O-Fucose and Fringe-Modified Recombinant Notch Peptides As Decoy Molecules to Promote Hematopoietic Progenitor Mobilization

Nearly all autologous hematopoietic cell therapy (HCT) procedures are performed using mobilized hematopoietic progenitor cells (HPCs). Successful outcome is dependent on infusing an adequate number of functionally active HPCs. Mobilization strategy of AMD3100, in combination with G-CSF, increases total CD34+ cells mobilized, and often is used for HPC mobilization in myeloma and non-Hodgkin lymphoma patients. Unfortunately, this approach does not always lead to adequate HPC collection in patients who previously have been exposed to extensive cytotoxic therapy. Furthermore, suboptimal number of HPCs mobilized in some patients may result in slower hematopoietic reconstitution and increased risk of complications during the transplant. Hence developing new mobilization strategy is highly desired for patients who are considered poor-mobilizers. Notch is a signaling molecule important for stem cell self-renewal and fate determination in many tissues including the hematopoietic system. An important feature of Notch is its adhesive nature which was first described by cell aggregation assays in Drosophila studies. We previously reported that hematopoietic stem cell and progenitors (HSPCs) with faulty Notch-ligand interaction display increased cell cycling, decreased adhesion to marrow osteoblastic lineage cells, and enhanced egress from the marrow.

Since Notch2 is the major Notch isotype expressed on HSPCs, we showed that prior treatment in mice with Notch2 blocking antibody but not Notch1 blocking antibody sensitizes HSPC to the mobilizing stimuli of G-CSF and AMD3100 with a 3~4 - fold increase in mobilization. We showed that transient Notch2 blockade leads to decreased CXCR4 expression by HSPCs with CXCR4 transcription being directly regulated by the Notch transcriptional protein RBPJ. Notch2 blockade also increases cell cycling of HPCs during mobilization temporarily without affecting hematopoietic stem cell (HSC) homeostasis and self-renewal. Moreover, transient Notch2 blockade leads to greater HSPC homing to the marrow that is accompanied with a faster hematopoietic recovery and a competitive repopulating advantage of HSPCs.

Herein, to search for alternative agents to Notch blocking antibody, we evaluate the ability of recombinant Notch peptides as decoy molecules to promote disengagement of Notch2-ligand interaction between HSPCs and niche supporting cells. By using OP9 cell-based binding assay and surface plasmon resonance (SPR) analyses, we showed that recombinant Notch1_11-13, Notch1_9-13, Notch1_8-13, Notch2_11-13, Notch2_9-13, and Notch2_8-13 peptide comprising EGF-like repeats 11-13, 8-13, and 9-13, of Notch1 or Notch2, respectively, all bind to Notch ligand Dll4, Dll1 and Jag2. Because EGF-like repeat 12 (EGF 12) has been shown located within the essential ligand binding domain, and the O -fucose attached to the threonine residue of Notch1 EGF12 acts as a surrogate amino acid to make specific and functional contact to residues of Notch ligand, we generated peptides in CHO cells with O -fucose attached to the threonine residue of EGF12 and further extended by Fringe. We found that binding affinities of the glycosylated peptides further increased compared to those of non-glycosylated peptides. Compared to Notch2 peptides, O -fucose and Fringe-modified Notch1_11-13 peptide shows the highest biding affinity to Notch ligand Dll4 with dissociation constant (K_d) reaching 34 nM. Similar to Notch2 blocking antibody, the O -fucose and Fringe-modified Notch1_11-13 peptide effectively outcompetes Notch2-ligand binding and thus blocks the adhesion of marrow HSPCs to the primary osteoblasts or OP9 cells expressing Notch ligands. Further, O -fucose and Fringe-modified Notch1_11-13 peptide can effectively induce HSPCs emigration from a 3D osteoblastic "spheroid niche", an in vitro model of HSC niche. Compared to Notch2 blocking antibody, O -fucose and Fringe-modified Notch1_11-13 peptide caused a further decrease of HSPC cells remained in the "spheroid niche" and more HSPC emigration out of the "niche".

In summary, our findings suggest that recombinant Notch decoy peptides may be considered as alternatives to Notch2 blockade to be combined with the mobilizing regimen of AMD3100 and G-CSF for more effective HSPC mobilization.