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We performed a phase 1 clinical trial to evaluate outcomes in patients receiving donor-derived CD19-specific chimeric antigen receptor (CAR) T cells for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene-capacity limitations of traditional viral vectors, CAR T cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, 1 patient developed a gradually enlarging retroperitoneal tumor due to a CAR-expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection, in an asymptomatic patient, of a second CAR T-cell tumor in thoracic para-aortic lymph nodes. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter–driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell–derived lymphoma progressed and 1 patient died. We describe the first 2 cases of malignant lymphoma derived from CAR gene–modified T cells. Although CAR T cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow-up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.

Introduction Invasive fungal infection (IFI) is a potentially devastating complication after allogeneic stem cell transplantation (aHSCT). Breakthrough infections are an increasing threat. The adoptive transfer of fungus-specific T cells (FSTs), analogous to virus specific T-cells for viral reactivation, may improve clinical outcomes but the delay involved in generating FSTs from the stem cell donor is problematic for this type of adoptive cell therapy. We created a bank of FSTs from normal donors for use in patients with post-transplant IFI. Here we describe the first use of partially HLA matched 3 rd party FSTs to treat IFI in a patient after aHSCT. Methods FSTs were manufactured by stimulating G-CSF primed apheresis products overnight with monocyte derived dendritic cells pulsed with lysates from Aspergillus terreus and Candida krusei. CD137 expressing cells were isolated and cultured with CD137 negative feeders in medium supplemented with IL-2, IL-7 and IL-15 for 12 days prior to assessment of target specificity and HLA restriction. Cells consisted principally of CD4 + lymphocytes secreting TNF-α, interferon-γ and IL-17 in response to fungal antigen stimulation. Standard release criteria were applied. We commenced a phase 1 cell dose escalation trial to assess the safety and efficacy of partially HLA-DR matched 3 rd party FSTs from a cryopreserved bank in patients with proven or probable IFI after aHSCT. Results Patient A is a 27 year old female who underwent a myeloablative sibling haploidentical transplant for Philadelphia negative B-ALL in second remission. At day 100 she developed grade III lower GI GVHD requiring methylprednisolone and ruxolitinib for steroid dependent disease. Subsequent CMV reactivation was treated with ganciclovir. Cough in association with pulmonary infiltrates followed, and a bronchoalveolar lavage grew Scedosporium aurantiacum, Clavispora lusitaniaand Aspergillus fumigatus. She developed severe headache and MRI imaging showed a thin rim-enhancing 38x37 mm lesion within the right frontal lobe with moderate vasogenic oedema. Surgical drainage yielded 20 ml of frank pus that grew Scedosporium aurantiacum. Antifungal therapy was started with vorinconazole, terbinafine, amphotericin and later caspofungin. Serial imaging after 12 days showed two new hyperdense foci representing extension of infection, consistent with worsening headaches. The patient received a single infusion of 3 rd party FSTs at a dose of 1 x10 6/m 2 at day 170 post-transplant while continuing antifungal therapy with vorinconazole and terbinafine. The product consisted of 94.1% CD4 +, 3.5% CD8 + T-cells and 1% NK cells and contained CD4 + T-cells responsive to both A. terreus and C. krusei antigens presented by HLA DR*03:01 shared between product and patient. There were no infusion related adverse events. Corticosteroids and calcineurin inhibitors were continued. Within one week of FST infusion there was an increase in the number of naïve and central memory CD4 + T-cells in blood and a fall in the number of CD4 + and CD8 + T-cells expressing Tim3. Over the following 3 months, there was a gradual rise in the number of CD4 + Tem and CD4 + Temra with a later and less pronounced rise in the analogous CD8 + populations. Serial imaging demonstrated rapid regression of the pulmonary abnormalities and gradual regression of the cerebral lesion at day 150 following FST infusion. 279 days after transplant and 109 days after infusion of FSTs, the patient developed worsening of headache and MRI confirmed rupture of the abscess into the right ventricle. Headache gradually resolved and the patient was discharged from hospital 329 days after transplant with ECOG 1 and no neurological abnormalities. However she was readmitted 13 days later with more severe headache with repeat imaging confirming raised intracranial pressure. CSF showed no evidence of fungi by PCR or culture. A CSF shunt was inserted and the patient remains well. Conclusion We report the first infusion of 3 rd party partially HLA DR matched fungus-specific T-cells for disseminated fungal infection following allogeneic stem cell transplantation. These data demonstrate the feasibility of this approach. The patient's favourable clinical outcome and phenotyping results suggest that the initial cell dose level is safe and may be associated with immune alterations that promote anti-fungal activity. Further trial recruitment is ongoing. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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.

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.

Hematopoietic stem cell transplantation (HSCT) represents the only crative treatment option for many hematological conditions but results in a profound T-cell deficiency in the post-HSCT period. Infections account for a significant proportion of non-relapse morbidity and mortality, and infections with multiple organisms either simultaneously or at different times after transplant are common. Adoptive cellular therapy (ACT) with prophylactic or therapeutic infusion of donor derived or third-party, pathogen-specific T-cells represents a novel methodology to rapidly reconstitute T-cell mediated immunity in this context. For cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infection, clear evidence of efficacy with limited toxicity has been observed, with response rates up to 90%. Infusion of third-party, partially human leukocyte antigen-matched pathogen-specific T-cells have also demonstrated remarkable efficacy with responses seen in up to 70% of patients with resistant CMV, EBV and adenoviral infection. This review addresses the nature of post-HSCT immune deficiency, the common infections that occur in the post-HSCT period and how advances in ACT manufacturing methodologies is allowing for wider implementation of T-cell therapies targeting multiple pathogens in HSCT recipients.

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 Introduction DNA transposon systems offer an alternative to viral vectors for genetic modification of eukaryotic cells. The Piggybac system is being used in CAR T-cell clinical trials presently, but its widespread use may be limited by licensing restrictions. Transposon mining of the little brown bat (Myotis lucifugus) genome has revealed the presence of an active DNA transposase named Piggybat. Aims To optimise manufacturing conditions and evaluate the genomic integration profile of the Piggybat transposase in the generation of CD19 specific CAR T-cells (CAR19-T) compared to Piggybac transposase. Methods, Results & Conclusion Methods CAR19-T were generated from healthy donors (n=3) by co-electroporation of two plasmids encoding the transposase and a CAR19 transposon. T-cells were co-cultured with irradiated feeder cells and cytokines. CAR19-T expansion, CAR expression, memory phenotype, CD19 specific activation and cytotoxicity were assessed after a 2-week culture period. CAR gene integration copy number was determined by droplet digital polymerase chain reaction and integration site analysis was conducted by next generation sequencing of PCR amplicons spanning genomic DNA/CAR gene intersection sites. Results Over 2 weeks, Piggybat CAR19-T expanded a mean of 26 fold with 52% CAR expression, and Piggybac CAR19-T expanded a mean of 11 fold with 67% CAR expression. Piggybat and Piggybac CAR19-T had a mean 59% and 48% CD8+ T-cells respectively, with a similar CAR19-T memory phenotype. Using 50ug/ml concentration of each transposon and transposase plasmids, Piggybat had fewer genomic integrants than Piggybac (mean 3.4 and 10.1 copies per cell respectively). Integrant copy number could be titrated by altering transposon plasmid concentration. Peak CAR19-T expansion, expression and MFI were achieved using 1ug Piggybat transposase plasmid and 5ug transposon plasmid. Piggybat and Piggybac CAR19-T demonstrated similar CD19 specificity and cytotoxicity. Piggybat CAR19-T showed 1920 unique genomic integration sites, 11.13% within 2.5kb of transcription start sites (TSS) and 43.72% being intragenic, compared to Piggybac with 2697 unique integration sites, 20.33% TSS localised and 47.77% intragenic. Conclusions Compared to Piggybac, Piggybat transposase can generate CD19 specific CAR T-cells with similar CAR expression, function and insertion site profile, but fewer integrants per cell. Thus, Piggybat is a new DNA transposon that may allow cost-effective CAR T-cell production for clinical use.