ALBERT

Description: Background Transfusion-dependent β-thalassemia (TDT) is a severe genetic disease caused by impaired β-globin production, leading to severe anemia, lifelong transfusion dependence with iron overload and serious comorbidities. Gene therapy (GT) offers a potentially transformative option for these patients. LentiGlobin GT contains autologous CD34+ hematopoietic stem cells (HSCs) transduced ex vivo with the BB305 lentiviral vector (LVV) encoding β-globin with a T87Q substitution. The safety and efficacy of LentiGlobin in patients with TDT was assessed in the phase 1/2 Northstar study in which 8/10 patients with non-β0/β0 genotypes and 3/8 patients with a β0/β0 genotype stopped transfusions. A refined manufacturing process to improve drug product (DP) characteristics is being evaluated in the studies presented here. Methods Northstar-2 (HGB-207; NCT02906202) and Northstar-3 (HGB-212; NCT03207009) are ongoing, international, single-arm, phase 3 studies in patients with TDT (≥ 100 mL/kg/yr of red blood cells [RBCs] or ≥ 8 RBC transfusions/yr) and non-β0/β0 genotypes or a β0/β0 genotype, respectively. HSCs were collected by apheresis after G-CSF and plerixafor mobilization. CD34+ HSCs were transduced with the BB305 LVV using a refined manufacturing process. Patients received single-agent, myeloablative busulfan conditioning and transduced cells were infused. The primary endpoint in Northstar-2 is the proportion of patients achieving transfusion independence (TI, weighted average hemoglobin [Hb] ≥ 9g/dL without RBC transfusions for ≥ 12 months continuously) and in Northstar-3 is the proportion of patients achieving transfusion reduction (≥ 60% reduction in transfused RBC volume post-DP infusion compared to pre-DP infusion). Patients were evaluated for engraftment, DP and peripheral blood vector copy number (VCN), GT-derived Hb (HbAT87Q), adverse events (AEs), vector integration, and evidence of replication competent lentivirus (RCL). Patients are followed for 2 years and offered participation in a long-term follow-up study. Results Eleven patients (median age 20 [min - max: 12 - 24] years) with TDT and non-β0/β0 genotypes (5 β+/β0, 4 βE/β0, 2 β+/β+) have been treated in Northstar-2 as of May 15, 2018 with a median follow-up of 8.5 (min - max: 0.3 - 16.2) months. DPs had a median cell dose of 7.4 x 106 (min - max: 5.0 - 19.4 x 106) CD34+ cells/kg, median VCN of 3.4 (min - max: 2.4 - 5.6) copies/diploid genome (c/dg) and a median of 82% (min - max: 53 - 90%) CD34+ cells were transduced. Median time to neutrophil and platelet engraftment was 21.5 (min - max: 16 - 28) and 44.5 (min - max: 34 - 84) days, respectively, in 10 patients; 1 patient was not yet evaluable. Serious AEs after DP infusion included 2 events of grade 4 liver veno-occlusive disease treated with defibrotide and 1 event each of hypotension, hypoxia, sepsis, and transfusion reaction, all resolved. Only 1 AE (grade 1 abdominal pain) was related to LentiGlobin. There were no deaths or graft failure and no evidence of vector-mediated RCL or clonal dominance. Of 8 patients with ≥ 6 months follow-up, 7 have stopped RBC transfusions. At last study visit, peripheral blood VCN was 1.1 - 5.0 c/dg and total Hb was 11.1 - 13.3 g/dL of which 7.6 - 10.2 g/dL (68 - 92%) was contributed by HbAT87Q. Median Hb at month 6 was 11.9 (min - max: 11.2 - 13.3) g/dL. The first treated patient achieved TI. The additional patient with ≥ 6 months follow-up had no transfusions for 11 months, however had a peripheral blood VCN of 0.2 c/dg and resumed transfusions due to symptomatic anemia. Bone marrow assessment of dyserythropoesis and data with longer follow-up will be presented. Two patients, 26- and 7- years old, have been treated in Northstar-3. Both had 2 DP lots manufactured with DP VCNs of 2.9/3.3 and 3.4/3.9 c/dg and 82%/85% and 78%/78% CD34+ cells were transduced, respectively. Both successfully engrafted. Additional data for these patients will be presented. Summary Seven of 8 patients with TDT and non-β0/β0 genotypes produced sufficient HbAT87Q to stop chronic transfusions following LentiGlobin GT in Northstar-2. The safety profile appears consistent with busulfan myeloablative conditioning with no grade ≥ 3 DP-related AEs. Initial results show DP characteristics in Northstar-3 are consistent with those in Northstar-2. Additional data from Northstar-3 will determine the impact of HbAT87Q production on transfusion reduction in patients without endogenous β-globin production. Disclosures Locatelli: 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; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Walters:AllCells Inc.: Other: Medical Director; ViaCord Processing Lab: Other: Medical Director; bluebird bio: Research Funding; Sangamo Therapeutics: Consultancy. Kwiatkowski:Terumo: Research Funding; Apopharma: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding; bluebird bio: Consultancy, Honoraria, Research Funding. Porter:Agios: Honoraria; Cerus: Honoraria; Novartis: Consultancy. Thuret:Addmedica: Research Funding; bluebird bio: Research Funding; Novartis: Research Funding. Kulozik:bluebird bio: Consultancy, Honoraria. Lal:Terumo Corporation: Research Funding; Celgene Corporation: Research Funding; Insight Magnetics: Research Funding; Bluebird Bio: Research Funding; La Jolla Pharmaceutical Company: Consultancy, Research Funding; Novartis: Research Funding. Thrasher:Orchard Therapeutics: Consultancy, Equity Ownership; Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Elliot:bluebird bio: Employment, Equity Ownership. Tao:bluebird bio: Employment, Equity Ownership. Asmal:bluebird bio: Employment, Equity Ownership. Thompson:Amgen: Research Funding; Baxalta/Shire: Research Funding; La Jolla Pharmaceutical: Research Funding; Novartis: Research Funding; bluebird bio: Consultancy, Research Funding; Celgene: Research Funding; Biomarin: Research Funding.

Description: Abstract 817 Strategies to suppress GVHD are often associated with broader suppression of the immune system leading to a compromised GVT effect. Using experimental models, we have demonstrated a novel strategy to enhance GVT effects and explicitly suppress GVHD using genetically engineered T lineage cells over-expressing TNF-Related Apoptosis Inducing Ligand (TRAIL). TRAIL can induce apoptotic signals through death receptor (DR) 4 and 5 molecules (only DR5 in mice) expressed on target cells. Expression of DR5 is higher on certain tumors and can be enhanced on others using small molecules rendering them susceptible to TRAIL mediated killing. TRAIL is therefore an attractive candidate for genetic engineering of donor T cells to enhance their GVT potential. We evaluated the effect of TRAIL over-expression (TRAIL+) in donor T cells (mature and precursor) on GVHD and GVT. Mature T cells derived from donor B6 splenocytes were transduced with a lentiviral TRAIL expression vector. The transduced TRAIL+ T cells were adoptively transferred on day 0 into lethally irradiated CBF1 recipients of T cell depleted allografts and LB27.4 tumor (B6 ^ CBF1+LB27.4) to assess their GVHD and GVT activity. TRAIL+ T cells displayed significantly enhanced antitumor immunity compared to T cells transduced with a control vector against LB27.4 tumor cell lines in vitro and upon transfer into tumor bearing allo-BMT recipients (p

Description: Abstract 3549 Poster Board III-486 BACKGROUND Adoptive transfer (AT) of TCR-gene transduced autologous T cells has become a potent therapeutic tool. We hypothesized that a) the use of allogeneic T cells as TCR vehicles after allogeneic HCT results in a reduced risk of graft versus host disease (GVHD) due to competitive downregulation of the alloreactive receptor, b) cell division of TCR-transduced T-cells by continuous allostimulation leads to prolonged in vivo persistence, and as a consequence to c) long lasting specific graft versus leukemia effects (GVL). METHODS Studies were performed using the OT-1 anti-ovalbumin (OVA) reactive TCR introduced in B10.A donor T cells for AT after MHC-mismatched HCT (B10.A mice (H-2a) into C57BL/6 (H-2b) mice). For direct quantification of an alloreactive endogenous receptor 2C mouse-derived T cells (endogenous TCR directed against H-2d) and anti-Ld antibodies were used in some experiments. After HCT, leukemia-bearing (C1498-OVA) mice (syngeneic HCT-recipients served as reference) received AT and were monitored for GVHD, GVL, and T cell persistence. Transferred T cells of surviving mice were retrieved for TCR-spectrotyping analysis and assessed for remaining alloreactivity. RESULTS 1) Up to 1×109 TCR-transduced CD8+ T cells were generated in vitro within 7 days using a retroviral vector system linking the genes for the α- and β-chain via a 2A sequence. 40% of CD8+ T cells coexpressed the respective TCR chains Vα2 and Vβ5. 2) The introduced TCR mediated comparable specific cytotoxicity against C1498-OVA in vitro being functional on autologous and allogeneic T cells. 3) After AT transduced T cells rescued up to 60% of mice in a dose dependent manner. (p = 0.001-0.01 versus mock-transduced controls). Minimal GVL-effects only were mediated by high doses of mock-transduced allogeneic T cells 4) After TCR-transfer the alloreceptor of 2C-derived T cells was downregulated by up to 60%. Increasing TCR transduction rates negatively correlated with the expression of the endogenous alloreactive receptor. Alloreactivity of B10.A-derived T cells against C57BL/6 stimulators was reduced by 50% in vitro. This translated into decreased GVHD-scores upon AT after allogeneic HCT (mock-transduced CD8+ cells served as controls (p=0.0011). 5) The degree of long-term persistence after AT was comparable to the autologous setting and correlated with tumor protection upon leukemia challenge after HCT. 13 weeks after AT the endogenous TCR-repertoire of TCR-engineered allogeneic T cells narrowed significantly as compared to their autologous counterpart. 100% of long term persisting T cells expressed the introduced TCR. CONCLUSION AT with TCR-engineered T cells after allogeneic HCT can decrease the risk of GVHD and provide potent GVL effects. Disclosures: No relevant conflicts of interest to declare.

Description: BACKGROUND: The co-transplantation of hematopoietic stem cells (HS) with those that have been engineered to express tumor-reactive T cell receptors (TCRs) and differentiated into precursor T cells (preTs) may optimize tumor reduction. Since expression of potentially self-(tumor-) reactive TCRs will lead to negative selection upon thymic maturation, we investigated whether preTs forced to express a leukemia-reactive TCR under the control of a tetracycline-inducible promoter would allow timely controlled TCR expression thereby avoiding thymic negative selection. METHODS: Using lentiviral vectors, murine LSK cells were engineered to express a Tetracycline-inducible TCR directed against a surrogate leukemia antigen. TCR-transduced LSK cells were co-cultured on T cell development-supporting OP9-DL1 cells to produce preTs. Lethally-irradiated B6/NCrl recipients received syngeneic T cell-depleted bone marrow and 8 × 106 syngeneic or allogeneic (B10.A) TCR-engineered preTs. An otherwise lethal leukemia cell (C1498) challenge was given 28 days later. RESULTS: After in vivo maturation and gene induction up to 70% leukemia free survival was achieved in recipients of syngeneic TCR-transduced preTs (p

Description: Adoptive transfer (AT) of T cells forced to express tumor-reactive T-cell receptor (TCR) genes is an attractive strategy to direct autologous T-cell immunity against tumor-associated antigens. However, clinical effectiveness has been hampered by limited in vivo persistence. We investigated whether the use of major histocompatibility complex–mismatched T cells would prolong the in vivo persistence of tumor-reactive TCR gene expressing T cells by continuous antigen-driven proliferation via the endogenous potentially alloreactive receptor. Donor-derived CD8+ T cells engineered to express a TCR against a leukemia-associated antigen mediated strong graft-versus-leukemia (GVL) effects with reduced graft-versus-host disease (GVHD) severity when given early after transplantation. AT later after transplantation resulted in a complete loss of GVL. Loss of function was associated with reduced expansion of TCR-transduced T cells as assessed by CDR3 spectratyping analysis and PD-1 up-regulation on T cells in leukemia-bearing recipients. PD-L1 blockade in allogeneic transplant recipients largely restored the GVL efficacy without triggering GVHD, whereas no significant antileukemia effects of PD-L1 blockade were observed in syngeneic controls. These data suggest a clinical approach in which the AT of gene-modified allogeneic T cells early after transplantation can provide a potent GVL effect without GVHD, whereas later AT is effective only with concurrent PD-L1 blockade.

Description: Abstract 4402 It is well established that patients with congenital neutropenia due to mutations of ELANE or HAX1 have a strongly increased risk of developing secondary leukemia. In contrast, the leukemia risk is not strongly increased in patients with specific other neutropenia subtypes (e. g. cyclic neutropenia with ELANE mutations). Here we report the first glycogen storage disease type Ib (GSD1b) patient in our registry who developed acute myelogenous leukemia (AML). GSD1b is an autosomal recessive disorder of glycogen metabolism with intracellular accumulation of glycogen caused by mutations in the glucose-6-phosphate translocase gene. Patients present with hepatomegaly, growth retardation, hypoglycemia, lactic acidosis as well as neutropenia and neutrophil dysfunction predisposing to bacterial infections. G-CSF has been shown to be an effective long-term treatment for GSD1b patients leading to an increase in neutrophil counts and enhancement of neutrophil function resulting in fewer infections and improved quality of life. The European Branch of the Severe Chronic Neutropenia International Registry (SCNIR) has collected long-term data from 23 patients with GSD1b. Median age was 17 years, 3 patients expired from infections at an age of 8; 9 and 26 years, respectively. All 23 patients received G-CSF treatment for a median duration of 9.5 years, 22 patients received continuous G-CSF with a median dose of 3.4 mcg/kg/day (range 0.8 to 5 mcg/kg/day), one patient received G-CSF interventionally only. Our patient was diagnosed with AML FAB classification M2 linked to cytogenetic aberrations involving trisomy 21. Leukemia cells also harboured a missense RUNX 1 mutation in exon 3. G-CSF treatment was initiated at age 5 months at a dose of 2.3 mcg/kg/d and was continued until AML diagnosis at age 10 years. The G-CSF dose was increased due to low ANCs and infectious episodes with otitis media and pneumonitis up to 4.05 mcg/kg/day. After initial AML treatment with a hypometylating chemotherapy consisting of azacytidine and cytarabine she developed partially reversible renal insufficiency and oxygen dependent pneumonitis which led to a reduction of chemotherapy despite persistence of AML blasts. Remission was achieved with the use of mylotarg. Subsequently, she received a conditioning regimen with melphalan, fludarabine and treosulfan plus ATG-Fresenius followed by infusion of 10/10 HLA matched peripheral blood stem cells from an unrelated donor. Leukocyte engraftment occurred on day +18 followed by neutrophil engraftment on day +21. Post-SCT she developed grade III skin GvHD on day +8 which was responsible to steroid treatment. Discussion: Up to date 3 GSD1b patients with development of AML have been reported in the literature. Two patients were on long-term G-CSF treatment (Pinsk et al. J Pediatr. 2002; Schroeder et al. J Medical Case Reports 2008), one patient transformed predating the availability of G-CSF (Simmons et al. J Pediatr. 1984). Conclusion: According to the literature, leukemic transformation in patients with GSD1b is a rare event. However, with a total of four patients reported with secondary AML with or without G-CSF treatment, GSD1b patients may have a higher leukemia risk than previously anticipated. Independent from G-CSF treatment, regular bone marrow examinations (morphology, cytogenetics, mutation analysis of candidate genes such as RUNX1) may help to detect malignant transformation early and therefore avoid chemotherapy prior to SCT. SCT is a treatment option for AML in GSD1b, but further data is required. Disclosures: No relevant conflicts of interest to declare.

Description: Background Transfusion-dependent β-thalassemia (TDT) is a severe genetic disease caused by impaired β-globin production leading to severe anemia and lifelong transfusion dependence. Autologous CD34+ cells encoding a βA-T87Q-globin gene (LentiGlobin for β-thalassemia) is currently being evaluated in patients with TDT. In the phase 1/2 Northstar study, 3/8 patients with β0/β0 genotypes became transfusion independent. The drug product (DP) manufacturing process (CD34+ cell transduction) was then refined to improve clinical outcomes. Interim results are presented here from the ongoing, international, single-arm, phase 3 Northstar-3 study (HGB-212; NCT03207009) evaluating LentiGlobin gene therapy in patients ≤50 years of age with TDT and either β0 or β+ IVS-I-110 mutations on both HBB alleles. Methods Patients with TDT undergo hematopoietic stem cell mobilization with G-CSF and plerixafor. CD34+ cells collected via apheresis are transduced with BB305 lentiviral vector. Patients undergo myeloablative, single-agent, pharmacokinetic-adjusted busulfan conditioning over 4 days and are infused with transduced cells. The primary efficacy endpoint is transfusion reduction (≥60% reduction in transfused red blood cell (RBC) volume post-DP infusion compared with pre-DP infusion). A key secondary endpoint is transfusion independence (TI; weighted average hemoglobin [Hb] ≥9 g/dL without RBC transfusions for ≥12 months). Patients are followed for 2 years and offered participation in a long-term follow-up study. Summary statistics presented as median (min - max). Results As of 12 April 2019, 11 patients (7 β0/β0, 2 β0/IVS-I-110, 2 homozygous IVS-I-110genotypes) were treated with LentiGlobin and have a median follow-up of 4.6 (1.5 - 15.7) months. Median age at enrollment was 17 (7 - 33) years; 3 patients were

Description: Background Transfusion-dependent β-thalassemia (TDT) is treated with regular, lifelong red blood cell (RBC) transfusions and despite iron-chelating therapy, carries a risk of serious organ damage from iron overload and other complications. Transplantation with autologous CD34+ cells encoding a βA-T87Q-globin gene (LentiGlobin for β-thalassemia) is being evaluated in patients with TDT. Interim results are presented here from the ongoing, international, single-arm, phase 3 Northstar-2 study (HGB-207; NCT02906202) of LentiGlobin gene therapy in pediatric, adolescent, and adult patients with TDT (defined by receiving ≥100 mL/kg/yr of RBCs or ≥8 RBC transfusions/yr) and non-β0/β0 genotypes. Methods Patients undergo hematopoietic stem cell (HSC) mobilization with G-CSF and plerixafor. Following apheresis, CD34+ cells are transduced with BB305 lentiviral vector and infused into patients after pharmacokinetic-adjusted, single-agent busulfan myeloablation. The primary efficacy endpoint is transfusion independence (TI; weighted average hemoglobin [Hb] ≥9 g/dL without RBC transfusions for ≥12 months). HSC engraftment, βA-T87Q-globin expression, Hb levels, detection of replication competent lentivirus (RCL), and adverse events (AE) are also assessed. Patients are followed for 2 years and offered participation in a long-term follow-up study. Summary statistics are presented as median (min - max). Results Twenty patients were treated in Northstar-2 as of 13 December 2018 and have been followed for a median of 8.1 (0.5 - 22.2) months. At enrollment, median age was 16 (8 - 34) years; 5 patients were 11 g/dL without transfusions. Five adolescent and adult patients were evaluable for the primary endpoint of transfusion independence, 4 (80%) of whom achieved TI. The median weighted average Hb during TI was 12.4 (11.5 - 12.6) g/dL which compared favorably to pre-transfusion nadir Hb levels before enrollment (median 9.1 g/dL [7.5 - 10.0 g/dL]). At time of analysis, the median duration of TI was 13.6 (12.0 - 18.2) months. One patient who did not achieve TI stopped transfusions for 11.4 months but resumed transfusions due to recurrent anemia. This patient had a 71.4% reduction in RBC transfusion volume from Month 6 to Month 18 compared to baseline. Marrow cellularity and myeloid:erythroid (M:E) ratios were evaluated in 8 adolescent and adult patients with ≥12 months follow-up to assess the effect of LentiGlobin treatment on dyserythropoiesis. Seven of 8 patients had improved marrow M:E ratios at Month 12 (0.63 - 1.90) compared with baseline (0.14 - 0.48). In patients who stopped transfusions, soluble transferrin receptor levels were reduced by a median of 72% (58% - 78%) at Month 12 (n=6). Updated outcomes in adolescents and adults and outcomes in pediatric patients will be reported. Summary In this update of the Northstar-2 study of LentiGlobin gene therapy in patients with TDT and non-β0/β0 genotypes, transfusion independence was observed in 4/5 evaluable adolescent and adults and 10/11 treated patients had total Hb of 〉11 g/dL without transfusion support 6 months after LentiGlobin infusion. HbAT87Q stabilized approximately 6 months after treatment and patients who stopped RBC transfusions had improved erythropoiesis. A safety profile consistent with busulfan conditioning was observed after LentiGlobin gene therapy. Disclosures Thompson: bluebird bio, Inc.: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Baxalta: Research Funding. Walters:TruCode: Consultancy; AllCells, Inc: Consultancy; Editas Medicine: Consultancy. Kwiatkowski:bluebird bio, Inc.: Consultancy, Research Funding; Terumo: Research Funding; Celgene: Consultancy; Agios: Consultancy; Imara: Consultancy; Apopharma: Research Funding; Novartis: Research Funding. Porter:Protagonism: Honoraria; Celgene: Consultancy, Honoraria; Bluebird bio: Consultancy, Honoraria; Agios: Consultancy, Honoraria; La Jolla: Honoraria; Vifor: Honoraria; Silence therapeutics: Honoraria. Thrasher:Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 4BIOCapital: Membership on an entity's Board of Directors or advisory committees. Thuret:BlueBird bio: Other: investigators for clinical trials, participation on scientific/medical advisory board; Celgene: Other: investigators for clinical trials, participation on scientific/medical advisory board; Novartis: Other: investigators for clinical trials, participation on scientific/medical advisory board; Apopharma: Consultancy. Elliot:bluebird bio, Inc.: Employment, Equity Ownership. Tao:bluebird bio, Inc.: Employment, Equity Ownership. Colvin:bluebird bio, Inc.: Employment, Equity Ownership. Locatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; 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: Consultancy; Miltenyi: Honoraria.