Abstract 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 (LinCD34+CD38CD90+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 (LinCD34+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.


Thway:Amgen, Inc.: Employment. Magana:Amgen, Inc.: Employment. Weissman:Amgen, Inc.: Equity Ownership.

Author notes


Asterisk with author names denotes non-ASH members.