Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy

Abstract

Hematopoietic stem cell (HSC) transplantation can cure diverse diseases of the blood system, including hematologic malignancies, anemias, and autoimmune disorders. However, patients must undergo toxic conditioning regimens that use chemotherapy and/or radiation to eliminate host HSCs and enable donor HSC engraftment. Previous studies have shown that anti-c-Kit monoclonal antibodies deplete HSCs from bone marrow niches, allowing donor HSC engraftment in immunodeficient mice. We show that host HSC clearance is dependent on Fc-mediated antibody effector functions, and enhancing effector activity through blockade of CD47, a myeloid-specific immune checkpoint, extends anti-c-Kit conditioning to fully immunocompetent mice. The combined treatment leads to elimination of >99% of host HSCs and robust multilineage blood reconstitution after HSC transplantation. This targeted conditioning regimen that uses only biologic agents has the potential to transform the practice of HSC transplantation and enable its use in a wider spectrum of patients.

Fig. 1.. Depletion of HSCs by anti–c-Kit antibody ACK2 is dependent on FcR activity.

(A) Total number of phenotypic Lin⁻c-Kit⁺Sca-1⁺CD150⁺Flt3⁻CD34⁻ LT-HSCs in wild-type (WT) mice as compared to immunocompromised Rag2^−/−cγ^−/− mice after treatment with anti–c-Kit antibody ACK2 (n = 3 to 5 per group; experiment was repeated three times). (B) Total frequency of Lin⁻c-Kit⁺Sca-1⁺CD150⁺Flt3⁻CD34⁻ LT-HSCs in Rag2^−/−cγ^−/− mice 6 days after treatment with increasing concentrations of ACK2 compared to 500 μg of ACK2 Fab (n = 3 per group). (C) Number of Lin⁻c-Kit⁺Sca-1⁺CD150⁺Flt3⁻CD34⁻ LT-HSCs in Fcer1g^−/− mice 6 days after ACK2 treatment as compared to untreated controls (n = 3; experiment was replicated in triplicate with similar results each time). (D) Frequency of donor-derived Mac-1⁺Gr-1⁺ granulocytes in peripheral blood of Rag2^−/−cγ^−/− animals after either ACK2 treatment or FcR blocking before ACK2 as compared to untreated recipients. Data and error bars in (B) to (D) represent means ± SEM. NS, not significant; ****P < 0.0001, ***P < 0.0005, **P < 0.005, *P < 0.05.

Fig. 2.. Engineering a CV1mb as an antagonist of murine CD47.

(A) Schematic of CV1 and CV1mb. CV1mb is a fusion of CV1 to the dimeric CH3 domain of human IgG1 linked by a disulfide-containing hinge. (B) Binding of CV1 and CV1mb to human and mouse blood, as determined by a flow cytometry–based receptor occupancy assay. Left: Blockade of allophycocyanin (APC)-labeled anti-mCD47 antibody MIAP301 binding to murine red blood cells (RBCs) by varying concentrations of CV1 and CV1mb. Right: Blockade of Alexa 488–labeled anti-hCD47 antibody Hu5F9-G4 binding to human RBCs by varying concentrations of CV1 and CV1mb. MFI, mean fluorescence intensity. (C) Phagocytosis of EGFR⁺ DLD-1 colon cancer cells by human macrophages after treatment with cetuximab (anti-EGFR) with and without CV1 and CV1mb, indicated as a percentage of maximal response (seen in the CV1 + cetuximab treatment group). PBS, phosphate-buffered saline. (D) Erythrocyte CD47 receptor occupancy from mice administered 200 mg of CV1 or CV1mb measured 1 hour before, 1 hour after, and 24 hours after protein injection. Data and error bars in (B) to (D) represent means ± SEM. ****P < 0.0001. All experiments were repeated in duplicate.

Fig. 3.. Combining anti–c-Kit antibodies with CD47 blockade produces profound depletion of HSCs and clearance of the bone marrow niche in immunocompetent mice.

(A) Total number of Lin⁻c-Kit⁺Sca-1⁺CD150⁺Flt3⁻CD34⁻ LT-HSCs in WT mice after 7 days of treatment with anti–c-Kit antibody ACK2, CD47 antagonist CV1mb, and combination of ACK2 and CV1mb as compared to untreated controls (n = 3; experiment was replicated four times). (B) Frequency of donor-derived HSCs in the bone marrow present 24 weeks after transplant into irradiated recipients. Recipients were transplanted with 1 million whole-bone marrow cells from treated donor mice and 1 million support bone marrow cells from GFP⁺ donors (n = 5). (C) Total numbers of downstream myeloid progenitors are decreased 7 days after treatment with ACK2 and CV1mb as compared to ACK2 alone. CMP, common myeloid progenitor (Lin⁻Sca-1⁺c-Kit⁺FcRg^loCD34⁺); GMP, granulocyte macrophage progenitor (Lin⁻Sca-1⁻c-Kit⁺FcRg^hiCD34⁺); MEP, megakaryocyte-erythroid progenitor (Lin⁻Sca-1⁻c-Kit⁺FcRg^loCD34⁻). (D) Hematoxylin and eosin staining of bone marrow section depicting loss of bone marrow cellularity at 7 days in ACK2- and CV1mb-treated mice as compared to mice treated with ACK2 alone. Data and error bars in (B) to (D) represent means ± SEM. ****P < 0.0001, ***P < 0.005, **P < 0.01, *P < 0.05.

Fig. 4.. Preconditioning with anti–c-Kit and CD47 blockade enables long-term engraftment of HSCs in immunocompetent mice.

(A) Schematic of protocol for conditioning of recipients with anti–c-Kit antibody ACK2 and CD47 antagonist CV1mb. F1 mice expressing both CD45.1 and CD45.2 alleles were treated with 500 μg of ACK2 once and 500 μg of CV1mb daily for 5 days. Six days after treatment, 1 million CD45.2⁺ donor Lin⁻ cells were transplanted daily for 3 days (n = 3 to 5; experiment was replicated in triplicate). BM, bone marrow. (B) Frequency of donor-derived Lin⁻c-Kit⁺Sca-1⁺CD150⁺ HSCs in the bone marrow 24 weeks after transplant in ACK2- and CV1mb-treated recipients as compared to mice treated with ACK2 alone (n = 3 to 5; experiment was replicated in triplicate). (C) Donor-derived blood chimerism of Gr-1⁺ Mac-1⁺ myeloid cells. (D) Donor-derived blood chimerism of CD19⁺ B cells. (E) Donor-derived blood chimerism of NK1.1⁺ NK cells. (F) Donor-derived blood chimerism of CD3⁺ T cells. All analyses were performed 24 weeks after transplant in ACK2- and CV1mb-treated recipients as compared to mice treated with ACK2 alone (n = 3 to 5; experiment was replicated in triplicate). Data and error bars in (B) to (F) represent means ± SEM. ****P < 0.0001, ***P < 0.005.

Fig. 5.. HSC allotransplantation in an mHC-mismatched model by conditioning with anti–c-Kit, CD47 blockade, and lymphocyte-depleting antibodies.

(A) Schematic of protocol for conditioning of recipients with anti–c-Kit antibody ACK2 and CD47 antagonist CV1mb or MIAP410. BALB/c mice expressing CD45.2 were treated with 500 μg of ACK2 once and 500 μg of CV1mb or MIAP410 daily for 5 days. Six days after treatment, 50,000 CD45.1⁺ donor B10D2 LSK HSCs were transplanted daily for 3 days. CD4 (GK1.5) and CD8 (YTS169.4) T cell–depleting antibodies were administered starting 1 day before transplant and during the course of the transplant as described in Materials and Methods. (B) Frequency of donor-derived Lin⁻c-Kit⁺Sca-1⁺CD150⁺ HSCs in the bone marrow 24 weeks after transplant. (C) Donor-derived bone marrow chimerism of Gr-1⁺Mac-1⁺ myeloid cells. (D) Donor-derived bone marrow chimerism of CD19⁺ B cells. (E) Donor-derived bone marrow chimerism of CD122⁺Dx5⁺ NK cells. (F) Donor-derived bone marrow chimerism of CD3⁺ T cells. Data and error bars in (B) to (E) represent means ± SEM. **P < 0.01, *P < 0.05; significance is compared to untreated controls.