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•Using WU-106/AMD3100 mobilization, in vivo transduction of long-term persisting HSCs in SCD mice can be done within 2 hours.•Compared with G-CSF/AMD3100 mobilization, the WU-106/AMD3100-based method is safer and as efficient for phenotypic correction of the disease in mice.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by high rate of relapse and mortality. Current chemotherapies whilst successful in eradicating blasts, are less effective in eliminating relapse-causing leukemic stem cells (LSCs). Although LSCs are usually identified as CD34+CD38- cells, there is significant heterogeneity in surface marker expression, and CD34- LSCs exist particularly in NPM1mut AMLs. By analyzing diagnostic primary DNMT3AmutNPM1mut AML samples, we suggest a novel flow cytometry sorting strategy particularly useful for CD34neg AML subtypes. To enrich for LSCs independently of CD34 status, positive selection for GPR56 and negative selection for NKG2D ligands are used. We show that the functional reconstitution capacity of CD34- and CD34+ LSCs as well as their transcriptomes are very similar which support phenotypic plasticity. Furthermore, we show that although CD34+ subpopulations can contain next to LSCs also normal and/or preleukemic hematopoietic stem cells (HSCs), this is not the case in CD34-GPR56+NKG2DL- enriched LSCs which thus can be isolated with high purity. Finally, we show that patients with AML, who retain at the time of diagnosis a reserve of normal and/or preleukemic HSCs in their bone marrow able to reconstitute immunocompromised mice, have significantly longer relapse-free and overall survival than patients with AML in whom functional HSCs are no longer detectable.

Interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 plays a critical role in the retention and migration of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) microenvironment. In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of the CXCR4/CXCL12 axis on the HSPC compartment were investigated by using 3 structurally unrelated small molecule CXCR4 antagonists. A >10-fold increase in mobilization efficiency was achieved by administering the antagonists as a subcutaneous continuous infusion for 2 weeks compared to a single bolus injection. A concurrent increase in self-renewing proliferation leading to a twofold to fourfold expansion of the HSPC pool in the BM was observed. The expanded BM showed a distinct repopulating advantage when tested in serial competitive transplantation experiments. Furthermore, major changes within the HSPC niche associated with previously described HSPC expansion strategies were not detected in bones treated with a CXCR4 antagonist infusion. Our data suggest that prolonged but reversible pharmacologic blockade of the CXCR4/CXCL12 axis represents an approach that releases HSPC with efficiency superior to any other known mobilization strategy and may also serve as an effective method to expand the BM HSPC pool.

Interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 plays a critical role in the retention and migration of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) microenvironment. In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of the CXCR4/CXCL12 axis on the HSPC compartment were investigated by using 3 structurally unrelated small molecule CXCR4 antagonists. A >10-fold increase in mobilization efficiency was achieved by administering the antagonists as a subcutaneous continuous infusion for 2 weeks compared to a single bolus injection. A concurrent increase in self-renewing proliferation leading to a twofold to fourfold expansion of the HSPC pool in the BM was observed. The expanded BM showed a distinct repopulating advantage when tested in serial competitive transplantation experiments. Furthermore, major changes within the HSPC niche associated with previously described HSPC expansion strategies were not detected in bones treated with a CXCR4 antagonist infusion. Our data suggest that prolonged but reversible pharmacologic blockade of the CXCR4/CXCL12 axis represents an approach that releases HSPC with efficiency superior to any other known mobilization strategy and may also serve as an effective method to expand the BM HSPC pool.

T-cell–directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123+ AML.

T-cell–directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VHof 1 antibody in tandem with the VLof the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123+AML.

Dominant, although nonexclusive roles of CXCR4 and its chief ligand CXCL12 in bone marrow (BM) retention and preservation of the relative quiescence of hematopoietic stem/progenitor cells (HSPCs), along with their involvement in human immunodeficiency virus infection, in trafficking of mature hematopoietic cells to sites of inflammation and in orderly migration of nonhematopoietic cells during embryogenesis, explain the significant interest of the scientific community in the mode of action of this receptor–ligand pair. In this focused review, we seek to distil from the large body of information that has become available over the years some of the key findings about the role of CXCR4/CXCL12 in normal immature hematopoiesis. It is hoped that understanding the mechanistic insights gained there from will help generate hypotheses about potential avenues in which cancer/leukemia cell behavior can be modified by interference with this pathway. StemCells2015;33:2391–2399

AML is a heterogeneous disease characterized by high rate of relapse and mortality. While chemotherapies may eradicate blasts, they are less effective in eliminating relapse-causing Leukemic Stem Cells (LSCs). Although LSCs are usually identified as CD34+CD38-cells, there is significant heterogeneity in surface marker expression and CD34-LSCs exist particularly in NPM1mutAMLs. By analyzing diagnostic primary DNMT3AmutNPM1mutAML samples, we suggest a novel flow cytometry sorting strategy particularly useful for CD34negAML subtypes. To enrich for LSCs independently of CD34 status, positive selection for GPR56 and negative selection for NKG2D-Ligands are employed. We demonstrate that the functional reconstitution capacity of CD34-and CD34+LSCs as well as their transcriptomes are very similar supporting phenotypic plasticity. Furthermore, we show that while CD34+subpopulations can contain next to LSCs also normal and/or pre-leukemic Hematopoietic Stem Cells (HSCs), this is not the case in CD34-GPR56+NKG2DL-enriched LSCs which thus can be isolated with high purity. Finally, we show that AML patients, who retain at time of diagnosis a reserve of normal and/or preleukemic HSCs in their bone marrow able to reconstitute immunocompromised mice, have significant longer relapse-free and overall survival compared to AML patients in whom functional HSCs are no longer detectable.

Background:

Rettig: WashU: Patents & Royalties: Patent Application 16/401,950. Karpova:WashU: Patents & Royalties: Patent Application 16/401,950. Ruminski:WahU: Patents & Royalties: Patent Application 16/401,950. Morrow:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. DiPersio:Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Equity Ownership; Incyte: Consultancy, Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Karyopharm Therapeutics: Consultancy; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; NeoImmune Tech: Research Funding.

Introduction:The integrin very-late antigen-4 (VLA-4) is comprised of an alpha4 chain (CD49d) that is non-covalently associated with the beta1 integrin chain (CD29). VLA-4 plays an important role in the homing and retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow (BM) microenvironment. We previously reported (Karpova et al. Blood 2016;128:659) a novel strategy and pathway for rapid and potent mobilization of HSPCs in mice using a combination of a VLA-4 inhibitor and the truncated isoform of the CXCR2 agonist Gro-beta (tGro-β, SB-251353). These studies were completed with a novel VLA-4 inhibitor, CWHM-823, that exhibited increased potency and/or solubility relative to the previously described comparators BIO5192 (α4β1-specific) and firategrast (α4β1 and α4β7 dual-specific). Here we extended these findings by generating and characterizing additional novel VLA-4 inhibitors.

No relevant conflicts of interest to declare.

Levesque: GlycoMimetics: Equity Ownership.

Introduction:

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.

Escot: Polyphor Ltd.: Employment. Douglas:Polyphor Ltd.: Employment. Romagnoli:Polyphor Ltd.: Employment. Chevalier:Polyphor Ltd.: Employment. Dembowsky:Polyphor Ltd.: Consultancy. Hooftman:Polyphor Ltd.: Employment. Bonig:Polyphor Ltd.: Research Funding.

Background:

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.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.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.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.Romagnoli: Polyphor Ltd.: Employment. Chevalier:Polyphor Ltd: Employment. Patel:Polyphor Ltd.: Employment.

Abstract 4100

CXCR4 mediates immature hematopoietic cell retention in marrow. As a G-protein coupled receptor, it is expected to rapidly adapt to continuously presented ligand concentrations. Thus how immature hematopoietic cells manage to retain sensitivity to its ligand, SDF-1, is unclear. Earlier studies demonstrated that perturbed CXCR4 internalization leads to abnormalities of mature and immature hematopoiesis. G-protein coupled receptor kinase 6 (GRK6) negatively modulates a different aspect of CXCR4 signalling, i.e. ligand-induced receptor desensitization. We therefore sought to explore immature murine hematopoiesis in mice deficient for GRK6.GRK6+/− mice were gifted by Robert Lefkowitz (Durham, NC). To generate GRK6−/− and WT littermates, GRK6+/− were bred and housed at the comparative medicine facility at Goethe University Frankfurt under non-SPF conditions. Young (8–12 wk) and old (60–100 wk) mice were studied. For transplantation, WT recipients were radio-conditioned with 950 cGy prior to i.v. transplantation of BM or spleen cells from WT and/or GRK6−/− donors. Stress hematopoiesis was induced with phenylhydrazine, 5-fluorouracil or sub-lethal irradiation. Mobilization was done with G-CSF or AMD3100, using standard dosing regimens. Where indicated, mice were splenectomized in deep anaesthesia using aseptic technique. All methods were approved by the IACUC. CBCs were done with an automatic hemacytometer. Progenitor cells were enumerated with cytokine-replete semi-solid media cultures. Surface and intracellular markers were studied by flow cytometry.GRK6−/− mice were fertile, had normal litter sizes, and pups were born at expected ratios. Weight and length were indistinguishable. GRK6−/− steady-state hematopoiesis was remarkable for lymphocytopenia in peripheral blood, more pronounced in young than in old mice. Circulating CFU-C were similar. Marrow cellularity and CFU-C contents and frequency increase with age was much more pronounced in WT mice. Marrow contents of LSK cells was the same for WT and GRK6−/− mice, young or old. Spleen size and leukocyte cellularity were similar. CFU-C contents of GRK6−/− spleens was almost two-fold greater. Accordingly, transplantation of similar numbers of WT or GRK6−/− CD45.2 spleen cells in competition with CD45.1 WT BM cells resulted in markedly higher donor chimerism for GRK6−/− than for WT cells. Despite this, splenectomy did not result in excessive numbers of circulating CFU-C in GRK6−/− mice. The immunophenotype of c-kit+ BM cells from WT and GRK6−/− mice was similar. Functionally, BM CFU-C were characterized by greater migration towards SDF-1 in transwell migration assays, as well as a greater sensitivity to lower doses of SDF-1. Erk1/2 phosphorylation was markedly stronger in SDF-1 treated GRK6−/− c-kit+ BM cells than in WT controls. This did not, however, translate into altered (improved) homing of transplanted cells to marrow or spleen, nor to altered engraftment kinetics. The same lymphocytopenia that was observed in naïve GRK6−/− mice was also seen 12 weeks after transplantation of WT mice with GRK6−/− cells, indicating cell-intrinsic effects. Serial competitive engraftment assays uncovered a modest engraftment defect; the kinetics of the loss of GRK6−/− contribution to hematopoiesis suggested defects in early niche interaction, but not in retention. Several additional models of stress hematopoiesis were also tested, including mobilization with G-CSF or the CXCR4 antagonist AMD3100, phenylhydrazine, 5-fluorouracil or sub-lethal irradiation; in all cases the response of GRK6−/− mice and WT mice was the same, i.e. did not uncover any marked defects associated with defective SDF-1 signalling.Given the prominent role of CXCR4 in immature hematopoiesis and observed in vitro hyperresponsiveness of GRK6−/− cells in response to SDF-1, the hematological phenotype of the GRK6−/− mouse is surprisingly modest. The data suggest that ligand-induced receptor phosphorylation is not a key modulator of SDF-1 responsiveness of hematopoietic stem/progenitor cells in the hematopoietic niche in marrow. Specifically, the data also add to the body of evidence indicating that the strength of in vitro SDF-1 responsiveness of cells does not predict homing efficiency nor engraftment. We conclude that GRK6 is a non-essential molecule for in vivo hematopoiesis.No relevant conflicts of interest to declare.

Abstract 2381