Umbilical cord blood (UCB) transplantation has a high early mortality rate primarily related to transplanted stem cell dose. To decrease early mortality and enhance engraftment, a portion of selected cord blood units (20% to 50%) was expanded with cytokines and the copper chelator tetraethylenepentamine (carlecortemcel-L) and transplanted with the unmanipulated fraction after myeloablative conditioning. The primary endpoint was 100-day survival, which was compared with a contemporaneous double-unit cord blood transplantation (DUCBT) group. We enrolled 101 patients at 25 sites; the DUCBT comparison (n = 295) was selected from international registries using study eligibility criteria. Baseline carlecortemcel-L study group unit nucleated cell (NC) and CD34 were 3.06 × 10 cell dose/kg and 1.64 × 10 cell dose/kg. Median NC and CD34 fold expansion were 400 and 77, with a mean total CD34 infused of 9.7 × 10/kg. The 100-day survival was 84.2% for the carlecortemcel-L study group versus 74.6% for the DUCBT group (odds ratio, .50; 95% CI, .26 to .95; P = .035). Survival at day 180 was similar for the 2 groups; the major cause of death after day 100 was opportunistic infections. Faster median neutrophil (21 days versus 28 days; P < .0001), and platelet (54 days versus 105 days; P = .008) engraftment was seen in the carlecortemcel-L study group; acute and chronic graft-versus-host disease rates were similar. In this multinational comparative study, transplanting expanded CD34 stem cells from a portion of a single UCB unit, with the remaining unmanipulated fraction improved 100-day survival compared with DUCBT control patients while facilitating myeloid and platelet engraftment. This trial was registered at www.clinicaltrials.gov as #NCT00469729.

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

CD38, originally described as a differentiation marker, has emerged as an important multifunctional protein. Its most well-characterized function is the ability to catalyze the synthesis of cyclic ADP-ribose (cADPR) from NAD. However, its major enzymatic activity is the hydrolysis of NAD (NADase) implicating it as the major regulator of cellular NAD levels. CD38 expression increases with commitment and differentiation. It is not clear, however, whether such changes in CD38 are merely phenotypic, or reflect an active role for CD38 in the regulation of cell differentiation. The regulation of CD38 gene expression is under the direct control of retinoid receptors (RAR). Antagonists to RAR abolish up-regulation of CD38 gene expression as well as RA induction of granulocytic differentiation down-stream of the myeloid compartment. In the present study we evaluated the involvement of CD38 in the regulation of HPC differentiation by treatment of ex-vivo cultures with LMW antagonists, targeted to either CD38 expression or to its biological activities. CB derived CD34+ cells were cultured with cytokines (S,T,F,6). Treatment of these cultures with an RAR-antagonist (AGN194310) abolished the expression of surface CD38. After 3 weeks in culture, the content of CFC was 3 ±1.1-fold higher, the content of CD34+ cells was 2.4 ± 0.24-fold higher and percentage CD34+ cells displaying CD34+Lin− phenotype was by 35 ± 10-fold higher (p<0.05, n= 14) in RAR-antagonist (10−6M) compared to cytokines-only treated cultures. Colonies derived from RAR-antagonist treated cultures sustained high re-plating capacity, a property that was lost during the first 3-weeks of expansion with cytokines only. In long-term cultures, the peak of CFUc and CD34+ cell expansion of RAR-antagonist treated cultures was 6–10 weeks later than control cultures. At the peak of expansion, cumulative numbers of CD34+ and CFUc were by 130- and 512-fold higher (p<0.05, n=4), respectively, in treated than in control cultures. CFU-MIX colonies were exclusively observed in RAR-antagonist treated cultures (between weeks 7–10). Interestingly, limited (1 week) exposure to the RAR-antagonist was sufficient for this long-term effect. Similarly, we tested the effect of an RXR antagonist (LGN 100754) (10−9 – 10−5 M) on short- and long-term cultures. Treatment with the RXR-antagonist did not down-regulate CD38 expression and only slightly improved ex-vivo expansion parameters over cytokines-only treated cultures. We next evaluated whether inhibition of CD38 enzymatic activities will also modulate in-vitro differentiation of cultured cells. To this end, CD34+ cell cultures were treated with nicotinamide (NA), a non-competitive inhibitor of CD38 NADase, previously demonstrated to abolish its enzymatic activities. 3-week treatment with NA (5mM) resulted in a marked decrease in CD38 expression and a marked increase in the fraction of CD34+Lin− cells as compared to cytokines-only treated cultures (48.0 ± 3.7% vs. 2.8 ± 0.7% and 18.6 ± 3% vs. 0.7 ± 0.06%, n=6, p<0.05, respectively). As with the RAR-antagonist, long-term expansion potential, as determined by CFC and CD34+ cell content, was significantly higher in cultures treated with NA relative to cytokines-only treated cultures. These results demonstrate that both a pan-RAR antagonist and NA inhibit differentiation and promote ex-vivo expansion of progenitor cells, suggesting the possible involvement of CD38 protein in these processes.

No abstract

Ex-vivo expansion strategies of cord blood (CB) derived human progenitor cells (HPC) have been developed to provide an answer to the delayed time to engraftment and to the extended periods of neutropenia and thrombocytopenia encountered. These problems occur in transplants of CB products performed in adults due to the low yield of HPC. Reports correlating the clinical outcome with the number of CD34+ cells suggest that the transplantation of ex vivo expanded CD34+ cells may shorten the time to engraftment. The use of copper chelators such as tetraethylenepentamine (TEPA) has been shown to prolong expansion of HPC by inhibiting cell differentiation and thus allowing self-renewal of primitive HPC (Exp Hematol.2004;32:547). The variability observed in the expansion results, caused by the intrinsic differences among the various sources of CB units and processing methodologies, complicates the interpretation of published results. In the present report we summarize our results of CD34+ cell ex-vivo expansion of over 100 units in the presence of IL-6, TPO, Flt-3 ligand and SCF with and without TEPA. After 3 weeks, the total nuclear cell (TNC), colony forming unit (CFU), and the total CD34+ cell fold expansion of TEPA-treated cultures were 424±10.5 (n=230), 104±7 (n=112) and 19±3.2 (n=113), respectively, with no significant differences compared to controls. However, the percentage of the primitive subset of HPC, CD34+/38− cells, significantly (p&lt;0.0001) increased in the TEPA-treated cultures (3.2%±0.2, n=59) vs. controls (1.6%±0.27, n=147). In contrast, after 5 weeks in culture, the TNC fold expansion was significantly (p&lt;0.05) higher in TEPA-treated cultures compared to the controls, 1471±63.5 (n=89) vs. 1270 ±240 (n=55), respectively. The increase in TNC in TEPA-treated cultures did not result in increased HPC differentiation, but was accompanied by an increased self-renew capacity of CD34+ cells as represented by a 57±5.9 fold (n=47) vs. a 32±3.5-fold (n=38) amplification in the controls (p&lt;0.0009). The overall fold expansion in culture analyzed by a Kaplan-Meier survival curve function demonstrate that the TNC, CFU, CD34+ and CD34+/38− cells derived from TEPA-treated cultures have higher in vitro survival probabilities than controls (p&lt;0.0014). Cumulative values of all parameters were calculated and a transformation performed using the rank procedure. The results underline that TEPA increases CFU potential and CD34+ and CD34+/38− content during the ex-vivo expansion (p&lt;0.01). The TEPA supplemented expansion technology was further tested after up-scaling of the processing and culturing procedures. AC133+ cells isolated by the CliniMACS device from frozen CB units obtained from 6 different banks. The expansion results of TNC, CD34+ cells, CFU and %CD34/38- were 337±23 (n=57), 21±4.3 (n=19), 133±27.5 (n=19) and 2.7% ±0.7 (n=19) fold, respectively. A clinical trial with TEPA expanded cultures for treatment of leukemia patients is currently ongoing at MD Anderson Cancer Center, USA.