Improving the outcome of umbilical cord blood transplantation through ex vivo expansion or graft manipulation

Cytotherapy, 2015; 0: 1e9

Improving the outcome of umbilical cord blood transplantation through ex vivo expansion or graft manipulation MITCHELL E. HORWITZ1 & FRANCESCO FRASSONI2 1

Adult Blood and Marrow Transplant Program, Duke University School of Medicine, Durham, North Carolina, USA, and 2Department of Hemato-Oncology and Center for Stem Cell and Cell Therapy, Istituto G. Gaslini Children’s Hospital Scientific Institute, Genova, Italy Abstract The outcome of umbilical cord blood transplantation for adult patients with hematologic malignancies now rivals that of matched unrelated donor transplantation. However, relatively low lymphocyte and hematopoietic stem and progenitor cell dose is a source of significant morbidity and mortality. Multiple strategies are now being studied to overcome these limitations. One strategy involves ex vivo expansion of the umbilical cord blood unit before transplantation. Ex vivo expansion has the potential to increase the number of lymphocytes, committed progenitors and long-term repopulating hematopoietic stem cells. Increasing the numbers of lymphocytes and committed progenitor cells will address the issue of delayed hematopoietic recovery after umbilical cord blood transplantation. Increasing the hematopoietic stem cell content will improve the availability of adequately sized and matched cord blood units for transplantation. It may also eliminate the need for dual umbilical cord blood transplantation for those without an adequately sized single umbilical cord blood graft. The second strategy involves exposure of the umbilical cord blood graft to compounds aimed at improving homing and engraftment following transplantation. Such a strategy may also address the problem of slow hematopoietic recovery as well as the increased risk of graft failure. Many of these strategies are now being tested in late-phase multi-center clinical trials. If proven cost-effective and efficacious, they may alter the landscape of donor options for allogeneic stem cell transplantation.

Introduction Allogeneic hematopoietic stem cell transplantation using umbilical cord blood (UCB) is a highly effective alternative to stem cells collected from healthy adult donors. There are significant qualitative and quantitative differences in the UCB graft compared with the adult donor graft. Most important, the Tcell and CD34þ hematopoietic stem and progenitor cell dose is significantly lower. This is particularly significant for adult recipients. Furthermore, the majority of T cells in the UCB graft are of the naive phenotype. As a result, three adverse ramifications arise from these differences: (i) increased incidence of graft failure, (ii) delayed hematopoietic recovery and (iii) slow immune reconstitution. Although there remains room for improvement, the advent of dual UCB transplantation has, in large part, addressed the problem of excessive graft failure. Strategies such as intrabone marrow injection and co-infusion of T-cell-depleted, haplo-identical mobilized peripheral blood have addressed the issue of delayed

hematologic recovery [1e3]. However, these techniques remain investigational. Despite the remaining limitations, the outcome of UCB transplantation rivals that of mobilized peripheral blood or bone marrow transplantation from unrelated adult donors [4,5]. Resolution of the remaining limitations could increase the desirability of the UCB transplantation for adult recipients, perhaps even escalating it to a preferred graft source in patients without a fully matched family member. Addressing the problem of suboptimal lymphocyte and stem cell dose within the UCB graft through ex vivo expansion has met with both practical and technical challenges. Before the advent of dual UCB transplantation, cells for ex vivo culture initiation were derived from a small aliquot of the transplanted single UCB graft. With the cell dose already marginal in most adult recipients, this strategy was only appropriate for the limited number of adult recipients with an available UCB unit that exceeded the minimum cell dose requirement. Furthermore,

Correspondence: Mitchell E. Horwitz, MD, Adult Blood and Marrow Transplant Program, Duke University School of Medicine, 2400 Pratt Street, DUMC 3961, Durham, NC 27710, USA. E-mail: [email protected] (Received 2 December 2014; accepted 14 February 2015) ISSN 1465-3249 Copyright Ó 2015, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2015.02.004

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Table I. Strategies for ex vivo manipulation of UCB stem cells.

Compound (Reference) Notch ligand [48] MSC co-culture [24] NiCord [21] copper chelation (StemEx) [19] StemRegenin 1 [26] PGE-2 [28] Fucosylation [49]

Modality

Phase I/II completed (no. of patients)

Lead institution/sponsor

Ex vivo expansion Ex vivo expansion Ex vivo expansion Ex vivo expansion Ex vivo expansion HSC modulation HSC modulation

Yes (15) Yes (32) Yes (11) Yes (101) Yes (17) Yes (12) No

FHCRC/— MDACC/Mesoblast Duke University/Gamida-Cell Gamida-Cell/Teva University of Minnesota/Novartis DFCI/Fate Therapeutics MDACC/—

Active multicenter phase II/III Ongoing Ongoing Ongoing Completed In development Ongoing No

DFCI, Dana-Farber Cancer Institute; FHCRC, Fred Hutchinson Cancer Research Center; HSC, hematopoietic stem cell; MDACC, MD Anderson Cancer Center.

there was no definitive method for assessing the impact of the manipulated cell fraction, particularly the ex vivo expanded hematopoietic stem cell. These hurdles compromised early attempts at ex vivo expansion [6]. Before discussing the potential for ex vivo expansion of UCB stem cells, it might be interesting to consider what occurs in vivo after transplantation of bone marrowederived stem cells. A typical bone marrow graft contains 1  108 CD34þ cells. Assuming that the total content of healthy bone marrow consists of 1  1012 cells [7], of which 0.5e1.2% are CD34þ, we can conclude that CD34þ cells expand approximately 2 logs after transplantation. Given that UCB typically contains 1 log fewer CD34þ cells than a bone marrow graft, we can calculate an approximate 3 log in vivo expansion of UCB CD34þ cells. This reveals the formidable proliferative capacity of UCB CD34þ cells [8], which forms the basis for ex vivo expansion. The current challenge is to develop methods to exploit this proliferative capacity of lymphocytes and hematopoietic stem cells in an ex vivo culture system. Recent years have brought forth a number of promising technologies that are now being tested in earlyand late-phase clinical trials. If proven safe, effective and practical, these strategies may indeed change the landscape of allogeneic stem cell transplantation. Hematopoietic stem/progenitor cell manipulation Until the advent of dual UCB transplantation, the biggest impediment to development of novel hematopoietic stem cell expansion technologies was the ability to safely test and assess the approach in the clinical setting. The emergence of dual UCB transplantation has changed this, paving the way for innovative clinical trials of novel methods of cord blood graft engineering (Table I) [9,10]. As long as the patient receives a single UCB unit of adequate size, a second experimentally manipulated graft can be ethically co-infused and assessed for efficacy.

Multiple strategies of ex vivo UCB manipulation have emerged. If committed hematopoietic progenitor cells are targeted for expansion, the period of bone marrow aplasia that complicates intensive chemotherapy can be reduced or eliminated. However, a second, unmanipulated UCB graft will always need to be co-infused to provide long-term hematopoietic recovery. If long-term repopulating cells are targeted for expansion, the manipulated graft can be expected to provide both short-term and longterm hematopoiesis, thereby eliminating the need for transplantation of a second unmanipulated unit. Another strategy aims not to expand hematopoietic stem/progenitor cells but to provide them with a homing and engraftment advantage. This advantage could both improve kinetics of engraftment as well as reduce the relatively high risk of graft failure that continues to plague the field of UCB transplantation. Notch-mediated expansion Since the early 1990s, the Notch gene family has been implicated in modulation of early hematopoietic stem cell fate [11]. In vitro studies suggested that activation of Notch signaling promotes retention of the most primitive hematopoietic stem cell phenotype during culture. This led investigators at the Fred Hutchinson Cancer Research Center in Seattle, Washington, to develop a serum free ex vivo hematopoietic stem cell culture system consisting of immobilized Delta1 Notch ligand along with earlyacting hematopoietic stem cell cytokines (stem cell factor, thrombopoietin, Flt3 ligand, interleukin (IL)3 and IL-6) [12]. Of note, cultures were initiated with CD34þ cells isolated from the UCB unit. The final expanded product contained no T cells. In the context of a phase I trial using a myeloablative dual UCB transplant configuration, patients received one unmanipulated UCB unit, and a second unit that was expanded for approximately 16 days in the presence of Notch ligand and cytokines. CD34þ hematopoietic stem-progenitor cells expanded a mean 164-fold during the culture period. In a report

Ex vivo expansion or graft manipulation describing the outcome of the first 10 patients treated on the trial, patients received an expanded cord blood graft containing an average of 6  106 CD34þ cells/kg. This compared with 0.24  106 from the unmanipulated cord blood graft. The large number of hematopoietic progenitor cells contained within the expanded cord blood graft resulted in a median time to neutrophil engraftment (ANC >500) of 16 days (range 7e34 days). This compared with 26 days for a historical control population that received standard myeloablative dual cord blood transplantation. Peripheral blood chimerism analysis confirmed that the expanded cord blood unit was responsible for early neutrophil engraftment. However, by 3 months after transplantation, hematopoiesis was derived by the unmanipulated cord blood unit in all but one of the evaluable subjects. Although it is possible that transient hematopoiesis from the expanded UCB graft is a consequence of loss of longterm repopulated cells during ex vivo culture, it is more likely that the T-cell-replete, unmanipulated unit mounted a cellular immune response leading to rejection of the expanded graft [13,14]. Because of the logistical complexity associated with production of a patient-specific expanded cord blood graft, Delaney and colleagues are currently assessing the safety and efficacy of an “off-the-shelf” graft. With this strategy, cord blood transplant recipients receive an unmanipulated single or double UCB transplantation as per standard of care. In addition, the patient receives a fully human leukocyte antigenemismatched third UCB graft that was previously expanded and cryopreserved [15]. The intent is for this mismatched third graft to provide early neutrophil recovery after which it is rejected by the incoming unmanipulated, T-cell-replete UCB graft. This approach is currently being studied in the context of a multi-center phase II trial (Clinicaltrials. gov NCT01175785). StemEx copper chelator expansion On the basis of pre-clinical evidence that low concentrations of copper prevents stem cell differentiation in an in vitro culture system, Gamida Cell developed StemEx, which combines the copper chelator tetraethylenepentamine with early- and lateacting hematopoietic cytokines [16,17]. The StemEx UCB graft is derived from a single UCB unit. A fraction of the unit (contained in a separate segment of the cryobag) is thawed and expanded for 21 days in the presence of tetraethylenepentamine. On the day of transplantation, the recipient receives the expanded fraction along with the unmanipulated fraction. StemEx was first studied and found to be safe and feasible in a phase I/II trial performed at the

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M.D. Anderson Cancer Center [18]. On the basis of this early experience, Gamida Cell launched a multinational phase II/III registration trial designed to test the hypothesis that more rapid neutrophil engraftment facilitated by StemEx would positively affect survival at 100 days after transplantation. The study accrued 101 subjects with high-risk leukemia and lymphoma from 25 stem cell transplant centers in the United States, Europe and Israel. Study patients were compared with a contemporaneous control group from the Center for International Blood and Marrow Transplant Research. The control population received myeloablative unmanipulated dual UCB transplantation between 2006 and 2010. The cumulative incidence of engraftment failure was 8.1% for the StemEx group and 14.5% for the controls (P ¼ 0.086). The median time to neutrophil engraftment (ANC >500) was 21 days in the StemEx group and 28 days in the control group (P < 0.0001). The survival at 100 days for StemEx recipients was 84.2% compared with 74.6% for conventional dual UCB transplant recipients (P ¼ 0.035) [19]. The study suggests that when compared with a registry-based retrospective patient cohort, transplantation of the StemEx single UCB graft results in improvement in a number of important clinical end points. Furthermore, Gamida Cell confirmed feasibility of conducting and completing a large, multinational cell therapy trial. NiCord expansion NiCord is an ex vivo expanded cell product from an entire unit of UCB that utilizes a small molecule, nicotinamide, as an epigenetic approach to inhibit differentiation and to increase the functionality of hematopoietic stem and progenitor cells expanded in ex vivo cultures. When nicotinamide is added to stimulatory hematopoietic cytokines, UCB-derived hematopoietic progenitor cell cultures demonstrate an increased frequency of phenotypically primitive CD34þCD38-cells. The cells also demonstrate increased migration toward stromal cell derived factore1. In a pre-clinical murine transplant model, they home to and engraft in the bone marrow with higher efficacy [20]. The unique feature of the NiCord UCB graft is that it consists of a hematopoietic stem-progenitor cell fraction that has been expanded for 21 days along with a non-cultured T-cell-containing fraction that is collected and re-frozen following thaw. Thus, the NiCord graft retains immunologic potency to augment both engraftment and immunologic recovery. The phase I study of NiCord commenced in 2010 at Duke University in Durham North Carolina

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Figure 1. Neutrophil engraftment in NiCord-engrafted patients compared with unmanipulated dual UCB transplantation.

and Loyola University in Chicago Illinois. Eligible patients had high-risk hematologic malignancies and were candidates for dual UCB transplantation. One unit was selected for NiCord expansion and the other to be infused without manipulation. Recipients were conditioned with total body irradiation (1350 cGy) and fludarabine with or without cyclophosphamide. Key end point measures were compared with a historical control cohort of patients who received myeloablative conditioning followed by an unmanipulated dual UCB transplantation. Eleven patients received the NiCord graft on this study. Full or partial neutrophil and T-cell engraftment derived from NiCord was observed in eight patients. With a median follow-up of 24 months, NiCord engraftment has remained stable in all patients. Two additional patients engrafted with the unmanipulated unit. Patients engrafting with NiCord achieved significantly earlier neutrophil recovery (median of 11 days versus 25 days, P ¼ 0.001) (Figure 1) and platelet recovery (30 versus 41 days, P ¼ 0.012) compared with controls [21]. This study confirms the presence of both longterm and short-term repopulating cells within the NiCord expanded cord blood graft. With this knowledge, Gamida Cell has launched and follow-up trial in which recipients receive myeloablative conditioning followed by a single NiCord expanded graft (Clinicaltrials.gov NCT01816230). To address practical and logistical concerns surrounding the transport of a fresh cell therapy product, the graft will be cryopreserved following expansion and before delivery to the transplant center.

Mesenchymal cell co-culture (Mesoblast) Ex vivo expansion of unfractionated, previously cryopreserved UCB is extremely challenging. Initiation of the culture with a purified population of CD34þ cells significantly enhances the expansion efficiency but also adds to the complexity of the assay [22]. To avoid CD34þ cell selection, McNiece and colleagues developed a culture system using mesenchymal stomal cells (MSCs) [23]. It is hypothesized that both contact-dependent and soluble factors provided by the MSCs more closely resemble the bone marrow micro-environment. Refinement of this technique ultimately led to development of a Good Manufacturing Practiceegrade mesenchymal precursor product that was brought to the clinic by the group at M.D. Anderson Cancer Center. The 14-day UCB expansion process consists of co-culture with the adherent MSCs for 7 days in the presence of hematopoietic cytokines and an additional 7 days in the presence of cytokines alone. Thirty-one adults with hematologic malignancies were treated with a dual UCB transplantation. The conditioning regimen was myeloablative, consisting of melphalan, thiotepa, fludarabine and rabbit antithymocyte globulin. One of the units was infused without manipulation and the other following expansion as described earlier. The outcome of these patients was compared with that of 80 historical controls who received a standard myeloablative dual UCB transplantation and whose outcome was reported to the Center for International Blood and Marrow Transplant Research data registry [24]. The

Ex vivo expansion or graft manipulation cultured unit expanded by a median factor of 12.2 and CD34þ cells were expanded by a factor of 30.1. The expanded unit provided a median CD34þ cell dose of 0.95  106/kg recipient body weight. In comparison, the unmanipulated unit provided a median CD34þ cell dose of 0.38  106/kg. The median time to neutrophil recovery was 15 days (range 9e42), and the median time to platelet recovery was 42 days (range 15e62). This compared favorably with the Center for International Blood and Marrow Transplant Research registry cohort, which engrafted neutrophils on median day 24 (range, 12e52) and platelets on median day 49 (range 18e264). Peripheral blood chimerism analysis performed in the peri-engraftment period demonstrated presence of the expanded unit in 46% of the subjects. By 6 months, the expanded unit was detectable in 13% of the subjects. By 1 year after transplantation, hematopoiesis was provided by the unmanipulated unit in all the subjects. HSC835 Aryl hydrocarbon Boitano and colleagues screened a library of 100,000 compounds resulting in identification of a purine derivative (StemRegenin 1 [SR1]) with marked ability to expand human CD34þ hematopoietic stem and progenitor cells ex vivo [25]. For this technology, a purified population of CD34þ cells is used to initiate the cell culture. During a 3-week culture in serum free media containing SR1 supplemented with thrombopoietin, stem cell factor, flt3 ligand and interleukin-6, the CD34þ cells expand 1118-fold relative to input cells. Removal of SR1 from the culture system led to rapid differentiation, highlighting the importance of SR1 in the culture system. Cultured cells also produced high-level engraftment in immunodeficient mice. A 3-week expansion of 300 UCB CD34þ cells provided a comparable level of human engraftment in NSG mice as 10,000 uncultured UCB CD34þ cells. These results demonstrate that SR1 promotes expansion of UCB CD34þ cells that contribute to both early and sustained engraftment. The first clinical results were presented at the 2014 annual meeting of the American Society of Hematology [26]. The phase I/II study enrolled recipients of myeloablative (TBI/fludarabine/cyclophosphamide) UCB transplantation. The first 15 patients received both an SR1-expanded and an unmanipulated UCB graft. An additional two patients received a single SR1-expanded graft. Recipients also received the CD34e, T-cell-containing fraction of the expanded UCB unit, similar to what was done in the NiCord trial. The SR1-expanded graft contained a median 12.3  106 CD34þ cells/

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kg (range 2.3e48.5  106/kg). This compares to recipients of standard double UCB graft without manipulation (n ¼ 111) who received a median of 0.5  106 CD34þ cells/kg (0.1e2.1). Eleven of the 17 patients demonstrated a predominance of the SR1-expanded unit. The remainder showed predominance of the unmanipulated unit. Neutrophils recovered at a median of 11 days [6e23] for those engrafting with the SR1 expanded unit compared with 23 days [14e30] for those in whom the unmanipulated unit predominated. Engraftment of the SR1 expanded unit has been stable, with the longest follow-up being 2 years. The SR1 expanded graft, now called HSC835 (Novartis Pharmaceuticals) continues to be studied in the context of single myeloablative UCB transplantation at the University of Minnesota (Clinicaltrials.gov NCT01930162). Prostaglandin e2 co-culture The previously discussed technologies are aimed at addressing the low stem cell content of the UCB by ex vivo expansion of both short-term and long-term hematopoietic progenitor cells. In contrast, the prostaglandin e2 co-culture methodology was conceived as an inexpensive, safe and practical method for augmenting the homing and engraftment potential of UCB stem cells without expansion. The method uses 16,16-dimethyl prostaglandin E2 (dmPGE2) previously identified in a zebra fish stem cell model to be a critical regulator of hematopoietic stem cell homeostasis [27]. From this pre-clinical work, it was hypothesized that brief ex vivo exposure of UCB to dmPGE2 could improve patient outcomes by increasing the “effective dose” of hematopoietic stem cells without significant toxicity. This hypotheses was tested in a phase I study conducted at the Dana Farber Cancer Institute in Boston. The investigators enrolled recipients of nonmyeloablative dual UCB transplantation. One unit was infused after brief exposure to dmPGE2 and the other was infused without manipulation. Two cohorts of patients were enrolled. In cohort 1, the investigational unit was incubated with 10 mmol/L of dmPGE2 for 60 min at 4 C (9 patients). Cohort 2 consisted of 12 patients in whom the larger of the 2 UCB units was incubated with the same concentration dmPGE2, but for 120 min instead of 60 min and at 37 C instead of 4 C. Although patients in cohort 1 experienced no safety concerns, there was no demonstrable benefit of the dmPGE2 treated unit. However, the optimized co-culture conditions used for patients in cohort 2 resulted in more rapid median time to neutrophil engraftment compared with historical controls (17.5 days versus 21 days, P ¼ 0.45). Furthermore, 10 of

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12 patients showed complete and sustained engraftment of the dmPGE2 treated units. Although it is possible the engraftment advantage afforded to the manipulated unit is a consequence of it being the larger of the two infused grafts, it is also possible that co-culture with PGE2 did indeed improve homing of the resident stem cells. No safety concerns emerged from this small phase I trial [28]. On the basis of the promising results emerging from this trial, the technology is now being studied in a randomized phase II trial supported by Fate Therapeutics (Clinicaltrials. gov NCT01627314).

This defect in bone marrow homing may, in part, be due to lack of binding to the critical adhesion molecules P- and E-selectin which are expressed on bone marrow endothelial cells [32]. Fucosylation of selectin ligands expressed on UCB stem cells increases affinity to P- and E-selectin. Thus, in preclinical models of UCB transplantation, engraftment of UCB stem cells into immunodeficient mice can be improved with pre-transplant ex vivo fucosylation [33,34]. This technique has now been translated to the bedside and is being tested in an ongoing phase I study at the M.D. Anderson Cancer Center (Clinicaltrials.gov NCT01471067).

C3a complement co-culture Complement fragment 3a (C3a) is a product of proteolytic cleavage of complement protein C3. Along with myriad immunoregulatory properties, C3a also sensitizes human hematopoietic stem and progenitor cells to homing properties of stromal derived factor 1 (SDF-1) through binding of the C3a receptor present on the cell surface. Pre-clinical murine transplant models demonstrated that incubation of hematopoietic stem cells with C3a before transplantation into lethally irradiated hosts accelerates engraftment kinetics [29,30]. On the basis of these promising pre-clinical data, a phase I/II study was performed at the University of Minnesota to assess safety and efficacy of C3a in promoting engraftment of UCB stem cells [31]. Adults receiving non-myeloablative dual UCB transplantation were eligible for the trial. One of the two UCB grafts was incubated at room temperature with Good Manufacturing Practiceegrade C3a before transplantation. Twenty-nine patients were treated on the study. Mild infusion-related side effects were observed after infusion of the C3a-primed units. Because the patients were conditioned with a non-myeloablative regimen and therefore experienced early autologous reconstitution, the impact of C3a on engraftment kinetics could not be assessed. Of the 27 patients who achieved neutrophil engraftment, nine had hematopoiesis derived from the C3aprimed unit and 18 from the unmanipulated unit. Thus, C3a treatment did not appear to provide an engraftment advantage. Fucosylation Like dmPGE2 and C3a co-culture, ex vivo fucosylation is a technique under development that is designed to augment homing of UCB stem cells to the bone marrow stroma. The rationale for this strategy hinges on the fact that UCB stem cells do not home as avidly to the bone marrow as those from adult bone marrow or mobilized peripheral blood.

UCB T-cell expansion Although the bulk of this review is dedicated to efforts to expand cord blood stem and progenitor cells, it is also important to acknowledge the initiative to expand cord blood T cells for the purpose of immune modulation. The therapeutic benefit of donor lymphocyte infusions after allogeneic stem cell transplantation is well established but is currently not an option for UCB transplant recipients because the donor is anonymous. The clinical need for donor cellular immune therapy is, in some respects, even more acute after UCB transplantation. First, there is the problem of a relative low number of T cells in the initial UCB inoculum. This, along with the naive phenotype of these cells explains the observed delay in immune recovery compared with recipients of adult donors grafts [35,36]. Despite the lower T-cell dose and slower immune reconstitution, the rate of post-transplant relapse does not appear to be appreciably higher after UCB transplantation [37]. However, relapse remains a major source for treatment failure and unlike recipients of adult donor transplantation, mature immunologically potent donor lymphocytes are not readily available for posttransplant immunotherapy. Thus, efforts are underway to address this deficiency. The link between cord blood donor and the cord blood graft is broken once a unit is donated to a public cord blood bank. Therefore, the only source for cord bloodederived post-transplant cellular therapy is residual cells left over from the original graft. Most cord blood units are stored in bags with small aliquots of cells that are contained in detached segments. This allows for additional testing to be done on the unit before it is thawed for transplantation. Unused detached segments could therefore be used for therapeutic purposes if expansion were deemed feasible. Alternatively, a small aliquot of the cord blood graft could be retained for expansion and transplantation at a later date.

Ex vivo expansion or graft manipulation Expansion of cord blood derived T-lymphocytes Berglund and colleagues from the Karolinska Institute in Sweden retained 5% of the cord blood inoculum for purposes of expanding donor T cells. The investigators used a clinical grade UCB T-cell culture system containing CD3/CD28 paramagnetic beads and media containing interleukin-2 and interleukin-7 [38,39]. This system allowed for expansion of polyspecific CD3þ T cells facilitating a donor lymphocyte infusion (DLI) dose of 1  106/kg recipient body weight. In a pilot phase I study, four patients received a cord bloodederived DLI at a median 87 days (range 48e137 days) after UCB transplantation. Safety was demonstrated in that there were no infusion-related toxicities and there was no clinically significant graft-versus-host disease (GvHD) arising as consequence of the DLI. Clinical activity of the cell product awaits larger study. Ex vivo development of virus-specific T cells is another of post-transplant immune modulation technique that is showing great promise. Developing such cells from virus-naive UCB T cells is particularly challenging. Pre-clinical studies using a variety of strategies suggests feasibility of such an approach [40e42]. A phase I study testing one of these strategies is ongoing at the Baylor College of Medicine.

Expansion of regulatory T cells GvHD remains the most vexing complication of allogeneic stem cell transplantation, regardless of the graft source. Up to 40% of UCB recipients experience acute and/or chronic GvHD. GvHD risk is augmented in recipients of double UCB transplantation [43]. Regulatory T cells (Tregs) are a subset of T cells involved in immunologic homeostasis. Pre-clinical models of stem cell transplantation suggest that Tregs given as a cellular therapy could be exploited to prevent or treat GvHD [44e46]. Investigators from the University of Minnesota have undertaken a strategy of ex vivo expansion of naturally occurring Tregs isolated from an UCB unit [47]. Recipients of non-myeloablative dual UCB transplantation were eligible for a phase I dose escalation study to assess safety and feasibility. The starting cell population consisted of CD25þ cells isolated from a third UCB unit. Stem cells from this third unit were not used for transplantation. These cells were cultured ex vivo for 18 days in the presence of anti-CD3/anti-CD28-coated beads and IL-2 and transfused into the patient on day þ1 of the UCB transplantation. Twenty-three subjects received a Treg product (CD4þ CD127- FoxP3þ) ranging from 0.1 to 30  105 cells. The infused Tregs, identifiable by their unique HLA type, were

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detectable in the peripheral blood for up to 14 days after the infusion. There were no demonstrable infusion-related or other serious complications attributable to the Treg infusion. The ability for this cell therapy to attenuate GvHD without compromising anti-tumor or anti-infective properties of the cellular immune system will need to be confirmed in a larger, ongoing trial at the University of Minnesota (Clinicaltrials.gov NCT00602693). Conclusion In the early 2000s, the advantages of UCB transplantation were only being appreciated in the pediatric population where outcomes were comparable to that of adult unrelated donor transplantation. Since then, a better understanding of the minimum safe transplantable cell dose, improved quality and characterization of the UCB inventory and the advent of dual UCB transplantation has broadened the applicability to the adult population. However, limitations remain, and chief among them is the kinetics of hematopoietic recovery following UCB transplantation. Ex vivo manipulation of the UCB graft before transplantation holds great promise in addressing these limitations. Clinical data are already available from multiple techniques demonstrating more rapid neutrophil and platelet recovery. Short-term safety has also been demonstrated. Confirmation of longterm safety and reduction in the graft failure rate will require longer follow-up and larger studies. With the development of improved techniques for the transplantation of haplo-identical related donor grafts, the major challenge going forward will be demonstration of cost-effectiveness and practicality of ex vivo UCB stem cell manipulation. Disclosure of interest: Mitchell Horwitz has received research support from, and has served as a consultant for Gamida Cell Ltd. Francesco Frassoni has no commercial, proprietary or financial interest in the products or companies described in this article. References [1] Frassoni F, Gualandi F, Podesta M, Raiola AM, Ibatici A, Piaggio G, et al. Direct intrabone transplant of unrelated cord-blood cells in acute leukaemia: a phase I/II study. Lancet Oncol 2008;9:831e9. [2] Brunstein CG, Barker JN, Weisdorf DJ, Defor TE, McKenna D, Chong SY, et al. Intra-BM injection to enhance engraftment after myeloablative umbilical cord blood transplantation with two partially HLA-matched units. Bone Marrow Transplant 2009;43:935e40. [3] Liu H, Rich ES, Godley L, Odenike O, Joseph L, Marino S, et al. Reduced-intensity conditioning with combined

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