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

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.

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 (Lin⁻cKit⁺Sca1⁺CD48⁻CD150⁺) 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.

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 10 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 CD48⁻CD150⁺ or LKS CD34⁻CD150⁺) 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.

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.

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.

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).