The novel CXCR4 antagonist POL5551 mobilizes hematopoietic stem and progenitor cells with greater efficiency than Plerixafor

Abstract Abstract 4100 INTRODUCTION: Mobilized hematopoietic stem/progenitor cells (HSPC) have become the favored cell source for stem cell transplantation. The current gold standard mobilizing agent is G-CSF, where a 5-day mobilization regimen precedes stem cell harvest. More fast-acting and potent mobilizing agents would be desirable in the interest of donor and recipient safety and convenience. AMD3100, a currently available fast-acting mobilizing agent has proven weak for clinical mobilization as a single agent, with an efficiency of less than 1/5th of G-CSF in humans. METHODS: Binding properties (position, selectivity, affinity) of the novel PEM CXCR4 antagonist POL5551 to its target receptor were analyzed. In vivo mobilization efficiency was studied after injection into C57Bl/6 or DBA/2 mice. Different administration modes (Bolus vs. continous infusion) were tested as well as a combination with a standard regimen (9×100 μg/kg q12h) of G-CSF or Cyclophosphamide. Progenitor cell mobilization was monitored using clonogenic in vitro assays. Properties of mobilized cells were tested by flow cytometry, in vitro transwell migration assays and in vivo (homing, engraftment kinetics, stem cell contents, secondary engraftment) in lethally irradiated CD45.1 or CD45.1/2 recipient mice, alone or with CD45.1 competitor bone marrow cells. RESULTS: POL5551 showed selective binding to CXCR4 with an affinity exceeding that of its natural ligand SDF1, albeit occupying the extracellular receptor domains only (Fig.1). Mobilization peaked 4 hours after i.p. injection and a positive but non-linear dose-response relationship was documented for doses between 0.5 and 100 mg/kg (6000 CFU-C/ml, Fig. 2). A dose of 15 mg/kg mobilized more than twice the number of CFU-C as an equimolar dose of AMD3100, and a single dose of POL5551 at 30 mg/kg mobilized as strongly as a standard 5-day course of G-CSF treatment. POL5551 synergized with G-CSF in that injection of 5 mg/kg POL5551 after G-CSF treatment increased mobilization by 10-fold (3,000 to approx. 30,000 CFU-C/mL); this represents a 2.5 fold increase compared to a similar treatment regimen with AMD3100. Similarly, synergism with Cyclophosphamide was observed (9,900 to 50,000 CFU-C/mL). Given as continous infusion, 5 mg/kg/day of POL5551 mobilized up to 8,000 CFU-C/ml, whereas at 30 mg/kg/day up to 40,000 CFU-C/ml were measured in circulation on day 3. Mobilized cells migrated efficiently in in vitro transwell assays and homed efficiently to the bone marrow of lethally irradiated recipients. Moreover POL5551 mobilized cells provided timely early engraftment and contained long-term engrafting stem cells with self-renewal capacity, including in serial transplantation. The immunophenotype of immature cells mobilized with POL5551 was characterized by low expression of several adhesion molecules. CONCLUSIONS: POL5551 mobilizes murine stem and progenitor cells with rapid kinetics and unprecedented efficiency, markedly exceeding that of G-CSF and AMD3100. The combination of POL5551 with G-CSF mobilized more strongly than G-CSF with other CXCR4 antagonists. Similar to what we previously described for other mobilized stem cell specimen, POL5551-mobilized cells homed to marrow and engrafted efficiently. Immunophenotype was similar to that of AMD3100 mobilized cells. If the data can be corroborated in humans, POL5551 has the potential to substitute for G-CSF as a mobilizing agent. Disclosures: Romagnoli: Polyphor Ltd.: Employment. Chevalier:Polyphor Ltd: Employment. Patel:Polyphor Ltd.: Employment.

Download Full-text

ALL Cells Mobilized by AMD3100 Are More Proliferative, and Remain In the Circulation Longer Than Normal HSC, Enhancing the Action of Vincristine

Blood ◽

10.1182/blood.v116.21.2137.2137 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2137-2137 ◽

Cited By ~ 1

Author(s):

Linda J. Bendall ◽

Robert Welschinger ◽

Florian Liedtke ◽

Carole Ford ◽

Aileen Dela Pena ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Bone Marrow Microenvironment ◽

Hematopoietic Progenitors ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Cell Mobilization ◽

Cycle Dependent

Abstract Abstract 2137 The chemokine CXCL12, and its receptor CXCR4, play an essential role in homing and engraftment of normal hematopoietic cells in the bone marrow, with the CXCR4 antagonist AMD3100 inducing the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. We have previously demonstrated that AMD3100 similarly induces the mobilization of acute lymphoblastic leukemia (ALL) cells into the peripheral blood. The bone marrow microenvironment is thought to provide a protective niche for ALL cells, contributing to chemo-resistance. As a result, compounds that disrupt leukemic cell interactions with the bone marrow microenvironment are of interest as chemo-sensitizing agents. However, the mobilization of normal hematopoietic stem and progenitor cells may also increase bone marrow toxicity. To better evaluate how such mobilizing agents affect normal hematopoietic progenitors and ALL cells, the temporal response of ALL cells to the CXCR4 antagonist AMD3100 was compared to that of normal hematopoietic progenitor cells using a NOD/SCID xenograft model of ALL and BALB/c mice respectively. ALL cells from all 7 pre-B ALL xenografts were mobilized into the peripheral blood by AMD3100. Mobilization was apparent 1 hour and maximal 3 hours after drug administration, similar to that observed for normal hematopoietic progenitors. However, ALL cells remained in the circulation for longer than normal hematopoietic progenitors. The number of ALL cells in the circulation remained significantly elevated in 6 of 7 xenografts examined, 6 hours post AMD3100 administration, a time point by which circulating normal hematopoietic progenitor levels had returned to baseline. No correlation between the expression of the chemokine receptor CXCR4 or the adhesion molecules VLA-4, VLA-5 or CD44, and the extent or duration of ALL cell mobilization was detected. In contrast, the overall motility of the ALL cells in chemotaxis assays was predictive of the extent of ALL cell mobilization. This was not due to CXCL12-specific chemotaxis because the association was lost when correction for background motility was undertaken. In addition, AMD3100 increased the proportion of actively cells ALL cells in the peripheral blood. This did not appear to be due to selective mobilization of cycling cells but reflected the more proliferative nature of bone marrow as compared to peripheral blood ALL cells. This is in contrast to the selective mobilization of quiescent normal hematopoietic stem and progenitor cells by AMD3100. Consistent with these findings, the addition of AMD3100 to the cell cycle dependent drug vincristine, increased the efficacy of this agent in NOD/SCID mice engrafted with ALL. Overall, this suggests that ALL cells will be more sensitive to effects of agents that disrupt interactions with the bone marrow microenvironment than normal progenitors, and that combining agents that disrupt ALL retention in the bone marrow may increase the therapeutic effect of cell cycle dependent chemotherapeutic agents. Disclosures: Bendall: Genzyme: Honoraria.

Download Full-text

Expansion and Maintenance of Hematopoietic Stem and Progenitor Cells in Course of Long-Term Inhibition of CXCR4/CXCL12 Signaling

Blood ◽

10.1182/blood.v128.22.2648.2648 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2648-2648 ◽

Cited By ~ 1

Author(s):

Darja Karpova ◽

Julie Ritchey ◽

Matthew Holt ◽

Darlene Monlish ◽

Laura G. Schuettpelz ◽

...

Keyword(s):

Cell Cycle ◽

Steady State ◽

Continuous Infusion ◽

Progenitor Cells ◽

Small Molecule ◽

Critical Role ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract During the past two decades peripheral blood stem cells have become the favored graft source for HSCT with 80 % of allogeneic and almost 100 % of autologous HSCT performed with mobilized blood. The critical role of the interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 for retention and migration of hematopoietic stem and progenitor cells (HSPC) has been well established. Interference with CXCR4/CXCL12 signalling iscurrentlybeing exploited as a strategy to mobilize HSPC indirectly with the most clinically relevant mobilizing agent to date, G-CSF as well as directly with the bicyclam CXCR4 antagonist Plerixafor (AMD3100).In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of CXCR4/CXCL12 axis within the HSPC compartment were investigated in healthy C57BL/6 mice using the non-peptidic small molecule CXCR4 antagonists Plerixafor and ALT1188 along with the Protein-EpitopeMimeticsInhibitor POL5551. Up to 12-14 fold higher mobilization efficiency was achieved by applying the antagonists via two weeks of continuous infusion (up to 8-10x104 CFU-C and LSK/ml) as compared to bolus treatment (4-6x103 CFU-C and LSK/ml) or 5-day course of G-CSF (3-6x103 CFU-C/ml).Despite dramatic increase in numbers of circulating HSPC, the BM HSPC pool dis not decrease; in fact it expanded up to 2-4-fold compared to steady state reservoir (sham-operated control mice) as measured by immunophenotypical (LSK SLAM) and functional (e.g. serial competitive transplantation) properties of the cells. Thus, in contrast to genetically CXCR4 ablatedHSPC, the reversible long-term blockade of the receptor did not diminish the long-term repopulating capacity of HSPC. Cell cycle analysis showed a 2-3-fold increase in cycling activity of BM HSPC: only 10-20% of LSK and 30-40 % of LSK SLAM cells were found to be quiescent (in G0 phase of the cell cycle) after two weeks of CXCR4 antagonist infusion versus 50-60 % of LSK and 70 % of LSK SLAM found in G0 under homeostatic conditions. This increased proliferation was very similar to the one induced transiently at day 3 G-CSF treatmentand would conceivably explain the sustained mobilization without concomitant depletion of the BM HSPC pool. Profiling of differentially treated BM HSC (LSK SLAM) via microarray analysis did not reveal substantial effects of CXCR4 inhibitor infusion on the expression signature. Ofnote, major cytological changes typically associated with G-CSF induced mobilization, e.g. depletion of bone lining osteoblast lineage cells and macrophages, were not detected in continuous infusion of POL5551 exposed BM suggesting limitedeffects within the BM niche compartment. Moreover analysis of the BM HSPC after different washout periods at the end of continuous infusion treatment revealed a rapid (within 1-3 days after discontinuation of infusion) reestablishment of steady state HSPC numbers in the BM.Our data suggest that prolonged pharmacologic blockade of the CXCR4/CXCL12 axis using multiple small molecule inhibitorsrepresents an approach thatreleasesHSPCwith efficiency superiorto any other knownmobilization strategybut also may serve as an effective method induce cell cycling and thus expand BM HSPCs. Figure Competitive transplantation of POL5551 treated andcontrol BM (n=5 recipients per group, mean±SEM) Figure. Competitive transplantation of POL5551 treated andcontrol BM (n=5 recipients per group, mean±SEM) Disclosures Levesque: GlycoMimetics: Equity Ownership.

Download Full-text