The CXCR4 Antagonist AMD3100 and the CXCR2 Agonist GROβ Synergistically Mobilize Hematopoetic Stem Cells (HSC) with Short and Long Term Repopulating Activity.

The CXCR4 antagonist AMD3100 mobilizes short and long term repopulating HSC peaking at 60 minutes post SC administration in mice. The CXCR2 agonist GROβ (CXCL2Δ4) similarly mobilizes HSC peaking at 15 minutes post SC administration, which is polymorphonuclear neutrophil (PMN) and active matrix metalloproteinase-9 (MMP-9) dependent. Based upon preliminary findings that CXCR4 signaling has a negative effect on MMP-9 release, we evaluated mobilization by the combination of AMD3100 plus GROβ. Initial sequential administration produced only additive mobilization in mice, however, simultaneous SC administration resulted in dramatic synergy in mobilization of CFU-GM, reaching >71 fold at 15 minutes post administration (8695±648/ml blood) compared to 6 fold by AMD alone (699±166) and 20 fold by GRO alone (2505±929) at 60 and 15 minutes, respectively. In addition, combination mobilization remained synergistically elevated for >2.5 hrs. Combination AMD plus GRO mobilization was significantly higher than a multiday regimen of G-CSF (100 ug/kg/day × 4 days)(2404±333 CFU-GM/ml blood). Extensive dose ranging and timing analysis confirm a requirement for early/simultaneous administration of both compounds for maximal mobilization response. Spearman’s Rank analysis showed a direct association between synergistic mobilization and synergistically elevated plasma proMMP-9 levels (r=0.84; P<0.001), suggesting that CXCR4 antagonism upregulates signaling through CXCR2 resulting in enhanced release of proMMP-9. Synergistic mobilization and elevaton of plasma MMP-9 by AMD plus GROβ, like GROβ, demonstrated inter-strain variability, being highest in DBA and lowest in C57Bl mice, while no strain variability was observed for AMD alone. Synergistic mobilization was absent in mice depleted of PMN with anti-GR-1 monoclonal antibody, in MMP-9 knockouot mice, and in normal mice treated with a monoclonal antibody that blocks MMP-9 activation, consistent with a requirement for synergistically enhanced PMN derived MMP-9 in response to AMD plus GROβ. A partial MMP-9 requirement was observed for mobilization induced by AMD alone. In limiting dilution transplant studies, peripheral blood mononuclear cells (PBMC) mobilized by GROβ or AMD alone resulted in 80% and 60% survival of lethally irradiated mice at 180 days, respectively. The combination of GRO plus AMD mobilized significantly more LTRC with 100% survival observed using 2×10⁶ cells and 50% survival at 2×10⁴ cells, whereas PBMC mobilized by GRO or AMD alone provided no survival at 2×10⁴ cells. In limiting dilution primary competitive congenic mouse transplant studies, both GRO and AMD alone mobilized competitive repopulating units (CRU) with the combination mobilizing significantly more CRU than expected, using nonparamatric ANOVA analysis. In addition, AMD plus GROβ mobilized significantly more CRU (2–4 fold) than G-CSF. A competitive advantage for PBSC mobilized by AMD plus GRO was more obvious in secondary non competitive transplants, where marrow cells from mice transplanted with PBSC mobilized by AMD plus GRO showed 89–99% engraftment in secondary recipients over a diluton range of 1:1 to 4:1 mobilized PBMC to congenic bone marrow. In summary, these studies suport a significant role of chemokines and chemokine receptors in HSC mobiliztion and identify a novel highly effective mobilizing regimen for rapid mobilization of HSC with enhanced repopulating activity.