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

Nature Biotechnology volume 34, pages 738–745 (2016)Cite this article

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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.

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Figure 1: CD45–SAP has potent cell-depletion activity.
Figure 2: CD45–SAP enables efficient donor-cell engraftment.
Figure 3: Differential effects of CD45–SAP and irradiation on bone marrow.
Figure 4: Differential effects of CD45–SAP and irradiation on blood and thymus.
Figure 5: Correction of sickle cell disease.

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Acknowledgements

We would like to thank R.T. Bronson (Harvard Medical School) for histology services, G. Sempowski (Duke University) for mouse sjTREC plasmid, and the Harvard Stem Cell and Regenerative Biology flow cytometry core. We would like to acknowledge N. Van Gastel and A. Papazian for technical assistance. R.P. was supported by a Life Science Research Foundation fellowship sponsored by the Jake Wetchler Foundation. B.S. was supported by an American Society of Hematology scholar award. J.H. was supported by an NIH NHLBI K99/R00 HL119559. M.K.M. was supported by NIAID NIH 1K08AI110655. D.T.S. was supported by the Gerald and Darlene Jordan Chair of Medicine of Harvard University. Grants from the Harvard Blavatnik Biomedical Accelerator Fund (D.T.S.), NIH NHLBI HL44851 (D.T.S.), HL129903 (D.T.S., D.J.R.), and HL107630 (D.J.R.) funded this work.

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Authors and 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. Division of Hematology/Oncology, Program in Cellular and Molecular Medicine, 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

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  1. Rahul Palchaudhuri
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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.

Corresponding authors

Correspondence to Rahul Palchaudhuri or David T Scadden.

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Competing 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.

Integrated supplementary information

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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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Palchaudhuri, R., Saez, B., Hoggatt, J. et al. Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin. Nat Biotechnol 34, 738–745 (2016). https://doi.org/10.1038/nbt.3584

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