Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin

Journal name:
Nature Biotechnology
Volume:
34,
Pages:
738–745
Year published:
DOI:
doi:10.1038/nbt.3584
Received
Accepted
Published online

Abstract

Hematopoietic stem cell transplantation (HSCT) offers curative therapy for patients with hemoglobinopathies, congenital immunodeficiencies, and other conditions, possibly including AIDS. Autologous HSCT using genetically corrected cells would avoid the risk of graft-versus-host disease (GVHD), but the genotoxicity of conditioning remains a substantial barrier to the development of this approach. Here we report an internalizing immunotoxin targeting the hematopoietic-cell-restricted CD45 receptor that effectively conditions immunocompetent mice. A single dose of the immunotoxin, CD45–saporin (SAP), enabled efficient (>90%) engraftment of donor cells and full correction of a sickle-cell anemia model. In contrast to irradiation, CD45–SAP completely avoided neutropenia and anemia, spared bone marrow and thymic niches, enabling rapid recovery of T and B cells, preserved anti-fungal immunity, and had minimal overall toxicity. This non-genotoxic conditioning method may provide an attractive alternative to current conditioning regimens for HSCT in the treatment of non-malignant blood diseases.

At a glance

Figures

  1. CD45-SAP has potent cell-depletion activity.
    Figure 1: CD45–SAP has potent cell-depletion activity.

    (a) Experimental outline for assessing ability of immunotoxins to deplete HSCs in immunocompetent C57BL/6 mice. HSCs were assessed by flow cytometry (LincKit+Sca1+CD48CD150+) and progenitor colony forming cells (CFCs) were assessed by colony forming assay. (b) Dose-dependent effects of CD45–SAP on HSCs and CFCs, assessed 8 d after administration in C57BL/6 mice. Non-treated mice served as the control. Data represent mean ± s.d. (n = 30 mice, 5 mice/group, assayed individually); all data points significant vs. control (P > 0.05). (c) CD45–SAP depletes HSCs in C57BL/6 mice whereas non-biotinylated CD45 antibody in the presence of streptavidin–SAP does not. Data represent mean ± s.d. (n = 5 mice/group, one of two independent experiments shown). (d) CD45–SAP clone 104 kills EML progenitor cells in vitro (72 h incubation) whereas non-biotinylated antibody in the presence of streptavidin–SAP does not affect cell viability. Data represent mean ± s.d. (n = 3 technical replicates) of one of three independent experiments. (e) Percent internalization of clone 104 antibody (Ab alone) or antibody–streptavidin complex (Ab–streptavidin) in EL4 cells over 24 h in vitro culture. Data represent mean ± s.d. of a single experiment with n = 6 replicates. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant (P > 0.05). Statistics calculated using unpaired t test.

  2. CD45-SAP enables efficient donor-cell engraftment.
    Figure 2: CD45–SAP enables efficient donor-cell engraftment.

    (a) Experimental outline for assessing transplantation window following 3 mg/kg CD45–SAP conditioning of C57BL/6 mice and transplantation of either CD45.1 or CD45.2–GFP 107 bone marrow cells. (b) Peripheral blood donor chimerism 4 months after transplantation of CD45.2 GFP or CD45.1 cells injected at various time points after 3 mg/kg CD45–SAP conditioning of C57BL/6 mice. Control represents non-conditioned mice receiving transplant. Data represent mean ± s.d. (n = 30 mice per donor cell type, 5 mice/time point, assayed individually); all data points significant vs. control (P < 0.05). (c) Representative flow cytometry plots illustrating donor cells in peripheral blood 8 months after transplantation in control or CD45–SAP conditioned C57BL/6 mice. (d) Long-term assessment of peripheral blood chimerism following CD45.2-GFP cell transplantation 8 d post CD45–SAP conditioning; all data points significant vs. control (P < 0.05). Data represent mean ± s.d. (n = 5 mice/group, assayed individually). (e) Contribution of donor cells to peripheral myeloid, B and T cells in CD45–SAP conditioned C57BL/6 mice 8 months after transplantation versus overall lineage distribution in non-treated control mice. Data represent mean ± s.d. (n = 5 mice/group, assayed individually) of one of two independent experiments. (f) Donor peripheral myeloid chimerism 4 months after transplantation of 2,000 purified HSCs (LKS CD48CD150+ or LKS CD34CD150+) in non-conditioned control and CD45–SAP conditioned C57BL/6 mice. Data represent mean ± s.e.m. (n = 5 mice/group, 2 independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant (P > 0.05). Statistics calculated using unpaired t test.

  3. Differential effects of CD45-SAP and irradiation on bone marrow.
    Figure 3: Differential effects of CD45–SAP and irradiation on bone marrow.

    (a) Relative bone marrow cellularity (total nucleated cells extracted from femur and tibia) at various time points after 3 mg/kg CD45–SAP or 5Gy TBI. (b) Relative CFC activity of bone marrow cells harvested from C57BL/6 mice at various times post 3 mg/kg CD45–SAP or 5Gy TBI. (c) Relative immunophenotypic quantification of HSCs in bone marrow harvested from C57BL/6 mice at various times post 3 mg/kg CD45–SAP or 5Gy TBI. Data in a, b, and c represent mean percentage relative to non-treated mice ± s.e.m. (n = 12 mice/group, n = 4 mice per time point, assayed individually). (d) Non-treated C57BL/6 control mice, or mice treated with 3 mg/kg CD45–SAP or 5Gy TBI (2 d after conditioning) were i.v. injected with high molecular weight (2 MDa) rhodamine–dextran to assess calvaria vascular integrity using intravital microscopy. Calvaria bone surface is shown in blue and rhodamine–dextran in red. Scale bars, 100 μm. Representative images captured 20 min after rhodamine–dextran administrations from independent experiments (n = 2 mice/group) are shown. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. indicates not significant (P > 0.05). Statistics calculated using unpaired t test.

  4. Differential effects of CD45-SAP and irradiation on blood and thymus.
    Figure 4: Differential effects of CD45–SAP and irradiation on blood and thymus.

    (a) Relative levels of peripheral myeloid cells in (non-transplanted) C57BL/6 mice at various times post 3 mg/kg CD45–SAP or 5Gy TBI. Data represent mean percentage relative to non-treated control mice ± s.e.m. (n = 20 mice/group, n = 4 mice per time point, assayed individually). (b) Kaplan-Meier survival curve following systemic Candida albicans infection 2 d after conditioning and in non-conditioned C57BL/6 control mice (n = 10 mice/group). (c) Relative levels of peripheral CD3+ T cells at various times after administration of 3 mg/kg CD45–SAP or 5Gy TBI in (non-transplanted) C57BL/6 mice. Data represent mean percentage relative to non-treated control mice ± s.e.m. (n = 20 mice/group, n = 4 mice per time point, assayed individually). (d) Hematoxylin and eosin staining of thymus (500 μm scale bars) and thymic cortex (50 μm scale bars) from non-treated control C57BL/6 mice and 3 mg/kg CD45–SAP or 5Gy TBI conditioned mice 2 d after conditioning. (e) Absolute number of T-cell receptor excision circles (TRECs) per mg of thymus tissue 3 d after conditioning with 3 mg/kg CD45–SAP or 5Gy TBI. Control represents non-treated mice. Data represent mean ± s.d. (n = 4 mice/group, assayed individually). *P < 0.05; **P < 0.01; ***P < 0.001; n.s. indicates not significant (P > 0.05). Statistics calculated using unpaired t test.

  5. Correction of sickle cell disease.
    Figure 5: Correction of sickle cell disease.

    (a) Experimental outline for CD45–SAP conditioning and transplantation in sickle disease mice. CD45–SAP dosing (in mg/kg) schedule is shown and BM represents transplantation of 10 million wild-type bone marrow cells. (b) Donor myeloid chimerism 4 months after transplantation of sickle mice transplanted under the conditions in a. Data represent the mean ± s.d. (n = 18 mice, n = 6 mice/group, assayed individually). (c) Assessment of red blood cell (RBC), hemoglobin (Hgb), hematocrit (Hct), and reticulocyte (Retic) numbers in wild-type control mice, sickle disease mice and group A (corrected sickle mice) 4 months after transplantation. Data represent the mean ± s.e.m. (n = 6 mice/group, assayed individually). (d) Native-PAGE analysis of normal (Hba) and sickle (Hbs) hemoglobin protein in blood from wild-type control mice, sickle mice and group A mice (two representative mice from each group). Statistics calculated using unpaired t test. Blot of additional groups shown in Supplementary Figure 10f. (e) Representative peripheral blood smears of wild-type mice, sickle disease mice, and group A mice, with sickle cells indicated by arrows (20 μm scale bars). (f) Representative spleens from wild-type control mice, sickle mice and group A mice (two representative mice from each group). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant (P > 0.05).

  6. Cell depletion activity of immunotoxins
    Supplementary Fig. 1: Cell depletion activity of immunotoxins

    (a) In vivo HSC depletion screen of candidate immunotoxins administered at 3 mg/kg in C57BL/6 mice. Bone marrow was harvested 8 days post-administration and HSCs quantified by flow cytometry. Non-treated C57BL/6 mice were used as controls. Data represent mean ± range (n = 2 mice/group, assayed individually). (b) Investigation of various ratios of biotinylated anti-CD45 antibody (clone 104) to streptavidin-saporin on in vivo HSC depletion activity assessed 8 days post-administration in C57BL/6 mice. Data represent mean ± SD (n = 4 mice/group, assayed individually). (c) Peripheral blood chimerism 4 months after competitive transplantation of bone marrow harvested from control or CD45-SAP conditioned mice demonstrates depletion of functional HSCs by CD45-SAP. Data represent mean ± SD (n = 5 mice/group, assayed individually). (d) CD45-SAP clone 104 does not deplete HSCs in congenic CD45.1 C57BL/6 mice. Data represent mean ± SD (n = 5 mice/group, assayed individually). (e) In vitro IC50 values against EL4 and EML cell lines after 72 hour incubation with CD45-SAP clones 104 and 30-F11. Data represent mean ± SD of 3 independent experiments. (f) In vivo HSC depletion by 3 mg/kg CD45-SAP created from clones 104 and 30-F11 assessed 8 days post-administration. Data represent mean ± SEM (n = 4 mice/group, assayed individually). (g) In vivo persistence 24 hours post-administration of AF488-labelled CD45 antibody clones 104 and 30-F11 in bone marrow progenitor (LKS) cells, peripheral blood white blood cells, and splenocytes. Data represent mean ± SD (n = 3 mice/group) of 1 of 2 independent experiments. * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  7. Donor engraftment post-administration of CD45-SAP
    Supplementary Fig. 2: Donor engraftment post-administration of CD45–SAP

    (a) Peripheral blood and bone marrow HSC chimerism 4 months post transplantation in 3 mg/kg CD45-SAP conditioned C57BL/6 mice transplanted with 107 GFP bone marrow cells. Data represent mean ± SD (n = 5 mice/group) of 1 of 2 independent experiments. (b) Long term peripheral blood chimerism following CD45.1 cell transplantation in 3 mg/kg CD45-SAP conditioned C57BL/6 mice; all data points significant vs. control (p values <0.05). (c) Donor chimerism within peripheral myeloid, B- and T-cell populations as a function of time in transplanted C57BL/6 mice conditioned with 3 mg/kg CD45-SAP. Data in (b) and (c) represent mean ± SD (n = 5 mice/group, assayed individually). (d) Blood chimerism 4 months post serial transplantation of marrow from primary CD45-SAP conditioned & transplanted mice into lethally irradiated secondary C57BL/6 recipients. Data represent mean ± SD (n = 5 mice/group) of 2 independent experiments. (e) HSC depletion in Balb/c mice 6 days post 3 mg/kg CD45-SAP. Data represent mean ± SEM. (f) Peripheral blood chimerism 4 months post-transplantation in Balb/c mice conditioned with 3 mg/kg CD45-SAP. Data represent in mean ± SD (n = 5 mice/group, assayed individually). (g) Multi-lineage distribution of the graft 4 months post-transplantation of purified HSCs (LKS CD34-CD150+ or LKS CD48-CD150+, 2000 cells each) in 3 mg/kg CD45-SAP conditioned C57BL/6 mice. Data represent mean ± SD (n = 5 mice/group). (h) 3 mg/kg CD45-SAP and 5Gy TBI achieve similar chimerism 4 months post-transplantation of 107 bone marrow cells in C57BL/6 mice, while 28 mg/kg ACK2 or 3 mg/kg ACK2-SAP conditioning fails to enable engraftment. Data represent mean ± SD (n = 5 mice/group) of 1 of 2 independent experiments, except for ACK2 and ACK2-SAP (n = 2 mice per group, assayed individually). (i) Multi-lineage distribution of the graft 4 months post-transplantation of 107 bone marrow cells in conditioned (3 mg/kg CD45-SAP or 5Gy TBI) C57BL/6 mice vs. overall lineage distribution in non-conditioned control mice. Data represent mean ± SD (n = 5 mice/group). (j) Four month peripheral chimerism following transplantation of 106 bone marrow cells in C57BL/6 mice conditioned with 3 mg/kg CD45-SAP, 5Gy TBI or the combination. Data represent mean ± SD (n = 5 mice/group, assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  8. Hematopoietic recovery kinetics post-transplantation
    Supplementary Fig. 3: Hematopoietic recovery kinetics post-transplantation

    (a) Myeloid, (b) T-cell, and (c) B-cell recovery post transplantation of 107 whole bone marrow cells in 3 mg/kg CD45-SAP or 5Gy TBI conditioned C57BL/6 mice. Data in (a-c) represent mean percentage relative to non-conditioned control ± SEM (n = 5 mice/group per time point, assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  9. Donor contribution to T-cell subsets
    Supplementary Fig. 4: Donor contribution to T-cell subsets

    Peripheral donor cell chimerism within various T-cell sub-populations 12 weeks post transplantation of 107 whole bone marrow cells in non-conditioned control or CD45-SAP conditioned C57BL/6 mice. Sub-populations were quantitated by flow cytometry using the immunophenotypic markers listed. Data represent mean ± SEM (n = 5 mice/group, assayed individually). *** indicates p value <0.001 vs control. Statistics calculated using two-sided unpaired t test.

  10. Impact on hematopoietic progenitors
    Supplementary Fig. 5: Impact on hematopoietic progenitors

    Immunophenotypic assessment of myeloid progenitors, granulocyte macrophage progenitors (GMP), common myeloid progenitors (CMP), megakaryocyte erythroid progenitors (MEP) and common lymphoid progenitors (CLP) in the bone marrow of C57BL/6 mice, 2 and 6 days after conditioning with 5Gy TBI or 3 mg/kg CD45-SAP. Data represent mean percentage relative to untreated control ± SEM (n = 6 mice/group, n = 3 mice per time point assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  11. Effects of CD45-SAP and irradiation on bone marrow histology
    Supplementary Fig. 6: Effects of CD45–SAP and irradiation on bone marrow histology

    Hematoxylin and eosin staining of femur marrow sections of non-treated control, 3 mg/kg CD45-SAP or 5Gy TBI conditioned C57BL/6 mice 2 days post-conditioning. Representative images from independent experiments (n = 2 mice/group) are shown. Scale bars in top and bottom images represent 500 μm and 20 μm, respectively.

  12. Effects on non-hematopoietic marrow composition
    Supplementary Fig. 7: Effects on non-hematopoietic marrow composition

    Composition of the non-hematopoietic bone marrow compartment 2 days post 3 mg/kg CD45-SAP or 5Gy TBI vs untreated control C57BL/6 mice. Enzymatically-dissociated marrow and bone-associated cells were characterized by immunophenotype with non-hematopoietic stromal and endothelial cells defined as CD45- Ter119- CD31- and CD45- Ter119- CD31+, respectively. Data represent mean ± SEM (n = 5 mice/group, assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  13. Effects on endosteal cells
    Supplementary Fig. 8: Effects on endosteal cells

    Hematoxylin and eosin staining of femur sections from non-treated control, 3 mg/kg CD45-SAP or 5Gy TBI conditioned C57BL/6 mice 2 days post-conditioning. Arrows indicate endosteal cells in the diaphysis section of the femur and scale bars represent 20 μm. Each image is from an independent mouse.

  14. Effects of CD45-SAP and irradiation on blood and thymus
    Supplementary Fig. 9: Effects of CD45–SAP and irradiation on blood and thymus

    (a) Relative levels of peripheral B-cells at various times post 3 mg/kg CD45-SAP or 5Gy TBI conditioning in non-transplanted C57BL/6 mice. Data represent mean ± SEM (n = 20 mice/group, n = 4 mice per time point, assayed individually). (b) Thymus mass of non-treated control, 3 mg/kg CD45-SAP or 5Gy TBI conditioned C57BL/6 mice harvested 3 days post-treatment. Data represents mean ± SD (n = 4 mice/group, assayed individually). Relative levels of (c) red blood cells (RBC), (d) hemoglobin, (e) hematocrit, and (f) platelets at various time points following 3 mg/kg CD45-SAP or 5Gy TBI conditioning in C57BL/6 mice. Data represent mean percentage relative to untreated control ± SEM (n = 20 mice/group, n = 4 mice per time point, assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  15. Sickle cell disease correction by HSCT post CD45-SAP conditioning
    Supplementary Fig. 10: Sickle cell disease correction by HSCT post CD45–SAP conditioning

    (a) HSC depletion in sickle disease mice 8 days post-administration of various doses of CD45-SAP. Data represent the mean ± SEM (n = 3 mice/dose, assayed individually). (b) Red blood cell (RBC) counts, (c) hemoglobin levels, (d) hematocrit levels, and (e) reticulocyte frequency for wild type control, sickle disease and the 3 groups of CD45-SAP conditioned and transplanted sickle mice 4 months post transplantation of 107 bone marrow cells from wild-type donor. Data in (b-e) represent the mean ± SEM (n = 6 mice/group, assayed individually). (f) Native-PAGE analysis of normal (Hba) and sickle (Hbs) hemoglobin protein in blood from wild-type control mice, sickle mice and groups A-C mice (two representative mice from each group). (g) Spleen mass 4 months post transplantation for wild type control, sickle disease and the 3 groups of CD45-SAP conditioned and transplanted sickle mice. Data represent the mean ± SEM (n = 3 mice/group, assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  16. Relative CD45 cell surface expression
    Supplementary Fig. 11: Relative CD45 cell surface expression

    Cell surface expression of CD45 on bone marrow HSCs and marrow progenitors as assessed by flow cytometry. The geometric mean fluorescence intensity of anti-CD45 antibody staining was measured and expression levels relative to HSCs are shown. Data represents mean ± SD (n = 3 mice, assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  17. Activity of anti-human CD45 immunotoxins
    Supplementary Fig. 12: Activity of anti-human CD45 immunotoxins

    CD45-SAP immunotoxins created from anti-human anti-CD45 monoclonal antibody clones MEM-28 and HI30 induce Jurkat cell death in vitro (72 hour incubation) with IC50 values of 130 and 200 pM, respectively; Data represents mean ± SD (n = 3 technical replicates) of a representative experiment.

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Author information

Affiliations

  1. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Rahul Palchaudhuri,
    • Borja Saez,
    • Jonathan Hoggatt,
    • Amir Schajnovitz,
    • David B Sykes,
    • Tiffany A Tate,
    • Agnieszka Czechowicz,
    • Youmna Kfoury,
    • FNU Ruchika,
    • Derrick J Rossi,
    • Gregory L Verdine &
    • David T Scadden
  2. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Rahul Palchaudhuri,
    • Borja Saez,
    • Jonathan Hoggatt,
    • Amir Schajnovitz,
    • David B Sykes,
    • Tiffany A Tate,
    • Youmna Kfoury,
    • FNU Ruchika &
    • David T Scadden
  3. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.

    • Rahul Palchaudhuri,
    • Borja Saez,
    • Jonathan Hoggatt,
    • Amir Schajnovitz,
    • David B Sykes,
    • Tiffany A Tate,
    • Agnieszka Czechowicz,
    • Youmna Kfoury,
    • FNU Ruchika,
    • Derrick J Rossi,
    • Gregory L Verdine &
    • David T Scadden
  4. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Rahul Palchaudhuri &
    • Gregory L Verdine
  5. Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.

    • Agnieszka Czechowicz &
    • Derrick J Rossi
  6. Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

    • Agnieszka Czechowicz &
    • Derrick J Rossi
  7. Department of Pediatric Oncology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.

    • Agnieszka Czechowicz
  8. Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Michael K Mansour

Contributions

R.P. conceived the study, designed and conducted the experiments, analyzed the data, and wrote the manuscript; B.S., J.H., A.S., D.B.S., Y.K., A.C., T.A.T., F.R., and M.K.M. conducted the experiments, analyzed the data, and reviewed the manuscript; D.J.R. and G.L.V. designed experiments and reviewed the manuscript; D.T.S. conceived the study, designed the experiments, analyzed the data, and wrote the manuscript.

Competing financial interests

The authors declare relevant competing financial interests as follows. Magenta Therapeutics, equity and consulting: R.P., J.H., A.C., D.J.R., and D.T.S. Fate Therapeutics, equity and consulting: D.T.S.; consulting: J.H. GlaxoSmithKline, consulting and sponsored research: J.H. and D.T.S. Intellia Therapeutics, equity and consulting: D.J.R. Moderna Therapeutics, equity: D.J.R. Inventors, US patent applications (US 62/143,642; US 62/220,204; US 62/221,595; and US 62/239,573): R.P. and D.T.S.

Corresponding authors

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Cell depletion activity of immunotoxins (232 KB)

    (a) In vivo HSC depletion screen of candidate immunotoxins administered at 3 mg/kg in C57BL/6 mice. Bone marrow was harvested 8 days post-administration and HSCs quantified by flow cytometry. Non-treated C57BL/6 mice were used as controls. Data represent mean ± range (n = 2 mice/group, assayed individually). (b) Investigation of various ratios of biotinylated anti-CD45 antibody (clone 104) to streptavidin-saporin on in vivo HSC depletion activity assessed 8 days post-administration in C57BL/6 mice. Data represent mean ± SD (n = 4 mice/group, assayed individually). (c) Peripheral blood chimerism 4 months after competitive transplantation of bone marrow harvested from control or CD45-SAP conditioned mice demonstrates depletion of functional HSCs by CD45-SAP. Data represent mean ± SD (n = 5 mice/group, assayed individually). (d) CD45-SAP clone 104 does not deplete HSCs in congenic CD45.1 C57BL/6 mice. Data represent mean ± SD (n = 5 mice/group, assayed individually). (e) In vitro IC50 values against EL4 and EML cell lines after 72 hour incubation with CD45-SAP clones 104 and 30-F11. Data represent mean ± SD of 3 independent experiments. (f) In vivo HSC depletion by 3 mg/kg CD45-SAP created from clones 104 and 30-F11 assessed 8 days post-administration. Data represent mean ± SEM (n = 4 mice/group, assayed individually). (g) In vivo persistence 24 hours post-administration of AF488-labelled CD45 antibody clones 104 and 30-F11 in bone marrow progenitor (LKS) cells, peripheral blood white blood cells, and splenocytes. Data represent mean ± SD (n = 3 mice/group) of 1 of 2 independent experiments. * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  2. Supplementary Figure 2: Donor engraftment post-administration of CD45–SAP (306 KB)

    (a) Peripheral blood and bone marrow HSC chimerism 4 months post transplantation in 3 mg/kg CD45-SAP conditioned C57BL/6 mice transplanted with 107 GFP bone marrow cells. Data represent mean ± SD (n = 5 mice/group) of 1 of 2 independent experiments. (b) Long term peripheral blood chimerism following CD45.1 cell transplantation in 3 mg/kg CD45-SAP conditioned C57BL/6 mice; all data points significant vs. control (p values <0.05). (c) Donor chimerism within peripheral myeloid, B- and T-cell populations as a function of time in transplanted C57BL/6 mice conditioned with 3 mg/kg CD45-SAP. Data in (b) and (c) represent mean ± SD (n = 5 mice/group, assayed individually). (d) Blood chimerism 4 months post serial transplantation of marrow from primary CD45-SAP conditioned & transplanted mice into lethally irradiated secondary C57BL/6 recipients. Data represent mean ± SD (n = 5 mice/group) of 2 independent experiments. (e) HSC depletion in Balb/c mice 6 days post 3 mg/kg CD45-SAP. Data represent mean ± SEM. (f) Peripheral blood chimerism 4 months post-transplantation in Balb/c mice conditioned with 3 mg/kg CD45-SAP. Data represent in mean ± SD (n = 5 mice/group, assayed individually). (g) Multi-lineage distribution of the graft 4 months post-transplantation of purified HSCs (LKS CD34-CD150+ or LKS CD48-CD150+, 2000 cells each) in 3 mg/kg CD45-SAP conditioned C57BL/6 mice. Data represent mean ± SD (n = 5 mice/group). (h) 3 mg/kg CD45-SAP and 5Gy TBI achieve similar chimerism 4 months post-transplantation of 107 bone marrow cells in C57BL/6 mice, while 28 mg/kg ACK2 or 3 mg/kg ACK2-SAP conditioning fails to enable engraftment. Data represent mean ± SD (n = 5 mice/group) of 1 of 2 independent experiments, except for ACK2 and ACK2-SAP (n = 2 mice per group, assayed individually). (i) Multi-lineage distribution of the graft 4 months post-transplantation of 107 bone marrow cells in conditioned (3 mg/kg CD45-SAP or 5Gy TBI) C57BL/6 mice vs. overall lineage distribution in non-conditioned control mice. Data represent mean ± SD (n = 5 mice/group). (j) Four month peripheral chimerism following transplantation of 106 bone marrow cells in C57BL/6 mice conditioned with 3 mg/kg CD45-SAP, 5Gy TBI or the combination. Data represent mean ± SD (n = 5 mice/group, assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  3. Supplementary Figure 3: Hematopoietic recovery kinetics post-transplantation (249 KB)

    (a) Myeloid, (b) T-cell, and (c) B-cell recovery post transplantation of 107 whole bone marrow cells in 3 mg/kg CD45-SAP or 5Gy TBI conditioned C57BL/6 mice. Data in (a-c) represent mean percentage relative to non-conditioned control ± SEM (n = 5 mice/group per time point, assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  4. Supplementary Figure 4: Donor contribution to T-cell subsets (226 KB)

    Peripheral donor cell chimerism within various T-cell sub-populations 12 weeks post transplantation of 107 whole bone marrow cells in non-conditioned control or CD45-SAP conditioned C57BL/6 mice. Sub-populations were quantitated by flow cytometry using the immunophenotypic markers listed. Data represent mean ± SEM (n = 5 mice/group, assayed individually). *** indicates p value <0.001 vs control. Statistics calculated using two-sided unpaired t test.

  5. Supplementary Figure 5: Impact on hematopoietic progenitors (324 KB)

    Immunophenotypic assessment of myeloid progenitors, granulocyte macrophage progenitors (GMP), common myeloid progenitors (CMP), megakaryocyte erythroid progenitors (MEP) and common lymphoid progenitors (CLP) in the bone marrow of C57BL/6 mice, 2 and 6 days after conditioning with 5Gy TBI or 3 mg/kg CD45-SAP. Data represent mean percentage relative to untreated control ± SEM (n = 6 mice/group, n = 3 mice per time point assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  6. Supplementary Figure 6: Effects of CD45–SAP and irradiation on bone marrow histology (770 KB)

    Hematoxylin and eosin staining of femur marrow sections of non-treated control, 3 mg/kg CD45-SAP or 5Gy TBI conditioned C57BL/6 mice 2 days post-conditioning. Representative images from independent experiments (n = 2 mice/group) are shown. Scale bars in top and bottom images represent 500 μm and 20 μm, respectively.

  7. Supplementary Figure 7: Effects on non-hematopoietic marrow composition (210 KB)

    Composition of the non-hematopoietic bone marrow compartment 2 days post 3 mg/kg CD45-SAP or 5Gy TBI vs untreated control C57BL/6 mice. Enzymatically-dissociated marrow and bone-associated cells were characterized by immunophenotype with non-hematopoietic stromal and endothelial cells defined as CD45- Ter119- CD31- and CD45- Ter119- CD31+, respectively. Data represent mean ± SEM (n = 5 mice/group, assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  8. Supplementary Figure 8: Effects on endosteal cells (1,375 KB)

    Hematoxylin and eosin staining of femur sections from non-treated control, 3 mg/kg CD45-SAP or 5Gy TBI conditioned C57BL/6 mice 2 days post-conditioning. Arrows indicate endosteal cells in the diaphysis section of the femur and scale bars represent 20 μm. Each image is from an independent mouse.

  9. Supplementary Figure 9: Effects of CD45–SAP and irradiation on blood and thymus (313 KB)

    (a) Relative levels of peripheral B-cells at various times post 3 mg/kg CD45-SAP or 5Gy TBI conditioning in non-transplanted C57BL/6 mice. Data represent mean ± SEM (n = 20 mice/group, n = 4 mice per time point, assayed individually). (b) Thymus mass of non-treated control, 3 mg/kg CD45-SAP or 5Gy TBI conditioned C57BL/6 mice harvested 3 days post-treatment. Data represents mean ± SD (n = 4 mice/group, assayed individually). Relative levels of (c) red blood cells (RBC), (d) hemoglobin, (e) hematocrit, and (f) platelets at various time points following 3 mg/kg CD45-SAP or 5Gy TBI conditioning in C57BL/6 mice. Data represent mean percentage relative to untreated control ± SEM (n = 20 mice/group, n = 4 mice per time point, assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  10. Supplementary Figure 10: Sickle cell disease correction by HSCT post CD45–SAP conditioning (236 KB)

    (a) HSC depletion in sickle disease mice 8 days post-administration of various doses of CD45-SAP. Data represent the mean ± SEM (n = 3 mice/dose, assayed individually). (b) Red blood cell (RBC) counts, (c) hemoglobin levels, (d) hematocrit levels, and (e) reticulocyte frequency for wild type control, sickle disease and the 3 groups of CD45-SAP conditioned and transplanted sickle mice 4 months post transplantation of 107 bone marrow cells from wild-type donor. Data in (b-e) represent the mean ± SEM (n = 6 mice/group, assayed individually). (f) Native-PAGE analysis of normal (Hba) and sickle (Hbs) hemoglobin protein in blood from wild-type control mice, sickle mice and groups A-C mice (two representative mice from each group). (g) Spleen mass 4 months post transplantation for wild type control, sickle disease and the 3 groups of CD45-SAP conditioned and transplanted sickle mice. Data represent the mean ± SEM (n = 3 mice/group, assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  11. Supplementary Figure 11: Relative CD45 cell surface expression (128 KB)

    Cell surface expression of CD45 on bone marrow HSCs and marrow progenitors as assessed by flow cytometry. The geometric mean fluorescence intensity of anti-CD45 antibody staining was measured and expression levels relative to HSCs are shown. Data represents mean ± SD (n = 3 mice, assayed individually). * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.

  12. Supplementary Figure 12: Activity of anti-human CD45 immunotoxins (200 KB)

    CD45-SAP immunotoxins created from anti-human anti-CD45 monoclonal antibody clones MEM-28 and HI30 induce Jurkat cell death in vitro (72 hour incubation) with IC50 values of 130 and 200 pM, respectively; Data represents mean ± SD (n = 3 technical replicates) of a representative experiment.

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    Supplementary Figures 1–12

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