Long-Acting, Oxidation-Resistant IL-33 Mobilizes High-Quality Hematopoietic Stem and Progenitor Cells More Efficiently Than Wild-Type IL-33, G-CSF or AMD3100

Mobilization and collection of peripheral blood hematopoietic stem and progenitor cells (HSPC) has become increasingly important for hematopoietic cell transplantation. Granulocyte-colony stimulating factor (G-CSF) alone or in combination with the CXCR4-inhibitor AMD3100 are the most common mobilization approaches. These are however limited since they (a) fail to mobilize a sufficient number of cells in a significant fraction of patients, and (b) are biased towards myeloid immune reconstitution. A novel, single drug mobilization agent that results in a more balanced - myeloid and lymphoid - reconstitution would therefore be highly favorable to improve transplantation outcome.

Interleukin (IL)-33 has recently been described as a potent mobilizer of HSPCs with long-term reconstitution potential. IL-33 is an intracellular protein that is believed to be enzymatically activated, released upon cell death, and inactivated upon oxidation. Whereas mobilization of HSPCs by G-CSF has been linked, at least in-part, to modulating the CXCR4-CXCL12 axis, the mobilizing function of IL-33 has been attributed to a different chemokine, CCL7.

Here we asked whether IL-33 mobilizes HSPCs better than current clinical mobilizing agents, G-CSF and/or AMD3100, and how the quality of the mobilized cells compares. By flow cytometry and colony-forming assays, we observed for the first time that IL-33 treatment in mice mobilized Lin^- Sca-1⁺ c-kit⁺ (LSK) cells and common myeloid progenitors (CMP/CFU-GEMMs) more efficiently than G-CSF or AMD3100 alone, and that IL-33 acts synergistically with both drugs. Next we engineered IL-33 variants lacking native cysteine residues, thereby rendering these variants resistant to oxidative inactivation. These variants had equal or better in vitro activity (a) in cell-based reporter assays and (b) in inducing CCL7 protein production by primary human umbilical vein endothelial cells (HUVECs). In vivo, these variants mobilized LSK cells and, interestingly also common lymphoid progenitor (CLP) and endothelial progenitor cells, more efficiently than wild-type IL-33 or G-CSF. We then engineered a one-armed IL-33-Fc fusion molecule comprising a single oxidation-resistant IL-33 fused to an Fc domain. Interestingly, a single dose of this oxidation-resistant IL-33-Fc variant was sufficient to significantly increase the mobilization of LSK, CLP, and endothelial progenitor cells, whereas oxidation-resistant IL-33 or G-CSF were not.

In conclusion, our findings suggest that a single dose of long-acting, oxidation-resistant IL-33 may allow efficient mobilization of high-quality HSPCs including CLP cells, and may thus represent a novel mechanism to improve bone marrow transplantation.

All authors were employed by Bayer at the time the studies were conducted.