ALBERT

Description: 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.

Description: Introduction Tyrosine Kinase Inhibitors (TKI) have completely changed the scenario of CML and dramatically improved the outcomes. Thus, early identification of patients expecting poor outcome is crucial to offer alternative TKI regimens or in some selected cases stem cell transplantation before disease progression may occur. The Evaluating Nilotinib Efficacy and Safety in Trial as First-Line Treatment (ENEST1st) is a phase 3b is an open-label study of nilotinib 300 mg twice daily (BID) in adults with newly diagnosed BCR-ABL positive CP-CML. Aim of the ENEST1st sub-study N10 was to investigate BM microenvironment markers that regulate leukemic stem cells in the bone marrow (BM) niche of Nilotinib-treated patients. Methods The study enrolled patients in 21 Italian ENEST1st participating centers. Response was based on ELN recommendations (Baccarani M, et al. Blood 2013 122:872-884). In an interim analysis, molecular and cytogenetic response by 24 months was assessed. Mononuclear cells were collected from BM and PB samples at the screening visit (V0) and after 3 months of treatment (V4). RT-qPCR for the expression of 10 genes (ARF, KIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the stemness and hematopoietic stem cells survival signaling regulation was conducted. RT-qPCR data were normalized by the expression of GUS mRNA (normalized copy number, NCN). Plasma samples were collected at different time points from both BM or PB samples. Concentrations of 20 different analytes, including IL-1a, IL-3, M-CSF, SCF, SDF1-a, TRAIL, HGF, PDGF-bb, IL1b, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, G-CSF, GM-CSF, MIP-1a, TNF-a, and VEGF, were simultaneously evaluated using commercially available multiplex bead-based sandwich immunoassay kits. Results 33 out of 37 patients enrolled were available for an interim molecular analysis at 24 months: an optimal response was achieved in 25 patients, a warning response in 5 patients and a failure response in 3 patients. We observed a significant correlation between the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) or cytokine signaling (SET) and patient outcome. Indeed, NANOg and SET mRNA were significantly down-regulated in PB samples at diagnosis of patients with optimal response compared to patients with warning/failure response (NANOg mRNA: 0.3±0.25 NCN vs 0.6±0.7 NCN, respectively; p=0.05; SET mRNA: 0.2±0.3 NCN vs 2.3±4.2 NCN, respectively; p=0.03). We also investigated the plasma level of several factors involved in the hematopoietic stem cells (HSCs). Some of these markers showed a significant correlation with patient's outcome when evaluated at diagnosis in either PB or BM samples. Indeed, high level of IL12 (in the BM samples), or HGF, mCSF and SCF (in the PB samples) were associated to a worst prognosis markers, since significantly correlating with no MMR@12months (IL12, p=0.03), intermediate/high Socal score (mCSF, p=0.03; SCF, p=0.03), no reduction of MMR below to 1 at 3 month (SCF, p=0.04) or warning/failure response to Nilotinib treatment (HGF, p=0.03; SCF, p=0.04). Indeed, we find a lower levels of PDGFb, SDF1, TNFa, TRAIL (in the BM samples), and HGF, SDF1, TRAIL (in the PB samples) in those patients with intermediate/high Hasford or Sokal score (PDGFb, p=0.0007; SDF1, p=0.02), warning/failure response to Nilotinib treatment (HGF, p=0.03) or lacking of MMR4.0 (SDF1, p=0.01; TNFa, p=0.02; TRAIL, p=0.05). Conclusion/Summary Taken together, our results suggest that the expression analysis of genes involved in cell pluripotency (NANOg) and/or cell signaling (SET) at baseline, may indicate early achievement of deep molecular response in shown CML-CP patients treated with nilotinib. In addition, in patients with optimal response to Nilotinib, high concentration of SDF-1, TRAIL (inversely correlated with BCR-ABL, and associated to an higher susceptibility to apoptosis in the leukemic blasts) were observed as well as BM TNF (cell-extrinsic and potent endogenous suppressor of HSC activity). A lower concentration of several factors associated to hematopoietic progenitor cell growth and survival (including HGF, SCF and IL12) were observed compared to patients failing to achieve response to Nilotinib. These data strongly suggest that stromal microenvironment supports the viability of BCR-ABL cells in BM niches through direct feeding, or environment releasing of survival factors. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Galimberti:Novartis: Employment. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Description: 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.

Description: 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.

Description: 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

Description: 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.

Description: In view of the exciting results reported in patients with CD19+ malignancies given CAR T cells, it is expected that a continuously growing number of patients will be offered this treatment and, thus, will be exposed to gene-modified products. Since the techniques of gene manipulation are relatively new, some of the risks associated to CAR T therapy may be unpredictable. Recently, two patients who relapsed with CD19-, CAR-expressing leukemia were reported, this observation being interpretable in light of an inadvertent leukemic cell transduction with the second generation CAR.CD19 lentivirus during CAR T cell manufacturing (Lacey, ASH, 2016 128:281). Immunoglobulin heavy chain sequencing analysis of 17 additional infusion products also identified the leukemic clonotypes in six additional products (86%). In vitro and in vivo experiments proved that these CAR+ leukemic clones were not killed by CAR.CD19 T cells (Ruella, ASH, 2017 130:4463). Since lentiviruses proved to be superior for transduction of quiescent hematopietic stem cells due to their ability to infect non-dividing cells, we hypothesized that CAR-T cell manufacturing based on the genetic modification of T cells by gammaretroviral vector could theoretically represent a safe approach. Peripheral blood or bone marrow (BM)-derived mononuclear cells of patients with 〉40% of blasts at diagnosis (CD45dim+/CD34+/CD19+/CD22+/CD10+), were transduced with a retrovirus encoding for a second generation CAR.CD19.41bb.z in frame with a suicide gene (i.e., inducible caspase 9, iC9) employed in the academic Clinical Trial (NCT03373071) run at the Bambino Gesù Children's Hospital, Rome, Italy. Patient-derived CAR-T cells showed a phenotype not significantly different from that found on CAR-T cells generated by healthy-donors (data not shown). In particular, we demonstrated that both flow-cytofluorimetry and RealTime-quantitative PCR (with a sensitivity up to 10-5) failed to identify leukemic cells in the final CAR-T cell products generated from Bcp-ALL patients. To generate an in vitro model of CAR+ leukemic cells, we genetically modified CD19+ RAJI and DAUDI cell lines with the bicistronic retroviral vector carrying both second generation CAR.CD19 and the suicide gene iC9 (iC9.CAR-RAJI and iC9.CAR-DAUDI). We demonstrated the possibility of promptlyeliminating CAR+ leukemic cells, through exposure to 20nM of AP1903 of iC9.CAR-DAUDI and iC9.CAR-RAJI cells. Indeed, very early activation (6 hours) of the suicide gene iC9 resulted into a significant reduction in the percentage of CAR+ RAJI leukemic cells (Fig.A). The presence of iC9.CAR.CD19 molecule on leukemic cells precluded the detection of the CD19 antigen, whereas cells retain the expression of all other specific B-lineage markers. CD19 antigen started to be detectable 72 hours after AP1903 exposurewhen CAR negative leukemic cells become preponderant. To demonstrate that CD19 antigen was not down-regulated, but only masked by CAR molecule in iC9.CAR-RAJI and iC9.CAR-DAUDI cell lines, we measured CD19 mRNA, showing no significant modification with respect to wild-type (WT) RAJI and DAUDI cell lines. Moreover, iC9.CAR-RAJI and iC9.CAR-DAUDI cell lines were effectively eliminated by CAR.CD19 T cells (12.5±13.7% and 3.4±4.3% residualleukaemia, respectively) at the same extent of WT cell line (0% and 0.08±0.1%, residual leukaemia, respectively; p〉0.05 Fig.B). To assess if patient-derived iC9.CAR.CD19 T cells were able to generate leukemia in vivo mouse model, NSG female mice were infused i.v. with 10x106 CAR-T cells and control NT-T cells. Mice were monitored for a total period of 250 days, by recurrent bleed. Simultaneously, another cohort of mice was infused with patient-derived BM cells (5x106) and monitored for the same time. Mice infused with Bcp-ALL BM cells developed leukemia-phenotype,with 82% of cells expressing hCD45dim and hCD19. By contrast, mice receiving patient-derived CAR-T cells showed a lowpercentage of CD45+ cells (0.1±0.01%), all CD3+. Despite the long period of observation, we did not detect any expansion of hCD19+ cells in this animal cohort. Taken together these data suggest that the use of a retroviral platform, associated with the presence of iC9 suicide gene, contributes to the genesis of a highly functional and safe CAR-T product, even when the production starts from a biological material characterized by high contamination of leukemic blasts. Disclosures Locatelli: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; bluebird bio: Consultancy; 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.

Description: 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

Description: Prognosis of a significant proportion of patients with chemotherapy-refractory or multiply-relapsed CD30+ Non-Hodgkin's Lymphoma (NHL) or Hodgkin lymphoma (HL) still remain poor. Targeting CD30 with monoclonal antibodies in HL and anaplastic large cell lymphoma was shown to induce remarkable clinical activity; however, occurrence of adverse events (mainly neuropathy) may result into treatment discontinuation in many patients. Immunotherapeutic approaches targeting CD30 by chimeric antigen receptor (CAR) has been demonstrated to be of value in two independent clinical trials, although clinical benefit was sub-optimal. We designed a new CAR construct characterized by an anti-CD30 single-chain variable-fragment cassette (AC10), linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. The inducible Caspase-9 (iCasp9) safety switch was included in both constructs with the goal of promptly controlling undue toxicity. As a selectable marker, we added in frame the CD34 antigen. The in vitro anti-tumor efficacy was evaluated by using either the NHL cell line: Karpas299, or the HL cell lines: L428, in both short-term cytotoxic assay (51Cr release assays) and long-term co-cultures for 6 days. Supernatant from co-culture experiments was analyzed by Elisa. We assessed the antitumor effect of CAR.CD30 T cells in a in vivo NSG mouse model engrafted i.v. with lymphoma FF-luciferase cell lines Karpas299 or L428, and monitored tumor growth by IVIS Imaging system. For tumor re-challenging, mice of the NHL model surviving until day +140, were i.v. infused with 0.2x106 Karpas299 cells, and subsequently followed for additional 110 days. Persistence of CAR.CD30 T cells was evaluated, together with a deep characterization of memory profile of T cells. Independently from the costimulatory domains CD28.OX40 or CD28.4-1BB, the generated retroviral vectors showed similar transduction efficiency of T cells (86.5±5.1% and 79.3±5.3%, respectively). Nevertheless, CD28.OX40 costimulatory domains was associated with more stable expression of the CAR over time, during extensive in vitro culture (84.72±5.30% vs 63.98±11.51% CD28.4-1BB CAR T cells at 30 days after transduction; p=0.002). For both CAR constructs, we did not observe any significant difference in the suicide gene iCasp9 activity, both in vitro and in vivo. In short-term cytotoxic assay, both CAR.CD30 T cells significantly and specifically lysed CD30+ NHL and HL tumor cell lines. In long-term co-culture, CD28.OX40 showed a superior anti-lymphoma in vitro activity as compared to CD28.41BB T cells, when challenged at very high tumor/effector ratio (8:1) (for Karpas 299; p=0.03). Moreover, the antigen stimulation was associated to higher levels of Th1 cytokine production, with CD28.OX40 T cells secreting a significantly higher amount of IFNγ, IL2 and TNFα as compared to CD28.41BB T cells (p= 0.040; p=0.008; p=0.02; respectively). Bioluminescence in HL (L428) tumor-bearing mice, treated with NT T cells, rapidly increased up to 5 log in less than 50 days and mice either died or were sacrificed due to morbidity. The best outcome was observed in mice treated with CD28.OX40, as three out of five mice were still alive at the experimental end-point of day+165, as compared with mice treated with CD28.4-1BB (60% vs 0%, p=0.0021). In NHL (Karpas 299) mouse models, CD28.OX40 had an extensive anti-tumor control superior to that of CD28.41BB T cells, leading to a significant reduction of tumor bioluminescence at day 45 (3.32x10 vs 2.29x10, p=0.04). The median survival of mice treated with NT and CD28.4-1BB CAR T cells was 45.5 and 58 days respectively, but undetermined for mice treated with CD28.OX40 CAR T cells (p=0.0002). After 140 days, cured mice were re-challenged with Karpas 299; mice were followed for other 100 days. Bioluminescence analysis showed rapid progression of the tumor in the control mice cohort, as well as in CD28.4-1BB treated mice. In contrast, in CD28.OX40 treated mice, at day+240 days, 4 out of 6 mice were tumor-free, resulting into a statistically significant survival benefit (p=0.0014). Only in mice treated with 28.OX40 T cells, we observed a long-lasting persistence of circulating CAR-T cells up to day +221. In summary, we have developed a novel CAR.CD30 construct displaying features that make it a particularly suitable candidate for a clinical trial in patients suffering from CD30+ tumors. Disclosures Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Locatelli: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, Speakers Bureau; BluebirdBio: Consultancy; Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: 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