PHYSICIANS ABSTRACTS

Van Bekkum Award

rapidly to IL-6-targeted anti-cytokine treatment. This approach has promise as a salvage therapy for patients who relapse after allo SCT, and collection of tolerized cells from the recipient appears to have a low risk of GVHD. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL. Disclosure of Interest: None Declared.

PH-O001 T CELLS ENGINEERED WITH A CHIMERIC ANTIGEN RECEPTOR (CAR) TARGETING CD19 (CTL019 CELLS) PRODUCE SIGNIFICANT IN VIVO PROLIFERATION, COMPLETE RESPONSES AND LONG-TERM PERSISTENCE WITHOUT GVHD IN CHILDREN AND ADULTS WITH RELAPSED, REFRACTORY ALL S. Grupp1,*, N. Frey2, R. Aplenc1, B. Levine3, S. Maude4, S. Rheingold1, C. Strait Barker4, D. Teachey1, Y. Mahnke3, D. Porter5, C. June3 1 Division of Oncology, Department of Pediatrics, Children’s Hospital and U. Penn School of Medicine, 2Heme Malignancies Program, Abramson Cancer Center and U. Penn School of Medicine, 3Pathology and Lab Medicine, U. Penn School of Medicine, 4Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, 5 Transplant Program, Abramson Cancer Center and U. Penn School of Medicine, Philadelphia, United States

Presidential Symposium PH-O002 RESULTS OF AN OPEN-LABEL, PROSPECTIVE, RANDOMIZED CLINICAL TRIAL ON TWO DIFFERENT DOSAGES OF RABBIT ANTI-THYMOCYTE GLOBULIN (RATG) IN CHILDREN WITH HEMATOLOGICAL MALIGNANCIES GIVEN ALLOGENEIC HSCT FROM AN UNRELATED VOLUNTEER F. Locatelli1,*, M. E. Bernardo2, C. Rognoni3, G. Giorgiani4, A. Rovelli5, A. Pession6, F. Fagioli7, C. Favre8, E. Lanino9, P. Comoli4, A. Bertaina2, A. Prete6, M. Zecca4 1 IRCCS Bambino Gesù Children’s Hospital and University of Pavia, 2 IRCCS Bambino Gesù Children’s Hospital, Rome, 3Laboratory of Medical Informatics, University of Pavia, 4IRCCS Fondazione Policlinico San Matteo, Pavia, 5Ospedale San Gerardo, Monza, 6 University of Bologna, Ospedale S. Orsola, Bologna, 7Ospedale Infantile Regina Margherita, Torino, 8Ospedale di Pisa, Pisa, 9Giannina Gaslini Institute, Genoa, Italy

Introduction: CARs combine antigen recognition and intracellular signaling domains into a single chimeric protein. We previously reported on CTL019 cells expressing a CAR with intracellular activation plus costimulatory domains. Infusion of CTL019 cells results in up to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence, including a sustained CR in 1 of 2 pts with ALL (Grupp, et al. NEJM 2013). We now report on outcomes and longer follow up from our pediatric and and now adult pilot studies treating 27 pts (22 children and 5 adults) with relapsed, refractory ALL with CD19-directed CAR-modified CTL019 T cells. Materials (or patients) and Methods: T cells were lentivirally transduced with a CAR composed of an anti-CD19 scFv plus 4-1BB/ CD3ζ signaling domains, activated/expanded ex-vivo with antiCD3/anti-CD28 beads, and then infused. The pediatric CTL019 range was 107 to 108 cells/kg with a transduction efficiency (TE) of 11–45%. On the adult protocol, the target dose was 5×109 total cells split over 3 days with TE of 6–31%. 15/27 pts had relapsed ALL after a prior allogeneic SCT. T cells were collected from the pt regardless of prior SCT status. Results: 22 children median age 9y and 5 adults median age 50y with CD19+ ALL were treated. One child had T cell ALL aberrantly expressing CD19. 24/27 pts had active disease immediately prior to CTL019 infusion, while 3 were CR and/or MRD(-). A median of 3.7×106 CTL019 cells/kg (0.7–18×106/kg) were infused over 1–3 days. 24/27 pts (89%) achieved a CR, including the pt with CD19+ T cell ALL. 13/19 pts have ongoing BM CR with median follow up 3.4 mo (2–18 mo), with 6 relapses, 2 with CD19(-) disease. All responding pts developed some degree of delayed cytokine release syndrome (CRS) coincident with peak T cell expansion, some severe. Cytokine analysis showed marked increases of IL-6 and IFNγ. Treatment for CRS that included significant hypotension or respiratory instability was required in ~30% of pts with the IL-6 receptor antagonist tocilizumab, with rapid resolution of CRS. No GVHD has been seen in pts who were s/p allo SCT, although the cells collected from pts s/p allo SCT were median 100% donor origin. Persistence of CTL019 cells was seen up to 18mo. The persistent CTL019 T cells included T central memory and T stem central memory cells out to at least 1 year. Discussion: CTL019 cells are T cells genetically engineered to express an anti-CD19 scFv coupled to CD3ζ signaling and 4-1BB costimulatory domains. These cells can undergo robust in-vivo expansion and can persist for >18mo in pts with relapsed ALL. CTL019 therapy is associated with a significant CRS that responds

Introduction: ATG is largely employed for preventing GvHD in patients given unrelated donor (UD) allogeneic HSCT. However, the optimal drug dose to be employed is still undefined. We conducted a prospective, open-label, multicenter, randomized clinical trial (RCT) on two different dosages of rATG in children with malignancies transplanted with either BM or PBSC from an UD, selected using high-resolution molecular typing for class I/II HLA loci. Materials (or patients) and Methods: Included in the study were 172 patients (median age 9 yrs, range 1–18) with hematological malignancies. Children were classified into standard and high-risk (SR and HR) groups. SR group included 107 children with ALL in 1st CR or 2nd CR belonging to S2 category of BFM classification, AML in 1st CR or in 2nd CR after a relapse occurring 6 months beyond treatment discontinuation, Ph+ CML in 1st chronic phase, refractory cytopenia (RC) and NHL in 2nd CR. HR included 65 patients with ALL in 2nd CR belonging to the S3 and S4 categories or more advanced CR, AML in 2nd CR after recurrence occurring within 6 months from treatment discontinuation and with MDS other than RC. The study aimed to test whether high-dose rATG (30 mg/Kg over days -4, -3 and -2) was superior to a lower dose (15 mg/Kg over the same days) in terms of grade II-IV acute GvHD prevention. Secondary end-points included: cumulative incidence (CI) of extensive chronic GvHD, transplantation-related mortality (TRM), disease recurrence and probability of DFS. Patients were stratified according to the stem cell origin used (BM vs. PBSC), the degree of HLA compatibility in the donor-recipient pair (donor with 0/1 allelic disparity in the GvHD direction vs. donor with 1 antigenic or 2 allelic disparities), and the classification of risk (SR vs. HR). All patients were given a myeloablative regimen, including TBI in 45% of patients. Cyclosporine-A and short-term MTX were employed for post-transplant GvHD prophylaxis in all patients.

S1

Discussion: In conclusion, SB system together with an optimized method of differentiation efficiently expand modified CD123.CAR+ and CD19.CAR+ CIK cells, redirect their activity towards AML and ALL cells, respectively, and retain a safe pattern of integrations in the genome. The development of an easy-clinical grade adoptive cell therapy protocol based on an innovative method of gene transfer will be fundamental to improve the range of applications of immunotherapy to control relapse in leukemic patients. Disclosure of Interest: None Declared.

Results: Clinical characteristics of patients allocated to the 2 randomization arms were comparable. With a median follow-up of 32 months (13–70), the 100-day CI of grade II-IV acute GvHD in the high and low rATG groups were 29% (confidence interval, 20–40) and 36% (28–48), respectively, p=0.27; the CI of extensive chronic GvHD in the two groups was 6% (2–15) and 10% (5–19), respectively, p=0.33. The CI of TRM was 21% (13–33) and 10% (5–19) in the high and low rATG groups, respectively (p=0.06). The CI of disease recurrence was 17% (12-24); it did not differ between high and low dose ATG (p=0.34). The 5-year DFS for the whole cohort of randomized patients was 67% (60–75); it was 59% (48–71) and 76% (67–85) for children given high and low dose rATG (p=0.03). DFS was 76% (67–85) and 54% (42–67) in the SR and HR groups, respectively (p=0.0011). DFS of the 136 children with acute leukemia given either high or low-dose rATG was 58% (44–71) and 76% (66–87), respectively (p=0.049). Discussion: These data indicate that, in an era in which high-resolution typing is used to select UDs, low dose rATG resulted into decreased incidence of TRM and better DFS, without an increased incidence of acute and chronic GvHD or leukemia recurrence in children with malignancies. Disclosure of Interest: None Declared.

PH-O004 UPFRONT MATCHED AND MISMATCHED UNRELATED TRANSPLANTATION IN PAEDIATRIC APLASTIC ANAEMIA: A UNITED KINGDOM MULTICENTRE RETROSPECTIVE EXPERIENCE N. Bhatnagar1,*, R. Wynn2, M. Velangi3, A. Vora4, D. Bonney2, B. Gibson5, R. Skinner6, A.-M. Ewins7, P. Amrolia8, R. Hough9, J. De La Fuente10, A. Shaw11, C. Steward12, P. Veys8, S. Samarasinghe6 1 Department of Haematology, Imperial College London, London, 2Blood and Marrow Transplant Unit, Royal Manchester Children’s Hospital, Manchester, 3Department of Haematology, Birmingham Children’s Hospital, Birmingham, 4Department of Paediatric Haematology, Sheffield Children’s Hospital, Sheffield, 5Department of Paediatric Haematology, Royal Hospital for Sick Children, Yorkhill, Glasgow, 6 Department of Paediatric Bone Marrow Transplantation, Royal Victoria Infirmary, Newcastle upon Tyne, 7Department of Paediatric Haematology, Royal Hospital for Sick Children, Yorkhill, Glasgow, 8 Department of Bone Marrow Transplantation, Great Ormond Street Hospital, 9Department of Adolescent Haematology, University College Hospital, 10Department of Paediatrics and Haematology, St. Mary’s Hospital and Imperial College, London, 11Bone Marrow Transplant Unit, Royal Hospital for Children, 12Bone Marrow Transplant Unit, Royal Hospital for Children, Bristol, United Kingdom

PH-O003 GENE THERAPY OF ACUTE LEUKEMIAS WITH CHIMERIC ANTIGEN RECEPTORS (CARS)-ENGINEERED CYTOKINE INDUCED KILLER (CIK) CELLS BY SLEEPING BEAUTY SYSTEM C. Francesca Magnani1,*, N. Turazzi1, F. Benedicenti2, S. Tettamanti1, G. Maria Paola Giordano Attianese1, V. Rossi1, E. Montini2, L. J. N. Cooper3, A. Aiuti2, A. Biondi1, E. Biagi1 1 Università Milano Bicocca, Osp. San Gerardo/Fondazione MBBM, Centro Ricerca Tettamanti, Clinica Pediatrica, Monza, 2San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy, 3University of Texas, MD Anderson Cancer Center, Houston, United States

Introduction: With the excellent outcomes currently seen in unrelated donor stem cell transplantation in idiopathic severe aplastic anaemia (SAA), it is unclear whether newly diagnosed children lacking a matched sibling donor (MSD) should receive immunotherapy with horse ATG or proceed directly to an unrelated donor stem cell transplant. We therefore retrospectively analysed the outcomes on 27 consecutive upfront matched unrelated (MUD) and mismatched unrelated donor (MMUDs) HSCT for idiopathic SAA using a Fludarabine, Cyclophosphamide and Alemtuzumab (FCC) regimen between 2000 and 2013. None of the 27 children had prior treatment with immunosuppressive therapy. Materials (or patients) and Methods: Data from children and adolescents with idiopathic SAA who received upfront MUD/MMUDs were collected retrospectively using standardised forms from 9 paediatric transplant centres. There was an upper age limit of 19 years inclusive. All patients lacked a MSD. Donor matching was performed by high resolution typing for HLA-A, -B, -C, -DRB1, and -DQB1. 22 patients received MUDs whilst 5 children received single antigen/allele MMUDs. The primary end points were eventfree survival (EFS) and overall survival (OS). Results: Patient /HSCT characteristics are shown in Table 1. There were 12 male (44.4%) and 15 female (55.5%) patients. The median year of diagnosis was 2011 (range 2005–2013). All patients required transfusion support prior to HSCT with a median of 5 red blood cell (range 0–236) and 13 platelet transfusions (range 1–35). The median follow-up was 22 months (range 5–95). The 2.5-year OS following HSCT was 95.7% (95% CI 72.9–99.4%). The 2.5-year EFS following HSCT was 92.11% (95% CI 71.9–98.0%). There was 1 death due to idiopathic pneumonia syndrome at 11 months post-HSCT. Primary graft failure occurred in 1 patient with a HLAA MMUD HSCT. Chimerism data was available for the remaining 25 patients; all had engrafted at last follow-up and 24 had normal blood counts. The median donor chimerism on unfractionated whole blood was 100% (range 88–100). Acute GVHD (grades I-II) was seen in 10 patients (37.0%) and grade III-IV was seen in 2 patients (7.4%). 6 patients developed chronic GVHD (22.2%) all with limited skin disease.

Introduction: T cell engineering with Chimeric Antigen Receptor (CARs) has been recently proved to be effective in redirecting effector activity towards leukemic blasts resistant to conventional chemotherapy and hematopoietic stem cell transplantation (HSCT). Since the profile of efficacy, safety and feasibility of cell manufacturing and gene therapy by viral vectors still remain major concerns, we explored here the use of Sleeping Beauty Transposon-mediated gene transfer in cytokine-induced killer (CIK) cells for targeting Acute Leukemias. Materials (or patients) and Methods: We generated and propagated CAR+ CIK cells using nucleofection and SB Transposase-Transposon system. Actually, current protocol of nucleofection associated with Transposon system impaired subsequent expansion and vitality of modified cells. With an optimized clinical-grade stimulation protocol, we genetically modified CIK cells to express two distinct 3rd generation CARs specific for myelogenous leukemia (AML) CD123+ or acute lymphoblastic leukemia (ALL) CD19+ blasts. Results: The nucleofection minimally affected the phenotype of CIK cells, and, most importantly, the optimized protocol was effective in inducing T-cell expansion, with a fold increase of 33.97 (CD123-CAR) and 32.56 (CD19-CAR) within 3 weeks, sufficient to be translated into adoptive cell therapy clinical protocols. Modified CIK cells displayed stable expression of CD123-CAR or CD19-CAR with a frequency of 51.43% (n=15) and 48.78% (n=7), respectively. Modified CIK cells exerted efficient lysis of leukemic cell lines and primary blasts, that was demonstrated to be driven by specific CAR-mediated target recognition. Interestingly, CAR triggering by the antigen expressed by leukemic cells promoted specific cytokine secretion and proliferation that was restricted to the modified fraction of CIK cells, suggesting activation and selection of modified CIK cells upon encounter with cancer cells in the patients. The loss of the expression of transposase during the differentiation was assessed to assure the genome stability of the cellular product treated by SB system by absolute quantification through RT-PCR. Finally, preliminary insertion-site analysis by LAM-PCR confirmed that the integrations in the genome of SB system do not correlate with the genes-enriched regions.

S2

[PH-O004] Table I Upfront MUD/MMUD HSCT (n=27) 7.6 (0.6-19.7) 22 (84.6%):4 (15.4%) 2012 (2005-2013) 4 (1-16) 18 (66.7%):9 (33.3%) 7.00 (2.4-50) 39 (17-105) 17.5 (9-29) 18 (10-40)

Median age at HSCT (range), years Ethnicity (Caucasian:Other) (n=26) Median year of HSCT (range) Median interval between diagnosis and HSCT (range), months Stem cell source: Bone marrow/PBSC Median CD34 cell dose (x106/kg) (n=23) Median In-patient hospital stay (range), days Median time to neutrophil recovery (>0.5x109/l), days (range) Median time to platelet recovery (>50x109/l), days (range)

There were 3 cases (11.1%) of adenoviraemia-1 treated with cidofovir. Epstein-Barr virus (EBV) viraemia was seen in 6 patients (22.2%), 2 treated with rituximab. There was 1 case of EBV associated post-transplant lymphoproliferative disease which was successfully treated with rituximab only. 7 children developed cytomegalovirus viraemia (25.9%), 5 required treatment. Discussion: Our data demonstrate that upfront MUD/MMUD in paediatric idiopathic SAA has excellent outcomes. An upfront MUD may be considered first line treatment when children lack a MSD and a MUD is readily available. Disclosure of Interest: None Declared.

risk groups. The desired features of the algorithm were clarity of risk stratification, simplicity, and reproducibility. We applied the algorithm in a first validation set from the two centers (n=164), and then in a second validation set of 303 samples obtained from pts enrolled in two multicenter trials of primary treatment for acute GVHD conducted by the BMT CTN (0302, 0802). In BMT CTN 0302 (n=109) pts were treated with steroids (2 mg/kg/d methylprednisolone or equivalent) and either MMF, etanercept, denileukin diftitox, or pentostatin; in BMT CTN 0802 (n=194) the investigational agents were MMF or placebo. There were no survival differences between any of the treatment groups, including placebo, and therefore all data were combined into a single validation set. Results: An algorithm using TNFR1, ST2, and REG3α performed as well as that incorporating all 5 biomarkers. The algorithm successfully divided pts in the training set into three grades with 6m NRM of 9%, 28%, and 47%, respectively (Panel A). In the validation set, the algorithm defined three grades with almost identical 6m NRM (Panel B). In the second validation set of BMT CTN pts, the algorithm again clearly identified the same three groups (Panel C), while Glucksberg grades of I, II and III/IV did not (14%, 20% and 26%, respectively). In both cohorts, AA3 pts were more likely to have steroid refractory GVHD than AA2 pts (52% vs 30%, p=0.004; 60% vs 34%, p3 months symptomatic remission as judged by a normal Crohn’s Disease Activity Index [CDAI], without immunosuppressive drugs and no endoscopic or radiological evidence of active disease at one year) by intention-to-treat analysis. Early dropouts were regarded as treatment failures with last assessments carried forward for numerical endpoints. Results: From July 2007 to March 2013, 23 patients underwent HSCT and 22 were controls. Only two SCT patients achieved all criteria for Sustained Disease Remission versus one in controls (p=1). However 14 HSCT patients (64%) versus four (19%) were off immunosuppressive drugs for >3 months (p=0.007). Ten versus two had a Crohn’s Disease Activity Index 3 months, p=0.07). Eight versus two patients were adjudicated endoscopically and radiologically free of active disease at final assessment (p=0.07). There were 76 serious adverse events in HSCT patients versus 38 in controls. One HSCT patient died. Off Rx last 3/12 Normal CDAI And off Rx last 3/12 Normal CDAI > last 3/12 And off Rx last 3/12 Free of active disease And off Rx 3/12 Sustained Disease Remission

HSCT Control 14 (61%) 4 (19%) 10 (43%) 2 (9%) 9 (39%) 2 (9%) 8 (35%) 2 (9%) 7 (30%) 2 (9%) 8 (35%) 2 (9%) 4 (17%) 0 (0%) 2 (9%) 1 (5%)

PH-O007 TRACKING T CELL DYNAMICS IN THE FIRST MONTH AFTER ALLOGENEIC HSCT OFFERS A UNIQUE OPPORTUNITY TO UNVEIL THE MECHANISM OF MEMORY STEM T CELL FORMATION IN HUMANS N. Cieri1,2,3,*, J. Peccatori2, G. Oliveira1,3, R. Greco2,3, C. Taccioli4, M. Forcato4, M. Noviello1, V. Valtolina1, A. Bondanza1, M. Morelli2, F. Lunghi2, S. Marktel2, L. Bellio5, S. Bicciato4, F. Ciceri2, C. Bonini1 1 Experimental Hematology Unit, 2Hematology and Bone Marrow transplantation Unit, San Raffaele Scientific Institute, 3Vita-Salute San Raffaele University, Milan, 4Center for Genome Research, Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, 5Immunohematology and Transfusion Medicine Unit, San Raffaele Scientific Institute, Milan, Italy Introduction: The mechanisms of T cell memory formation in vivo have been an area of intense research, both because they concern a fundamental aspect of adaptive immunity and because the induction of robust memory is the goal of any immunotherapeutic strategy. However, which is the smallest unit from which simultaneous generation of distinct T cell subsets occurs and what are the environmental or cell-intrinsic requisites for diversification remain largely unknown, especially in humans. With this respect, allogeneic hematopoietic stem cell transplantation (HSCT) offers a unique opportunity to address these issues directly in vivo. Materials (or patients) and Methods: We characterized T cell dynamics during the first month post HSCT in 20 patients receiving myeloablative conditioning, T-replete haploidentical peripheral blood stem cell graft, and GvHD prophylaxis consisting of cyclophosphamide (PT-Cy day 3,4), MMF and sirolimus from day 5. Results: Upon infusion, naïve T cells (TN) cells gradually disappeared from circulation, and by day 8 post HSCT the peripheral T cell compartment was composed by memory lymphocytes, including the recently described stem memory T cells (TSCM). Strikingly, at day 8, TSCM cells were the most represented circulating subset, and their frequency was significantly higher than within the infused graft. To investigate whether such high TSCM frequency was due to expansion of TSCM cells infused within the graft or to their direct in vivo

P value 0.006 0.02 0.04 0.07 0.11 0.07 0.11 1

S4

TCRB sequencing. Sequences uniquely harbored by TN cells within apheresis were retrieved in all memory subsets at day 30 after HSCT, demonstrating that TN cells were able to generate the entire spectrum of immunological memory, including TSCM. We next validated these results at the antigen-specific level. In 5 patients suitable for dextramer analysis, we recorded the presence of WT1 or PRAME-specific TN cells within donor apheresis. Of notice, upon HSCT, tumor-specific T cells increased in frequency and converted to a memory phenotype, including TSCM. Finally, by quantifying patient serum cytokines, we found that the degree of IL-7 availability at day 3 post HSCT dictated the extent of TSCM expansion at day 8. Discussion: These data unveil, for the first time in humans, the mechanisms of TSCM generation in vivo. Ongoing analyses will define correlations with clinical events. Disclosure of Interest: None Declared.

generation, T cell proliferation was analyzed. At day 3 upon in vivo infusion (and in the absence of immunosuppressive agents), all memory subsets robustly proliferated, with TSCM cells displaying significantly higher frequencies of Ki-67+ cells compared to all other subsets. In sharp contrast, TN cells were scantly Ki-67+, in line with the longer timeframe required for their priming. PT-Cy abrogated proliferation in all subsets, suggesting effective Cy-mediated killing of cycling T lymphocytes. T cell counts however did not drop between day 5 and 8 post HSCT, and TSCM cells increased in percentages and absolute numbers. Thus, we reasoned that only direct conversion of TN into TSCM could explain TSCM expansion observed at day 8 post HSCT. To prove the in vivo generation of TSCM from TN cells, we exploited the unique TCR sequences harbored by each individual T lymphocyte as unambiguous clonal markers. Purified T cell subsets from apheresis and harvested 30 days post HSCT in 3 consecutive patients were subjected to CDR3

S5