Successful Plerixafor-Mediated Mobilization, Apheresis, And Lentiviral Vector Transduction Of Hematopoietic Stem Cells In Patients With Severe Sickle Cell Disease

Abstract Background Patients with severe sickle cell disease (SCD) may benefit from β-globin gene transfer into autologous hematopoietic stem cells (HSC). Successful HBB gene transfer requires vector-mediated transduction of primitive HSCs. Steady-state bone marrow (BM) is the default HSC source in patients with SCD. Normal human BM contains up to 30% CD34+CD19+ pro-B cells and other lineage-committed cell types (CD34dim) that will not contribute to improved long-term erythropoiesis via gene therapy; these cells mobilize at low rates. CD34+ cell yields from BM harvest (BMH) are typically lower than those after mobilization and peripheral blood (PB) apheresis; multiple rounds of BMH may be required to obtain adequate cell doses for autologous gene therapy (GT) protocols. As G-CSF can cause life-threatening SCD complications and is contraindicated, plerixafor, a CXCR4 receptor antagonist, may accomplish HSC mobilization without the neutrophil or endothelial activation that elicit vaso-occlusion. We modified the protocol for the HGB-206 phase 1 study of LentiGlobin GT in severe SCD (NCT02140554) to assess HSC mobilization with plerixafor alone, followed by apheresis and transduction of mobilized cells. We also characterized BM-derived and plerixafor-mobilized HSC populations from patients with SCD. Methods HGB-206 is a phase 1 study of LentiGlobin Drug Product (DP), which contains autologous HSCs transduced ex vivo with the betibeglogene darolentivec (BB305) lentiviral vector, in patients with severe SCD (defined as a history of recurrent vaso-occlusive crisis [VOC], acute chest syndrome, stroke, or tricuspid regurgitant jet velocity of >2.5 m/s). Patients in group B receive 240 µg/kg plerixafor followed 4-6 hours later by apheresis, processing ~3 total blood volumes to collect backup HSCs. If < 1.5 x 106 CD34+ cells are collected, patients undergo a second day of apheresis. Cells collected in excess of those required for backup in case of graft failure are transduced with BB305 lentiviral vector for exploratory analyses. Group B patients then proceed to BMH to obtain cells for clinical DP manufacture. Group C will receive DP manufactured from mobilized PB. Mass cytometry (CyTOF) was used to analyze ex vivo cultured CD34+ cells with over 35 cell surface markers. Results To date, 3 patients have undergone plerixafor mobilization. Patients had a transient 1.5- to 3-fold increase in peak white blood cell and absolute neutrophil levels after plerixafor. Peak absolute CD34+ cell counts in PB were 170, 58, and 160 x 106 CD34+ cells/liter. A total of 15.3, 5.6, and 9.0 x 106 CD34+ cells/kg were collected in a single day of apheresis, and no subsequent apheresis or mobilization was required. In the same study, a mean of 5.0 (range 0.3-10.8) x 106 CD34+ cells/kg were collected per BMH (N=21). The mobilization and apheresis procedures had an acceptable toxicity profile. No dose-limiting toxicities were observed after plerixafor dosing. One patient had a single VOC approximately 48 hours after receiving plerixafor; this patient also experienced VOCs of similar severity after BMH. In contrast, after 21 BMHs in 9 patients, 18 ≥ grade 3 AEs were reported in 6 patients, primarily pain. Ex vivo cultured CD34+ cells isolated from BMH consisted of an average of 41.0% (17.3%-50.7%) CD34dim cells, with 16%-50% of the CD34dim cells expressing lymphoid markers. In contrast, ex vivo cultured CD34+ cells isolated from plerixafor mobilized PB contained an average of 8.2% (1.5-19.5%) CD34dim cells. Similar drug product vector copy numbers were obtained after research-scale transduction of CD34+ cells from marrow and PB from the same patient. Conclusion Initial results suggest that obtaining adequate doses of CD34+ cells from plerixafor-mobilized PB of patients with SCD may be safe and feasible, without the life-threatening complications associated with G-CSF, and with fewer, less invasive procedures compared with BMH. PB-derived CD34+ cells may contain lower proportions of lineage-committed CD34+ cells than BM-derived cells from patients with SCD. Cells collected by BMH and PB mobilization/apheresis appear to have an equivalent transduction efficiency. Together these results indicate that it may be possible to use plerixafor-only mobilization in clinical studies of autologous HSC GT in SCD. Results of mobilization, apheresis, and DP manufacturing at clinical scale for additional patients will be available for presentation. Disclosures Pierciey: bluebird bio: Employment. Kanter: American Society of Hematology (Sickle Cell Disease Guideline Panel): Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; MUSC: Other: The site PI for sponsored research conducted at MUSC who receives funds from: Novartis, bluebird bio, GBT, Sancillo, Apopharma, Pfizer; NHLBI (sickle cell disease research advisory committee): Membership on an entity's Board of Directors or advisory committees, Research Funding; Sancillo: Research Funding; Apopharma: Research Funding; Pfizer: Research Funding; GBT: Research Funding; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kwiatkowski: Novartis: Research Funding; Bluebird Bio: Research Funding; Apopharma: Research Funding; Agios: Consultancy, Honoraria; Ionis: Consultancy, Honoraria. Thompson: Novartis: Consultancy, Research Funding; bluebird bio: Consultancy, Research Funding; Baxalta: Research Funding; Celgene: Consultancy, Research Funding. Shestopalov: bluebird bio: Employment, Equity Ownership. Bonner: bluebird bio: Employment, Equity Ownership. Joseney-Antoine: bluebird bio: Employment, Equity Ownership. Asmal: bluebird bio: Employment, Equity Ownership. Walters: bluebird bio: Research Funding; ViaCord Processing Lab: Other: Medical Director; Sangamo Therapeutics: Consultancy; AllCells, Inc: Other: Medical Director.