Chimeric antigen receptor (CAR) T-cell immunotherapy has shown great success in clinical cancer, bringing hope to apply CAR strategies to other clinical settings. Here we developed a CAR macrophage (CAR-M) that recognizes the major inflammatory molecule tumour necrosis factor (TNF) and activates an intracellular IL-4 signalling pathway, thereby programming engineered macrophages for an anti-inflammatory function. CAR-M therapy has exhibited efficacy in mouse models of both acute and chronic inflammatory diseases. In kidney ischaemia reperfusion injury (IRI), infused CAR-Ms switched to an anti-inflammatory phenotype in inflamed kidney and attenuated kidney IRI. The anti-inflammatory phenotype of infused CAR-Ms switched off during the recovery phase of kidney IRI, coinciding with the disappearance of TNF. In Adriamycin-induced nephropathy, a model of chronic inflammatory disease, infused CAR-Ms maintained an anti-inflammatory phenotype for several weeks in response to sustained high levels of TNF and improved kidney function and structure. CAR-Ms also effectively reduced tissue injury in another organ, the liver. Human anti-TNF CAR-Ms exhibit anti-inflammatory phenotype and function in response to TNF. The CAR-M design, using signal switching, holds promise for the treatment of a broad range of acute and chronic inflammatory diseases.

Abstract Disease relapse and infections cause significant morbidity and mortality post-allogeneic stem cell transplant (HSCT), which remains the only cure for many myeloid malignancies. We investigated a novel combination of donor-derived, tumour-associated, antigen-specific T-cells targeting Wilm’s tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME), and multipathogen T-cells targeting CMV, EBV, Adenovirus and Aspergillus given prophylactically post-HSCT. Ten patients with acute myeloid leukaemia (n=6) or high risk myelodysplasia (n=4) who overexpressed WT1 and/or PRAME on diagnostic tumour samples received 1 infusion of multipathogen and 1-4 infusions of tumour-specific T-cells (all at 2x107cells/m2). There were no infusion-related severe adverse events. Low level viral reactivations occurred (CMV n=5, EBV n=7, Adenovirus n=1), however none required treatment. There were no cases of viral tissue disease or invasive fungal infections. At a median 2 years post-transplant, overall survival was 80%, all surviving patients were in complete remission and 6/8 patients had ECOG 0/1. Acute GVHD occurred in 2/10 patients, chronic GVHD in another 2/10. Infusion was associated with rapid, sustained reconstitution of pathogen- and tumour-specific immunity as measured by MHC tetramer for CMV and T-cell receptor based clone tracking. This novel combination of T-cell therapies was safe and associated with excellent clinical outcomes.

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.

7039 Background: Disease relapse and infection cause significant morbidity and mortality after allogeneic HSCT for acute myeloid leukemia (AML) and myelodysplasia (MDS). Wilms’ tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) are both commonly overexpressed in these conditions. We assessed the safety of a novel combination of tumour associated antigen (TAA) and multipathogen (MP) specific T cells administered prophylactically after HSCT in a phase 1 trial. Methods: Patients with overexpression of WT1 or PRAME by ddPCR on diagnostic tumour samples were eligible. TAA and MP specific T cells were ex vivo expanded from stem cell donors by stimulating apheresis derived mononuclear cells with tumour, viral or fungal peptides. T cells specific for CMV, EBV, Adenovirus (AdV) and Aspergillus (Asp) antigens were produced separately and pooled in equal parts into a MP product. Patients received 1 infusion of MP specific and up to 4 infusions of TAA specific T cells at 4-weekly intervals from 28 days post HSCT (cell dose 2x107/m2 per infusion). Results: Ten HSCT recipients have received a total of 38 infusions. Median age was 48 years (17-67), disease AML (n = 6) or high risk MDS (n = 4), DRI intermediate (n = 4) or high (n = 6), conditioning myeloablative (n = 8) or reduced intensity (n = 2), donor source sibling (n = 7) or matched unrelated (n = 3). Median expression of WT1 on diagnostic bone marrow was 1442 copies/104 copies ABL (0-3870), PRAME 131 copies/104 copies ABL (4-12300). Patients received WT1 (n = 4), PRAME (n = 5) or both WT1 and PRAME specific T cells (n = 1). Mean tumour antigen specificity in the TAA product was 1.4% and 13.3% of CD3+ cells for WT1 and PRAME respectively. Mean total pathogen specificity in the MP product was approximately 44% (CMV = 14.0%, EBV = 14.8% and AdV = 11.6% of CD3+ cells, Asp = 14.8% of CD4+ cells). All patients received MP specific T cells. No immediate infusion related adverse events were reported. At a median 540 days post transplant (80-1265), 8 of 10 patients remain alive and in complete disease remission. Two patients did not proceed after completing 3 of 5 infusions due to the development of graft versus host disease (GVHD); both are in remission. There were 2 deaths; one patient with progressive disease who had persistent high risk MDS pre and post HSCT, and one with multiorgan failure who had multiple post transplant complications (venoocclusive disease, sepsis, GVHD), both prior to infusion. Low level viral reactivations occurred (CMV n = 5, EBV n = 7, BKV n = 3, HHV6 n = 4, AdV n = 1), however none required treatment and there were no cases of viral tissue disease or EBV PTLD. There were no invasive fungal infections. Conclusions: Prophylactic infusions of donor derived WT1/PRAME and multipathogen specific T cells post HSCT are well tolerated and associated with low rates of infection and relapse. Clinical trial information: NCT02895412.

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(+) stem cell isolation was used to minimize the development of acute and chronic GVHD. Two prophylactic infusions, one combining donor-derived cytomegalovirus, Epstein-Barr virus, and Aspergillus fumigatus specific T-cells and the other comprising donor-derived CD19 directed chimeric antigen receptor (CAR) bearing T-cells, were given 21-28 days after transplant. Two patients were transplanted for acute lymphoblastic leukemia from HLA identical siblings using standard doses of cyclophosphamide and total body irradiation without antilymphocyte globulin. Patients received no post-transplant immune suppression and were given no pre-CAR T-cell lymphodepletion. Neutrophil and platelet engraftment was prompt. Following adoptive T-cell infusions, there was rapid appearance of antigen-experienced CD8(+) and to a lesser extent CD4(+) T-cells. Tetramer-positive T-cells targeting CMV and EBV appeared rapidly after T-cell infusion and persisted for at least 1 year. CAR T-cell expansion occurred and persisted for up to 3 months. T-cell receptor tracking confirmed the presence of product-derived T-cell clones in blood targeting all three pathogens. Both patients are alive over 3 years post-transplant without evidence of GVHD or disease recurrence. Combining robust donor T-cell depletion with directed T-cell adoptive immunotherapy targeting infectious and malignant antigens permits independent modulation of GVHD, infection, and disease recurrence. The combination may separate GVHD from the graft versus tumor effect, accelerate immune reconstitution, and improve transplant tolerability.

Abstract 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.

Abstract 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.

Post-transplant lymphoproliferative disorder (PTLD) is an uncommon complication of allogeneic haematopoietic stem cell transplantation (HSCT). Incidence ranges from 1% to 11%, with higher rates seen with mismatched and unrelated donors.1 Plasma-cell PTLD is a rare subtype of monomorphic PTLD, accounting for 4–6% of cases in solid-organ transplant (SOT) recipients;2-4 incidence is not characterised in the post-HSCT setting. Plasma-cell PTLD presents a particular treatment quandary. The mainstay of PTLD therapy comprises reduction of immunosuppression (RI) and rituximab alone or in combination with chemotherapy. In plasma cell PTLD, RI is not associated with long-term survival,4, 5 rituximab is not effective given CD20 negativity, and treatments incorporating chemotherapy have reduced overall survival (OS) compared with non-chemotherapy-based regimens.5 Proteasome inhibitor-based regimens are being increasingly used with improving survival; however, prognosis remains poor with median a OS of 2·4 years.5 Novel approaches to treatment are needed in this space. In the present study, we describe a particularly unusual case of plasma cell PTLD successfully treated with one such novel approach to treatment – adoptive cell therapy with Epstein–Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs). A 24-year-old female underwent fludarabine/melphalan/anti-thymocyte globulin conditioned matched unrelated donor HSCT for EBV-negative Hodgkin lymphoma. EBV serostatus was donor negative/recipient positive. After an uncomplicated transplant admission, she was readmitted on day+45 with rapidly enlarging cervical lymphadenopathy, abdominal pain and melaena. The EBV viral load (VL) had risen from undetectable to 4164 iu/ml. A positron emission tomography (PET) scan demonstrated intense fluorodeoxyglucose-avid uptake in nodes above and below the diaphragm, with extra-nodal uptake in the bowel and spleen (Fig 1B). Right cervical lymph node core biopsy revealed effacement by plasma-cell PTLD, with immunohistochemistry demonstrating CD138 and CD20 negativity, CD38 and EBV positivity by Epstein–Barr encoding region in situ hybridisation, and cytoplasmic kappa light chain restriction; biopsy of the small and large bowel identified the same population, with no features of graft-versus-host disease (GVHD). Plasma cell flow cytometry was not performed on nodal and gut tissue. Bone marrow biopsy demonstrated a clonal plasma cell infiltrate accounting for 20% of cellularity, which in contradistinction to node and gut biopsy was CD138 positive by immunohistochemistry and flow cytometry. Inverse EBV seromismatch and early post-transplant presentation suggested recipient-origin PTLD; molecular chimerism by short tandem repeat analysis on nodal tumour tissue confirmed this, with 84% recipient origin in tumour on day+50 post-transplant, while peripheral blood donor chimerism was 100%. Initial management comprised a single dose of rituximab 375 g/m2 prior to demonstration of CD20 negativity and concomitant rapid wean of cyclosporin immunosuppression from 225 mg twice daily to cessation at 14 days. No response was achieved, with twice-weekly monitoring of EBV demonstrating ongoing rise in VL to a peak of 92 648 iu/ml. Worsening abdominal pain requiring total parenteral nutrition ensued alongside a rise in total protein and globulins to 105 g/l and 88 g/l respectively, and hyperviscosity syndrome developed with headaches and visual impairment. Ophthalmoscopy demonstrated dilated central retinal veins, cotton wool spots and intra-retinal haemorrhages. Serum protein electrophoresis revealed an immunoglobulin M (IgM) paraprotein of 67g/l; myeloid differentiation factor 88 (MYD88)L265P mutation was negative. Urgent plasmapheresis was performed with resolution of abdominal pain, headache and visual impairment. Given the clinical response of abdominal pain to plasmapheresis, this was favoured to be related to evolving PTLD and hyperviscosity rather than emergent gastrointestinal GVHD; endoscopy was not repeated. The patient was enrolled in a clinical trial of partially human leucocyte antigen (HLA)-matched third-party donor EBV-specific CTLs and infused with 2 × 107 CTL/m2 on day+17 following presentation with PTLD (day+62 post-transplant). Clinical and biochemical response occurred within days, with fall in EBV VL, resolution of lymphadenopathy and reduction in paraprotein. Persisting low-level EBV viraemia was noted 12 weeks after infusion, with 5% residual disease seen on repeat bone marrow biopsy, and restaging PET demonstrating partial response with resolution of nodal disease and ongoing albeit improved bowel activity (Fig 1C). A second CTL infusion (same donor/dose) for residual disease was given on day+86 following presentation with PTLD (day+131 post-transplant). PET complete metabolic response was subsequently achieved (Fig 1D) and the EBV VL fell below the detection threshold. At 18 months post-transplant the patient remains in ongoing PET complete metabolic response, with morphological and immunophenotypic complete remission on bone marrow biopsy, undetectable EBV and no GVHD. However, evidence of minimal residual disease is noted biochemically, with IgM kappa paraprotein remaining detectable below the level of quantitation. Serial PET-computed tomography and EBV VL/paraprotein trends relative to CTL infusion are depicted in Figs 1, 2. The present case is particularly unusual in several aspects. In HSCT patients, PTLD typically derives from the donor graft and recipient-derived plasma-cell PTLD is exceedingly rare.6 Furthermore, plasma cell PTLD is nearly always CD138 positive,3, 4, 7 with only one prior published case of CD138 negativity.3 The immunophenotypically distinct CD138-negative gut/nodal disease identified in the present case may indicate evolution of a more aggressive subclone; indeed, in de novo myeloma CD138-negative populations are characterised as more immature and proliferative than CD138-positive populations.8 While false-negative immunostaining cannot be excluded, this was thought to be less likely given CD138 negativity by immunohistochemistry across multiple separately fixed gut and nodal samples with positive staining of control tissue. Finally, while plasma-cell PTLD is commonly associated with paraproteinaemia, typically only modest elevations are seen.2, 4 De novo IgM myeloma accounts for <0·5% of cases,9 and only rare cases of IgM secretory plasma-cell PTLD are reported.4, 10 The high-level IgM paraprotein and hyperviscosity syndrome seen in the present case is atypical although not undescribed, as a previous case of plasma-cell PTLD presenting with IgM hyperviscosity syndrome has been reported in a paediatric liver transplant recipient.10 With half of plasma-cell PTLD cases expressing EBV,3, 4, 7 adoptive immunotherapy with EBV-CTLs is a promising emerging treatment. EBV-CTLs may be delivered within an unmanipulated donor lymphocyte infusion (DLI) or as a selectively expanded line of EBV-CTLs of donor, recipient, or third-party origin. In HSCT-recipients EBV-CTLs have been shown to both prevent and successfully treat EBV-PTLD.11-13 Outcomes vary by CTL source, with response rates of 70% for DLI, 70–90% for donor-derived EBV-CTLs, and 50–70% for third-party CTLs.12 Third party ‘off-the-shelf’ CTLs have the benefit of rapid availability and negligible GVHD risk.12 A prospective phase II trial of third-party EBV-CTLs in 31 SOT and two HSCT recipients with EBV-PTLD refractory to conventional therapy demonstrated 64% and 52% response rates at 5-weeks and 6-months, with better outcomes seen in best-HLA-matched donor CTLs.13 No studies to date have reported outcomes of EBV-CTL therapy by subtype of PTLD. This is to our knowledge the first reported case of successful treatment with EBV-CTLs in the plasma-cell subtype of PTLD, highlighting EBV-CTLs as a promising therapeutic option for future cases. Jessica Elliott and Adrian G. Selim completed the case summary and literature review; Jessica Elliott wrote the manuscript; Adrian G. Selim, David Ritchie, David Gottlieb and Ashish Bajel revised and reviewed the manuscript; Selmir Avdic, Leighton Clancy, Elissa Atkins, Emily Blyth, and David Gottlieb researched and supplied clinical, scientific and trial-related information.

Primary central nervous system lymphoma (PCNSL) occurring following organ transplantation (post-transplantation lymphoproliferative disorder [PTLD]) is a highly aggressive non-Hodgkin lymphoma. It is typically treated with high-dose methotrexate-based regimens. Outcomes are dismal and clinical trials are lacking. It is almost always Epstein-Barr virus (EBV) associated. Two patients (CA1-2) presented with EBV-associated PCNSL after renal transplant. CA1 was on hemodialysis and had prior disseminated cryptococcus and pseudomonas bronchiectasis, precluding treatment with methotrexate. CA2 was refractory to methotrexate. Both were treated off-label with the first-generation Bruton's tyrosine kinase inhibitor ibrutinib for 12 months. Cerebrospinal fluid penetration at therapeutic levels was confirmed in CA1 despite hemodialysis. Both patients entered remission by 2 months. Sequencing confirmed absence of genetic aberrations in human leukocyte antigen (HLA) class I/II and antigen-presentation/processing genes, indicating retention of the ability to present EBV-antigens. Between Weeks 10 and 13, they received third-party EBV-specific T cells for consolidation with no adverse effects. They remain in remission ≥34 months since therapy began. The strength of these findings led to an ongoing phase I study (ACTRN12618001541291).

We performed a Phase I clinical trial of donor derived CD19-specific chimeric antigen receptor T-cells (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, one patient developed a gradually enlarging retroperitoneal tumor due to a CAR expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection of a second CAR T-cell tumor in thoracic para-aortic lymph nodes in an asymptomatic patient. 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 one patient died. We describe the first two 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. The trial was registered at www.anzctr.org.au as ACTRN12617001579381.

Abstract 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.

Introduction Disease relapse and infection cause significant morbidity and mortality after allogeneic HSCT for AML and MDS. Wilms' tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) are both commonly overexpressed in these conditions, and are attractive targets for immunotherapy. We have assessed the safety of a novel combination of tumour associated antigen (TAA) specific and multipathogen (MP) specific T cells administered prophylactically after HSCT in a phase 1 trial. Methods Patients were eligible for the study if WT1 or PRAME gene expression was elevated as determined by droplet digital PCR on diagnostic tumour samples. TAA and MP specific T cells were generated from stem cell donors by stimulating apheresis-derived mononuclear cells with autologous antigen presenting cells expressing tumour, viral or fungal antigens. T cells specific for CMV, EBV and Aspergillus antigens were produced separately and pooled in equal parts into a MP product. Patients received 1 infusion of MP specific T cells and up to 4 infusions of TAA specific T cells at 4-weekly intervals dosed at 2x107/m2, from 28 days post HSCT. Results Seven HSCT recipients have received a total of 26 T cell infusions to date. Median age was 49 years (range 26-67), disease AML (n=4) or high risk MDS (n=3), conditioning myeloablative (n=6) or reduced-intensity (n=1), donor source sibling (n=4) or matched unrelated (n=3). Median expression of WT1 on diagnostic bone marrow tissue was 1464 copies/104 copies of ABL (0-3870), PRAME 131 copies/104 copies of ABL (4-1670). Mean tumour antigen specificity in the TAA product was 2.2% of CD3+ cells for WT1 and 7.3% of CD4+ cells for PRAME. Mean total pathogen specificity in the MP product was approximately 15% (CMV=4.7% and EBV=5.4% of CD3+ cells, Aspergillus=5.3% of CD4+ cells). Patients received WT1 specific (n=3), PRAME specific (n=3) or both WT1 and PRAME specific T cells (n=1). All patients received MP specific T cells. No immediate infusion-related adverse events were reported. At the time of report, at a median of 375 days post-transplant (80-847), 5 out of 7 patients remain alive. Four patients remain in complete disease remission without graft versus host disease (GVHD). One patient did not proceed after 3 of 5 planned infusions after developing chronic lung GVHD but remains in disease remission. There have been 2 deaths (progressive disease and multiorgan failure). The patient with progressive disease had MDS with complex cytogenetics with evidence of persistent disease pre and post-HSCT, prior to T cell infusions. The patient with multiorgan failure had multiple post-transplant complications including bacterial sepsis, hepatic venoocclusive disease and grade 3 acute GVHD of the gut prior to infusion. Patients had low level viral reactivation (CMV n=3, EBV n=5, BKV n=3, HHV6 n=2), however none required treatment and there were no cases of viral tissue disease or EBV post-transplant lymphoproliferative disorder. There were no invasive fungal infections. Conclusion Prophylactic infusions of donor derived WT1/PRAME specific and multipathogen specific T cells post HSCT are well-tolerated and associated with low rates of infection and relapse in patients treated to date. Disclosures No relevant conflicts of interest to declare.

Viral infections, principally cytomegalovirus, Epstein Barr virus (EBV) and adenovirus, are a leading cause of morbidity and mortality after allogeneic stem cell transplantation. The use of systemic antivirals is limited by limited efficacy and organ toxicities. Inability to clear infection is exacerbated by transplant‐related immunosuppression and prophylaxis or treatment of acute graft versus host disease. We report the first patient to clear three serious viral infections after stem cell transplant using third‐party donor partially human leukocyte antigen (HLA) matched virus‐specific cytotoxic T cells. The patient, a 53 year old female with transplanted for relapsed leukemia, with severe graft versus host disease received five T cell infusions from three separate donors that ultimately cleared serious systemic infections with cytomegalovirus and adenovirus, and an EBV‐driven lymphoma. Systemic antivirals had resulted in failed clinical responses. Use of repeated infusions of partially HLA matched virus‐specific T cells from banks containing cryopreserved cells should be strongly considered in transplant recipients with single or multiple refractory viral infections.

Invasive fungal infections are a major cause of disease and death in immunocompromised hosts, including patients undergoing allogeneic hematopoietic stem cell transplant (HSCT). Recovery of adaptive immunity after HSCT correlates strongly with recovery from fungal infection. Using initial selection of lymphocytes expressing the activation marker CD137 after fungal stimulation, we rapidly expanded a population of mainly CD4(+) T cells with potent antifungal characteristics, including production of tumor necrosis factor alpha, interferon gamma, interleukin-17, and granulocyte-macrophage colony stimulating factor. Cells were manufactured using a fully good manufacturing practice-compliant process. In vitro, the T cells responded to fungal antigens presented on fully and partially HLA-DRB1 antigen-matched presenting cells, including when the single common DRB1 antigen was allelically mismatched. Administration of antifungal T cells lead to reduction in the severity of pulmonary and cerebral infection in an experimental mouse model of Aspergillus. These data support the establishment of a bank of cryopreserved fungus-specific T cells using normal donors with common HLA DRB1 molecules and testing of partially HLA-matched third-party donor fungus-specific T cells as a potential therapeutic in patients with invasive fungal infection after HSCT.

Abstract Objective Adoptive immunotherapy with ex vivo expanded tumor‐specific T cells has potential as anticancer therapy. Preferentially expressed antigen in melanoma (PRAME) is an attractive target overexpressed in several cancers including melanoma and acute myeloid leukaemia (AML), with low expression in normal tissue outside the gonads. We developed a GMP‐compliant manufacturing method for PRAME‐specific T cells from healthy donors for adoptive immunotherapy. Methods Mononuclear cells were pulsed with PRAME 15‐mer overlapping peptide mix. After 16 h, activated cells expressing CD137 were isolated with immunomagnetic beads and cocultured with irradiated CD137neg fraction in medium supplemented with interleukin (IL)‐2, IL‐7 and IL‐15. Cultured T cells were restimulated with antigen‐pulsed autologous cells after 10 days. Cellular phenotype and cytokine response following antigen re‐exposure were assessed with flow cytometry, enzyme‐linked immunospot (ELISPOT) and supernatant cytokine detection. Detailed phenotypic and functional analysis with mass cytometry and T‐cell receptor (TCR) beta clonality studies were performed on selected cultures. Results PRAME‐stimulated cultures (n = 10) had mean expansion of 2500‐fold at day 18. Mean CD3+ percentage was 96% with CD4:CD8 ratio of 4:1. Re‐exposure to PRAME peptide mixture showed enrichment of CD4 cells expressing interferon (IFN)‐γ (mean: 12.2%) and TNF‐α (mean: 19.7%). Central and effector memory cells were 23% and 72%, respectively, with 24% T cells expressing PD1. Mass cytometry showed predominance of Th1 phenotype (CXCR3+/CCR4neg/CCR6neg/Tbet+, mean: 73%) and cytokine production including IL‐2, IL‐4, IL‐8, IL‐13 and GM‐CSF (2%, 6%, 8%, 4% and 11%, respectively). Conclusion PRAME‐specific T cells for adoptive immunotherapy were enriched from healthy donor mononuclear cells. The products were oligoclonal, exhibited Th1 phenotype and produced multiple cytokines.

AbstractObjectivesCytomegalovirus (CMV) is known to have a significant impact on immune recovery post‐allogeneic haemopoietic stem cell transplant (HSCT). Adoptive therapy with donor‐derived or third‐party virus‐specific T cells (VST) can restore CMV immunity leading to clinical benefit in prevention and treatment of post‐HSCT infection. We developed a mass cytometry approach to study natural immune recovery post‐HSCT and assess the mechanisms underlying the clinical benefits observed in recipients of VST.MethodsA mass cytometry panel of 38 antibodies was utilised for global immune assessment (72 canonical innate and adaptive immune subsets) in HSCT recipients undergoing natural post‐HSCT recovery (n = 13) and HSCT recipients who received third‐party donor‐derived CMV‐VST as salvage for unresponsive CMV reactivation (n = 8).ResultsMass cytometry identified distinct immune signatures associated with CMV characterised by a predominance of innate cells (monocytes and NK) seen early and an adaptive signature with activated CD8+ T cells seen later. All CMV‐VST recipients had failed standard antiviral pharmacotherapy as a criterion for trial involvement; 5/8 had failed to develop the adaptive immune signature by study enrolment despite significant CMV antigen exposure. Of these, VST administration resulted in development of the adaptive signature in association with CMV control in three patients. Failure to respond to CMV‐VST in one patient was associated with persistent absence of the adaptive immune signature.ConclusionThe clinical benefit of CMV‐VST may be mediated by the recovery of an adaptive immune signature characterised by activated CD8+ T cells.

Infectious diseases contribute significantly to morbidity and mortality in recipients of allogeneic haematopoietic stem cell transplantation (aHSCT), particularly in the era of highly immunosuppressive transplant regimens and alternate donor transplants. Delayed cellular immune recovery is a major mechanism for the increased risk in these patients. Adoptive cell therapy with ex vivo manipulated pathogen-specific T cells (PSTs) is increasingly taking its place as a treatment strategy using donor-derived or third party–banked cells. The majority of clinical trial data in the form of early-phase studies has been in the prophylaxis or treatment of cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV). Advancements in methods to select and enrich PSTs offer the opportunity to target the less common viral pathogens as well as fungi with this technology. Early clinical studies of PSTs targeting polyomaviruses (BK virus and JC virus), human herpesvirus 6 (HHV6), varicella zoster virus (VZV) and Aspergillus spp. have shown promising results in small numbers of patients. Other potential targets include herpes simplex virus (HSV), respiratory viruses and other invasive fungal species. In this review, we describe the burden of disease of this wider spectrum of pathogens, the progress in the development of manufacturing capability, early clinical results and the opportunities and challenges for implementation in the clinic.

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.