Omidubicel (nicotinamide-expanded cord blood) is a potential alternative source for allogeneic hematopoietic cell transplantation (HCT) when an HLA-identical donor is lacking. A phase I/II trial with standalone omidubicel HCT showed rapid and robust neutrophil and platelet engraftment. In this study, we evaluated the immune reconstitution (IR) of patients receiving omidubicel grafts during the first 6 months post-transplant, as IR is critical for favorable outcomes of the procedure. Data was collected from the omidubicel phase I-II international, multicenter trial. The primary endpoint was the probability of achieving adequate CD4+ T-cell IR (CD4IR: > 50 × 10 6 /L within 100 days). Secondary endpoints were the recovery of T-cells, natural killer (NK)-cells, B-cells, dendritic cells (DC), and monocytes as determined with multicolor flow cytometry. LOESS-regression curves and cumulative incidence plots were used for data description. Thirty-six omidubicel recipients (median 44; 13–63 years) were included, and IR data was available from 28 recipients. Of these patients, 90% achieved adequate CD4IR. Overall, IR was complete and consisted of T-cell, monocyte, DC, and notably fast NK- and B-cell reconstitution, compared to conventional grafts. Our data show that transplantation of adolescent and adult patients with omidubicel results in full and broad IR, which is comparable with IR after HCT with conventional graft sources.

Omidubicel is an ex vivo expanded hematopoietic progenitor cell and nonexpanded myeloid and lymphoid cell product derived from a single umbilical cord blood unit. We report results of a phase 3 trial to evaluate the efficacy of omidubicel compared with standard umbilical cord blood transplantation (UCBT). Between January 2017 and January 2020, 125 patients age 13 to 65 years with hematologic malignancies were randomly assigned to omidubicel vs standard UCBT. Patients received myeloablative conditioning and prophylaxis with a calcineurin inhibitor and mycophenolate mofetil for graft-versus-host disease (GVHD). The primary end point was time to neutrophil engraftment. The treatment arms were well balanced and racially diverse. Median time to neutrophil engraftment was 12 days (95% confidence interval [CI], 10-14 days) for the omidubicel arm and 22 days (95% CI, 19-25 days) for the control arm (P < .001). The cumulative incidence of neutrophil engraftment was 96% for patients receiving omidubicel and 89% for patients receiving control transplants. The omidubicel arm had faster platelet recovery (55% vs 35% recovery by 42 days; P = .028), had a lower incidence of first grade 2 to 3 bacterial or invasive fungal infection (37% vs 57%; P = .027), and spent more time out of hospital during the first 100 days after transplant (median, 61 vs 48 days; P = .005) than controls. Differences in GVHD and survival between the 2 arms were not statistically significant. Transplantation with omidubicel results in faster hematopoietic recovery and reduces early transplant-related complications compared with standard UCBT. The results suggest that omidubicel may be considered as a new standard of care for adult patients eligible for UCBT. The trial was registered at www.clinicaltrials.gov as #NCT02730299.

Historical efforts at expansion of umbilical cord blood (UCB) derived CD34+ hematopoietic stem cells (HSCs) ex vivo with cytokines yielded large numbers of progenitors for transplantation but impaired their long-term engraftment. We used nicotinamide (NAM), an allosteric inhibitor of NAD-enzymes, to create omidubicel, an investigational cell therapy designed to improve the expansion of CD34+ HSCs for bone marrow transplant. A Phase 1/2 clinical study in patients with high-risk hematologic malignancies showed rapid neutrophil engraftment in patients compared to historical controls [1]. We hypothesized that NAM treatment maintains the stemness and engraftment potential of omidubicel, which is associated with clinical benefit [2].We performed transcriptome analysis to discern the mechanism of action of NAM after ex vivo expansion of CD34+ cells in omidubicel compared to CD34+ controls. Analysis revealed that HSCs expanded with cytokines alone led to an increase in genes responsible for cell proliferation, differentiation, and apoptosis, and in the production of reactive oxygen and nitrogen species (ROS, RNS), and matrix metalloproteinases (MMPs), all of which were attenuated by NAM.Transcription factor (TF) enrichment analysis demonstrated that NAM modulated TFs critically involved in activation of pathways for HSC differentiation, apoptosis, and migration. Specifically, NF-kB, C-Jun, LXR/RXR and PPARα/RXRα, and mTor signaling were all reduced in NAM-treated CD34+ cells compared to controls.NAM treatment upregulated genes linked to cellular metabolism including the Sirtuin family genes, TCA cycle genes, and HIF1a. NAM upregulated genes responsible for telomerase expression further validating our hypothesis that NAM preserves cell stemness.In summary, these data provide further scientific rationale for the favorable clinical engraftment and patient outcomes observed in the Phase 1/2 clinical study of omidubicel. An international, randomized, multi-center Phase 3 study of omidubicel in patients with high-risk hematologic malignancies is underway [2].[1] Horwitz ME. et. al., J Clin Oncol. 2019 Feb 10;37(5):367-374.[2] ClinicalTrials.gov identifier NCT02730299.

BackgroundAdoptive transfer of cytolytic natural killer (NK) cells is limited by the short-term persistence of NK cells and their impaired effector function after infusion. We have previously shown that ex-vivo expansion of NK cells with nicotinamide (NAM) resulted in potent cytotoxicity against multiple tumor cell lines, and robust homing, proliferation and retention in vivo (Blood 2017 130:657). We now report the first-in-human experience of NAM-NK in patients with relapsed/refractory non-Hodgkin lymphoma (NHL) and multiple myeloma (MM).MethodsFollowing donor apheresis, CD3-depleted mononuclear cells were cultured for 2 weeks in the presence of NAM (5mM) and IL-15 (20ng/ml). Patients received lymphodepleting chemotherapy followed by two doses of NAM-NK (Days 0 and 2) and low-dose IL-2. Rituximab or elotuzumab was administered to patients with NHL or MM, respectively, to facilitate tumor targeting and antibody-dependent cellular cytotoxicity.ResultsAs of Oct 2018, 9 patients were enrolled. Of 7 evaluable patients, 5 had refractory NHL (3 follicular and 2 diffuse large cell lymphoma) and 2 had MM. Final NAM-NK product (n=7) contained a median of 98% NK cells. In vitro culture with NAM and IL15 resulted in a 3.8-fold increase in TNC and 40-fold increase in NK cells after 14-16 day culture; expression of homing receptor CD62L increased from 2.9% in apheresis to 21% in final product. CD3 content was kept <0.5% (<5 × 105/kg/dose). NAM-NK was infused in 3 patients at the initial dose (2 × 107/kg) without dose-limiting toxicity (DLT). Transient neutropenia was observed in all 3 with neutropenic fever in 1 patient. The 2nd dose level of NAM-NK (1 × 108/kg) was well-tolerated in 4 patients without DLT or grade 3 or 4 adverse events; no cytokine release syndrome or neurotoxicity were observed. Response assessment at 2 months showed that 3 patients achieved complete metabolic remission (follicular lymphoma 2 patients (Figure 1b), transformed lymphoma 1 patient). One patient with MM had stable disease at 2 months and the second with MM experienced progressive disease. Flow cytometry analysis of peripheral blood showed proliferation of NAM-NK in blood between days 2 and 7 in all tested patients (range 2-55% donor NK cells; Figure 1a). Compared to host NK cells, donor NAM-NK cells in blood demonstrated higher CD16 expression (median 68% vs 82%) and enhanced proliferation (median Ki67 81% vs 99%).ConclusionsNAM-NK cells have been safely administered, were well tolerated and proliferated and persisted in vivo. Promising early evidence of clinical activity was observed in patients with advanced disease. Dose escalation will be followed by an expansion cohort at the MTD. Adoptive transfer of cytolytic natural killer (NK) cells is limited by the short-term persistence of NK cells and their impaired effector function after infusion. We have previously shown that ex-vivo expansion of NK cells with nicotinamide (NAM) resulted in potent cytotoxicity against multiple tumor cell lines, and robust homing, proliferation and retention in vivo (Blood 2017 130:657). We now report the first-in-human experience of NAM-NK in patients with relapsed/refractory non-Hodgkin lymphoma (NHL) and multiple myeloma (MM). Following donor apheresis, CD3-depleted mononuclear cells were cultured for 2 weeks in the presence of NAM (5mM) and IL-15 (20ng/ml). Patients received lymphodepleting chemotherapy followed by two doses of NAM-NK (Days 0 and 2) and low-dose IL-2. Rituximab or elotuzumab was administered to patients with NHL or MM, respectively, to facilitate tumor targeting and antibody-dependent cellular cytotoxicity. As of Oct 2018, 9 patients were enrolled. Of 7 evaluable patients, 5 had refractory NHL (3 follicular and 2 diffuse large cell lymphoma) and 2 had MM. Final NAM-NK product (n=7) contained a median of 98% NK cells. In vitro culture with NAM and IL15 resulted in a 3.8-fold increase in TNC and 40-fold increase in NK cells after 14-16 day culture; expression of homing receptor CD62L increased from 2.9% in apheresis to 21% in final product. CD3 content was kept <0.5% (<5 × 105/kg/dose). NAM-NK was infused in 3 patients at the initial dose (2 × 107/kg) without dose-limiting toxicity (DLT). Transient neutropenia was observed in all 3 with neutropenic fever in 1 patient. The 2nd dose level of NAM-NK (1 × 108/kg) was well-tolerated in 4 patients without DLT or grade 3 or 4 adverse events; no cytokine release syndrome or neurotoxicity were observed. Response assessment at 2 months showed that 3 patients achieved complete metabolic remission (follicular lymphoma 2 patients (Figure 1b), transformed lymphoma 1 patient). One patient with MM had stable disease at 2 months and the second with MM experienced progressive disease. Flow cytometry analysis of peripheral blood showed proliferation of NAM-NK in blood between days 2 and 7 in all tested patients (range 2-55% donor NK cells; Figure 1a). Compared to host NK cells, donor NAM-NK cells in blood demonstrated higher CD16 expression (median 68% vs 82%) and enhanced proliferation (median Ki67 81% vs 99%). NAM-NK cells have been safely administered, were well tolerated and proliferated and persisted in vivo. Promising early evidence of clinical activity was observed in patients with advanced disease. Dose escalation will be followed by an expansion cohort at the MTD.

Umbilical cord blood (UCB) transplantation has a high early mortality rate primarily related to transplanted stem cell dose. To decrease early mortality and enhance engraftment, a portion of selected cord blood units (20% to 50%) was expanded with cytokines and the copper chelator tetraethylenepentamine (carlecortemcel-L) and transplanted with the unmanipulated fraction after myeloablative conditioning. The primary endpoint was 100-day survival, which was compared with a contemporaneous double-unit cord blood transplantation (DUCBT) group. We enrolled 101 patients at 25 sites; the DUCBT comparison (n = 295) was selected from international registries using study eligibility criteria. Baseline carlecortemcel-L study group unit nucleated cell (NC) and CD34(+) were 3.06 x 10(7) cell dose/kg and 1.64 x 10(5) cell dose/kg. Median NC and CD34(+) fold expansion were 400 and 77, with a mean total CD34 infused of 9.7 x 10(5)/kg. The 100-day survival was 84.2% for the carlecortemcel-L study group versus 74.6% for the DUCBT group (odds ratio, .50; 95% Cl, .26 to .95; P= .035). Survival at day 180 was similar for the 2 groups; the major cause of death after day 100 was opportunistic infections. Faster median neutrophil (21 days versus 28 days; P <.0001), and platelet (54 days versus 105 days; P = .008) engraftment was seen in the carlecortemcel-L study group; acute and chronic graft-versus-host disease rates were similar. In this multinational comparative study, transplanting expanded CD34(+) stem cells from a portion of a single UCB unit, with the remaining unmanipulated fraction improved 100-day survival compared with DUCBT control patients while facilitating myeloid and platelet engraftment. (C) 2018 American Society for Blood and Marrow Transplantation.

Adoptive transfer of cytolitic Natural Killer (NK) cells is a promising immunotherapeutic modality for hematologic and other malignancies. However, limited NK cell in vivo persistence and proliferation have been challenging clinical success of this therapeutic modality. Here we present a reliable, scalable and GMP-compliant culture method for the expansion of highly functional donor NK cells for clinical use.

Background: Patients with severe sickle cell disease (SCD) experience organ damage, poor quality of life, and are at high risk of premature mortality. To date, allogeneic hematopoietic stem cell transplant remains the only available curative therapy for SCD. However, patients with SCD have difficulties finding matched related or unrelated donors for transplantation. UCB could be an alternative graft option for most of the SCD patients but, to date, results with unrelated UCB have not been satisfactory for these hard to engraft patients. NiCord is an ex vivo expanded product manufactured from an entire UCB unit which has been shown to produce rapid and sustained engraftment in combination with a second unmanipulated CB unit or as a standalone graft in adult patients with high-risk hematologic malignancies. We hypothesized that the combination of NiCord with an unmanipulated UCB unit might overcome the engraftment barriers which have limited the success of UCBT in patients with SCD. This strategy is currently evaluated for safety and efficacy in a phase I/II multi-center study in pediatric patients with SCD undergoing myeloablative conditioning therapy. Here we report the results of the first 8 patients in the study.

The influence of TNF-α and Fas-ligand (FasL) on viability and function was evaluated in fresh- and expanded-umbilical cord blood (UCB) cells. CD34+ progenitors and T cells display outstanding survival, whereas ~30% and >50% B lymphocytes and myeloid cells undergo spontaneous apoptosis within 24 and 48 h, respectively. Although the impact of exposure to toxic doses of FasL and TNF-α was undetectable in measurements of apoptosis; removal of dead cells after 2 days of incubation with the ligands revealed a twofold increase in frequency of colony-forming cells (CFU). The sensitivity of progenitors to apoptosis was also unaffected by Fas cross-linking following TNF-induced upregulation of the receptor, increasing CFU frequency without impairing SCID repopulating cell (SRC) activity. Most significant enrichment in CD34+ progenitors and corresponding increase in CFU frequency were observed when FasL was applied during the final week of ex vivo expansion under the influence of nicotinamide, without impairing SRC activity. These data emphasize differential sensitivities of UCB progenitors and lineage-positive cells to apoptotic signaling mediated by the Fas and TNF receptors, which might be useful in improving the efficiency of ex vivo expansion and improving UCB cell engraftment.

BACKGROUND:Delayed hematopoietic recovery is a major drawback of umbilical cord blood (UCB) transplantation. Transplantation of ex vivo-expanded UCB shortens time to hematopoietic recovery, but long-term, robust engraftment by the expanded unit has yet to be demonstrated. We tested the hypothesis that a UCB-derived cell product consisting of stem cells expanded for 21 days in the presence of nicotinamide and a noncultured T cell fraction (NiCord) can accelerate hematopoietic recovery and provide long-term engraftment.METHODS:In a phase I trial, 11 adults with hematologic malignancies received myeloablative bone marrow conditioning followed by transplantation with NiCord and a second unmanipulated UCB unit. Safety, hematopoietic recovery, and donor engraftment were assessed and compared with historical controls.RESULTS:No adverse events were attributable to the infusion of NiCord. Complete or partial neutrophil and T cell engraftment derived from NiCord was observed in 8 patients, and NiCord engraftment remained stable in all patients, with a median follow-up of 21 months. Two patients achieved long-term engraftment with the unmanipulated unit. Patients transplanted with NiCord achieved earlier median neutrophil recovery (13 vs. 25 days, P < 0.001) compared with that seen in historical controls. The 1-year overall and progression-free survival rates were 82% and 73%, respectively.CONCLUSION:UCB-derived hematopoietic stem and progenitor cells expanded in the presence of nicotinamide and transplanted with a T cell-containing fraction contain both short-term and long-term repopulating cells. The results justify further study of NiCord transplantation as a single UCB graft. If long-term safety is confirmed, NiCord has the potential to broaden accessibility and reduce the toxicity of UCB transplantation.TRIAL REGISTRATION:Clinicaltrials.gov NCT01221857.FUNDING:Gamida Cell Ltd.

Abstract Background UCBT has proven effective in treating patients with hematologic malignancies (HM),but engraftment is slower and graft failure rates higher compared to other unrelated transplants. We have previously shown that incubation of UCB CD133+ cells with cytokines and the copper chelator TEPA (5µM), inhibits stem cell differentiation and results in a median of 89 and 30 fold expansion of CD34+ and CD34+CD38- cells, respectively (Cytotherapy 2004;6:344). Using this technology (StemEx®) we performed a prospective multicenter myeloablative UCBT trial in patients with HM. Methodology Patients were transplanted with a single CBU of which CD133+ cells from a segregated portion of the CBU (20-50%) were cultured for 21 days with hematopoietic cytokines and TEPA and transplanted along with a minimum of 107nucleated cells (NC)/kg from the un-manipulated (UM) portion of the same unit (NCT00469729). The primary endpoint of this study was 100 day overall survival. Using an intent to treat design, outcome was compared to a 2006-2010 double UCBT (dUCBT) control group (n = 295) collected from and by the CIBMTR and Eurocord registries using identical eligibility criteria to the StemEx® study: lack of a 5/6 or 6/6 matched sibling donor, age 12-55 years with high risk AML or ALL in 1stCR or subsequent, advanced CML or after failing TKI, MDS with Int-2 or high risk features or chemosensitive relapsed lymphoma. GvHD prophylaxis included a calcineurin inhibitor and mycophenolate mofetil. Comparison of the primary endpoint was based on a logistic regression model that adjusts the treatment comparison for imbalance between the groups in important prognostic factors found to impact mortality: age, sex, CMV status and disease risk. Results 25 centers in US, EU and Israel enrolled 101 eligible patients between Oct 2007-Feb 2012 with AML-43, ALL-30, MDS and CML-8 each, and lymphoma-12. Median age was 37 (12.6-55.8); median weight was 68 kg (42.5-128.5). The baseline NC and CD34 cell dose/kg were 3.06 (1.29-11.0) x 107 and 1.64 (0.24-9.23) x 105. 70% of the units were matched at 4/6 loci. Median NC and CD34 fold expansion were 400 (0-764) and 77 (6-280), respectively. StemEx yielded a median of 14-fold increase in the number of CD34+ infused, in comparison to the number of CD34+ cells the patients could have received from the entire UM CBU. In total, patients received a median dose of 2.2 x 107 NC/kg and 9.7 x 105CD34/kg. No significant acute toxicity was seen with the expanded cell infusions. The primary endpoint of this study has been met: 100 day survival was significantly higher in the StemEx® vs control group; 84.2 vs 74.6 % [95% CI 0.5 (0.26-0.95); p = 0.035]. Neutrophil and platelet engraftment rates were faster in the StemEx® vs control group: 21 vs 28 days (p< 0.0001) and 54 vs 105 days (p = 0.008), respectively. There was a trend in the reduction of engraftment failure from 14.4% in the control to 8.1% in the StemEx® group, p=0.086. Early engraftment (EE) (ANC ≤ day 20 and platelets ≤ day 60) was achieved more frequently in StemEx® (39.4%) than in the control arm (12.4%) , p<0.001. EE was associated with improved 100 day survival (p = 0.0028). Grade III/IV aGvHD and cGvHD rates did not differ between the study and control groups (19.4 vs 16.9%; p = 0.107) and 18.4 vs 16.0% (p = 0.731), respectively. Of the 101 patients, 16 patients died in the first 100 days. Causes of death were infection (5), multiorgan failure/ARDS (4), VOD (2) and relapse, acute GvHD, graft failure, hemorrhagic CVA and DIC (1 each). Importantly, the CD34+ cell dose from the StemEx expanded fraction was associated with time to ANC and platelet engraftment (p<0.001, 0.011, respectively) and inversely associated with grade III/IV infections during 100 day post-transplant (p = 0.023). Conclusions This multi-international study demonstrated the advantage of StemEx® over dUCBT historical controls,measured by a significant improvement in day 100 survival and faster engraftment of ANC and platelets. The robust associations obtained between CD34+ cell dose derived from the expanded portion and graft functionality provide additional support for the clinical benefit obtained over the dUCBT historical control arm. This technology holds the promise of increasing the number of UCBT being performed while potentially reducing its short term morbidity and mortality. Disclosures: Stiff: Gamida Cell Ltd: Consultancy, Honoraria, Research Funding. Peled:Gamida Cell: Employment. Landau:Gamida Cell Ltd: Employment. Rosenheimer:Gamida Cell Ltd: Employment. Mandel:Gamida Cell LTd: Employment. Hasson:Gamida Cell Ltd: Employment. Olesinski:Gamida Cell Ltd: Employment. Glukhman:Gmaida Cell Ltd: Employment. Snyder:Gamida Cell Ltd: Employment. Galamidi Cohen:Gamida Celll Ltd: Employment. Kidron:Gamida Cell Ltd: Employment. Bracha:Gamida Cell Ltd: Employment. Harati:Gamida Cell Ltd: Employment. Ben-Abu:Gamida Cell Ltd: Employment. Freind:Gamida Cell Ltd: Employment. Freedman:Gamida Cell Ltd: Consultancy. Olmer:Gamida Cell Ltd: Consultancy. Barishev:Gamida Cell Ltd: Consultancy. Nagler:Gamida Cell Ltd: Consultancy, Research Funding. Sanz:Gamida Cell Ltd: Consultancy, Honoraria, Research Funding.

Clinical results with NK cells in investigational tumor immunotherapy protocols have at best resulted in partial responses only. The inability of ex vivo expanded NK cells to proliferate in vivo, as well as to home to and be retained in the tumor micro-environment, likely plays a role in their limited efficacy to date.

Human transplantation of expanded HPCs resulting in durable, robust donor myeloid and T-cell engraftment has yet to be reported. A pilot study of myeloablative dual UCB transplantation where one UCB unit is expanded ex vivo using NiCord technology has completed accrual. The NiCord UCB graft consisted of an expanded CD133+ and an unexpanded CD133- T-cell fraction. HPCs were expanded for 21 days in media containing cytokines supplemented with nicotinamide (NAM). All patients were conditioned with TBI (1350cGy), fludarabine 160mg/m2 ±cyclophosphamide 120mg/kg (n=2). GvHD prophylaxis consisted of tacrolimus and MMF. Eleven patients (med. age 45; range 21-61) with high-risk malignancies received NiCord and an UM graft (Table). Both units were comparably HLA-matched with the recipient; 4/6 (n=7), 5/6 (n=3) or 6/6 (n=1) vs. 4/6 (n=8), 5/6 HLA (n=3), respectively. However, the UM unit contained a larger pre-cryopreserved cell dose (3 x 107/kg [range 1.9-3.9] vs. 2.5 x 107/kg [range 1.7-3.8]). After expansion (CD133+ fraction), NiCord contained a median TNC and CD34+ cell dose of 2.7 x 107/kg (1.0-6.4) and 3.5 x 106/kg (0.9-18.3), respectively. The NiCord T-cell dose was substantially smaller than the UM graft. Eight patients engrafted with NiCord (one of which is a mixed donor chimera) and two with the UM graft (Table). One patient experienced primary graft failure. The median time to neutrophil engraftment was 12.5 days (7-26) for the entire cohort, and 10.5 (7-18) days for those engrafting with NiCord. Three patients experienced grade I/II acute GvHD. There were no cases of Grade III/IV acute GvHD. No safety concerns were raised. The estimated 100-day treatment-related mortality is 10%. With a median follow-up of 8 months, the progression-free and overall survival are both 90%. NiCord expanded HPC's are capable of out-competing those from the UM unit and predominate in the majority of patients. NiCord expanded HPC's reduce the time to hematopoietic recovery and are capable of long term (>22 months) neutrophil and T-cell engraftment. Stem cell transplantation using NiCord is feasible, and may provide a potent cord blood graft enabling transplantation of a single expanded unit, without co-infusion of UM cells.TablePatient NumberDisease Stage/AgeEngrafted CBUEngraftment DayNiCord Chimerism∗Performed at date of last follow-up from peripheral blood.Months post TransplantANC >500Platelet >20,000CD15 (%)CD3 (%)1AML (CR1)/61NiCord + UM1433422222MDS (Int-2)/43NiCord1130100100173MDS (Int-2)/59NiCord10309793154AML (CR2)/41UM183600145AML (CR1)/57UM264900116AML (CR2)/45NiCord1030100% (Whole Blood)87HL/21NiCord + UM7261003168NHL/46Graft failure----5‡Re-transplanted with haploidentical donor.9AML(CR1)/45NiCord14419761210AML (PR)/59NiCord18->98 (Whole Blood)2†Death Day 47 Pneumonia.11ALL (CR1)/44NiCord7N/A1001001∗ Performed at date of last follow-up from peripheral blood.† Death Day 47 Pneumonia.‡ Re-transplanted with haploidentical donor. Open table in a new tab