Successful Plerixafor-Mediated Mobilization, Apheresis, and Lentiviral Vector Transduction of Hematopoietic Stem Cells in Patients with Severe Sickle Cell Disease

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 (CD34^dim) 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%) CD34^dim cells, with 16%-50% of the CD34^dim 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%) CD34^dim 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.