Preclinical optimization and validation
Our earlier published studies utilized enriched CD34+
CB progenitors as the starting cell population for Notch-mediated expansion7
. To limit cell separation procedures, we first determined whether isolation of the CD38−
subset of CD34+
cells was required. We compared growth characteristics and generation of SCID repopulating cells (SRC) of CD34+
CB cells. Cells were cultured for 17–21 days in the presence of fibronectin fragments and immobilized engineered Notch ligand (Delta1ext-IgG
) or control human IgG in serum free conditions supplemented with cytokines (SCF 300 ng ml−1
, Flt3L 300 ng ml−1
, TPO 100 ng ml−1
, IL–6 100 ng ml−1
, and IL–3 10 ng ml−1
, denoted as “5GF”). Delta1ext-IgG
consists of the extracellular domain of Delta1 fused to the Fc domain of human IgG1. We observed no significant difference in absolute numbers of CD34+
cells generated, with a CD34+
cell fold expansion of 138 ±64 and 163±64, (mean±sem, p
=0.1612, data not shown) for the CD34+
versus the CD34+
starting cell population, respectively. Assessment of in vivo
NOD/SCID repopulating ability at 3, 6 and 10 weeks post infusion revealed enhanced human engraftment in the marrow of recipient mice when a CD34+
compared to CD34+
starting cell population was used (mean CD45% in CD34+
starting cell populations cultured in the presence of Delta1ext-IgG
: 3 weeks; 6.7% versus 1.6%, p=0.02 and 10 weeks: 1.0% vs. 0.2%, p
=0.1). We further determined that the 5GF combination was superior to use of fewer cytokines for in vitro
generation of CD34+
cells and SRC frequency determined by limiting dilution analysis (Supplementary Fig. 1
We evaluated multiple, closed system tissue culture bags, tissue culture and non-tissue culture treated flasks for ligand binding and growth of cells (see Methods). We also evaluated commercially available clinical grade serum-free media. Based on in vitro
generation of CD34+
cells and NOD/SCID repopulating ability, StemSpan SFEM media and X-fold tissue culture bags (Baxter) and Nunc flasks were found to be superior (data not shown). Details regarding the methods for large scale production of cGMP engineered Delta1ext-IgG
can be found in Supplementary Methods
Reproducibility of this optimized culture system under cGMP conditions was validated in 19 runs using CB units previously cryopreserved as part of the NHLBI Cord Blood Transplantation (COBLT) Study (obtained via the NHLBI repository). We observed consistent growth, averaging greater than 150 fold expansion of CD34+ cells regardless of the starting purity which ranged from 19 to 95% (mean 64% ± 5.2%, sem), with the low 19% purity value being an outlier (). All validation run experiments resulted in detectable engraftment of human CD45+ cells in immunodeficient mice.
Preclinical Full-Scale Validation Runs (N=19)
We further evaluated the functional properties of cells generated using CD34+
cells selected from pooled fresh CB and cryopreserved until use. We found a 222 ± 57 (mean ± sem, n
=5) fold expansion of CD34+
cells cultured in the presence of Delta1ext-IgG
versus a 68±30 fold expansion for cells cultured in the presence of control IgG (p
=0.006). Repopulating ability and SRC frequency was significantly higher in mice receiving cells cultured with Delta1ext-IgG
<0.0001) and included both myeloid and lymphoid cells (). The observed increase in overall human hematopoietic reconstitution at 9 versus 3 weeks after culture with Delta1ext-IgG
resulted from an increase in engrafted lymphoid cells. Myeloid engraftment decreased somewhat, suggesting at least a portion of the starting engrafting cells were short term in nature. However, the continued presence of at least some longer-term repopulating cells after ex vivo
culture was suggested by secondary transplantation of whole marrow from NOD/SCID IL-2Rγnull;
mice transplanted 17–22 weeks prior with expanded cells that revealed human engraftment (defined as ≥0.5% human CD45+
cells) in the marrow of 7/11 secondary recipient mice, also NOD/SCID IL-2Rγnull;
mice (Supplementary Fig. 2
). Moreover, SRC frequency was found to be markedly enhanced with limit dilution analysis demonstrating a 15.6 fold increase in SRC frequency in mice that received Delta1ext-IgG
-cultured cells as compared to those receiving non-cultured cells at 3 weeks post transplant (p
=0.0001) and a 6.2 fold increase in SRC frequency at 9 weeks (p
=0.0001) (). To our knowledge, this is the highest reported expansion of human SRC frequency reported to date 9–11
. Furthermore, the fold-increase in repopulating cells revealed in these limit dilution assays suggest that the observed increase in overall human engraftment in mice receiving Delta1-cultured cells resulted mainly or completely from the generation of repopulating cells as a direct result of culture with Notch ligand.
Culture of CD34+ CB progenitors with Notch ligand results in enhanced and more rapid human engraftment as well as increased SRC frequency
SRC Frequency Determined by Limiting Dilution Analysis
Critical to future clinical efficacy of this approach was whether more rapid engraftment could be observed after Notch-mediated expansion. In three independent experiments, measurable human engraftment at 10 days post transplant (defined as ≥0.5% human CD45+ cells in the marrow of recipient mice) was seen in all mice receiving Delta1-cultured cells, whereas no mice receiving non-cultured CB cells showed human engraftment. This engraftment consisted of > 95% myeloid cells as measured by co-expression of the human antigens, CD33 and CD45 ( and data not shown), demonstrating the ability of Notch-expanded CB progenitors to provide markedly accelerated hematopoietic repopulation in this animal model system.
Proof of principal: preliminary results of phase I trial
A phase I trial involving transplantation of a non-manipulated unit along with CB progenitors from a second CB unit that have undergone Notch-mediated ex vivo expansion is ongoing. The primary objective is evaluation of safety of infusing the ex vivo expanded CB progenitors, while secondary objectives include evaluation of the kinetics and durability of hematopoietic reconstitution and the relative contribution to engraftment as provided by the expanded and non-manipulated CB units.
To date, we have enrolled ten patients with high risk acute leukemias in morphologic remission at the time of transplant (Supplementary Table 1
) with a median age of 27.5 years (range, 3 to 43) and median weight of 61.5 kilograms (range, 16 to 79). Based on studies by Wagner and colleagues at the University of Minnesota demonstrating safety of double CB unit infusion, patients receive a myeloablative preparative regimen (1320 cGy TBI, 120mg/kg cytoxan and 75mg/m2
Fludarabine) followed by infusion of one non-manipulated and one ex vivo
expanded CB graft. CB donors are chosen on the basis of cell dose and HLA, with all units being ≥ 4/6 matched to the recipient (intermediate resolution at HLA-A and B, and high resolution at DRB1) and at least 3/6 matched to each other (Supplementary Table 1
). Additional criteria for donor CB selection include a minimum requirement of a total nucleated cell (TNC) dose in the non-manipulated graft of at least 2.5 × 107
TNC/kg (based on pre-cryopreservation numbers), independent of the match grade (HLA) of the unit to the recipient. All patients receive prophylaxis for graft-versus-host-disease (GVHD) consisting of cyclosporine and mycophenolate mofetil beginning on day –3.
Sixteen days prior to the stem cell infusion date, we thawed the unit selected for ex vivo
expansion, selected for CD34using the Isolex 300i and initiated cultures as described above. Cultures were expanded as required to maintain cell densities at ≤1×106
total cells ml−1
. On the day of transplant, the cultures were harvested and infused 4 hours after infusion of a non-cultured, unrelated donor CB graft. At harvest, there was an average fold expansion of CD34+
cells of 164 (±48 s.e.m, range 41–471) and an average fold expansion of total cell numbers of 562 (±134 s.e.m, range 146–1496) (). Additional details of the final harvested product can be found in the supplemental data (Supplementary Tables 2 and 3)
. Of note, there were no mature T cells infused with the expanded graft. Despite cell losses that occur with CD34 cell selection, in all cases the absolute number of CD34+
cells at the end of culture greatly exceeded the pre-cryopreservation CD34 cell number. The infused CD34+
cell dose derived from the expanded CB graft averaged 6 ×106
CD34/kg (range 0.93 to 13 ×106
) versus 0.24 ×106
CD34/kg (range 0.06 to 0.54 ×106
= 0.0004) from the non-manipulated CB graft. There was no significant difference in the average TNC/kilogram dose infused between the non-manipulated and expanded cell grafts.
Clinical grade culture of CB progenitors with Delta1ext-IgG results in significant in vitro expansion of CD34+ cells and more rapid neutrophil recovery in a myeloablative double CBT setting
The kinetics of hematopoietic recovery and the relative contribution of the expanded and non-manipulated CB grafts to engraftment were determined beginning 7 days post transplant. Time to ANC ≥500 μl−1
was evaluable in nine out of ten patients in whom time to achieve an ANC of ≥500 μl−1
was shortened significantly, with a median time of 16 days (range, 7–34 days). This compares quite favorably with a median time of 26 days (range, 16–48 days; p
= 0.002) in a concurrent cohort of 20 patients undergoing double CB transplantation at our institution with identical conditioning and post-transplant immunosuppressive regimen (). This cohort also did not differ significantly in age, weight, diagnosis or infused cell doses as provided by the non-manipulated units (Supplemental Table 1
). Furthermore, prior publications suggest that an ANC threshold of >100 μl−1
is strongly associated with a survival benefit post allogeneic stem cell transplant12
. Among enrolled patients median time to achieve an ANC >100 μl−1
was 9 days versus 19 days in the conventional setting (as above) (p
=0.006, data not shown). One patient experienced primary graft rejection. Of note, we have observed no infusional toxicities or other safety concerns. Average follow-up time for this set of 10 patients is currently 354 days (range 77–806), and 7 out of 10 patients remain alive with no evidence of disease, and sustained complete donor engraftment. Acute grade II GVHD has been observed in all evaluable patients, except for one who had overall grade III acute GVHD. All patients responded to therapy. No chronic extensive GVHD has been observed and three patients have been diagnosed with chronic limited GVHD.
Contribution to donor engraftment by the expanded or non-manipulated grafts was determined weekly in the first month beginning at day 7 post-transplant on peripheral blood sorted cell fractions. In eight of the nine engrafted patients there were sufficient numbers of peripheral blood sorted myeloid cells for evaluation and in each of these patients revealed a predominance of donor cell engraftment derived from the expanded cell graft in both the CD33 and CD14 cell fractions (). The one patient that was not evaluable at day 7 due to an insufficient quantity of cells had concurrent reactivation of HHV6, a virus that has been shown to inhibit progenitor cell proliferation 13
. This patient was also the only patient with neutrophil engraftment occurring after day 21 post transplant and the only engrafted patient with no contribution to engraftment observed from the expanded graft.
In four patients, an ANC of ≥500 μl−1 was attained at a time when the predominant contribution to myeloid engraftment was from the expanded cell graft (81, 96, 100 and 100% contribution to sorted CD33 and CD14 cell fractions (, left column). Three of these four patients reached an ANC of ≥500 μl−1 exceptionally early (7, 7 and 9 days), with the fourth at day 16. However, in the remaining four patients who all engrafted early (≤21 days), ANC ≥500 occurred at a time when contribution to myeloid engraftment was derived from the non-manipulated graft. This may suggest a potential facilitating effect exerted by the ex vivo expanded cells on the non-manipulated CB graft (, right column).
We observed longer-term in vivo persistence of the expanded cell graft in two patients. In one patient, analysis at day 240 post transplant revealed a portion (10–15%) of the donor CD14, CD56 and CD19 cells were derived from the expanded graft but was no longer present by one year. In the second patient at day 180 post transplant, contribution to engraftment from the expanded cell population at day 180 post transplant in CD33, CD14, CD56 and CD19 cells ranged from 25 to 66% of total donor engraftment (). However, the expanded graft did not contribute to CD3+ cell engraftment.
Long-term Persistence in Patients 2 and 6: Percent Peripheral Blood Cells Derived from the Expanded Cell Graft
Our prior reports demonstrated that expansion of mouse and human HSPC can result from constitutive activation of Notch signaling, and subsequent studies used recombinant ligands to activate endogenous Notch receptors, with the goal of enhancing ex vivo
generation of non-genetically altered hematopoietic repopulating cells for therapeutic intent 5–8
. Here we have shown that Notch-mediated expansion of CB stem/progenitor cells results in a dramatic expansion of hematopoietic precursors capable of rapid multi-lineage in vivo
NOD/SCID reconstitution, while possibly retaining longer-term repopulating ability. Moreover, preliminary results from our Phase I trial provide the first evidence that a key regulator of cell fate, the Notch pathway, can be used in a clinically compliant manner to generate a safe and potentially efficacious cell therapy product.
Significantly enhanced rates of myeloid engraftment have been seen, with an overall median time to neutrophil recovery (ANC 500 μl−1
) of 16 days, with six of nine patients achieving neutrophil engraftment between 7 and 17 days, faster than would be expected using 2 non-manipulated units (median time 23 to 26 days or longer in the published literature) 14,15
. Of note, early (day 7) myeloid engraftment was derived almost entirely from the expanded cell graft (). In four patients the expanded cell graft remained the dominant donor graft when an ANC ≥500 μl−1
was attained early (≤16 days) post-transplant. Furthermore, all but one of the remaining patients achieved an ANC of ≥500 μl−1
by day 21 post-transplant, despite loss of contribution to engraftment from the expanded cell graft. This is highly suggestive of a facilitating effect of the cultured cells in promoting engraftment from the non-manipulated CB unit, an hypothesis currently being explored.
The expanded cell population may also have retained a portion of longer-term repopulating stem/progenitor cells as suggested in the two patients with in vivo
persistence of cultured donor cells. The lack of in vivo
persistence in the remaining patients could either be due to loss of stem cell self-renewal capacity during culture, or to immune mediated rejection. It has been well documented that in most patients who receive two non-manipulated units for CBT, only one unit contributes to persistent long-term engraftment14,16,17
. The mechanisms responsible for this single donor dominance remain ill-defined; however, one possibility is immune mediated rejection of the losing unit by the winning graft. If this is correct, even if longer-term repopulating cells were present after culture of CB progenitor cells in the presence of Notch ligand, immune cells present in the non-manipulated unit would be expected to reject the expanded cell graft, which itself does not contain either mature or newly generated T cells. This hypothesis is further supported by recent studies demonstrating an IFN-γ response by CD8+
T cells in the “winning” unit against the rejected one in patients undergoing double cord blood unit transplantation20
. Included in these studies were four patients in the present trial which showed an IFN-γ response by cells derived from the non-cultured cord blood unit in response to cells derived from the cultured unit in three of the patients and in whom only cells from the non-cultured unit survived long-term. In the fourth patient, failure to detect an IFN-γ response was associated with persistence of the cultured as well as non-cultured unit. Consequently, persistence of long-term engrafting potential retained within the expanded progenitor cell population was not expected and the >180 day in vivo
persistence of progeny derived from the cultured cells observed in the two CBT patients despite these barriers was surprising, but consistent with our pre-clinical data in immunodeficient mice in which ex vivo
expanded CB progenitors proved capable of secondary transplantation.
To our knowledge, this is the first demonstration of rapid hematopoietic engraftment derived from ex vivo expanded hematopoietic progenitors. However, while rapid neutrophil engraftment has been demonstrated in the patients treated to date, larger phase II/III studies are required to test the hypothesis that enhanced kinetics of engraftment observed will improve patient outcomes, including effects on overall survival, incidence of clinically significant infections, and days in the hospital.