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We designed a trial to simultaneously address the problems of graft versus host disease (GVHD), infection, and recurrence of malignancy after allogeneic stem cell transplantation. CD34

Virus-specific T-cells (VSTs) from third-party donors mediate short- and long-term antiviral effects in allogeneic hematopoietic stem cell transplant (HSCT) recipients with relapsed or refractory viral infections. We investigated early administration of third-party VSTs, together with antiviral therapy in patients requiring treatment for first cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. Thirty HSCT patients were treated with 1 to 4 VST infusions (2 × 107 cells/m2; CMV n=27, EBV n=3) at a median of 4 days after initiation of antiviral treatment. The overall viral response rate was 100%, with a complete response (CR) rate of 94%. Of the 28 patients who achieved a CR, 23 remained virus PCR negative (n=9) or below quantitation limit (n=14) for the duration of follow-up. Four patients had brief episodes of quantifiable reactivation not requiring additional therapy, and one required a second infusion after initial CR, remaining PCR negative thereafter. All 3 patients treated for EBV post-transplant lymphoproliferative disorder achieved sustained CR. Rates of aGVHD and cGVHD after infusion were 13% and 23%, respectively. There were no serious infusion-related adverse events. VST infusion was associated with rapid recovery of CD8+CD45RA−CD62L− and a slower recovery of CD4+CD45RA−CD62L− effector memory T-cells; CMV-specific T-cells comprised up to 13% of CD8+ cells. At 1 year post-transplant, non-relapse mortality was 10%, cumulative incidence of relapse was 7%, overall survival was 88% and 25 of 27 patients had ECOG status of 0 or 1. Early administration of third-party VSTs in conjunction with antiviral treatment appears safe and leads to excellent viral control and clinical outcomes. Registered on Australian New Zealand Clinical Trials Registry as #ACTRN12618000343202.

We base this work on central tenets of Black feminist thought. The core objectives of Black feminist thought are to clarify Black women's experiences and ideas through self-definition, to refute stereotypical depictions, to validate Black women's situated knowledge, and to resist marginalization that occurs as a result of our intersectional identities (Hill Collins, 2000). Our work as academics is informed by our identities as Black women, and these identities continue to be shaped by our work as academics. Last, our narrative examines how both mentorship from Black and non-Black academics, and sisterhood among Black women scholars, sustain and inspire the work to which we are committed.

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.

Introduction: Reactivation of viruses such as cytomegalovirus (CMV) or Epstein Barr virus (EBV) after allogeneic hemopoietic stem cell transplant (aHSCT) is associated with increased non relapse mortality and a requirement for antivirals with mainly hemopoietic and renal toxicities that can further compromise transplant outcomes and increase health care costs. The use of 3 rd party virus specific T cells (VSTs) has been effective in treating recurrent and refractory viral reactivation after transplant and leads to rapid restoration of viral immunity. We investigated whether early administration of 3 rd party VSTs together with antiviral therapy could safely enhance immune recovery and improve viral control in patients requiring treatment for their initial CMV and EBV infection after aHSCT. Methods: We performed a single arm phase 1 clinical trial in which aHSCT patients requiring treatment for their first CMV or EBV reactivation (or EBV driven malignancy) received infusions of partially HLA matched 3 rd party VSTs within 7 days of commencing standard antiviral treatment. Patients were required to have a viral copy number of at least 1,000 copies/mL for CMV, 10,000 copies/mL of blood for EBV, or proven tissue infection irrespective of copy number for treatment initiation. T cell products were expanded from G-CSF stimulated aphereses from normal donors following peptide stimulation and CD137 magnetic bead selection. T cells were cultured for up to 12 days before specificity testing and cryopreservation. Patients were eligible to receive up to 4 doses of VSTs at 4 week intervals with products selected with a minimum of 1 of 6 HLA matches at HLA-A, -B and -DRB1 with antiviral activity demonstrated through the shared HLA molecule. The primary endpoint of the study was infusion safety. Results: Thirty aHSCT patients were treated with 1-4 VST infusions (27 CMV, 3 EBV) commencing at a median of 4 days after initiation of antiviral treatment. 27 patients were transplanted for hematological malignancies, 3 for immune deficiencies. Conditioning was myeloablative in 12 patients and the majority of patients (22/30) received in vivo T cell depletion. 7/27 CMV seropositive recipients were transplanted from CMV seronegative donors. A total of 41 infusions were given, most frequently targeting antigens presented through shared HLA molecules A2 and/or B7. All infusions were administered at a cell dose of 2 x 10 7/m 2. VST products were CD3 + (median 97.9%, range 96.2 - 99.4%), with median percentage of CD3 +CD8 + 85.8% (range 23.2 - 95.5%). There was one infusion related adverse event consisting of fever that resolved rapidly after admission and antibiotics. Overall viral response rate was 100% with a complete response rate of 94% (Figure 1). Of the 28 patients who achieved a CR (after either 1 or 2 infusions), 22 remained virus PCR negative (n = 8) or below the limit of quantitation (n = 14) for the duration of follow up. 3 patients had brief episodes of quantifiable reactivation not requiring additional therapy and 3 patients required a second infusion following initial CR. All remained PCR negative after their 2 nd CR. All 3 patients treated for EBV PTLD achieved sustained CR. Overall rates of acute and chronic GVHD post-infusion were 33% (10/30) and 20% (6/30) respectively (grade IIII/IV aGVHD 10%, severe cGVHD 7%). VST infusion was associated with a reduction in activation and inhibitory marker expression on CD4 + and CD8 + lymphocytes within the first 30 days and recovery of CD8 + (and more slowly CD4 +) CD45RA -CD62L - effector memory cells. Within the first 100 days after infusion there was an increase in interferon-γ responsiveness of blood lymphocytes. CMV and EBV specific tetramer positive T cells were detected comprising up to 13% of CD8 + cells for up to 6 months post infusion. At 1 year post transplant, non relapse mortality was 10%, cumulative incidence of relapse was 7% and overall survival was 87%. Conclusion: The combination of traditional antiviral treatment and early administration of 3 rd party VSTs is safe and achieves high rates of viral control without evidence of increased acute or chronic GVHD and with evidence of enhanced immune responses to viral antigens. 1 year overall survival was high with low rates of non relapse mortality and relapse. These encouraging results require confirmation in a prospective randomized study comparing best available therapy with best available therapy combined with early VST administration. Figure 1 Figure 1. Disclosures Ritchie: Novartis: Honoraria; BMS: Research Funding; Takeda: Research Funding; CRISPR Therapeutics: Research Funding; Amgen Inc: Honoraria, Research Funding; CSL: Honoraria.

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.

Background & Aim Disease relapse, graft versus host disease (GVHD) and infection are responsible for the majority of deaths after HSCT. We assessed a cellular engineering approach to simultaneously reduce the incidence of these complications using CD34+ stem cell selection combined with post-transplant infusion of pathogen-specific and leukemia-specific T cells. Methods, Results & Conclusion METHODS We conducted a pilot study in which patients with ALL in remission were treated with a CD34-selected stem cell transplant followed by two planned prophylactic infusions of donor-derived T cells targeting opportunistic pathogens (CMV, EBV and Aspergillus), and leukaemia (CD19 CAR T cells). Cellular products were manufactured locally. Patients did not receive anti-thymocyte globulin or post-transplant GVHD prophylaxis. RESULTS We performed matched sibling donor transplants on two patients aged 45 and 27 years with ALL in morphological CR. Patient 1 had Ph1 positive ALL with detectable BCR-ABL at the time of transplant (0.001%). Patient 2 was Ph1 negative and had no detectable disease by flow cytometry pre-transplant. Conditioning was cyclophosphamide 120mg/kg and TBI 1200cGy. Patients received CD34 selected stem cells (total CD34+ doses 3.5 and 3.6 × 106/kg; total CD3+ cell doses 1.3 and 0.2 × 104/kg). Neutrophil engraftment (>0.5) occurred on days 11 and 12, platelet engraftment (>20) on days 8 and 13. Patients received infusions of pathogen specific T cells (day 21) and CD19 CAR T cells (days 27 and 21). CRS (grades 1 and 2) and neurotoxicity (grade 1) developed in both patients; treated with tocilizumab, patient 1 also received dexamethasone. CAR T cells proliferated in vivo despite low disease burden and persisted in blood for at least 6 weeks in both patients (Figure 1). Patient 1 received foscarnet for 9 days and a second pathogen-specific T cell infusion on day 47 post-transplant. Patient 2 developed asymptomatic reactivation of HHV6 and BK virus. At 364 and 350 days post-transplant, both patients are well, free of GVHD and remain in remission. CONCLUSION This is the first description of a strategy that combines prophylactic donor-derived CAR T cell and pathogen-specific T cell administration after HSCT in the context of CD34+ selection to minimise development of GVHD. Our results demonstrate absence of GVHD, minimal treatment for viral reactivation and remission at 12 months post-transplant suggesting that this approach has promise for improving GVHD relapse free survival following HSCT.

Background Administration of partially HLA-matched third party virus-specific T cells (VST) from a cryopreserved cell bank is safe and effective after failure of standard antiviral therapy to resolve viral infection occurring after allogeneic stem cell transplantation (HSCT). Aim In this phase I trial, we assessed the safety and efficacy of administering partially HLA-matched third party VST at the time of initial antiviral therapy following HSCT rather than waiting for failure of at least two weeks of standard antiviral treatment. Methods A cryopreserved cell bank of VST directed at cytomegalovirus (CMV), Epstein Barr virus (EBV) or adenovirus (Adv) was established using G-CSF mobilised peripheral blood from healthy stem cell donors. After stimulation with peptide mixes, VST were selected by expression of CD137+ cells and cultured with cytokines. HSCT recipients were treated with up to 4 doses of 2x107 of VST/m2, the first commencing within 7 days of initial antiviral treatment for viral reactivation. Results A total of 188 doses of VST were manufactured from 7 donors with 12 product manufacturing runs (CMV n=3, EBV n=4 and Adv n=5). Median virus specificity was 75% for CMV, 83% for EBV and 37% for Adv. Thirty HSCT recipients were treated with VST a median of 55 days post-transplant. Data from 25 patients treated for initial viral reactivation were available for analysis (CMV n=22, EBV n=2, Adv n=1). Median age was 58 years (0-71). Patients underwent transplant for myeloid malignancies (n=16), lymphoid malignancies (n=5) and non-malignant conditions (n=4). Patients with malignant disease were transplanted in CR1 (n=8), CR2 (n=3), >CR2 (n=3) or with active disease (n=7). Conditioning was myeloablative in 11 patients and reduced intensity in 14 patients. Donors were matched unrelated (n=20), haploidentical (n=4) or siblings (n=1). 21 patients received some form of T cell depletion (most commonly pre-transplant thymoglobuline in vivo). All patients received VST within 7 days of commencing initial antiviral therapy. 18 patients received a single VST infusion, 6 received 2 and 1 received 4 VST infusions. There were 3 mild infusion related adverse events (vomiting, hypertension, fever). 3 patients had aGVHD pre-infusion (2 grade 1 skin, 1 grade 3 GI). Two patients died of acute GVHD (1 patient with resolved grade 3 GI GVHD pre-VST infusion developed grade 4 GI GVHD 89 days post- infusion as immunosuppression was weaned; the other patient developed de novo liver and GI GVHD 30 days post infusion in the context of a rapid wean in immunosuppression for severe BK virus haemorrhagic cystitis). 2 patients developed de novo grade 1 GVHD post-infusion. 2 patients developed mild limited cGVHD and 1 patient developed extensive cGVHD after VST infusion. 23/25 patients (92%) had complete viral clearance of the infection for which VST were given, 2 had a partial viral response. Median time to best viral response was 20 days. There were 5 deaths (refractory aGVHD in 2 patients, pulmonary VOD/CMV pneumonitis, disease relapse, and sepsis/aspiration pneumonia). 4 of 25 patients died within 12 months of transplant for a 1 year NRM of 12%. At a median follow up of 431 days (112-1391) post-transplant, 20 of 25 patients (80%) remain alive (Figure 1). Conclusion Infusion of third party partially HLA-matched donor-derived VST at the time of first antiviral treatment for CMV, EBV and Adv post HSCT is associated with minimal infusion toxicity, a low rate of moderate to severe GVHD and complete viral clearance in 92% of recipients. Overall survival in this group of high-risk patients requiring treatment for viral reactivation after HSCT is high. A randomised trial will be performed to determine whether administration of third-party VST in addition to standard anti-viral treatment improves transplant outcomes. Figure 1 Disclosures Gottlieb: Haemalogix P/L: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy; Gilead: Consultancy; AbbVie: Consultancy; University of Sydney: Employment; Merck: Consultancy. Ritchie:Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; BMS: Research Funding; Takeda: Research Funding; Beigene: Research Funding; Imago: Research Funding; Novartis: Honoraria; Sanofi: Honoraria.

Background & Aim Aim The non-viral piggyBac transposon system reduces the complexity and expense of CAR T cell manufacture. piggyBac CAR19 T cells showed potent pre-clinical activity, so we now assess safety and clinical activity in a first-in-human study. Methods, Results & Conclusion Methods Eight patients (age 18-66 years, median 30; male n=7, female n=1) with relapsed/refractory CD19+ malignancies post HLA-matched sibling HSCT were treated with donor-derived T cells genetically modified using piggyBac to express a second-generation 4-1BB-based CAR19. The trial was conducted as an investigator-initiated study at a university hospital with local CAR19 T cell manufacture. CAR19 T cells were expanded ex vivo from 50 × 106 peripheral blood mononuclear cells, with CD19 stimulation and IL-15 support. Final products contained median 1.9 × 109 (range 0.8-2.5 × 109) total cells with 80.6% (39-93%) CAR T cells, and were generated in 15 (n=7) or 23 (n=1) days. Patients received up to 3 escalating doses of CAR19 T cells (10, 50 and 100 × 106/m2) following lymphodepleting cyclophosphamide and fludarabine (3 doses, n=5; 1 dose, n=3). Results Diagnoses were B-ALL (n=6), DLBCL (n=1), and Burkitt Lymphoma (n=1). Four patients each had a high or low burden of disease prior to treatment. The patient with Burkitt Lymphoma died of complications unrelated to CAR T cells 307 days with 3 doses. Toxicity included: cytokine release syndrome (n=3), B-cell aplasia with hypogammaglobulinaemia (n=7), prolonged cytopenias (n=6), bacterial infection (n=3) and exacerbation of chronic GVHD (n=1). No CAR T cell-related encephalopathy syndrome occurred. Conclusions Manufacture of piggyBac CAR19 T cells for clinical use is rapid, robust and uncomplicated, permitting application of this technology to multiple healthcare institutions. Early results suggest similar safety and activity to that of CAR19 T cells generated with viral vectors. The optimal CAR19 T cell dosing strategy remains to be determined.

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.

Uncontrolled cytomegalovirus (CMV) reactivation after allogeneic hematopoietic stem cell transplantation causes significant morbidity and mortality. Adoptive transfer of CMV-specific cytotoxic T lymphocytes (CTLs) is a promising therapy to treat reactivation and prevent viral disease. In this article, we describe the generation of clinical-grade CMV-specific CTLs directly from granulocyte colony-stimulating factor–mobilized hemopoietic progenitor cell (G-HPC) products collected for transplantation. This method requires less than 2.5% of a typical G-HPC product to reproducibly expand CMV-specific CTLs ex vivo. Comparison of 11 CMV CTL lines generated from G-HPC products with 52 CMV CTL lines generated from nonmobilized peripheral blood revealed similar expansion kinetics and phenotype. G-HPC–derived CTLs produced IFN-γ after reexposure to CMVpp65 antigen and exhibited CMV-directed cytotoxicity but no alloreactivity against transplantation recipient–derived cells. Seven patients received CMV-specific CTL lines expanded from G-HPC products in a prophylactic adoptive immunotherapy phase I/II clinical trial. Use of G-HPC products will facilitate integration of CTL generation into established quality systems of transplantation centers and more rapid inclusion of T cell therapies into routine clinical care.

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.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.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.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.

Background aims Virus-specific T-cell immunotherapy is emerging as a promising management strategy for virus infections in patients after hematopoietic stem cell transplant (HSCT). Here we present outcomes of 10 adult patients who received multi-virus-specific T cells prophylactically after HSCT. Methods Donor-derived cytomegalovirus (CMV)-, Epstein-Barr virus (EBV)-, adenoviral- and varicella zoster virus (VZV)-specific T cells were generated in a single culture and administered to HSCT patients at a dose of 2 × 10 7 /m 2 virus-specific T cells at a median of 63 days post-transplant. Patients were monitored for 12 months for evidence of viral reactivation and graft-versus-host disease. Results There was no acute infusion-related toxicity. Six patients developed CMV reactivation after T-cell infusion with a median peak CMV DNA titer of 600 copies per milliliter, and 1 received CMV-specific pharmacotherapy post-infusion. No EBV, adenoviral or VZV reactivation or disease was reported. Using interferon-γ Elispot analysis on post-infusion samples, we identified anti-viral immunity against all viruses including VZV. Three patients (30%) developed grade II–IV acute graft-versus-host disease. Conclusions This is the first description of the use of a multi-virus-specific T-cell product containing cells specific for VZV after allogeneic HSCT. The T-cell product appears safe in the setting of HSCT and confirms our previous findings regarding CMV control and treatment. A larger study with longer follow-up is required to determine the efficacy of VZV-specific T cells given prophylactically in controlling episodes of herpes zoster and disseminated varicella infection after cessation of prophylactic anti-viral treatment.

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.

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.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.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.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.

The efficacy of adoptive transfer of stem cell donor-derived virus-specific T cells (VST) to prevent or treat viral infection in allogeneic HSCT is well established. However, this approach has some limitations. It is prerequisite on the donor cells being accessible, and virus seropositive. The generation of a product applicable to a single intended recipient is cost- and labour-intensive, and may not be immediately available in urgent clinical scenarios. Creation of a bank of cryopreserved third party donor-derived VST addresses these limitations. We established such a bank of 177 VST, specific for cytomegalovirus (CMV n=77), Epstein Barr virus (EBV n=55), and adenovirus (AdV n = 47). To date, 19 allogeneic HSCT patients have been treated with partially HLA-matched VST for viral reactivation or disease (CMV=17 (2 with CMV colitis), EBV=1, AdV=1); persistent despite at least 14 days of antiviral treatment. Patients were transplanted from unrelated (n=12), sibling (n=4), or haploidentical (n=3) donors. The graft source was PBSC (n= 15), cord (n=1), or bone marrow (n=3), and 12/19 patients underwent in vivo T cell depletion. The 17 patients treated for CMV received a median of 31 (15-113) days of antiviral therapy prior to infusion, and 14/17 were transplanted from a CMV negative donor. Patients were eligible for up to 4 infusions of 2x107/m2 VST if viral persistence was documented 2 weeks after initial infusion. VST were matched at a minimum of one HLA allele between VST and recipient, and chosen on the basis of greatest number of HLA matches with preference for products with viral activity restricted through a shared HLA. A total of 31 VST have been infused; 1 patient received 4 infusions, 1 patient received 3 infusions, 7 patients received 2 infusions, and 10 patients received 1 infusion. VST were matched at 1/6 (n=4) to 4/6 HLA alleles, with 24/31 infusions matched at both a class I and class II HLA allele. The antiviral activity of VST products (determined by MHC tetramer staining or cytokine response to epitope stimulation) generated from 12 third-party donors was predominantly restricted through class I HLA alleles: A1 (n=4), A2 (n=2), A2 + B7 (n=1), A2 + DRB04:01 (n=1), A2 + B35 + DRB01:01 (n=1), A24 (EBV-specific, n=1), B7 (n=1), and DRB01:01 (Adv-specific, n=1). Best viral response post-initial VST was assessed in 18 evaluable patients. A complete response (CR) was documented if virus PCR negativity was achieved at any time, and partial response (PR) for a greater than 50% reduction from the pre-administration titre. At a median follow-up of 6.3 (1-14.8) m, a CR was attained in 14/18 (78%) patients and PR in 4/18 (22%) patients. Both patients with CMV colitis achieved CR and symptoms resolved. Reinitiation of antiviral therapy after cessation was not required in 15/18 patients post-final VST infusion. The median number of months free from antiviral therapy was 5.1 (0-14.6). No immediate infusion-related toxicities have occurred. One patient with skin GVHD prior to enrolment developed grade II acute skin GVHD post-infusion. Chronic GVHD has been documented in 4 patients (2 had preceding acute GVHD pre-infusion, 1 occurred following donor lymphocyte infusion with no preinfusion GVHD, 1 developed mild skin cGVHD with no preceding GVHD). One patient treated with EBV-specific VST was not evaluated for response as he died from relapsed disease 14 days after VST infusion. A further 4 deaths have occurred; 1 due to presumed CMV disease following a PR to VST; 1 bacterial pneumonia; 1 relapsed disease; 1 due to CNS EBV PLTD in a patient with a CR to CMV-specific VST. T cell receptor deep sequencing was performed to investigate the persistence of infused cells in treated patients, but no clones detected in individual VST products were shown to persist beyond 24hrs post-infusion in the 3 patients examined. Overall, third party VST appear to be safe and efficacious with a 100% overall response rate and durable responses demonstrated in the 18 evaluable patients reported. The use of an 'off the shelf' product has allowed prompt treatment of viral infection in a number of of allogeneic HSCT patients in whom the HSCT donor would not have been suitable or available for generation of T cells. It appears that expansion and persistence of infused cells may not be the predominant mechanism for achieving the demonstrated clinical responses, and investigations are underway to examine alternative potential mechanisms of VST mediated immune modulation. Disclosures No relevant conflicts of interest to declare.

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$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.

Introduction Adoptive transfer of donor derived virus specific T cells (VST) can be effective therapy for infections in allogeneic HSCT recipients. However, this is not a practical strategy to treat acute infections due to the time required to prepare products and potential unavailability of transplant donors. To overcome this, treatment with cryopreserved partially HLA-matched T cells from third-party donors is being investigated. A recent report described disease resolution using cells matched at only one or two HLA alleles (Leen et al., (2013) Blood 121(26):5113-23). This less stringent requirement for matching would allow a small bank of cells to provide most patients with a therapeutic product. We describe the establishment of a virus specific T cell bank in Australia with centralized manufacturing by the Westmead Hospital BMT laboratory. The bank has been used to treat patients in multiple states in a Phase I clinical trial to treat patients who have failed antiviral pharmacotherapy. Aim To assess the safety and feasibility of treatment with partially HLA-matched VSTs derived from third-party donors for refractory cytomegalovirus (CMV), Epstein-Barr Virus (EBV), or adenoviral (AdV) infection in allogeneic HSCT patients. Methods We generated a bank of cryopreserved VSTs from peripheral blood or G-CSF mobilized stem cell product of healthy donors. Products were generated by co-culturing PBMC with dendritic cells loaded with overlapping peptides covering CMV pp65, AdV hexon or EBV BZLF1, LMP2A and EBNA1 proteins. Cultures were re-stimulated once with peptide loaded DC and cultured for 14 days with IL-2. Products were assessed for phenotype, sterility and specificity by MHC multimer staining where applicable or production of interferon-gamma in response to peptides by flow cytometry. Patients with persistent viral reactivation/infection after 2 weeks of standard therapy were eligible to receive up to 4 fortnightly infusions of 2x107 cells/m2partially HLA matched (minimum 1/6) CMV, EBV, or AdV specific T cells, and were followed for up to 12 months. Results T cell products were expanded from 25 donors to create a bank of 177 bags of VSTs (75 CMV, 47 AdV and 55 EBV). CMV specific products were predominantly T cells (mean 95.8±3%) with a higher proportion of CD8+ compared to CD4+ T cells (mean 66.6±23.9% versus 20.1±6.2%). Specificity was mapped by MHC multimer staining to epitopes restricted to common HLA types including HLA-A*0101, HLA-A*0201, HLA-A*2402, HLA-B*0702 and HLA-B*3501. AdV specific T cells had a higher proportion of CD4+ T cells (mean 64.6±23.8% versus 34.2±20.1% CD8 T cells). Specificity was mapped to CD8 epitopes restricted to HLA-A*0101 and HLA-A*2402 as well as 10 CD4 T cell epitopes restricted to three HLA-DRB1 alleles (DRB1*0301, DRB1*0701, DRB1*1501). EBV specific products contained a mix of CD8+ and CD4+T cells (mean 38.9%±18% AND 42.5±23.1% respectively). The antigen specificity of EBV products showed high variability between donors. Dominant responses to known MHC class I restricted epitopes were infrequent though responses were mapped to HLA-A*0201 and HLA-A*2402 restricted LMP2A epitopes, a HLA-B*0801 restricted BZLF1 epitope and a HLA-B*0702 restricted EBNA1 epitope. Based on HLA frequency analysis in the Australian recipient population we estimate 94%, 89% and 74% of patients would have access to a CMV, AdV and EBV specific product respectively with the current bank. To date nine patients have received VSTs, with median follow-up of 5.5 months (0-12 months). All patients had treatment resistant CMV after a median of 26 days (19-116 days) prior therapy. Six patients received a single infusion and 3 patients received 2, 3 and 4 infusions respectively. HLA matching ranged from 2-4/6 HLA match. There were no instances of 24hr infusion related toxicity. Follow-up data is available for 7 patients. One patient with chronic hepatitis C developed abnormal liver function tests 3 months post-infusion. One patient died from presumed progressive CMV disease 6 months post-enrolment. Five patients achieved a best response of CMV PCR negativity (2 with complete resolution of CMV-colitis). One patient has shown >50% reduction in CMV copy number over 3 weeks. Conclusion The infusion of third party CMV specific T cells is a promising therapy that offers the advantage of rapid availability, centralized manufacturing and relatively low cost per dose when produced on a large scale. Disclosures No relevant conflicts of interest to declare.

Aim Adoptive antiviral immunotherapy has recently gained popularity as a prophylactic and therapeutic strategy for viral infections post allogeneic stem cell transplant (HSCT). We report the outcomes of 10 patients who received multi-virus specific T cells (CTL) prophylactically post HSCT, and compare the outcomes of these patients to a contemporaneous cohort control. Methods Donor derived CMV, EBV, adenoviral and VZV specific T cells were generated in a 21-day culture, which involved stimulation of peripheral blood mononuclear cells by antigen-pulsed monocyte derived dendritic cells (DC) on days 0 and 7 and culture with 20-50units/ml of IL2. DC were transfected with an adenoviral vector encoding the CMV immune-dominant antigen pp65 or epitopes of EBV antigens EBNA1, LMP1 and LMP2. A commercial VZV vaccine (Varivax®, Merck & Co) was used to stimulate VZV specific T cells. Patients undergoing matched sibling HSCT at Westmead Hospital from Feb 2011 to Jun 2012 were recruited. Patients with no active acute graft versus host disease were given 2x107/m2 multi-virus CTLs from day 35 onwards. After CTL infusion, patients were monitored for 12 months. Clinical outcome measures included adverse events related to CTL infusion, incidence of acute and chronic graft versus host disease and the incidence of CMV, EBV, adenoviral and VZV reactivations and infection. Patients were treated for viral reactivation according to standard institutional guidelines. A contemporaneous cohort of matched sibling transplant recipients treated during the same period at Westmead Hospital was used for comparison. A landmark analysis was performed on both groups using day 35 as a landmark to focus on outcome events related to CTL infusion. Results 10 patients who underwent matched sibling donor HSCT from Feb 2011 to Jun 2012 were given multi-virus specific T cells. There were no adverse events in any of the patients within 24 hours of T cell infusion. Three patients developed grade II-IV acute graft versus host disease (aGVHD) after transplant, 1 prior to CTL infusion, and 2 post CTL infusion. 7 patients developed chronic graft versus host disease. 8 patients reactivated CMV at any time post-transplant, 6 prior to CTL infusion of whom 2 required ganciclovir therapy; 5 patients developed low level CMV reactivation (median peak titre 600 copies/ml) post CTL infusion. 1 patient received ganciclovir for CMV PCR positivity on colonic tissue. No CMV disease developed and no evidence of EBV, VZV or adenoviral reactivation was noted during 12 months follow up. Using the day 35 landmark to exclude patients with grade II-IV acute GVHD, CMV reactivation and premature death in both CTL and control cohorts, 9 patients in the study cohort and 21 patients in the control cohort were available for comparison. 22% in the study cohort and 19% in the control cohort developed grade II-IV acute GVHD. 67% in the CTL cohort and 57% in the control cohort developed chronic GVHD. 7 of 9 patients in the study cohort and 9 of 21 patients in the control cohort had a CMV reactivation post transplant. The median peak CMV copy number was lower in the study cohort (600 vs 2840 copies/ml). 29% and 67% of patients received ganciclovir in the CTL and control cohort respectively. There was no clinically documented CMV disease in either cohort. 1 EBV, 1 adenoviral and 1 VZV reactivation were identified in the control cohort. No reactivations were identified in the study cohort. Conclusion Multi-virus T cell therapy appears safe, with no evidence of increased GVHD, or excess of adverse events observed as a result of CTL administration. Patients receiving T cells in this study had a lower median peak CMV copy number during reactivation episodes and fewer required anti-CMV pharmacotherapy. These results confirm out previous data on the efficacy of prophylactically administered CMV specific T cells given post HSCT but a larger study is required to determine their efficacy in preventing EBV, adenovirus and varicella zoster virus disease. Disclosures: No relevant conflicts of interest to declare.

Aim: We present the preliminary data on a phase I/II clinical trial administering multi-virus specific T cells prophylactically to patients who have undergone HSCT. Methods and Results: Donor derived CMV, EBV, adenoviral and VZV specific T cells were generated according to previously described standard operating procedures. HSCT recipients received 2 10/m virus specific T cells on or after day 28 post transplant and were monitored for evidence of viral reactivation and graft versus host disease. 10 patients who underwent sibling donor HSCT from Feb 2011 to Jun 2012 were given multi-virus specific T cells. Of the 8 patients who received CMV specific T cells, 3 (37.5%) patients had CMV reactivation post T cell infusion with a median peak CMV DNA titre of 600 copies/ml. No patient developed CMV disease but one patient received valganciclovir at the discretion of the treating physician. No EBV, adenoviral or VZV reactivation or disease was seen. 3 patients (30%) developed grade IIeIV acute graft versus host disease (aGVHD). We used a landmark analysis at day 35 to compare these patients with a contemporaneous control cohort of 21 sibling HSCT recipients. CMV reactivation developed in 9 (42%) control patients with a median peak CMV DNA titre of 2840 copies/ml. 67% of the control group who reactivated CMV required ganciclovir and/or foscarnet therapy. Adenoviral, EBV and VZV reactivation was detected in 1, 1 and 1 control patients respectively. 4 patients (19%) in the control group developed grade IIeIV aGVHD. Conclusion: Mutli-virus specific T cell therapy appears safe in the setting of HSCT. There is a trend towards a reduction in the need for antiviral treatment for CMV reactivation. A larger study is required to determine the safety and efficacy of multivirus specific T cells given prophylactically following HSCT to rapidly reconstitute immunity.

Abstract 4040 Influenza viruses cause fatal respiratory infections in stem cell transplant patients. Specific T cells provide long-lived adaptive immunity to influenza and the potential for generating such cells for clinical use was investigated. The inactivated influenza vaccine (Fluvax, CSL Australia) was used as the antigen source. Only reagents and culture media approved for clinical manufacture were used. Monocyte-derived dendritic cells (MoDC) pulsed with Fluvax were used to stimulate autologous PBMC at a responder to stimulator ratio of 10:1. On Day 7, a second stimulation was performed. 20U/ml IL-2 was added from Day 7 and 50U/ml IL-2 from Day 14 onwards. Media exchanges were performed as required using fresh medium containing IL-2. Over 21 days of culture, a mean fold increase of 26.3 in cell number was observed (n=7). Cultures were mainly T cells (mean 92.9%) with effector and central memory phenotypes. CD4 cells dominated (mean 78.1%) with a lower percentage of CD8 cells (mean 14.9%). Following expansion, CD154 was expressed on 8.1% of CD4 cells ( In conclusion, we demonstrate a clinically applicable method that yields high numbers of highly reactive T cells specific for influenza viruses including the H1N1 strain. Cells express a phenotype of activated antigen specific cells capable of B cell helper function. We propose that reconstructing host immunity through adoptive transfer of donor derived influenza virus specific T cells possibly combined with early post-transplant influenza vaccination will reduce the frequency of influenza-related deaths in the period of severe immune suppression that follows allogeneic stem cell transplant. Disclosures: Off Label Use: Use of influenza vaccine for in vitro cell generation.