The Combination of GROβ and AMD3100 Leads to Rapid and Robust Mobilization of Hematopoietic Stem Cells in Nonhuman Primates

Introduction: Mobilized peripheral blood grafts are currently the predominant source of hematopoietic stem and progenitor cells (HSPC) for both autologous and allogeneic transplantation. The most common clinical hematopoietic stem cell mobilization protocol is five days of Filgrastim (G-CSF). This regimen requires daily injections, has been associated with bone pain and often results in unpredictably low yields. A rapid mobilization method that ideally only required a single treatment and had robust and predictable kinetics would be a significant improvement over the current standard of care. In mice, a unique CXCR2 agonist, GROβ, induces rapid mobilization of stem and progenitor cells 15 minutes after a single injection. When co-administered with plerixafor (AMD3100), an inhibitor of CXCR4, a synergistic increase in mobilization results, with a graft enriched in highly engraftable hematopoietic stem cells (Hoggatt et al., Blood 2016 128:368). Here, we present data demonstrating that combination treatment with GROβ and AMD3100 synergistically mobilizes CD34+ cells and colony forming units (CFU) in nonhuman primates (NHP).

Methods: Cynomolgus macaques were injected with G-CSF (10 µg/kg/day, s.c. for four days), AMD3100 (1 mg/kg, s.c. once) or GROβ (various doses and routes) alone and in combination with AMD3100. Blood was collected at various times post treatment and analyzed by multicolor flow cytometry to quantitate HSPC numbers. Additional aliquots of mobilized blood were plated in methylcellulose and CFU were enumerated seven days later.

Results: We tested the effect of GROβ alone or in combination with AMD3100 in Cynomolgus monkeys. As published previously, administration of G-CSF, AMD3100 or G-CSF+AMD3100 induced significant mobilization of hematopoietic stem and progenitor cells. In comparison, a single injection each of GROβ+AMD3100 yielded 4-fold more CD34+ cells compared to four days of G-CSF (p < 0.01). While the combination of G-CSF+AMD3100 mobilized significantly more CD34+ cells compared to GROβ+AMD3100 (p < 0.0001), the numbers of CD34+ CD90+ CD45RA- cells, representing the most primitive of hematopoietic stem and progenitor cells, were equivalent between treatment groups. Similarly, GROβ+AMD3100 treatment elicited 7-fold more CFU compared to animals treated with G-CSF (p < 0.01), 3.5-fold more CFU compared to AMD3100 (p < 0.01) and equivalent CFU compared to G-CSF+AMD3100. Importantly, the frequency of CFU among the CD34+ cells was significantly greater with GROβ+AMD3100 (1 in 4) compared to G-CSF (1 in 10) or G-CSF+AMD3100 (1 in 25). In contrast to the increased CD34+ numbers and CFU, white blood cell (WBC) and neutrophil counts were significantly lower with GROβ+AMD3100 compared to AMD3100 alone or G-CSF+AMD3100.

Conclusions: We describe a rapid mobilization method that within four hours of a single treatment results in robust hematopoietic mobilization in nonhuman primates. The GROβ+AMD3100 regimen enriches for primitive CD34+ CD90+ CD45RA- stem and progenitor cells with increased colony forming capacity compared to CD34+ cells mobilized with G-CSF+AMD3100, suggesting a significant graft quality difference with the new regimen. The ability to rapidly mobilize primitive HSC with increased CFU frequency may allow effective single day mobilization of HPSCs for patients requiring a HSCT and be especially useful for HSC based gene therapy and gene editing protocols where HSPC grafts enriched for primitive HSC may reduce manufacturing costs and ensure durability of the gene modified HSC. The ability to achieve increased HSPC numbers without dramatically increasing WBC and neutrophils may be especially advantageous in patient populations where G-CSF mobilization is not well tolerated such as Sickle Cell Disease, or where G-CSF leads to increased disease activity such as in autologous transplant for Multiple Sclerosis. A single treatment that results in robust mobilization that is equivalent to G-CSF may allow for a one-day collection method of donor stem cells, and these findings in nonhuman primates support clinical exploration.