted, addition of cells targeting malignant antigens and combination of these therapies with CD34+ stem cell selection. Developing progressively greater selectivity through graft engineering and immunotherapy will improve outcomes of transplantation in the future.