Pegylated Recombinant Murine GM-CSF Is a Potent Mobilizer of Murine Bone Marrow Progenitors, Synergizes with BIO5192 and Plerixafor (AMD3100), and Skews Mobilized Cells to a Tolerogenic Phenotype.

Abstract 2432

Poster Board II-409

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a naturally occurring acidic glycoprotein growth factor that regulates proliferation and differentiation of hematopoietic stem and progenitor cells. Inflammation results in the release of GM-CSF from numerous cell types including T-cells, macrophages, and neutrophils. Murine GM-CSF (mGM-CSF), unlike human GM-CSF, has a very short half-life in vivo. Pegylation of mGM-CSF (peg-mGM-CSF) has been shown to prolong its half-life.

There are numerous reports of GM-CSF use in humans to mobilize stem cells. It was observed by our group that patients who were transplanted with stem cell products mobilized by GM-CSF had a lower incidence of acute GVHD (Devine et al. BBMT 2005; 36:531). The reason for this is unclear. A recent report suggested that regulatory T-cells (T-regs) express the low affinity alpha subunit of the mGM-CSF receptor and can be expanded ex vivo in the presence of GM-CSF and independent of IL-2 (Kared et al. Blood 2008;112:2575). We hypothesized that peg-mGM-CSF may expand T-regs in vivo and act synergistically with the CXCR4 antagonist Plerixafor (AMD3100) and a small molecule inhibitor of VLA-4, BIO5192, to generate optimal peripheral blood stem cell products enriched in hematopoietic progenitors and T-regs.

Peg-mGM-CSF was manufactured in a bioreactor as previously described (Sainathan et al. Prot Express and Purification 2005;44:94). C57/Bl6 mice were injected with peg-mGM-CSF 5ug IP, daily x 4 days then bled and analyzed for CFU progenitor assays (CFU-C) and FACS analysis. The same mice were then injected with AMD3100 (5mg/kg SC) with or without BIO5192 (1mg/kg IV) and serially bled at 30 minutes, 1 hour, 3 hours, and 6 hours. CFU-C were performed and blood and spleen were analyzed for T-cell and dendritic cell subsets by FACS. We observed that peg-mGM-CSF was a potent mobilizer of hematopoietic progenitors and resulted in leukocytosis (mean difference 17.98 K/uL +/- 2.15, 95%CI 13.4 - 22.53, p<0001, n=6) and splenomegally. Peg-mGM-CSF increased CFU-C by 26.5 fold compared to PBS treated mice (n=6, 95% CI = 10.25-42.73). Peg-mGM-CSF had synergistic effects on progenitor mobilization when combined with AMD3100 (89.8 fold, 95% CI 61.6-118, n=6) or BIO5192 (94 fold, 95% CI 46.5-141.6, n=6) compared to PBS treated mice. The combination of peg-mGM-CSF, AMD3100 and BIO5192 resulted in 119.7 fold (95% CI 63.7-175.6, n=6) increase in progenitor mobilization over PBS treated mice and peaked at 30 minutes. In addition, peg-mGM-CSF increased circulating CD4+ FoxP3+ T-regs from a mean of 10.66% +/- 2.65 to 23.86% +/- 4.7 at 5 days (n=15, p=0.001 by paired t-test). Splenic T-regs were also increased from 16.09% +/- 2.7 to 31.56% +/- 4.34 (n=9, p=0.001 by paired t-test). This effect was sustained out to 4 days after the last dose of peg-mGM-CSF. Furthermore, there was no significant change in thymic T-regs, and this effect was not significantly affected by the addition of AMD3100 or BIO5192. The function of these peg-mGM-CSF T-regs, as measured in a MLR was found to be equivalent to T- regs from PBS treated control mice (n=3, p=0.27, by paired t-test). Interestingly, analysis of the spleens from animals on day 5 after peg-mGM-CSF demonstrated an increase in absolute CD11c+ cells, as expected, and a 4 fold increase in the absolute number of plasmacytoid dendritic cells as determined by CD11c+ SiglecH+ staining. Additional studies are underway to assess the functional impact of murine peripheral blood stem cells mobilized with peg-mGM-CSF in combination with small molecule inhibitors of the CXCR4 and VLA-4 axes on engraftment and graft versus host disease in a murine acute GVHD parent to F1 model.