Anti-CD117 antibody depletes normal and myelodysplastic syndrome human hematopoietic stem cells in xenografted mice

Abstract

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.

Graphical abstract

Figure 1.

Anti-human CD117 mAb SR-1 blocks SCF binding to CD117 on human HSCs and inhibits human HSC proliferation in vitro. (A) Relative CD117 expression of HSCs (Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻) and progenitors (Lin⁻CD34⁺) from normal BM samples and MDS BM samples from all IPSS-R risk groups. CD117 expression was assessed by mean fluorescence intensity (MFI) by flow cytometry using a commercially available anti-human CD117 mAb, clone 104D2. MFI was normalized to BM CD3⁺ T cells from healthy subjects, which served as staining control. *P < .001 compared with control CD3⁺ T cells (Student t test). (B) Pretreatment with SR-1 mAb inhibits binding of fluorescently labeled SCF (SCF-A488) to human CD117. A mixture of CD117-expressing and untransfected fibroblasts were stained with SR-1 mAb (left), SCF-A488 alone (center), or first stained with SR-1 and then subsequently stained with SCF-A488 (right). SR-1 mAb was detected using a fluorescently labeled goat-anti-mouse secondary mAb. (C) SR-1 mAb inhibits proliferation of FACS-purified human UCB-derived HSCs (Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻) in liquid culture. UCB HSCs were plated via plate-sorting at a density of 25 cells per well in StemSpan media supplemented with SCF, TPO, FLT3L, IL-3, and IL-6, and treated with varying concentrations of SR-1. Cell counts were performed on days 2, 4, and 5 postplating. *P < .001 comparing 100 μg/mL, 10 μg/mL, and 1 μg/mL conditions compared with untreated on day 7 (Student t test). Error bars indicate 1 standard error of the mean. (D) SR-1 mAb inhibits proliferation of FACS-purified human BM-derived HSCs (Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻) in liquid culture. BM HSCs were plated via plate sorting at a density of 25 cells per well in StemSpan media supplemented with SCF, TPO, FLT3L, IL-3, and IL-6, and treated with varying concentrations of SR-1. Cell counts were performed on days 2, 4, 5, and 7 postplating. *P < .001 comparing 100 μg/mL, 10 μg/mL, 1 μg mL, and 0.1 μg/mL conditions compared with untreated on day 7 (Student t test). Error bars indicate 1 standard error of the mean. (E) SR-1 mAb inhibits colony formation of FACS-purified human UCB-derived HSCs (Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻) in methylcellulose supplemented with G-CSF and treated with indicated concentrations of SR-1. CFU-GM colony counts were performed 2 weeks after culture initiation. Representative images of the colonies present at 2 weeks are shown (for untreated and 1 μg/mL SR-1). *P < .001 compared with day 0 (Student t test). Error bars indicate 1 standard error of the mean.

Figure 2.

Anti-human CD117 antibodies deplete normal and MDS HSCs in vivo. (A) SR-1 depletes normal human UCB-derived HSCs in vivo, as shown by percent depletion from baseline of human myeloid (human CD45⁺CD13/33⁺) chimerism on day 8 and 8 weeks after completion of SR-1 treatment; baseline human myeloid chimerism was determined on day 0, prior to treatment. FACS-purified human UCB HSCs (Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻) were transplanted into sublethally irradiated NSG pups and baseline myeloid chimerism was obtained on day 0 of the second graft administration, which was ∼12 weeks after initial establishment of xenografts. Xenografted mice were treated with 500 μg of SR-1 or control isotype IgG antibody administered IV on day 1, day 3, day 5, and day 7. *P < .001 compared with IgG control at same time point (Student t test) (n = 3, IgG treated; n = 3, SR-1 treated). (B) SR-1 depletes low-risk MDS HSCs in vivo, as shown by percent depletion from baseline of human myeloid (human CD45⁺CD13/33⁺) chimerism on day 8 and 8 weeks after completion of SR-1 treatment; baseline human myeloid chimerism was determined on day 0, prior to treatment. FACS-purified low-risk MDS HSCs (Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻) were transplanted into sublethally irradiated NSG pups and baseline myeloid chimerism was obtained on day 0 of the second graft administration, which was ∼12 weeks after initial establishment of xenografts. Xenografted mice were treated with 500 μg of SR-1 or control isotype IgG antibody administered IV on day 1, day 3, day 5, and day 7. *P < .001 compared with IgG control at same time point (Student t test) (n = 4, IgG treated; n = 4, SR-1 treated). (C) AMG 191 depletes low-risk MDS HSCs in vivo, as shown by human myeloid (human CD45⁺CD13/33⁺) chimerism on prior to treatment on day 0 and then at day 21 and 12 weeks after completion of treatment with AMG 191. FACS-purified low-risk MDS HSCs (Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻) were transplanted into sublethally irradiated NSG pups and baseline myeloid chimerism was obtained on “day 0,” which was ∼12 weeks after initial establishment of xenografts. Xenografted mice were treated with 75 μg of AMG 191 administered IV on day 1. *P < .001 compared with pretreatment day 0 (Student t test; n = 6, AMG 191 treated).

Figure 3.

SR-1 permits engraftment of second human UCB HSC graft in low-risk MDS-xenografted mice. (A) Total human chimerism (human CD45⁺) as well as distribution of myeloid (human CD45⁺CD13/33⁺), B-cell (human CD45⁺CD19⁺), and T-cell (human CD45⁺CD3⁺) chimerism in BM of low-risk MDS-xenografted mice treated with control isotype IgG antibody or SR-1 mAb on day 1, 3, 5, and 7, and then transplanted on day 15 with a second normal human UCB HSC graft. Chimerism was measured 12 weeks after second normal human UCB HSC transplant (n = 4, IgG treated; n = 4, SR-1 treated). (B) Frequency of cytogenetically abnormal clones (−Y, del(20q), or +8), as detected by FISH within human cells FACS-isolated from BM of low-risk MDS-xenografted mice treated with IgG control antibody or SR-1 mAb on days 1, 3, 5, and 7 and then transplanted on day 15 with a second normal human UCB HSC graft. Cells were analyzed 12 weeks after second normal human UCB HSC transplant (n = 4, IgG treated; n = 4, SR-1 treated).

Figure 4.

SR-1 transiently depletes high-risk MDS cells and permits engraftment of second human UCB HSC graft in high-risk MDS-xenografted mice. (A) SR-1 transiently depletes human blood cells derived from high-risk MDS HSCs in vivo, as shown by chimerism of human myeloid (hCD13/CD33⁺) cells on pretreatment day 0 and 8 days and 8 weeks after completion of treatment with 500 μg of SR-1 administered IV on days 1, 3, 5, and 7. *P < .001 compared with day 0 (Student t test) (n = 4, SR-1 treated). (B) Total human chimerism (human CD45⁺) as well as distribution of myeloid (human CD45⁺CD13/33⁺), B-cell (human CD45⁺CD19⁺), and T-cell (human CD45⁺CD3⁺) chimerism in BM of high-risk MDS-xenografted mice treated with SR-1 mAb on days 1, 3, 5, and 7, and then transplanted on day 15 with a second normal human UCB HSC graft. Chimerism was measured 12 weeks after second normal human UCB HSC transplant (n = 4, SR-1 treated). (C) Frequency of cytogenetically abnormal clone (−7, del(5q), or +8), as detected by FISH within human cells FACS-isolated from BM of high-risk MDS-xenografted mice treated with SR-1 mAb on days 1, 3, 5, and 7 and then transplanted on day 15 with a second normal human UCB HSC graft. Cells were analyzed 12 weeks after second normal human UCB HSC transplant (n = 4, SR-1 treated).