Rapid and Sustained Engraftment of a Single Allogeneic Ex-Vivo Expanded Cord Blood Unit (CBU) After Reduced Intensity Conditioning (RIC) in Adults. Preliminary Results of a Prospective Trial

Abstract Abstract 486 CBU is a widely used source of stem cells for allogeneic transplantation (SCT). Engraftment rate and speed of a single CBU in adults remains unsatisfactory. Transplantation of 2 CBU may overcome this problem to the expense of an increased incidence of GVHD. Until now, attempts at using ex-vivo expanded CBU have been unsuccessful to promote long term engraftment of the expanded product. We report the results achieved in the first 8 Pts included in a PCT of transplantation of a single ex-vivo expanded allogeneic CBU. Eudract 2008–006665–81, Clinicaltrials.gov NCT 01034449. Methods: Adults patients with an indication for SCT and unable to tolerate MAC (age>45, comorbidities, previous high-dose therapy) were included after inform consent if no Id sibling, no MUD 9 (C or DQ mismatch accepted) to 10/10 HLA matches and no CBU fulfilling the HLA matching (≥ 4/6) and richness (≥ 3 to 4 × 107 TNC/kg before thawing) criteria were available. RIC consisted of Flu (40 mg/m2/d × 5d), Cyclophosphamide (50 mg/kg × 1d) ICT 2 Gy. GVHD prevention consisted of MMF (d-3 to d28) and CSA from d-3. Graft engineering: 1 CBU with > 2 TNC/kg < 3 and 4 to 6 HLA compatibilities was thawed, CD34+ cells were selected through magnetic device (Miltenyi) and submitted to ex-vivo expansion in SF medium ( HPO1-Macopharma) supplemented with SCF, Flt3l, G-CSF and TPO during 12 days, starting d-12 of the transplantation (Ivanovic, Cell Transplant 2011) CD34 neg cells were cryopreserved. On d0, expanded cells were washed and resuspended in HSA 4% and upon viability and sterility were injected to the pt. Cd34 neg cells were thawed and injected to the pt 3 h later. Results: From 03/2010 to 06./2011 8 pts have been included, med age 55y.o. (26–64) with AL: 3, Hodgkin's: 2, MDS: 3. Pts had received 1 to 3 lines of Tx (med:2). For 1 pt the expanded product was contaminated and this pt then received a back-up unmanipulated CBU. He engrafted correctly and is AW at 14 m with full donor chimerism. For the 7 other pts, the ex-vivo median fold expansion of CD34+ cells and TNC was 39 (29–75) and 390 (127–526) respectively, leading to a graft that contained 1.3 to 13 × 106 CD34+ cells/kg (med: 2 × 106/kg). The CD34 neg counterpart contained 3 × 106 CD3+/kg (1–5) and 0,9 × 106 CD19+ cells/kg (0,3–1,5). At d42, 6/7 pts who received the expanded graft engrafted with ≥99% donors cells. A 2d RIC was performed followed by a double CBU transplant in the patient who did not engraft. That 2d graft again failed to engraft. However the patient remains alive at 9m. For the 6 pts who engrafted with the expanded product the time to reach 500, 1000 PMN's and 20 000 plts/mm3 was 7d (6–19), 8d (6–21) and 24d (0–39) respectively. The chimerism on WBC and CD3+cells (evaluated on d 15, 42, 60, 100, 180, 365) remains full donor up to 1 year + after transplant (1y+: 2 pts; 6m+: 1pt; 180d+: 1 pt; 60d+: 1pt) or to relapse (at 1y) in the one pt who relapsed. Five pts experienced an AGVHd (grade III-IV: 1 pt). With a median FU of 10m (2 to 18m) 7 pts are alive, 6 wo disease. One pt died 1y after transplant from relapse. Conclusion: Ex-vivo expansion of a single CBU is feasible and reproducible. Transplantation of the expanded product together with the CD34 neg counterpart of the same CBU produces rapid, complete and sustained donor engraftment after RIC in adults. Disclosures: No relevant conflicts of interest to declare.

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Clinical Grade Ex-Vivo Expansion of Granulomonocytic Cells and Progenitors Using High-Dose SCF and G-CSF without Exhaustion of Stem Cell Activity.

Blood ◽

10.1182/blood.v104.11.2850.2850 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2850-2850

Author(s):

Marie-Laure Bonnet ◽

Martine Guillier ◽

Natacha Greissinger ◽

Marie-Catherine Marracho ◽

Muriel Bedouel ◽

...

Keyword(s):

Stem Cell ◽

Ex Vivo ◽

Hematopoietic Cells ◽

Ex Vivo Expansion ◽

Cell Populations ◽

High Dose ◽

Clinical Grade ◽

Cell Numbers ◽

Cd34 Cells ◽

High Concentrations

Abstract Ex-vivo expansion of mature hematopoietic cells and progenitors is a major cell therapy technology aiming to shorten chemotherapy-induced cytopenias. The goal of amplifying the most primitive hematopoietic stem cell populations in clinically acceptable conditions is currently difficult, due to the absence of clinical grade early acting-cytokines and lack of adequate stem cell assays. Stem cell factor (SCF) and granulocyte colony stimulating factor (G-CSF) are both clinically applicable and they have been shown to induce a dose-response effect with regard to the expansion of mature cell populations when used in combination with early- acting cytokines. The combined effects of high concentrations of G-CSF and SCF on hematopoietic cells and progenitors and the effects of this procedure on the more primitive stem cell subsets have not been studied. We have developed a preclinical protocol to evaluate the feasibility of expansion of granulomonocytic cells using SCF and G-CSF with the goal of translating this protocol to the clinical application. In preliminary assays, the effects of low and high concentrations of both cytokines on total nucleated cell numbers and progenitors from purifed CD34+ cells (50 104cells/ml) have been tested. As compared to low concentrations (G-CSF 10 ng/ml and SCF 50 ng/ml) high concentrations of G-CSF and SCF (100 ng/ml and 300 ng/ml respectively) induced a two-fold increase of the nucleated cells and progenitors after 9 days of culture (n=7 experiments). High concentrations were therefore chosen for further experiments. CD34+ cells purified from mobilized peripheral blood grafts (purity 99%) have been cultured in a clinically acceptable medium containing clinical grade fetal calf serum ( 10 %), G-CSF (100 ng/ml) and SCF (300 ng/ml) for 9 days. Cells were analyzed using apoptosis tests, immunophenotyping and clonogenic assays at day 0 and day+9. In several experiments ( n= 9) a mean 21-fold expansion of total viable cell numbers was obtained, with a mean 4-fold expansion of clonogenic progenitors. Expanded cells had consistently CD11b+, CD13+, CD15+ phenotype and 2–4% of them remained CD34+. In a scale-up experiment started with 23.106 CD34+ cells, the total cell numbers expanded 16-fold at day+9, with generation of high numbers of CD11b + (18%), CD13+(98%) and CD15+ cells (91%) demonstrating the feasibility of the protocol in clinical scale. To determine if ex-vivo expansion would lead to a significant exhaustion of more primitive stem cells, we have evaluated the NOD/SCID-repopulating cell (RC) contents of the cultures before (day 0) and after ex-vivo expansion (day 9), in mice transplanted with day 0 and day+9 cells. In two experiments, the numbers of NOD/SCID mice engrafted with CD45+ human cells have been found increase from 30% ( 3/10) at day 0 to 100 % ( 10/10 ) at day 9 (Exp1 ) and from 50 % (3/6) at day 0 to 90 % ( 7/8 ) at day9 (Exp 2), demonstrating the persistence of NOD/SCID-RC potential after ex-vivo expansion. Overall the large-scale granulomonocytic cell production protocol that we developed could be of major interest in order to maintain the dose-intensity in high dose chemotherapy regimens and to shorten neutropenia after autologous PBSC transplantation while maintaing the stem cell potential of the graft.

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Kinetics of Engraftment and Graft Versus Host Disease After Cotransplantation of Ex Vivo Expanded and Unexpanded Cord Blood Units In Immunodeficient Mice.

Blood ◽

10.1182/blood.v116.21.3722.3722 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3722-3722

Author(s):

Li Ming Ong ◽

Xiubo Fan ◽

Pak Yan Chu ◽

Florence Gay ◽

Justina Ang ◽

...

Keyword(s):

Cord Blood ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Insulin Growth Factor ◽

Nsg Mice ◽

Nonobese Diabetic ◽

Cd34 Cells

Abstract Abstract 3722 Ex vivo expansion of cord blood (CB) hematopoietic stem cells (HSCs) and cotransplantation of two CB units can enhance applicability of CB transplants to adult patients. This is the first study on cotransplantation of ex vivo expanded and unexpanded human CB units in immunodeficient mice, simulating conditions for ex vivo CB expansion clinical trials. CB units were cultured in serum-free medium supplemented with Stem Cell Factor, Flt-3 ligand, Thrombopoietin and Insulin Growth Factor Binding Protein-2 with mesenchymal stromal co-culture. Cotransplantation of unexpanded and expanded CB cells was achieved by tail vein injection into forty-five sublethally irradiated nonobese diabetic SCID-IL2γ−/− (NSG) mice. Submandibular bleeding was performed monthly and mice were sacrificed 4 months following transplantation to analyze for human hematopoietic engraftment. CB expansion yielded 40-fold expansion of CD34+ cells and 18-fold expansion of HSCs based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts had 4.30% human cell repopulation, compared to 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses (p = 0.07). Ex vivo expanded grafts with lower initiating cell doses also had equivalent engraftment to unexpanded grafts with higher cell dose (8.0% vs 7.9%, p= 0.93). However, the unexpanded graft, richer in T-cells, predominated in final donor chimerism. Ex vivo expansion resulted in enhanced CB engraftment at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis. The expanded graft with increased stem/progenitor cells enhanced initial engraftment despite eventual rejection by the unexpanded graft. Disclosures: No relevant conflicts of interest to declare.

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Mitoxantrone and Etoposide with or without Intermediate-Dose Cytarabine for the Treatment of Patients with Primary Induction Failure or Relapsed Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.2174.2174 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2174-2174

Author(s):

Steven Trifilio ◽

Alfred Radamaker ◽

Diane Newman ◽

Kathryn Coyle ◽

Katrin Carlson Leuer ◽

...

Keyword(s):

Conflicts Of Interest ◽

Prospective Trial ◽

Response To Treatment ◽

High Dose ◽

Cell Transplant ◽

Significant Difference ◽

Primary Induction ◽

Induction Failure ◽

Acute Myeloid ◽

Intermediate Dose

Abstract Abstract 2174 Introduction: The prognosis for patients with primary induction failure (PIF) or relapsed acute myeloid (Rel-AML) is poor. Mitoxantrone (M) plus etoposide (E)- based salvage regimens (ME), in particular, either alone or with intermediate dose cytarabine (MEC) are effective in these high risk patients; However, these regimens have not been directly compared. Heterogeneity in chemotherapy dose, dose escalation and age have been important limitations in the evaluation of previous studies. Also problematic, historically patients were classified according to FAB criteria. Since then, karyotype has been shown to be a main determinant of prognosis. The influence of karyotype on response to ME or MEC is currently unknown. Herein, we report the response to treatment with a fixed dosing schedule of ME or MEC in 66 patients treated for PIF or Rel-AML with intermediate(intermed-) or unfavorable(unfav-) risk cytogenetics. Differences in CR between ME and MEC subsets were analyzed to determine the effect of adding of C to ME. Methods: 66 consecutive patients with PIF or Rel-AML treated with either ME(n=37)or MEC(n=29) between 10/2004-12/2008 were evaluated. All patients had received initial induction therapy with daunorubicin 45–60mg/m2 IV bolus d1-3 and cytarabine 100mg/m2 CI d1-7(7+3), and consolidation with HIDAC if CR was achieved. ME and MEC were dosed according to previously published studies. The decision to use a given salvage was left to the discretion of the treating physician. Chi-Square test was used for statistical analysis. Results: Table1 shows there was no difference between the ME and MEC groups with regards to age, sex, % blasts at initial diagnosis, and %CR after initial induction with 7+3, or % patients who received inter- to high dose C prior to ME or MEC. Length of CR (after 7+ 3 and consolidation) was significantly longer in the MEC group. A significantly higher number of CR's was observed in the MEC group compared to ME(p=0.05). Within the MEC group, no difference in CR was observed between patients with intermed- and unfav-risk cytogenetics(p=0.96). The same was true within the ME group(p=0.13). When MEC was compared to ME, a significant difference in CR was observed in patients with unfav-risk cytogenetics(p=0.044) and patients <60years old. Prior to therapy, MEC patients had higher number of blasts. MEC patients had a significantly longer duration of remission. One patient in both the ME and MEC group died before hematopoetic reconstitution. Conclusion: In clinical practice, as observed in the present study, we observed a greater overall CR rate in patients who received MEC compared to those treated with ME, particularly in younger patients with unfav-risk cytogenetics. For those who achieved CR after MEC or ME, a longer duration of CR was observed in the MEC group compared to ME. These results could be particularly beneficial for those patients receiving salvage therapy as a temporizing measure prior to allogeneic hematopoetic stem cell transplant ion, and encourages confirmation from a prospective trial. Disclosures: No relevant conflicts of interest to declare.

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Osteogenic- and Adipogenic- Differentiated Bone Marrow-Derived Mesenchymal Stem Cells Fail to Support Expansion of Adult Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.4810.4810 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4810-4810

Author(s):

Olga Kulemina ◽

Izida Minullina ◽

Sergey Anisimov ◽

Renata Dmitrieva ◽

Andrey Zaritskey

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Cord Blood ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Feeder Layers

Abstract Abstract 4810 Ex vivo expansion and manipulation of primitive hematopoietic cells has become a major goal in the experimental hematology, because of its potential relevance in the development of therapeutic strategies aimed at treating a diverse group of hematologic disorders. Osteoblasts, mesenchymal stem/progenitor cells (MSC/MPC), adipocytes, reticular cells, endothelial cells and other stromal cells, have been implicated in regulation of HSC maintenance in endosteal and perivascular niches. These niches facilitate the signaling networks that control the balance between self-renewal and differentiation. In the present study, we evaluated and compared the effects of three different stromal feeder layers on expansion of HSPC derived from BM and cord blood (CB): BM mesenchymal stem cells (MSC), osteoblast-differentiated BM mesenchymal stem cells (Ost-MSC) and adipocyte-differentiated BM mesenchymal stem cells (Ad-MSC). BM-MSC cultures were established from plastic adherent BM cell fractions and analyzed for immunophenotype, frequency of colony forming units (CFU-F), frequency of osteo- (CFU-Ost) and adipo- (CFU-Ad) lineage progenitors. Cultures with similar clonogenity (CFU-F: 26,4 ± 4,5%) and progenitors frequency (CFU-Ost: 14,7 ± 4,5%; CFU-Ad: 13,3 ± 4,5%) were selected for co-culture experiments. All MSC were positive for stromal cell-associated markers (CD105, CD90, CD166, CD73) and negative for hematopoietic lineage cells markers (CD34, CD19, CD14, CD45). CD34+ cells were separared from BM and CB samples by magnetic cell sorting (MACS) and analyzed for CD34, CD38 and CD45 expression. Feeder layers (MSC, Ost-MSC, Ad-MSC) were prepared in 24-well plates prior to co-culture experiments: MSCs (4×104 cells/well) were cultured for 24 h and either used for following experiments or stimulated to differentiate into either osteoblasts or adipoctes according to standard protocols. CD34+ cells (3500-10000 cells per well) were co-cultured in Stem Span media with or without a feeder layers and in the presence of cytokines (10 ng/mL Flt3-L, 10 ng/mL SCF, 10ng/mL IL-7) for 7 days. Expanded cells were analyzed for CD34, CD38 and CD45 expression. Results are shown on figures 1 and 2. As expected, CB-derived HSPC expanded much more effectively than BM-derived HSPC. The similar levels of expansion were observed for both, the total number of HSPC, and more primitive CD34+CD38- fraction in the presence of all three feeder layers. Ost-MSC supported CB-derived HSPC slightly better than MSC and Ad-MSC which is in a good agreement with data from literature (Mishima et.al., European Journal of Haematology, 2010), but difference was not statistically significant. In contrast, whereas BM-MSC feeder facilitated CD34+CD38- fraction in BM-derived HSPC, Adipocyte-differentiated MSC and osteoblast-differentiated MSC failed to support BM-derived CD34+CD38- expansion (11,4 ±.4 folds for MSC vs 0,9 ±.0,14 for Ad-MSC, n=5, p<0,01 and 0,92 ±.0,1 for Ost-MSC, n=5, p<0,01).Figure 1.Cord Blood HSPC ex vivo expansionFigure 1. Cord Blood HSPC ex vivo expansionFigure 2.Bone Marrow HSPC ex vivo expansionFigure 2. Bone Marrow HSPC ex vivo expansion Conclusion: BM- and CB-derived CD34+CD38- cells differ in their dependence of bone marrow stroma. Coctail of growth factors facilitate CB HSPC expansion irrespective of lineage differentiation of supporting MSC feeder layer. In contrast, primitive BM CD34+CD38- HSPC were able to expand only on not differentiated MSC. Disclosures: No relevant conflicts of interest to declare.

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Preclinical Development of a Cord Blood (CB)-Derived Hematopoietic Stem Cell (HSC) Product for Allogeneic Transplantation in Patients with Hematological Malignancies

Blood ◽

10.1182/blood.v128.22.818.818 ◽

2016 ◽

Vol 128 (22) ◽

pp. 818-818 ◽

Cited By ~ 2

Author(s):

Camelia Iancu-Rubin ◽

Helen Fong ◽

Goar Mosoyan ◽

Ami Patel ◽

Rachel Sabado ◽

...

Keyword(s):

Cord Blood ◽

Board Of Directors ◽

Hematological Malignancies ◽

Ex Vivo ◽

Allogeneic Transplantation ◽

Ex Vivo Expansion ◽

Cell Selection ◽

Advisory Committees ◽

Cd34 Cells

Abstract Umbilical CB is an established source of HSC for allogeneic transplantation in patients lacking HLA-matched donors. The major limitation of CB transplantation is the relatively low number HSCs within a single CB unit (CBU) resulting in delayed engraftment, infections and ultimately increased mortality. Because of this, double CBU transplantation has been used to reach the required cell doses but this approach has not led to an improved overall outcome and often results in an increased rate of GVHD. These findings have prompted the development of ex vivo expansion strategies to increase the number of HSCs, so that a single CBU can be transplanted. Most of these techniques, however, result in enrichment of short term marrow repopulating cells (ST-RC) at the expense of long term (LT)-RC which may impact durable long term engraftment. In addition, they require 2-3 weeks of culture which complicates the timing of transplant and increases the risk of contamination. Our laboratory has developed a novel approach to expand the numbers of functional HSCs, by transiently influencing the epigenetic determinants of HSCs self-renewal. A 7-day treatment of CB CD34+ cells with valproic acid (VPA) results in a dramatic increase in the number of HSCs capable of durable hematopoietic engraftment in animal model recipients (Chaurasia et al. JCI, 2014). Here we report the pre-clinical development of a VPA-expanded HSC product for utilization in the treatment of patients with hematological malignancies. In the place of using freshly collected CBU as starting sources of CD34+ cells, we validated, optimized and scaled-up the expansion procedure utilizing cryopreserved CBU procured from FDA-licensed Cord Blood Banks and clinically relevant GMP reagents and materials. CBUs from 5 different donors were subjected to thawing followed by positive CD34+ cell selection using a Miltenyi CliniMACS Prodigy®. The total number of nucleated cells (TNC), CD34+ cells, viability, clonogenic potential (i.e. CFU number) and the frequency of various HSC sub-classes were determined post-thawing after which each CBU was subjected to CD34+ cell selection. CD34+ cells counts varied between 1.6 and 13.6x106 (mean of 4.5x106/CBU) and had a purity ranging from 69.2-82.8%. CD34+ cells were treated with cytokines for 16-18h, followed by addition of VPA and ex vivo expansion for 7 days. The generated cell product was characterized phenotypically and functionally and the results were compared to the unmanipulated CBU (uCBU) (Table 1). First, the expanded grafts had greater than 90% viability (range 91.4 to 97%) as compared to 68.2% in the uCBUs after thawing. The average number of CD34+ cells generated was 494.8x106 CD34+ cells (i.e. 126-fold greater than uCBU) which is the equivalent of 61.8x105/Kg/ body weight from a single CBU for an 80 kg individual. The fraction of CD34+ cells, which represented over 60% of the expanded graft, was further assessed for the presence ST-RC, intermediate-term (IT)-RC and LT-RC defined phenotypically as CD34+/CD45RA-/CD90-/CD49f-, CD34+/CD45RA-/CD90+/CD49f-, and CD34+/CD45RA-/CD90+/CD49f+, respectively. The average numbers of each of these HSC sub-classes per expanded CBU were 64, 217 and 265 fold higher than their respective numbers found in the uCBUs. Notably, the expanded grafts contained the equivalent of 22.38x105 IT-RC/kg and 16.98x105 LT-RC/kg where as uCBUs contained only 0.1x105 IT-RC/kg and 0.06x105 LT-RC/kg. Considering the ability of these HSC sub-classes to contribute to intermediate and long term hematopoietic engraftment, their presence in such high number gives the VPA-expanded grafts improved potential to lead to durable hematopoietic and immune reconstitution after transplantation. In addition, the expanded graft has a phenotype which would also be anticipated to lead to rapid hematopoietic recovery since lineage committed precursors (i.e. CD33+, CD15+, CD235a+ and CD41+ cells) represented 35-45% of its composition. Finally, as compared to uCBUs, the expanded HSC product contained 20 times more assayable CFUs consisting predominately of CFU-GEMM which are capable of contributing to multilineage engraftment. In summary, we report the generation of an ex vivo expanded CB HSCs product highly enriched in primitive HSCs sub-classes and which is currently being developed for a Phase I clinical trial for allogeneic CB transplantation in patients with hematological malignancies. Disclosures Bhardwaj: Parker Institute of Cancer Immunotherapy: Membership on an entity's Board of Directors or advisory committees; Checkpoint Sciences: Membership on an entity's Board of Directors or advisory committees.

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High-Dose Cyclophosphamide In Combination With G-CSF Versus G-CSF Alone For Mobilization Of Hematopoietic Stem Cells After Induction Therapy Including Velcade, Cyclophosphamide and Dexacort

Blood ◽

10.1182/blood.v122.21.5369.5369 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5369-5369

Author(s):

Noam Benyamini ◽

Irit Avivi ◽

Eldad J Dann ◽

Tsila Zuckerman ◽

Lavi Noa ◽

...

Keyword(s):

Stem Cell ◽

Induction Therapy ◽

Conflicts Of Interest ◽

Young Patients ◽

High Dose ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Significant Difference ◽

The Difference

Abstract Introduction Even in the era of novel agents, high-dose chemotherapy followed by autologous stem cell transplant (ASCT) is considered to be an essential part of treatment for young patients with multiple myeloma (MM), providing durable responses. Currently, VCD (velcade, cyclophosphamide and dexacort) is one of the most commonly employed induction regimens. High-dose cyclophosphamide (HDC), often used in stem cell (SC) mobilization in conjunction with G-CSF, is associated with adverse events and only modest efficacy against myeloma. An alternative mobilization regimen, using G-SCF alone, has been recently suggested to provide adequate SC collection with less toxicity. Nevertheless, the efficacy and safety of using G-SCF alone after VCD induction have not been fully explored. The current study compares the safety and efficacy of mobilization using HDC-G-CSF versus G-CSF alone in MM patients treated with VCD as induction therapy. Methods The study was approved by the Institutional Review Board of the Rambam Medical Center (Approval # 0110-13 RMB). Data on all consecutive newly diagnosed transplant-eligible MM patients, treated with VCD between 2009 and 2012, were retrospectively reviewed. Eligibility criteria were: VCD induction followed by SC mobilization, either with G-CSF or HDC-G-CSF, with subsequent high-dose melphalan (200 mg/m2) and ASCT. The mobilization protocol was chosen at the discretion of the treating physician. Evaluated data included patient characteristics, SC collection and engraftment related parameters. For statistical analysis, Mann-Whitney non-parametric test for 2 independent groups was used. Results 79 patients were included: 50 mobilized with HDC-G-CSF, and 29 with G-CSF alone. There were no statistically significant differences in terms of patient demographic and MM-related characteristics (MM type, ISS, number of VCD cycles, and disease status at the end of induction) between the 2 cohorts. The first day of SC collection yielded a median of 14.6x106 (range 1.9 -10.1) vs 5.3x106 CD34 cells/Kg (range 0.6-37.7) in the HDC-G-CSF vs the G-CSF groups (p=<0.001). A significantly higher total CD34 collection was obtained in the HDC-G-CSF treated patients (15.9 x 106 vs 8.1x106 CD34 cells/Kg, respectively, P<0.001). Additionally, a bivariate analysis showed that male gender and platelet count (>150,000/mL) prior to mobilization had a significant impact on the outcome of SC collection. The percentage of patients needing more than one day of leukopheresis following HDC-G-CSF and G-CSF was 42% and 83%, respectively. During treatment and mobilization, 20% of patients in the HDC-G-CSF cohort were hospitalized due to neutropenic fever, while none of the patients from the G-CSF group required hospitalization (P<0.011). In all patients apart from one (G-CSF group), at least the minimum of CD34 cells/Kg required to perform a transplant (2x106 CD34 cells/Kg) was collected. Moreover, most patients succeeded in collecting >5x106 CD34 cells/Kg (96% and 93.1% in HDC-G-CSF and G-CSF groups, respectively). Notably, the difference between the groups achieved statistical significance only in collection of >8x106 CD34 cells/Kg (88% and 55.2% of patients treated with HDC-G-CSF and G-CSF, respectively). The median amount of cells administered at transplantation was 7.9x106 and 4.9x106 for patients mobilized with HDC-G-CSF vs G-CSF, respectively, reflecting the difference in the total amount of collected cells. Despite the variation in the amount of transplanted cells, no significant difference in parameters of the transplant outcome was revealed between the 2 cohorts: time to neutrophil engraftment (>500 cells/µl) at a median of 12 days in both groups and platelets engraftment (>25,000 cells/µl) at a median of 14.5 vs 13 days in the HDC-G-CSF and G-CSF groups, respectively. The length of hospitalization, approaching 17 days, did not differ between the 2 groups. Conclusions Mobilization using HDC-G-CSF results in a higher total amount of collected CD34 cells and requires less days of leukophersis. Nevertheless, G-CSF alone provides a sufficient number of SC for transplantation in almost all patients, and this approach is much safer than treatment with HDC-G-CSF. Since engraftment results are identical with the 2 mobilization methods, the use of G-CSF alone could be considered as a preferable cell mobilization protocol in patients previously exposed to VCD induction. Disclosures: No relevant conflicts of interest to declare.

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Antagonizing PPARγ Expands Human Hematopoietic Stem and Progenitor Cells By Switching on FBP1-Repressed Glycolysis and Preventing Differentiation

Blood ◽

10.1182/blood.v130.suppl_1.709.709 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 709-709

Author(s):

Bin Guo ◽

Xinxin Huang ◽

Hal E. Broxmeyer

Keyword(s):

Ex Vivo ◽

Ex Vivo Expansion ◽

Metabolic Reprogramming ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Ppar Γ ◽

Control Shrna ◽

Cd34 Cells ◽

Human Hscs

Abstract Allogeneic hematopoietic cell transplantation (HCT) is widely used as a life-saving treatment for malignant and non-malignant blood disorders. Hematopoietic stem cells (HSCs) are a major contributing cell population for a successful HCT. While cord blood (CB) is an acceptable source of HSCs for clinical HCTbecause of its many advantages including prompt availability, lower incidence of GvHD and virus infection, CB HCT is usually associated with slower time to engraftment especially in adult patients when compared with other cell sources; this is partly due to limiting numbers of HSCs in single cord units. In order to overcome this limitation, ex vivo expansion of CB HSCs has been evaluated in preclinical and clinical studies for improvement of the clinical efficacy of CB HCT. While a number of different ways have been evaluated to ex-vivo expand human HSCs, little is known about the mechanisms involved, and whether efficient expansion of CB HSCs could be achieved by metabolic reprogramming. In a compound screen for potential candidates which could promote ex vivo expansion of CB HSCs, we found that PPARγ antagonist GW9662 treatment significantly enhanced ex vivo expansion of CB phenotypic HSCs (~5 fold) and progenitor cells (HPCs) (~6.8 fold) in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and cytokines (SCF, FL, TPO) when compared with vehicle control. GW9662 significantly increased numbers of CB colony-forming unit (CFU) granulocyte/macrophage (GM) (~1.8 fold) and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) (~3.2 fold) progenitors after 4 days ex vivo culture. To assess whether the ex vivo expanded CB HSCs enhanced by the PPARγ antagonist were functional in vivo, we performed both primary and secondary transplantation in immunocompromised NSG mice. Engraftment of CB CD34+ cells in primary recipients was significantly increased (~3 fold) both in bone marrow (BM) and peripheral blood (PB) by the cultured cells treated with GW9662. The percentages of both myeloid and lymphoid lineages were enhanced in BM of primary recipients transplanted with GW9662-treated CB CD34+ cells. We also transplanted CB CD34+ cells transfected with control shRNA or PPAR γ shRNA into NSG mice, and consistently found that both myeloid and lymphoid chimerism was enhanced in BM of recipients which were infused with PPAR γ shRNA transfected-CD34+ cells compared with control shRNA transfected-CD34+ cells. Long term reconstituting and self-renewing capability of GW9662-treated CB CD34+ cells with both enhanced myeloid and lymphoid chimerism, was confirmed in PB and BM in secondary recipients. Limiting dilution analysis was performed to calculate SCID-repopulating cells (SRC), a measure of the number of functional human HSCs. The SRC frequency of GW9662-cultured CB CD34+ cells was 4 fold greater than that of day 0 uncultured CD34+ cells, and 5 fold increased above that of vehicle-treated CD34+ cells with cytokines alone. To gain mechanistic insight into how PPARγ antagonism enhances expansion of human CB HSCs and HPCs, we performed RNA-seq analysis. Antagonizing PPARγ in CB CD34+ cells resulted in downregulation of a number of differentiation associated genes, including CD38, CD1d, HIC1, FAM20C, DUSP4, DHRS3 and ALDH1A2, which suggests that PPARγ antagonist may maintain stemness of CB CD34+ cells partly by preventing differentiation. Of interest, we found that FBP1, encoding fructose 1, 6-bisphosphatase, a negative regulator of glycolysis, was significantly down-regulated by GW9662, which was further confirmed by RT-PCR, western blot and flow cytometry analysis. GW9662 significantly enhanced glucose metabolism in CB HSCs and HPCs without compromising mitochondrial respiration. Enhanced expansion of CB HSCs by antagonizing PPARγ was totally suppressed by removal of glucose or by inhibition of glycolysis. Importantly, suppression of FBP1 greatly promoted glycolysis and ex vivo expansion of long-term repopulating CB HSCs (~3.2 fold). Overexpression of FBP1 significantly suppressed enhancedexpansion and engraftment of CB HSCs by PPARγ antagonist. Our study demonstrates that PPARγ antagonism drives ex vivo expansion of human CB HSCs and HPCs by switching on FBP1 repressed glucose metabolism and by preventing differentiation. This provides new insight into human HSC self-renewal, and suggests a novel and simple means by which metabolic reprogramming may improve the efficacy of CB HCT. Disclosures No relevant conflicts of interest to declare.

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Elimination of FVIII-Specific B Cells By Immunotoxins Comprised of a Single FVIII Domain Fused to Pseudomonas Exotoxin a

Blood ◽

10.1182/blood.v128.22.561.561 ◽

2016 ◽

Vol 128 (22) ◽

pp. 561-561

Author(s):

Kerstin Brettschneider ◽

Anja Schmidt ◽

Joerg Kahle ◽

Aleksander Orlowski ◽

Diana Stichel ◽

...

Keyword(s):

B Cells ◽

Knockout Mice ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Coagulation Factor ◽

High Dose ◽

Dependent Manner ◽

C2 Domain ◽

Exotoxin A

Abstract The development of inhibitory antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most serious complication for patients with hemophilia A that undergo FVIII replacement therapy. In addition, healthy individuals can spontaneously develop inhibitory anti-FVIII auto-antibodies, which results in acquired hemophilia A. The current standard therapy for patients with hemophilia A and inhibitors, named immune tolerance induction (ITI), is based on frequent and mostly high dose administrations of FVIII. Unfortunately, the eradication of inhibitors can only be achieved in about 70% of patients. Alternative treatment of inhibitor patients with the monoclonal anti-CD20 antibody rituximab results in complete eradication of inhibitors; however, depletion of the entire CD20-positive B cell population is potentially accompanied by severe side effects. Recent studies in hemophilic FVIII knockout mice showed that the application of a FVIII-toxin conjugate resulted in (i) prevention of inhibitor development in naïve mice and (ii) long-term eradication of inhibitors in FVIII-immunized mice. As the use of FVIII for cell targeting of immunotoxins is presumably limited by its high molecular weight (250 kDa) and adhesiveness (off-target reactivity) we explored the potential use of alternative immunotoxins in the current study. The introduced immunotoxins are comprised of a single FVIII domain fused to the Exotoxin A (ETA) from Pseudomonas aeruginosa.The rationale for the use of a single domain instead of full length FVIII as cell-binding component is that immunodominant domains like A2 and C2 might still allow targeting of sufficient amounts of FVIII-specific B-cells by immunotoxins. For proof of concept studies, we generated a histidine-tagged C2 domain-ETA fusion protein (C2-ETA) that was bacterially expressed and purified by affinity chromatography. Purified C2-ETA was recognized by a panel of commercially available monoclonal anti-C2 antibodies in ELISA suggesting proper folding of the C2 domain in the bacterially expressed protein. To test the capacity of C2-ETA to eliminate FVIII-specific B-cells, splenocytes of FVIII-immunized FVIII knockout mice were re-stimulated with FVIII ex vivo in presence and absence of different concentrations of C2-ETA and ETA alone (as control). Re-stimulation of FVIII-specific memory B cells to FVIII- and C2-specific antibody secreting cells (ASC) was analyzed in anEnzyme linked immunospot (ELISPOT) assay using FVIII and C2 as antigens. While differentiation to FVIII-specific ASC was only partially inhibited by C2-ETA, differentiation to C2-specific ASC was completely blocked in a dose-dependent manner. In contrast, the use of ETA alone had no effect. Further analysis of the FVIII domain specificity of antibodies in plasma of FVIII-immunized FVIII knockout mice used for depletion studies revealed a strong contribution of C2-specific antibodies to the overall FVIII-specific immune response. In summary, our results show that the developed C2-ETA immunotoxin is able to specifically eliminate FVIII C2 domain-specific B cells ex vivo. Currently, C2-ETA is tested for its capacity to eliminate FVIII-specific B cells in FVIII knockout mice and additional FVIII domain-ETA immunotoxins are developed. Disclosures No relevant conflicts of interest to declare.

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