Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation

Abstract

Hematopoietic stem cell transplantation (HSCT) is a curative therapy for blood and immune diseases with potential for many settings beyond current standard-of-care. Broad HSCT application is currently precluded largely due to morbidity and mortality associated with genotoxic irradiation or chemotherapy conditioning. Here we show that a single dose of a CD117-antibody-drug-conjugate (CD117-ADC) to saporin leads to > 99% depletion of host HSCs, enabling rapid and efficient donor hematopoietic cell engraftment. Importantly, CD117-ADC selectively targets hematopoietic stem cells yet does not cause clinically significant side-effects. Blood counts and immune cell function are preserved following CD117-ADC treatment, with effective responses by recipients to both viral and fungal challenges. These results suggest that CD117-ADC-mediated HSCT pre-treatment could serve as a non-myeloablative conditioning strategy for the treatment of a wide range of non-malignant and malignant diseases, and might be especially suited to gene therapy and gene editing settings in which preservation of immunity is desired.

Fig. 1

One-time, intravenous treatment with CD117-ADC potently depletes hematopoietic stem cells (HSCs). a Experimental outline for assessing the ability of antibody-drug-conjugates to deplete HSCs in immunocompetent, wild-type C57BL/6 mice. HSCs and progenitors were assessed through flow cytometric (FACS) phenotypic analysis and colony-forming cell-forming assays, and in vivo reconstitution potential was assessed by competitive transplantation assays. b Representative FACS plots of bone marrow from untreated control and CD117-ADC-treated animals (2B8 clone bound to saporin toxin). c, d Dose-dependent phenotypic depletion of HSCs (Lin−cKit+Sca1+CD150+CD48−) by CD117-ADC and lack thereof by naked CD117 mAb as assessed 8 days after IV administration, displaying decreased HSC frequency (c) and total HSC number (d). Non-treated mice served as controls. e, f In vivo depletion of HSCs affirmed by lack of long-term reconstitution activity of treated WBM as assessed by donor total blood chimerism (e) and donor granulocyte chimerism (f) in competitive transplantation assays. Statistics calculated using unpaired t test. Data represent mean ± SEM. (n = 3–5 mice/group, assayed individually); all data points significant as indicated vs. untreated control (**P < 0.01, ***P < 0.001, ****P < 0.0001)

Fig. 2

CD117-ADC conditioning durably and robustly enhances donor murine whole bone marrow (WBM) engraftment. a Experimental outline for assessing the ability of antibody-drug-conjugates to condition immunocompetent, wild-type C57BL/6 mice allowing for efficient engraftment of donor murine WBM. b CD117-ADC pre-treatment 8 days before infusion of 10 × 10⁶ CD45.1+ donor whole bone marrow cells leads to robust turnover of recipient peripheral blood as assessed through flow cytometric (FACS) analysis unlike naked CD117 pre-treatment. Representative FACS plots of peripheral blood showing >80% total donor peripheral blood CD45.1+ cells in otherwise CD45.2+ host. c CD117-ADC conditioning with WBM transplantation results in rapid multi-lineage engraftment with kinetics paralleling lifespan of cell populations. d Almost complete donor HSC (Lin−cKit+Sca+CD150+CD48−) and HPC (Lin−cKit+Sca+) chimerism post CD117-ADC treatment and donor WBM transplantation confirmed by phenotypic bone marrow analysis of transplanted animal 20 weeks post transplantation. Representative FACS plots of bone marrow HSCs showing >99.9% donor HSC chimerism. e Donor HSC engraftment confirmed through secondary transplantation of WBM into lethally irradiated recipients. Data represent mean ± SEM (n = 5 mice/group assayed individually). Statistics calculated using unpaired t test; all data points significant as indicated vs. untreated control (****P < 0.0001)

Fig. 3

CD117-ADC conditioning leads to comparable total multi-lineage engraftment as alternative conditioning regimens post WBM transplantation with improved engraftment kinetics. a CD117-ADC conditioning pre-WBM transplantation leads to comparable total peripheral blood chimerism as CD45.2-ADC and 5 Gy TBI conditioning. Robust multi-lineage donor engraftment with rapid donor granulocyte chimerism (b) and slower donor B-cell (c) and T-cell turnover (d). e–g No significant leukopenia observed post CD117-ADC treatment and WBM transplantation, as opposed to CD45.2-ADC and 5 Gy TBI conditioning with grossly normal absolute myeloid cells (e), absolute B-cells (f), and absolute T-cells (g). Data represent mean ± SEM (n = 5 mice/group, assayed individually). Statistics calculated using unpaired t test; all data points significant as indicated vs. untreated control (**P < 0.01; ***P < 0.001; ****P < 0.0001)

Fig. 4

CD117-ADC conditioning effectively enhances purified mouse and human HSC engraftment, with increased HSC cell dose resulting in increased multi-lineage reconstitution. a Experimental outline for assessing the ability of antibody-drug-conjugates to condition immunocompetent, wild-type C57BL/6 mice allowing for efficient engraftment of donor murine HSC. b, c CD117-ADC pre-treatment 8 days before infusion of FACS-purified CD45.1+ donor HSCs leads to robust total donor peripheral blood (b) and donor granulocyte chimerism (c). Rapid donor B-cell (d) and T-cell (e) reconstitution post transplantation of purified HSCs into CD117-ADC-conditioned animals as compared to 5 Gy TBI controls. f Similarly enhanced donor HSPC (Lin−cKit+Sca+) and HSC (Lin−cKit+Sca+CD150+CD48−) chimerism post CD117-ADC treatment and transplantation of 2000 purified HSCs confirmed by phenotypic bone marrow analysis of transplanted animal 20 weeks post transplantation. g Donor HSC engraftment confirmed through secondary transplantation of WBM into lethally irradiated recipients. h Experimental outline for assessing the ability of antibody-drug-conjugates to condition immunocompromised, NSG mice for efficient engraftment of donor human CD34+ cord blood cells. i Single treatment of CD117-ADC effectively enhances donor human cord blood HSPC engraftment in NSG mice, enabling creation of irradiation-free xenografts with multi-lineage reconstitution with total donor chimerism nearing similar levels as 2 Gy TBI. j Multi-lineage human chimerism observed in all xenografted mice, with B-cell engraftment predominant regardless of the conditioning method. Data represent mean ± SEM (n = 3–5 mice/group, assayed individually). Statistics calculated using unpaired t test; all data points significant as indicated vs. untreated control (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

Fig. 5

CD117-ADC is uniquely non-ablative to the peripheral blood with no clinically significant cytopenias, and results in preservation of phenotypic and functional immunity. a, b Despite HSC depletion, only very minor and not clinically significant decreases in hemoglobin (a) and platelet counts (b) were observed 8 days post CD117-ADC treatment. c Unlike TBI, ADC treatments did not lead to neutropenia, and myeloid counts were increased 8 days post CD117-ADC treatment at the time of transplantation. Unlike CD45.2-ADC, peripheral B-cell (d) and T-cell counts (e) remained largely intact 8 days post administration of CD117-ADC. f T-cell numbers not only remained intact post CD117-ADC treatment, but animals were uniquely able to mount viral immune responses to LCMV infection post CD117-ADC as indicated in the experimental outline with results showing present LCMV-specific activated CD8 T-cells. g Additionally, post CD117-ADC treatment LCMV-specific activated CD8 T-cells generated from prior LCMV infection uniquely remained, as indicated in the experimental outline with results showing similar numbers of LCMV-specific activated CD8 T-cells CD117-ADC similar to post no conditioning. h Animals were also able to mount functional immune response to Candida challenge post CD117-ADC treatment, as indicated in the experimental outline with animals showing persistent neutrophils, control of Candida load, and increased survival compared to other treatment groups. Data represent mean ± SEM (n = 3–5 mice/group, assayed individually). Statistics calculated using unpaired t test; all data points significant as indicated vs. untreated control (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

Fig. 6

Human CD117-ADC inhibits human HSCs in vitro and robustly depletes human HSCs in vivo. Human CD117-ADCs were prepared similarly by conjugating biotinylated anti-human CD117 (104D2 clone) with streptavidin–saporin toxin. a Human CD117-ADC inhibited human cord blood growth in vitro, as compared to unconjugated antibody controls. b Experimental outline for assessing the ability of anti-CD117 antibodies and antibody-drug-conjugates to deplete human HSCs in xenograft mice. c Only antibody conjugated to saporin is effective at eliminating human myeloid cells. Data represent mean ± SEM (n = 3–5 samples/group, assayed individually). Statistics calculated using unpaired t test; all data points significant as indicated vs. unconjugated antibody-treated control animals (*P < 0.05; **P < 0.01; ****P < 0.0001)