Unmanipulated Cord Blood Research Articles

UM171 Expansion of Cord Blood Improves Donor Availability and HLA Matching For All Patients, Including Minorities

Cord blood (CB) stem cell transplantation offers a greater tolerance to HLA mismatches compared to adult-derived stem cell transplants (i.e., bone marrow or peripheral blood stem cells). Indeed, 4/6 or 5/8 HLA-matched CB transplantations are regularly performed for patients lacking a matched unrelated donor. Unfortunately, most banked CB units contain a stem cell dose that is too small to treat adult patients, resulting in only 4% to 5% of available CB units offering an adequate cell dose for prompt engraftment for adult patients. Ex vivo stem cell expansion appears to be an attractive strategy to circumvent this cell dose issue, while also enabling the selection of better HLA-matched CB units. In this study, we retrospectively performed HLA matching simulations to assess how the minimal cell content requirements associated with UM171 CB expansion may improve usability of existing CB unit inventories and donor availability for patients of different races and ethnicities. We analyzed a dataset of 58,971 adults for whom a donor search was initiated through the National Marrow Donor Program Be The Match registry against 142,942 CB units from major U.S. public CB banks listed on the Be The Match registry. Our results show that by enabling selection of smaller CB units, UM171-expanded CB transplantation increases donor availability from 72% to 84% for all patients compared to single unmanipulated CB transplantation. Furthermore, the low cell dose criteria for UM171-expanded CB also increases donor availability compared to double CB transplantation, while enabling better HLA matching between donor and recipient. UM171 expanded CB appears particularly beneficial for racial and ethnic minority patients as CB availability increases from 53% to 78% for African Americans, from 66% to 85% for Hispanics, and from 68% to 84% for Asians and Pacific Islanders, compared to single unmanipulated CB transplantation. In addition, UM171 expansion dramatically improves usability of CB units currently in inventories, as only 4.3% and 0.6% of banked CBs have sufficient cell doses for a 70 kg and 100 kg patient, respectively. UM171 raises this proportion to 53.8% and 20.2%, respectively, making CB banks potentially more cost effective. In conclusion, UM171 expansion allows the use of smaller CB units while also improving access to transplantation for racial and ethnic minorities.

Allogeneic Stem Cell Transplantation with Omidubicel: Long-Term Follow-up from a Single Center

Allogeneic Stem Cell Transplantation with Omidubicel: Long-Term Follow-up from a Single Center

UM171-Expanded Cord Blood Transplants Support Robust T Cell Reconstitution with Low Rates of Severe Infections

Rapid T cell reconstitution following hematopoietic stem cell transplantation (HSCT) is essential for protection against infections and has been associated with lower incidence of chronic graft-versus-host disease (cGVHD), relapse, and transplant-related mortality (TRM). While cord blood (CB) transplants are associated with lower rates of cGVHD and relapse, their low stem cell content results in slower immune reconstitution and higher risk of graft failure, severe infections, and TRM. Recently, results of a phase I/II trial revealed that single UM171-expanded CB transplant allowed the use of smaller CB units without compromising engraftment (www.clinicaltrials.gov, NCT02668315). We assessed T cell reconstitution in patients who underwent transplantation with UM171-expanded CB grafts and retrospectively compared it to that of patients receiving unmanipulated CB transplants. While median T cell dose infused was at least 2 to 3 times lower than that of unmanipulated CB, numbers and phenotype of T cells at 3, 6, and 12 months post-transplant were similar between the 2 cohorts. T cell receptor sequencing analyses revealed that UM171 patients had greater T cell diversity and higher numbers of clonotypes at 12 months post-transplant. This was associated with higher counts of naive T cells and recent thymic emigrants, suggesting active thymopoiesis and correlating with the demonstration that UM171 expands common lymphoid progenitors in vitro. UM171 patients also showed rapid virus-specific T cell reactivity and significantly reduced incidence of severe infections. These results suggest that UM171 patients benefit from rapid T cell reconstitution, which likely contributes to the absence of moderate/severe cGVHD, infection-related mortality, and late TRM observed in this cohort.

UM171-Expanded Cord Blood Transplants Support Robust T-Cell Reconstitution with Low Rates of Severe Infections

Introduction Rapid T cell reconstitution following hematopoietic stem cell transplantation is essential for protection against infections and has been associated with lower incidence of chronic graft-vs-host disease (cGVHD), relapse and transplant-related mortality (TRM). While cord blood (CB) transplants are associated with lower rates of cGVHD and relapse, their low stem cell content results in slower immune reconstitution and higher risk of graft failure, severe infections and TRM. Recently, results of a Phase I/II trial revealed that single UM171-expanded CB transplant allowed the use of smaller CB units without compromising engraftment. We now report on T cell reconstitution and immune function in patients transplanted with UM171-expanded CB grafts. Methods We performed a retrospective analysis of 20 patients treated with UM171-expanded CB and compared it to a contemporary cohort of 12 patients treated in the same institution who received unmanipulated CB transplant with similar conditioning regimens. Of note, no patient received ATG as part of the conditioning in either cohort. We used flow cytometry and TCR sequencing to evaluate T cell reconstitution, and virus-specific ELISpot assays to evaluate T cell function in the first year post-transplantation. We also categorized infectious events as per definitions of infection severity in the BMT CTN Technical MOP Version 3.0 and report the mean cumulative count of infectious events for each cohort. Results While median T cell dose in graft was at least 2-3x lower for the cohort of patients treated with UM171-expanded CB due to the selection of smaller cords and to cell loss occurring during CD34 selection process, numbers and phenotype of T cells at 3, 6 and 12 months post-transplant were similar in patients treated with UM171-expanded or unmanipulated CB transplant. TCR sequencing analyses revealed that UM171 patients had greater T cell diversity and higher numbers of T cell clonotypes at 12 months post-transplant compared to patients who received unmanipulated CB. Younger UM171 patients (i.e. <40 years old) also showed a more pronounced increase in naïve T cells and recent thymic emigrants (RTE) between 3- and 12-months post-transplant compared to age-matched unmanipulated CB patients, suggesting that UM171-expansion improves thymopoiesis at least in the young patients. This also correlated with the demonstration that UM171 expands common lymphoid progenitors in vitro. ELISpot assays revealed that UM171 patients showed early virus-specific T cell reactivity, at 2- and 3-months post-transplant. Most importantly, UM171 patients had a 2-fold lower frequency of severe (i.e. grade 2-3) infections at 1 year post-transplant, even though time to engraftment of 500 neutrophils was similar between the two cohorts (17 and 20 days for the UM171-expanded and unmanipulated CB cohorts respectively, p=0.94). Conclusion Our data show that the relative T-cell paucity of the UM171 graft is rapidly compensated after transplant with no significant difference observed between the two cohorts in terms of numbers and phenotypes of T cells at 3, 6 or 12 months post-transplant. Although it is difficult to dissect the relative contribution of homeostatic expansion and de novo thymopoiesis, recipients of UM171 grafts had a greater TCR diversity at one year, which was more evident among patients younger than 40 years of age. The prompt immune reconstitution observed in UM171 patients translated into a low rate of severe (grade 2-3) infections and no infection-related mortality. These results support rapid and functional T cell reconstitution following UM171 expanded CB transplantation, which likely contributes to the absence of moderate/severe cGVHD, infection-related mortality and late TRM observed in this cohort. Figure legend: Mean cumulative counts of infectious events in patients transplanted with UM171-expanded (blue) or unmanipulated (red) CB. Mean cumulative counts are shown for all infectious events (A), bacterial (B) and viral (C) infections. Events were categorized by type and severity as per BMT CTN guidelines (Appendix 4A). Infectious events of grade 1-3 are shown in pale colors, while more severe events (grade 2-3) are shown in dark colors. Censored patients (including those who relapsed) are indicated with white circles. Figure 1 Disclosures Dumont-Lagacé: ExCellThera: Current Employment. Busque:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria. Sauvageau:ExCellThera: Current equity holder in private company, Other: CEO, Patents & Royalties. Cohen:ExCellThera: Consultancy, Other: principal investigator of an ongoing UM171 clinical trial.

Hematopoietic stem cell transplantation using single UM171-expanded cord blood: a single-arm, phase 1–2 safety and feasibility study

Benefits of cord blood transplantation include low rates of relapse and chronic graft-versus-host disease (GVHD). However, the use of cord blood is rapidly declining because of the high incidence of infections, severe acute GVHD, and transplant-related mortality. UM171, a haematopoietic stem cell self-renewal agonist, has been shown to expand cord blood stem cells and enhance multilineage blood cell reconstitution in mice. We aimed to investigate the safety and feasibility of single UM171-expanded cord blood transplantation in patients with haematological malignancies who do not have a suitable HLA-matched donor. This single-arm, open-label, phase 1-2 safety and feasibility study was done at two hospitals in Canada. The study had two parts. In part 1, patients received two cord blood units (one expanded with UM171 and one unmanipulated cord blood) until UM171-expanded cord blood demonstrated engraftment. Once engraftment was documented we initiated part 2, reported here, in which patients received a single UM171-expanded cord blood unit with a dose de-escalation design to determine the minimal cord blood unit cell dose that achieved prompt engraftment. Eligible patients were aged 3-64 years, weighed 12 kg or more, had a haematological malignancy with an indication for allogeneic hematopoietic stem cell transplant and did not have a suitable HLA-matched donor, and a had a Karnofsky performance status score of 70% or more. Five clinical sites were planned to participate in the study; however, only two study sites opened, both of which only treated adult patients, thus no paediatric patients (aged <18 years) were recruited. Patients aged younger than 50 years without comorbidities received a myeloablative conditioning regimen (cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2, and 12 Gy total body irradiation) and patients aged older than 50 years and those with comorbidities received a less myeloablative conditioning regimen (cyclophosphamide 50 mg/kg, thiotepa 10 mg/kg, fludarabine 150 mg/m2, and 4 Gy total body irradiation). Patients were infused with the 7-day UM171-expanded CD34-positive cells and the lymphocyte-containing CD34-negative fraction. The primary endpoints were feasibility of UM171 expansion, safety of the transplant, kinetics of hematopoietic reconstitution (time to neutrophil and platelet engraftment) of UM171-expanded cord blood, and minimal pre-expansion cord blood unit cell dose that achieved prompt engraftment. We analysed feasibility in all enrolled patients and all other primary outcomes were analysed per protocol, in all patients who received single UM171-expanded cord blood transplantation. This trial has been completed and was registered with ClinicalTrials.gov, NCT02668315. Between Feb 17, 2016, and Nov 11, 2018, we enrolled 27 patients, four of whom received two cord blood units for safety purposes in part 1 of the study. 23 patients were subsequently enrolled in part 2 to receive a single UM171-expanded cord blood transplant and 22 patients received a single UM171-expanded cord blood transplantation. At data cutoff (Dec 31, 2018), median follow-up was 18 months (IQR 12-22). The minimal cord blood unit cell dose at thaw that achieved prompt engraftment as a single cord transplant after UM171 expansion was 0·52 × 105 CD34-positive cells. We successfully expanded 26 (96%) of 27 cord blood units with UM171. Among the 22 patients who received single UM171-expanded cord blood transplantation, median time to engraftment of 100 neutrophils per μL was 9·5 days (IQR 8-12), median time to engraftment of 500 neutrophils per μL was 18 days (12·5-20·0), and no graft failure occurred. Median time to platelet recovery was 42 days (IQR 35-47). The most common non-haematological adverse events were grade 3 febrile neutropenia (16 [73%] of 22 patients) and bacteraemia (nine [41%]). No unexpected adverse events were observed. One (5%) of 22 patients died due to treatment-related diffuse alveolar haemorrhage. Our preliminary findings suggest that UM171 cord blood stem cell expansion is feasible, safe, and allows for the use of small single cords without compromising engraftment. UM171-expanded cord blood might have the potential to overcome the disadvantages of other cord blood transplants while maintaining the benefits of low risk of chronic GVHD and relapse, and warrants further investigation in randomised trials. Canadian Institutes of Health Research, Canadian Cancer Society and Stem Cell Network.

Double-Unit Cord Blood (CB) Transplantation (dCBT) Supplemented with Haplo-Identical CD34+ Cells May be Associated with Enhanced Neutrophil Recovery but Successful Myeloid Bridging Is Strongly Influenced By Haplo CD34+ Cell Dose and Haplo-Winning CB Unit HLA-Match

Background While dCBT is associated with high rates of sustained donor engraftment, delayed neutrophil recovery is frequent. Methods We investigated the addition of CD34+ selected haplo-identical PBSC to double unit CB grafts (haplo-dCBT) in patients (pts) undergoing ablative dCBT with the aim to provide a myeloid bridge prior to CB engraftment. ATG was not used due to its adverse effects on immune recovery & potential to increase relapse risk. Results 78 adult pts [median 48.5 years (range 21-68), median 82 kgs (range 48-138), 3 with prior allografts, 54 (69%) acute leukemias or 10 (13%) MDS/ MPN (all 4/8 haplo-recipient HLA-match & ≥ 3/8 winning unit-haplo HLA-match were associated with higher likelihood of bridging (Table 2). Haplo CD34+ dose & winning unit-haplo HLA-match remained significant in multivariate analysis. While there was no difference in the day 100 TRM in the 34 optimal bridge pts vs others [9% (95%CI 2-21) vs 15% (95%CI 6-27), p = 0.388], optimal bridge pts had earlier hospital discharge [28.5 (range 20-60) vs 36 days (range 28-98)]. Conclusions ATG-free haplo-dCBT can speed neutrophil recovery but this benefit is highly variable, and a successful myeloid bridge is not guaranteed due to early haplo rejection. For these reasons our data does not support pursuit of haplo-dCBT. It is likely that similar limitations will also apply to ATG-free single CB unit-haplo transplants. Our findings also have significance for strategies that combine unmanipulated CB with third-party or ex vivo expanded T-cell depleted products given higher CD34+ cell dose & better HLA-match to the unmanipulated CB unit could improve successful bridging.

Rapid and Robust CD4+ and CD8+ T-, NK-, B-Cell, Dendritic Cell, and Monocyte Reconstitution after Nicotinamide-Expanded Cord Blood Transplantation

Introduction Nicotinamide-expanded cord blood (NiCord) is a potential alternative source for allogeneic hematopoietic cell transplantation (HCT) when an HLA-id donor is lacking. A phase 1/2 trial with standalone NiCord HCT showed rapid neutrophil- (11 days) and platelet engraftment (34 days). We previously reported that successful CD4+ immune reconstitution (IR) is crucial for infectious and relapse control associated with favorable survival (JACI 2017) and is a better predictor for event-free survival than neutrophil reconstitution. We performed unique in-depth immune monitoring to evaluate and compare IR after NiCord and conventional HCT. Methods In this phase1/2 international multicenter trial, we compared IR after NiCord HCT to cohorts of adolescent and young adult (AYA) patients receiving either unmanipulated cord blood transplantation (unCBT) or T-repleted-unrelated bone marrow transplantation (BMT). All patients received HCT for a hematologic malignancy with myeloablative conditioning without serotherapy. Immune monitoring was performed (harmonized sampling, handling and analyses) in a central lab. The primary endpoint was probability of achieving CD4+ IR (>50*106/L within 100 days). Secondary endpoints were IR of B-cells, CD4+ and CD8+ T-cells, natural killer (NK)-cells, monocytes, and dendritic cells (DC) 7-365 days after HCT. In addition, TREC analyses were performed on CD3+ MACs-sorted cells. Linear-mixed effects modelling in LOESS-regression curves and two-sided log-rank test for univariate comparisons in cumulative incidence plots were used. Results 27 NiCord recipients (median 41.5; 13.4-61.7yrs) were included. NiCord cell dose consisted of median 6.4*106 CD34+/kg, and 2.3*106 CD3+T-cells/kg of the co-infused negative fraction (following CD133+ selection). Of these patients, 91% achieved successful CD4+ IR, which was comparable (p=0.76, Figure 1) to the 27 unCBT (median 15.4; 12.2-22.1 yrs) and 20 BMT (median 14.3; 12.1-19.7 yrs) recipients included in this study. We observed similar reconstitution of T-cells (p=0.15), monocytes (p=0.94), conventional DCs (p=0.41), and plasmacytoid DCs (p=0.52). Interestingly, reconstitution of NK-cells (p Conclusions In-depth immune monitoring reveals fast and full IR after NiCord HCT in adult patients, which is equal or even faster to IR after unCBT or BMT, despite the younger age of the AYA cohorts (expected to reconstitute faster). This may be explained by the higher stem cell dose and higher proliferative capacity of the NiCord-expanded product. Optimal comparison of IR in NiCord vs. unCBT in a randomized phase 3 trial is underway.

Double-Unit Cord Blood (CB) Transplantation with Haplo-Identical CD34+ Cells (haplo-dCBT) May Speed Neutrophil Recovery Although Successful Bridging Is Contingent on Close Haplo-Winning CB Unit HLA-Match

Background: While dCBT is associated with high rates of sustained donor engraftment, delayed neutrophil recovery in adults is frequent and can contribute to extended hospitalization and early transplant-related mortality.Methods: We investigated engraftment after myeloablative dCBT supplemented with CD34+ selected haploidentical PBSC (haploCD34+) in patients (pts) with high risk hematologic malignancies or aplastic anemia in a phase II clinical trial. The aim was to abrogate neutropenia (ANC >/= 500 within 14 days) with a haplo myeloid bridge prior to CB engraftment. Pts did not receive ATG due to the increased mortality risk reported in adult CBT. Double unit CB grafts allowed comparison to dCBT controls without haploidentical graft supplementation.Results: 78 adult pts [median age 48.5 years (range 21-68), median weight 82 kgs (range 48-138), 44 (56%) CMV+, 3 with prior allografts] underwent haplo-dCBT between 9/2012-12/2017. Diagnoses included 54 (69%) acute leukemias, 10 (13%) MDS/ MPN (all ≤ 10% blasts at work-up), 10 (17%) NHL/ HD and 1 aplastic anemia. Conditioning was myeloablative (1 Cy 120/ Flu 75/ TBI 1375, 77 intermediate intensity Cy 50/ Flu 150/ Thio 5-10/ TBI 400) with CSA/ MMF. CB units had a median infused TNC of 2.3 (range 1-5.7) x 107/kg/unit & median infused viable CD34+ cell dose of 1.1 (range 0.1-3.1) x 105/kg/unit with a median 5/8 (range 2-7) unit-recipient HLA-allele match. Haplo CD34+ grafts [procured from children (46%), siblings (31%), parents (13%) or extended family (10%)] had a median infused CD34+ dose of 5.2 x 106/kg (range 1.1-16.8) and a median infused CD3+ dose of 1.6 x 103/kg (range 0.3-13.7). Sixty-one (78%) haplos were 4/8 and 17 (22%) were 5-7/8 HLA-matched to the pt. In 77 evaluable pts (1 pt died on day 14), 4 engraftment patterns were observed (Table 1). All but 2 pts had sustained CB engraftment with either an optimal haplo-bridge (Gp. 1, 34/77, 44%), a transient bridge with a second nadir preceding CB engraftment (Gp. 2, 20/77, 26%), or no bridge (Gp. 3, 21/77, 27%). The 2 remaining pts had CB/ haplo graft failure (Gp. 4, 2/77, 3%); both were successfully re-transplanted with single CB units. While there was no difference in the day 100 TRM in the 34 optimal bridge pts vs pts with transient or no bridge [9% (95%CI 2-21) vs 15% (95%CI 6-27), p = 0.388], optimal bridge pts had faster platelet recovery [19 (range 14-41) vs 44.5 days (range 14-67)] and earlier hospital discharge [28.5 (range 20-60) vs 36 days (range 28-98)]. Similar to dCBT alone, chimerism analysis revealed sustained engraftment in haplo-dCBT is mediated by a “winning” CB unit. This was heralded by winning unit-derived T-cells seen as early as day +28. Although universal, the speed of haplo rejection varied, and a high haplo chimerism percentage early post-transplant did not guarantee successful bridging. Analysis of factors potentially predicting an optimal bridge is shown in Table 2. The median winning CB unit-haplo 8-allele HLA-match was 3/8 (range 1-7/8). In univariate analysis, higher haplo CD34+ dose/kg, > 4/8 haplo-recipient HLA-match and ≥ 3/8 winning unit-haplo HLA-match were associated with a higher likelihood of bridging. Haplo CD34+ dose and winning unit-haplo HLA-match remained significant in multivariate analysis.Conclusions: While haplo-dCBT can be associated with enhanced neutrophil recovery, this platform does not guarantee a successful myeloid bridge due to early haplo rejection by the winning CB unit. This universal haplo rejection highlights the importance of the CB graft dose and quality with this ATG-free strategy as the CB will mediate sustained engraftment. Our findings have significance for strategies that combine unmanipulated CB with any third-party or ex vivo expanded T-cell depleted product given higher product CD34+ cell dose and better HLA-match to the unmanipulated CB unit could improve the likelihood of successful myeloid bridging. The data also support alternative approaches to improve myeloid recovery after CBT such as optimized unit selection and vivo expansion. [Display omitted] DisclosuresO'Reilly:Atara Biotherapeutics: Consultancy, Patents & Royalties, Research Funding. Perales:Takeda: Other: Personal fees; Novartis: Other: Personal fees; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support; Merck: Other: Personal fees; Abbvie: Other: Personal fees. Sauter:Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Precision Biosciences: Consultancy; Kite: Consultancy. Shah:Janssen: Research Funding; Amgen: Research Funding.

Expansion of Cord Blood Stem Cells and Enhancing Their Mobilization and Homing Potential Using Mesenchymal Stromal Cells

Umbilical cord blood (CB) contains hematopoietic stem cells (HSC). CB can be collected easily, cryopreserved and be readily available from CB banks when needed. Transplantation with CB is associated with less graft-versus-host disease (GvHD) when compared to peripheral blood (PB) and bone marrow (BM) HSCT. Attempts have been made to overcome the issue of low and insufficient number of CB stem cells.Mesenchymal stromal cells (MSCs) has been proven as immune modulators in GvHD and other immunological diseases. MSCs may enhance HSC engraftment to improve hematopoietic recovery. There is compelling evidence that MSCs produce a wide range of cytokines that are capable of maintaining HSC in a quiescent state and other cytokines which induces proliferation and self-renewal of HSC.Recently, it has been shown that expansion of HSC in unfractionated CB is markedly enhanced by co-culture with MSCs, with the time to neutrophil engraftment and platelet recovery markedly shortened. CB stem cells expanded with allogeneic MSCs then transplanted into patients led to multiple times expansion for both total nucleated cells and CD34+ cells in vivo when compared to unmanipulated CB stem cells. Therefore, expansion of CB stem cells with MSCs has great potential. The exact mechanism of how CB cells expand at the molecular level remains unknown. This study's aim was to expand CB in an ex vivo coculture system using MSCs and selected growth factors (GFs) and to investigate expansion at the molecular level.In this study, CB cells were expanded in a coculture system for two weeks using BM-derived MSCs with and without GFs (SCF, TPO, Flt3-L, G-CSF and IL6). A 69.9 fold expansion in CB total nucleated cells and 53.2 fold expansion in CD34+ cells was achieved after 12 days using MSCs as feeders in 50 ng/ml Flt3-L, SCF, TPO, G-CSF, and IL6.The viability of CB cells was significantly higher when cocultured with MSCs regardless of GFs addition. CB expanded on MSCs expressed higher percentages of CD45+CD34+CXCR4+ and CD45+CD34+EpHB4+ populations. Interestingly, the HSC maintenance marker EpHB4 expressed by MSCs at low levels decreased significantly in the coculture system.To investigate MSCs effect on CB expression levels of other genes that are important for expansion, stemness and pluripotency hematopoietic lineage commitment, RUNX1, SOX17, HOXC8, Myc, TP53, SOX9, FOXO1, FOXO4, GATA1 genes were examined. The study demonstrated that HOXC8, SDF-1, SOX17 and SOX9 expression was suppressed in CB cells, while the expression of other genes such as CXCR4, EpHB4, FOXO1, Myc, and HPRT1 increased.The undetectable level of HOXC8 expression, which regulates self-renewal and differentiation of stem cells, may have caused less CB stem cell differentiation, favoring an increase in CD34+ cells. Equally, SOX17 and SOX9 expression, which also decreased to undetectable levels in CB cells post coculture with MSCs, plays a significant role in CB expansion. SOX17 is known as a primer of hemogenic potential, regulating hematopoietic development from hESCs/iPSCs, whereas SOX7 plays an important role in HSC differentiation. Therefore, we speculate that MSCs-CB coculture dampens the differentiation of adjacent CB cells, associated with changes of signaling in BM-like niches.Simultaneously, MSCs may be sending signalling messages to CB cells in the ex vivo niche to maintain quiescence or self-renewal capacity through other signalling pathways. FOXO family has the ability to regulate stem cells and program them to remain quiescent through cell-cycle repression via the oxidative stress-activated P66shc-Akt-FOXO pathway. In this study, FOXO1 was upregulated in CB cells cultured with MSCs supporting our hypothesis and other earlier findings (decrease in the expression of EpHB4 on MSCs and a decline in the expression of SDF1 in CB cells), suggesting that MSCs have the ability to induce stem cell quiescence and maintain stemness.In conclusion BM-derived MSCs support CB viability, expansion, and increased stemness potential, by modifying key hematopoietic, progenitor, differentiation and stemness signaling pathways. The study identified HOXC8, SDF-1, SOX17, SOX9, CXCR4, EpHB4, FOXO1, Myc, and HPRT1 as potential factors involved in CB expansion signaling pathways. Using MSCs as a feeder layer resulted in higher CB viability and proliferation rates which may increase the potential use of CB in hematopoietic stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

MGTA-456, a First-in-Class Cell Therapy Produced from a Single Cord Blood Unit, Enables a Reduced Intensity Conditioning Regimen and Enhances Speed and Level of Human Microglia Engraftment in the Brains of NSG Mice

Background. Allogeneic bone marrow transplant (BMT) is a promising, curative approach for patients with inherited metabolic disorders (IMDs), a class of pediatric diseases characterized by a single enzyme deficiency. The goal of transplant is to provide cells that produce functional enzymes otherwise deficient in these patients, and thereby prevent or ameliorate neurological complications associated with selected IMDs. Donor-derived microglial cells are protective, limiting neurological disease progression. For IMD patients who do not have an HLA matched, non-carrier related donor, cord blood (CB) is the preferred HSPC source given its rapid availability and superior clinical outcomes compared to other graft sources. CB, however, is associated with delayed hematopoietic recovery and relatively poor engraftment due to the limited numbers of hematopoietic stem cells (HSCs) in a CB unit, delaying enzyme/protein reconstitution and cross-correction of non-hematopoietic cells.An aryl hydrocarbon receptor antagonist (AHRa)-based culture has been shown to expand CB CD34+ and CD34+CD90+ cells 330-fold and 100-fold, respectively, leading to rapid hematopoietic recovery after infusion of the clinical product, MGTA-456 (Wagner et al., Cell Stem Cell 2016 and Orchard et al., ASH 2018). As microglia are thought to be derived from HSCs, we hypothesized that MGTA-456 might lead to faster and greater microglial engraftment and potentially enable reduced intensity conditioning. Here, we assessed human hematopoietic and brain engraftment in NSG mice after transplant with MGTA-456 and showed that microglia engrafted faster with MGTA-456, less conditioning was needed, and that, mechanistically, these cells are derived from the CD34+CD90+ cell fraction.Methods. CB CD34+ cells were expanded in growth factor-supplemented media with or without an AHRa for 10 days. NSG mice were transplanted with unmanipulated CB CD34+ cells or the expanded product after 200 cGy total body irradiation or busulfan (BU) dosed at 20 or 40 mg/kg ip. Microglial engraftment was measured by flow cytometry of homogenized brains, quantitating the number of CD45+CD11b+Iba1+ cells, and by immunohistochemistry of brain sections.Results. Relative to naïve, unmanipulated CB CD34+ cells, transplant of MGTA-456 into sublethally irradiated mice led to an 8-fold increase in hematopoietic engraftment and a 10-fold increase in microglial engraftment in the brain (p<0.0001, n=15 mice), with histology consistent with engrafting microglia. As high dose BU enables enhanced microglia engraftment relative to irradiation by crossing the blood brain barrier and clearing host microglia (Capotondo et al., PNAS 2013), we evaluated the effectiveness of MGTA-456 after BU conditioning at 20 or 40 mg/kg. Transplant of MGTA-456 led to a 37-fold increase in engraftment relative to mice transplanted with unmanipulated CB CD34+ cells (p<0.001, n=8). Notably, transplant of MGTA-456 into mice conditioned with low-dose BU (20 mg/kg) led to a 15-fold increase in engraftment relative to high-dose BU (40 mg/kg)-conditioned animals transplanted with unmanipulated CB CD34+ cells (p<0.001, n=8). To evaluate speed of microglial engraftment, we evaluated brains weekly to 16 weeks after transplant. A 28-fold increase in microglial engraftment was demonstrated as early as 2 weeks post-transplant with MGTA-456 (p<0.0001, n=8). Number of engrafting hematopoietic cells in the periphery correlated with number of engrafting microglia in the brain (p<0.0001). Lastly, subpopulations of MGTA-456 were evaluated to determine the source of microglial engraftment. Only CD34+CD90+ cells, but not CD34+CD90- or CD34- cells, led to brain engraftment, consistent with the subpopulation of cells that result in hematopoietic engraftment following transplant of unexpanded cells (Radtke et al., Sci Trans Med 2017 and Goncalves et al., Blood 2017 130:659).Conclusions. These studies demonstrate that microglial engraftment is faster and greater in recipients of MGTA-456 even after lower dose BU conditioning, that microglial engraftment correlates with peripheral blood recovery, and that microglia cells are derived from CD34+CD90+ cells. These results suggest that lower dose BU may improve safety without jeopardizing efficacy in IMD recipients of MGTA-456. A Phase 2 clinical trial is ongoing to evaluate transplant of MGTA-456 in patients with select IMDs. [Display omitted] DisclosuresGoncalves:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Li:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Rapid and Robust CD4+ and CD8+ T-, NK-, B- and Monocyte Cell Reconstitution after Nicotinamide-Expanded Cord Blood Transplantation

IntroductionNicotinamide-expanded cord blood (NiCord) is a promising alternative source for allogeneic hematopoietic cell transplantation (HCT) when an HLA-matched donor is unavailable. A phase 1/2 trial with standalone NiCord HCT showed rapid neutrophil engraftment (median 11 days) and platelet engraftment (median 34 days). However, successful CD4+ immune reconstitution (IR) has shown to be crucial for infectious and relapse control associated with favorable survival (Admiraal JACI 2017). We performed unique in-depth immune monitoring to evaluate and compare the recovery of immune subsets after NiCord and conventional HCT.MethodsIn the phase1/2 multicenter trial, patients (n=36) with hematologic malignancies received NiCord-HCT after myeloablative (MA) conditioning without antithymocyte globulin (ATG). Immune monitoring was performed (harmonized sampling, handling and analyses in a central lab) in a random subgroup. The primary endpoint was probability of achieving CD4+ IR (>50*106/L within 100 days). Secondary endpoints were subset recovery over time of B-cells, CD4+ and CD8+ T-cells, natural killer (NK), monocytes, and dendritic cells (DC), during 7-365 days after HCT. In addition, TREC analyses are pending and will be available at the meeting. Data were compared with IR in cohorts of adolescent and young adult (AYA) patients at the UMC Utrecht receiving either unmanipulated cord blood transplantation (unCBT) or T-repleted unrelated bone marrow transplantation (BMT) for hematological malignancy after MA conditioning without ATG. Linear-mixed effects modelling in LOESS-regression curves and two-sided log-rank test for univariate comparisons in cumulative incidence plots were used.Results24 NiCord recipients (median 41.5; 13.4-61.7 yrs) had blood samples available for in-depth early immune monitoring. NiCord cell dose consisted of median 6.4*106 CD34+/kg, and 2.3*106 CD3+T-cells/kg of the co-infused negative fraction (following CD133+-selection). 91% of patients achieved successful early CD4+ IR after NiCord (Fig 1). When comparing the NiCord with 27 unCBT (median age 15.4; range 12.2-22.1 yrs) and 20 BMT (median age 14.3; range 12.1-19.7 yrs), no difference in probability of early CD4+ IR was noted (p=0.76: Fig 1). Overall T-cell reconstitution was similar; CD3+ (p=0.99), CD4+ (p=0.71), CD8+ (p=0.08), although effector and central memory CD4+ and CD8+ T-cells, Tregs, gamma-delta T-cells, Th2, and Th17 recovered somewhat faster after NiCord. Recovery of conventional- (p=0.41) and plasmacytoid DCs (p=0.52) was similar as well. Overall reconstitution of NK-cells (p<0.001); especially naïve NK-cells, monocytes (p<0.001); mostly classical, and B-cells (p=0.026) was faster after HCT with NiCord, compared to unCBT and BMT cohorts (Fig 2). In B-cell recovery, strikingly faster early recovery of follicular B-cells (p=0.04), memory B-cells (p=0.003), and plasma cells (p=0.003) was observed in NiCord recipients.ConclusionsIn-depth immune monitoring reveals rapid and robust immune reconstitution in NiCord recipients, with high early CD4+ IR probability, and comparable recovery of T-cell-, NK-cell-, monocyte-, and DC-subsets to AYA controls receiving unCBT and BMT. Interestingly, B-cell recovery consisted of markedly higher follicular B-cell, memory B-cell, and plasma cell levels in NiCord recipients. Next to higher B-cell recovery, monocyte and NK-cells also recovered faster after NiCord transplantation, despite the younger age of the AYA cohort (expected to reconstituted faster). This may be explained by the higher stem cell dose and higher proliferative capacity of the NiCord- expanded product. Optimal comparison of IR in NiCord vs. unCBT in a randomized phase 3 trial is underway. [Display omitted] DisclosuresHorwitz:Gamida Cell: Research Funding. Jagasia:Incyte Corporation: Membership on an entity's Board of Directors or advisory committees. Wagner:Magenta Therapeutics: Consultancy, Research Funding; Novartis: Research Funding. Cilloni:Janssen: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees. Nierkens:Gamida: Research Funding.

Ex Vivo Mesenchymal Precursor Cell–Expanded Cord Blood Transplantation after Reduced-Intensity Conditioning Regimens Improves Time to Neutrophil Recovery

We previously showed the safety of using cord blood (CB) expanded ex vivo in cocultures with allogeneic mesenchymal precursor cells (MPC) after myeloablative conditioning with faster recovery of neutrophils and platelets compared with historical controls. Herein, we report the transplantation outcomes of 27 patients with hematologic cancers who received 1 CB unit expanded ex vivo with MPCs in addition to an unmanipulated CB (MPC group) after reduced-intensity conditioning (RIC). The results in this group were compared with 51 historical controls who received 2 unmanipulated CB units (control group). The analyses were stratified for 2 RIC treatment groups: (1) total body irradiation 200 cGy + cyclophosphamide + fludarabine) (TCF), and (2) fludarabine + melphalan (FM). Coculture of CB with MPCs led to an expansion of total nucleated cells by a median factor of 12 and of CD34+ cells by a median factor of 49. In patients in whom engraftment occurred, the median time to neutrophil engraftment was 12 days in the MPC group, as compared with 16 days in controls (P = .02). The faster neutrophil engraftment was observed in both RIC groups. The cumulative incidence of neutrophil engraftment on day 26 was 75% with expansion versus 50% without expansion in patients who received FM as the RIC regimen (P = .03). Incidence of neutrophil engraftment was comparable in MPC and control groups if treated with TCF (82% versus 79%, P = .40). Transplantation of CB units expanded with MPCs is safe and effective with faster neutrophil engraftment even after RIC regimens.

Infusion of Ex-Vivo Expanded Cord Blood Progenitor Cells Reduces the Time to Platelet Engraftment and the Number of Platelet and Red Blood Cell Transfusions Required in Patients Receiving Myleloblative Cord Blood Transplants

BackgroundPatients undergoing cord blood transplants (CBT) for hematologic malignancies are known to be at risk for significantly delayed neutrophil and platelet engraftment, resulting in an increased risk of transplant related mortality from infections and bleeding complications. We have previously shown that infusion of CD34+ hematopoietic stem/progenitor cells (HSPC) expanded ex-vivo significantly shortens the time to neutrophil engraftment when compared to conventional CBT. Here, we retrospectively analyzed whether infusion of ex-vivo expanded CD34+ cells with one or two unmanipulated cord blood (CB) units reduces both the time to platelet engraftment and the number of platelet and red blood cell transfusions required within the first 100 days post-transplant for patients undergoing myeloblative CBT.MethodsBetween April 2006 and December 2015, 123 patients on research protocols underwent a CBT with myeloblative conditioning (fludarabine 75 mg/kg, cyclophosphamide 120 mg/kg, and 13.2 Gy TBI) with or without infusion of an ex-vivo expanded cord blood (CB) CD34+ HSPC product. Time to platelet engraftment, units of platelets transfused, and units of red blood cells transfused through 100 days post-transplant were determined for each patient. The time to platelet engraftment was defined as the first day the post-transplant platelet count was greater than 20,000 without a platelet transfusion for 7 consecutive days. A two sample t-test was applied to compare the time to platelet engraftment and the number of platelet and red blood cell transfusions through 100 days post-transplant between two cohorts, conventional and expanded. Cumulative incidence of platelet engraftment was calculated using the Fine and Gray hazard regression model and considered death and relapse as competing risks, controlled for weight, gender, and CBT group.ResultsOne hundred twenty-three consecutive patients, median age 22 years (range 7 mos - 45 yrs), being treated for AML (n=54), ALL (n=49), MDS/myeloproliferative disorders (n=9), and other malignancies (n=11) were included in the study. No significant differences between the two cohorts were found with respect to weight, age, sex, disease, and diagnosis. Seventy-four (60%) patients received a conventional CBT with 1 (n=18) or 2 (n=56) unmanipulated CB units, while 49 (40%) patients received a CBT with ex-vivo expanded CD34+ HSPC in addition to 1 (n=14) or 2 (n=35) unmanipulated CB units. Median time to platelet engraftment for the conventional vs the expanded cohorts was 43 days and 35 days, respectively (p=0.0009). Median number of platelet transfusions through day 100 for the conventional vs the expanded cohorts was 18.5 and 12.5, respectively (p=0.028) (Figure 1A). Median number of red blood cell transfusions through day 100 for the conventional vs the expanded cohorts was 13.5 and 9.5, respectively (p=0.039) (Figure 1B). From the competing risk hazard regression model, patients in the expanded cohort vs the conventional cohort had significantly higher likelihood of reaching platelet engraftment (HR 2.45 - 95% CI: 1.66 to 3.60 and p-value <0.0001). At day 40, the cumulative incidence of platelet engraftment was 69% for the expanded cohort and 38% for the conventional cohort. At day 60, the cumulative incidence of platelet engraftment was 98% for the expanded cohort and 71% for the conventional cohort (Figure 2).ConclusionThe infusion of ex-vivo expanded CD34+ HSPC in conjunction with one or two unmanipulated CB units significantly decreases the time to platelet engraftment as well as the number of platelet and red blood cell transfusions required within the first 100 days post-transplant. By reducing the duration of thrombocytopenia, administration of ex-vivo expanded CD34+ HSPC may protect patients from potentially fatal bleeding complications, improve their quality of life, and importantly reduce the cost of post-transplant transfusion requirements. [Display omitted] [Display omitted] DisclosuresDelaney:Nohla Therapeutics: Employment.

Successful Engraftment of Donor Umbilical Cord Blood (UCB) Cells after Co-Transplantation of Nicord® (Ex Vivo Expanded UCB Progenitor Cells with Nicotinamide) and a Second Unmanipulated Cord Blood Unit after Myeloablative Chemotherapy in Pediatric Patients with Severe Sickle Cell Disease

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.Methods: Patients with SCD and high risk features were eligible if they were between 2 and 45 years of age and if they had adequate performance status and organ function, 2 suitable unrelated UCB units that minimally matched the patient at 4/6 HLA alleles, and if they did not have a fully HLA matched related or unrelated adult donor. The NiCord graft, manufactured by the study sponsor, consisted of a CD133+ expanded cell fraction and an uncultured CD133- T-cell containing fraction. All patients received hydroxyurea beginning on day -35. Patients were subsequently prepared for transplantation with myeloablative chemotherapy: Busulfan/Cyclophosphamide and Anti Thymocyte Globulin (ATG). After the first 3 patients an adverse effect of ATG on the expanded graft was suspected and ATG was replaced by Fludarabine for the subsequent 5 patients. On day 0, the unmanipulated CB unit and NiCord graft were infused. Engraftment, GVHD incidence, treatment related mortality and survival were assessed by conventional parameters.Results: Eight patients, at a median age of 12 years (range 3-16 years) and a median weight of 41 kg (range 17-67 kg) were transplanted. HLA matching between patients and NiCord units was 4/6 (n=7) and 5/6 (n=1); and that between patients and unmanipulated units was 4/6 (n=5) and 5/6 (n=3). Median CD34+ cell dose infused in the NiCord graft was 107 x 105/kg. All 8 transplanted patients initially engrafted neutrophils at a median of 7 days (range 6-20 days) with the NiCord graft. One patient experienced secondary graft failure and died after a second transplant. Ultimately, long term engraftment was derived from NiCord in 2 patients, from the unmanipulated unit in 4 patients and mixed donor chimerism is still sustained in one patient. Patients were hospitalized for initial transplant for a median of 55 days (range 43-100 days). The 7 patients with sustained full donor cell engraftment are currently alive at a median follow-up of 33 months (range 3-45 months), transfusion free and without sickle cell related symptoms. Engraftment and clinical outcomes of the 8 patients are shown in Table 1.Conclusions: NiCord appears to overcome the engraftment barriers of UCB in patients with SCD. Remarkably, sustained donor cell engraftment was obtained in 7/8 patients, despite 4/6 matched UCB units and 5 patients without ATG. NiCord has the potential to increase access to transplantation for patients with SCD by enabling the successful use of unrelated UCB donors. Further optimization of this approach with strategies to further decrease GVHD is warranted. [Display omitted] DisclosuresMartin:Novartis: Other: Support of clinical trials; Jazz Pharmaceuticals: Other: One time discussion panel. Galamidi:Gamida Cell Ltd.: Employment. Peleg:Gamida Cell Ltd.: Employment. Goshen:Gamida Cell Ltd.: Employment. Schwarzbach:Gamida Cell Ltd.: Employment. Snyder:Gamida Cell Ltd.: Employment. Peled:Gamida Cell Ltd.: Employment.

Ex Vivo Mesenchymal Precursor Cell-Expanded Cord Blood Transplantation Following Reduced Intensity Conditioning Regimens

Introduction: Double-unit cord blood (CB) transplantation (DCBT) after myeloablative conditioning, where one CB unit is expanded ex-vivo with mesenchymal precursor cells (MPC)-co-culture led to significantly improved neutrophil and platelet engraftment compared to historical controls.1 Our aim was to determine the efficacy of this technique after reduced-intensity conditioning (RIC) regimens.Methods: We evaluated consecutive adult patients with hematological malignancies who received RIC regimens followed by DCBT where one unit was infused unmanipulated and second unit was expanded ex-vivo with MPC prior to infusion ("MPC group" n=27), as described previously,1 andcompared themwith historical cohort who received two unmanipulated CB units ("controls" n= 51) from 2003-2015. Two RIC regimens were used - Flu/Cy/TBI (flu 40 mg/m2, cy 50 mg/kg and TBI 200cGy) (n=40) and Flu/Mel (flu 40 mg/m2 and mel 140 mg/m2) (n=38) with rabbit ATG 3mg/kg. Tacrolimus and MMF were used for GVHD prophylaxis. Primary endpoint was median time to engraftment of neutrophils (≥ 0.5 X 109/L X 3 consecutive days) and platelets (≥ 20 X 109/L X 7 consecutive days without transfusion).Results: Diagnoses were AML/MDS (N=38; 49%), ALL (N=13; 17%),NHL (N=18; 23%), HD (N=3; 4%), CML (N=1; 1%), and CLL (N=4; 5%). Baseline patient characteristics were similar among the groups [Table 1]. Majority of CB units (66%) were 5-6/8 matched at HLA-A, B, C and DRB1 by high resolution testing. Infused median total nucleatedcells (TNC) (x107/Kg) were 4 and 8, and median CD34 cells (x105/Kg)were 4.3 and 19.7, respectively for control and MPC groups. Co-culture with MPCs led to expansion of TNC by a median of 11.1 and of CD34+ cells by a median of 49.3.Among engrafted patients, median time to neutrophil engraftment was 12 (range 1-28) days in MPC group as compared with 16 (range 5-48) days in controls (p=0.02); the median time to platelet engraftment was 31 days and 37 days, respectively (P=0.3). On day 26, the cumulative incidence of neutrophil engraftment was 78% in MPC group versus 67% in controls (P=0.1). On day 60, the cumulative incidence of platelet engraftment was 74% and 74%, respectively (P=0.7). [Figure 1]Conditioning regimen also affected the time to neutrophil recovery. Among patients with Flu/Mel, the median time to neutrophil engraftment was 14 days in MPC-group (n=13) compared with 22 days in controls (n=19) (p=0.001). Patients with Flu/Cy/TBI regimen had faster time to engraftment; median time to neutrophil engraftment was 6 days in MPC group (n=10) and 11 days in controls (n=27) (p=0.04). However, the median time to platelet engraftment was similar in MPC- and control groups in patients who received either Flu/Mel (37 vs 44 days, p=0.1) or Flu/Cy/TBI regimen (29 vs 31 days, p=0.5).There was no difference in the cumulative incidence of day 100 non-relapse mortality (4% vs. 11%, p=0.6), 6th month mortality (25% vs 24%, p=0.6), grade II-IV acute GVHD (28% vs 27%, p=0.9), 2-year chronic GVHD (29% vs 20%; p=0.9) and estimates of 2-year OS (32% vs. 34%) between patients with ex-vivo expanded and unmanipulated CB units.Conclusions: Transplantation of CB units expanded with MPC appeared to be safe and effective. Using MPC-expanded CB units significantly improved time to engraftment following Flu/Mel or Flu/Cy/TBI RIC regimens as compared with unmanipulated units.Table 1Patient characteristicsControl(N=51)MSC group(N=27)P valueAge, years (median, interquartile range)57 (48, 63)59 (49, 67)0.3Disease StatusCR1/CR220 (40%)12 (45%)Advanced31 (61%)15 (56%)0.7Disease Risk Index, n (%)V. High/High8 (16%)9 (33%)Intermediate38 (75%)16 (59%)Comorbidity index0-124 (47%)13 (48%)2-422 (43%)11 (41%)>45 (10%)3 (11%)0.97Flu/Mel regimen22 (43%)16 (59%)0.2

Distinct Patterns of Early Hematopoietic Reconstitution in Patients Receiving Notch Expanded Non HLA-Matched Off-the-Shelf Cord Blood Derived Hematopoietic Stem and Progenitor Cells

Introduction: Delayed hematopoietic recovery and increased risk of transplant related mortality (TRM) continue to be major risk factors for patients undergoing myeloablative cord blood transplant (CBT). To overcome this issue, our group has developed methods for the ex vivo expansion of cord blood (CB) derived hematopoietic stem and progenitor cells (HSPCs) using an engineered Delta-1 Notch ligand. This expanded CB HSPC product has now been developed for use as a cryopreserved, universal donor (non-HLA matched), off-the-shelf (OTS) cell therapy to be infused as an adjuvant graft in CBT recipients. We have previously reported the promising clinical outcomes observed after infusion of the expanded OTS cell product after myeloablative CBT for faster neutrophil and platelet recovery, reduced TRM and grade III-IV acute GVHD and higher overall survival rate in comparison to the conventional CBT patients1. Based on these results, a multicenter Phase II randomized clinical trial is now underway. Herein, we sought to identify factors that contributed to the improved outcome in these patients by defining the patterns of early hematopoietic recovery in the peripheral blood of patients during the first four weeks after myeloablative CBT.Method: Between February 2013 and June 2015, 27 patients were enrolled in this study to assess the efficacy of our OTS product as an adjuvant graft to improve hematopoietic reconstitution and reduce regimen related toxicities in recipients of myeloablative CBT. Fourteen of the 27 patients were randomized to receive the OTS product. The frequencies of monocytes, T cells and NK cells (defined as CD14+, CD3+ and CD56+ respectively) within the freshly isolated peripheral blood mononuclear cells were determined by real-time immunophenotyping using multi-color flow cytometry on days 7, 14, 21, and 28 following CBT 2.Results: Strikingly, on day 7 the median absolute number of monocytes was ~100-fold higher in the OTS group as compared to the conventional group (4.7 cells/μL, 95% CI: 2.0 - 13 cells/μL vs 0.04 cells/µL, 95% CI: 0.01- 0.2 cells/μL), however by day 14 the absolute number of monocytes was similar in the two groups. Chimerism studies further confirmed that on day 7 nearly all (95-100%) of the circulating myeloid cells were derived from the OTS graft, whereas on day 14 the CD14 fraction was predominantly from the unmanipulated conventional graft. Furthermore, we anticipated that infusion of a non-HLA matched cell product would induce enhanced alloreactivity of the unmanipulated CB unit in vivo. Thus, we set out to analyze the frequency and composition of the donor derived peripheral blood T cells in both groups. We did not observe any differences in the absolute number of T cells present on day 7, but on day 14, we documented a robust expansion of circulating T cell in OTS patients (median=26 cells/µL, 95% CI: 12 - 50 cells/μL in the OTS group vs 5.4 cells/µL, 95% CI: 1.7 - 12 cells/μL, in the conventional group, p=0.0015). T cell subset analysis revealed that it is CD8+ T cells that are the primary contributor to the observed total T cell burst on day 14, with an absolute median number of 20.2/μL (OTS) vs 1.29/ μL (conventional), CI 95%: 10.7 - 36.6 vs 0.53 - 8.75, p = 0.0003. Additionally, a small, ~3- fold, increase in numbers of CD4+ T cells from day 7 to 14 was seen in the OTS patients but not in the conventional group (p = 0.03), resulting in a significantly lower median day 14 CD4:CD8 ratio in OTS patients than in conventional patients (0.17 vs 1.4, 95% CI: 0.1- 0.5 vs 0.4 - 3.9, p =0.004). Moreover, the analysis of NK cells at any time point and monocytes and T cells on day 21 and 28 did not yield any statistically significant changes in patients in each groupConclusion: Our findings show that patients receiving a non-HLA matched OTS product display a distinct pattern of early immune cell recovery characterized by an early monocyte burst on day 7 as "myeloid bridge" and a CD8 T cell burst on day 14. Better understanding of how these observations could impact the overall clinical outcomes in myeloablative CBT recipients will potentially expand the future applications of the non-HLA matched OTS product. SHAPE \\* MERGEFORMAT1. Milano F. et al. Blood. 2014;124(21):46.2. Li J. et al. Blood. 2014;124(21):3897. [Display omitted] DisclosuresDelaney:Biolife Solutions: Membership on an entity's Board of Directors or advisory committees; medac: Research Funding; Novartis: Other: Chair, DSMB.

CMV-specific T cells generated from naïve T cells recognize atypical epitopes and may be protective in vivo.

Adoptive transfer of cytomegalovirus (CMV)-specific T cells derived from adult seropositive donors can effectively restore antiviral immunity after transplantation. However, CMV-seronegative donors lack CMV-specific memory T cells, which restricts the availability of virus-specific T cells for immunoprophylaxis. We demonstrate the feasibility of deriving CMV-specific T cells from naïve cells for T cell therapy. Naïve T cells primed to recognize CMV were restricted to different, atypical epitopes than T cells derived from CMV-seropositive individuals; however, these two cell populations had similar avidities. CMV-seropositive individuals also had T cells recognizing these atypical epitopes, but these cells had a lower avidity than those derived from the seronegative subjects, which suggests that high-avidity T cells to these epitopes may be lost over time. Indeed, recipients of cord blood (CB) grafts who did not develop CMV were found by clonotypic analysis to have T cells recognizing atypical CMVpp65 epitopes. Therefore, we examined unmanipulated CB units and found that T cells with T cell receptors restricted by atypical epitopes were the most common, which may explain why these T cells expanded. When infused to recipients, naïve donor-derived virus-specific T cells that recognized atypical epitopes were associated with prolonged periods of CMV-free survival and complete remission. These data suggest that naïve-derived T cells from seronegative patients may be an additional source of cells for CMV immunoprophylaxis.

Human adipose-tissue derived stromal cells in combination with hypoxia effectively support ex vivo expansion of cord blood haematopoietic progenitors.

The optimisation of haematopoietic stem and progenitor cell expansion is on demand in modern cell therapy. In this work, haematopoietic stem/progenitor cells (HSPCs) have been selected from unmanipulated cord blood mononuclear cells (cbMNCs) due to adhesion to human adipose-tissue derived stromal cells (ASCs) under standard (20%) and tissue-related (5%) oxygen. ASCs efficiently maintained viability and supported further HSPC expansion at 20% and 5% O2. During co-culture with ASCs, a new floating population of differently committed HSPCs (HSPCs-1) grew. This suspension was enriched with СD34+ cells up to 6 (20% O2) and 8 (5% O2) times. Functional analysis of HSPCs-1 revealed cobble-stone area forming cells (CAFCs) and lineage-restricted colony-forming cells (CFCs). The number of CFCs was 1.6 times higher at tissue-related O2, than in standard cultivation (20% O2). This increase was related to a rise in the number of multipotent precursors - BFU-E, CFU-GEMM and CFU-GM. These changes were at least partly ensured by the increased concentration of MCP-1 and IL-8 at 5% O2. In summary, our data demonstrated that human ASCs enables the selection of functionally active HSPCs from unfractionated cbMNCs, the further expansion of which without exogenous cytokines provides enrichment with CD34+ cells. ASCs efficiently support the viability and proliferation of cord blood haematopoietic progenitors of different commitment at standard and tissue-related O2 levels at the expense of direct and paracrine cell-to-cell interactions.

CMVpp65-Specific T cells generated from naïve T cell populations recognize atypical but not canonical epitopes and may be protective in Vivo

CMVpp65-specific T-cells (cCTL) from CMV-seropositive (CMVpos) donors effectively restore antiviral-immunity after stem cell transplantation. However, naïve T-cells cord blood (CB) and CMV-seronegative (CMVneg) donors) require different culture systems. With a novel protocol we have generated cCTL from CB and CMVnegs and found that of >20 T-cell lines, all recognized atypical epitopes of pp65. HLA-A2+ cCTL derived from naive T-cells recognized novel pp65 epitopes (e.g. LQT) but not the typical epitope NLV. Naïve donor CTL specific for the atypical CMV epitopes had similar avidity to CMVpos CTL recognizing typical epitopes (mean: 300pM), but 30x more avid than CMVpos CTL recognizing atypical epitopes (P<0.05). TCR sequencing performed on T-cells specific for typical (CMVpos) and atypical (CMVpos,CMVneg, and CB) epitopes revealed that CMVpos donor cCTL recognizing typical epitopes were less polyclonal(P=0.02). To test if atypical epitopes are targeted in CMVpos donors in response to virus infection, we tested whether cCTL generated using CMV-infected fibroblasts recognize the same epitopes. CMVpos cCTL recognized typical epitopes while CB/CMVneg cCTL recognized atypical epitopes. Using TCR sequencing we identified TCRs recognizing typical and atypical epitopes. In unmanipulated CB units, 5 different TCRs recognizing atypical epitopes were found in 5/5 CB units whereas of 22 NLV sequences, only 1 sequence was found in 1/5 CB units suggesting that T-cells recognizing atypical epitopes are more prevalent during the primary infection and are later replaced by T-cells recognizing typical epitopes. These results reveal major differences in the naïve/memory T-cell repertoire and atypical epitopes may be important. Indeed, in a clinical trial of CB-derived virus-specific T cells, one patient with active CMV viremia received virus-specific T-cells and was ultimately able to clear the virus with detectable CMV-specific T cells in the peripheral blood of the patient post-T-cells.

Infusion of Ex Vivo Expanded Cord Blood Progenitor Cells Reduces the Risk of Bacteremia after Myeloablative Cord Blood Transplant

▪Background: Delayed hematopoietic recovery after myeloablative cord blood transplantation (CBT) is associated with higher infection-related morbidity and mortality when compared to recipients of conventional donor grafts. In particular, prolonged neutropenia, related to low cell doses provided by the cord blood (CB) graft, results in higher rates of infection in the first 100 days post-transplant. To overcome this issue, our group has explored ex vivo expansion of CB-derived CD34+ cells in the presence of Notch ligand. We have previously demonstrated that infusion of this expanded progenitor cell product leads to a significant reduction in the time to neutrophil engraftment in the myeloablative CBT setting [Delaney C, et al, Nat Med 2010]. We now hypothesize that faster neutrophil engraftment is associated with lower risk of bloodstream infections in the first 100 days post-transplant in patients receiving ex-vivo expanded progenitors (EXP) compared to patients receiving conventional CBT (CON).Methods: Between April 2006 and June 2014, a total of 93 consecutive patients were enrolled on multicenter studies coordinated by our Institution. Of these 93, 55 (59%) received single (n=9) or double (n=46) unmanipulated CB graft, while 38 (41%) patients received 1 or 2 unmanipulated CB units plus infusion of either a fresh partially HLA-matched (n=23) or a cryopreserved non-HLA matched (n=15) ex-vivo expanded CB unit. All patients received the same conditioning regimen of Fludarabine (75mg/m2), Cytoxan (120mg/kg) and TBI (13.2 Gy) over 8 days. GVHD prophylaxis consisted of cyclosporine and mycophenolate mofetil. Cox regression model was used to assess the risk of bacterial infection where "failure" was defined as the first occurrence of bloodstream infection within 100 days following CBT.Results: The median patient age was 22 years (range 0.6-45) and 26 (range 3-45) for CON and EXP group respectively (p=0.51); gender, race and CMV serostatus were not different between the 2 groups. Disease distribution was similar in the two groups, with most patients undergoing CBT for AML (n=47), ALL (n=28) and MDS (n=10). Median pre-thaw TNC/kg x107 was 4.97 (range 3.4-16.6) and 11.2 (range 2.7-30.4) for the CON and EXP groups respectively, and pre-thaw CD34 cells/kg x106 was 0.19 for the CON (range 0.04-0.98) and 6.3 for the EXP group (range 0.12-49.7). As expected, time to engraftment was significantly faster for patients receiving the EXP units at 17 days (range 6-41) vs. 25 days in the CON group (range 14-45, p<0.01). Only 1 case of graft failure was observed in the EXP group. The median time to first positive bloodstream infection in the EXP cohort was 36 days (range 3-98) vs. 8 days (range 1-88) in the CON group. The cumulative incidence of bacteremia was significantly lower in the EXP group (n=14) compared to the CON group (n=36) at 37% vs. 65% respectively (p=0.005, Figure 1). The hazard ratio (HR) of bacteremia episodes was significantly lower among patients receiving the expanded cells (HR 0.43; 95% CI 0.23-0.79; p=0.007). Among first documented bacteremic episodes, 5 of 50 (10%) were gram-negative rod bacteremias, all of which were in the CON group.Conclusions: Infections remain a significant risk for morbidity and mortality after myeloablative CBT and are the leading cause of early treatment related mortality. Our data suggest that there was a significant reduction in risk of bacteremia in the first 100 days post myeloablative CBT and a longer time to first infection when patients received ex vivo expanded progenitor cells, either as a partially matched fresh infusion or as an off-the-shelf, non-HLA matched expanded product. Infusion of the expanded cells resulted in reduced time to myeloid engraftment and offered a protective effect against episodes of bacteremia, but the exact mechanisms are presently unknown. It is possible that the expanded cells may home to regions of mucosal injury post TBI and mitigate breakdown thus reducing bacterial translocation. Further research to further define the expanded cells’ mechanism of action with regards to infection reduction is currently underway in animal models. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.