Safety and feasibility of hematopoietic progenitor stem cell collection by mobilization with plerixafor followed by apheresis vs bone marrow harvest in patients with sickle cell disease in the multi‐center <scp>HGB</scp> ‐206 trial
Authors: John F. Tisdale, Francis J. Pierciey, Melissa Bonner, Alexis A. Thompson, Lakshmanan Krishnamurti, Markus Y. Mapara, Janet L. Kwiatkowski, Ilya Shestopalov, Jean‐Antoine Ribeil, Wenmei Huang, Mohammed Asmal, Julie Kanter, Mark C. Walters
Journal: American Journal of Hematology (2020)

Introducing functional anti-sickling hemoglobin (Hb) via gene transfer into autologous hematopoietic stem and progenitor cells (HSPCs) has a strong therapeutic potential for sickle cell disease (SCD). Typically, HSPCs are obtained by bone marrow harvest (BMH) or apheresis post-mobilization with granulocyte-colony stimulation factor (G-CSF). While SCD bone marrow (BM) is a suitable HSPC source, fewer lineage-negative HSPCs are harvested vs mobilization/apheresis.1 However, a moratorium on G-CSF use in SCD was proposed given the risk of significant complications, including acute chest syndrome (ACS), multi-organ failure, and death.2 Single-agent plerixafor, instead of G-CSF, has recently been described as a safe and effective alternative for HSPC mobilization in SCD.1, 3-5 Here, we report results from the first multi-center experience of collecting HSPCs for gene therapy manufacture by apheresis post-mobilization with a single-daily dose of plerixafor compared with BMH. A multi-center, phase 1/2 study (HGB-206; NCT02140554) is evaluating the safety and efficacy of LentiGlobin (a drug product [DP] containing autologous CD34+ HSPCs transduced with BB305 lentiviral vector [LVV] encoding human β-globin gene with anti-sickling βA-T87Q) in patients with SCD. Patients were initially treated with DP made from BM-harvested HSPCs using the original (Group A) and then refined (Group B), manufacturing process. In Group C, patients received DPs made from plerixafor-mobilized HSPCs (Figure S1). Hydroxyurea was discontinued ≥30 days pre-cell collection (all patients). A transfusion regimen (Hb target of 10 g/dL and HbS <30%) was instituted ~60 days pre-HSPC collection in Groups B and C. Multiple BMHs (total nucleated cell target/BMH of ≥6 × 108/kg) and mobilization cycles (≤2 consecutive apheresis days/cycle separated by ≥14 days; total target of ≥10 × 106 CD34+ cells/kg, with 1.5 × 106 CD34+ cells/kg reserved for rescue) were permitted. Data are shown as median (min-max). Further details about HSPC collection, selection, transduction, and immunophenotyping of CD34+ cells are described in the Supplementary Methods. As of September 14, 2018, Groups A and B completed enrollment; 11 patients (9 in Group A, 2 in Group B) underwent BMH. Group C enrollment was ongoing, and 14/18 enrolled patients had initiated mobilization/apheresis. Given Group B patient 1 also underwent plerixafor mobilization/apheresis (for research purposes), the total of patients who had mobilization/apheresis was 15 (Figure S1). The median age was 26 years and all patients had βSβS genotype (Table S1). Overall, 11/11 (100%) and 7/15 (47%) patients had an adverse event (AE) attributed to BMH and plerixafor mobilization/apheresis, respectively (Table S2; additional details on AEs and serious AEs are in the Supplementary Results). Within 7 days of BMH, ≥Grade 3 AEs were reported in 55% of patients, including procedural pain (55%; deemed serious in 2 patients), vaso-occlusive pain (18%; considered serious), and anemia and postoperative anemia (9% each) (Table S2). These events were attributed to BMH, and all patients recovered without sequelae. Seven days post-BMH and through conditioning, 2 additional patients (from 9 who received DP following BMH, Figure S1) had ≥Grade 3 vaso-occlusive pain events (Table S3). Post-mobilization/apheresis, 4/15 patients (27%) had ≥Grade 3 AEs within 7 days after last plerixafor administration; 3 of these (20%) reported serious vaso-occlusive pain attributed to mobilization/apheresis. These 3 serious AEs occurred within 2 days, post-plerixafor mobilization/apheresis, and resolved within 5 days (Supplementary Results). Three patients were reported to have hypomagnesemia (2 Grade 1, 1 Grade 3), and two had hypocalcemia (1 Grade 1, 1 Grade 2) within 7 days of plerixafor mobilization/apheresis (Table S2). No additional patients (from 10 Group C patients who had DP manufactured as of the data cut-off; Figure S1) had ≥Grade 3 AEs beyond 7-days post plerixafor and through the initiation of conditioning (Table S3). Opioid use for pain management was reported in 5/11 patients (46%) within 7 days pre-BMH and in 11/11 patients (100%) in the 7 days post-BMH. In patients who had plerixafor mobilization/apheresis, 8/15 patients (53%) received opioids for pain management 7 days pre- and 7 days post-HSPC collection. A median of two BMHs (1–4) or one mobilization cycle yielded enough cells for DP manufacturing and rescue (Figure S2A). Ten of the 11 patients who had HSPCs collected by BMH needed multiple procedures to meet the DP manufacturing cell dose. In contrast, all patients had ≤2 mobilization cycles to satisfy the DP manufacturing requirement; a third cycle was completed in one patient for rescue HSPC harvesting (Figure S2B). The yields of 5.1 (0.6-9.2) × 106 CD34+ cells/kg per BMH/patient vs 10.1 (3.8-20.0) × 106 CD34+ cells/kg were achieved per mobilization cycle/patient (Table S5). The CD34+ yield/mobilization cycle was significantly higher vs a single BMH (P < .0001; Figure 1A). No prominent changes in electrolyte levels or markers of hemolysis or inflammation were observed on Day 1 post-plerixafor mobilization/apheresis (Table S4). The HbS fraction at the last measurement before mobilization cycle was <30% in 23/26 mobilization cycles (with maximum of 33.1%) for 13/15 patients in Groups B and C for whom the data were available (Table S5). One patient with 31.2% HbS on a day pre-HSPC collection was among the 3 described earlier who developed vaso-occlusive pain post-plerixafor mobilization/apheresis (Supplementary Results). The mean platelet count decreased from 344.7 (SD of 136.4) × 109/L pre-mobilization to 136.3 (SD of 42.5) × 109/L at Day 1 post-apheresis (Table S4). Platelet transfusions were not required for mobilization/apheresis (N = 15) or through conditioning for 9 treated Group C patients. For 15 patients who had plerixafor mobilization/apheresis, the total blood volume (TBV) processed/patient/apheresis was 14 555 (10 751-25 000) mL, and the collection efficiencies (CE) 1 and 2 per patient were 49 (17-83)% and 33 (16-62)%, respectively (Tables S5, S6). Three patients had ≥25 000 mL TBV processed (Table S6), with two developing vaso-occlusive pain following those HSPC collections (Supplementary Results). The collected product hematocrit was 7 (4-13)% for 14 Group C patients who had initiated cell collection. Esrick et al. previously suggested that a collection interface with a higher hematocrit (deeper than the manufacturer-recommended interface of 3%) could increase CD34+ yields.3 In our study, product hematocrit was also >3%, with a 5-10% range for 7 patients who had data availablefor a single day of collection (in other cases, product hematocrit was measuredwhen products from 2 collection days were combined). The resultingCEs were favorable (CEs >40%) in 5/7 (71.4%) patients; however, nocorrelation between product hematocrit was observed with CE1 (rp = −0.12, P = .78)or CE2 (rp = −0.17, P = .69). WBC and ANC count increased from 11.3 (7.4-17.8) × 109/L and 6.1 (2.4-14.5) × 109/L at baseline to 42.0 (22.7-45.8) × 109/L and 16.0 (12.9-32.6) × 109/L immediately prior to apheresis on Day 1/Cycle 1, respectively, with near resolution within 24 hours post-plerixafor; magnitudes of increases were lower for Day 2/Cycle 1 (Figure S3A, S3B; Table S7). For WBC and ANC levels, the pattern of changes was similar between Cycle 1 and subsequent cycles (Supplementary Results). Baseline CD34+ counts were 7.2 (2.9-52.0) cells/μL and increased to 99.0 (45.0-627.0) cells/μL immediately before apheresis on Day 1/Cycle 1 and to 62.4 (36.0-129.0) cells/μL on Day 2/Cycle 1 before returning to baseline by 24 hours post-plerixafor on Day 2 (Figure 1B). The CD34+ dynamics during mobilization/apheresis for Cycles 2 and 3 were similar to Cycle 1. The proportion of CD34bright (long-term repopulating)5, 6 cells in LVV-transduced CD34+ cells collected by apheresis (median 96%) was significantly higher (P < .0001) vs BMH collections (median 58%), and was not significantly different vs HSPC collection by plerixafor mobilization/apheresis in healthy donors (P = .18) (Figure 1C). CD34dim (lineage-committed) cells from LVV-transduced CD34+ cells consisted primarily of myeloid progenitors and pro-B cells, and the ratio of myeloid, lymphoid, and plasmacytoid dendritic cells (pDC) among the CD34dim populations was similar with the two harvesting methods (Figure 1D). CyTOF immunoprofiling was performed on CD34+ cells from Group B patient 1 (who had cells collected by both harvesting methods). Compared with BMH, CD34+ HSPCs collected by plerixafor mobilization/apheresis had a lower proportion of CD34dim cells (0.5% vs 16.5%; mean of 2 replicates) and cells expressing CD19 within the CD34dim population (5.5% vs 36.6%; Figure 1E), illustrating the superior yield of CD34bright cells as determined by both inter- and intra-subject comparisons. Transduction efficiencies associated with both cell collection methods were also assessed, at the same multiplicity of infection, for Group B patient 1. The mean vector copy numbers (VCNs) ± standard deviations in DPs from HSPCs collected by plerixafor mobilization/apheresis and BMH were comparable at 1.7 ± 0.2 and 1.5 ± 0.1 per diploid genome respectively. Thus, plerixafor mobilization and apheresis did not appear to alter the ability of HSPCs to be transduced by LVV. These data show that plerixafor mobilization/apheresis provides an HSPC harvesting method that appears safer than BMH for patients with SCD. Based on the experience in the HGB-206 study thus far, the following mitigation steps may help to reduce the risk of vaso-occlusive pain events during plerixafor mobilization/apheresis in patients with SCD: ~2 months of regular RBC transfusion to achieve HbS <30% of total Hb before plerixafor mobilization, strict fluid and electrolyte management during apheresis, and avoidance of apheresis exceeding 4–5 hours or processing more than four times TBV. Our experience adds substantially to the previous evidence that plerixafor mobilization/apheresis is superior to BMH for harvesting high-quality, CD34bright HSPCs suitable for ex vivo manipulation in SCD. The authors thank the patients, their families, and caregivers for participating in the study. They would like to acknowledge staff members at all the HGB-206 clinical sites: K. Hammond and J. Schneiderman at Ann and Robert H. Lurie Children's Hospital of Chicago; H.C. Kim, K. Smith-Whitley, D. Teachey, T. Olson, J. Freedman, S. Grupp, Y. Wang, I. Sommers, and P. Venkatapuram at Children's Hospital of Philadelphia; and C. Bascon, A. Garcia, M. Moriarty, and A. Lal at UCSF Benioff Children's Hospital, Oakland. They would also like to acknowledge the following former and current bluebird bio employees and consultants: Alexandra Miller, Becky Price, Craig Jones, Erin Meister, Erin Whitney, Gretchen Lewis, Iva Kronja, Kyla MacLeod, Min Luo, Olivier Negre, Ren Chen, and Stef De Reys. Funding for this research was provided by bluebird bio, Inc. Medical writing support was provided by Patrice Ferriola (KZE PharmAssociates, LLC) and Peloton Advantage, LLC, and funded by bluebird bio. John F. Tisdale and Francis J. Pierciey Jr contributed equally to this work. A.A.T.: consultancy for bluebird bio, Inc; Celgene; and Novartis; research funding from Baxalta; bluebird bio, Inc; Celgene; and Novartis. I.S., M.B., F.J.P., and J.A.R. are employees of bluebird bio, Inc, and own bluebird bio, Inc, stock. M.A. and W.H. were employed by and own stock in bluebird bio, Inc. J.K.: consultancy for bluebird bio, Inc; Cowen; Guidepoint Global; GLG; Imara; Jeffries; Modus; Novartis; and Sangamo; honoraria from Medscape, Novartis, Peerview, and Rockpointe; membership on Board of Directors or advisory committees for NHLBI and SCDAA. J.L.K: consultancy for Agios; bluebird bio, Inc; Celgene; and Imara; and research funding from Apopharma; bluebird bio, Inc; Novartis; Terumo; Sangamo. L.K., M.M., and J.F.T.: no disclosures. M.C.W.: consultancy for Editas, All Cells, Inc, Veevo, and TruCode, bluebird bio, Inc, Cambridge, MA. Figure S1. Disposition of Study Participants. Figure S2. Number of BMH procedures and plerixafor mobilization/apheresis. Figure S3. Cell counts over time during plerixafor mobilization/apheresis. Supplementary_Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.