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Objectives Adoptive immunotherapy using donor‐derived antigen‐specific T‐cells can prevent and treat infection after allogeneic haemopoietic stem cell transplant (HSCT). Methods We treated 11 patients with a prophylactic infusion of 2 × 107 cells per square metre donor‐derived T‐cells targeting seven infections (six viral and one fungal) following HSCT. Targeted pathogens were cytomegalovirus (CMV), Epstein–Barr virus (EBV), adenovirus, varicella zoster virus, influenza, BK virus (BKV) and Aspergillus fumigatus. Results T‐cell products were successfully generated in all patients with 10 products responsive to 6 or 7 infections. T‐cell infusions were associated with increases in antigen‐experienced activated CD8+ T‐cells by day 30. CMV, EBV and BKV reactivation occurred in the majority of patients and was well controlled except where glucocorticoids were administered soon after T‐cell infusion. Three patients in that circumstance developed CMV tissue infection. No patient required treatment for invasive fungal infection. The most common CMV and EBV TCR clonotypes in the infusion product became the most common clonotypes seen at day 30 post‐T‐cell infusion. Donors and their recipients were recruited to the study prior to transplant. Grade III/IV graft‐versus‐host disease developed in four patients. At a median follow‐up of 390 days post‐transplant, six patients had died, 5 of relapse, and 1 of multi‐organ failure. Infection did not contribute to death in any patient. Conclusion Rapid reconstitution of immunity to a broad range of viral and fungal infections can be achieved using a multi‐pathogen‐specific T‐cell product. The development of GVHD after T‐cell infusion suggests that infection‐specific T‐cell therapy after allogeneic stem cell transplant should be combined with other strategies to reduce graft‐versus‐host disease., In this study, 12 patients were treated with ex vivo expanded donor‐derived T‐cells specific for seven virus and fungal pathogens. There was no acute infusion‐related toxicity. CD8 T‐cell numbers in the peripheral blood rose within 30 days of infusion. Graft‐versus‐host disease and corticosteroid use were associated with viral reactivation; no patient developed fungal disease. Relapse (n = 5) and multi‐organ failure (n = 1) were the causes of death. Infection did not contribute to death in any patient.

Background: Adaptive resistance mechanisms leading to treatment failure have been identified in older patients receiving venetoclax (VEN) in combination with either azacitidine or low dose cytarabine (LDAC) as frontline therapy for acute myeloid leukemia (AML). These include the expansion or secondary emergence of kinase activating mutations, including FLT3-ITD in patients with non-adverse karyotype (NON-ADV), as well as TP53 mutations among patients with adverse karyotype (ADV)(DiNardo & Tiong et al, Blood 2020). INTERVENE is a phase 2 study evaluating the safety and efficacy of the "risk-stratified" addition of a novel third agent to VEN-LDAC, delivered in tandem to LDAC to minimize the risk of myelotoxicity (Figure 1A). To mitigate VEN resistance associated with activated kinases in NON-ADV risk AML, midostaurin (MIDO), a FLT3/multi-kinase inhibitor, was incorporated in combination with VEN. To address VEN resistance associated with TP53 defects in ADV risk AML, a HDAC inhibitor pracinostat (PRAN) was incorporated in accordance with pre-clinical studies suggesting synergistic induction of TP53 independent cell death with VEN plus HDAC inhibition (Salmon et al, ASH 2018). We hereby report the results of the dose-finding safety run-in phase of the study. Methods: Eligibility: Patients with treatment naïve AML (excluding APL), aged ≥60 years and unfit for intensive chemotherapy were included. Prior hypomethylating agents for antecedent myeloid neoplasms were permitted with a 14-day washout. Patients were stratified according to cytogenetic risk, as per Medical Research Council 2010 criteria. Treatment: VEN D1-28 (with dose ramp-up in cycle 1) was combined with LDAC (20mg/m 2 SC D1-10), with the third agent starting after/on the last day of LDAC (Fig 1A). Each cycle was 28 days. In the NON-ADV stratum (VEN-LDAC-MIDO), 2 dose levels were explored: (L1) VEN 400mg + LDAC + MIDO 50mg BD D11-28; (L2) VEN 600mg + LDAC + MIDO 50mg. In the ADV stratum (VEN-LDAC-PRAN), 3 dose levels were tested: (L1) VEN 400mg + LDAC + PRAN 45mg starting D10 and given 3x/week orally for a total of 9 doses; (L2) VEN 600mg + LDAC + PRAN 45mg; (L3) VEN 600mg + LDAC + PRAN 60mg. Azole antifungals were prohibited in cycle 1 but allowed from cycle 2 with VEN dose modification. Endpoints (safety run-in): Primary: occurrence of dose-limiting toxicities (DLT) during cycle 1 and determination of recommended phase 2 doses (RP2D) using a Bayesian Logistic Regression Model. Secondary: Preliminary response rates. Molecular studies: Next generation sequencing using a custom 48-gene Roche KAPA HyperCapture myeloid panel and FLT3-ITD targeted amplicon sequencing were performed on baseline bone marrow samples. First patient enrolled: 7SEP2020. Data cut-off: 29JUN2021. Results: 32 patients were enrolled: 18 in NON-ADV and 14 in ADV strata, respectively. Two patients in the NON-ADV stratum withdrew within the first 7 days due to non-therapy related reasons (1=personal, 1=incidental lung lesion) and were not DLT/response evaluable. Median age was 77 years (68-87; 69% ≥75 years). 43.8% (14/32) had secondary/therapy related AML. Although gastrointestinal adverse events (AE) during cycle 1 were more common in VEN-LDAC-PRAN arm with nausea (57 vs 17%), vomiting (36% vs 6%) and diarrhea (50% vs 22%), grade 3+ toxicities were uncommon (0-7%)(Table 2). Occurrence of febrile neutropenia was similar between the two arms. 30-day mortality was 0% and 14% (2/14: 1=infection, 1=disease progression) for NON-ADV and ADV strata, respectively. No DLTs were observed in either stratum across all dose levels, thus the RP2D was the highest dose level explored for both triplet combinations. The intention-to-treat overall response rate CR+CRi+CRh was 72.2% (13/18) in the NON-ADV arm and 57.1% (8/14) in ADV arm. The expanded response rate including PR and MLFS was 77.8% (14/18) and 71.4% (10/14) in the NON-ADV and ADV strata, respectively. Median time to best response was 1 cycle (range 1-6). Updated response and survival outcomes will be presented at the meeting. Conclusion: The addition of MIDO or PRAN to VEN-LDAC was tolerable in older/unfit patients with treatment naïve AML. Preliminary efficacy with this risk-stratified approach compared favorably to prior studies with VEN-LDAC alone (Wei et al Blood 2020: CR+CRi 56% in NON-ADV, 28% in ADV). The randomized phase 2 part of this tandem triplet strategy with the goal of preventing adaptive resistance is underway. Figure 1 Figure 1. Disclosures Chua: Abbvie: Other: Conference travel and accommodation . Reynolds: Alcon: Current equity holder in publicly-traded company; Abbvie: Research Funding; Novartis AG: Current equity holder in publicly-traded company. Enjeti: Astra Zeneca: Honoraria; Sanofi: Honoraria; AbbVie: Honoraria; Roche: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hiwase: AbbVie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Marlton: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Queensland Health: Current Employment; BeiGene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bajel: Abbvie, Amgen, Novartis, Pfizer: Honoraria; Amgen: Speakers Bureau. Grove: Astellas: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Cooney: Amgen: Other: Travel, accommodation, expenses ; Roche: Other: Travel, accommodation, expenses ; Novartis: Other: Online conference registration . Beligaswatte: Astellas: Membership on an entity's Board of Directors or advisory committees. Anstee: Walter and Eliza Hall Institute: Patents & Royalties: Dr Anstee was a former employee of the Walter and Eliza Hall Institute and is eligible for a fraction of the royalty stream related to Venetoclax. Perera: Abbvie: Speakers Bureau; BMS: Speakers Bureau. Ritchie: Takeda: Research Funding; BMS: Research Funding; Novartis: Honoraria; CRISPR Therapeutics: Research Funding; Amgen Inc: Honoraria, Research Funding; CSL: Honoraria. Wei: Genentech: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees; Macrogenics: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: This presentation will focus on the ALLG INTERVENE clinical trial combining venetoclax+LDAC+midostaurin or venetoclax+LDAC+pracinostat. Although venetoclax and midostaurin are individually FDA-approved in some indications, the combinations examined in this clinical trial have not been approved by FDA.

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.

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.