Fresh or Cryopreserved CD34+-Selected Mobilized Peripheral Blood Stem and Progenitor Cells for the Treatment of Poor Graft Function after Allogeneic Hematopoietic Cell Transplantation.
ABSTRACT: CD34+-selected stem cell boost (SCB) without conditioning has recently been utilized for poor graft function (PGF) after allogeneic hematopoietic stem cell transplantation with promising results. Unfortunately, many patients have been unable to receive the boost infusion as their donors were unwilling or unable to undergo an additional stem cell collection. Therefore, we conducted this study utilizing either fresh or cryopreserved peripheral blood stem cell products to create CD34+-selected boost infusions for the treatment of PGF. Additionally, to explore relationship of CD34+ dose and response, we included a cohort of donors mobilized with plerixafor in addition to the standard granulocyte colony-stimulating factor (G-CSF). Twenty-six patients with PGF were included in this study. Seventeen donor-recipient pairs were enrolled onto the prospective study; an additional 9 patients treated off protocol were reviewed retrospectively. Three different donor products were used for CD34+ selection: (1) fresh mobilized product using G-CSF only, (2) fresh mobilized products using G-CSF and plerixafor, and (3) cryopreserved cells mobilized with G-CSF. CD34+ cell selection was performed using a CliniMACS. The infusion was not preceded by administration of any chemotherapy or conditioning regimen. The primary objective was hematologic response rate and secondary objectives included CD34+ yields, incidence and severity of acute and chronic graft-versus-host disease (GVHD), overall survival (OS), and relapse-free survival (RFS). The median post-selection CD34+ counts per kilogram of recipient weight were 3.1 × 106, 10.9 × 106, and 1 × 106 for G-CSF only, G-CSF plus plerixafor, and cryopreserved products, respectively. The median CD34+ yields (defined as the number of CD34+ cells after selection/CD34+ cells before CD34+ selection) were 69%, 66%, and 28% for G-CSF only, G-CSF plus plerixafor, and cryopreserved products, respectively. After SCB, 16 of the 26 recipients (62%) had a complete response, including 5 of 8 (63%) who received cryopreserved products. Five had a partial response (19%), resulting in an overall response rate of 81%. One-year RFS and OS were 50% and 65%, respectively. There was no treatment-related toxicity reported other than GVHD: 6 (23%) developed acute GVHD (2 grade I and 4 grade II) and 8 (31%) developed chronic GVHD (2 limited and 6 extensive). Cryopreserved products are viable alternatives to create SCB for the treatment of PGF. When collecting fresh products is an option, the addition of plerixafor increases CD34+ yield over G-CSF alone; however, it is currently unclear if the CD34+ cell dose impacts the efficacy of the SCB.
Project description:Successful stem cell gene therapy requires high numbers of genetically engineered hematopoietic stem cells collected using optimal mobilization strategies. Here we focus on stem cell mobilization strategies for thalassemia and present the results of a plerixafor-based mobilization trial with emphasis on the remobilization with granulocyte-colony stimulating factor (G-CSF)+plerixafor in those patients who had previously failed mobilization. Plerixafor rapidly mobilized CD34(+) cells without inducing hyperleukocytosis; however, 35% of patients failed to reach the target cell dose of ≥6×10(6) CD34(+) cells/kg. Four subjects who failed on either plerixafor or G-CSF were remobilized with G-CSF+plerixafor. The combination proved highly synergistic; the target cell dose was readily reached and the per-apheresis yield was significantly increased over initial mobilization, ultimately resulting in single-apheresis collections, despite a more than 50% reduction of the dose of G-CSF in splenectomized patients to avoid hyperleukocytosis. The total stem and progenitor cells mobilized in G-CSF+plerixafor patients were higher than in patients treated by plerixafor alone. Importantly, the G-CSF+plerixafor-mobilized cells displayed a primitive stem cell phenotype and higher clonogenic capacity over plerixafor-mobilized cells. G-CSF+plerixafor represents the optimal strategy when very high yields of stem cells or a single apheresis is required. The high yields and the favorable transplantation features render the G-CSF+plerixafor-mobilized cells the optimal CD34(+) cell source for stem cell gene therapy applications.
Project description:Plerixafor (AMD3100) and G-CSF mobilize peripheral blood stem cells (PBSCs) by different mechanisms. A rhesus macaque model was used to compare plerixafor and G-CSF-mobilized CD34+ cells. Three PBSC concentrates were collected from 3 macaques treated with G-CSF, plerixafor, or plerixafor plus G-CSF. CD34+ cells were isolated by immunoselection and were analyzed by global gene and micro RNA (miR) expression microarrays. Unsupervised hierarchical clustering of the gene expression data separated the CD34+ cells into three groups based on mobilization regimen. Plerixafor-mobilized cells were enriched for B cells, T cells and mast cells genes and G-CSF-mobilized cells were enriched for neutrophils, and mononuclear phagocytes (MPs) genes. Genes up-regulated in plerixafor plus G-CSF-mobilized CD34+ cells included many that were not up-regulated by either agent alone. Two hematopoietic progenitor cell miR, miR-10 and miR-126, and a dendritic cell miR, miR-155, were up-regulated in G-CSF-mobilized CD34+ cells. A pre-B cell acute lymphocytic leukemia miR, miR-143-3p, and a T cell miR, miR-143-5p, were up-regulated in plerixafor plus G-CSF-mobilized cells. The composition of CD34+ cells is dependent on the mobilization protocol. Plerixafor-mobilized CD34+ cells include more B, T, and mast cell precursors while G-CSF-mobilized cells have more neutrophil and MP precursors. Overall design: Three rhesus macaques were given all three mobilization protocols and PBSCs were collected from each of these three animals. Two monkeys were given plerixafor first, one was given G-CSF first and all three were given plerixafor plus G-CSF last. This resulted in 6 samples (CD34+ and CD34- cells from each of the 3 mobilizations) from each of the 3 individual animals (biological replicates).
Project description:BACKGROUND: Granulocyte-colony-stimulating factor (G-CSF) is routinely used for mobilization of hematopoietic stem and progenitor cells preceding autologous transplantation after high-dose chemotherapy in hematologic malignancies. However, due to high mobilization failure rates, alternative mobilization strategies are required. STUDY DESIGN AND METHODS: Patients who poorly mobilized CD34+ hematopoietic cells (HCs) with G-CSF additionally received the CXCR4 antagonist plerixafor. The phenotype of CD34+ HCs collected after this plerixafor-induced "rescue" mobilization, in regard to adhesion molecule and CD133, CD34, and CD38 expression in comparison to CD34+ HCs collected after traditional G-CSF administration in good mobilizers, was analyzed flow cytometrically. To confirm previous studies in our patient cohort, the efficiency of mobilization and subsequent engraftment after this "on-demand" plerixafor mobilization were analyzed. RESULTS: Pronounced mobilization occurred after plerixafor administration in poor mobilizers, resulting in similar CD34+ cell yields as obtained by G-CSF in good mobilizers, whereby plerixafor increased the content of primitive CD133+/CD34+/CD38- cells. The surface expression profiles of the marrow homing and retention receptors CXCR4, VLA-4, LFA-1, and CD44 on mobilized CD34+ cells and hematopoietic recovery after transplantation were similar in patients receiving G-CSF plus plerixafor or G-CSF. Unexpectedly, the expression levels of respective adhesion receptors were not related to mobilization efficiency or engraftment. CONCLUSION: The results show that CD34+ HCs collected by plerixafor-induced rescue mobilization are qualitatively equivalent to CD34+ HCs collected after traditional G-CSF mobilization in good mobilizers, in regard to their adhesive phenotype and engraftment potential. Thereby, plerixafor facilitates the treatment of poor mobilizers with autologous HC transplantation after high-dose chemotherapy.
Project description:Plerixafor (AMD3100) and G-CSF mobilize peripheral blood stem cells (PBSCs) by different mechanisms. A rhesus macaque model was used to compare plerixafor and G-CSF-mobilized CD34+ cells. Three PBSC concentrates were collected from 3 macaques treated with G-CSF, plerixafor, or plerixafor plus G-CSF. CD34+ cells were isolated by immunoselection and were analyzed by global gene and micro RNA (miR) expression microarrays. Unsupervised hierarchical clustering of the gene expression data separated the CD34+ cells into three groups based on mobilization regimen. Plerixafor-mobilized cells were enriched for B cells, T cells and mast cells genes and G-CSF-mobilized cells were enriched for neutrophils, and mononuclear phagocytes (MPs) genes. Genes up-regulated in plerixafor plus G-CSF-mobilized CD34+ cells included many that were not up-regulated by either agent alone. Two hematopoietic progenitor cell miR, miR-10 and miR-126, and a dendritic cell miR, miR-155, were up-regulated in G-CSF-mobilized CD34+ cells. A pre-B cell acute lymphocytic leukemia miR, miR-143-3p, and a T cell miR, miR-143-5p, were up-regulated in plerixafor plus G-CSF-mobilized cells. The composition of CD34+ cells is dependent on the mobilization protocol. Plerixafor-mobilized CD34+ cells include more B, T, and mast cell precursors while G-CSF-mobilized cells have more neutrophil and MP precursors. Three rhesus macaques were given all three mobilization protocols and PBSCs were collected from each of these three animals. Two monkeys were given plerixafor first, one was given G-CSF first and all three were given plerixafor plus G-CSF last. This resulted in 6 samples (CD34+ and CD34- cells from each of the 3 mobilizations) from each of the 3 individual animals (biological replicates).
Project description:We hypothesized that during conditioning chemotherapy for allogeneic stem cell transplant (allo-SCT), the disruption of stromal-leukemia interactions using G-CSF in combination with the CXCR4-specific inhibitor, plerixafor, may promote the release of leukemic cells from the niche and increase tumor elimination. In a phase 1/2 investigation, we treated 45 AML/myelodysplastic syndrome (MDS)/CML patients (34 AML, 7 MDS and 4 CML) with G-CSF (10 ?g/kg daily for 6 days starting on day -9) plus plerixafor (doses of 0, 80, 160 or 240 ?g/kg daily for 4 days starting on day -7) along with the busulfan-fludarabine (Bu-Flu) conditioning regimen. In the phase 1 part, we determined that G-CSF plus plerixafor is safe in this setting. We compared the clinical effects and outcomes of AML/MDS study patients (n=40) with 164 patients from a historical data set who received Bu-Flu alone before allo-SCT by stratifying on cytogenetics and disease status to correct for bias. Study patients had increased myeloid chimerism and lower rates of GvHD. There was no significant difference in relapse-free survival or overall survival. The G-CSF plus plerixafor combination increased circulating WBCs, CD34+ cells and CXCR4+ cells, and preferentially mobilized FISH+ leukemic cells.
Project description:Plerixafor, a direct antagonist of CXCR4/stromal-derived factor 1, can safely and rapidly mobilize allografts without the use of granulocyte colony-stimulating factor (G-CSF). We conducted a phase 2, multicenter, prospective study of plerixafor-mobilized HLA-identical sibling allografts for allogeneic hematopoietic cell transplantation in recipients with hematological malignancies. Donors (n = 64) were treated with subcutaneous plerixafor (240 µg/kg) and started leukapheresis (LP) 4 hours later. The primary objective was to determine the proportion of donors who were successfully mobilized: defined as collection of ≥2.0 × 106 CD34+ cells per kilogram recipient weight in ≤2 LP sessions. Recipients subsequently received reduced intensity (RIC; n = 33) or myeloablative (MAC; n = 30) conditioning. Sixty-three of 64 (98%) donors achieved the primary objective. The median CD34+ cell dose per kilogram recipient weight collected within 2 days was 4.7 (0.9-9.6). Plerixafor was well tolerated with only grade 1 or 2 drug-related adverse events noted. Bone pain was not observed. Plerixafor-mobilized grafts engrafted promptly. One-year progression-free and overall survivals were 53% (95% confidence interval [CI], 36% to 71%) and 63% (95% CI, 46% to 79%) for MAC and 64% (95% CI, 47% to 79%) and 70% (95% CI, 53% to 84%) for RIC recipients, respectively. Donor toxicity was reduced relative to G-CSF mobilized related donors. This is the first multicenter trial to demonstrate that, as an alternative to G-CSF, plerixafor rapidly and safely mobilizes sufficient numbers of CD34+ cells from matched sibling donors for HCT. Engraftment was prompt, and outcomes in recipients were encouraging. This trial was registered at clinicaltrials.gov as #NCT01696461.
Project description:Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34(+) cells into circulation. Recently, plerixafor has been used as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression levels in CD4(+) T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8(+) T cells and altered expression levels of 16 cytokine-associated genes in CD3(+) T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC-matched, minor histocompatibility-mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell-mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF.
Project description:High numbers of genetically modified hematopoietic stem cells (HSCs) equipped with enhanced engrafting potential are required for successful stem cell gene therapy. By using thalassemia as a model, we investigated the functional properties of hematopoietic stem and progenitor cells (HSPCs) from Hbb(th3)/45.2(+) mice after mobilization with G-CSF, plerixafor, or G-CSF+plerixafor and the engraftment kinetics of primed cells after competitive primary and noncompetitive secondary transplantation. G-CSF+plerixafor yielded the highest numbers of HSPCs, while G-CSF+plerixafor-mobilized Hbb(th3)/45.2(+) cells, either unmanipulated or transduced with a reporter vector, achieved faster hematologic reconstitution and higher levels of donor chimerism over all other types of mobilized cells, after competitive transplantation to B6.BoyJ/45.1(+) recipients. The engraftment benefit observed in the G-CSF+plerixafor group was attributed to the more primitive stem cell phenotype of G-CSF+plerixafor-LSK cells, characterized by higher CD150(+)/CD48 expression. Moreover, secondary G-CSF+plerixafor recipients displayed stable or even higher chimerism levels as compared with primary engrafted mice, thus maintaining or further improving engraftment levels over G-CSF- or plerixafor-secondary recipients. Plerixafor-primed cells displayed the lowest competiveness over all other mobilized cells after primary or secondary transplantation, probably because of the higher frequency of more actively proliferating LK cells. Overall, the higher HSC yields, the faster hematological recovery, and the superiority in long-term engraftment indicate G-CSF+plerixafor-mobilized blood as an optimal graft source, not only for thalassemia gene therapy, but also for stem cell gene therapy applications in general.
Project description:Hematopoietic stem cells can be mobilized from healthy donors using single-agent plerixafor without granulocyte colony-stimulating factor and, following allogeneic transplantation, can result in sustained donor-derived hematopoiesis. However, when a single dose of plerixafor is administered at a conventional 240 ?g/kg dose, approximately one-third of donors will fail to mobilize the minimally acceptable dose of CD34+ cells needed for allogeneic transplantation. We conducted an open-label, randomized trial to assess the safety and activity of high-dose (480 ?g/kg) plerixafor in CD34+ cell mobilization in healthy donors. Subjects were randomly assigned to receive either a high dose or a conventional dose (240 ?g/kg) of plerixafor, given as a single subcutaneous injection, in a two-sequence, two-period, crossover design. Each treatment period was separated by a 2-week minimum washout period. The primary endpoint was the peak CD34+ count in the blood, with secondary endpoints of CD34+ cell area under the curve (AUC), CD34+ count at 24 hours, and time to peak CD34+ following the administration of plerixafor. We randomized 23 subjects to the two treatment sequences and 20 subjects received both doses of plerixafor. Peak CD34+ count in the blood was significantly increased (mean 32.2 versus 27.8 cells/?L, P=0.0009) and CD34+ cell AUC over 24 hours was significantly increased (mean 553 versus 446 h cells/?L, P<0.0001) following the administration of the 480 ?g/kg dose of plerixafor compared with the 240 ?g/kg dose. Remarkably, of seven subjects who mobilized poorly (peak CD34+ ?20 cells/?L) after the 240 ?g/kg dose of plerixafor, six achieved higher peak CD34+ cell numbers and all achieved higher CD34+ AUC over 24 hours after the 480 ?g/kg dose. No grade 3 or worse drug-related adverse events were observed. This study establishes that high-dose plerixafor can be safely administered in healthy donors and mobilizes greater numbers of CD34+ cells than conventional-dose plerixafor, which may improve CD34+ graft yields and reduce the number of apheresis procedures needed to collect sufficient stem cells for allogeneic transplantation. (ClinicalTrials.gov, identifier: NCT00322127).
Project description:A single subcutaneous (SC) injection of plerixafor results in rapid mobilization of hematopoietic progenitors, but fails to mobilize 33% of normal allogeneic sibling donors in 1 apheresis. We hypothesized that changing the route of administration of plerixafor from SC to IV may overcome the low stem cell yields and allow collection in 1 day. A phase 1 trial followed by a phase 2 efficacy trial was conducted in allogeneic sibling donors. The optimal dose of IV plerixafor was determined to be 0.32 mg/kg. The primary outcome of reducing the failure to collect ≥2 × 106 CD34+/kg recipient weight in 1 apheresis collection to ≤10% was not reached. The failure rate was 34%. Studies evaluating the stem cell phenotype and gene expression revealed a novel plasmacytoid dendritic cell precursor preferentially mobilized by plerixafor with high interferon-α producing ability. The observed cytomegalovirus (CMV) viremia rate for patients at risk was low (15%), as were the rates of acute grade 2-4 graft-versus-host disease (GVHD) (21%). Day 100 treatment related mortality was low (3%). In conclusion, plerixafor results in rapid stem cell mobilization regardless of route of administration and resulted in novel cellular composition of the graft and favorable recipient outcomes. These trials were registered at clinicaltrials.gov as #NCT00241358 and #NCT00914849.