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Graphical Abstract Highlights d Six antibodies suppress a mouse's HSCs, T cells, and NK cells in 8 days d Antibody treatment enables HSC transplants without radiation and/or chemotherapy d Transplants can be performed with fully MHC mismatched donors d Following HSC transplants, animals tolerate solid organs from the same donor

The myelodysplastic syndromes (MDS) represent a group of clonal disorders that result in ineffective hematopoiesis and are associated with an increased risk of transformation into acute leukemia. MDS arises from hematopoietic stem cells (HSCs); therefore, successful elimination of MDS HSCs is an important part of any curative therapy. However, current treatment options, including allogeneic hematopoietic cell transplantation (HCT), often fail to ablate disease-initiating MDS HSCs, and thus have low curative potential and high relapse rates. Here, we demonstrate that human HSCs can be targeted and eliminated by monoclonal antibodies (mAbs) that bind cell-surface CD117 (c-Kit). We show that an anti-human CD117 mAb, SR-1, inhibits normal cord blood and bone marrow HSCs in vitro. Furthermore, SR-1 and clinical-grade humanized anti-human CD117 mAb, AMG 191, deplete normal and MDS HSCs in vivo in xenograft mouse models. Anti-CD117 mAbs also facilitate the engraftment of normal donor human HSCs in MDS xenograft mouse models, restoring normal human hematopoiesis and eradicating aggressive pathologic MDS cells. This study is the first to demonstrate that anti-human CD117 mAbs have potential as novel therapeutics to eradicate MDS HSCs and augment the curative effect of allogeneic HCT for this disease. Moreover, we establish the foundation for use of these antibody agents not only in the treatment of MDS but also for the multitude of other HSC-driven blood and immune disorders for which transplant can be disease-altering.

Key Points After TLI, donor blood stem cells first engraft in irradiated marrow and gradually redistribute into unexposed sites. Long-term donor cell engraftment requires the presence of regulatory host cells that promote host stem-cell cycling.

4099 Engraftment of allogeneic hematopoietic stem cells (HSC) requires conditioning to overcome immunologic and anatomic barriers preventing access to the marrow niche. Most patients who undergo allogeneic hematopoietic cell transplantation (allo-HCT) are prepared with cytotoxic chemotherapy and/or radiation to eliminate these barriers, and to facilitate eradication of malignant cells, if present. Many non-malignant conditions, such as primary immunodeficiencies, hemoglobinopathies, and autoimmune diseases may be successfully treated by transplantation of allogeneic HSC, but the toxicity of conventional conditioning regimens is, in many cases, prohibitive. Targeted elimination of barriers to the HSC niche would be a preferable approach. Signaling via the c-Kit receptor (CD117) is critical for the maintenance of pluripotent HSC. Anti-CD117 monoclonal antibodies (mAbs) deplete HSC and facilitate engraftment of donor HSC in a mouse model of severe combined immunodeficiency (SCID) (Czechowicz et al., Science, 2007). Patients with SCID are highly susceptible to infections, but also have limited immunologic barriers to alloengraftment, making this patient population ideal for studying targeted stem cell depletion to facilitate allo-HSC engraftment. We identified a clinical grade humanized anti-human CD117 mAb (anti-hCD117) as a potential candidate for this purpose. Anti-hCD117 significantly inhibited mitosis in human cord blood and bone marrow derived HSC (Lin−CD34+CD38−CD90+CD45RA−) in liquid and methylcellulose culture containing Flt3 ligand, stem cell factor (SCF), thrombopoietin (TPO), IL-3, and IL-6. To assess in vivo activity of anti-hCD117, we employed it alone, or in combination with alemtuzumab (anti-CD52), to deplete human stem and differentiated cells from hematopoietically humanized NOD/scid/IL2Rg−/− (HuNSG) mice. Pups were conditioned with 100cGy and then humanized by injection of 2000–4000 human HSC into the facial vein on day p2 or intrahepatically on day p4–5. After permitting hematopoietic stabilization for 4–6 months, we confirmed multi-lineage xenochimerism in the peripheral blood (PB) and bone marrow (BM) prior to mAb treatment. After a single treatment with anti-hCD117, mice were depleted of total human leukocytes a median 60% (35–100%; n=11) in the PB and 100% (84–100%; n=10) in the BM at 6 weeks after treatment, with >80% depletion of human myeloid cells in both compartments. Partial recovery of human chimerism was observed at 16 weeks, consistent with recovery of some LT-HSC after anti-hCD117 therapy. The addition of anti-CD52 facilitated clearance of human lymphoid cells not eradicated by anti-hCD117. Human HSC and progenitor cells (Lin−CD34+CD117+; HS/PC) in the bone marrow decreased from 0.4% (0–1.7%) to 0% (0–0.1%; n=10) 6 weeks after treatment with anti-hCD117. We then modeled a human transplant by treating HuNSG mice with anti-hCD117, anti-CD52, or both, to deplete their primary human graft. After monitoring mAb catabolism by ELISA, mice received a second (non-HLA matched) human CD34+ HS/PC graft modified to express the green fluorescent protein using a lentivector. After overnight prestimulation in XVIVO-15 supplemented with SCF, Flt3 ligand, TPO, and IL-3, human CD34+ HS/PC were exposed for 18 hours to lentivector at 1×108 TU/mL. Cells were washed and 80,000 transduced CD34+ HS/PC were injected IV into untreated and mAb-conditioned HuNSG mice. After 6 weeks, PB was evaluated and demonstrated GFP+hCD45+cells in 3/5 (60%) mice treated with anti-hCD117 + anti-CD52, 0/5 mice treated with either anti-hCD117 or anti-CD52 alone, and 1/5 untreated mice. Anti-hCD117 is a promising reagent for depletion of human HSC and facilitation of allo-HSC engraftment. Although anti-hCD117 alone capably depletes human CD34+CD117+ HS/PC and myeloid chimerism in HuNSG mice, the addition of anti-CD52 facilitates engraftment, possibly by reducing alloreactive rejection by T cells from the primary graft. Additional HuNSG mice are receiving second human transplants following mAb conditioning to further explore the utility of combining anti-hCD117 and anti-CD52 for this purpose. These studies will lead the way to minimally toxic allogeneic HSC transplant regimen, and in a broader view, to the application of targeted biological therapies that deplete endogenous stem cells and facilitate their replacement with allogeneic or gene-corrected stem cells. Disclosures: Thway: Amgen, Inc.: Employment. Magana:Amgen, Inc.: Employment. Weissman:Amgen, Inc.: Equity Ownership.