doi: 10.1182/blood-2018-06-853879.
Epub 2019 Jan 7.
Anti-human CD117 antibody-mediated bone marrow niche clearance in nonhuman primates and humanized NSG mice
Affiliations
- PMID: 30617195
- PMCID: PMC6509543
- DOI: 10.1182/blood-2018-06-853879
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Anti-human CD117 antibody-mediated bone marrow niche clearance in nonhuman primates and humanized NSG mice
Blood.
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No abstract available
Conflict of interest statement
Conflict-of-interest disclosure: I.L.W. and A. Czechowicz are inventors on patents that include the use of anti-CD117 antibodies in HCT conditioning, and I.L.W., J.A.S., and A. Chhabra are inventors on patents that pair anti-CD47 agents and anti-CD117 antibodies for HCT conditioning. I.L.W. is a cofounder, stockholder, and Director of Forty Seven, Inc, which has licensed these patents from Stanford University. J.A.S., A. Chhabra, and S.S.P. have equity ownership in Forty Seven, Inc. A. Czechowicz has equity ownership in Forty Seven, Inc; Magenta Therapeutics; Beam Therapeutics; Editas Medicines; and Global Blood Therapeutics. The remaining authors declare no competing financial interests.
Figures
Effects of in vivo administration of AMG 191 on CD34+ HSPCs and peripheral blood of cynomolgus macaques. (A) AMG 191 is cleared in the serum of NHPs in a dose-dependent manner. The highest levels of AMG 191 in serum of all animals were observed at the first time point collection (5 minutes after dose administration on day 1). Antibody levels measured on day 4 in the 0.1 mg/kg group fell below the assay detection limit of 50 ng/mL. The level of AMG 191 in serum was analyzed and terminal elimination half-life (t1/2) was determined. The averages of half-life (t1/2) obtained from 2 animals of each group are presented. (B) Representative fluorescence-activated cell sorter (FACS) plot showing CD34+ cells in BM of NHP #4 on days 0 (before AMG 191 treatment), 10, and 42 postadministration of 1.0 mg/kg AMG 191. CD34+ cells were gated from live cells. (C) Frequency of CD34+ cells among live cells in BM aspirates obtained from each NHP. BM aspirates were collected from individual NHPs (2 animals for each dose group) prior to the administration of AMG 191 (baseline, designated as day 0), and on days 4, 7, 10, 21, and 42 postadministration. AMG 191 was infused into animals on day 1. *Note: There was a technical inconsistency with the staining of the baseline cell samples collected from NHPs #5 to #8 (day 0). Red cell lysis was performed after the cells were stained, leading to a lower baseline of the CD34+ frequency of the stained populations. In all subsequent analyses, red cell lysis was performed before staining with the marker antibodies. Colors correspond to days relative to infusion. Animal identification (#1-#8) and dose level are shown on the x-axis. (D) RBC parameters affected by AMG 191 treatment. Hemoglobin and absolute reticulocyte count from individual NHPs taken before treatment (day 0) and postinfusion of AMG 191. Colored lines correspond to individual NHPs with doses as shown in the legend on the right. NHPs #3 to #8 had statistically significant decreases in hemoglobin from baseline compared with day 21 (P = .001; paired Student t test).
AMG 191 depletes human HSPCs engrafted in NSG mice and second human donor engraftment level correlates with AMG 191 dose administered. (A) Representative FACS plots showing hCD34+hCD117+ cell population gated on hCD45+ cells present in BM aspirates obtained from humanized NSG mice before (Pre-AMG 191) and 2 weeks posttreatment of AMG 191 (1.0 mg/kg; Post-AMG 191). (B) Frequency of human hematopoietic progenitor cells (hCD45+hCD34+hCD117+) among all live cells present in BM aspirates obtained from humanized NSG mice treated with 0.3 mg/kg (left panel) and 1.0 mg/kg AMG 191 (right panel). P values were obtained using the paired Student t test. (C) A second human donor cell engraftment is facilitated by AMG 191 conditioning. Pooled cord blood CD34+ cells transduced with mCitrine-expressing lentivirus were transplanted into unconditioned and AMG 191–treated humanized NSG mice (0.3 and 1.0 mg/kg, see supplemental Materials and methods for details). Second donor grafts were infused on days 21 and 25 post–AMG 191 administration, days when the PK level was predicted to fall below 2000 ng/mL (supplemental Figure 5). The choice of this threshold level was based on PK studies in mice that showed that serum levels <2200 ng/mL of the anti-mouse CD117 antibody had no effect on donor mouse HSC engraftment (supplemental Figure 6). Six weeks after transplantation, chimerism was accessed in BM of transplanted humanized NSG mice. Secondary engraftment was measured by the frequency of mCitrine-expressing cells in each cell subset. Cell frequency was analyzed by FACS and FlowJo software. P values were obtained using the unpaired Student t test and Prism software. Data and error bars in panels B and C represent the mean plus or minus standard error of the mean (sem).
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