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Key Points TCRαβ/CD19-depleted HLA-haploidentical HSCT is an effective strategy for children with several nonmalignant disorders.Patients given this type of transplant benefit from a low incidence of GVHD and TRM, with graft failure being the main obstacle., Visual Abstract, Several nonmalignant disorders (NMDs), either inherited or acquired, can be cured by allogeneic hematopoietic stem cell transplantation (HSCT). Between January 2012 and April 2020, 70 consecutive children affected by primary immunodeficiencies, inherited/acquired bone marrow failure syndromes, red blood cell disorders, or metabolic diseases, lacking a fully matched donor or requiring urgent transplantation underwent TCRαβ/CD19-depleted haploidentical HSCT from an HLA-partially matched relative as part of a prospective study. The median age at transplant was 3.5 years (range 0.3-16.1); the median time from diagnosis to transplant was 10.5 months (2.7 for SCID patients). Primary engraftment was obtained in 51 patients, while 19 and 2 patients experienced either primary or secondary graft failure (GF), the overall incidence of this complication being 30.4%. Most GFs were observed in children with disease at risk for this complication (eg, aplastic anemia, thalassemia). All but 5 patients experiencing GF were successfully retransplanted. Six patients died of infectious complications (4 had active/recent infections at the time of HSCT), the cumulative incidence of transplant-related mortality (TRM) being 8.5%. Cumulative incidence of grade 1-2 acute GVHD was 14.4% (no patient developed grade 3-4 acute GVHD). Only one patient at risk developed mild chronic GVHD. With a median follow-up of 3.5 years, the 5-year probability of overall and disease-free survival was 91.4% and 86.8%, respectively. In conclusion, TCRαβ/CD19-depleted haploidentical HSCT from an HLA-partially matched relative is confirmed to be an effective treatment of children with NMDs. Prompt donor availability, low incidence of GVHD, and TRM make this strategy an attractive option in NMDs patients. The study is registered at ClinicalTrial.gov as NCT01810120.

Background: allogeneic HSCT is the only potentially curative treatment for many non-malignant diseases (NMD), either inherited or acquired. However, many patients lack an HLA-matched donor (familiar (MFD) or unrelated (MUD)) and the outcome of children transplanted from an HLA-haploidentical relative (haplo) was historically inferior to that of transplants from a MFD or a MUD. We previously published promising results in a cohort of 23 children with NMD given this type of allograft (Bertaina et al., Blood 2014), demonstrating a low transplant-related mortality (TRM) and high cure rates. Here, we report the outcome of a large cohort of children affected by NMD who received a TBdepl-haploHSCT at our Center (NCT01810120). Patients and methods: Between February 2011 and June 2020, 80 consecutive patients affected by NMD received TBdepl-haploHSCT from an HLA-partially matched relative at Ospedale Pediatrico Bambino Gesù in Rome, Italy. Patients had many different disorders (see Table for details on patient- and transplant-related characteristics). Median time from diagnosis to transplant for the whole cohort was 12 months (range 1-177), while it was 2.5 months (range 1.3-11.2) for SCID patients. All patients, including children with SCID, received a conditioning regimen, which varied according to the original disease. Pre-transplant anti-thymocyte globulins (from day -4 to day -2) were given to modulate bi-directional donor/recipient alloreactivity, while rituximab (on day -1) was administered to prevent PTLD. Moreover, no post-transplant pharmacological GvHD prophylaxis was given. Results: fifty-eight patients (72.5%) achieved primary donor cell engraftment, while 3 patients experienced secondary graft failure (GF); the cumulative incidence of either primary or secondary GF was 27.8% (95% CI 17.2-37.0). Median time to neutrophil and platelet recovery was 13.5 (range 9-33) and 10 days (range 7-51), respectively. As expected, GF occurred more frequently in children with disorders known to be associated with an increased GF risk (i.e., HLH, thalassemia, SAA or osteopetrosis) (see also Figure 1A). Three children (4%) experiencing GF died because of infectious complications before retransplant. Sixteen of the 22 patients with either primary or secondary GF were successfully retransplanted (2 with a mismatched unrelated cord blood unit, the other having received a second TBdepl-haploHSCT from either the same donor or the other parent). Since 3 other patients died [all because of infectious complications, 2 due to disseminated adenovirus infection and 1 to CMV pneumonia)], TRM is 7.8% (95% CI 1.6-13.7). Eighteen patients experienced acute GVHD of any grade, the cumulative incidence of this complication being 22% (95% CI 13.5-31.8); 10/18 patients developed grade II acute GVHD (no patient developed grade III or IV aGVHD), this resulting into a cumulative incidence of 12.9% (95% CI 6.6-21.4). Only one patient at risk developed mild chronic GVHD. Twenty-two and 7 patients developed clinically-relevant (i.e., with a viral load > 1000 copies/ml and/or requiring specific antiviral-treatment) CMV and adenovirus infection, respectively, at a median time of 4 (range 0-16) and 1 (range 1-4) weeks from HSCT. Time averaged area under the curve (i.e., viral burden under the curve/weeks at risk for infection) for CMV and ADV are reported in Figure 1B. With a median follow-up of 36 months (range 2 - 110), the 5-year probability of overall survival and event-free survival for the entire cohort of patients is 92.1% (95% CI 83.3-96.4) (Figure 1C) and 68.1% (95% CI 56.4-77.2), respectively. Considering the 16/22 given a successful 2nd allograft, the 5-year disease-free survival is 88.4% (95% CI 78.9-93.8). Details on reconstitution of CD3+, CD4+ and CD8+ lymphocytes are reported in Figure 1D. Conclusions: TBdepl-haploHSCT is an effective option for children with different NMD. GF (either primary or secondary) is a challenging problem in a sub-group of patients at risk (i.e., those with HLH, thalassemia, SAA or osteopetrosis): thus, new strategies to overcome this problem are desirable. However, a second transplant is able to rescue most of these patients. Prompt availability of this type of transplant, limiting infectious risk, low incidence of both acute and chronic GvHD preserving a good quality of life in patients makes this strategy an attractive choice in patients with NMD. Figure 1 Disclosures Merli: Bellicum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; SOBI: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria; Sanofi-Genzyme: Honoraria; Atara Therapeutics: Honoraria. Algeri:BlueBird Bio: Membership on an entity's Board of Directors or advisory committees; Atara Therapeutics: Membership on an entity's Board of Directors or advisory committees. Locatelli:Jazz Pharmaceeutical: Speakers Bureau; Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Neuromyelitis optica is an immune‐mediated disease characterized by a relapsing course, resulting in progressive disability. In children, given the long life expectancy, a disease‐modifying treatment could be particularly desirable. Unfortunately, the currently available treatment strategies with this potential are scarce. Very limited data are available about the use of allogeneic hematopoietic stem cell transplantation (HSCT) for autoimmune neurological diseases. In this report, we present a pediatric case successfully treated with allogeneic HSCT from an HLA‐haploidentical donor, after ex vivo TCR/CD19‐depletion of the graft. To the best of our knowledge, this is the first case of a pediatric patient to benefit from such a treatment.

We report on the outcome of 24 patients with Fanconi anemia (FA) lacking an HLA matched related or unrelated donor, given an HLA-haploidentical T-cell receptor αβ (TCRαβ+) and CD19+ cell-depleted hematopoietic stem cell transplantation (HSCT) in the context of a prospective, single-center phase 2 trial. Sustained primary engraftment was achieved in 22 (91.6%) of 24 patients, with median time to neutrophil recovery of 12 days (range, 9-15 days) and platelet recovery of 10 days (range, 7-14 days). Cumulative incidences of grade 1 to 2 acute graft-versus-host disease (GVHD) and chronic GVHD were 17.4% (95% confidence interval [CI], 5.5%-35.5%) and 5.5% (95% CI, 0.8%-33.4%), respectively. The conditioning regimen, which included fludarabine, low-dose cyclophosphamide and, in most patients, single-dose irradiation was well tolerated; no fatal transplant-related toxicity was observed. With a median follow-up of 5.2 years (range, 0.3-8.7 years), the overall and event-free survival probabilities were 100% and 86.3% (95% CI, 62.8%-95.4%), respectively (2 graft failures and 1 case of poor graft function were considered as events). The 2 patients who experienced primary graft failure underwent a subsequent successful HSCT from the other parent. This is the first report of FA patients given TCRαβ+/CD19+-depleted haplo-HSCT in the context of a prospective trial, and the largest series of T-cell–depleted haplo-HSCT in FA reported to date. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

Background: WAS is a rare X-linked recessive disorder, characterized by thrombocytopenia with low platelet volume, recurrent infections, eczema, autoimmunity, vasculitis and increased incidence of malignancies. Patients with classical WAS have chronic morbidities, severely impaired quality of life and a decreased life expectancy. Allogeneic HSCT is the only well-established curative treatment, gene therapy being still an experimental approach. Excellent outcome have been reported in patients transplanted at early age from either matched related and unrelated donors, with 5-year overall survival (OS) exceeding 90%. However, experience with HLA-haploidentical HSCT is limited and has been historically associated with inferior results. Haploidentical HSCT after selective depletion of α/β+ T-cells and CD19+ B-cells (TBdepl-haploHSCT) was shown to be safe and effective in children with multiple types of non-malignant disorders (Merli et al, Blood Adv 2021). To further optimize this approach and accelerate the recovery of adaptive immunity, we conducted a phase I/II trial evaluating the safety and efficacy of post-transplant infusion of a titrated number of donor T-cells transduced with the inducible caspase-9 (iC9) suicide gene (BPX-501, or rivogenlecleucel, cells) in children with either malignant or non-malignant disorders (ClinicalTrials.gov identifier: NCT02065869). We report the outcome of a cohort of 12 children affected by WAS who received a TBdepl-haploHSCT at our Center. Patients and methods: Between 2014 and 2021, 12 patients affected by WAS received a TBdepl-haploHSCT from an HLA-partially matched relative at Ospedale Pediatrico Bambino Gesù in Rome, Italy. Patients and transplant characteristics are depicted in Table 1. All patients received a myeloablative conditioning regimen, combining pharmacokinetic-adjusted busulfan with Thiotepa and Fludarabine. Pre-transplant anti-thymocyte globulins (from day -4 to day -2) were given to modulate bi-directional donor/recipient alloreactivity, while rituximab was administered on day -1 to prevent PTLD. No post-transplant pharmacological GvHD prophylaxis was employed. Eight subjects, enrolled in NCT02065869 trial, additionally received post-transplant infusion of BPX-501 cells (dose: 1x10 6 cells/kg) at a median time of 16 days after HSCT (range 12-20). Results: 11 patients achieved primary donor cell engraftment, while one patient experienced secondary graft failure (GF), likely triggered by CMV reactivation. Median time to neutrophil and platelet recovery was 15 (range 8-33) and 10 days (range 9-16), respectively. The patient with secondary GF was successfully re-transplanted with a second TBdepl-haploHSCT from the same donor. Grade I/II skin acute GvHD (aGvHD) occurred in 3 patients, the cumulative incidence of aGvHD being 25.9% (95% CI 0-47.7). None of these 3 patients required activation of iC9 with rimiducid and no cases of chronic GVHD (cGvHD) were observed. No patient died. With a median follow-up of 58 months (range 1 - 78), the 5-year probability of OS and event-free survival is 100% and 90.7% (95% CI 50.8-98.7), respectively. Considering the successful second allograft, the 5-year disease-free survival is 100%. Platelet recovery is reported in Figure 1A. Two months after the allograft, all evaluable subjects had a platelet count above 50.000/microL (median 202.000, range 53.000-353.000). All subjects but one have full donor chimerism at last follow-up, the remaining patient having stable mixed chimerism (40% of donor cells) without any WAS manifestation. All subjects with a follow-up of at least 6 months are independent of immunoglobulin replacement. Details on reconstitution of lymphocytes subsets are reported in Figure 1B,C,D. Conclusions: TBdepl-haploHSCT after Bu-based conditioning regimen is an highly-effective curative option for children with WAS, being characterized by high-engraftment rate with fast recovery of both neutrophils and platelets, low incidence of aGvHD and no occurrence of cGVHD. Given the prognostic impact of age in determining HSCT outcome in WAS and prompt availability of haploidentical family donors, our data suggest that this approach should be offered without delay to those patients with WAS who lack a matched donor. Infusion of BPX-501 cells contribute to accelerate the recovery of adaptive T-cell immunity, further increasing the safety of the procedure. Figure 1 Figure 1. Disclosures Merli: SOBI: Consultancy; JAZZ: Consultancy. Locatelli: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc.: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyl: Honoraria.

Background: Pediatric myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal disorders, accounting for less than 5% of childhood hematologic malignancies. Usual indications to HSCT are: MDSs with excess of blasts, MDSs secondary to previously administered chemoradiotherapy and RCC associated with monosomy 7, complex karyotype, severe neutropenia, or erythrocyte/platelet transfusion dependence [Locatelli & Strahm, Blood 2018]. We previously demonstrated that TBdepl-haploHSCT is a suitable option for children with acute leukemia, with outcomes comparable to those reported in studies using either an HLA-identical sibling or an unrelated volunteer as donor. Here we present the results of this approach in children with MDSs. Patients and methods: Between February 2013 and February 2021, 23 children with MDSs other than juvenile myelomonocytic leukemia received TBdepl-haploHSCT from an HLA-partially matched relative at Ospedale Pediatrico Bambino Gesù, Rome, Italy or at IRCCS Fondazione Policlinico San Matteo, Pavia, Italy as part of a prospective study (#NCT01810120). All patients were prepared to the allograft using a fully-myeloablative conditioning regimen including a combination of cytotoxic drugs and/or total body irradiation (TBI). Anti-T-lymphocyte globulin (ATLG) was used before transplantation (12 mg/kg total dose, from days -5 to day -3) to modulate bi-directional donor/recipient alloreactivity. Rituximab (200 mg/sqm) was administered on day -1 to prevent post-transplantation EBV-induced lymphoproliferative disorders (PTLD). No patient received any post-transplant pharmacological GvHD prophylaxis. Results: Characteristics of patients enrolled in the study are shown in Table 1 (which reports also donor and graft characteristics). Median follow-up of surviving patients is 4.2 years (range: 0.5 - 8.5 years). Seventeen children were affected by refractory cytopenia of childhood (RCC) (2 cases occurring in the context of inherited bone marrow failure syndromes: one had GATA2 deficiency and the other SAMD9L mutation), while 1 and 5 were affected by MDS with excess of blasts 1 (EB1) and EB2 (one had GATA2 deficiency), respectively. Median time to neutrophil and platelet recovery was 14 (range 10-19) and 11 (range 9-14) days, respectively, with four patients (3 with RCC and 1 with EB2) experiencing primary graft failure, the cumulative incidence of this complication being 17.3% (95% CI 0.3-31.5). All these 4 patients were rescued with a second TBdepl-haploHSCT from the same or the other parent. Cumulative incidence of grade II-III acute GvHD was 11.4% (95% CI 0-25.2). One patient developed skin and gut GvHD after the second TBdepl-haploHSCT, while for all other patients skin was the sole organ involved; no case of grade IV GvHD was observed. One patient developed moderate chronic GvHD [cumulative incidence 5.2% (95% CI 0-14.8)], which completely resolved with low-dose steroids and ruxolitinib. Notably, no patient died for transplant-related complications. Six patients experienced CMV, 2 HHV-6 and 1 adenoviral infection/reactivation; one patient developed lung aspergillosis, which resolved with specific treatment. One patient affected by EB2, not in remission at time of transplant, relapsed 27 months after HSCT, the 5-year cumulative incidence of relapse being 7.1% (95% CI, 0-19.7); she eventually died after failing a second HSCT. The 5-year probability of overall and event-free survival were 92.3% (95% CI 56.6 -98.9) and 76.3% (95% CI 51.3-89.6) (Figure 1A and B), respectively. Five-year disease-free-survival was 90% (95% CI 47.3-98.5). Because of the low number of events, no prognostic factor related to OS and EFS was found. In particular, the MDS variant did not influence the patient's outcome. The median CD3+ cell count on day +30, +90, +180 and +360 were 113, 171, 558 and 1307/mcl, respectively. Conclusions: These data indicate that TBdepl-haploHSCT is a safe and effective transplant option also in children with MDS. Indeed, the low risk of both non-relapse mortality and acute/chronic GvHD makes this approach particularly attractive in the pediatric setting. Moreover, this haplo strategy compares favorably with T-cell replete approaches [Suo et al., 2020]. Figure 1 Figure 1. Disclosures Merli: JAZZ: Consultancy; SOBI: Consultancy. Locatelli: Miltenyl: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc.: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Based on the clinical success observed in acute lymphoblastic leukemia (ALL) with chimeric antigen receptor engineered T (CAR T), we hypothesized that combining the specificity of a CAR with the innate allo-reactivity of KIR-mismatched NK cells might provide a powerful tool for adoptive cell therapy. The use of a third-party bank of CAR-NK cells offers the advantage of an immediate availability to be exploited in the allogenic setting and could be associated with a lower toxicity profile than CAR-T cells. In order to overcome regulatory and manufacturing hurdles associated with generation of CAR-NK cells, we developed a feeder-free culture resulting in a 3.2-log expansion after 20 days of culture. Specifically, natural cytotoxicity receptors (NCR) expressed on NK cells are stimulated in the presence of pleiotropic cytokines and expanded in GMP grade bioreactors. Expanded NK cells from healthy donors preserve a high percentage of CD56+ CD57- cells (85±13%), associated with high proliferative capability, and maintain the surface expression and the responsiveness of NCR and CD16. We proved that NK cells generated from 10 different healthy donors have high ability to recognize and eliminate different tumor types, including acute myeloid leukemia (AML) and ALL. After genetic modification with a retroviral vector encoding a CAR specific for CD19 antigen, transduction of activated NK cells averaged 38%±15% and the CAR.CD19 expression was stable over extended in vitro culture (60 days). Detailed phenotypic characterization of CAR-NK cells showed that CAR expression was not limited to the more mature NKG2A-/KIR+ cells, but rather was distributed across different NK subsets. We also demonstrated that NK and CAR-NK cells display significant anti-leukemia activity towards CD19+ leukemia and lymphoma cell lines (LCL 721.221, DAUDI and BV173) and primary blasts obtained from patients with B-cell precursor ALL (Bcp-ALL). Co-culture experiments using a 1:5 E/T ratio, showed that, while the anti-tumor activity was already remarkable with non-modified effector NK cells (60±30%, 71±33% and 54±23% of residual LCL 721.221, DAUDI and BV173 cells, respectively; p An in vivo model of leukemia xenograft immunodeficient mice was used to evaluate whether CAR-NK cells are associated with a lower toxicity profile compared to CAR-T cells. While the in vivo antileukemia activity was superimposable between CAR-T and CAR-NK cells (mouse bioluminenscence at 20 days, 4.9x105 vs 6.6x105 photons/second, respectively; p=n.s. Figure A), mice treated with two i.v. infusions (day 0 and day 15) of 10x106 CAR.CD19 NK cells had a 100% overall survival (OS of 5 out of 5 mice) at 50 days compared to 20% of mice (1 out of 5) receiving 10x106 CAR.CD19 T cells (Figure B; p=0.01). Cytokine plasma level monitoring, performed on day +7 and +30 after effector cell infusion in the absence of leukemia persistence (as evidenced by a lack of bioluminescence signal), showed that mice engrafted with CD19+ leukemia and treated with CAR.CD19-NK cells have lower levels of circulating hIFN-g cytokine compared to mice treated with CAR.CD19-T cells at both day 7 (42±82 vs 330±346 ng/ml; p=0.05) and day 30 (0.9±0.7 vs 4148±667 ng/ml; p=0.05). These in vitro and in vivo data demonstrate the feasibility of clinical scale feeder-free expansion of non-modified NK cells and stably transduced CAR-NK cells. Both non-modified and gene-modified cells were capable of significant tumor killing, suggesting a multi-modal adoptive cell approach to treatment of leukemia. Since NK cells have been shown to be safely used in third-party setting (St. Jude Children's Research Hospital, USA; NCT00640796), we suggest that ex-vivo expanded, feeder-free NK cells can be universally applied for 'off-the-shelf' immuno-gene-therapy, and that their innate allo-reactivity can be safely harnessed to potentiate allogeneic cell therapy. Figure. Figure. Disclosures Locatelli: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Hematopoietic stem cell transplantation (HSCT) is standard therapy for numerous hematological diseases. The use of haploidentical donors, sharing half of the HLA alleles with the recipient, has facilitated the use of this procedure as patients can rely on availability of a haploidentical donor within their family. Since HLA disparity increases the risk of graft-versus-host disease, T-cell depletion has been used to remove alloreactive lymphocytes from the graft. Selective removal of αβ T cells, which encompass the alloreactive repertoire, combined with removal of B cells to prevent EBV-related lymphoproliferative disease, proved safe and effective in clinical studies. Depleted αβ T cells and B cells are generally discarded as by-products. Considering the possible use of donor T cells for donor lymphocyte infusions or for generation of pathogen-specific T cells as mediators of graft-versus-infection (GvI) effect, we tested whether cells in the discarded fractions were functionally intact. Response to alloantigens and to viral antigens comparable to that of unmanipulated cells indicated a functional integrity of αβ T cells, in spite of the manipulation used for their depletion. Furthermore, B cells proved to be efficient antigen-presenting cells, indicating that antigen uptake, processing and presentation were fully preserved. Therefore, we propose that separated αβ T lymphocytes could be employed for obtaining pathogen-specific T cells, applying available methods for positive selection, which eventually leads to indirect allodepletion. In addition, these functional T cells could undergo additional manipulation, such as direct allodepletion or genetic modification.

Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative (haplo-HSCT) is a suitable option for children with acute leukemia (AL) either relapsed or at high-risk of treatment failure. We developed a novel method of graft manipulation based on negative depletion of αβ T and B cells and conducted a prospective trial evaluating the outcome of children with AL transplanted with this approach. Eighty AL children, transplanted between September 2011 and September 2014, were enrolled in the trial. All children were given a fully myeloablative preparative regimen. Anti-T-lymphocyte globulin from day -5 to -3 was used for preventing graft rejection and graft-versus-host disease (GVHD); no patient received any posttransplantation GVHD prophylaxis. Two children experienced primary graft failure. The cumulative incidence of skin-only, grade 1-2 acute GVHD was 30%; no patient developed extensive chronic GVHD. Four patients died, the cumulative incidence of nonrelapse mortality being 5%, whereas 19 relapsed, resulting in a 24% cumulative incidence of relapse. With a median follow-up of 46 months for surviving patients, the 5-year probability of chronic GVHD-free, relapse-free survival (GRFS) is 71%. Total body irradiation-containing preparative regimen was the only variable favorably influencing relapse incidence and GRFS. The outcomes of these 80 patients are comparable to those of 41 and 51 children given transplantation from an HLA-identical sibling or a 10/10 allelic-matched unrelated donor in the same period. These data indicate that haplo-HSCT after αβ T- and B-cell depletion represents a competitive alternative for children with AL in need of urgent allograft. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

Background Virus-specific CD4 and CD8 T lymphocytes from HLA-matched donors are effective for treatment and prophylaxis of viral infections in immune-compromised recipients of hematopoietic stem cell transplant recipients. Adoptive immune reconstitution is based on selection of specific T cells or on generation of specific T-cell lines from the graft donor. Unfortunately, the graft donor is not always immune to the relevant pathogen or the graft donor may not be available (registry-derived or cord blood donors). Study Design and Methods Since the possibility of using T cells from a third-party subject is now established, we screened potential donors for T-cell responses against cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus, the viruses most frequently targeted by adoptive immune reconstitution. Specific T-cell responses against viral antigens were analyzed in 111 donors using a miniaturized interferon-γ release assay. Results Responders to CMV were 64%, to EBV 40%, and to adenovirus 51%. Simultaneous responders to the three viruses were 49%. CMV-specific CD4 and CD8 T-cell lines could be generated from 11 of 12 donors defined as positive responders according to the T-cell assay. Conclusions These data demonstrate that a large fraction of volunteers can be recruited in a donor registry for selection or expansion of virus specific T cells and that our T-cell assay predicts the donors' ability to give rise to established T-cell lines endowed with proliferative potential and effector function for adoptive immune reconstitution.

Background: For children with AL candidate to receive an allograft and lacking a suitable HLA-matched donor, HLA-haplo-HSCT after abTCD may represent a valid alternative. Due to delayed recovery of adaptive T-cell immunity with abTCD-haplo-HSCT alone, we conducted a phase I/II trial testing the safety and efficacy of post-transplant infusion of a titrated number of donor-derived T cells transduced with the iC9 gene (rivogenlecleucel; ClinicalTrials.gov id: NCT02065869) in children with malignant and non-malignant diseases. Here, we report on the cohort of 70 patients with AL treated in Italy with abTCD-haplo-HSCT+rivogenlecleucel, comparing the results with those of 88 patients given abTCD-haplo-HSCT alone and previously published by our group (Blood 2018; 132:2594-2607). Patients and methods: Patients (age < 18 years) were transplanted between 2010 and 2018. Patient and disease characteristics are shown in Table 1. Median age at HSCT was 6 years (range 0.3-18), and median follow-up was 71 and 31 months for abTCD-haplo-HSCT and abTCD-haplo-HSCT+rivogenlecleucel, respectively. Compared to the control group, abTCD-haplo-HSCT+rivogenlecleucel recipients were transplanted more recently and from a younger donor, and received a higher number of CD34+ cells (Table 1). Diagnosis did not differ between the 2 groups, acute lymphoblastic leukemia (ALL) being the most frequent diagnosis. All patients were transplanted in morphological complete remission (CR1 and CR2) and received myeloablative preparation. Graft composition is reported in Table 1; notably all patients received >10x106 CD34+cells/Kg and Results: Graft failure occurred in 2% of patients in each group. Median time to neutrophil and platelet engraftment was 14 (6-23) and 11 (5-56) days, with no differences between groups (p=0.28). Rivogenlecleucel were infused at a median time of 21 days (range 11-59). Treatment was well tolerated; no infusion-related side effects were recorded. Cumulative incidence (CI) of 100-day grade II-IV acute GvHD was 18.9% vs 15.9% (p=0.77) and CI of 1-year chronic GvHD was 6.9% vs 5.7% (p=0.56) in abTCD-haplo-HSCT and abTCD-haplo-HSCT+rivogenlecleucel, respectively. The 4-year non-relapse-mortality (NRM) was significantly lower in abTCD-haplo-HSCT+rivogenlecleucel (1.4% vs 8%, p=0.05) (Figure 1). There was no statistically significant difference in the 4-year CI of relapse (RI) (17% vs. 25%, p=0.30), respectively. Disease recurrence was the most common causes of death in both groups, viral and fungal infections being the most frequent non-relapse fatalities. The 4-year overall survival (OS) and leukemia-free survival (LFS) was 70% vs 87%, p=0.01 (Figure 2) and 67% vs 82%, p=0.05, for abTCD-haplo-HSCT and abTCD-haplo-HSCT+rivogenlecleucel, respectively. There was no difference in 4-year CI of CMV reactivation between the 2 groups (p=0.68), median time to CMV reactivation being 29 and 30 days (p=0.29), respectively. Once infused, rivogenlecleucel expanded (mainly in the CD8+ subset), reaching a peak at 9 months after infusion. At 6-months, median CD3+, CD3/CD4, CD3/CD8, CD3-/CD56 and CD20/CD19 count/microL were 820, 265, 225, 141, 171, for abTCD-haplo-HSCT and 898, 294, 288, 214, and 161 for abTCD-haplo-HSCT+rivogenlecleucel, (p=ns, p=ns, p=0.02, p=0.03, p=ns), respectively. The advantage in the recovery of CD3/CD8 and CD3-/CD56 after abTCD-haplo-HSCT+rivogenlecleucel persisted at 1 year (p=0.01, p=0.03, respectively). In multivariable analysis, abTCD-haplo-HSCT+rivogenlecleucel was associated with better OS (HR 0.27, p=0.003) and LFS (HR 0.40, p=0.001); there was also a trend for lower relapse risk (HR 0.50, p=0.098). Age below the median value at HSCT (HR 2.62, p=0.01), CR1 at HSCT (HR 0.35, p=0.03) and use of irradiation in the conditioning regimen (HR 0.32, p=0.02) were other factors correlating with OS and LFS. Conclusions: These data confirm that the infusion of donor-derived rivogenlecleucel is safe and well tolerated. Rivogenlecleucel cells infusion contributed to enhance recovery of cytotoxic T and NK cells, improving patients NRM and OS/LFS. Rivogenlecleucel (with the possibility of inducing apoptosis of donor T cells) may facilitate the cellular therapy approaches aimed at optimizing immune recovery after HSCT. Disclosures Merli: Amgen: Honoraria; Bellicum: Consultancy; Novartis: Honoraria; Sobi: Consultancy. Algeri:Bluebird bio: Consultancy, Honoraria; Miltenyi: Honoraria; Atara Biotherapeutics: Consultancy, Honoraria. Woodard:Bellicum Pharmaceuticals, Inc: Employment, Other: Stock, Stock options. Locatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BluebirdBio: Consultancy.

β-thalassemia is one of the most common monogenic blood disorders worldwide, and is highly prevalent in Mediterranean countries. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been the only curative treatment for transfusion-dependent β-thalassemia (TDT; the most severe disease form) for many years, but it is limited by donor availability and has a significant risk of morbidity and mortality. We conducted a chart review of patients with β-thalassemia who underwent allo-HSCT (N=80) at the Bambino Gesù Children's Hospital, Rome, Italy, between March 2011 and August 2018. Median (range) age at allo-HSCT was 5.5 (0.3-20.0) years [ In total, 18 (22.5%), 28 (35.0%), and 34 (42.5%) patients received allo-HSCT from human leukocyte antigen (HLA)-identical sibling donors, HLA-haploidentical donors, and unrelated donors (fully matched donor: n=28, donor with a single HLA disparity: n=6), respectively. Of these donors, 42 (52.5%) were carriers for thalassemia-associated mutations. In total, 53 (66.3%) donors and 35 (43.8%) recipients were cytomegalovirus-positive. Bone marrow was the stem cell source in 51 cases (63.8%), while 28 patients received an alphabeta T-cell depleted peripheral blood haploidentical HSCT (35.0%); the remaining child (1.3%) received both bone marrow and cord blood from the same related donor. All patients continued to receive transfusions immediately after allo-HSCT; however, only 7 (8.8%) received a transfusion in the 3 to 12-month post-transplantation period (2 due to underlying disease; 5 due to other reasons including GI bleeding). Median (range) time to reach transfusion-free status was 3.8 (1.1-47.8) weeks. Median (interquartile range) hemoglobin levels at 6 and 12 months after allo-HSCT were 10.9 (10.2-11.9) and 11.9 (10.6-13.0) g/dL, respectively. The cumulative incidences of primary and secondary graft failure were 10.0% and 12.5% at 24 months (HLA-identical donor: 0% and 11.1%, haploidentical donor: 17.9% and 3.6%, unrelated donor: 8.8% and 20.6%). Eleven out of 14 patients experiencing graft failure were successfully rescued with a second allograft, while 2 patients were not retransplanted due to parental decision and 1 patient died after the engraftment of the second allograft. Eight patients developed grade II-IV acute graft-versus-host disease (GVHD) and one patient developed moderate chronic GVHD. Cumulative incidence rates of grades II-IV and III-IV acute GVHD were 12.7% and 8.0% at 24 months (HLA-identical donor: 0% and 0%, haploidentical donor: 7.3% and 0%, unrelated donor: 23.8% and 18.8%). Three patients (3.8%) died of transplant-related causes (1 case each of hemophagocytic lymphohistiocytosis, sepsis, and multi-organ failure [the patient receiving the second allograft]) with a median (range) time from transplantation to death of 8.7 (3.7-11.0) months. Of these patients, all had been transplanted from an unrelated donor and 2 had reached sustained full-donor chimerism. The probability of overall and event-free (event defined as either death or primary/secondary graft failure) survival was 96.2% and 81.2% at 24 months (HLA-identical sibling donor: 100% and 88.9%, haploidentical donor: 100% and 78.6%, unrelated donor: 91.2% and 79.4%). The probability of thalassemia-free survival (event defined as either death or primary/secondary graft failure not rescued by a second allograft) was 93.7% at 24 months (HLA-identical sibling donor: 100%, haploidentical donor: 92.9%, unrelated donor: 91.2%). In this large single-center cohort of children with predominantly TDT, allo-HSCT led to beneficial outcomes for most patients, resulting in the discontinuation of transfusions with 93.7% of patients being thalassemia-free. Nevertheless, HSCT is still associated with GVHD, graft failure, and mortality, and only 22.5% of patients had an HLA-identical sibling donor, illustrating a key limitation of allo-HSCT. Emerging research is addressing such barriers to treatment. Disclosures Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Algeri:Miltenyi: Honoraria; Atara Biotherapeutics: Consultancy, Honoraria; Bluebird bio: Consultancy, Honoraria. Gruppioni:Bluebird bio: Employment, Equity Ownership. Kommera:Bluebird bio: Employment, Equity Ownership. Maa:Bluebird bio: Employment, Equity Ownership. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

Survival rates of children with relapsed/refractory (r/r) BCP-ALL remain unsatisfactory and little progress has been made in the past 2 decades. Similarly, relapse of childhood B-NHL is usually associated with an aggressive disease and poor outcomes. Targeted immunotherapy with T-cells genetically modified to express a CD19-directed CAR showed an unprecedented antitumor efficacy, leading to the recent FDA and EMA approval of two CD19-CAR products for treatment of BCP-ALL and B-NHL. Relevant toxicities have, however, been reported, mainly related to the development of severe Cytokine Release Syndrome (CRS) and/or of neurotoxicity. At Ospedale Pediatrico Bambino Gesù (OPBG) in Rome, we developed a clinical-grade, 2nd generation, CD19-specific CAR construct, including 4.1bb as costimulatory domain and the inducible caspase-9 safety switch (iC9-CD19-CAR), vehiculated by a retroviral vector, to conduct an academic, phase I/II clinical trial in patients (age 1-25 yrs) affected by BCP-ALL or B-NHL. We now report on the results of the phase I and of the first 8 patients treated in the phase II portion of the study, in terms of feasibility, toxicity, maximum tolerated/recommended dose (MTD/RD) and data on response rate and biological correlates. The phase I, dose-escalation portion of the study included 3 dose levels (DL), namely: DL1, 0.5×106; DL2, 1.5×106; DL3, 3.0×106 CAR+ T cells per kg of recipient body weight. In the phase II portion, patients were treated at the RD identified in the phase I, namely 3.0×106 CAR+cells/kg. All patients received a lymphodepleting regimen consisting of fludarabine and cyclophosphamide for 3 days and iC9-CD19-CAR T cells were subsequently administered as single infusion. Patients were monitored for toxicity, expansion and persistence of iC9-CD19-CAR T cells. Seventeen children were enrolled into the trial and received iC9-CD19-CAR T cells between January 2018 and June 2019. Data were analyzed as of July 20, 2019. The characteristics of the patients are detailed in table 1. The designed dose concentration was successfully produced for all the enrolled patients and we did not observe any production failure. The median transduction rate in the drug product was 54% (range 21-73), while the median vector copy number was 3.8 (range 2.8-6.2). During the phase I portion of the study, no dose limiting toxicities (DLTs) have been recorded, defining the MTD as 3.0×106 CAR+ T cells per kg of recipient body weight. The treatment was overall tolerated and all the toxicities were reversible, the most severe being grade 3-4 neutropenia, thrombocytopenia and/or anemia, occurring in 16/17 (94.1%) patients; in 13/16 patients (81.2%) the hematological toxicity developed before the infusion and persisted after the administration of CAR T cells. Cytokine release syndrome (CRS) occurred in 10/17 patients (58.8%) and was overall moderate, reaching grade 3 (Lee criteria) in one patient only. Notably, none of the patients developed neurotoxicity and no activation of the safety switch was required. All patients were assessed for response at 4 weeks from iC9-CD19-CAR T cell infusion and 13/15 (86.7%) patients with ALL achieved complete remission (CR) with negativity of minimal residual disease (MRD), including 2/3 patients receiving the DL1, 9 patients who had failed a previous allogeneic haematopoietic stem-cell transplantation (HSCT) and 6 patients that had previously received blinatumomab, as CD19-directed immunotherapy. The iC9-CD19-CAR T cells expanded in vivo and were detectable by both flow-cytometry and molecular biology in the blood (Fig.1), bone marrow and cerebrospinal fluid of the responders. One CD19-negative relapse 3 months after infusion was recorded, while 3 additional patients relapsed with CD19+ leukemia blasts. Four patients received HSCT while in CR with MRD negativity because of regrowth of normal CD19+ B cells. The 18-month probability of overall survival for the BCP-ALL cohort is 72.2% (Fig.2). One of the 2 B-NHL patients showed a partial response. Our data indicate that iC9-CD19-CAR T cell in an academic setting is feasible, safe and extremely effective in treating highly resistant/relapsed BCP-ALL. In our trial, no major or life-threatening toxicities were observed and, despite the moderate CRS recorded, high rates of CR were achieved, suggesting that the combination of a retroviral platform and 4.1bb as costimulation is able to mediate a potent antitumor effect Disclosures Merli: Amgen: Honoraria; Novartis: Honoraria; Sobi: Consultancy; Bellicum: Consultancy. Algeri:Bluebird bio: Consultancy, Honoraria; Atara Biotherapeutics: Consultancy, Honoraria; Miltenyi: Honoraria. Locatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

We report on the successful infusion of human adenovirus (HAdV)-specific T cells in a child with congenital amegakaryocytic thrombocytopenia, given T-cell-depleted hematopoietic stem cell transplantation (HSCT) from the HLA-haploidentical mother during extracorporeal membrane oxygenation (ECMO) for severe HAdV-related respiratory failure. Donor-derived, interferon (IFN)-γ-secreting HAdV-specific T cells were enriched using the cytokine capture assay, after in vitro stimulation with overlapping peptides from the immunodominant HAdV5 hexon protein. Two weeks after T-cell transfer, viral load decreased and ECMO was discontinued. T-cell responses to HAdV antigens were documented after four weeks and were associated with viral clearance, immune reconstitution and clinical amelioration. Pediatr Blood Cancer 2014;61:376–379. © 2013 Wiley Periodicals, Inc.

Hematopoietic Stem Cells Transplantation (HSCT) is an effective treatment for hematological and non-hematological diseases. The main challenge in autologous HSCT is purging of malignant cells to prevent relapse. In allogeneic HSCT graft-versus-host disease (GvHD) and opportunistic infections are frequent complications. Two types of graft manipulation have been introduced: the first one in the autologous context aimed at separating malignant cells from hematopoietic stem cells (HSC), and the second one in allogeneic HSCT aimed at reducing the incidence of GvHD and at accelerating immune reconstitution. Here we describe the manipulations used for cell purging in autologous HSCT or for T Cell Depletion (TCD) and T cell selection in allogeneic HSCT. More complex manipulations, requiring a Good Manufacturing Practice (GMP) facility, are briefly mentioned.

HLA-haploidentical family donors represent a valuable option for children requiring allogeneic hematopoietic stem cell transplantation (HSCT). Because graft-versus-host diseases (GVHD) is a major complication of HLA-haploidentical HSCT because of alloreactive T cells in the graft, different methods have been used for ex vivo T cell depletion. Removal of donor αβ T cells, the subset responsible for GVHD, and of B cells, responsible for post-transplantation lymphoproliferative disorders, have been recently developed for HLA-haploidentical HSCT. This manipulation preserves, in addition to CD34+ progenitors, natural killer, γδ T, and monocytes/dendritic cells, contributing to anti-leukemia activity and protection against infections. We analyzed depletion efficiency and cell yield in 200 procedures performed in the last 3 years at our center. Donors underwent CD34+ hematopoietic stem cell (HSC) peripheral blood mobilization with granulocyte colony–stimulating factor (G-CSF). Poor CD34+ cell mobilizers (48 of 189, 25%) received plerixafor in addition to G-CSF. Aphereses containing a median of 52.5 × 109 nucleated cells and 494 × 106 CD34+ HSC were manipulated using the CliniMACS device. In comparison to the initial product, αβ T cell depletion produced a median 4.1-log reduction (range, 3.1 to 5.5) and B cell depletion led to a median 3.4-log reduction (range, 2.0 to 4.7). Graft products contained a median of 18.5 × 106 CD34+ HSC/kg recipient body weight, with median values of residual αβ T cells and B cells of 29 × 103/kg and 33 × 103/kg, respectively. Depletion efficiency monitored at 6-month intervals demonstrated steady performance, while improved recovery of CD34+ cells was observed after the first year (P = .0005). These data indicate that αβ T cell and B cell depletion of HSC grafts from HLA-haploidentical donors was efficient and reproducible.

Hemopoietic stem cell transplantation (HSCT) is a standard procedure for treatment of malignant and non-malignant hematological diseases. HSCT donors include HLA-identical siblings, matched or mismatched unrelated donors and haploidentical related donors. Graft-versus-host disease (GvHD), mediated by donor alloreactive T-cells in the graft, can be triggered by minor histocompatibility antigens in HLA-identical pairs, by alleles at loci not considered for MUD-matching or by the mismatched haplotype in haplo-HSCT. Therefore, removal of donor T-cells, that contain the alloreactive precursors, is required, but T-cell depletion associates with opportunistic infections and with reduced graft-versus-leukemia effect. Selective T-cell depletion strategies have been introduced, like removal of αβ T-lymphocytes and of naive T-cells, two subsets including the alloreactive precursors, but the ultimate goal is specific removal of alloreactive T-cells. Here we review the different approaches to deplete alloreactive T-cells only and discuss pros and cons, specificity, efficiency and efficacy. Combinations of different methods and innovative approaches are also proposed for depleting specific alloreactive T-cells with high efficiency.

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative (haplo-HSCT) is a suitable option for children/young adults with acute leukemia (AL) either relapsed or at high-risk of treatment failure and in urgent need of an allograft. A novel method of graft manipulation based on the selective, negative depletion of αβ T and B cells has been recently developed. We published the results of a prospective trial (ClinicalTrial.gov identifier: NCT01810120) enrolling 80 children with AL transplanted until September/2014 using this approach (Locatelli, Blood 2017). In the present analysis, we update those results, evaluating also additional patients given haplo-HSCT after that date. Patients and methods: Analyzed are 111 children with AL enrolled in the trial; median age is 10 years (range 0.9-22.2). Eighty-two (74%) and 29 (26%) patients had acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML), respectively; they were transplanted between 09/2011 and 05/2018. All children were transplanted in complete morphological remission and received a fully myeloablative preparative regimen. Details on patients' characteristics, as well as on the number of HSC and lymphocyte subsets infused, are shown in Table 1. The donor was mainly chosen according to immunological criteria, giving priority to NK-cell alloreactivity, evaluated according to the killer immunoglobulin-like receptor (KIR)/KIR-Ligand mismatch in graft-versus-host direction model, KIR B haplotype, higher B-content score and size of NK alloreactive subset. Anti-T lymphocyte globulin (ATLG Grafalon®, Neovii Biotech) was administered at a dose of 12 mg/Kg from day -5 to -3 for preventing graft rejection and graft-versus-host disease (GvHD). Moreover, to reduce the risk of EBV-related post-transplant lymphoproliferative disorder (PTLD), on day -1, patients received rituximab (200 mg/m2) for in vivo depletion of both donor and recipient B cells. No patient was given any post-transplantation pharmacological GvHD prophylaxis. Results: Median follow-up of surviving patients is 47 months (range: 2 months - 7.7 years). All patients but two successfully engrafted and the median time to neutrophil and platelet recovery was 13 (range 9-22) and 11 (range 8-20) days, respectively. Acute GvHD occurred in 28 patients; it was of grade I and grade II severity in 9 and 19 patients, respectively. Skin was the sole organ involved in all patients but one, who had gut involvement. The cumulative incidence of grade I-II acute GvHD was 25% (95% confidence interval, CI, 17-33). Four out of the 91 patients at risk developed chronic GvHD, in all cases of limited severity, the cumulative incidence of this complication being 5% (95% CI, 1-9). Six patients died for transplant-related complications, this resulting into a 5-year cumulative incidence of transplant-related mortality (TRM) of 6% (95% CI, 2-11). Twenty-three patients relapsed at a median time of 186 days (range 60-1012) after transplantation, the 5-year cumulative incidence of relapse being 24% (95% CI, 16-33). The 5-year probability of overall and leukemia free survival (LFS) were both above 70%, as shown in Figure 1A and 1B, respectively. The 5-year probability of LFS in children with ALL and AML was 69% (95% CI, 57-79) and 73% (95% CI, 52-86), respectively (Figure 1C). Use of total body irradiation (TBI) during the preparative regimen was associated with better patient's outcome (Figure 1D), since it protected against the risk of leukemia recurrence [18% (95% CI, 10-28) vs. 45% (95% CI, 22-66) in patients who did or did not receive TBI, respectively, p Conclusions: This study, reporting long-term outcome of a large population of children/young adults with AL, confirms that αβ T- and B-cell depleted haplo-HSCT is an effective option for patients in need of an urgent allograft and lacking an HLA-identical donor. While TRM is impressively low, the main cause of treatment failure is leukemia recurrence, whose incidence can be lowered by the use of TBI during the conditioning regimen. The remarkably low risk of chronic GvHD renders the approach attractive also in terms of patient's quality of life. Figure 1 Figure 1. Disclosures Locatelli: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

Cellular immune functions are impaired in hemopoietic stem cell and solid organ transplantation or in cancer and autoimmune diseases treated with intensified immunosuppression. Thus, control of opportunistic pathogens is lost and severe infections break out. Defective cellular immunity can be restored upon endogenous immunoreconstitution or, if delayed, exogenous immunoreconstitution with pathogen specific T-lymphocytes selected or expanded from appropriate donors can be applied. Here we describe how recent developments in basic immunology knowledge and techniques have accelerated progresses of clinical trials in this attractive field. In particular, methods for the identification of appropriate antigens, for selection and expansion of specific T-cells and for safer manipulation of cellular products have been applied with promising advances. Finally, the development of biobanks of specific T-cells is described as an attractive perspective to reconstruct pathogen specific cellular immunity.

Twenty-three children with nonmalignant disorders received HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) after ex vivo elimination of αβ(+) T cells and CD19(+) B cells. The median number of CD34(+), αβ(+)CD3(+), and B cells infused was 16.8 × 10(6), 40 × 10(3), and 40 × 10(3) cells/kg, respectively. No patient received any posttransplantation pharmacologic prophylaxis for graft-versus-host disease (GVHD). All but 4 patients engrafted, these latter being rescued by a second allograft. Three patients experienced skin-only grade 1 to 2 acute GVHD. No patient developed visceral acute or chronic GVHD. Cumulative incidence of transplantation-related mortality was 9.3%. With a median follow-up of 18 months, 21 of 23 children are alive and disease-free, the 2-year probability of disease-free survival being 91.1%. Recovery of γδ(+) T cells was prompt, but αβ(+) T cells progressively ensued over time. Our data suggest that this novel graft manipulation strategy is safe and effective for haplo-HSCT. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

Background: Allogeneic HSCT from either an HLA-identical sibling or an unrelated donor is a potentially curative treatment for patients with hemoglobinopathies and erythroid disorders (ED), such as Thalassemia Major (TM), Sickle Cell Disease (SCD) and Diamond-Blackfan Anemia (DBA). The limited historical experience with HLA-haploidentical HSCT in this setting has reported a disease-free survival probability lower than that reported using HLA-matched donors. In the last few years, we have developed a novel method of graft manipulation, based on the selective depletion of α/β+ T-cells and CD19+ B-cells (ClinicalTrial.gov identifier: NCT01810120), which was shown to be safe and effective in children with multiple types of non-malignant disorders (Bertaina el al, Blood 2014). To further optimize this approach through the acceleration of the recovery of adaptive immunity, we designed an ongoing phase I/II trial aimed to test the safety and efficacy of post-transplant infusion of donor T-cells transduced with the iC9 suicide gene (BPX-501 cells) in children with either malignant or non-malignant disorders (ClinicalTrials.gov identifier: NCT02065869). As the transduced gene contains sequences for the CD19 marker, BPX-501 cells (CD3+/CD19+) can be easily tracked in peripheral blood. We report on 10 children with hemoglobinopathies and ED who were enrolled in the phase II portion of the study. Patients and methods: Five patients were males and 5 were females, and median age at diagnosis and at HSCT was 5.34 and 9.52 years (range 2.33-11.71), respectively. Seven patients had TM (all bo/bo), 2 DBA and 1 SCD. All 10 patients were transfusion-dependent and receiving iron-chelation therapy before haplo-HSCT. Among the thalassemia patients, 4 patients belonged to class I and 3 to class II of the Pesaro classification. All patients were transplanted from a parent. Median number of CD34+ and αβ+ T-cells infused was 22.5 x 106/kg and 0.3 x 105/kg, respectively. In all patients, conditioning regimen included busulfan (16 mg/Kg), thiotepa (10 mg/Kg) and fludarabine (160 mg/m2). Rabbit ATG (12 mg/Kg over 3 days, from day -4 to day -2) was administered to prevent graft-versus-host disease (GvHD) and graft failure and Rituximab (200 mg/ m2 on day -1) was administered to prevent EBV-related lymphoproliferative disorders. No post-transplantation GvHD prophylaxis was given. Median follow-up is 301 days (range 41-420 days). Basic phenotyping of circulating lymphocytes was assessed by flow cytometry on fresh heparinized peripheral blood samples at 10, 20, 30, 60, 90, 120 and 150 days post haplo-HSCT. Results: After haplo-HSCT, the median time to reach neutrophil and platelet recovery was 14 days (range 11-28) and 10 days (range 8-12), respectively. After engraftment of the allograft, BPX-501 cells were infused (dose: 1x106 cells/kg) at a median time of 13.5 days after HSCT (range 10-26). Nine of the 10 patients maintained sustained donor engraftment, reaching full chimerism. The patient who experienced secondary graft failure was successfully re-transplanted from the same parent and he is full donor chimeric with transfusion-independence. Grade I/II skin acute GvHD occurred in 2 patients (at 31 and 59 days after HSCT, respectively). There was no occurrence of chronic GVHD. Remarkably, no patient has died and none of the patients have been re-hospitalized after initial discharge. The last erythrocyte transfusion was administered on day +7 post-transplant (range 4-33 days). At last follow-up, the median hemoglobin value of these patients was 11.35 gr/dL (range 10.2-13.4). BPX-501 cells expanded after infusion and still persist in all patients at last follow-up. All children are alive and transfusion-independent. Details on T cell, NK cell and B cell recovery are shown in Figure 1 (Panel A-D). Conclusions: Children with hemoglobinopathies and DBA can benefit from curative haplo-HSCT after depletion of α/β T-cells followed by infusion of BPX-501 cells, which, expanding and persisting over time, contribute to speed immune recovery of adaptive T-cell immunity, thus rendering the procedure safer. Figure 1 Figure 1. Disclosures French: Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees. Moseley:Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees.

Background: Haplo-HSCT after depletion of α/β T and B cells is a suitable and effective option for those children with acute leukemia (AL) who need an allograft and lacking an immediately available HLA-identical donor. With this approach, recipients can benefit immediately after transplantation from the anti-leukemia effect mediated by donor natural killer (NK) and γd T cells, which can also protect against infections. A further improvement of the results achievable with this platform could achieved with a faster adaptive T-cell immunity recovery, which play a key role to augment the graft-versus-leukemia effect and the capacity to fight infections. In light of these considerations, we designed a phase I/II trial aimed at testing the safety and efficacy of post-transplant infusion of donor-derived T cells transduced with the new iC9 suicide gene (BPX-501) in children with either malignant or non-malignant disorders (NCT02065869). Remarkably, after the activation and transduction with the retroviral iC9 construct, BPX501 cells switch the phenotype towards a preferential CD45RO pattern. Patients and methods: The phase I portion of the trial consisted of a classical 3+3 design with 3 cohorts, receiving escalating doses of BPX-501 cells of 2.5x105, 5x105, and 1x106 cells/kg, respectively. Patients included in the phase II portion were planned to receive the recommended dose identified during the phase I part of the study.Enrollment of patients started in December 2014; so far, 25 patients with AL in morphological complete remission (CR) have been enrolled. Twenty patients had acute lymphoblastic leukemia (ALL) and 5 acute myeloid leukemia (AML). Details on patient, donor and transplant characteristics are reported in table 1. All patients transplanted in CR1 had either poor cytogenetic/molecular characteristics or high levels of minimal residual disease at the end of induction therapy, both factors predicting a high relapse rate. All patients were given a fully myeloablative conditioning regimen (table 1). Before haplo-HSCT, children received rabbit anti-thymocyte globulin (ATG NEOVII, 12 mg/Kg over 3 days, from day -4 to day -2) to prevent both graft-versus-host disease (GvHD) and graft failure, and Rituximab (200 mg/ m2 on day -1) to prevent EBV-related lymphoproliferative disorders. No post-transplantation GvHD prophylaxis was administered. Results: All patients engrafted and no secondary graft failure was recorded. Median time to neutrophil and platelet recovery was 18 days (range 10-22) and 11 days (range 9-13), respectively. Once documented the engraftment of donor cells, BPX-501 T lymphocytes were infused at a median time of 17 days (range 13-52) after the allograft. Two patients were enrolled in the phase I portion of the study; one each received 2.5x105 and 1x106 cells/kg. The remaining 23 children were treated in the phase II, where the recommended dose was 1x106 cells/kg. However, since we did not observe any acute GvHD requiring the infusion of the dimerizing agent (Rimiducid/AP1903) activating iC9 gene in the first 15 children receiving 1x106 cells/kg, we decided to emend the protocol to further increase the BPX501 cell dose infused to 2 and 4x106 cells/kg. Thus, the last 6 patients were enrolled in these 2 last dose levels (3 patients each). Six and 3 patients developed grade II-IV acute and chronic GvHD, respectively. In one child, given 4x106 cells/kg, we infused rimiducid for steroid-resistant grade II skin acute GvHD, with complete resolution of the disease in 24 hours. The cumulative incidence of grade II-III acute and chronic GvHD are shown in figure 1A and B, respectively. Median follow-up of these 25 children is 8 months (range 1-19 months). One of them died due to chronic GvHD-associated bronchiolitis obliterans and one child with ALL transplanted in CR2 relapsed; the cumulative incidence of non-relapse mortality and leukemia recurrence are shown in figure 1C. The probability of disease-free survival at 15 months is 87% (figure 1D). Once infused, BPX501 cells expanded and persisted over time in both peripheral blood and bone marrow. Conclusion: Overall, these data indicate that the infusion of BPX-501 cells in children with AL given selectively manipulated haplo-HSCT results in low non-relapse mortality and chronic GvHD. Although the median observation time is still limited, the cumulative incidence of disease recurrence is promising. Table 1 Table 1. Figure 1 Figure 1. Disclosures Stanson: Bellicum pharmaceuticals: Employment. Moseley:Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees.

Alterations in hematopoietic microenvironment of acute lymphoblastic leukemia patients have been claimed to occur, but little is known about the components of marrow stroma in these patients. In this study, we characterized mesenchymal stromal cells (MSCs) isolated from bone marrow (BM) of 45 pediatric patients with acute lymphoblastic leukemia (ALL-MSCs) at diagnosis (day+0) and during chemotherapy treatment (days: +15; +33; +78), the time points being chosen according to the schedule of BM aspirates required by the AIEOP-BFM ALL 2009 treatment protocol. Morphology, proliferative capacity, immunophenotype, differentiation potential, immunomodulatory properties and ability to support long-term hematopoiesis of ALL-MSCs were analysed and compared with those from 41 healthy donors (HD-MSCs). ALL-MSCs were also genetically characterized through array-CGH, conventional karyotyping and FISH analysis. Moreover, we compared ALL-MSCs generated at day+0 with those isolated during chemotherapy. Morphology, immunophenotype, differentiation potential and in vitro life-span did not differ between ALL-MSCs and HD-MSCs. ALL-MSCs showed significantly lower proliferative capacity (p

T-cell depleted HLA-haploidentical hematopoietic stem cell transplantation (HSCT) is a suitable option for patients in need of an allograft who lack a HLA-matched donor. Although it offers the advantage of being immediately applicable to virtually all patients, so far, graft manipulation with removal of all T lymphocyte subsets and of natural killer (NK) cells has been associated with an increased risk of life-threatening infections, as well as, in some studies, of leukemia recurrence. We recently developed a new method of graft manipulation based on the physical removal of αβ+ T cells and CD19+ B cells, which permits to leave mature NK cells and γδ+ T cells in the graft. We, thus, started a formal study (NCT01810120) in children with acute leukemia aimed at evaluating the safety and efficacy of this approach. As of April 2013, we enrolled 45 patients (pts; 29 males, 16 females). Median age at HSCT was 10.1 years (range 0.9-17.9). Thirty-five pts had acute lymphoblastic leukemia (ALL) and 10 acute myeloid leukemia (AML); all children were transplanted in morphological complete remission (CR). Fifteen pts were transplanted in first CR, 27 in second CR and 3 in more advanced CR. All pts transplanted in CR1 had either poor cytogenetic/molecular characteristics or high levels of minimal residual disease (MRD) at the end of induction therapy. The donor was either the mother (n=25) or the father (n=20); according to the model of KIR/KIR ligand disparity, 22 pts were transplanted from an NK-alloreactive donor. The median number of CD34+ cells, NK cells, γδ+ T cells, B cells and αβ+ T cells were 14.6, 31.7, 7.8, 0.08 and 0.04x106/kg, respectively. A myeloablative regimen, containing Total Body Irradiation in 34 cases, was given to all children, who also received anti-thymocyte globulin (12 mg/kg over 3 days, from -5 to -3). Rituximab (200 mg/m2) was administered on day -1 to further prevent EBV-related lymphoproliferative disorders. No pharmacological graft-versus-host disease (GVHD) prophylaxis was employed after transplantation. Sustained primary engraftment occurred in 44/45 pts, the remaining child being successfully re-transplanted from the other parent. The median time to reach an absolute neutrophil count >0.5x109/L and a platelet count >50x109/L was 13 days (9-18) and 11 days (8-20). No child developed gut or liver acute GVHD. Thirteen pts experienced skin-only grade I-II GVHD, this leading to a cumulative incidence (CI) of 29% (95% confidence interval, CO. IN., 18-45). Only 2 pts developed skin limited chronic GVHD. Two pts died for causes other than disease relapse (both in the first 60 days after HSCT), the CI of transplantation-related mortality (TRM) being 4% (CO. IN. 1-16). Seven pts relapsed, the CI of disease recurrence being 16% (CO. IN. 6-32). With a median follow-up of 11 months (range 2-30), the 2-year Kaplan Meier estimate of leukemia-free survival (LFS) was equal to 75% (CO. IN. 57-86); this value was 73% (CO. IN. 52-85) for pts with ALL. LFS of pts who did or did not experience skin-only acute GVHD was 83% (CO. IN. 48-96) and 72% (CO. IN. 50-86), p=NS. The probability of LFS of the 39 pts with either negative or low ( These data indicate that a selective graft manipulation results into effective prevention of both acute and chronic GVHD, rapid recovery of neutrophil and platelet counts and low TRM. Although the median observation time is still limited, also the CI of disease recurrence is encouraging. Post-transplant add-back of limited numbers of αβ+ T cells transduced with the iCasp9 suicide gene could optimize the recovery of this subset in early weeks after HSCT, maximizing immune-mediated viral and leukemia control, without an unacceptable risk of GVHD. Disclosures: No relevant conflicts of interest to declare.

Background: We recently completed a prospective study (ClinicalTrial.gov identifier: NCT01810120) which showed that haplo-HSCT after depletion of α/β T cells is an effective option for those children in need of an allograft and lacking an immediately available HLA-identical related or unrelated donor. However, recovery of adaptive T-cell immunity remains suboptimal and some patients died due to viral infections in the early post-transplant period. Thus, strategies aimed at accelerating early recovery of adaptive T-cell immunity are desirable. Study design and patients: We designed a phase I/II trial aimed at testing the safety and the efficacy of post-transplant infusion of donor-derived T cells transduced with the new iC9 suicide gene (BPX-501) in children with malignant or non-malignant disorders (ClinicalTrials.gov identifier: NCT02065869); enrollment started in December 2014. Cells are administered within 14 + 4 days after haplo-HSCT. The phase I portion of the trial consists of a classical 3+3 design with 3 cohorts, receiving escalating doses of BPX-501 cells of 2.5 x 105, 5 x105, and 1x106 cells/kg, respectively. Patients included in the phase II portion received the highest dose identified during the phase I portion of the study for a maximum of 60 children in both phase I/II portions of the study. As of July 25th 2015, 25 children have been screened and included in the study: 23 have been infused with BPX-501 cells. The analysis refers to the 16 patients with a minimum follow-up of 90 days after transplantation; they had acute lymphoblastic leukemia (ALL, 6), acute myeloid leukemia (1), severe combined immune-deficiency (4), Wiskott-Aldrich syndrome (3) and Fanconi Anemia (2). All children with acute leukemia were transplanted in morphological complete remission (CR). Median age at haplo-HSCT was 3.5 years (range, 03-17.8); 7 patients (44%) were females. All children received >10x106 CD34+ cells/Kg and Results: BPX-501 cells were infused at a median time of 16 days (range 13-18); median cell viability post-thaw was 91% (range 65-97). Treatment was well tolerated and no infusion-related side effects were recorded. The recommended dose identified during the phase I of the trial to be used for the phase II portion was 1x106 cells/kg. Four children developed grade I-II skin only acute graft-versus-host disease (GvHD) at 16, 20, 22 and 34 days after haplo-HSCT, respectively, which resolved with topical steroids; no patient had either gut or liver acute GvHD. The 100-day cumulative incidence (CI) of skin-only grade I-II acute GvHD was 25% (SE 3.6); it was 30% (SE 2.1) in the historical controls (Figure 1 - Panel A). No patient developed chronic GvHD. In 4 patients, mixed chimerism present at time of BPX-501 cell infusion completely reverted to full donor chimerism. None of the 16 patients included in the analysis had graft failure or died of transplant-related complications. Two patients, both with ALL transplanted in CR3, relapsed at 86 and 153 days after the allograft, respectively. Median time to discharge after haplo-HSCT was 28 days (range, 19-86) as compared to 38 days (range, 18-174) in the historical controls (p=0.08). Four patients were re-hospitalized due to: cytomegalovirus (CMV) infection (2), fever of unknown origin (1) and valganciclovir-induced neutropenia (1). BPX-501 cells progressively expanded over time and are still persisting, potentially contributing to the recovery of adaptive T-cell immunity. The mean number of both CD3+ and BPX-501 cells at the different time-points are reported in Figure 1 - Panel B, which also details the data of historical controls. Conclusions: Overall, these data indicate that the infusion of BPX-501 cells is safe and well tolerated. The 100-day CI of skin-only grade I-II acute GvHD observed in these patients is similar to that of children included in the previous trial of haplo-HSCT after depletion of α/β T cells. BPX-501 cells expand in vivo and persist over time, potentially contributing to accelerate the recovery of adaptive T-cell immunity, with improved clinical outcome. The iC9 cell-suicide system may increase the implementation of cellular therapy approaches aimed at optimizing immune recovery after transplantation. Figure 1. Figure 1. Disclosures Qasim: Cell Medica: Research Funding; Autolus Ltd: Consultancy, Equity Ownership, Research Funding; Miltenyi Biotec GmbH: Research Funding; Cellectis: Research Funding. Moseley:Bellicum Pharmaceuticals: Employment, Equity Ownership.

Background: Immune recovery is crucial for patients treated with allogeneic HSCT and in particular of those receiving a T-cell depleted haplo-HSCT. We recently developed a novel method of graft manipulation based on physical elimination of α/β T cells and B-lymphocytes for preventing graft-versus-host disease (GvHD) and EBV-related lymphoproliferative disorders, respectively. Thanks to this approach, we successfully conducted a prospective trial in children with malignant or non-malignant disorders (ClinicalTrial.gov identifier: NCT01810120). Although patients enrolled in this trial had faster immune recovery and lower incidence of infections than those given haplo-HSCT after infusion of positively selected CD34+ cells, reconstitution of adaptive T-cell immunity remains suboptimal. We therefore designed a phase I/II trial aimed at testing the effect on post-transplant immune recovery of adoptive infusion (within 14 + 4 days after transplantation) of BPX-501 cells in children given haplo-HSCT after depletion of α/β T and B cells (ClinicalTrials.gov identifier: NCT02065869). Patients and methods: As of July 25th 2015, 23 children have been infused with BPX-501 cells. The 9 children included in the phase I portion of the study were given 2.5x105, 5x105, and 1x106 BPX-501 cells/kg, respectively, while the 14 included in the phase II received 1x106 BPX-501 cells/kg. This analysis refers to the 16 patients with a minimum follow-up of 90 days; 7 children had acute leukemia and 9 non-malignant disorders. Basic phenotype of circulating lymphocytes was assessed by flow cytometry on fresh heparinized peripheral blood samples at 10, 20, 30, 60, 90, 120 and 150 days post haplo-HSCT, respectively. The following antibodies were used: anti-TCRαβ FITC/anti-TCRγδ PE/anti-CD3 PerCP-Cy™5.5 (WT31, 11F2, SK7), anti-CD4 APC Cy7 (RPA-T4), anti-CD19 BV 510 (SJ25C1), anti-CD3 BV 421 (UCHT1), anti-CD56 PeCy7 (B159), anti-CD16 APC (B73.1), anti-CD8 APC (RPA-T8) from BD Biosciences (San Diego, CA, USA). Antigen-driven activation of peripheral mononuclear cells was evaluated by standard lymphoproliferation assay (LPA) with 3H-thymidine pulsing on day 4 and harvesting 18 hours later. Antigens included PHA or CMV, EBV and AdV whole viral lysate. Results were scored positive with stimulation indexes (SI) >10 for PHA and >3 for viral antigens. Results: None of the patients died from transplant-related complications. Chimerism analysis investigated through short tandem repeats showed that in all but 4 patients, cells were of donor origin before the infusion of BPX-501 cells. In the 4 patients, there was a reversion to complete donor chimerism after infusion of BPX-501 cells. At early time points after haplo-HSCT, gδ T cells predominated over αβ T lymphocytes; subsequently, this latter population became the more largely represented. The number of both CD3+ T lymphocytes and of BPX-501 cells is shown in Panel A of Figure 1, reconstitution of whole T cells in historical children given haplo-HSCT after depletion of α/β T cells is also shown. The number of CD3+ T lymphocytes reached greater than 0.5x109/L 2 months after infusion of BPX-501 cells. Remarkably, while usually immune recovery after transplantation is characterized by prevalence of CD8+ cells, in our patients the physiological predominance of CD4+ lymphocytes was maintained (Panel B of Figure 1. Reconstitution of natural killer cells (NK) is shown in Panel C of Figure 1. As compared to patients receiving CD34+ selected cell haplo-HSCT, children included in this study had a faster reconstitution of mature KIR+/NKG2A- NK cells. Serum levels of IgA and IgM over time are shown in Panel D of Figure 1: there was a recovery of newly synthetized Ig at 3 months. The analysis of the function of T cells showed that the proliferative response to a polyclonal mitogen or to CMV lysate was comparable to that of a healthy control in 50% of patients as early as day + 60 after haplo-HSCT and BPX-501; on day +150, all patients reached a normal SI. Response to both EBV and AdV antigens was slightly delayed, but progressively improved over time (see also Figure 2). Conclusions: Overall, these data indicate that infusion of BPX-501 cells is able to accelerate the recovery of adaptive T-cell immunity since these cells, once infused, expand in vivo and persist over time, potentially contributing to protect patients from infections. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Moseley: Bellicum Pharmaceuticals: Employment, Equity Ownership.

Background: Allogeneic HSCT is a widely used treatment for children with acute leukemia (AL) either relapsed or at high risk of treatment failure. However, an HLA-identical sibling is available for only 20-25% of patients and an UD can be located in a suitable time only for a portion of the remaining population. HSCT from an HLA-haploidentical relative (haplo-HSCT) is now considered an alternative option, especially in view of the recent insights in graft manipulation. We recently developed a novel method of more selective T-cell depletion based on physical elimination of α/β T cells (ClinicalTrial.gov identifier: [NCT01810120][1]), shown to be effective for both preventing graft-versus-host disease (GvHD) and for conferring improved protection against infections in comparison to haplo-HSCT performed through the infusion of positively selected CD34+ cells. The initial results on 40 patients with AL were reported at the ASH Meeting in 2013 (Bertaina et al). We now present the comparison of the outcome of 80 children with AL given haplo-HSCT after α/β T-cell depletion (group 1) with that of patients transplanted from an HLA-identical sibling (group 2) or an UD (group 3) in the same time period. Patients and methods: All patients with AL were transplanted at the Bambino Gesu Children's Hospital in Rome, Italy, between December 2010 and September 2014; 80 patients were included in group 1, 41 in group 2 and 51 in group 3. Patients were offered α/β T-cell-depleted haplo-HSCT in the absence of suitable conventional donor (HLA identical sibling or 10/10 UD evaluated using high resolution typing) or if affected by rapidly progressive disease not permitting time to identify an UD. Clinical characteristics of patients assigned to the 3 groups and those of their donor are shown in Table1. All children were given a fully myeloablative regimen. No group 1 patient was given any post-transplantation GvHD prophylaxis, while patients of group 2 and 3 were given Cyclosporine-A and short-term methotrexate. Group 1 and 3 patients received ATG Fresenius® (4 mg/Kg/day) from day -5 to -3 for preventing both graft rejection and GvHD. Results: All group 2 and 3 patients had sustained engraftment of donor cells, while 1 of the 80 patients included in group 1 experienced primary graft failure and was rescued by haplo-HSCT from the other parent. The cumulative incidence (CI) of acute GvHD was 30%, 41% and 42%, respectively. Remarkably, all children of the group 1 who developed acute GvHD had a skin-only involvement, while 17% and 16.3% of those of group 2 and 3 had either gut or liver involvement (p

Background: T-cell depleted haplo-HSCT is an established treatment for children with primary immune deficiencies (PID). However, children given this type of allograft are exposed to the risk of fatal events due to viral infections because of the prolonged impairment of adaptive immunity. We recently developed a novel method of selective T-cell depletion based on physical elimination of α/β T cells (ClinicalTrial.gov identifier: NCT01810120), which was shown to be safe and more effective than transplantation of positively-selected CD34+ cells for preventing life-threatening infections. However, we recorded some severe and even fatal viral infections, which prompted us to explore innovative approaches to accelerate the recovery of adaptive immunity. For this purpose, we designed an ongoing phase I/II trial aimed at testing the safety and the efficacy of post-transplant infusion of BPX-501 cells in children with malignant or non-malignant disorders (ClinicalTrials.gov identifier: NCT02065869). We report 3 cases of children with either severe combined immune deficiency (SCID) or Wiskott-Aldrich syndrome (WAS), who were enrolled in the dose escalation phase of the study and who cleared cytomegalovirus (CMV) or Adenovirus (AdV) infections likely due to the contribution of the BPX-501 cells. Patients and methods: Patient #1, affected by SCID, was transplanted from the HLA-haploidentical father. Before transplantation she had CMV-DNAemia which was treated with ganciclovir until donor stem cell infusion. She was given 2.5 x 105/kg BPX-501 cells on day 17 after transplantation. Patient #2, also affected by SCID, was transplanted from the HLA-haploidentical mother. Before transplantation she had AdV-DNAemia and high load of the virus in stools. She was given 5 x 105/kg BPX-501 cells on day 15 after transplantation. Patient #3 was affected by WAS and referred to the transplant unit; in the months preceding haplo-HSCT the child had developed CMV retinitis and hepatitis with high levels of CMV-DNAemia. This patient was transplanted from the father and received 1 x 106/kg BPX-501 cells on day 15 after haplo-HSCT. Basic phenotype of circulating lymphocytes was assessed by flow cytometry on fresh heparinized peripheral blood samples at 10, 20, 30, 60, 90, 120 and 150 days post haplo-HSCT, respectively. Since BPX-501 cells are CD3+/CD19+, it was easy to track the presence of these genetically modified cells. CMV specific reconstitution was also monitored through the INF gamma ELISPOT assay. In particular, peripheral blood mononuclear cells were stimulated for 16hrs in the presence of peptide libraries derived from pp65, IE1 and IE2 CMV-specific antigens. Results: The increase in the number of both CD3+ T lymphocytes and BPX-501 cells over time after transplantation together with the modifications of DNAemia of both CMV and AdV in the 3 patients are reported in Panel A, B and C, respectively, of Figure 1. In all of these patients, the pre-existing viral infection was progressively cleared once the BPX-501 cells were infused. These cells expanded in vivo and are still persisting, contributing to the recovery of adoptive immunity. The median time to reach an absolute number of α/β CD3+ cells greater than 0.5x109/L was 90, 90 and 30 days, respectively. None of these patients experienced either acute or chronic Graft-versus-Host Disease (GvHD) and no organ inflammatory-related toxicity was recorded. All children are alive and disease free, without infections, at day +200, +180 and +160, respectively. The 2 patients with CMV infection showed a specific response for at least one CMV-derived antigen; indeed, one patient showed a prevalence in pp65 response, whereas in the second one, we observed a specific anti-CMV response against all three tested antigens (Figure 1 - Panel D). Conclusions: Infusion of BPX-501 cells is able to accelerate the recovery of adaptive T-cell immunity in children with PID given haplo-HSCT after depletion of α/β T cells, thus rendering the procedure safer even in children with active infections at time of transplantation. These cells, once infused, expand in vivo and persist over time, contributing to the clearance of viral infections, without inducing GvHD. Figure 1. Figure 1. Disclosures Moseley: Bellicum Pharmaceuticals: Employment, Equity Ownership.

Adherent antigen presenting cells (APC) pulsed with protein or peptide antigens were used to capture specific CD4 or CD8 T-cells derived from established T-cell lines or from PBMC of immune subjects based on physiological interaction between TCR and MHC-peptide complex. This method could be applied independently of epitope specificity, HLA restriction alleles, activation markers and secreted cytokines, parameters required by other methods for selection of specific T cells. Non specific T-cells were removed by applying a 1g force that did not affect binding of specific T-lymphocytes. Lymphocyte selection was specific and the average recovery was 36% for CD4 T-cells. CD8 T-cells proved trickier to purify, since solid phase APC were recognized as targets for cytotoxicity. Specificity was comparable to CD4 cells, but the average recovery for CD8 cells was 26%. No residual alloreactivity was detected in expanded T-cells. Frequency and recovery of specific T-cells were comparable to other current technologies, such as generation of T-cell lines and cytokine capture method. Since antigen and IL2 are the only reagents added to the cultures, this physiological procedure can be proposed for selection and expansion of pathogen specific T-cells not only for research purposes, but also for adoptive reconstitution of immunocompromised subjects if performed under GMP conditions.

The evaluation of antigen-specific T-cell responses is helpful for both research and clinical settings. Several techniques can enumerate antigen-responsive T cells or measure their products, but they require remarkable amounts of peripheral blood mononuclear cells (PBMCs). Since screening numerous antigens or testing samples from pediatric or lymphopenic patients is hampered in clinical practice, we refined a miniaturized, high-throughput assay for T-cell immunity. Antigens and cells in 10-μl volumes were dispensed into 1,536-well culture plates precoated with anti-gamma interferon (anti-IFN-γ) antibodies. After being cultured, the wells were developed by enzyme-linked immunosorbent assay for bound cytokine. Miniaturization and automation allowed quantitation of antigen-specific responses on 104PBMCs. This method was applied for epitope mapping of mycobacterial antigens and was used in the clinic to evaluate T-cell immunity to relevant opportunistic pathogens by using small blood samples. A comparison with conventional methods showed similar sensitivity. Therefore, current flow cytometric methods that provide information on frequency and phenotype of specific T cells can be complemented by this assay that provides extensive information on cytokine concentrations and profiles and requires 20- to 50-fold fewer PBMCs than other analytical methods.

Objective Reconstitution of cellular immunity by infusion of cytomegalovirus (CMV)-specific T lymphocytes is an attractive alternative to drugs currently used to control CMV reactivation in immunocompromised patients. For this purpose, we established a method for generating both anti-CMV CD4 and CD8 T cells following Good Manufacturing Practice indications, and we extensively characterized their immune functions. Materials and Methods For generating CD4 and CD8 CMV-specific lymphocytes, T cells from 11 CMV-seropositive donors were stimulated three times with dendritic cells (DC) pulsed with a library of selected CMV peptides, recognized by >85% of the Caucasian population. At the end of the culture, T cells were analyzed for their specificity, cytotoxicity, chemotactic migration, proliferation, and cytokine production. Results T cells were successfully expanded and enriched in CMV-specific subsets with an effector memory or an effector memory CD45 RA + phenotype. CMV-specific T-cell lines showed specific cytotoxicity (average lysis: 47%) against CMV peptides-pulsed DCs, and were depleted of auto- and alloreactivity. Moreover, the ability to proliferate following antigenic stimulation and the presence of functional CD4 lymphocytes producing Th1 and Th2 cytokines can ensure long-term antiviral immunity after in vivo injection. CMV-specific T lymphocytes also proved to be fully equipped to reach CMV-infected tissues, because they expressed CD49d and CCR1, CXCR3, CXCR4, necessary to recruit effector cells to inflamed sites. In accordance with this profile, they significantly migrated towards inflammatory chemokines and towards the supernatant collected from inflamed lung fibroblasts, frequently involved in CMV pathology. Conclusion This strategy allows expansion of effector T cells capable to exert CD8 and CD4-mediated immune functions and, thus, is suitable for clinical use.

In addition to HIV infection, several acquired immunodeficiencies lead to depletion of CD4 lymphocytes. These include immunosuppression resulting from high dose cancer chemotherapy or induced to control graft rejection, as well as in autoimmune diseases. The consequence of this depletion is an increased susceptibility to opportunistic infections or the inability to control primary infection in the case of HIV infection. In all instances a full or partial immunoreconstitution is desirable. In order to monitor the cellular immune state of a patient, rational information cannot be simply derived from phenotypic quantification of T lymphocytes. Instead loss or recovery of CD4 cells should be monitored by defining the specificity, the function and the clonality of the relevant cell population. Several methods are now available for this type of investigation. Here we describe an approach for the definition of clonal heterogeneity of antigen specific CD4 lymphocytes, a parameter that may help monitor loss or reconstitution in acquired immunodeficiencies. As examples of antigen specific CD4 T cell responses we focused on Pneumocystis carinii and on cytomegalovirus, as prototypic opportunistic pathogens which are responsible for severe infections in AIDS and in other immunosuppressive conditions which arise for instance following transplantation. Specific CD4 T cell lines were generated from normal controls and from seropositives in order to select antigen specific lymphocytes. The cells were subsequently analyzed for clonal diversity according to TCR BV gene family usage and according to TCR CDR3 size heterogeneity (spectratyping).

Abstract 2018 Introduction. Although HLA haploidentical hematopoietic stem cell transplantation (HSCT) has been largely employed in children with life-threatening non-malignant disorders, the survival of patients given this type of allograft has been reported to be inferior to that of patients transplanted from a compatible, unrelated volunteer (UV). We have recently developed a novel method of ex vivo T-cell depletion based on the selective elimination of αβ+ T cells through labeling with a biotinylated anti-TCR αβ antibody, followed by incubation with an anti-biotin antibody conjugated to paramagnetic beads (CliniMACS®, Miltenyi Biotec, Germany). We also remove B cells through an anti-CD19 monoclonal antibody, to prevent post-transplant EBV-associated lymphoproliferative disease (PTLD). Here, we report the results on 13 patients given this type of allograft. Patients and Methods. Seven patients were males and 6 females, median age at transplantation being 3 yr and 9 mo (range 0.3–28.7). Four patients had severe combined immune deficiency (SCID), three Fanconi anemia (FA), 2 severe aplastic anemia (SAA) and 1 each immune deficiency with polyendocrinopathy, enteropathy, X-linked (IPEX), congenital amegakaryocytic thrombocytopenia (CAMT), hemophagocytic lymphohistiocytosis (HLH) and thalassemia with autoimmune hemolytic anemia. All patients were transplanted from 1 parent (10 from the mother and 3 from the father), the median number of CD34+ cells, αβ CD3+ cells and B cells infused being 17.8×106/kg, 0.64×105/kg and 5.3×106/kg, respectively. Conditioning regimen consisted of treosulfan and fludarabine (FLU) in 7 children (4 SCID, 1 IPEX, 1 HLH and 1 CAMT), FLU and cyclophosphamide in 5 (3 FA and 2 SAA) and busulphan, FLU and thiotepa in 1 child (thalassemia). No patient received pharmacological prophylaxis for graft-versus-host disease (GVHD) after the allograft. All patients were given anti-thymocyte globulin (ATG Fresenius; 3 mg/kg/day) on days -5 through -3 before allografting and, to prevent PTLD, rituximab (200 mg/m2) on day -1. Results. All patients but 2 engrafted, the median time to reach neutrophil and platelet recovery being 13 days (range 8–19) and 11 days (range 7–40), respectively. The 2 patients (1 each with SAA and thalassemia) who had primary graft failure were successfully re-transplanted, the first one from the same parent and the second one from the other parent. No patient experienced secondary graft failure. Grade I/II skin acute GVHD occurred in 3 patients, while no patient had visceral acute GVHD. Limited skin chronic GVHD occurred in 1 of the 8 patients at risk. Two patients (1 with SAA and 1 with CAMT) died from respiratory failure secondary to cytomegalovirus and adenovirus infection at 60 and 80 days after transplantation. Two more patients had episodes of cytomegalovirus re-activation, which were successfully treated with ganciclovir. No patient had EBV-related PTLD. With a median follow-up of 209 days (range 37–543), 11 out of the 13 (84%) patients are alive and disease-free, the Karnofsky/Lansky score being 100. The median chimerism is 100% (range 85–100). Noteworthy, all patients with primary immune disorders and FA are alive and disease-free. T-cell recovery was initially sustained by gd T cells, while, after 45 days from the allograft, αβ T cells predominated. Conclusions. The infusion of B-cell and αβ+ T-cell-depleted hematopoietic progenitors from a HLA-haploidentical parent is an effective treatment option for children with life-threatening congenital or acquired non-malignant disorders. The engraftment rate was high and incidence of acute GVHD was low. The transplantation-related mortality of 15% observed in this cohort is comparable to that observed using a HLA-matched UV. If confirmed in a larger cohort of patients and with a longer follow-up, these results suggest that this transplant option be offered to any child lacking a HLA-identical sibling. Disclosures: No relevant conflicts of interest to declare.

Abstract 343 Introduction. Allogeneic hematopoietic stem cell transplantation (HSCT) from a HLA-haploidentical relative is a suitable option for patients (pts) lacking a compatible donor, either related or unrelated. The two main approaches for overcoming the obstacles of HLA barriers are based either on the infusion of large numbers of T-cell-depleted HSC or on intensive pharmacological prevention of graft-versus-host disease (GVHD). While for many years T-cell depletion (TCD) of the graft has been based on either immunomagnetic positive selection of CD34+ cells or on physical removal of all subsets of T cells by virtue of mAb, we and other groups have recently developed a novel method of ex vivo TCD based on the selective elimination of αβ+ T cells through labeling with a biotinylated anti-TCRαβ Ab, followed by incubation with an anti-biotin Ab conjugated to paramagnetic beads (Miltenyi Biotec, Germany). This approach also allows the removal of B cells to prevent post-transplant EBV-associated lymphoproliferative disease (PTLD). Here, we report the results of graft manipulation using this approach. Methods. Twenty-two children entered the study, 15 with hematological malignancies and 7 with non-malignant disorders. No post-transplant GvHD prophylaxis was employed. HLA-haploidentical family donors received G-CSF (12–16 μg/kg of body weight) to mobilize HSC prior to large-volume leukapheresis, which was commenced when circulating CD34+ HSC were >20 cells/μl. Cell therapy products containing up to 60×109 white blood cells (WBC) were processed according to the manufacturer's protocol. In some cases, leukapheresis bags were stored overnight at 4°C in appropriate media at a WBC concentration Results. Median recovery of CD34+ HSC and median number of infused CD34+ HSC were 99.3% (range 55.4–100) and 14.7×106/kg (range 7.9–37), respectively. The graft contained 3.9×106 CD3+ T cells/kg (range 0.9–30.9) and 0.08×106 B cells/kg (range 0.002–0.32). The log-depletion of αβ+ T cells was 4.5 (range 3.2–6.1), with a median number of transplanted αβ+ T cells equal to 36×103/kg (range 1–139.9). Patients received 36×106 CD56+ NK cells/kg (range 1.6–80.3) and 4.1×106 γδ+ T cells/kg (range 0.9–30.8). A median of 40.8% NK cells (range 30.3–60.2) co-expressed Tim-3, an activating co-receptor promoting IFN-γ production and being scarcely expressed on NK cells that reconstitute after allogeneic HSCT. The αβ/CD19-depleted grafts also contained DC1 and DC2 precursors, which were identified based on BDCA-1 (0.23% of all nucleated cells, range 0.02–2.4), BDCA-3 (0.93%, range 0.57–9.4) and BDCA-2 (0.66%, range 0.49–1.28) or BDCA-4 expression (0.69%, range 0.02–1.5), respectively. Invariant NKT cells were enumerated using mAb reacting against the TCR Vα24-Jα18 chain and were detected in minute percentages ( Conclusions. The immunomagnetic removal of αβ+ T cells and B cells was effective and gave reproducible results. In all cases, a remarkable depletion of unwanted T and B cells was attained. The depleted graft contained high numbers of CD34+ HSC, as well as of immune effector cells implicated in the control of leukemia growth and GVHD, such as γδ+ T cells, NK cells, DC1, DC2 and non-classical CD14+CD16+ monocytes. From a clinical standpoint, the infusion of αβ/CD19-depleted grafts from HLA-haploidentical family donors was safe, resulting into sustained donor engraftment without life-threatening complications. Disclosures: No relevant conflicts of interest to declare.

Background HLA-haploidentical hematopoietic stem cell transplantation (HSCT) is suitable for patients lacking related or unrelated HLA-matched donors. Herein, we investigated whether plerixafor (MZ), as an adjunct to G-CSF, facilitated the collection of mega-doses of hematopoietic stem cells (HSC) for TCR-αβ/CD19-depleted haploidentical HSCT, and how this agent affects the cellular graft composition. Methods Ninety healthy donors were evaluated. Single-dose MZ was given to 30 ‘poor mobilizers’ (PM) failing to attain ≥40 CD34+ HSCs/μL after 4 daily G-CSF doses and/or with predicted apheresis yields ≤12.0x106 CD34+ cells/kg recipient’s body weight. Results MZ significantly increased CD34+ counts in PM. Naïve/memory T and B cells, as well as natural killer (NK) cells, myeloid/plasmacytoid dendritic cells (DCs), were unchanged compared with baseline. MZ did not further promote the G-CSF-induced mobilization of CD16+ monocytes and the down-regulation of IFN-γ production by T cells. HSC grafts harvested after G-CSF + MZ were enriched in myeloid and plasmacytoid DCs, but contained low numbers of pro-inflammatory 6-sulfo-LacNAc+ (Slan)-DCs. Finally, children transplanted with G-CSF + MZ-mobilized grafts received greater numbers of monocytes, myeloid and plasmacytoid DCs, but lower numbers of NK cells, NK-like T cells and Slan-DCs. Conclusions MZ facilitates the collection of mega-doses of CD34+ HSCs for haploidentical HSCT, while affecting graft composition. Electronic supplementary material The online version of this article (doi:10.1186/s12967-014-0240-z) contains supplementary material, which is available to authorized users.