Methods for manipulating CD47-mediated phagocytosis

Abstract

To provide methods for manipulating phagocytosis of cells, including hematopoietic cells, such as bone marrow cells and hematopoietic cells in circulating blood, and solid tumor cells.SOLUTION: The invention provides methods for protection from phagocytosis in circulating blood by providing a host animal with a CD47 mimetic molecule, which interacts with SIRPα on phagocytic cells, such as macrophages, and decreases phagocytosis. In some embodiments, cells in circulating blood are hematopoietic stem cells, or hematopoietic progenitor cells, particularly in a transplantation environment where protection from phagocytosis is desirable. In other embodiments, cells in circulating blood are leukemia cells, particularly acute myeloid leukemia (AML), where increased phagocytosis is desirable.SELECTED DRAWING: None

Description

[Background technology]

[0001] background The reticuloendothelial system (RES) is a part of the immune system. The RES consists of phagocytic cells, primarily monocytes and macrophages, located in the reticular connective tissue. The RES consists of 1) monocytes in the circulating blood; 2) resident macrophages in the liver, spleen, lymph nodes, thymus, submucosa of the airway and digestive tract, bone marrow, and connective tissue; and 3) macrophage-like cells, including dendritic cells in lymph nodes, Langerhans cells in the skin, and microglial cells in the central nervous system. These cells accumulate in the lymph nodes and spleen. The RES functions to clear pathogens, particulate matter in the blood circulation, and aged or damaged hematopoietic cells.

[0002] To eliminate foreign cells or particles in the innate immune response, macrophage-mediated phagocytosis is induced when phosphatidylserine receptor (PSR) reacts with phosphatidylserine (PS), which may be presented on the outer membrane of dead cells, such as apoptotic and necrotic cells. Then, the interaction between PS and PSR plays a very important role in the clearance of apoptotic cells by macrophages. Once phagocytosis is performed by macrophages, the inflammatory response is downregulated by the increase of factors such as IL-10, TGF-β, and prostaglandin E2 (PGE2). The strict balance between inflammatory and anti-inflammatory responses in both innate and adaptive immunity plays a crucial role in maintaining cellular homeostasis and protecting the host from foreign invasions.

[0003] The causal relationship between inflammation and tumor progression is a widely accepted concept. Data now support the concept of cancer immune surveillance, in which one of the physiological functions of the immune system is to recognize and destroy transformed cells. However, some tumor cells can evade recognition and destruction by the immune system. Once tumor cells escape, the immune system can participate in their proliferation, for example by promoting tumor angiogenesis.

[0004] Although both adaptive and innate immune cells are involved in the surveillance and elimination of tumor cells, monocytes / macrophages may be the first line of defense in tumors because they can rapidly form colonies and secrete cytokines that attract and activate dendritic cells (DCs) and NK cells, and thus mount adaptive immune responses against transformed cells.

[0005] Tumors that escape the immune system may be the result of alterations that occur during the immune surveillance phase. As an example, some tumor cells become defective in antigen processing and presentation pathways, such as the absence or abnormal function of components of the IFN-γ receptor signaling pathway, which facilitates their evasion from the adaptive immune response. Other tumors suppress the induction of proinflammatory danger signals, leading, for example, to impaired DC maturation. Finally, inhibition of the defense functions of the immune system, such as the overproduction of the anti-inflammatory cytokines IL-10 and TGF-β, which can be produced by many tumor cells themselves but also by macrophages or regulatory T cells, may also promote tumor escape.

[0006] Tumors can be thought of as abnormal organs initiated by tumorigenic cancer cells that have acquired the capacity for unlimited proliferation through accumulated mutations. When viewed in this perspective of tumors as abnormal organs, principles of normal stem cell biology can be applied to better understand how tumors develop. A number of observations suggest that the similarity between normal stem cells and tumorigenic cells is appropriate. Both normal stem cells and tumorigenic cells have extensive proliferative capacity and the ability to give rise to new (normal or abnormal) tissues. Both tumors and normal tissues are composed of heterogeneous combinations of cells with distinct phenotypic characteristics and differing proliferative capacities.

[0007] Stem cells are defined as cells that have the ability to perpetuate themselves by self-renewal and generate mature cells of a particular tissue by differentiation. In most tissues, stem cells are rare. As a result, to study the properties of stem cells, they must be prospectively identified and carefully purified. Perhaps the most important and useful property of stem cells is their property of self-renewal. Through this property, striking similarities can be found between stem cells and cancer cells: tumors can often arise from the transformation of normal stem cells, similar signaling pathways can regulate the self-renewal of stem cells and cancer cells, and cancers can contain rare cells with unlimited self-renewal capacity that drive tumorigenesis.

[0008] The study of cell surface markers that are specific to cancer cells or specifically upregulated in cancer cells is crucial in providing targets for reducing cancer cell proliferation or depleting cancer cells.Provided herein is a marker for myeloid leukemia, particularly for acute myeloid leukemia (AML).The inventors' research has revealed the role of this marker in helping AML stem cells escape phagocytosis clearance.Provided is a method for using this marker to increase phagocytosis of AML stem cells (AML SC) and improve hematopoietic stem cell and progenitor stem cell transplantation.

[0009] Interestingly, certain markers have been shown to be common to leukemic stem cells and hematopoietic stem cells (HSCs). During normal development, HSCs migrate via the bloodstream to ectopic microenvironments in fetal and adult life. Once in the bloodstream, HSCs must pass through the vascular beds of the spleen and liver before settling into the microenvironment. In these vascular beds, macrophages function to remove damaged cells and foreign particles from the bloodstream. Furthermore, during inflammatory conditions, macrophage phagocytosis becomes more active. Thus, newly arriving stem cells face the possibility of being phagocytosed on the way, unless additional protection can be generated. Examining the mechanisms by which endogenous HSCs escape phagocytic clearance may provide insight into methods to improve the transplant success of hematopoietic and progenitor stem cells. The present invention meets these and other needs. Summary of the Invention

[0010] Methods are provided for manipulating the phagocytosis of circulating hematopoietic cells, including bone marrow cells. In some embodiments of the invention, the circulating cells are hematopoietic stem or progenitor cells, particularly in transplantation settings where protection from phagocytosis is desired. In other embodiments, the circulating cells are leukemia cells, particularly acute myeloid leukemia (AML), where increased phagocytosis is desired. In certain embodiments of the invention, methods are provided for manipulating macrophage phagocytosis of circulating hematopoietic cells. In yet another embodiment of the invention, methods are provided for manipulating the phagocytosis of solid tumors.

[0011] In some embodiments of the present invention, hematopoietic stem or progenitor cells are protected from phagocytosis in the circulation by providing the host animal with a CD47 mimetic molecule that interacts with SIRPα on phagocytic cells such as macrophages and reduces phagocytosis. The CD47 mimetic can be soluble CD47, CD47 coated on the surface of the cells to be protected, and CD47 mimetic that binds to the CD47 binding site of SIRPα. In some embodiments of the present invention, CD47 is provided as a fusion protein, e.g., soluble CD47 fused to an Fc fragment, e.g., IgG1 Fc, IgG2 Fc, IgA Fc, etc.

[0012] In other embodiments, tumor cells, such as solid tumor cells, leukemia cells, etc., are targeted for phagocytosis by blocking CD47 on the cell surface. It has been shown that leukemia cells, particularly AML cells, avoid macrophage surveillance by upregulating CD47 expression. When a patient is administered an agent that blocks CD47 protein, such as an antibody that binds to CD47 and prevents the interaction of CD47 with SIRPα, the clearance of AML cells via phagocytosis is increased. In other aspects, the agent that blocks CD47 is combined with a monoclonal antibody that targets one or more other AMLSC markers, such as CD96, and these compositions can be synergistic in enhancing the phagocytosis and elimination of AMLSC compared to using a single agent. In other embodiments, cells of solid tumors are targeted for phagocytosis by blocking CD47 present on the cell surface.

[0013] In another embodiment, a method is provided for targeting or depleting AML cancer stem cells, comprising contacting a cell population, for example, the blood of a leukemia patient, with a reactant that specifically binds to CD47 to target or deplete AMLSC.In certain aspects, the reactant is an antibody that is conjugated to a cytotoxic agent, for example, a radioisotope, a chemotherapeutic agent, a toxin, etc.In some embodiments, depletion, for example, purification of autologous stem cell products (mobilized peripheral blood or bone marrow) for use in autologous transplantation for acute myeloid leukemia patients, is performed on an ex vivo population of cells.In another embodiment, a method is provided for targeting cancer cells of solid tumors in a human subject by administering an antibody against CD47 to the subject. [The present invention 1001] A method for manipulating the phagocytosis of hematopoietic cells in a human subject by introducing an agent that modulates signaling through CD47. [The present invention 1002] The method of claim 10, wherein the hematopoietic cells are circulating hematopoietic cells. [The present invention 1003] The method of claim 1001, comprising the step of administering to a subject a composition comprising a population of hematopoietic cells and a CD47 mimetic, wherein the CD47 mimetic binds to a SIRPα receptor and downregulates phagocytosis. [The present invention 1004] The method of claim 1003, wherein the CD47 mimetic is a soluble CD47. [The present invention 1005] The method of claim 1003, wherein the CD47 mimetic comprises a soluble CD47 fused to IgG1 Fc. [The present invention 1006] The method of claim 1003, wherein said cells are hematopoietic stem cells. [The present invention 1007] The method of claim 1003, wherein said cells are hematopoietic progenitor cells. [The present invention 1008] The method of any one of claims 1 to 10, comprising the step of contacting blood cells of an AML patient with a CD47 inhibitor that upregulates phagocytosis. [The present invention 1009] The method of claim 1008, wherein the CD47 inhibitor is an antibody that specifically binds to CD47 and inhibits the interaction of the SIRPα receptor with CD47. [The present invention 1010] A method of targeting or depleting AML cancer stem cells comprising contacting reactant blood cells with an antibody that specifically binds CD47 to target or deplete AMLSC. [The present invention 1011] The method of claim 10, wherein said antibody is conjugated to a cytotoxic agent. [The present invention 1012] The method of any one of claims 1 to 11, wherein the cytotoxic agent is selected from the group consisting of a radioisotope, a chemotherapeutic agent, and a toxin. [The present invention 1013] The method of claim 1012, wherein said depletion is performed on said blood cells ex vivo. [The present invention 1014] A method for increasing phagocytosis of cancer cells of a solid tumor in a human subject, comprising administering to the subject a composition comprising a CD47 inhibitor that upregulates phagocytosis of the cancer cells. [The present invention 1015] 1. A method for targeting cancer cells of a solid tumor in a human subject, the method comprising administering to the subject a composition comprising an antibody that specifically binds to CD47 to target the cancer cells. [The present invention 1016] The method of the present invention, wherein the CD47 inhibitor is an antibody that specifically binds to CD47 and inhibits the interaction of the SIRPα receptor with CD47. [The present invention 1017] The method of claim 1015, wherein said antibody is conjugated to a cytotoxic agent. [The present invention 1018] The method of the present invention, wherein the antibody is a bispecific antibody. [Brief description of the drawings]

[0014] [Figure 1]FACS analysis of CD47 expression in human HSC and progenitor cells derived from myelodysplastic syndromes (MDS, blue), chronic myeloid leukemia, accelerated phase (CML AP, green), and normal bone marrow (red). [Diagram 2] ET vs. PV. FACS analysis of CD47 expression by HSC, progenitor, and lineage positive cells in human myeloproliferative disorders, including essential thrombocythemia (ET, blue) and polycythemia vera (PV, green), compared to human normal bone marrow (red). [Diagram 3] Figure 3A: Progenitor cell profiles in normal bone marrow (left), postpolycythmic myelofibrosis with myeloid metaplasia (PPMM), and CML blast crisis.Figure 3B: FACS analysis of CD47 expression in HSC, progenitor, and lineage positive cells in human normal bone marrow (red), UMPD (green), PV (blue=ML), and atypical CML (orange). [Figure 4] CD47 expression by CMML progenitors (blue) increased with disease progression compared to normal bone marrow (red). [Diagram 5] Figures 5A-5B: (A) Progenitor cell profiles of normal bone marrow (left) and AML (right). (B) FACS analysis of CD47 expression of HSCs, progenitors, and lineage positive cells (blasts) in human normal bone marrow (red) and AML (blue). [Figure 6]CD47 is more highly expressed in AML LSCs than in their normal counterparts. A: Relative CD47 expression was measured by flow cytometry in HSCs (Lin-CD34+CD38-CD90+) and MPPs (Lin-CD34+CD38-CD90-CD45RA-) of normal bone marrow, and in LSCs (Lin-CD34+CD38-CD90-) and bulk leukemia cells derived from human AML samples. To account for the analysis of different days, mean fluorescence intensity was normalized to cell size and lineage positive cells. The same samples of normal bone marrow (red, n=3) or AML (blue, n=13) are displayed with the same symbols in different populations. Using a two-tailed Student's t-test, the differences in mean expression between HSC and LSC (p=0.003), HSC and bulk leukemia (p=0.001), MPP and LSC (p=0.004), and MPP and bulk leukemia (p=0.002) were statistically significant. Using a two-tailed paired Student's t-test, the differences in mean expression between AML LSC compared to bulk AML were not statistically significant (p=0.50). B: Clinical and molecular characteristics of primary human AML samples engineered in vitro and / or in vivo. [Figure 7] Anti-CD47 antibodies stimulate in vitro macrophage phagocytosis of primary human AML LSCs. AML LSCs were purified by FACS from two primary human AML samples, labeled with the fluorescent dye CFSE, and incubated with mouse bone marrow-derived macrophages in the presence of an isotype control antibody (A) or an anti-CD47 antibody (B). These cells were assessed for the presence of fluorescently labeled LSCs inside the macrophages by immunofluorescence microscopy. (C) The phagocytic index in each condition was determined by calculating the number of ingested cells per 100 macrophages. [Figure 8]Figure 8A-C: Monoclonal antibodies against human CD47 preferentially enable phagocytosis of human AML LSCs by human and mouse macrophages. A, B. CFSE-labeled AML LSCs were incubated with human peripheral blood-derived macrophages (A) or mouse bone marrow-derived macrophages (B) in the presence of IgG1 isotype control antibody, anti-CD45 IgG1 antibody, or anti-CD47 (B6H12.2) IgG1 antibody. These cells were assessed for the presence of fluorescently labeled LSCs inside the macrophages (indicated by arrows) by immunofluorescence microscopy. C. CFSE-labeled AML LSCs or normal bone marrow CD34+ cells were incubated with human macrophages (left) or mouse macrophages (right) in the presence of the indicated antibodies and then assessed for phagocytosis by immunofluorescence microscopy. The phagocytic index in each condition was determined by calculating the number of ingested cells per 100 macrophages. For AML LSCs, the differences between isotype or anti-CD45 antibody treatment and anti-CD47 blocking antibody (B6H12.2 and BRIC126) treatment were statistically significant in all pairwise comparisons with human and mouse macrophages (p<0.001).For human macrophages, the differences between AML LSCs and normal CD34+ cells were statistically significant for B6H12.2 (p<0.001) and BRIC126 (p=0.002). [Figure 9]Anti-CD47 antibodies stimulate in vitro macrophage phagocytosis of primary human AML LSCs. AML LSCs were purified by FACS from four primary human AML samples, labeled with the fluorescent dye CFSE, and incubated with human peripheral blood macrophages in the presence of isotype control, isotype-matched anti-CD45, or anti-CD47 antibodies. (A) Fluorescently labeled LSCs were assessed for presence inside macrophages by immunofluorescence microscopy. The phagocytic index in each condition was determined by calculating the number of ingested cells per 100 macrophages. (B) Macrophages were harvested, stained with fluorescently labeled anti-human macrophage antibodies, and analyzed by flow cytometry. Double-positive events, hMac+CFSE+, confirm macrophages that have phagocytosed CFSE-labeled LSCs. Each sample is represented by a different color. [Figure 10] Figure 10A-B: Monoclonal antibodies against human CD47 inhibit AML LSC engraftment in vivo. Three primary human AML samples were incubated with an IgG1 isotype control antibody, an anti-CD45 IgG1 antibody, or an anti-CD47 IgG1 antibody (B6H12.2) prior to transplantation into neonatal NOG mice. A portion of these cells were analyzed for coating by staining with an anti-mouse IgG secondary antibody and analyzed by flow cytometry (A). After 13 weeks, the mice were sacrificed and bone marrow was analyzed by flow cytometry to determine the percentage of human CD45+CD33+ myeloid leukemia cells (B). The difference in mean engraftment between anti-CD47-coated cells and both isotype-coated cells (p<0.001) and anti-CD45-coated cells (p=0.003) was statistically significant. [Figure 11]CD47 is upregulated in acute myeloid leukemia in mice. Representative stem and progenitor plots of leukemic hMRP8bcrabl×hMRP8bcl2 cells compared to control non-leukemic animals are shown. Gated Lin-c-Kit+Sca-1+ cells from control bone marrow (a) and leukemic spleen (b), and gated Lin-c-Kit+Sca-1- cells from control bone marrow (c) and leukemic spleen (d) show perturberance of normal hematopoiesis in leukemic mice. Prevalence is shown as a percentage of total bone marrow or spleen mononuclear fraction. (e) Quantitative RT-PCR shows that CD47 is upregulated in leukemic BM cells. Data are shown from three sets of mice transplanted with either leukemic BM cells or control hRMP8bcrabl×hMRP8bcl2 BM cells and then sacrificed 2–6 weeks later. Results were normalized to β-actin and 18S rRNA expression. Percent change was determined compared to controls transplanted with intact Bcl-2+ BM cells. Error bars represent 1 sd. (f) Histograms show CD47 expression in gated populations of leukemic (grey) and control (black) mice. [Figure 12A] GMP expansion and CD47 upregulation in human myeloid leukemia. a) Representative FACS plots of myeloid progenitors (CD34+CD38+Lin-), including common myeloid progenitors (CMPs), megakaryocyte erythroid progenitors (MEPs), and granulocyte macrophage progenitors (GMPs), in normal bone marrow (BM), and in aCML, BC CML, and AML. [Figure 12B] GMP proliferation and CD47 upregulation in human myeloid leukemia. b) Comparative FACS histograms of CD47 expression by normal (red; n=6) and acute myeloid leukemia (AML, blue; n=6) hematopoietic stem cells (HSC; CD34+CD38-CD90+Lin-) and progenitor cells (CD34+CD38+Lin-). [Figure 12C]GMP expansion and CD47 upregulation in human myeloid leukemia. c) Comparative FACS histograms of CD47 expression by hematopoietic stem cells (HSC; CD34+CD38-CD90+Lin) and committed progenitor cells (CD34+CD38+Lin-) from normal (red) and chronic myeloid leukemia. Upper panels: HSC, progenitor, and lineage positive cells from normal (n=7) and chronic phase CML (n=4). Middle panels: HSC, progenitor, and lineage positive cells from normal (n=7) and accelerated phase CML (n=7). Lower panels: HSC, progenitor, and lineage positive cells from normal (n=7) and blast crisis CML (n=4). [Figure 13A] Overexpression of mouse CD47 enhances the tumorigenic potential of MOLM-13 cells. a) MOLM-13 cells were transduced with either a control virus or a virus expressing mouse CD47 cDNA (type 2). The resulting cell lines, designated Tet or Tet-CD47, were competitively transplanted into RAG / common gamma chain-deficient mice with non-transduced MOLM-13 cells (5x105 Tet cells (n=6) or Tet-47 cells (n=8) with 5x105 MOLM-13). Upon moribundity, mice were analyzed for GFP and human CD45 chimerism. [Figure 13B] Overexpression of mouse CD47 enhances the tumorigenic potential of MOLM-13 cells. b) MOLM-13 chimerism in hematopoietic tissues was determined based on human CD45 chimerism and measurement of tumor lesion size. [Figure 13C] Overexpression of mouse CD47 increases the tumorigenic potential of MOLM-13 cells. c) Survival of mice competitively transplanted with MOLM-13 cells and Tet MOLM-13 cells or Tet-CD47 MOLM-13 cells is plotted. Control mice died due to heavy tumor burden at the injection site, but no engraftment in hematopoietic tissues. [Figure 13D] Overexpression of mouse CD47 enhances the tumorigenic potential of MOLM-13 cells. d) Hematoxylin and eosin section (200x) of a Tet-CD47 MOLM-13 transplanted liver, demonstrating periportal (arrows) and sinusoidal (arrowhead) tumor infiltration. [Figure 13E] Overexpression of mouse CD47 increases the tumorigenicity of MOLM-13 cells. e) 1x106 Tet MOLM-13 cells (n=5) or Tet-CD47 MOLM-13 cells (n=4) were injected into the right thigh of RAG2- / - Gc- / - mice, and tissues were analyzed 50-75 days later to determine chimerism of MOLM-13 cells in the bone marrow. [Figure 13F] Overexpression of mouse CD47 enhances the tumorigenicity of MOLM-13 cells. f) Survival curves of mice implanted intrafemorally with Tet or Tet-CD47 MOLM-13 cells. [Figure 13G] Overexpression of mouse CD47 enhances the tumorigenic potential of MOLM-13 cells. g) Examples of liver tumor formation and hepatomegaly in mice implanted with Tet-CD47 MOLM-13 and control. GFP fluorescence shows tumor nodule formation and diffuse infiltration. [Figure 14A] Overexpression of CD47 prevents phagocytosis of non-opsonized hepatic MOLM-13 cells. a) Tet or Tet-CD47 MOLM-13 cells were incubated with bone marrow-derived macrophages (BMDMs) for 2, 4, or 6 hours and the phagocytic index was measured. Error bars represent 1 sd. (n=6 per time point). [Figure 14B] Overexpression of CD47 prevents phagocytosis of non-opsonized hepatic MOLM-13 cells. b) FACS analysis of BMDM incubated with either Tet or Tet-CD47 cells. [Figure 14C] Overexpression of CD47 prevents phagocytosis of non-opsonized hepatic MOLM-13 cells. c) Photomicrographs (400x) of BMDM incubated with Tet or Tet-CD47 MOLM-13 cells at 2 and 24 hours. [Figure 14D]Overexpression of CD47 prevents phagocytosis of non-opsonized hepatic MOLM-13 cells. d) Tet or Tet-CD47 MOLM-13 cells were transplanted into RAG2- / - Gc- / - mice and macrophages in bone marrow, spleen, and liver were analyzed 2 hours later. Gating was done on the GFP+ fraction of macrophages. Results are representative of three experiments. [Figure 15A] High expression of CD47 in MOLM-13 cells correlates with tumorigenic potential and evasion of phagocytosis. a) Tet-CD47 MOLM-13 cells were separated into high and low expressing clones as previously described. Histograms show CD47 expression in MOLM-13 high expressing cells (black), MOLM-13 low expressing cells (gray), and mouse bone marrow cells (shaded). Values ​​obtained from MFI / FSC2 (x109) are shown. [Figure 15B] High expression of CD47 in MOLM-13 cells correlates with tumorigenic potential and evasion of phagocytosis. b) Mice implanted with CD47hi MOLM-13 cells were given doxycycline for 2 weeks. Histograms show CD47 expression levels in untreated (shaded) and treated (shaded) mice, with MFI / FSC2 (x109) values ​​displayed. [Figure 15C] High expression of CD47 on MOLM-13 cells correlates with tumorigenic potential and evasion of phagocytosis. c) Survival of RAG2- / - Gc- / - mice implanted with 1x106 CD47hi MOLM-13 cells, CD47lo MOLM-13 cells, or CD47hi MOLM-13 cells and treated with doxycycline 2 weeks after implantation. [Figure 15D] High expression of CD47 on MOLM-13 cells correlates with tumorigenic potential and evasion of phagocytosis. d) Liver and spleen size of mice at necropsy or 75 days after transplantation of 1x106 CD47hi MOLM-13 cells, CD47lo MOLM-13 cells, or CD47hi MOLM-13 cells with doxycycline treatment 2 weeks after transplantation. [Figure 15E] High expression of CD47 on MOLM-13 cells correlates with tumorigenic potential and evasion of phagocytosis. e) Human cell bone marrow and spleen chimerism in mice at necropsy or 75 days after transplantation of 1x106 CD47hi MOLM-13 cells, CD47lo MOLM-13 cells, or CD47loi MOLM-13 cells with doxycycline treatment 2 weeks after transplantation. [Figure 15F] High expression of CD47 in MOLM-13 cells correlates with tumorigenic potential and evasion of phagocytosis. f) Mouse CD47 expression in CD47lo MOLM-13 cells engrafted in bone marrow (open) compared to the parent cell line (shaded). MFI / FSC2 (x109) values ​​are shown. [Figure 15G] High expression of CD47 in MOLM-13 cells correlates with tumorigenic potential and evasion of phagocytosis. g) 2.5x105 CD47hi or CD47lo MOLM-13 cells were incubated with 5x104 BMDMs for 2 hours. Phagocytic index is shown. [Figure 15H] High expression of CD47 in MOLM-13 cells correlates with tumorigenic potential and evasion of phagocytosis. h) 2.5x105 CD47hi MOLM-13 RFP and CD47lo MOLM-13 GFP cells were incubated with 5x104 BMDMs for 2 hours. Phagocytosis index of three individual samples of CD47hi MOLM-13 RFP cells (red) and CD47lo MOLM-13 GFP cells (green) are shown. [Figure 15I] High expression of CD47 in MOLM-13 cells correlates with tumorigenic potential and evasion of phagocytosis. i) 2.5x105 CD47hi MOLM-13 RFP and CD47lo MOLM-13 GFP cells were incubated with 5x104 BMDMs for 24 hours. Photomicrographs show brightfield (top left), RFP (top right), GFP (bottom left), and merged (bottom right) images. [Figure 16]a) FACS analysis of CD47 expression on bone marrow hematopoietic stem cells (c-kit+Sca+Lin-), myeloid progenitors (c-kit+Sca-Lin-) or blasts (c-kit lo Sca-Lin-) of non-leukemic Fas lpr / lpr hMRP8bcl-2 (blue) and leukemic Fas lpr / lpr hMRP8bcl-2 (green). b) Mouse bone marrow was transduced with a p210 bcr / abl-containing retrovirus as previously described24. When moribund, mice were sacrificed and spleens were analyzed. CD47 expression is shown on c-Kit+Mac-1+ cells in the spleens of two leukemic mice (unshaded histograms) and in bone marrow from a wild-type mouse (shaded histograms). c) Histograms show CD47 expression in gated populations of hMRP8bcrabl x hMRP8bcl2 leukemic mice (red), hMRP8bcl2 non-leukemic mice (blue), and wild-type mice (green). CD47 was stained using FITC-conjugated anti-mouse CD47 (Pharmingen). [Figure 17A] a) Human CD47 expression in human leukemia cell lines and cord blood HSCs (black histograms). Isotype control staining is shown in grey. [Figure 17B] b) CD47 MFI above background was normalized to cell size by dividing by FSC2. The values ​​obtained for each cell type are shown above the bars. [Figure 17C] c) HL-60 cells engraft in mouse bone marrow. 5x105 cells were injected intravenously into RAG2- / - Gc- / - animals and the mice were analyzed 4 weeks later. [Figure 17D] d) Cells were stained with CFSE and co-cultured with BMDM. Phagocytosis events were counted after 2 hours. For irradiation, Jurkat cells were given a dose of 2 Gray and incubated for 16 hours prior to the phagocytosis assay. [Figure 18A](a) Analysis of stem and progenitor cells derived from bone marrow of IAP+ / +, IAP+ / -, and IAP- / - mice. Stem cells (left) are gated on lineage-c-Kit+Sca-1+ cells. Myeloid progenitor cells (right) are gated on lineage-c-Kit+Sca-1+ cells. Total bone marrow frequency is shown near each gated population. [Figure 18B] (b) Colony formation yields of individually sorted LT-HSCs on day 7. G-granulocytes; M-macrophages; GM-granulocytes and macrophages; GEMM-granulocytes, macrophages, erythrocytes, and megakaryocytes; Meg-megakaryocytes. [Figure 18C] (c) Survival curves of recipient mice that received a radiation dose of 9.5 Gray and were transplanted with the cells shown in the figure. All irradiated control mice died within 12-15 days. n=5 for each group. [Figure 18D] (d) Example plot of CD45.1 / CD45.2 chimerism at 4 weeks post-transplant. CD45.1 mice were transplanted with 50 LT-HSC (CD45.2) and 2x105 CD45.1 helper bone marrow. Cells are gated on B220-CD3-Mac-1+side scatter mid / hi cells. IAP- / - cells fail to engraft. [Figure 18E] (e) Summary of chimerism analysis of mice transplanted with 50 or 500 IAP+ / + or IAP- / - cells. [Figure 18F] (f) IAP+ / + or IAP- / - c-Kit enriched cells were incubated with wild-type BMDM. Results show the mean phagocytic index calculated from three individual samples. Error bars correspond to 1 s.d. [Figure 18G] (g) Photomicrograph of the phagocytosis assay taken after 2 h, showing the genotype of -Kit-enriched cells. [Figure 19A] (a) Mice were mobilized with Cy / G and bone marrow was analyzed on day 2. CD47 expression levels in c-Kit+ cells are shown. [Figure 19B](b) Myeloid progenitor gate and stem cell gate are shown for bone marrow at day 2 post-mobilization. The histograms on the left show CD47 expression levels in bone marrow LT-HSCs and GMPs at steady state (shaded histogram), day 2 post-mobilization (black line), and day 5 post-mobilization (grey line). [Figure 19C] (c) Relative MFI of CD47 in GMPs from days 0 to 5 after Cy / G mobilization. Results were normalized so that steady-state GMPs equaled 100. [Figure 19D] (d) Myeloid progenitor gate and stem cell gate are shown for bone marrow after LPS treatment at day 2. Histograms show CD47 expression levels in LT-HSCs and GMPs at day 2 after LPS (black line), day 5 after LPS (dark grey shaded histogram), steady state (light grey shaded histogram), and IAP- / - (black shaded histogram). [Figure 19E] (e) Evaluation of KLS cells in hematopoietic organs of mobilized IAP+ / + and IAP- / - mice from days 2 to 5. Two mice per genotype per day were analyzed. [Figure 20] (a) CD47 expression levels of IAP+ / +, IAP+ / -, and IAP- / - LT-HSCs. MFI values ​​are shown for each group. (b) Donor chimerism analysis of grafts from IAP+ / + (top) or IAP+ / - (bottom) mice. Mice were bled 2, 8, and 40 weeks after transplantation. 2 × 106 donor cells were transplanted into sublethally irradiated congenic recipients. [Figure 21A] Identification and separation of normal and leukemic progenitors from the same patient based on differential CD47 expression. A: CD47 expression in the Lin-CD34+CD38- LSC enriched fraction of specimen SU008 was measured by flow cytometry. CD47 high and low expressing cells were identified and purified using FACS. In the left panel, lineage negative cells are gated, whereas in the right panel, Lin-CD34+CD38- cells are gated. [Figure 21B]Identification and isolation of normal and leukemic progenitors from the same patient based on differential CD47 expression. B: Lin-CD34+CD38-CD47lo and Lin-CD34+CD38-CD47hi cells were plated in complete methylcellulose, which can support the growth of all myeloid colonies. After 14 days, myeloid colony formation was determined by morphological assessment. Representative CFU-G / M (left) and BFU-E (right) are shown. [Figure 21C] Identification and separation of normal and leukemic progenitors from the same patient based on differential CD47 expression. C: Lin-CD34+CD38-CD47lo cells were transplanted into two newborn NOG mice. After 12 weeks, the mice were sacrificed and bone marrow was analyzed by flow cytometry for the presence of human CD45+CD33+ myeloid and human CD45+CD19+ lymphoid cells. [Figure 21D] Identification and separation of normal and leukemic progenitors from the same patient based on differential CD47 expression. D: Normal bone marrow HSCs, bulk SU008 leukemic cells, Lin-CD34+CD38-CD47hi cells, Lin-CD34+CD38-CD47lo cells, or human CD45+ cells purified from bone marrow of mice transplanted with Lin-CD34+CD38-CD47lo cells were assessed by PCR for the presence of FLT3-ITD mutations. Wild-type FLT3 and FLT3-ITD products are shown. [Figure 22] Increased CD47 expression in human AML is associated with poor clinical outcome. Relapse-free survival (A,C) and overall survival (B,D) of 132 cytogenetically normal AML patients (A,B) and a subset of 74 patients without FLT3-ITD mutations (C,D). Patients were stratified into CD47 low and CD47 high expressers (28% high expressers, 72% low expressers) based on optimal thresholds determined by microarray analysis from an independent training dataset. Significance measures are based on log-likelihood estimates of p-values ​​when treating the CD47 expression model as a binary classification. [Figure 23A]Figure 23A-E: Monoclonal antibodies against human CD47 eliminate AML in vivo. Neonatal NOG mice were transplanted with AML LSCs and 8-12 weeks later peripheral blood (A, B) and bone marrow (C-E) were analyzed for baseline engraftment prior to treatment with anti-CD47 antibody (B6H12.2) or control IgG antibody (day 0). Mice were treated with daily intraperitoneal injections of 100 μg for 14 days, at the end of which mice were sacrificed and peripheral blood and bone marrow were analyzed for percentage of human CD45+CD33+ leukemia. A: Pre- and post-treatment human leukemic chimerism in peripheral blood from representative mice treated with anti-CD47 antibody and control IgG, as measured by flow cytometry. [Figure 23B] Figure 23A-E: Monoclonal antibodies against human CD47 eliminate AML in vivo. Neonatal NOG mice were transplanted with AML LSCs and 8-12 weeks later, peripheral blood (A, B) and bone marrow (C-E) were analyzed for baseline engraftment before treatment with anti-CD47 antibody (B6H12.2) or control IgG antibody (day 0). Mice were treated with daily intraperitoneal injections of 100 μg for 14 days, at the end of which mice were sacrificed and peripheral blood and bone marrow were analyzed for the percentage of human CD45+CD33+ leukemia. B: Summary of human leukemic chimerism in peripheral blood assessed on multiple days during the course of treatment demonstrated clearance of leukemia in mice treated with anti-CD47 antibody compared to control IgG treatment (p=0.007). [Figure 23C] Figure 23A-E: Monoclonal antibodies against human CD47 eliminate AML in vivo. Neonatal NOG mice were transplanted with AML LSCs and 8-12 weeks later peripheral blood (A, B) and bone marrow (C-E) were analyzed for baseline engraftment prior to treatment with anti-CD47 antibody (B6H12.2) or control IgG antibody (day 0). Mice were treated for 14 days with daily intraperitoneal injections of 100 μg, at the end of which mice were sacrificed and peripheral blood and bone marrow were analyzed for percentage of human CD45+CD33+ leukemia. C: Pre- and post-treatment human leukemic chimerism in bone marrow from representative mice treated with anti-CD47 antibody and control IgG, as measured by flow cytometry. [Figure 23D] FIG. 23A-E: Monoclonal antibodies against human CD47 eliminate AML in vivo. Neonatal NOG mice were transplanted with AML LSCs and 8-12 weeks later, peripheral blood (A, B) and bone marrow (C-E) were analyzed for baseline engraftment before treatment with anti-CD47 antibody (B6H12.2) or control IgG antibody (day 0). Mice were treated with daily intraperitoneal injections of 100 μg for 14 days, at the end of which mice were sacrificed and peripheral blood and bone marrow were analyzed for the percentage of human CD45+CD33+ leukemia. D: Overview of human leukemic chimerism in bone marrow at day 14 compared to day 0 demonstrated a dramatic reduction in leukemic burden in mice treated with anti-CD47 antibody compared to control IgG treatment (p<0.001). [Figure 23E] Figure 23A-E: Monoclonal antibodies against human CD47 eliminate AML in vivo. Neonatal NOG mice were transplanted with AML LSCs and 8-12 weeks later, peripheral blood (A, B) and bone marrow (C-E) were analyzed for baseline engraftment before treatment with anti-CD47 antibody (B6H12.2) or control IgG antibody (day 0). Mice were treated with daily intraperitoneal injections of 100 μg for 14 days, at the end of which mice were sacrificed and peripheral blood and bone marrow were analyzed for the percentage of human CD45+CD33+ leukemia. E: Hematoxylin-eosin stained sections of representative mouse bone marrow cavities from mice transplanted with SU004 after treatment with either control IgG (panels 1, 2) or anti-CD47 antibody (panels 4, 5). The IgG-treated bone marrow was filled with monomorphic leukemic blasts, whereas the anti-CD47-treated bone marrow was hypocellular, demonstrating the disappearance of human leukemia. In several anti-CD47-treated mice in which leukemia persisted, macrophages containing engulfed pyknotic cells were detected, capturing the disappearance of human leukemia (arrows in panels 3 and 6). [Figure 24] Increased CD47 expression predicts poor overall survival in DLBCL and ovarian cancer (A) Cohort of 230 patients with diffuse large B-cell lymphoma (p=0.01), (B) Cohort of 133 patients with advanced stage (III / IV) ovarian cancer (p=0.04). [Diagram 25] Anti-CD47 antibodies enable phagocytosis of solid tumor stem cells in vitro. The indicated cells were incubated with human macrophages in the presence of IgG1 isotype, anti-HLA, or anti-CD47 antibodies, and the phagocytic index was determined by immunofluorescence microscopy. Statistics: For bladder cancer cells, IgG1 isotype was compared to anti-HLA (p=0.93) and anti-CD47 (p=0.01); for normal bladder urothelium, IgG1 isotype was compared to anti-HLA (p=0.50) and anti-CD47 (p=0.13); for ovarian cancer cells, IgG1 isotype was compared to anti-HLA (p=0.11) and anti-CD47 (p<0.001). Each data point represents a separate tumor or normal tissue sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] DETAILED DESCRIPTION OF EMBODIMENTS Methods for manipulating hematopoietic cells, including hematopoietic cells in the circulation, are provided. In some embodiments of the present invention, hematopoietic stem cells or hematopoietic progenitor cells are protected from phagocytosis in the circulation by providing a host animal with a CD47 mimetic molecule that interacts with SIRPα on phagocytic cells such as macrophages and reduces phagocytosis. In other embodiments, leukemia cells are targeted for phagocytosis by blocking CD47 on the cell surface. In other embodiments, cells of solid tumors are targeted for phagocytosis by blocking CD47 on the cell surface. In another embodiment, a method for targeting or depleting AML cancer stem cells is provided, comprising contacting reactant blood cells with an antibody that specifically binds to CD47 to target or deplete AMLSC. In another embodiment, a method for targeting cancer cells of solid tumors in a human subject is provided by administering an antibody against CD47 to the subject.

[0016] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary, and the scope of the present invention will be limited only by the appended claims, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0017] Where a range of values ​​is provided, unless the context dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in those smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of these limits, ranges excluding either or both of those included limits are also included in the invention.

[0018] Methods recited herein may be carried out in any order of the recited events which is logically possible, as well as in the recited order of events.

[0019] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described below.

[0020] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0021] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. Thus, this specification is intended to serve as a predicate for using exclusive language such as "solely" and "only" in conjunction with the recitation of claim elements or for using a "negative" limitation.

[0022] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0023] definition CD47 Polypeptides. Three transcriptional variants of human CD47 (variant 1, NM 001777; variant 2, NM 198793; and variant 3, NM 001025079) encode three isoforms of the CD47 polypeptide. CD47 isoform 1 (NP 001768), the longest of the three isoforms, is 323 amino acids long. CD47 isoform 2 (NP 942088) is 305 amino acids long. CD47 isoform 3 is 312 amino acids long. The three isoforms are identical in sequence in the first 303 amino acids. Amino acids 1-8 contain the signal sequence, amino acids 9-142 contain the soluble fragment, the CD47 immunoglobulin-like domain, and amino acids 143-300 are the transmembrane domain.

[0024] "CD47 mimetics" include molecules that function similarly to CD47 by binding and activating the SIRPα receptor. Molecules useful as CD47 mimetics include derivatives, variants, and biologically active fragments of naturally occurring CD47. By "variant" polypeptide is meant a biologically active polypeptide, as defined below, that has less than 100% sequence identity with a native sequence polypeptide. Such variants include polypeptides in which one or more amino acid residues have been added to the N-terminus or C-terminus of the native sequence or internally to the native sequence; polypeptides in which about 1-40 amino acid residues have been deleted and optionally replaced with one or more amino acid residues; and derivatives of the above polypeptides in which amino acid residues have been covalently modified such that the resulting product has a non-naturally occurring amino acid. Typically, biologically active variants have an amino acid sequence that has at least about 90% amino acid sequence identity with the native sequence polypeptide, preferably at least about 95%, and more preferably at least about 99%. Variant polypeptides may be naturally or non-naturally glycosylated. That is, the polypeptide has a glycosylation pattern that differs from the glycosylation pattern found in the corresponding naturally occurring protein. The variant polypeptides may have post-translational modifications not found in the native CD47 protein.

[0025] Fragments of soluble CD47 are of interest, particularly biologically active fragments and / or fragments corresponding to functional domains. Fragments of interest are typically at least about 10 amino acids long to at least about 15 amino acids long, usually at least about 50 amino acids long, but usually not more than about 142 amino acids long, where the fragment is considered to have an amino acid stretch identical to CD47. For example, a fragment "at least 20 amino acids long" is intended to include, for example, 20 or more consecutive amino acids from a polypeptide encoded by a CD47 cDNA. In this context, "about" includes the specifically recited value or a few amino acids (5, 4, 3, 2, or 1) larger or smaller. The protein variants described herein are encoded by polynucleotides within the scope of the invention. The genetic code can be used to select the appropriate codons to construct the corresponding variants. These polynucleotides can be used to generate polypeptides, and these polypeptides can be used to generate antibodies by known methods.

[0026] A "fusion" polypeptide is a polypeptide that comprises a polypeptide or a portion thereof (e.g., one or more domains) fused or conjugated to a heterologous polypeptide. For example, a fused soluble CD47 protein will have at least one biological property in common with a native-sequence soluble CD47 polypeptide. Examples of fusion polypeptides include immunoadhesins, which combine a portion of a CD47 polypeptide with an immunoglobulin sequence, as described above, and epitope-tagged polypeptides that comprise a soluble CD47 polypeptide or a portion thereof fused to a "tag polypeptide." A tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough so as not to interfere with biological activity of the CD47 polypeptide. Suitable tag polypeptides generally have at least six amino acid residues, and usually between about 6 and 60 amino acid residues.

[0027] A "functional derivative" of a native sequence polypeptide is a compound that has qualitative biological properties in common with the native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of the native sequence, derivatives of native sequence polypeptides and their fragments, provided that they have a biological activity in common with the corresponding native sequence polypeptide. The term "derivative" encompasses both amino acid sequence variants of the polypeptide and covalent modifications thereof. Derivatives and fusions of soluble CD47 are used as CD47 mimetic molecules.

[0028] The first 142 amino acids of the CD47 polypeptide comprise the extracellular region of CD47 (SEQ ID NO: 1). The amino acid sequences of the extracellular regions of the three isoforms are identical, and therefore any of the isoforms can be used to make soluble CD47. "Soluble CD47" is a CD47 protein that lacks a transmembrane domain. Soluble CD47 is secreted outside of the cell that expresses it, instead of being localized on the cell surface. Soluble CD47 may be fused to another polypeptide to add functionality, e.g., to increase stability in vivo. In general, such fusion partners are stable plasma proteins that, when present as a fusion, can extend the in vivo plasma half-life of the soluble CD47 protein, and in particular such stable plasma proteins are immunoglobulin constant domains. In most cases, stable plasma proteins are usually present in multimeric form, e.g., as immunoglobulins or lipoproteins, in which the same or different polypeptide chains are usually linked by disulfide bonds and / or non-covalent bonds to form assembled multi-chain polypeptides. Soluble CD47 fused to human Ig G1 has been described (Motegi S. et al. EMBO J. 22(11): 2634-2644).

[0029] Stable plasma proteins are typically proteins having about 30-2,000 residues and exhibit a long circulating half-life in the native environment, i.e., greater than about 20 hours. Examples of suitable stable plasma proteins are immunoglobulins, albumins, lipoproteins, apolipoproteins, and transferrin. Typically, the extracellular region of CD47 is fused to a plasma protein at the N-terminus of the plasma protein or a fragment thereof that can confer a long half-life to the soluble CD47. It is sufficient if the plasma half-life of the soluble CD47 is greater than about 100%.

[0030] Usually, the C-terminus of soluble CD47 is fused to the N-terminus of the constant region of an immunoglobulin instead of the variable region, but N-terminus fusions can also be used. Typically, such fusions retain at least the functionally active hinge, CH2 domain, and CH3 domain of the constant region of an immunoglobulin heavy chain, where the heavy chain can include one or a combination of proteins of the IgG1, IgG2a, IgG2b, IgG3, IgG4, IgA, IgM, IgE, and IgD, usually IgG class. They can also be fused to the C-terminus of the Fc portion of the constant domain, or directly to the N-terminus of the CH1 of the heavy chain or the corresponding region of the light chain. Usually, this is accomplished by constructing the appropriate DNA sequence and expressing it in recombinant cell culture. Alternatively, the polypeptide can be synthesized according to known methods.

[0031] The precise site at which the fusion is made is not critical; a particular site can be selected in order to optimize the biological activity, secretion, or binding characteristics of CD47. Optimal sites will be determined by routine experimentation.

[0032] In some embodiments, hybrid immunoglobulins are assembled as monomers or hetero- or homo-multimers, and in particular as dimers or tetramers.Generally, these assembled immunoglobulins have known unit structures. The basic four-chain structural unit is the form in which IgG, IgD, and IgE exist.In higher molecular weight immunoglobulins, the four-chain unit is repeated; generally, IgM exists as a pentamer of basic four-chain units linked by disulfide bonds.IgA immunoglobulins, and occasionally IgG immunoglobulins, can also exist in serum in multimeric form.In the case of multimers, each four-chain unit can be the same or different.

[0033] Suitable CD47 mimics and / or CD47 fusion proteins can be identified by compound screening to detect the ability of the agent to mimic the biological activity of CD47. One biological activity of CD47 is the activation of SIRPα receptor on macrophages. In vitro assays can be performed as a first screen for the effectiveness of candidate agents, and in vivo assays are usually performed to support biological assays. Desirable agents are effective in temporarily blocking SIRPα receptor activation. Desirable agents are essentially temporary, for example, due to biological degradation.

[0034] In vitro assays for CD47 bioactivity include, for example, inhibition of phagocytosis of porcine cells by human macrophages, binding to SIRPα receptors, SIRPα tyrosine phosphorylation, and the like. An exemplary assay for CD47 bioactivity involves contacting a human macrophage composition in the presence of a candidate agent. The cells are incubated with the candidate agent for about 30 minutes and lysed. The cell lysate is mixed with an anti-human SIRPα antibody to immunoprecipitate SIRPα. The precipitated proteins are separated by SDS PAGE, then transferred to nitrocellulose and probed with an antibody specific for phosphotyrosine. Candidate agents useful as CD47 mimetics increase SIRPα tyrosine phosphorylation by at least 10%, or up to 20%, or 50%, or 70%, or 80%, or up to about 90%, compared to the phosphorylation level observed in the absence of the candidate agent. Another exemplary assay for CD47 bioactivity involves measuring phagocytosis of hematopoietic cells by human macrophages. Candidate agents useful as CD47 mimetics down-regulate phagocytosis by at least about 10%, at least about 20%, at least about 50%, at least about 70%, at least about 80%, or up to about 90% compared to the level of phagocytosis observed in the absence of the candidate agent.

[0035] The polynucleotide encoding soluble CD47 or soluble CD47-Fc can be introduced into a suitable expression vector. The expression vector is introduced into a suitable cell. In general, the expression vector has a convenient restriction site located near the promoter sequence, which allows for the insertion of the polynucleotide sequence. A transcription cassette can be prepared, which includes a transcription initiation region, a CD47 gene or a fragment thereof, and a transcription termination region. The transcription cassette can be introduced into various vectors, such as plasmids; retroviruses, such as lentiviruses; and adenoviruses, which can be maintained transiently or stably in cells, usually for at least about a day, and more usually for at least several days to several weeks.

[0036] The various manipulations may be carried out in vitro or in a suitable host, such as E. coli. After each manipulation, the resulting construct may be cloned, the vector isolated, and DNA screened or sequenced to ensure the accuracy of the construct. The sequence may be screened by restriction analysis or sequencing, etc.

[0037] Soluble CD47 can be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, protein G affinity chromatography, e.g., hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is used for purification.

[0038] Soluble CD47 can also be recovered from purified cellular products, whether directly isolated or cultured; from products of chemical synthetic procedures; and from products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, bacterial cells, yeast, higher plant cells, insect cells, and mammalian cells.

[0039] To obtain differential responses to different concentrations, multiple assays may be performed in parallel using different concentrations.As known in the art, typically, a concentration range obtained from 1:10 or other logarithmic scale dilutions is used to determine the effective concentration of an agent.If necessary, these concentrations may be further refined by a second series of dilutions.Typically, one of these concentrations serves as a negative control.That is, it is a concentration of zero or below the detection level of the agent, or a concentration of the agent that does not cause detectable changes in binding.

[0040] Compounds of interest for screening include biologically active agents from many chemical classes, primarily organic molecules, but in some cases inorganic molecules, organometallic molecules, immunoglobulins, chimeric CD47 proteins, CD47-related proteins, gene sequences, and the like. Also of interest are small organic molecules that contain functional groups necessary for structural interactions with proteins, particularly hydrogen bonding, and typically contain at least one amine, carbonyl, hydroxyl, or carboxyl group, and often at least two chemical functional groups. Candidate agents often contain cyclic carbon or heterocyclic structures, and / or aromatic or polyaromatic structures, substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0041] Compounds are obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, natural compound libraries in the form of bacterial, fungal, plant, and animal extracts are also available or readily produced. Furthermore, natural or synthetically produced libraries and compounds can be easily modified by conventional chemical, physical, and biochemical means and used to produce combinatorial libraries. Known pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, and the like, to produce structural analogs.

[0042] By "manipulating phagocytosis" is meant up-regulating or down-regulating phagocytosis by at least about 10%, or up to 20%, or 50%, or 70%, or 80%, or up to about 90% compared to the level of phagocytosis observed in the absence of intervention. Thus, in the context of reducing phagocytosis of circulating hematopoietic cells, particularly in the transplant context, manipulating phagocytosis means down-regulating phagocytosis by at least about 10%, or up to 20%, or 50%, or 70%, or 80%, or up to about 90% compared to the level of phagocytosis observed in the absence of intervention.

[0043] CD47 inhibitors. Agents of interest as CD47 inhibitors include specific binding members that prevent CD47 from binding to SIRPα receptor. The term "specific binding member" or "binding member" as used herein refers to a member of a specific binding pair, i.e., two molecules, usually two different molecules, where one of these molecules (i.e., the first specific binding member) specifically binds to the other molecule (i.e., the second specific binding member) by chemical or physical means. CD47 inhibitors useful in the methods of the present invention include analogs, derivatives, and fragments of the original specific binding member.

[0044] In a preferred embodiment, the specific binding member is an antibody. The term "antibody" or "antibody portion" is intended to include any molecular structure, including a polypeptide chain, that has a specific shape that fits and recognizes an epitope, and one or more non-covalent interactions stabilize the complex between the molecular structure and the epitope. The antibody used in the present invention may be a polyclonal antibody, but monoclonal antibodies are preferred because they can be replicated in cell culture or recombinantly and can be modified to reduce antigenicity.

[0045] Polyclonal antibodies can be raised by standard protocols by injecting production animals with antigenic compositions. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. When using complete proteins or larger protein portions, antibodies can be raised by immunizing production animals with the protein and a suitable adjuvant (e.g., Freund's adjuvant, Freund's complete adjuvant, oil-in-water emulsion, etc.). When using small peptides, it is advantageous to conjugate the peptide to a larger molecule to create an immunostimulatory conjugate. Commonly used conjugate proteins that are commercially available for such applications include bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). To raise antibodies against specific epitopes, peptides derived from the complete sequence can be utilized. Alternatively, to generate antibodies against relatively short peptide portions of protein targets, excellent immune responses can be elicited when the polypeptide is linked to a carrier protein such as ovalbumin, BSA, or KLH. Alternatively, in the case of monoclonal antibodies, hybridomas can be formed by isolating stimulated immune cells, for example, immune cells from the spleen of the inoculated animal. These cells are then fused to immortalized cells, such as myeloma cells or transformed cells, which can replicate indefinitely in cell culture, thereby generating immortal immunoglobulin-secreting cell lines. Furthermore, antibodies or antigen-binding fragments can also be produced by genetic engineering. Humanized, chimeric, or xenohuman antibodies, which provoke less immune response when administered to humans, are preferred for use in the present invention.

[0046] In addition to complete immunoglobulins (or their recombinant counterparts), immunoglobulin fragments (e.g., Fab', F(ab')2, or other fragments) that contain the epitope binding site are useful as antibody moieties in the present invention. Such antibody fragments can be generated from complete immunoglobulins by cleavage with ricin, pepsin, papain, or other proteases. Fragments or minimal immunoglobulins can be engineered using recombinant immunoglobulin technology. For example, "Fv" immunoglobulins for use in the present invention can also be generated by linking the light chain variable region to the heavy chain variable region by a peptide linker (e.g., polyglycine, or another sequence that does not form an alpha-helix or beta-sheet motif).

[0047] The effectiveness of CD47 inhibitors is evaluated by analyzing CD47 activity. The above-mentioned assay or its modified version is used. In an exemplary assay, AML SCs are incubated with bone marrow-derived macrophages in the presence or absence of candidate agents. Inhibitors of cell surface CD47 are expected to upregulate phagocytosis by at least about 10%, or up to 20%, or 50%, or 70%, or 80%, or up to about 90%, compared to phagocytosis in the absence of candidate agents. Similarly, in vitro assays for tyrosine phosphorylation levels of SIRPα show a decrease in phosphorylation by at least about 10%, or up to 20%, or 50%, or 70%, or 80%, or up to about 90%, compared to phosphorylation observed in the absence of candidate agents.

[0048] In one embodiment of the present invention, the agent or pharmaceutical composition comprising the agent is provided in an amount effective to detectably inhibit the binding of CD47 to SIRPα receptor present on the surface of phagocytic cells.Effective amount is determined by routine experimental testing in the art.Effective amount may vary according to the number of cells to be transplanted, transplantation site, and transplantation patient specific factors.

[0049] The terms "phagocytic cell" and "phagocyte" are used interchangeably herein and refer to cells capable of phagocytosis. There are three major categories of phagocytic cells: macrophages, mononuclear cells (histiocytes and monocytes); polymorphonuclear leukocytes (neutrophils) and dendritic cells.

[0050] The term "biological sample" encompasses a variety of sample types obtained from an organism and can be used in diagnostic or monitoring assays. The term encompasses blood and other liquid samples derived from an organism, solid tissue samples, such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny. The term encompasses samples that have been manipulated in any way after procurement, such as by treatment with reagents, solubilization, or enrichment for specific components. The term encompasses clinical samples, and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0051] Hematopoietic stem cells (HSCs), as used herein, refer to a cell population that has the ability to self-renew and give rise to all hematopoietic lineages. Such cell populations have been described in detail in the art. Hematopoietic progenitor cells include myeloid committed progenitors (CMPs), lymphoid committed progenitors (CLPs), megakaryocytic progenitors, and multipotent progenitors. The first lymphoid committed cells in adult mouse bone marrow to be described were common lymphoid progenitors (CLPs), and the first myeloid committed cells to be described were common myeloid progenitors (CMPs). Importantly, these cell populations have an extremely high level of lineage fidelity in in vitro and in vivo developmental assays. A comprehensive description of these cell subsets can be found in Akashi et al. (2000) Nature 404(6774):193, U.S. Patent No. 6,465,247; and published application USSN 09 / 956,279 (common myeloid progenitor cells); Kondo et al. (1997) Cell 91(5):661-7, and international application WO99 / 10478 (common lymphoid progenitor cells), reviewed in Kondo et al. (2003) Annu Rev Immunol. 21:759-806 (each of which is specifically incorporated herein by reference). The composition may be frozen at liquid nitrogen temperature for long-term storage, and can be thawed and used. For such compositions, cells are typically stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0052] The population of interest for use in the method of the present invention may be a substantially pure composition, for example, at least about 50% HSC, at least about 75% HSC, at least about 85% HSC, at least about 95% or more HSC; or may be a combination of one or more stem cell populations and progenitor cell populations, such as white blood cells obtained by apheresis. If a purified cell population is desired, the target population may be purified according to known techniques. For example, a population containing white blood cells, including particularly blood or bone marrow samples, is stained with a reactant specific to markers present on hematopoietic stem cells and hematopoietic progenitor cells, and these markers are sufficient to distinguish the main stem cell and progenitor cell populations. These reactants, such as antibodies, may be detectably labeled or may be indirectly labeled in the staining procedure.

[0053] Any combination of markers may be used that is sufficient to select the stem / progenitor cells of interest. A combination of markers of interest may include CD34 and CD38, which may be used to select hematopoietic stem cells (CD34 + , CD38 - ) to progenitor cells (CD34 + , CD38 + HSCs are negative for lineage markers and positive for CD90 expression.

[0054] The myeloid lineage has three cell populations, termed CMPs, GMPs, and MEPs. These cells express CD34 + CD38 + are negative for multiple mature lineage markers, including early lymphoid markers such as CD7, CD10, and IL-7R, and are further distinguished by the markers CD45RA (an isoform of CD45 that can downregulate at least some classes of cytokine receptor signaling) and IL-3R. These characteristics are - IL-3Rα lo (CMP), CD45RA + IL-3Rα lo (GMP), and CD45RA -IL-3Rα - (MEP). - IL-3Rα lo The cells give rise to GMPs and MEPs, with at least one third giving rise to both GM and MegE colonies at the single cell level. All three myeloid progenitors stain negatively for the markers Thy-1 (CD90), IL-7Rα (CD127); and a panel of lineage markers that may include CD2; CD3; CD4; CD7; CD8; CD10; CD11b; CD14; CD19; CD20; CD56; and glycophorin A (GPA) in humans, and CD2; CD3; CD4; CD8; CD19; IgM; Ter110; Gr-1 in mice. Apart from the mouse MEP subset, all progenitor cells are CD34 positive. In mice, all progenitor subsets can be further characterized as Sca-1 (Ly-6E and Ly-6A) negative, and c-kit high. In humans, all three subsets are CD38 + It is.

[0055] Common lymphoid progenitor cells, or CLPs, express low levels of c-kit (CD117) on the cell surface. Antibodies that specifically bind to c-kit in humans, mice, rats, etc. are known in the art. Alternatively, the c-kit ligand, steel factor (Slf), can be used to identify cells expressing c-kit. CLP cells express high levels of IL-7 receptor alpha chain (CDw127). Antibodies that bind to human CDw127 or mouse CDw127 are known in the art. Alternatively, the cells are identified by binding a ligand to the receptor IL-7. Human CLPs express low levels of CD34. Antibodies specific to human CD34 are commercially available and well known in the art. See, for example, Chen et al. (1997) Immunol Rev 157:41-51. Human CLP cells are also characterized as CD38 positive and CD10 positive. The CLP subset also has a phenotype in which it does not express lineage-specific markers, such as B220, CD4, CD8, CD3, Gr-1, and Mac-1. CLP cells are characterized as not expressing Thy-1, a marker characteristic of hematopoietic stem cells. Furthermore, the CLP phenotype is characterized by the lack of expression of Mel-14. - , CD43 lo , H.S.A. lo , CD45 + , and can be characterized as common cytokine receptor gamma chain positive.

[0056] Megakaryocyte progenitor (MKP) cells are positive for CD34 expression and the tetraspanin CD9 antigen. The CD9 antigen is a 227 amino acid molecule with four hydrophobic domains and one N-glycosylation site. This antigen is widely expressed but is absent on certain progenitor cells of the hematopoietic lineage. MKP cells express CD41, also called glycoprotein IIb / IIIa integrin, which is the platelet receptor for fibrinogen and several other extracellular matrix molecules, and antibodies thereto are commercially available, for example, from BD Biosciences, Pharmingen, San Diego, CA. under catalog numbers 340929, 555466. MKP cells are positive for the expression of CD117, which recognizes the receptor tyrosine kinase c-Kit. Antibodies are commercially available, for example, from BD Biosciences, Pharmingen, San Diego, CA under catalog number 340529. MKP cells are also lineage negative and negative for Thy-1 (CD90) expression.

[0057] The phrase "solid tumor" as used herein refers to an abnormal mass of tissue that does not usually contain cysts or liquid areas. Solid tumors can be benign or malignant. Various types of solid tumors are named based on the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, lymphomas, etc.

[0058] Anti-CD47 antibodies. Certain antibodies that bind to CD47 prevent the interaction of CD47 with SIRPα receptor. Antibodies include free antibodies and antigen-binding fragments derived therefrom, as well as conjugates, such as pegylated antibodies, drug conjugates, radioisotope conjugates, or toxin conjugates.

[0059] Monoclonal antibodies against specific epitopes or combinations of epitopes allow targeting and / or depletion of cell populations expressing markers.Monoclonal antibodies can be used to screen cell populations expressing markers using a variety of techniques, including magnetic separation using antibody-coated magnetic beads, "panning" using antibodies bound to solid matrices (i.e. plates), and flow cytometry (see, for example, U.S. Patent No. 5,985,660 and Morrison et al. Cell, 96:737-49 (1999)).These techniques allow screening of specific populations of cells in immunohistochemistry of biopsy specimens; and in detecting the presence of markers released by cancer cells into blood and other biological fluids.

[0060] Humanized versions of such antibodies are also within the scope of the present invention. Humanized antibodies are particularly useful for in vivo applications in humans due to their reduced antigenicity.

[0061] The phrase "bispecific antibody" refers to a synthetic or recombinant antibody that recognizes multiple proteins. Examples include bispecific antibodies 2B1, 520C9xH22, mDX-H210, and MDX447. Bispecific antibodies against a combination of epitopes allow targeting and / or depletion of cell populations that express the combination of epitopes. Exemplary bispecific antibodies include those that target a combination of CD47 and cancer cell markers such as CD96, CD97, CD99, PTHR2, HAVCR2, etc. The creation of bispecific antibodies is described in the literature, for example, USPN5989830, USPN5798229, which are incorporated herein by reference.

[0062] Terms such as "treatment", "treating" and "treat" are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. This effect may be preventative in that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic in that it partially or completely stabilizes or cures the disease and / or the adverse effects that may result from the disease. As used herein, "treatment" encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease or condition from occurring in a subject who may be susceptible to the disease or condition but has not yet been diagnosed as having it; (b) suppressing the disease symptoms, i.e., preventing their development; or (c) alleviating the disease symptoms, i.e., causing regression of the disease or condition.

[0063] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired.

[0064] "Host cell," as used herein, means a microorganism or eukaryotic cell or cell line cultured as a single-cell entity that can be or has been used as a recipient of a recombinant vector or other transfer polynucleotide, and includes the progeny of the original transfected cell. It will be understood that due to natural, accidental, or deliberate mutations, the morphology or genomic or total DNA complement of the progeny of a single cell may not necessarily be completely identical to the original parent.

[0065] The terms "cancer," "neoplasm," "tumor," and "carcinoma" are used interchangeably herein to refer to cells that exhibit relatively autonomous growth and, as a result, an abnormal growth phenotype characterized by a marked loss of cell growth control. In general, cells of interest for detection or treatment in this application include precancerous (e.g., benign), malignant, premetastatic, metastatic, and nonmetastatic cells. Detection of cancerous cells is of particular interest. The term "normal" as used in the context of "normal cells" is intended to refer to cells of a non-transformed phenotype or cells that exhibit the morphology of non-transformed cells of the tissue type being examined. "Cancerous phenotype" generally refers to any of a variety of biological phenomena characteristic of cancerous cells, which may vary depending on the type of cancer. In general, the cancerous phenotype is identified based on, for example, abnormalities in cell growth or proliferation (e.g., uncontrolled growth or proliferation), abnormalities in cell cycle regulation, abnormalities in cell motility, abnormalities in cell-cell interactions, or metastasis.

[0066] By "therapeutic target" is meant a gene or gene product whose activity, upon modulation (e.g., by modulation of expression, biological activity, etc.), can modulate the cancerous phenotype. As used throughout, "modulation" is intended to mean an increase or decrease in the specified phenomenon (e.g., modulation of biological activity means an increase in biological activity or a decrease in biological activity).

[0067] How to transplant Methods for manipulating phagocytosis of circulating hematopoietic cells are provided. In some embodiments of the present invention, the circulating cells are hematopoietic stem or progenitor cells, particularly in transplantation settings where protection from phagocytosis is desirable. In other embodiments, the circulating cells are leukemia cells, particularly acute myeloid leukemia (AML), where increased phagocytosis is desirable.

[0068] In some embodiments of the present invention, hematopoietic stem or progenitor cells are protected from phagocytosis in the circulation by providing the host animal with a CD47 mimetic molecule that interacts with SIRPα on macrophages and reduces macrophage phagocytosis. The CD47 mimetic can be soluble CD47, CD47 coated on the surface of the cells to be protected, and CD47 mimetic that binds to the CD47 binding site of SIRPα. In some embodiments of the present invention, CD47 is provided as a fusion protein, e.g., soluble CD47 fused to an Fc fragment, e.g., IgG1 Fc, IgG2 Fc, IgA Fc, etc.

[0069] Methods for generating proteins that lack transmembrane regions are well known in the art. For example, soluble CD47 can be generated by introducing a stop codon immediately before the polynucleotide sequence that codes for the transmembrane region. Alternatively, the polynucleotide sequence that codes for the transmembrane region can be replaced with a polynucleotide sequence that codes for a fusion protein such as IgG1 Fc. The sequences of Fc fragments from various sources are available through publicly accessible databases, including Entrez, Embl, etc. For example, the mRNA that codes for human IgG1 Fc fragment is provided under accession number X70421.

[0070] The present invention provides a method for transplanting hematopoietic stem or progenitor cells into a mammalian recipient. The need for transplantation may arise due to genetic or environmental conditions, such as exposure to chemotherapy, radiation, etc. The cells for transplantation may be a mixture of cells, such as buffy coat lymphocytes derived from a donor, or may be partially or substantially pure. These cells may be autologous (especially if removed prior to cytoreductive or other therapy) or allogeneic cells, and may be used for the isolation and subsequent transplantation of hematopoietic stem or progenitor cells.

[0071] The cells may be combined with a soluble CD47 mimic prior to administration. For example, the cells may be combined with the mimic at a concentration of about 10 μg / ml, about 100 μg / ml, about 1 mg / ml, about 10 mg / ml, etc., at a temperature of about 4° C., about 10° C., about 25° C., about 37° C., for a period of time sufficient to coat the cells, and in some embodiments, the cells are maintained on ice. In other embodiments, the cells are contacted with the CD47 mimic immediately prior to introduction into the recipient, with the mimic at a concentration as described above.

[0072] Compositions containing hematopoietic stem or progenitor cells and a CD47 mimetic are administered in a physiologically acceptable medium, usually intravascularly, but may also be introduced into bone or other convenient sites where the cells may find a suitable site for regeneration and differentiation. Typically, at least 1×10 5 pcs, preferably 1 x 10 6 One or more cells are administered. The composition may be introduced by injection or catheter, etc.

[0073] Myeloproliferative disorders, leukemia, and myelodysplastic syndromes Acute leukemias are rapidly progressive leukemias characterized by replacement of normal bone marrow by clones of blast cells that appear as a result of malignant transformation of hematopoietic cells. Acute leukemias include acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). ALL frequently affects the CNS, whereas acute monoblastic leukemia affects the gingiva and AML affects localized populations at any site (granulocytic sarcoma or chloroma).

[0074] Usually, the presenting symptoms are nonspecific (e.g., fatigue, fever, malaise, weight loss) and reflect failure of normal hematopoiesis. Anemia and thrombocytopenia are very common (75-90%). WBC counts may be decreased, normal, or increased. If the WBC count is not significantly decreased, blast cells are usually present in blood smears. ALL blasts can be differentiated from AML blasts by histochemical, cytogenetic, immunophenotypic, and molecular biological studies. In addition to smear examinations with the usual stains, terminal transferase, myeloperoxidase, Sudan black B, and specific and nonspecific esterases are used.

[0075] ALL is the most common malignancy in children, with a peak incidence between 3 and 5 years of age. ALL also occurs in adolescents, with a second, lower peak in adulthood. Typical treatment emphasizes early initiation of an intensive multidrug treatment program that may include prednisone, vincristine, anthracyclines, or asparaginase. Other drugs and combinations are cytarabine and etoposide, and cyclophosphamide. Relapse usually occurs in the bone marrow, but may also occur in the CNS or testes, alone or in combination with the bone marrow. A second remission can be induced in many children, but subsequent remissions tend to be brief.

[0076] The incidence of AML increases with age; it is the more common acute leukemia in adults. AML may be associated with chemotherapy or radiation (secondary AML). Remission induction rates are lower than for ALL, and long-term disease-free survival reportedly occurs in only 20-40% of patients. Treatment differs significantly from that for ALL, as AML responds to fewer drugs. The basic induction treatment program includes cytarabine with daunorubicin or idarubicin. Some treatment programs include 6-thioguanine, etoposide, vincristine, and prednisone.

[0077] Polycythemia vera (PV) is an idiopathic chronic myeloproliferative disorder characterized by increased Hb concentration and RBC mass (erythrocytosis). PV occurs in approximately 2.3 per 100,000 cases per year, with men more frequently affected (approximately 1.4:1). The mean age at diagnosis is 60 years (range 15-90 years; rare in childhood), with 5% of patients being younger than 40 years at onset. The bone marrow may appear normal but is usually hypercellular; hyperplasia involves all marrow elements and replaces marrow fat. There is increased production and turnover of RBCs, neutrophils, and platelets. Increased megakaryocytes may be present in the mass. Bone marrow iron is absent in more than 90% of patients, even when phlebotomy is not performed.

[0078] Studies of women with PV who are heterozygous at the X-chromosome-linked G6PD locus have shown that RBCs, neutrophils, and platelets have the same G6PD isoenzyme, supporting a clonal origin of the disorder at the pluripotent stem cell level.

[0079] Ultimately, approximately 25% of patients exhibit poor RBC survival and are unable to adequately mount erythropoiesis; anemia and myelofibrosis develop. Extramedullary hematopoiesis occurs in the spleen, liver, and other sites that have the potential to form blood cells.

[0080] Without treatment, 50% of symptomatic patients die within 18 months of diagnosis. With treatment, median survival is 7 to 15 years. Thrombosis is the most common cause of death, followed by complications such as myeloid metaplasia, hemorrhage, and leukemogenesis.

[0081] The incidence of transformation to acute leukemia was significantly higher in patients treated with radioactive phosphate ( 32 P) or alkylating agents. PV that transforms to acute leukemia is more resistant to induction chemotherapy than de novo leukemia.

[0082] Accurate diagnosis is essential because PV is the only form of erythrocytosis for which myelosuppressive therapy may be indicated for treatment. Treatment must be individualized according to age, sex, medical condition, clinical symptoms, and hematological findings.

[0083] Myelodysplastic syndromes (MDS) are a group of syndromes (preleukemia, refractory anemia, Ph-negative chronic myeloid leukemia, chronic myelomonocytic leukemia, myeloid metaplasia) commonly seen in older patients. Exposure to carcinogens may be implicated. MDS is characterized by clonal proliferation of hematopoietic cells, including erythroid, myeloid, and megakaryocytic types. The bone marrow is normal or hypercellular, and ineffective hematopoiesis causes various cytopenias. Anemia is most frequent. Impaired cell production is also associated with morphologic cellular abnormalities in the bone marrow and blood. Extramedullary hematopoiesis may occur, leading to hepatomegaly and splenomegaly. Myelofibrosis may occasionally be present at diagnosis or develop during the course of MDS. MDS clones are unstable and tend to progress to AML.

[0084] Anemia is the most common clinical feature and is usually associated with macrocytosis and anisocytosis. Some degree of thrombocytopenia is normal; on blood smear, platelets vary in size and some appear hypogranular. WBC counts may be normal, increased, or decreased. The granular state of the neutrophil cytoplasm is abnormal, with anisocytosis and a variable number of granules. Eosinophils may also have abnormal granular state. Monocytosis is characteristic of the chronic myelomonocytic leukemia subgroup, and immature myeloid cells may occur in the less differentiated subgroup. Prognosis is highly dependent on classification and any associated diseases. The response of MDS to AML chemotherapy is similar to that of AML, taking into account age and karyotype.

[0085] cancer treatment The present invention provides a method for reducing the proliferation of cancer cells by increasing the clearance of cancer cells by phagocytosis through the introduction of CD47 blocking agents, such as anti-CD47 antibodies.In certain embodiments, cancer cells can be AML stem cells.In other embodiments, cancer cells can be solid tumor cells, such as glioblastoma, melanoma, etc.By blocking the activity of CD47, the downregulation of phagocytosis found in some tumor cells, such as AML cells, is prevented.

[0086] In addition to CD47, the inventors have discovered several markers specific to AML SC, including CD96, CD97, CD99, PTHR2, HAVCR2, etc. These markers are disclosed in U.S. Patent Application No. 61 / 011,324, filed Jan. 15, 2008, which is incorporated herein by reference.

[0087] "Reducing the proliferation of cancer cells" includes, but is not limited to, reducing the proliferation of cancer cells and reducing the incidence of non-cancerous cells becoming cancerous cells. Whether or not a reduction in cancer cell proliferation has been achieved may be determined, but is not limited to, by the presence or absence of a known mechanism of action of the present invention. 3 This can be readily determined using any known assay method, including [H]-thymidine incorporation, counting cell numbers over a period of time, detection and / or measurement of AML-associated markers, and the like.

[0088] Whether a substance, or a particular amount of a substance, is effective in treating cancer can be evaluated using any of a variety of known cancer diagnostic assays, including but not limited to biopsy, contrast radiographic studies, CAT scans, and detection of cancer-related tumor markers in the blood of an individual. Substances can be administered systemically or locally, usually systemically.

[0089] In an alternative embodiment, an agent, for example, a chemotherapeutic drug that reduces cancer cell proliferation, can be targeted to cancer cells by binding to a CD47-specific antibody. Thus, in some embodiments, the present invention provides a method for delivering a drug to cancer cells, comprising administering a drug-antibody complex to a subject, where the antibody is specific for a cancer-associated polypeptide, and the drug is a drug that reduces cancer cell proliferation, and a variety of these drugs are known in the art. Targeting can be achieved by binding the drug to an antibody specific for a cancer-associated polypeptide (e.g., covalently or non-covalently linked directly or via a linker molecule to form a drug-antibody complex). Methods for binding a drug to an antibody are well known in the art and need not be detailed herein.

[0090] In certain embodiments, bispecific antibodies may be used, for example, bispecific antibodies in which one antigen-binding domain is directed to CD47 and the other antigen-binding domain is directed to a cancer cell marker such as CD96, CD97, CD99, PTHR2, HAVCR2, etc.

[0091] Depletion of AMLSC is useful in AML treatment. Depletion can be achieved by several methods. Depletion is defined as target population reduction of up to about 30%, or up to about 40%, or up to about 50%, or up to about 75% or more. Effective depletion is usually determined based on the sensitivity of a particular disease state to the level of target population. Thus, even about 20% depletion can be beneficial in the treatment of certain conditions.

[0092] The CD47-specific agent that specifically depletes targeted AMLSC is used to contact with patient blood in vitro or in vivo, and after the contact step, the number of viable AMLSC in the targeted population is reduced.The effective amount of the antibody for such purpose is sufficient to reduce the targeted population to a desired level, for example, as described above.The antibody for such purpose may have low antigenicity in humans or may be a humanized antibody.

[0093] In one embodiment of the invention, the antibodies for depleting the target population are added to the patient's blood in vivo. In another embodiment, the antibodies are added to the patient's blood ex vivo. Beads coated with the antibody of interest may be added to the blood, and then the target cells bound to these beads can be removed from the blood using procedures common in the art. In one embodiment, the beads are magnetic and are removed using a magnet. Alternatively, if the antibody is biotin-labeled, it is possible to indirectly immobilize the antibody on a solid phase to which avidin or streptavidin, etc., is adsorbed. The solid phase, usually agarose or sepharose beads, is separated from the blood by brief centrifugation. There are numerous methods common in the art for tagging antibodies and removing such antibodies and any cells bound to them. Once the desired degree of depletion is achieved, the blood is returned to the patient. Depleting the target cells ex vivo reduces side effects such as infusion reactions associated with intravenous administration. An additional advantage is that this procedure significantly expands the repertoire of available antibodies, as it need not be limited to antibodies that are poorly antigenic in humans or to humanized antibodies. EXAMPLES

[0094] Example 1 CD47 is a marker for myeloid leukemia material and method Immunohistochemistry. Cytospins of double sorted myeloid progenitor populations (CMP, GMP), IL-3Rα high CD45 RA+ cells, and CD14+c-kit+lin- cells were performed using a Shandon cytospin apparatus. Cytospins were stained with Giemsa 1 / 5 diluted in H20 for 10 min, followed by May-Grünwald for 20 min. Cytospins were analyzed using a Zeiss microscope.

[0095] Human bone marrow and peripheral blood samples. Normal bone marrow samples were obtained with informed consent (whole cells) from paid donors aged 20-25 years who were negative for hepatitis A, hepatitis B, hepatitis C, and HIV by serology. CMML bone marrow samples were obtained with informed consent from previously untreated patients at Stanford University Medical Center.

[0096] Flow cytometric analysis and cell sorting of human bone marrow HSCs and myeloid progenitors. Mononuclear fractions were extracted following Ficoll density centrifugation by standard methods and analyzed fresh or after rapid thawing of samples previously frozen in 90% FCS and 10% DMSO in liquid nitrogen. In some cases, CD34+ cells were enriched from the mononuclear fraction with the aid of immunomagnetic beads (Progenitor Isolation Kit, Miltenyi Biotec, Bergisch-Gladbach, Germany). Prior to FACS analysis and sorting, cells were incubated with phycoerythrin (PE)-Cy5-conjugated antibodies specific for lineage markers, including CD2 RPA-2.10; CD11b, ICRF44; CD20, 2H7; CD56, B159; GPA, GA-R2 (Becton Dickinson-PharMingen, San Diego); CD3, S4.1; CD4, S3.5; CD7, CD7-6B7; CD8, 3B5; CD10, 5-1B4; CD14, TUK4; CD19, SJ25-C1 (Caltag, South San Francisco, CA) and APC-conjugated anti-CD34, HPCA-2 (Becton Dickinson-ParMingen), in addition to PE-conjugated anti-IL-3Rα, 9F5 (Becton Dickinson-ParMingen) and FITC-conjugated anti-CD45RA, MEM56 (Caltag). Myeloid progenitor cells were stained with biotin-labeled anti-CD38, HIT2 (Caltag), followed by streptavidin-Texas Red to visualize CD38-BIO stained cells and resuspended in propidium iodide to exclude dead cells. Unstained samples and isotype controls were included to assess background fluorescence.

[0097] After staining, cells were analyzed and sorted using a modified FACS Vantage equipped with a 599 nm dye laser and a 488 nm argon laser (Becton Dickinson Immunocytometry Systems, Mountain View, CA). Double-sorted progenitor cells (HSCs) were confirmed to be CD34+CD38+ and lineage negative. Common myeloid progenitors (CMPs) were identified based on CD34+CD38+IL-3Rα+CD45RA-lin- staining, and their progeny, including granulocyte / macrophage progenitors (GMPs), were CD34+CD38+IL-3Rα+CD45RA+. Meanwhile, megakaryocyte / erythroid progenitors (MEPs) were identified based on CD34+CD38+IL-3Rα-CD45RA-lin- staining (Manz, PNAS 11872).

[0098] CD47 expression by normal progenitor cells and myeloproliferative and AML progenitor cells Peripheral blood and bone marrow samples were obtained from patients with myeloproliferative disorders and acute myeloid leukemia at Stanford University Medical Center with informed consent and in accordance with Stanford IRB and HIPAA regulations. Peripheral blood mononuclear cells or bone marrow mononuclear cells (1–5 × 10 cells) were cultured with a lineage cocktail as previously described, except for CD7, CD11b, and CD14. 6 Samples were subsequently stained for 45 min with CD14 PE (1 / 25), CD47 FITC (1 / 25), CD38 Bio (Bio) and c-kit APC (1 / 25) or CD34 APC or FITC (1 / 50), then washed and stained for 45 min with streptavidin-Texas Red (1 / 25) and finally resuspended in propidium iodide.

[0099] Consideration We show herein that CD47 overexpression is a hallmark of the progression of human myeloproliferative disorders to AML (see Figures 1-5B). CD47 controls not only integrin function but also the ability of macrophages to phagocytose cells, depending on the level of CD47 expression. Thus, aberrant CD47 expression may enable LSCs to evade both the host's innate and adaptive immunity.

[0100] Analysis of human CD47 expression was performed by FACS in human normal, preleukemic myeloproliferative disorder (MPD), or AML HSC, progenitor, and lineage-positive cells derived from bone marrow or peripheral blood. MPD samples (n=63) included polycythemia vera (PV; n=15), postpolycythemia myeloid metaplasia / myelofibrosis (PPMM / MF; n=5), essential thrombocythemia (ET; n=8), atypical chronic myeloid leukemia (aCML; n=2), CML (n=7), chronic eosinophilic leukemia (CEL; n=1), chronic myelomonocytic leukemia (CMML; n=13), and acute myeloid leukemia (AML; n=12). Using leukemia transgenic mouse models, we observed that the progression of human myeloproliferative disorders to AML (n=12) was accompanied by an increased GMP pool (70.6%+ / -SD 2.15) compared to normal bone marrow (14.7%+ / -SD 2.3). Furthermore, FACS analysis revealed that CD47 expression was first increased 1.7-fold in AML compared to normal HSCs, and then increased 2.2-fold compared to normal, with the commitment of AML progenitors to the myeloid lineage. CD47 was overexpressed by AML undifferentiated progenitors and their progeny, but not by the majority of MPDs (MFI 2.3+ / -SD 0.43), compared to normal bone marrow (MFI 1.9+ / -SD 0.07). Thus, increased CD47 expression is a useful diagnostic marker for progression to AML and, moreover, represents a novel therapeutic target.

[0101] Example 2 Human and murine leukemias upregulate CD47 to avoid killing by macrophages CD47 facilitates engraftment, inhibits phagocytosis, and is more highly expressed in AML LSCs. We measured CD47 expression in human AML LSCs and normal HSCs by flow cytometry. HSCs (Lin-CD34+CD38-CD90+) from three samples of normal mobilized human peripheral blood and AML LSCs (Lin-CD34+CD38-CD90-) from seven samples of human AML were analyzed for surface expression of CD47 (Figure 6). CD47 was expressed at low levels on the surface of normal HSCs; however, CD47 was expressed on average about 5-fold more highly in AML LSCs as well as bulk leukemic blasts.

[0102] Anti-human CD47 monoclonal antibodies stimulate phagocytosis and inhibit engraftment of AML LSCs. To test the model in which CD47 overexpression on AML LSCs prevents phagocytosis of these cells through interaction with SIRPα on effector cells, we used a monoclonal antibody against CD47 that is known to disrupt the CD47-SIRPα interaction. A hybridoma producing a mouse anti-human CD47 monoclonal antibody called B6H12 was obtained from ATCC and used to obtain purified antibody. First, we performed an in vitro phagocytosis assay. Primary human AML LSCs from two samples of human AML were purified by FACS and then loaded with the fluorescent dye CFSE. These cells were incubated with mouse bone marrow-derived macrophages and monitored using immunofluorescence microscopy (Figure 7) and flow cytometry (Figure 9) to identify phagocytosed cells. In both cases, no phagocytosis was observed in the presence of an isotype control antibody; however, significant phagocytosis was detected when anti-CD47 antibodies were added (Figure 9). Thus, blockade of human CD47 with monoclonal antibodies can stimulate phagocytosis of these cells by mouse macrophages.

[0103] Next, we investigated the ability of anti-CD47 antibodies to inhibit AML LSC engraftment in vivo. Two primary human AML samples were either untreated or coated with anti-CD47 antibodies before transplantation into NOG neonatal mice. After 13 weeks, the mice were sacrificed and analyzed for bone marrow engraftment of human leukemia by flow cytometry (Figure 10). Control mice showed leukemia engraftment, whereas mice transplanted with anti-CD47-coated cells showed little or no engraftment. These data suggest that blocking human CD47 with monoclonal antibodies can inhibit AML LSC engraftment.

[0104] CD96 is a cell surface molecule specific for human acute myeloid leukemia stem cells. Initially called Tactile, CD96 was first identified as a T cell surface molecule that is significantly upregulated upon T cell activation. CD96 is expressed at low levels on resting T cells and NK cells and is strongly upregulated upon stimulation in both cell types. It is not expressed on other hematopoietic cells, and examination of its expression pattern has shown that it is otherwise present only in some intestinal epithelia. The cytoplasmic domain of CD96 contains a putative ITIM motif, but it is not known whether this functions in signal transduction. CD96 promotes the adhesion of NK cells to target cells expressing CD155 and stimulates the cytotoxicity of activated NK cells.

[0105] Preferential cell surface expression of molecules confirmed by gene expression analysis. In addition to CD47 and CD96, several molecules described in U.S. Patent Application No. 61 / 011,324 are known to be expressed on AML LSCs, including CD123, CD44, CD99, and CD33.

[0106] Tumor progression is characterized by several features, including growth signal independence, inhibition of apoptosis, and evasion from the immune system, among others. Herein, we show that the expression of CD47, the ligand of macrophage inhibitory signal-regulatory protein alpha (SIRPα) receptor, is increased in human and mouse myeloid leukemia, allowing cells to evade phagocytosis and increase tumorigenicity. CD47, also known as integrin-associated protein (IAP), is an immunoglobulin-like transmembrane pentaspanin that is widely expressed in mammalian tissues. We provide evidence that CD47 is upregulated in stem and progenitor cells of mouse and human myeloid leukemia, as well as in leukemic blasts. Consistent with the biological role of CD47 in the development and maintenance of myeloid leukemia, we demonstrate that ectopically overexpressing CD47 allows myeloid leukemia cell lines to grow in mice lacking T cells, B cells, and NK cells, but are otherwise rapidly cleared when transplanted into these recipients. We also show that the leukemogenic potential of CD47 is dose-dependent, as high-expressing clones have greater tumorigenic potential than low-expressing clones. We also show that CD47 functions in promoting leukemogenesis by inhibiting phagocytosis of leukemic cells by macrophages.

[0107] CD47 is a leukemic Fas lpr / lprCD47 is significantly upregulated in leukemic hMRP8bcr / abl×hMRP8bcl2 transgenic bone marrow and in leukemic hMRP8bcr / abl×hMRP8bcl2 mice. Quantitative RT-PCR revealed that CD47 transcripts were increased 3-4 fold in leukemic hMRP8bcr / abl×hMRP8bcl2 bone marrow and 6-7 fold in c-Kit-enriched leukemic bone marrow compared to healthy hMRP8bcl2+ bone marrow (Fig. ​(Fig.11e).11e). In leukemic spleens, the granulocyte macrophage progenitor (GMP) population and c-Kit+Sca-1+Lin- stem and progenitor cell subsets were expanded compared to control mice of the same genotype as the leukemic mice but did not develop the disease (Fig.11a-d). We found that CD47 protein expression levels began to increase at the Flk2-CD34-c-Kit+Sca-1+Lin- long-term hematopoietic stem cell (LT-HSC) stage in leukemic mice compared to control mice (Fig. 11f). This increased expression level was maintained in GMP and Mac-1+ blasts, but not in megakaryocyte / erythroid committed progenitors (MEPs) (Fig. 11f). The increase in CD47 in leukemic cells compared to normal cells ranged from 3- to 20-fold. hMRP8bcr / abl×hMRP8bcl2 primary (n=3) and secondary (n=3) transplanted mice, Fas lpr / lpr CD47 expression was increased in all mice that developed leukemia that we tested, derived from hMRP8bcl2 × hMRP8bcl2 primary (n = 14) and secondary (n = 19) mice, and from hMRP8bcl2 × hMRP8bcl2 primary (n = 3) and secondary (n = 12) mice. We also found that CD47 expression was increased in mice that developed leukemia after receiving mouse bone marrow cells retrovirally transduced with p210bcr / abl.

[0108] Analysis of human hematopoietic progenitor cell populations by FACS was performed in blood and bone marrow from normal umbilical cord blood and mobilized peripheral blood (n=16) and myeloproliferative disorders (MPDs), including polycythemia vera (PV; n=16), myelofibrosis (MF; n=5), essential thrombocythemia (ET; n=7), chronic myelomonocytic leukemia (CMML; n=11), and atypical chronic myeloid leukemia (aCML; n=1), as well as blast phase chronic myeloid leukemia (CML; n=19), chronic phase CML (n=7), and acute myeloid leukemia (AML; n=13). This analysis demonstrated that granulocyte-macrophage progenitors (GMPs) are expanded in MPDs with myeloid skewed differentiation potential, including atypical CML, proliferation-phase CMML, and acute leukemias, including blast crisis-phase CML and AML (Fig. 12a). AML HSCs and AML progenitors uniformly showed high levels of CD47 expression compared to normal controls (Fig. 12b); CD47 levels were elevated in all samples derived from BC-CML and AML. Furthermore, progression from chronic phase CML to blast crisis was associated with a marked increase in CD47 expression (Fig. 12c). Using the methods described in this study, we found that human CD47 protein expression in CML-BC was 2.2-fold higher in CD90+CD34+CD38-Lin- cells compared to normal cells (p=6.3×10 -5 ), and in CD90-CD34+CD38-Lin- cells, it was 2.3-fold higher than in normal cells (p=4.3×10 -5 ), and 2.4-fold in CD34+CD38+Lin- cells (p=7.6×10 -6 ) was increased (Figures 12b-12c); however, using a newer optimized staining protocol, we observed that CD47 was increased approximately 10-fold in AML and BC-CML compared to normal human HSC and progenitor cells.

[0109] We then investigated whether forced expression of mouse CD47 in human leukemia cells would confer a competitive advantage in forming tumors in mice. MOLM-13 cells derived from an AML 5a patient were transduced with Tet-MCS-IRES-GFP (Tet) or Tet-CD47-MCS-IRES-GFP (Tet-CD47) (Fig. 13a) and stable integrants were propagated based on GFP expression. These cells were then transplanted intravenously into T, B, and NK deficient, recombination activating gene 2, common gamma chain deficient (RAG2- / -, Gc- / -) mice in a competitive setting with non-transduced MOLM-13 cells. Only Tet-CD47 transduced cells were able to form tumors in these mice, efficiently engrafting the bone marrow, spleen, and peripheral blood (Fig. 13a-b). These tumors were also characterized by a large tumor burden in the liver (Fig. 13b, 13g). This is particularly striking since the liver is believed to have the largest number of macrophages of any organ, and it has been estimated that Kupffer cells may comprise 80% of the total tissue macrophage population. These cells also occupy 30% of the sinusoidal lining, thereby strategically positioning them at the site of entry into the liver. Thus, significant engraftment at that location should disable the cytotoxic response of macrophages. In addition to developing tumor nodules, Tet-CD47 MOLM-13 cells displayed a pattern of liver involvement typically seen in human AML, with leukemic cells infiltrating the liver in a sinusoidal and perivenous pattern (Figure 13d). Overall, Tet-CD47 MOLM-13-implanted mice died faster than Tet MOLM-13-implanted mice, which did not substantially engraft leukemic cells in hematopoietic tissues (Figure 13c). Tet-MOLM-13 mice still exhibited a significant mortality rate, likely due to focal growth at the injection site (retro-orbital sinus) with spread into the brain.

[0110] Because CD47 has been shown to be important for hematopoietic cell migration and is known to regulate binding to extracellular matrix proteins by direct interaction or through its action on integrins, one possibility for the lack of proliferation of Tet MOLM-13 cells in mice was their inability to migrate into the microenvironment. To test this possibility, Tet MOLM-13 cells or Tet-CD47 MOLM-13 cells were injected directly into the femoral cavity of immunodeficient mice. Tet-CD47 MOLM-13 cells were able to engraft all bones and other hematopoietic tissues of recipient mice, whereas Tet MOLM-13 cells showed minimal, if any, engraftment only at the site of injection (Figure 13e). Mice transplanted in this manner with Tet-CD47 MOLM-13 cells died approximately 50-60 days after transplantation (n=4), whereas mice receiving Tet MOLM-13 cells (n=5) continued to survive for at least 75 days without signs of disease, at which point they were euthanized for analysis. These results suggest a function for CD47 in MOLM-13 engraftment other than or in addition to migration or homing.

[0111] It has been shown that the complete absence of CD47 results in phagocytosis of transplanted mouse red and white blood cells by abolishing the interaction with SIRPα on macrophages. Therefore, we tested whether phagocytosis of unopsonized hepatic MOLM-13 cells could be prevented by overexpression of CD47. We incubated Tet MOLM-13 cells or Tet-CD47 MOLM-13 cells with bone marrow-derived macrophages (BMDM) for 2-24 h and assessed phagocytosis by counting the number of ingested GFP+ cells under a microscope or by assessing the frequency of GFP+ macrophages by flow cytometry. Expression of CD47 dramatically reduced the clearance of these cells by macrophages at all time points tested, whereas Tet-MOLM-13 was rapidly phagocytosed in a manner that increased over time (Figure 14a-c). We also injected mice with MOLM-13 cells and analyzed hematopoietic organs 2 hours later to obtain evidence of macrophage phagocytosis. Macrophages in bone marrow, spleen, and liver all had a greater proportion of GFP+ cells when Tet MOLM-13 cells were injected compared to CD47-expressing cells. This indicates that CD47 overexpression can counteract pro-phagocytic signals already present on leukemia cells, allowing them to survive when they would otherwise be cleared by macrophages.

[0112] Recent reports have shown that lack of CD47 reactivity across species may result in xeno-rejection of transplanted cells. Furthermore, recent studies have demonstrated that human CD47 cannot interact with SIRPα from C57Bl / 6 mice, but can react with receptors from non-obese diabetic (NOD) mice, which are more permissive to human cell engraftment than C57Bl / 6 mice. Furthermore, we also observed that HL-60 cells, a human promyelocytic cell line that has higher levels of human CD47 expression than MOLM-13, can engraft and cause leukemia in mice. Jurkat cells, a human T-lymphocyte cell line, are highly enriched in human CD47 and are phagocytosed by mouse macrophages in vitro at a much slower rate than MOLM-13. Thus, our data indicate that the ability of cells to engraft in mice in vivo or to evade phagocytosis by mouse macrophages in vitro correlates with the expression level of human CD47.

[0113] To model the tumorigenic effects of high and low CD47 expression, we classified MOLM-13 cell clones expressing mouse CD47 into high and low expressers. When corrected for cell size, CD47 lo The CD47 density on MOLM-13 cells was similar to that on mouse bone marrow cells, whereas CD47 hi MOLM-13 cells showed approximately 9-fold higher expression, an increase commensurate with the changes observed in CD47 expression on primary leukemia cells compared to their normal counterparts (Fig. 15a). When high- or low-expressing cells were transplanted into recipients, only mice transplanted with high-expressing cells succumbed to disease by 75 days of age (Fig. 15c). Furthermore, organ hypertrophy was more pronounced in mice transplanted with high-expressing cells (Fig. 15d). CD47 loMice receiving MOLM-13 cells still had notable liver masses. However, these masses were always one or two large nodules that were well encapsulated and physically separated from the liver parenchyma, in stark contrast to the tumor masses derived from CD47hi MOLM-13 cells, which consisted of hundreds of small masses scattered throughout the parenchyma. Thus, these large tumor masses consisted of cells that had found a macrophage-free microenvironment away from the main organ body in which to grow. As expected, the extent of infiltration of MOLM-13 cells in the bone marrow and spleen of recipient mice was similarly consistent with that of CD47hi MOLM-13 cells. hi The results showed that the CD47 lo We examined CD47 expression levels in two mice that received MOLM-13 cells but showed significant bone marrow engraftment. In both cases, cells surviving after day 75 had much higher CD47 levels than the original line (Figure 15f), suggesting that there is strong in vivo selective pressure for high levels of CD47 expression in leukemic cells. Together, these data suggest that CD47 expression levels are an important factor in tumorigenic potential and that there is strong selection for clones that highly express CD47 in heterogeneous populations of leukemic cells.

[0114] We then investigated whether higher levels of CD47 expression conferred greater protection from macrophage phagocytosis. hi MOLM-13 red fluorescent protein (RFP) expressing cells and CD47 lo An in vitro phagocytosis assay was performed using MOLM-13 red fluorescent protein (RFP) expressing cells. After incubation with macrophages, CD47 hi Much more CD47 than cells lo The cells were phagocytosed (FIG. 15g). Phagocytosis index was calculated for control MOLM-13 cells, bulk (unsorted) CD47 MOLM-13 cells, and CD47 lo MOLM-13 cells, and CD47 hiWhen compared to MOLM-13 cells, a direct correlation between CD47 expression levels and the ability to evade phagocytosis can be seen (Fig. 14a, Fig. 15f). lo RFP MOLM-13 cells and CD47 hi When GFP MOLM-13 cells were co-incubated with macrophages in the same wells, low expressing cells were much more likely to be phagocytosed (Fig. S15h, i). Thus, in a mixed population of cells with varying levels of CD47 expression, low expressing cells are more likely to be cleared by phagocytic clearance over time.

[0115] We also measured CD47 expression using another method. Because CD47 is expressed in MOLM-13 cells using the Tet-OFF system, we utilized the Tet-inducible promoter element to control CD47 expression in MOLM-13 cells. hi Starting two weeks after MOLM-13 cell transplantation, a cohort of mice was given doxycycline and followed until day 75 after transplantation. During this time course, none of the doxycycline-fed mice died from disease, and there was no extensive infiltration of MOLM-13 cells in hematopoietic organs (Figure 15b-d). At the dose of doxycycline used in this experiment, muCD47 expression in MOLM-13 cells was reduced to levels below those in normal mouse bone marrow, but notably was not abolished (Figure 15b). Thus, sustained high levels of CD47 expression are necessary for robust MOLM-13 survival in hematopoietic organs.

[0116] Many examples of tumor clearance by T, B, and NK cells have been described in the literature, indicating that a healthy immune system is essential to regulate nascent tumor growth. However, to date, few examples have been shown to show that macrophage-mediated phagocytosis can check tumor development. Taken together, our studies reveal that ectopic expression of CD47 can enable otherwise immunogenic tumor cells to rapidly grow in hosts that are deficient in T, B, and NK cells. Furthermore, CD47 is consistently upregulated in murine and human myeloid leukemias, including all forms of chronic and acute myeloid leukemias tested to date, so this likely reflects a mechanism used by human myeloid leukemias to evade the host immune system. For example, tumor cells can be recognized as targets by activated macrophages and likely cleared by phagocytosis. By upregulating CD47, cancers can evade this form of innate immune tumor surveillance.

[0117] This form of immune evasion is particularly important because these cancers often occupy sites of high macrophage infiltration. CD47 was first cloned as an ovarian tumor cell marker, suggesting that it may play a role in preventing phagocytosis of other tissue cancers as well. Furthermore, solid tumors often metastasize to macrophage-rich tissues such as liver, lung, bone marrow, and lymph nodes, suggesting that they must be able to evade macrophage-mediated killing in these tissues. Thus, finding ways to disrupt CD47-SIRPα interaction may prove broadly useful in developing novel therapies for cancer. Preventing CD47-SIRPα interaction is doubly effective, since antigens derived from phagocytosed tumor cells can be presented by macrophages to activate adaptive immune responses to further destroy tumors.

[0118] method Mice. hMRP8bcrabl transgenic mice, hMRP8bcl2 transgenic mice, and Fas lpr / lpr Transgenic mice were generated and crossed to obtain double transgenics. hMRP8bcl2 homozygotes were obtained by crossing heterozygous mice with each other. C57Bl / 6 Ka mice from our colony were used as a source of wild-type cells. For transplantation experiments, C57Bl / 6 RAG2 mice were irradiated with 4 Gy of gamma radiation from a cesium irradiator (Phillips). - / - Common γ chain (Gc) - / - Cells were transplanted into mice. Primary mouse leukemias were transplanted into CD45.2 C57Bl6 / Ka mice that were given a radiation dose of 9.5 Gy. Mice were euthanized when moribund.

[0119] Mouse tissues. Long bones were flushed with PBS supplemented with 2% fetal bovine serum staining media (SM). Spleen and liver were isolated in SM using frosted glass slides and then passed through a nylon mesh. All samples were treated with ACK lysis buffer to lyse red blood cells before further analysis.

[0120] Analysis by quantitative RT-PCR. Bone marrow was obtained from leukemic hMRP8bcr / abl×hMRP8bcl2 mice or hMRP8bcl2 control mice. Cellular c-Kit was enriched using c-Kit microbeads and autoMACS columns (Miltenyi). RNA was extracted using Trizol reagent (Invitrogen) and reverse transcribed using SuperScriptII reverse transcription polymerase (Invitrogen). cDNA equivalent to approximately 1000 cells was used per PCR reaction. Quantitative PCR was performed using a SYBR green kit on an ABI Prism 7000 PCR (Applied Biosystems) instrument at 50°C for 2 min, followed by 95°C for 10 min, then 95°C for 15 min followed by 60°C for 1 min for 40 cycles. β-actin and 18S RNA were used as controls for cDNA amount to normalize the results for CD47 expression. The sequences of the forward and reverse primers for 18S RNA were, respectively: TIFF0007638360000001.tif5131 and for β-actin TIFF0007638360000002.tif6128 and for CD47 The file was TIFF0007638360000003.tif5128.

[0121] Human bone marrow and peripheral blood samples. Normal bone marrow samples were obtained with informed consent (whole cells) from paid donors aged 20-25 years who were negative for hepatitis A, hepatitis B, hepatitis C, and HIV by serology. Blood and bone marrow cells were obtained with informed consent from patients with chronic myelomonocytic leukemia (CMML), chronic myelogenous leukemia (CML), and acute myeloid leukemia (AML) who were previously untreated.

[0122] Cell lines. MOLM-13 cells were obtained from DSMZ. HL-60 and Jurkat cells were obtained from ATCC. Cells were maintained in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal bovine serum (FBS) (Hyclone). To separate MOLM-13 cells into CD47 high and low expressing cells, Tet-CD47 MOLM-13 cells were stained with anti-mouse CD47 Alexa-680 antibody (mIAP301). The top and bottom 5% of mouse CD47 expressing cells were sorted by BD FACSAria and grown again for 2 weeks in IMDM + 10% FCS. These cells were selected and sorted three more times following the same protocol to obtain high and low expressing cells used in this study. To obtain red fluorescent protein (RFP) constructs, mCherry RFP DNA was cloned into Lentilox 3.7 (pLL3.7) empty vector. Lentivirus derived from this construct was then used to infect cell lines.

[0123] Cell staining and flow cytometry. Staining of mouse stem and progenitor cells was performed using the following monoclonal antibodies: Mac-1, Gr-1, CD3, CD4, CD8, B220, and Ter119 conjugated to Cy5-PE (eBioscience) were used in the lineage cocktail, c-Kit PE-Cy7 (eBioscience), Sca-1 Alexa680 (e13-161-7, generated in our laboratory), CD34 FITC (eBioscience), CD16 / 32 (FcGRII / III) APC (Pharmingen), and CD135 (Flk-2) PE (eBioscience) were used to stain mouse stem and progenitor subsets as previously described. Mouse CD47 antibody (clone mIAP301) was evaluated using a biotin-labeled antibody generated in our laboratory. The cells were then stained with streptavidin-conjugated Quantum Dot 605 (Chemicon), and the samples were analyzed using a FACSAria (Beckton Dickinson).

[0124] For human samples, mononuclear fractions were extracted following Ficoll density centrifugation by standard methods and analyzed fresh or after rapid thawing of samples previously frozen in 90% FCS and 10% DMSO in liquid nitrogen. In some cases, CD34+ cells were enriched from the mononuclear fraction with the aid of immunomagnetic beads (Progenitor Isolation Kit, Miltenyi Biotec, Bergisch-Gladbach, Germany). Prior to FACS analysis and sorting, PE-Cy5-conjugated antibodies specific for lineage markers (including CD2 RPA-2.10; CD11b, ICRF44; CD20, 2H7; CD56, B159; GPA, GA-R2 (Becton Dickinson-PharMingen, San Diego); CD3, S4.1; CD4, S3.5; CD7, CD7-6B7; CD8, 3B5; CD10, 5-1B4; CD14, TUK4; CD19, SJ25-C1 (Caltag, South San Francisco, CA)) and APC-conjugated anti-CD34, HPCA-2 (Becton Dickinson-ParMingen) were used in addition to PE-conjugated anti-IL-3Rα, 9F5 (Becton Dickinson-ParMingen) and FITC-conjugated anti-CD45RA, MEM56 (Caltag). Myeloid progenitor cells were stained with biotin-conjugated anti-CD38, HIT2 (Caltag), followed by streptavidin-Texas Red staining to visualize CD38-BIO stained cells.

[0125] After staining, cells were analyzed using a modified FACS Vantage (Becton Dickinson Immunocytometry Systems, Mountain View, CA) or FACSAria equipped with a 599 nm dye laser and a 488 nm argon laser. Hematopoietic stem cells (HSCs) were confirmed to be CD34+CD38+CD90+ and lineage negative. CD47 expression was assessed in all human samples using anti-human CD47 FITC (clone B6H12, Pharmingen). Percent change in CD47 expression was determined by dividing the average mean fluorescence intensity of CD47 in all CML-BC, CML-CP, or AML samples by the mean fluorescence intensity of normal cells of a given cell population. Common myeloid progenitors (CMPs) were identified based on CD34+CD38+IL-3Rα+CD45RA-Lin- staining, and their progeny, including granulocyte / macrophage progenitors (GMPs), were CD34+CD38+IL-3Rα+CD45RA+Lin-, whereas megakaryocyte / erythroid progenitors (MEPs) were identified based on CD34+CD38+IL-3Rα-CD45RA-Lin- staining.

[0126] To determine the density of mouse CD47 or human CD47, cells were stained with saturating amounts of anti-CD47 antibody and analyzed using a FACSAria. Because forward scatter is directly proportional to cell diameter and density is equivalent to the expression level per unit of surface area, we calculated the geometric mean fluorescence intensity of the CD47 pathway using FloJo software and the forward scatter squared (FSC). 2 ) to obtain an approximation of the density of CD47 expression at the membrane.

[0127] Engraftment of MOLM-13 cells was assessed by using anti-human CD45 PE-Cy7 (Pharmingen), anti-mouse CD45.2 APC (clone AL1-4A2), and anti-mouse CD47 Alexa-680 (mIAP301). All samples were resuspended in propidium iodide-containing buffer prior to analysis to exclude dead cells. FACS data were analyzed using FloJo software (Treestar).

[0128] Lentivirus preparation and transduction. pRRL.sin-18.PPT.Tet07.IRES.GFP.pre plasmid, CMV plasmid, VSV plasmid, and tet transactivator (tTA) plasmid were obtained from Luigi Naldini. Full-length mouse cDNA of CD47 (type 2) was provided by Eric Brown (UCSF). CD47 cDNA constructs were ligated into the BamHI / NheI sites of Tet-MCS-IRES-GFP. Plasmid DNA was transfected into 293T cells using standard protocols. Supernatants were collected and concentrated using a Beckman LM-8 centrifuge (Beckman). Cells were transduced with Tet or Tet-CD47-MCS-IRES-GFP and tTA lentivirus for 48 h. GFP+ cells were sorted and purified and propagated for several generations to ensure transgene stability.

[0129] Injection. Cells were injected intravenously into the retro-orbital sinus or via the tail vein of recipient mice as described above. For intrafemoral injections, a volume of 20 μl of cells was injected into the femoral cavity of anesthetized mice using a 27-gauge needle. Mice were anesthetized using an isofluorane gas chamber, if necessary.

[0130] MOLM-13 cell engraftment. Animals were euthanized if moribund, and bone marrow, spleen, and liver were harvested. One hour prior to euthanasia, animals were tail bled to obtain peripheral blood. MOLM-13 cell engraftment was determined in bone marrow, spleen, and peripheral blood as previously described. Tumor burden in the liver was determined by calculating the area of ​​each visible tumor nodule using the formula ((length (mm) + width (mm)) / 2) x π. The areas of each nodule per liver were then added together.

[0131] Doxycycline administration. Doxycycline hydrochloride (Sigma) was added to drinking water to a final concentration of 1 mg / mL. Drinking water was changed every 4 days and protected from light. Additionally, mice received a bolus of 10 μg doxycycline via intraperitoneal injection once a week.

[0132] Bone marrow-derived macrophages (BMDM). Femurs and tibias were harvested from C57Bl / 6 Ka mice, and bone marrow was flushed and placed in a sterile suspension of PBS. Bone marrow suspensions were expanded for 7–10 days in IMDM containing 10% FBS supplemented with 10 ng / mL recombinant mouse macrophage colony-stimulating factor (MCSF, Peprotech).

[0133] In vitro phagocytosis assay. BMDMs were harvested by incubation in trypsin / EDTA (Gibco) for 5 min and gentle scraping. 5 × 10 cells were cultured per well in 24-well tissue culture plates (Falcon). 4 Macrophages were seeded at a concentration of 2.5 × 10 cells. After 24 h, the medium was replaced with serum-free IMDM. After an additional 2 h, the wells containing macrophages were seeded with 2.5 × 10 cells. 5Tet MOLM-13 cells or Tet-CD47 MOLM-13 cells were added and incubated at 37°C for the indicated times. After co-incubation, wells were thoroughly washed three times with IMDM and examined by an Eclipse T5100 (Nikon) using enhanced green fluorescent protein (GFP) or Texas Red filter sets (Nikon). The number of GFP+ or RFP+ cells within the macrophages was counted and the phagocytic index was calculated using the following formula: phagocytic index = number of ingested cells / (number of macrophages / 100). At least 200 macrophages were counted per well. To analyze phagocytosis by flow cytometry, macrophages were harvested after incubation with MOLM-13 cells by trypsin / EDTA and gentle scraping. Cells were stained with anti-Mac-1 PE antibody and analyzed using a BD FACSAria. Fluorescent and bright-field images were taken separately using an Eclipse T5100 (Nikon), an extra-high pressure mercury lamp (Nikon), an endow green fluorescent protein (eGFP) bandpass filter (Nikon), a Texas Red bandpass filter (Nikon), and a RT Slider (Spot Diagnostics) camera. Images were merged using Photoshop software (Adobe).

[0134] For in vivo assays, RAG2 - / - Gc - / - Two hours after injection of target cells into mice, bone marrow from the long bones of the legs, spleen, and liver were harvested. They were prepared into single cell suspensions in PBS containing 2% FCS. Cells were labeled with anti-human CD45 Cy7-PE and anti-mouse F4 / 80 biotin (eBiosciences). Secondary staining was performed with streptavidin-APC (eBiosciences). Cells that were human CD45-, F4 / 80+ were considered to be macrophages, and GFP+ cells in this fraction were assessed.

[0135] Example 3 Hematopoietic stem and progenitor cells upregulate CD47 to promote mobilization and homing to hematopoietic tissues The inventors herein report the discovery of a novel CD47-deficient (IAP - / - ) mice fail to engraft in wild-type recipients. As expected, these cells are rapidly cleared by host macrophages, whereas IAP + / + This is not the case for HSCs. When stem and progenitor cells are forcibly separated and transferred into the circulation using cyclophosphamide / G-CSF or lipopolysaccharide, CD47 is rapidly upregulated in these cells. We propose that the high levels of CD47 in stem cells during the stresses of hematopoiesis and mobilization provide additional protection from phagocytosis by activated macrophages of the reticuloendothelial system. In support of this hypothesis, we show that IAPs transplanted into wild-type recipients are more likely to be involved in the development of endothelial cell death. + / - We show that CD47-dependent endothelial cell proliferation and engraftment in hematopoietic progenitor cells increases with time, whereas wild-type donor cells do not. We conclude that phagocytosis is an important physiological mechanism for the clearance of hematopoietic progenitor cells over time, and that overexpression of CD47 is required to prevent phagocytic clearance.

[0136] HSCs have the ability to migrate via the bloodstream to ectopic microenvironments in fetal and adult life. Furthermore, HSCs can be driven into the circulation using a combination of cytotoxic agents and cytokines that initially increase the number of HSCs in situ. Once in the bloodstream, HSCs must pass through the vascular beds of the spleen and liver. Macrophages at these sites function to remove damaged cells and foreign particles from the bloodstream. Furthermore, during inflammatory conditions, macrophage phagocytosis becomes more active. Thus, additional protection from phagocytosis may be required for newly arriving stem cells at these sites.

[0137] We determined whether CD47 expression on bone marrow stem and progenitor cells plays a role in regulating normal hematopoiesis. CD47 expression has been shown to be essential to prevent phagocytosis of red blood cells, T cells, and whole bone marrow cells in the transplant environment. Therefore, we investigated whether the lack of CD47 would prevent the engraftment of HSCs after intravenous delivery. To test this, we generated CD47 knockout mice (IAP - / - ) were used. These mice develop normally and do not show any gross abnormalities. However, they die very rapidly after intraperitoneal bacterial challenge because neutrophils do not rapidly migrate to the intestine. Furthermore, cells derived from these mice cannot be transplanted into wild-type recipients, but appear to engraft in IAP- / - recipients.

[0138] The present inventors have + / - Mice and IAPs - / - We first examined the incidence of stem and progenitor cells in mice. When examining cells in the stem cell compartment and myeloid progenitor compartment, there was no difference between these mice and wild-type mice (Figure 18a). We then tested the ability of stem cells derived from these mice to form colonies in an in vitro assay. We selected highly purified Flk2-CD34-KLS stem cells from these mice and seeded them on methylcellulose in the presence of a standard cytokine cocktail. We examined colony formation on day 7 and found no significant difference in the number and type of colonies formed between wild-type and IAP- / - stem cells (Figure 18b).

[0139] Next, the inventors - / - We investigated whether bone marrow cells derived from mice could rescue recipient mice from the effects of lethal radiation. Typically, 2 × 10 5This dose of bone marrow cells is expected to rescue 100% of wild-type recipient mice in this assay. - / - We found that bone marrow could not rescue these recipients (Figure 18c). However, administration of these cells extended life span; mice usually died between 12 and 15 days after radiation, whereas IAP - / - Mice that received bone marrow lived approximately 7-10 days longer (Figure 18c). Although we do not yet know the reason for the extended life span in this case, we observed that multipotent progenitor cells and megakaryocytic erythroid progenitor cells can extend survival after lethal radiation, and that the contribution of these cells after transplantation of whole bone marrow may have contributed to the extended survival time.

[0140] Next, we investigated wild-type cells and IAP - / - Flk-2 from cells - CD34 - KLS stem cells were sorted and cultured at 2 x 10 5 They were transplanted into wild-type recipients along with competitor cells. - / - None of the mice given HSCs engrafted any of the donor cells, indicating that CD47 is indeed required for the ability of these cells to engraft (Figure 18d-e). We speculated that this was due to phagocytosis of cells lacking CD47, as has been shown for red blood cells and T cells. To test this, we used wild-type and IAP - / - c-Kit from mouse bone marrow + We enriched the cells and co-incubated them with bone marrow-derived macrophages. - / - Stem cells and IAPs - / - Progenitor cells were rapidly phagocytosed in this assay, whereas wild-type cells were only minimally phagocytosed (Figure ​(Figure18f-g). - / - When incubated with macrophages, IAP - / -The cells were significantly less phagocytosed, confirming that the phagocytic capacity of macrophages from these mice was indeed abnormal.

[0141] Because stem and progenitor cell mobilization involves several steps where they come into contact with macrophages (egress from bone marrow sinusoids, bone marrow and liver sinusoids, and entry into the splenic marginal zone), we investigated whether CD47 is upregulated in the bone marrow of mice induced to undergo mobilization. The most commonly used protocol involves administration of the drug cyclophosphamide (Cy), which kills dividing cells (mainly myeloid progenitor cells), followed by treatment with granulocyte colony-stimulating factor (G-CSF). This involves administering cyclophosphamide on the first day, then giving G-CSF every day thereafter. By convention, the first day after cyclophosphamide administration is referred to as day 0. The number of stem cells in the bone marrow peaks on day 2; on days 3-4, they leave the bone marrow into the periphery, and their numbers in the spleen and liver peak on day 5; myeloerythroid progenitor cells are also mobilized. During the mobilization response, there is a characteristic increase in the frequency of stem and myeloid progenitor cells.

[0142] Therefore, we subjected wild-type mice to this mobilization protocol and sacrificed them on days 2 to 5. We found that at day 2, c-Kit +We found a significant increase in CD47 in bone marrow cells (Figure 19a). We found that CD47 levels were elevated approximately 4-fold in stem and progenitor cells on day 2 of mobilization (Figure 19b). This increase in CD47 expression was seen at all levels of the myeloid progenitor hierarchy, as not only GMPs but also LT-HSCs showed this increase in CD47 expression (Figure 19b). By day 5, when most of the egress from the bone marrow had ceased, CD47 levels had returned to near normal levels. In Figure 19c, we show the mean fluorescence intensity of CD47 expression in GMPs from days 0 to 5 of mobilization. CD47 levels are actually subnormal after myeloablation on day 0, but then rise rapidly to a peak on day 2. Expression then declines rapidly, with levels equaling steady state by day 5.

[0143] Endotoxin is also thought to contribute to bone marrow mobilization. This may represent a physiological response to infection that requires an increase in normal bone marrow production of immune cells to clear untoward pathogens. Lipopolysaccharide (LPS) is a cell wall component of Gram-negative bacteria. It binds to lipid-binding protein (LBP) in serum, which can then form a complex with CD1411 and toll-like receptor 4 (TLR-4)12 on monocytes, macrophages, and dendritic cells. This activates macrophages, resulting in a proinflammatory response. LPS administration has also been shown to increase the phagocytic capacity of macrophages. This may be due to the LBP-LPS complex acting as an opsonin.

[0144] We tested whether LPS administration to mice affected CD47 expression in stem and progenitor cells. Recapitulating the pattern seen in Cy / G-induced mobilization, LPS caused proliferation of stem and progenitor cells by day 2 after treatment, followed by migration to the spleen and liver (Fig. 19d). Two days after LPS administration, stem and progenitor cells in bone marrow had upregulated CD47 to a similar extent as in Cy / G mobilization. By day 5, when the inflammatory response had disappeared, protein levels had fallen to steady-state levels (Fig. 19d).

[0145] Since CD47 was consistently upregulated during the mobilization response, we decided to test the ability of stem and progenitor cells to mobilize after Cy / G. CD47 knockout mice are defective in neutrophil migration to sites of inflammation8 and dendritic cell migration to secondary lymphoid organs. The exact role of CD47 in the migration of these cells is unclear, but may be related to poor integrin binding in the blood circulation (CD47 binds to several integrins) or lack of interaction with SIRPα on endothelial cells. Therefore, we reasoned that if CD47 is involved in the migratory ability of these cells in the mobilization response, IAP- / - mice should show reduced cell numbers in peripheral organs after Cy / G.

[0146] To test this hypothesis, we administered Cy / G to both wild-type and knockout mice and sacrificed them on days 2-5. For each mouse, we analyzed the number of stem and progenitor cells in the bone marrow, spleen, and liver. Because the number of CD34- cells was significantly reduced during the expansion state, making it difficult to accurately calculate the number of LT-HSCs, we decided to use the crude KLS population as a surrogate for HSCs. Since GMPs expanded the most among all populations upon mobilization, we decided to analyze their numbers as well. To calculate absolute progenitor cell counts, the overall cellularity of the bone marrow, spleen, and liver was estimated by counting mononuclear cells in intact organs with a hemocytometer. For bone marrow, we assumed that the long leg bones represented 15% of the total bone marrow. This number was then multiplied by the occurrence of the cell population to determine the absolute counts.

[0147] The inventors compared wild-type mice and IAP - / - We found little difference in the mobilization of KLS or GMP between the IAP mice (Fig. 19e). - / -The ability of the mice to mobilize progenitor cells to the spleen was somewhat reduced by day 3, but normal numbers of cells returned to the periphery by days 4 and 5. The bone marrow and liver compartments appeared similar to wild-type mice. Thus, IAP - / - The mice do not exhibit significant recruitment defects.

[0148] Heterozygous IAP + / - The amount of CD47 on erythrocytes is approximately half that of wild-type erythrocytes and platelets. + / - The amount of phagocytosis that occurs in red blood cells and platelets is increased in a dose-dependent manner compared to wild type. Given our observations that CD47 levels are elevated under stress and mobilization conditions, we hypothesized that cells that are genetically hemizygous for CD47 may be susceptible to phagocytosis and clearance by macrophages over a period of time. Thus, we hypothesize that IAPs may play a key role in long-term contribution to hematopoiesis. + / - We investigated whether stem cells would be at a disadvantage compared to wild-type stem cells.

[0149] First, we investigated the IAP + / + stem cells, IAP + / - Stem cells and IAPs - / - The levels of CD47 expressed on stem cells were analyzed. - Flk-2 - FACS analysis of KLS stem cells revealed that the MFI of CD47 on heterozygous HSCs was indeed nearly half the level of wild-type stem cells (Figure 20a). We then transplanted these cells and tested their ability to engraft and produce hematopoietic cells in recipients. We sublethally irradiated congenic wild-type recipient mice with 475 Gy. We then irradiated one cohort of recipients with 2 × 10 6 A separate cohort was transplanted with complete wild-type bone marrow cells and the same amount of IAP + / -Bone marrow cells were transplanted. Such a volume is expected to contain approximately 50-100 HSCs. Because granulocyte chimerism in peripheral blood is a good surrogate marker of stem cell health, we analyzed cells derived from the blood of these recipients at regular intervals. When wild-type bone marrow was transplanted into wild-type recipients, granulocyte chimerism was maintained for up to 40 weeks. However, IAP + / - When cells were transplanted, despite initially showing successful engraftment, 3 out of 5 mice lost donor chimerism over time (Figure 20b).

[0150] We observed that CD47 is upregulated on the surface of hematopoietic cells during leukemia progression. We also found that the level of CD47 expression was similarly elevated when mice were stimulated with Cy / G to mobilize stem and progenitor cells to the periphery or challenged with LPS. But why is CD47 upregulated in these conditions? Various studies have described a dose-dependent effect of CD47 in preventing phagocytosis. IAPs with CD47 levels half that of wild-type cells + / - Erythrocytes and IAPs + / - Platelets are phagocytosed more rapidly than their normal counterparts. Evidence also indicates that the level of CD47 expression on cells correlates well with the ability of the cells to bind to the SIRPα inhibitory receptor on macrophages. Recently, Danska et al. reported that the ability of NOD-SCID mice to support the engraftment of human hematopoietic cells correlates with mutations in the SIRPα receptor in these mice. Herein, we show that stem and progenitor cells expressing higher levels of CD47 are less likely to be cleared by phagocytosis.

[0151] These studies suggest a role for CD47 upregulation in protecting hematopoietic stem cells during conditions when they are more prone to be phagocytosed by macrophages, such as after myeloablation and during mobilization. Macrophages have the ability to remove senescent or damaged cells when they encounter them; they appear to be able to eliminate damaged stem cells as well. Thus, stem cells that are recovering to a healthy state may upregulate CD47 during the mobilization response to prevent clearance, whereas damaged stem cells cannot and are cleared. We speculate that this is a mechanism by which the hematopoietic system self-regulates itself to ensure that only healthy undamaged cells are given the opportunity to survive and proliferate and utilize resources during high stress conditions. The mobilization of HSCs and progenitor cells into the bloodstream and then to sites of hematopoiesis following LPS-induced inflammation is of great interest; HSCs use integrin α4β1 (Wagers and Weissman, Stem Cells 24(4):1087-94, 2006) and the chemokine receptor CXCR4 (Wright DE et al., J Exp Med 195(9): 1145-54, 2002) to migrate from the blood to the bone marrow. We have previously shown that integrin α4β1 binds to VCAM1 on hematopoietic stroma (Mikaye K et al. J Exp Med 173(3):599-607, 1991); VCAM1 is also a vascular addressin on blood vessels that inflammatory T cells use to recognize and invade local sites of cell death and inflammation. In addition to expressing the integrin-associated protein CD47, migrating HSCs express functional integrin α4β1, suggesting that hematopoietic stem and progenitor cells that migrate during inflammatory conditions may not only re-seed bone marrow hematopoiesis but also participate in local inflammation as well.

[0152] material and method Mice. C57Bl / 6 CD45.1 and C57Bl / 6 CD45.2 (wild type) mice were maintained in our colony. IAP- / - mice were obtained from Eric Brown (University of California, San Francisco). They were bred on a C57Bl6 / J background and crossed with our wild type colony.

[0153] Screening. IAP+ / - were crossed with each other to generate offspring IAP- / - and IAP+ / -. Mice were screened by PCR of tail DNA. The following primers were used: TIFF0007638360000004.tif20164

[0154] Cell staining and sorting. Staining of mouse stem and progenitor cells was performed using the following monoclonal antibodies: Mac-1, Gr-1, CD3, CD4, CD8, B220, and Ter119 conjugated to Cy5-PE (eBioscience) were used in the lineage cocktail, c-Kit PE-Cy7 (eBioscience), Sca-1 Alexa680 (e13-161-7, generated in our laboratory), CD34 FITC (eBioscience), CD16 / 32 (FcGRII / III) APC (Pharmingen), and CD135 (Flk-2) PE (eBioscience) were used to stain mouse stem and progenitor subsets as previously described2122. Mouse CD47 antibody (clone mIAP301) was evaluated using a biotin-labeled antibody generated in our laboratory. The cells were then stained with streptavidin-conjugated Quantum Dot 605 (Chemicon). Samples were analyzed using a FACSAria (Beckton Dickinson).

[0155] CD34-Flk2-KLS stem cells were double sorted using a BD FACSAria. Peripheral blood cells were obtained by tail vein blood collection and red blood cells were removed by Dextran T500 (Sigma) precipitation and ACK lysis. Cells were stained with anti-CD45.1 APC, anti-CD45.2 FITC, anti-Ter119 PE (Pharmingen), anti-B220 Cy5-PE (eBiosciences), anti-CD3 Cascade Blue, and anti-Mac-1 Cy7-PE (eBiosciences). Granulocytes were Ter119- B220- CD3- Mac-1+ SSC hi. Cells were analyzed using a BD FACSAria.

[0156] All samples were resuspended in propidium iodide-containing buffer prior to analysis to exclude dead cells. FACS data were analyzed using FloJo software (Treestar).

[0157] In vitro colony formation assay. LT-HSCs were clonally selected directly into 96-well plates containing methylcellulose medium (Methocult 3100) prepared as described. This medium was also supplemented with recombinant mouse stem cell factor (SCF), interleukin (IL)-3, IL-11, granulocyte-macrophage colony-stimulating factor (GM-CSF), thrombopoietin (Tpo), and erythropoietin (Epo). Colonies were scored for CFU-G, CFU-M, CFU-GM, CFU-GEMM, and Meg.

[0158] Cell transfer. For complete bone marrow transfer, IAP+ / +, IAP+ / -, or IAP- / - cells were freshly isolated from leg long bones. Cells were counted using a hemocytometer and resuspended in 100 uL of PBS+2% FCS. For some experiments, CD45.1 cells from C57Bl / 6 Ka CD45.1 mice were used as donors into CD45.2 wild-type mice.

[0159] For sorted cells, cells were sorted in precise amounts (i.e., 50 or 500 cells per tube) into PBS buffer and resuspended in 100 uL of PBS+2% FCS. For competition experiments, 2×10 freshly isolated complete bone marrow cells from C57Bl / 6 CD45.1 were 5 Each piece was added to 100 uL of stem cell suspension.

[0160] C57Bl / 6 Ka CD45.1 or C57Bl / 6 J CD45.2 recipient mice were irradiated with the indicated doses using a cesium source. The sublethal dose was 4.75 Gy, and the lethal dose was a fractionated dose of 9.5 Gy. Cells were transferred into the retro-orbital sinus of isofluorane-anesthetized mice using a 27-gauge syringe.

[0161] Mobilization assay. Mice were mobilized with cyclophosphamide (Sigma) (200 mg / kg) and G-CSF (Neupogen) (250 μg / kg) as previously described. Bacterial LPS from E. coli 055:B5 (Sigma) was administered into the peritoneal cavity at a dose of 40 mg / kg.

[0162] For analysis of mobilized organs, whole spleen, whole liver, and long leg bones were prepared into single cell suspensions. Cell concentrations were measured using a hemocytometer to determine the overall cellularity of hematopoietic cells in these organs.

[0163] c-Kit + Cell enrichment. Mouse whole bone marrow was stained with CD117 microbeads (Miltenyi). c-Kit was isolated using a magnetic separator on an AutoMACS Midi column (Miltenyi). + The cells were sorted.

[0164] In vitro phagocytosis assay. BMDMs were prepared as previously described. Prior to the assay, c-Kit enriched bone marrow cells were stained with CFSE (Invitrogen). 2.5 × 10 c-Kit enriched cells were 5 5 × 10 macrophages4 Macrophages and c-Kit cells were co-seeded with IAP + / + Mouse or IAP - / - Cells were obtained from either mouse or mouse. Cells were incubated for 2 hours and the phagocytic index was determined. Photographs were taken as previously described.

[0165] Example 4 CD47 is an independent prognostic factor and therapeutic antibody target on human acute myeloid leukemia cells Acute myeloid leukemia (AML) is organized as a cellular hierarchy that is initiated and maintained by a subset of self-renewing leukemia stem cells (LSCs). We hypothesized that increased CD47 expression on AML LSCs leads to pathogenesis by inhibiting phagocytosis through the interaction of CD47 with inhibitory receptors on phagocytes. We found that AML LSCs express CD47 more than their normal counterparts, and that increased CD47 expression predicted worse overall survival in three independent cohorts of adult AML patients. Furthermore, a blocking monoclonal antibody against CD47 preferentially allowed phagocytosis of AML LSCs by macrophages in vitro and inhibited their engraftment in vivo. Finally, treatment of mice engrafted with human AML with anti-CD47 antibodies eliminated AML in vivo. In summary, increased CD47 expression is an independent poor prognostic factor that can be targeted on human AML stem cells with a monoclonal antibody capable of stimulating phagocytosis of LSCs.

[0166] result CD47 is more highly expressed in AML LSCs than in their normal counterparts and is associated with FLT3-ITD mutations. We have studied several mouse models of myeloid leukemia and confirmed that CD47 expression is increased on mouse leukemia cells compared to normal bone marrow. This prompted us to investigate CD47 expression in human AML LSCs and their normal counterparts. Using flow cytometry, CD47 was more highly expressed in multiple AML LSC specimens than in HSCs and MPPs in normal bone marrow (Figure 6). This increased expression extended to bulk leukemia cells, which expressed CD47 similarly to LSC-enriched fractions.

[0167] Examination of a subset of these samples showed that CD47 surface expression correlated with CD47 mRNA expression. To investigate CD47 expression across morphologic, cytogenetic, and molecular subgroups of AML, gene expression data were analyzed from a cohort of 285 adult patients previously described (Valk et al., 2004 N Engl J Med 350, 1617-1628). No significant differences in CD47 expression were found among the FAB (French-American-British) subtypes. CD47 was expressed at similar levels in most cytogenetic subgroups, except in cases with t(8;21)(q22;q22), a favorable risk group that showed statistically significantly lower CD47 expression. In molecularly characterized AML subgroups, no significant association was found between CD47 expression and mutations in the tyrosine kinase domain of FLT3 (FLT3-TKD), overexpression of EVI1, or mutations in CEBPA, NRAS, or KRAS. However, higher CD47 expression was strongly correlated with the presence of FLT3-ITD (p<0.001). FLT3-ITD has been observed in nearly one-third of AML with normal karyotype and is associated with worse overall survival. This finding was confirmed separately in two independent datasets of 214 and 137 AML patients (Table 1).

[0168] Table 1. Clinical and molecular characteristics of AML samples from the validation cohort and comparison of CD47 low and high expression groups. TIFF0007638360000005.tif128128 Clinical and molecular features at diagnosis are tabulated. WBC indicates white blood cell count; FAB indicates French-American-British ancestry; FLT3-ITD indicates internal tandem duplication of the FLT3 gene (in 10 cases where FLT3-ITD status was absent, predicted FLT3-ITD status based on gene expression was imputed using the method of Bullinger et al., 2008); FLT3-TKD indicates tyrosine kinase domain mutation of the FLT3 gene; NPM1 indicates mutation in the NPM1 gene; MLL-PTD indicates partial tandem duplication of the MLL gene; CEBPA indicates mutation in the CEBPA gene. CR indicates complete remission. CR was assessed after both the first and second induction treatment programs, which included ICE (idarubicin, etoposide, cytarabine) or A-HAM (all-trans retinoic acid and high-dose cytarabine plus mitoxantrone). Autologous-HSCT indicates autologous transplant; allogeneic-HSCT indicates allogeneic transplant. †P values ​​compare differences in molecular and clinical features at diagnosis between patients with low and high CD47 mRNA expression. CD47 expression was dichotomized based on optimal cut points for overall survival stratification, identified by us based on an independent published microarray dataset (Valk et al, 2004), as described in the Supplementary Methods.

[0169] Identification and separation of normal and leukemic progenitor cells from the same patient based on differential CD47 expression. In the LSC-enriched Lin-CD34+CD38- fraction of specimen SU008, the majority of CD47 hiIn addition to CD47lo expressing cells, rare populations of CD47lo expressing cells were detected (Figure 21A). These populations were isolated to greater than 98% purity by fluorescence-activated cell sorting (FACS) and transplanted into neonatal NOG mice or seeded in complete methylcellulose. As observed in some AML specimens, CD47 hi The cells were unable to engraft in vivo or form any colonies in vitro.

[0170] However, CD47 lo The cells engrafted with normal myelo-lymphoid hematopoiesis in vivo and formed numerous morphologically normal myeloid colonies in vitro (Fig. 21B, C). This specimen contained a FLT3-ITD mutation, which was detected in the bulk leukemia cells (Fig. 21D). Purified CD47 hi The cells contained the FLT3-ITD mutation and were therefore part of the leukemic clone, but expressed CD47 lo The cells did not contain this mutation. lo Human cells isolated from engrafted mice contained only wild-type FLT3, indicating that CD47 lo The cells were shown to contain normal hematopoietic progenitor cells.

[0171] Increased CD47 expression in human AML is associated with poor clinical outcome. We hypothesized that increased CD47 expression in human AML leads to pathogenesis. From this hypothesis, we predicted that AML with higher CD47 expression would be associated with worse clinical outcome. Consistent with this hypothesis, analysis of a group of 285 previously described adult AML patients with various cytogenetic and molecular abnormalities (Valk et al., 2004) revealed that dichotomizing patients into low and high CD47 expression groups was associated with a significantly increased risk of death in the high expression group (p=0.03). The association of this dichotomizing stratification of CD47 expression with overall survival was confirmed in a second study cohort of 242 adult patients with normal karyotype (NK-AML) (Metzeler et al., 2008 Blood) (p=0.01).

[0172] By applying this stratification to a separate validation cohort of 137 adult patients with normal karyotype (Bullinger et al., 2008 Blood 111, 4490-4495), we confirmed the prognostic value of CD47 expression for both overall and relapse-free survival (Figure 22). Analysis of clinical characteristics of CD47 low and CD47 high expressers in this cross-validation cohort also confirmed statistically significant differences in white blood cell (WBC) counts and FLT3-ITD status, but no differences in rates of complete remission and type of consolidation therapy, including allogeneic transplant (Table 1). Kaplan-Meier analysis demonstrated that high CD47 expression at diagnosis was significantly associated with poor relapse-free and overall survival (Figure 22A,B). The median relapse-free survival of patients in the CD47 low expresser group was 17.1 months compared to 6.8 months in the CD47 high expresser group. This corresponds to a hazard ratio of 1.94 (95% CI 1.30-3.77, p=0.004). Regarding overall survival, the median survival time for patients with low CD47 expression was 22.1 months compared with 9.1 months for those with high CD47 expression. This corresponds to a hazard ratio of 2.02 (95% CI 1.37-4.03, p=0.002). When CD47 expression was considered as a continuous variable, increased expression was also associated with worse recurrence-free (p=0.02) and overall survival (p=0.02).

[0173] Despite its association with FLT3-ITD (Table 1), high CD47 expression at diagnosis was also significantly associated with worse recurrence-free survival and overall survival in the subgroup of 74 patients without FLT3-ITD, when considered either as a dichotomous classification (Fig. 2C,D) or as a continuous variable (p=0.02 for both recurrence-free survival and overall survival). In multivariable analysis considering age, FLT3-ITD status, and CD47 expression as continuous variables, increased CD47 expression remained associated with worse recurrence-free survival (hazard ratio 1.33 (95% CI 1.03-1.73, p=0.03)) and overall survival (hazard ratio 1.31 (95% CI 1.00-1.71, p=0.05)) (Table 2).

[0174] (Table 2) TIFF0007638360000006.tif35128

[0175] Monoclonal antibodies against human CD47 preferentially enable phagocytosis of AML LSCs by human macrophages. We hypothesized that increased CD47 expression in human AML leads to pathogenesis by inhibiting phagocytosis of leukemic cells, leading us to predict that disrupting the CD47-SIRPα interaction with monoclonal antibodies against CD47 would preferentially enable phagocytosis of AML LSCs. Several anti-human CD47 monoclonal antibodies have been generated, including some that can block the CD47-SIRPα interaction (B6H12.2 and BRIC126) and others that cannot (2D3) (Subramanian et al., 2006 Blood 107, 2548-2556). We tested the ability of these antibodies to enable phagocytosis of AML LSCs or normal human bone marrow CD34+ cells by human macrophages in vitro. Incubation of AML LSCs with human macrophages in the presence of an IgG1 isotype control antibody or a mouse anti-human CD45 IgG1 monoclonal antibody did not result in significant phagocytosis as measured by either immunofluorescence microscopy (Figure 8A) or flow cytometry. However, addition of the anti-CD47 blocking antibodies B6H12.2 and BRIC126, but not the non-blocking antibody 2D3, allowed phagocytosis of AML LSCs (Figure 8A,C). Phagocytosis of normal CD34+ cells was not observed with any of the antibodies (Figure 8C).

[0176] Monoclonal antibodies against human CD47 enable phagocytosis of AML LSCs by mouse macrophages. CD47-SIRPα interaction has been implicated as a critical regulator of xenograft rejection in some xenotransplants; however, there are conflicting reports regarding the ability of CD47 from one species to bind and stimulate SIRPα of another species. To directly evaluate the effect of inhibiting the interaction of human CD47 with mouse SIRPα, we performed an in vitro phagocytosis assay as described above with mouse macrophages. Incubation of AML LSCs with mouse macrophages in the presence of an IgG1 isotype control antibody or a mouse anti-human CD45 IgG1 monoclonal antibody did not result in significant phagocytosis as measured by either immunofluorescence microscopy (Figure 8B) or flow cytometry. However, addition of the anti-CD47 blocking antibodies B6H12.2 and BRIC126, but not the non-blocking antibody 2D3, enabled phagocytosis of AML LSCs (Figure 8B,C).

[0177] Monoclonal antibodies against human CD47 inhibit AML LSC engraftment and eliminate AML in vivo. We tested the ability of the anti-CD47 blocking antibody B6H12.2 to target AML LSC in vivo. We first utilized a pre-coating strategy in which AML LSC were purified by FACS and incubated with IgG1 isotype control antibody, anti-human CD45 antibody, or anti-human CD47 antibody. Aliquots of cells were analyzed for coating by staining with secondary antibodies to demonstrate that both anti-CD45 and anti-CD47 antibodies bound to the cells (Figure 10A). The remaining cells were transplanted into neonatal NOG mice and analyzed for leukemic engraftment 13 weeks later (Figure 10B). In all but one mouse, cells coated with isotype control and anti-CD45 antibody showed long-term leukemic engraftment. However, the majority of mice transplanted with anti-CD47 antibody-coated cells showed no detectable leukemic engraftment.

[0178] Next, we utilized a therapeutic strategy in which mice were first transplanted with human AML LSCs and then administered 100 μg of either mouse IgG or anti-CD47 antibody by daily intraperitoneal injection for 14 days, and measured leukemic engraftment pre- and post-treatment. Analysis of peripheral blood demonstrated that mice treated with anti-CD47 antibody often had near-complete clearance of circulating leukemia after a single dose, whereas control mice showed no response (Figure 23A,B). Similarly, bone marrow from mice treated with anti-CD47 antibody showed a significant reduction in leukemic engraftment, whereas mice treated with control IgG showed increased leukemic involvement (Figure 23C,D). Histological analysis of bone marrow demonstrated monomorphic leukemic blasts in mice treated with control IgG (Figure 23E, panels 1,2) and loss of hypocellular areas in mice treated with anti-CD47 antibody (Figure 23E, panels 4,5). Macrophages containing engulfed pyknotic cells were detected in the bone marrow of several mice treated with anti-CD47 antibodies in which leukemia persisted, capturing the disappearance of human leukemia (Figure 23E, panels 3 and 6).

[0179] We report herein that CD47 is more highly expressed in AML LSCs than in their normal counterparts, and hypothesize that increased CD47 expression in human AML leads to pathogenesis by inhibiting phagocytosis of these cells via the interaction of CD47 with SIRPα. Consistent with this hypothesis, we demonstrate that increased CD47 expression in human AML is associated with decreased overall survival. We also demonstrate that disrupting the CD47-SIRPα interaction with monoclonal antibodies against CD47 preferentially allows phagocytosis of AML LSCs by macrophages in vitro, inhibits AML LSC engraftment, and results in the elimination of AML in vivo. Collectively, these results establish the rationale for exploring the use of anti-CD47 monoclonal antibodies as a novel therapeutic approach for human AML.

[0180] The pathogenic effects of CD47 appear to be mechanistically distinct from the two major complementary classes of mutations in the proposed model of AML pathogenesis. According to this model, class I mutations (mainly affecting proliferation and apoptosis (e.g., FLT3 and NRAS)) and class II mutations (mainly impairing hematopoietic cell differentiation (e.g., CEBPA, MLL, and NPM1)) cooperate during leukemogenesis. As demonstrated herein, CD47 mediates pathogenesis by a unique mechanism, giving LSC and progeny blasts a survival advantage by evading phagocytosis by the innate immune system. Strategies to evade immune responses have been described for many human tumors, and we believe that increased CD47 expression represents the first such immune evasion mechanism, which is relevant for the prognosis and treatment of human AML.

[0181] High CD47 expression is a marker of leukemic stem cells and predicts overall survival in AML. AML LSCs are enriched in the Lin-CD34+CD38- fraction, which contains HSCs and MPPs in normal bone marrow. Identification of cell surface molecules that can distinguish leukemic cells from normal stem cells is essential for flow cytometric assessment of minimal residual disease (MRD) and for the development of prospective separation strategies for use in cell therapy. Several candidate molecules have been recently identified, including CD123, CD96, CLL-1, and now CD47. CD123 was the first molecule demonstrated to be more highly expressed in AML LSCs compared to normal HSC-enriched populations. We previously confirmed the AML LSC-specific expression of CD96 compared to normal HSCs and demonstrated that only CD96+ leukemic cells, but not CD96- leukemic cells, can engraft in vivo.

[0182] CLL-1 was identified as an AML LSC-specific surface molecule expressed in the majority of AML samples but not in normal HSCs; importantly, the presence of Lin-CD34+CD38-CLL-1+ cells in the bone marrow of some patients in hematological remission was predictive of relapse. Herein, we demonstrate not only that CD47 is more highly expressed in AML LSCs compared to normal HSCs and MPPs, but also that this differential expression can be exploited to separate normal HSCs / MPPs from LSCs. This provides the first demonstration of prospective separation of normal stem cells from leukemic stem cells in the same patient sample, offering the possibility of an autologous HSC transplantation therapy to deplete LSCs.

[0183] We first identified high expression of CD47 in AML LSCs, but noted that expression in bulk blasts was the same. Therefore, we utilized publicly available gene expression data in bulk AML to investigate the relationship between CD47 expression and clinical outcome. Consistent with our hypothesis, we found that increased CD47 expression independently predicted worse clinical outcome in karyotypically normal AML patients, including the largest subgroup of AML patients, the subset without FLT3-ITD mutations. Because this analysis relied on the relative expression of CD47 mRNA, quantitative PCR assays for AML prognosis could be based on the level of CD47 expression. Such assays could be used in risk-adapted therapy decision-making, particularly in the large subgroup of AML patients without FLT3-ITD mutations and with normal karyotypes.

[0184] Targeting CD47 on AML LSCs with therapeutic monoclonal antibodies. Cell surface molecules preferentially expressed on AML LSCs over their normal counterparts are candidates for targeting with therapeutic monoclonal antibodies. So far, several molecules on AML have been targeted, including CD33, CD44, CD123, and now CD47. CD33 is the target of the monoclonal antibody conjugate gemtuzumab ozogamicin (Mylotarg), which is approved for the treatment of relapsed AML in elderly patients. Targeting CD44 with monoclonal antibodies has been shown to significantly reduce AML engraftment in mice, providing evidence that monoclonal antibodies specifically act on LSCs to induce their differentiation. Monoclonal antibodies against CD123 have recently been reported to be effective in reducing AML LSC function in vivo. Herein, we report that monoclonal antibodies against CD47 can stimulate phagocytosis of AML LSCs in vitro and inhibit engraftment in vivo.

[0185] Several lines of evidence suggest that targeting CD47 with monoclonal antibodies likely acts by disrupting the CD47-SIRPα interaction, thereby interfering with the phagocytosis inhibitory signal. First, two anti-CD47 blocking antibodies enabled AML LSC phagocytosis, whereas one non-blocking antibody did not, even though all three antibodies bound to the cells similarly. Second, in the case of the B6H12.2 antibody used in the majority of our experiments, an isotype-matched anti-CD45 antibody that also bound to LSC did not have the same effect. Indeed, the B6H12.2 antibody is a mouse isotype IgG1 that cannot bind mouse Fc receptors as effectively as antibodies of isotype IgG2a or IgG2b.

[0186] For human clinical therapy, blocking CD47 on AML LSCs with humanized monoclonal antibodies promotes LSC phagocytosis by a similar mechanism, as shown by in vitro phagocytosis by human macrophages (Figure 8A, C). Higher CD47 expression is detected on AML LSCs; however, CD47 is expressed on normal tissues, including bone marrow HSCs. We confirmed that anti-CD47 antibodies have a preferential effect over normal bone marrow CD34+ cells in enabling phagocytosis of AML LSCs by human macrophages in vitro. Indeed, we did not detect increased phagocytosis of normal CD34+ cells compared to isotype control, demonstrating that blocking CD47 with monoclonal antibodies is a practical therapeutic strategy for human AML.

[0187] The experimental evidence presented herein provides a rationale for anti-CD47 monoclonal antibodies as a monotherapy for AML. However, such antibodies may be as effective, or even more effective, as part of a combination strategy. Combining an anti-CD47 antibody that can block strong inhibitory signals for phagocytosis with a second antibody that can bind to an LSC-specific molecule (e.g., CD96) and bind to Fc receptors on phagocytes, thereby delivering strong positive signals for phagocytosis, may provide a synergistic stimulation for phagocytosis and specific elimination of AML LSC. Furthermore, a combination of monoclonal antibodies against AML LSC, including anti-CD47 blocking antibodies against two other cell surface antigens and a human IgG1 antibody, is believed to be more likely to eliminate leukemia cells with existing epitope mutations or antigenic defects that are likely to relapse in patients treated with a single antibody.

[0188] Experimental procedure Human samples. Normal human bone marrow mononuclear cells were purchased from AllCells Inc. (Emeryville, CA). Human acute myeloid leukemia samples (Figure 1A) were obtained with informed consent from patients at Stanford University Medical Center according to IRB-approved protocols (Stanford IRB numbers 76935 and 6453). Human CD34-positive cells were enriched using magnetic beads (Miltenyi Biotech).

[0189] Flow cytometry and cell sorting. An antibody panel was used for analysis and sorting of AML LSCs (Lin-CD34+CD38-CD90-, lineage included CD3, CD19, and CD20), HSCs (Lin-CD34+CD38-CD90+), and MPPs (Lin-CD34+CD38-CD90-CD45RA-) as previously described (Majeti et al., 2007). Analysis of CD47 expression was performed using anti-human CD47 PE antibody (clone B6H12, BD Biosciences, San Jose CA).

[0190] Genomic DNA preparation and analysis of FLT3-ITD by PCR. Genomic DNA was isolated from cell pellets using the Gentra Puregene Kit according to the manufacturer's protocol (Gentra Systems, Minneapolis, MN). FLT3-ITD status was screened by PCR using primers that generate a 329 bp wild-type product and various larger ITD products.

[0191] Anti-human CD47 antibodies. Monoclonal mouse anti-human CD47 antibodies included BRIC126, IgG2b (Abcam, Cambridge, MA), 2D3, IgG1 (Ebiosciences. San Diego, CA), and B6H12.2, IgG1. The B6H12.2 hybridoma was obtained from the American Type Culture Collection (Rockville, MD). Antibodies were purified from hybridoma supernatants using protein G affinity chromatography according to standard procedures or obtained from BioXCell (Lebanon, NH).

[0192] Methylcellulose colony assay. Methylcellulose colony formation was analyzed by seeding sorted cells into 6-well plates with each well containing 1 ml of complete methylcellulose (Methocult GF+ H4435, Stem Cell Technologies). Plates were incubated at 37° C. for 14 days and then scored based on morphology.

[0193] In vitro phagocytosis assay. Human AML LSC or normal bone marrow CD34+ cells were labeled with CFSE and incubated with either mouse or human macrophages in the presence of 7 μg / ml of IgG1 isotype control, anti-CD45 IgG1 antibody, or anti-CD47 antibody (clones B6H12.2, BRIC126, or 2D3) for 2 hours. Cells were then analyzed by fluorescence microscopy to determine the phagocytic index (number of ingested cells per 100 macrophages). In some cases, cells were then harvested and stained with either mouse or human macrophage markers, and macrophage+CFSE+ were identified as phagocytosed cells by flow cytometry. Statistical analysis using Student's t-test was performed using GraphPad Prism (San Diego, CA).

[0194] In vivo pre-coating engraftment assay. LSCs isolated from AML specimens were incubated with 28ug / mL of IgG1 isotype control, anti-CD45 IgG1 antibody, or anti-CD47 IgG1 antibody (B6H12.2) for 30 minutes at 4°C. Aliquots of cells were then stained with donkey anti-mouse PE secondary antibody (Ebioscience) and analyzed by flow cytometry to assess coating. Approximately 10 5 Coated LSCs were transplanted into each neonatal irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NOG) mouse. Mice were sacrificed 13 weeks after transplantation and bone marrow was analyzed for human leukemia engraftment (hCD45+hCD33+) by flow cytometry (Majeti et al., 2007 Cell Stem Cell 1, 635-645). The presence of human leukemia was confirmed by Wright-Giemsa staining of hCD45+ cells and FLT3-ITD PCR. Statistical analysis using Student's t-test was performed using GraphPad Prism (San Diego, CA).

[0195] In vivo antibody treatment of AML-engrafted mice. FACS-purified 1–25 × 10 5 LSCs were transplanted into NOG pups. After 8–12 weeks, human AML engraftment (hCD45+CD33+ cells) in peripheral blood and bone marrow was assessed by tail bleed and femoral puncture, respectively. Transplanted mice were then treated with daily intraperitoneal injections of 100 μg of anti-CD47 antibody or control IgG for 14 days. Mice were sacrificed on day 15, and peripheral blood and bone marrow were analyzed for AML.

[0196] AML Patients, Microarray Gene Expression Data, and Statistical Analyses. Gene expression and clinical data were analyzed for three previously described cohorts of adult AML patients: (1) a training dataset of 285 patients with diverse cytogenetic and molecular abnormalities as described by Valk et al., (2) a testing dataset of 242 patients with normal karyotypes as described by Metzeler et al., and (3) a validation dataset of 137 patients with normal karyotypes as described by Bullinger et al. Clinical endpoints analyzed included overall survival and relapse-free survival. An event was defined as the interval between study entry and withdrawal from the study due to lack of complete remission, relapse, or death from any cause, and data were censored for patients without an event at the last follow-up visit.

[0197] FLT3-ITD PCR. All reactions contained 5 μl of 10× PCR buffer (50 mM KCL / 10 nM Tris / 2 mM MgCl2 / 0.01% gelatin), 1 μl of 10 mM dNTPs, 2 units of Taq polymerase (Invitrogen), 1 ul of 10 μM forward primer 11F TIFF0007638360000007.tif5128 and reverse primer 12R TIFF0007638360000008.tif5128, and 10–50 ng of genomic DNA. PCR conditions for amplifying the FLT3 gene were 40 cycles of denaturation (95°C for 30 s), annealing (62°C for 30 s), and extension (72°C for 30 s).

[0198] Preparation of mouse and human macrophages. BALB / c mouse bone marrow mononuclear cells were harvested and grown in IMDM containing 10% FBS supplemented with 10 ng / mL recombinant mouse macrophage colony-stimulating factor (M-CSF, Peprotech, Rocky Hill, NJ) for 7–10 days to allow terminal differentiation of monocytes into macrophages. Human peripheral blood mononuclear cells were prepared from discarded normal blood obtained from Stanford University Medical Center. Monocytes were isolated by allowing mononuclear cells to adhere to culture plates for 1 h at 37°C, after which nonadherent cells were removed by washing. More than 95% of the remaining cells were CD14 positive and CD11b positive. Adherent cells were then incubated in IMDM containing 10% human serum (Valley Biomedical, Winchester, VA) for 7–10 days to allow terminal differentiation of monocytes into macrophages.

[0199] In vitro phagocytosis assay. BMDM or peripheral blood macrophages were harvested by incubation in trypsin / EDTA (Gibco / Invitrogen) for 5 min followed by gentle scraping. Macrophages 5 × 10 4 10 cells were seeded into each well of a 24-well tissue culture plate containing 10% IMDM containing 10% FBS. After 24 hours, the medium was replaced with serum-free IMDM and the cells were cultured for an additional 2 hours. LSCs were fluorescently labeled with CFSE according to the manufacturer's protocol (Invitrogen). 2 × 10 cells labeled with CFSE were 4LSCs were added to wells containing macrophages with 7 μg / mL of IgG1 isotype antibody (Ebiosciences), anti-CD45 antibody (clone HI30, Ebiosciences), or anti-CD47 antibody and incubated for 2 h. The wells were then washed three times with IMDM and examined by an Eclipse T5100 immunofluorescence microscope (Nikon) using an enhanced green fluorescent protein filter capable of detecting CFSE fluorescence. The number of CFSE-positive cells in the macrophages was counted, and the phagocytic index was determined as the number of ingested cells per 100 macrophages. At least 200 macrophages were counted per well. Fluorescent and bright-field images were taken separately and merged using Image Pro Plus (Media Cybernetics, Bethesda, MD). In Figure 22A,B, the left three images are shown at 200x magnification, and the right anti-CD47 image is shown at 400x magnification. Cells were then harvested from each well using trypsin / EDTA for flow cytometric analysis of phagocytosis. Cell suspensions were then stained with mouse macrophage antibodies anti-mouse F4 / 80-PECy7 (Ebiosciences) or anti-human CD14-PECy7 (Ebiosciences) and analyzed using a FACSAria. Phagocytosed LSCs were defined as either CFSE+F4 / 80+ or ​​CFSE+CD14+ cells when incubated with mouse or human macrophages, respectively.

[0200] Microarray gene expression data. Panel A of Supplementary Figure 22 describes the main microarray datasets analyzed herein, including the training, testing, and validation cohorts. Training set: Gene expression, cytogenetic, and molecular data of 285 and 465 AML patients profiled by Valk et al. and Jongen-Lavrencic et al. using Affymetrix HG-U133A and HG-U133 Plus 2.0 microarrays, respectively, were obtained from Gene Expression Omnibus with corresponding accession numbers (GSE1159 and GSE6891). Outcome data were only available for the former dataset, and corresponding clinical information was kindly provided by the authors. This cohort is denoted as the "training" dataset. The latter dataset was used to confirm the univariate associations of karyotype and molecular mutations described in the former. However, these two datasets overlap in that 247 of the 285 patients from the first study were included in the second and were therefore excluded when examining the association between FLT3-ITD and CD47 expression in the second dataset.Using NetAffx4, RefSeq5, and UCSC Genome Browser6, we identified 211075_s_at and 213857_s_at as Affymetrix probe sets on U133 Plus 2.0 microarrays that map exclusively to constitutively transcribed CD47 exons.The geometric mean base 2 logarithms of these two probe sets were used to estimate the corresponding statistical measures of association with CD47 mRNA expression levels and FAB classification, karyotype, and molecular variants. Because the data provided by Valk et al. as GSE1159 are Affymetrix intensity measurements, we transformed these intensities to the ratio-normalized base-2 logarithms to allow comparison with corresponding measurements derived from cDNA microarrays using conventional schemes.Specifically, we first (1) normalized the raw data using CEL files from all 291 microarrays in this dataset using gcRMA8, then (2) divided the intensity measurement for each gene on a given array by the average intensity of the gene across all arrays to obtain a ratio, (3) transformed the resulting ratios to logarithms (base 2), and (4) median centered the expression data across arrays and then across genes. To assess the prognostic value of CD47, we used probe set 213857_s_at from Affymetrix HG-U133A and HG-U133 Plus 2.0 microarrays, given their similar expression distribution (Supplementary Figure 3B), and taking into account their location within the mRNA transcripts compared to cDNA clones on annotated Stanford cDNA microarrays in the NetAffx resource.

[0201] Test set: Gene expression and clinical data of 242 adult NKAML patients profiled by Metzeler et al. using Affymetrix HG-U133A and HG-U133 Plus 2.0 microarrays were obtained from Gene Expression Omnibus using the corresponding accession number (GSE12417). As the raw data of this dataset was not available, to evaluate the prognostic value of CD47, we used the normalized dataset (base 2 logarithm) provided by the authors and evaluated CD47 expression on the corresponding microarrays using probe set 213857_s_at.

[0202] Validation Set: Gene expression data of 137 normal karyotype AML patients profiled by Bullinger et al. using cDNA microarrays were obtained from the Stanford Microarray Database10. Corresponding clinical information, including outcome data and FLT3 mutation status, was kindly provided by the authors. Using microarray features and initial annotations from SOURCE11, RefSeq5, and UCSC Genome Browser6, we confirmed that IMAGE:811819 is a sequence-confirmed cDNA clone that maps to the constitutively transcribed 3'-end exon of CD47 on the corresponding cDNA microarray.

[0203] Treatment details: AML patients described by Valk et al. (training set) were treated according to several protocols of the Dutch-Belgian Hematology-Oncology Cooperative Group. The majority (90%) of NK-AML patients described by Metzeler et al. (test set) were treated according to the German AML Cooperative Group protocol AMLCG-1999, and all patients received intensive double induction and consolidation chemotherapy. All 137 NK-AML patients described by Bullinger et al. (validation set) received an intensive standard-of-care treatment program (protocol AML HD98A). The protocol included two courses of induction therapy with idarubicin, cytarabine, and etoposide, one consolidation cycle of high-dose cytarabine and mitoxantrone (HAM), followed by random assignment to a later consolidation cycle of HAM for autologous hematopoietic cell transplantation if an HLA-identical family donor was not available for allogeneic hematopoietic cell transplantation.

[0204] Statistical analysis. We used two-tailed t-tests and analysis of variance to estimate significant differences in CD47 expression levels in AML subgroups based on morphological, cytogenetic, and molecular categorization. Associations of high and low CD47 groups with baseline clinical, demographic, and molecular characteristics were analyzed using Fisher's exact test and Mann-Whitney rank sum test for categorical and continuous variables, respectively. A two-tailed p-value of <0.05 was considered to indicate statistical significance.

[0205] The prognostic value of CD47 expression was measured by comparing relapse-free and overall survival of patients using the Kaplan-Meier product limit method and the estimation of survival curves by the log-rank test. In this analysis, we first stratified AML patients into two groups, CD47 high and CD47 low, by comparing CD47 expression (measured by 213857_s_at in GSE1159) against an optimal threshold. This threshold was determined using X-Tile16. We used this method to maximize the chi-square statistic between the two groups in terms of expected and observed deaths. This stratification distinguishes the 261 AML patients with available outcome data into two groups of different sizes. The 72% of patients with the lowest expression were considered to be CD47 low, and the 28% with the highest expression were considered to be CD47 high. The overall survival of these two groups differed, with a hazard ratio of 1.42 in the high CD47 group and a corresponding uncorrected p-value of 0.033, necessitating cross-validation to avoid the risk of overfitting.

[0206] Therefore, we assessed the validity and robustness of risk stratification using CD47 expression by applying this optimal threshold to an independent test cohort of 242 NK-AML patients described by Metzeler et al. Notably, despite the presence of other variables potentially confounding the association with survival (including older age and different treatments), deriving the optimal cut-point using the 242 NK-AML patients in the test dataset resulted in a similar stratification, with 74% of patients with the lowest expression considered to be CD47 low and 26% with the highest expression considered to be CD47 high.

[0207] We next evaluated the validity of this stratification in a cross-validation cohort of 137 uniformly treated NK-AML patients described by Bullinger et al. Within this validation dataset, we were able to similarly define two groups of similar size (i.e., 72% and 28% with lowest and highest CD47 levels, respectively), which showed significantly different outcomes when assessed for overall survival (Figure 22B, p=0.002, hazard ratio 2.02, 95% CI 1.37-4.03) and relapse-free survival (Figure 223A, p=0.004, hazard ratio 1.94, 95% CI 1.30-3.77). Five of the 137 patients did not provide reliable CD47 measurements using the data selection and normalization criteria described by the authors.

[0208] To determine the robustness of this association, we also examined the predictive value of CD47 expression when dividing the validation cohort into CD47-low and CD47-high expression groups based on expression relative to the median, or as a continuous variable. As noted above, higher CD47 expression was associated with worse recurrence-free and overall survival. Of the 137 patients studied, a subset of 123 patients had available survival data, CD47 expression data, and FLT3-ITD status reported. Within this cohort, we assessed the relationship between CD47 expression level as a continuous variable and outcome using univariate Cox proportional hazards analysis with recurrence-free survival or overall survival as the dependent variable. We used multivariate Cox proportional hazards analysis with recurrence-free survival or overall survival as the dependent variable and FLT3-ITD status, age, and continuous CD47 expression level as independent variables assessed directly.

[0209] Associations between CD47 and other covariates (e.g., NPM1, CEBPA) were limited by sample size and missing data on covariates. Wald tests were used to assess the significance of each covariate in multivariate analyses. Univariate and multivariate proportional hazards analyses were performed using the coxph function in the statistical package R.

[0210] Example 5 CD47 is a prognostic factor and a therapeutic antibody target on solid tumor cancer stem cells The present inventors have found that increased CD47 expression is associated with worse clinical outcomes in diffuse large B-cell lymphoma (DLBCL) and ovarian cancer (Figure 24). Furthermore, the present inventors have now found that anti-CD47 antibodies enable phagocytosis by human macrophages of cancer stem cells derived from bladder cancer, ovarian cancer, and medulloblastoma in vitro (Figure 25).

[0211] Sequence information SEQUENCE LISTING <110> The Board of Trustees of the Leland Stanford Junior University <120> Methods for manipulating phagocytosis mediated by CD47 <150> US 61 / 011,324 <151> 2008-01-15 <150> US 61 / 189,786 <151> 2008-08-22 <160> 18 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 142 <212> PRT <213> Homo sapiens <400> 1 Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn 130 135 140 <210> 2 <211> 5346 <212> DNA <213> Homo sapiens <400> 2 ggggagcagg cgggggagcg ggcgggaagc agtgggagcg cgcgtcgcg cggcgtgca 60 gcctgggcag tgggtcctgc ctgtgacgcg cggcggcggt cggtcctgcc tgtaacggcg 120 gcggcggctg ctgctccaga cacctgcggc ggcggcggcg acccgcggc gggcgcggag 180 atgtggcccc tggtagcggc gctgttgctg ggctcggcgt gctgcggatc agctcagcta 240 ctatttaata aaacaaaatc tgtagaattc acgttttgta atgacactgt cgtcattcca 300 tgctttgtta ctaatatgga ggcacaaaac actactgaag tatacgtaaa gtggaaattt 360 aaaggaagag atattacac ctttgatgga gctctaaaca agtccactgt ccccactgac 420 tttagtagtg caaaaattga agtctcacaa ttactaaaag gagatgcctc tttgaagatg 480 gataagagtg atgctgtctc acacacagga aactacactt gtgaagtaac agaattaacc 540 agagaaggtg aaacgatcat cgagctaaaa tatcgtgttg tttcatggtt ttctccaaat 600 gaaaatattc ttattgttat tttcccaatt tttgctatac tcctgttctg gggacagttt 660 ggtattaaaa cacttaaata tagatccggt ggtatggatg agaaaacaat tgctttactt 720 gttgctggac tagtgatcac tgtcattgtc attgttggag ccattctttt cgtcccaggt 780 gaatattcat taaagaatgc tactggcctt ggtttaattg tgacttctac agggatatta 840 atattacttc actactatgt gtttagtaca gcgattggat taacctcctt cgtcattgcc 900 atattggtta ttcaggtgat agcctatatc ctcgctgtgg ttggactgag tctctgtatt 960 gcggcgtgta taccaatgca tggccctctt ctgatttcag gtttgagtat cttagctcta 1020 gcacaattac ttggactagt ttatatgaaa tttgtggctt ccaatcagaa gactatacaa 1080 cctcctagga aagctgtaga ggaacccctt aatgcattca aagaatcaaa aggaatgatg 1140 aatgatgaat aactgaagtg aagtgatgga ctccgatttg gagagtagta agacgtgaaa 1200 ggaatacact tgtgtttaag caccatggcc ttgatgattc actgttgggg agaagaaaca 1260 agaaaagtaa ctggttgtca cctatgagac ccttacgtga ttgttagtta agttttatt 1320 caaagcagct gtaatttagt tataaaata attatgatct atgttgtttg cccaattgag 1380 atccagtttt ttgttgttat ttttaatcaa ttaggggcaa tagtagaatg gacaatttcc 1440 aagaatgatg cctttcaggt cctagggcct ctggcctcta ggtaaccagt ttaaattggt 1500 tcagggtgat aactacttag cactgccctg gtgattaccc agagatatct atgaaaacca 1560 gtggcttcca tcaaaccttt gccaactcag gttcacagca gctttgggca gttatggcag 1620 tatggcatta gctgagaggt gtctgccact tctgggtcaa tggaataata attaagtac 1680 aggcaggaat ttggttggga gcatcttgta tgatctccgt atgatgtgat attgatggag 1740 atagtggtcc tcattcttgg gggttgccat tcccacattc ccccttcaac aaacagtgta 1800 acaggtcctt cccagattta gggtactttt attgatggat atgttttcct tttattcaca 1860 taaccccttg aaaccctgtc ttgtcctcct gttacttgct tctgctgtac aagatgtagc 1920 accttttctc ctctttgaac atggtctagt gacacggtag caccagttgc aggaaggagc 1980 cagacttgtt ctcagagcac tgtgttcaca cttttcagca aaaatagcta tggttgtaac 2040 atatgtattc ccttcctctg atttgaaggc aaaaatctac agtgtttctt cacttctttt 2100 ctgatctggg gcatgaaaaa agcaagattg aaatttgaac tatgagtctc ctgcatggca 2160 acaaaatgtg tgtcaccatc aggccaacag gccagccctt gaatggggat ttattactgt 2220 tgtatctatg ttgcatgata aacattcatc accttcctcc tgtagtcctg cctcgtactc 2280 cccttcccct atgattgaaa agtaaacaaa acccacattt cctatcctgg ttagaagaaa 2340 attaatgttc tgacagttgt gatcgcctgg agtactttta gacttttagc attcgttttt 2400 tacctgtttg tggatgtgtg tttgtatgtg catacgtatg agataggcac atgcatcttc 2460 tgtatggaca aaggtggggt acctacagga gagcaaaggt taattttgtg cttttagtaa 2520 aaacatttaa atacaaagtt ctttattggg tggaattata tttgatgcaa atatttgatc 2580 acttaaaact tttaaaactt ctaggtaatt tgccacgctt ttgactgct caccaatacc 2640 ctgtaaaaat acgtaattct tcctgtttgt gtaataagat attcatattt gtagttgcat 2700 tataatagt tatttcttag tccatcagat gttcccgtgt gcctctttta tgccaaattg 2760 attgtcatat ttcatgttgg gaccaagtag tttgcccatg gcaaacctaa attatgacc 2820 tgctgaggcc tctcagaaaa ctgagcatac tagcaagaca gctcttttg aaaaaaaaa 2880 tatgtataca caaatatata cgtatatcta tatacgta tgtataca cacatgtata 2940 ttcttccttg attgtgtagc tgtccaaaat aataacatat atagagggag ctgtattcct 3000 ttacaaat ctgatggctc ctgcagcact ttttccttct gaaaatatt acattttgct 3060 aacctagttt gttactttaa aaatcagttt tgatgaaagg agggaaaagc agatggactt 3120 gaaaaagatc caagctccta ttagaaaagg tatgaaaatc tttatagtaa aattttttat 3180 aaactaaagt tgtacctttt aatatgtagt aaactctcat ttatttgggg ttcgctcttg 3240 gatctcatcc atccattgtg ttctctttaa tgctgcctgc cttttgaggc attcactgcc 3300 ctagacaatg ccaccagaga tagtggggga aatgccagat gaaaccaact cttgctctca 3360 ctagttgtca gcttctctgg ataagtgacc acagaagcag gagtcctcct gcttgggcat 3420 cattgggcca gttccttctc tttaaatcag atttgtaatg gctcccaaat tccatcacat 3480 cacatttaaa ttgcagacag tgttttgcac atcatgtatc tgttttgtcc cataatatgc 3540 tttttactcc ctgatcccag tttctgctgt tgactcttcc attcagtttt atttatgtg 3600 tgttctcaca gtgacaccat ttgtccttt ctgcaacaac ctttccagct acttttgcca 3660 aattctattt gtttctcct tcaaaacatt ctcctttgca gttcctcttc atctgtgtag 3720 ctgctctttt gtctcttaac ttaccattcc tatagtactt tatgcatctc tgcttagttc 3780 tattagtttt ttggccttgc tcttctcctt gattttaaaa ttccttctat agctagagct 3840 tttctttctt tcattctctc ttcctgcagt gttttgcata catcagaagc taggtacata 3900 agttaaatga ttgagagttg gctgtattta gatttatcac tttttaatag ggtgagcttg 3960 agagttttct ttctttctgt tttttttttt tgtttttttt tttttttttt ttttttttt 4020 ttttgactaa tttcacatgc tctaaaaacc ttcaaaggtg attatttttc tcctggaaac 4080 tccaggtcca ttctgtttaa atccctaaga atgtcagaat taaaataaca gggctatccc 4140 gtaattggaa atatttcttt tttcaggatg ctatagtcaa tttagtaagt gaccaccaaa 4200 ttgttatttg cactaacaaa gctcaaaaca cgataagttt actcctccat ctcagtaata 4260 aaaattaagc tgtaatcaac cttctaggtt tctcttgtct taaaatgggt attcaaaaat 4320 ggggatctgt ggtgtatgta tggaaacaca tactccttaa tttacctgtt gttggaaact 4380 ggagaaatga ttgtcgggca accgtttatt ttttattgta ttttatttgg ttgagggatt 4440 tttttataaa cagttttact tgtgtcatat tttaaaatta ctaactgcca tcacctgctg 4500 gggtcctttg ttaggtcatt ttcagtgact aatagggata atccaggtaa ctttgaagag 4560 atgagcagtg agtgaccagg cagtttttct gcctttagct ttgacagttc ttaattaaga 4620 tcattgaaga ccagctttct cataaatttc tctttttgaa aaaaagaaag catttgtact 4680 aagctcctct gtaagacaac atcttaaatc ttaaaagtgt tgttatcatg actggtgaga 4740 gaagaaaaca ttttgttttt attaaatgga gcattattta caaaaagcca ttgttgagaa 4800 ttagatccca catcgtataa atatctatta accattctaa ataaagagaa ctccagtgtt 4860 gctatgtgca agatcctctc ttggagcttt tttgcatagc aattaaaggt gtgctatttg 4920 tcagtagcca ttttttgca gtgatttgaa gaccaaagtt gttttacagc tgtgttaccg 4980 ttaaaggttt tttttttat atgtattaaa tcaatttatc actgtttaaa gctttgaata 5040 tctgcaatct ttgccaaggt actttttat ttaaaaaaaa acataacttt gtaaatatta 5100 ccctgtaata ttatatac ttaataaaac atttaagct attttgttgg gctatttcta 5160 ttgctgctac agcagaccac aagcacattt ctgaaaaatt taatttatta atgtattttt 5220 aagttgctta tattctaggt aacaatgtaa agaatgattt aaaatattaa ttatgaattt 5280 tttgagtata atacccaata agcttttaat tagagcagag ttttaattaa aagttttaaa 5340 tcagtc 5346 <210> 3 <211> 5288 <212> DNA <213> Homo sapiens <400> 3 ggggagcagg cgggggagcg ggcgggaagc agtgggagcg cgcgtcgcg cggcgtgca 60 gcctgggcag tgggtcctgc ctgtgacgcg cggcggcggt cggtcctgcc tgtaacggcg 120 gcggcggctg ctgctccaga cacctgcggc ggcggcggcg acccgcggc gggcgcggag 180 atgtggcccc tggtagcggc gctgttgctg ggctcggcgt gctgcggatc agctcagcta 240 ctatttaata aaacaaaatc tgtagaattc acgttttgta atgacactgt cgtcattcca 300 tgctttgtta ctaatatgga ggcacaaaac actactgaag tatacgtaaa gtggaaattt 360 aaaggaagag atattacac ctttgatgga gctctaaaca agtccactgt ccccactgac 420 tttagtagtg caaaaattga agtctcacaa ttactaaaag gagatgcctc tttgaagatg 480 gataagagtg atgctgtctc acacacagga aactacactt gtgaagtaac agaattaacc 540 agagaaggtg aaacgatcat cgagctaaaa tatcgtgttg tttcatggtt ttctccaaat 600 gaaaatattc ttattgttat tttcccaatt tttgctatac tcctgttctg gggacagttt 660 ggtattaaaa cacttaaata tagatccggt ggtatggatg agaaaacaat tgctttactt 720 gttgctggac tagtgatcac tgtcattgtc attgttggag ccattctttt cgtcccaggt 780 gaatattcat taaagaatgc tactggcctt ggtttaattg tgacttctac agggatatta 840 atattacttc actactatgt gtttagtaca gcgattggat taacctcctt cgtcattgcc 900 atattggtta ttcaggtgat agcctatatc ctcgctgtgg ttggactgag tctctgtatt 960 gcggcgtgta taccaatgca tggccctctt ctgatttcag gtttgagtat cttagctcta 1020 gcacaattac ttggactagt ttatatgaaa tttgtggctt ccaatcagaa gactatacaa 1080 cctcctagga ataactgaag tgaagtgatg gactccgatt tggagagtag taagacgtga 1140 aaggaataca cttgtgttta agcaccatgg ccttgatgat tcactgttgg ggaagaaa 1200 caagaaaagt aactggttgt cacctatgag acccttacgt gattgttagt taagttttta 1260 ttcaaagcag ctgtaattta gttaataaaa taattatgat ctatgttgtt tgcccaattg 1320 agatccagtt ttttgttgtt atttttaatc aattaggggc aattagtagaa tggacaattt 1380 ccaagaatga tgcctttcag gtcctagggc ctctggcctc taggtaacca gtttaaattg 1440 gttcagggtg ataactactt agcactgccc tggtgattac ccagagatat ctatgaaaac 1500 cagtggcttc catcaaacct ttgccaactc aggttcacag cagctttggg cagttatggc 1560 agtatggcat tagctgagag gtgtctgcca cttctgggtc aatggaataa taaattaagt 1620 acaggcagga atttggttgg gagcatcttg tatgatctcc gtatgatgtg atattgatgg 1680 agatagtggt cctcattctt gggggttgcc attcccacat tcccccttca acaaacagtg 1740 taacaggtcc ttcccagatt tagggtactt ttattgatgg atatgttttc cttttattca 1800 cataacccct tgaaaccctg tcttgtcctc ctgttacttg cttctgctgt acaagatgta 1860 gcaccttttc tcctctttga acatggtcta gtgacacggt agcaccagtt gcaggaagga 1920 gccagacttg ttctcagagc actgtgttca cacttttcag caaaaatagc tatggttgta 1980 acatatgtat tcccttcctc tgatttgaag gcaaaaatct acagtgtttc ttcacttctt 2040 ttctgatctg gggcatgaaa aaagcaagat tgaaatttga actatgagtc tcctgcatgg 2100 caacaaaatg tgtgtcacca tcaggccaac aggccagccc ttgaatgggg atttattact 2160 gttgtatcta tgttgcatga taaacattca tcaccttcct cctgtagtcc tgcctcgtac 2220 tccccttccc ctatgattga aaagtaaaca aaacccacat ttcctatcct ggttagaaga 2280 aaattaatgt tctgacagtt gtgatcgcct ggagtacttt tagactttta gcattcgttt 2340 tttacctgtt tgtggatgtg tgtttgtatg tgcatacgta tgagataggc acatgcatct 2400 tctgtatgga caaaggtggg gtaccttacag gagagcaaag gttaattttg tgcttttagt 2460 aaaaacattt aaatacaaag ttctttattg ggtggaatta tatttgatgc aaatatttga 2520 tcacttaaaa cttttaaaac ttctaggtaa tttgccacgc tttttgactg ctcaccaata 2580 ccctgtaaaa atacgtaatt cttcctgttt gtgtaataag atattcatat ttgtagttgc 2640 attaataata gttattctt agtccatcag atgttcccgt gtgcctcttt tatgccaaat 2700 tgattgtcat atttcatgtt gggaccaagt agtttgccca tggcaaacct aaatttatga 2760 cctgctgagg cctctcagaa aactgagcat actagcaaga cagctcttct tgaaaaaaaa 2820 aatatgtata cacaaatata tacgtatatc tatatatacg tatgtatata cacacatgta 2880 tattcttcct tgattgtgta gctgtccaaa atataacat atatagaggg agctgtattc 2940 ctttatacaa atctgatggc tcctgcagca cttttcctt ctgaaaatat ttacattttg 3000 ctaacctagt ttgttacttt aaaaatcagt tttgatgaaa ggagggaaaa gcagatggac 3060 ttgaaaaaga tccaagctcc tattagaaaaa ggtatgaaaa tctttatagt aaaattttt 3120 ataaactaaa gttgtacctt ttaatatgta gtaaactctc attatttgg ggttcgctct 3180 tggatctcat ccatccattg tgttctcttt aatgctgcct gccttttgag gcattcactg 3240 ccctagacaa tgccaccaga gatagtgggg gaaatgccag atgaaaccaa ctcttgctct 3300 cactagttgt cagcttctct ggataagtga ccacagaagc aggagtcctc ctgcttgggc 3360 atcattgggc cagttccttc tctttaaatc agatttgtaa tggctcccaa attccatcac 3420 atcacattta aattgcagac agtgttttgc acatcatgta tctgttttgt cccataatat 3480 gctttttact ccctgatccc agttctgct gttgactctt ccattcagtt ttatttattg 3540 tgtgttctca cagtgacacc atttgtcctt ttctgcaaca acctttccag ctacttttgc 3600 caaattctat ttgtcttctc cttcaaaaca ttctcctttg cagttcctct tcatctgtgt 3660 agctgctctt ttgtctctta acttaccatt cctatagtac tttatgcatc tctgcttagt 3720 tctattagtt ttttggcctt gctcttctcc ttgattttaa aattccttct atagctagag 3780 cttttctttc tttcattctc tcttcctgca gtgttttgca tacatcagaa gctaggtaca 3840 taagttaaat gattgagagt tggctgtatt tagatttatc actttttaat agggtgagct 3900 tgagagttt ctttctttct gttttttttt tttgtttttt tttttttttt ttttttttt 3960 ttttttgact aatttcacat gctctaaaaa ccttcaaagg tgattatttt tctcctggaa 4020 actccaggtc cattctgttt aaatccctaa gaatgtcaga attaaaataa cagggctatc 4080 ccgtaattgg aaatatttct tttttcagga tgctatagtc aatttagtaa gtgaccacca 4140 aattgttatt tgcactaaca aagctcaaaa cacgataagt ttactcctcc atctcagtaa 4200 taaaaattaa gctgtaatca accttctagg tttctcttgt cttaaaatgg gtattcaaaa 4260 atggggatct gtggtgtatg tatggaaaca catactcctt aatttacctg ttgttggaaa 4320 ctggagaaat gattgtcggg caaccgttta ttttttattg tattttattt ggttgaggga 4380 tttttttata aacagtttta cttgtgtcat attttaaaat tactaactgc catcacctgc 4440 tggggtcctt tgttaggtca ttttcagtga ctaataggga taatccaggt aactttgaag 4500 agatgagcag tgagtgacca ggcagttttt ctgcctttag ctttgacagt tcttaattaa 4560 gatcattgaa gaccagcttt ctcataaatt tctctttttg aaaaaaagaa agcatttgta 4620 ctaagctcct ctgtaagaca acatcttaaa tcttaaaagt gttgttatca tgactggtga 4680 gagaagaaaa cattttgttt ttattaaatg gagcattatt tacaaaaagc cattgttgag 4740 aattagatcc cacatcgtat aaatatctat taaccattct aaataaagag aactccagtg 4800 ttgctatgtg caagatcctc tcttggagct tttttgcata gcaattaaag gtgtgctatt 4860 tgtcagtagc catttttttg cagtgatttg aagaccaaag ttgttttaca gctgtgttac 4920 cgttaaaggt tttttttttt atatgtatta aatcaattta tcactgttta aagctttgaa 4980 tatctgcaat ctttgccaag gtactttttt atttaaaaaa aaacataact ttgtaaatat 5040 taccctgtaa tattatatat acttaataaa acattttaag ctattttgtt gggctatttc 5100 tattgctgct acagcagacc acaagcacat ttctgaaaaa tttaatttat taatgtattt 5160 ttaagttgct tatattctag gtaacaatgt aaagaatgat ttaaaatatt aattatgaat 5220 tttttgagta tatacccaa tagactttta attagagcag agttttaatt aaaagtttta 5280 aatcagtc 5288 <210> 4 <211> 5313 <212> DNA <213> Homo sapiens <400> 4 ggggagcagg cgggggagcg ggcgggaagc agtgggagcg cgcgtcgcg cggcgtgca 60 gcctgggcag tgggtcctgc ctgtgacgcg cggcggcggt cggtcctgcc tgtaacggcg 120 gcggcggctg ctgctccaga cacctgcggc ggcggcggcg acccgcggc gggcgcggag 180 atgtggcccc tggtagcggc gctgttgctg ggctcggcgt gctgcggatc agctcagcta 240 ctatttaata aaacaaaatc tgtagaattc acgttttgta atgacactgt cgtcattcca 300 tgctttgtta ctaatatgga ggcacaaaac actactgaag tatacgtaaa gtggaaattt 360 aaaggaagag atattacac ctttgatgga gctctaaaca agtccactgt ccccactgac 420 tttagtagtg caaaaattga agtctcacaa ttactaaaag gagatgcctc tttgaagatg 480 gataagagtg atgctgtctc acacacagga aactacactt gtgaagtaac agaattaacc 540 agagaaggtg aaacgatcat cgagctaaaa tatcgtgttg tttcatggtt ttctccaaat 600 gaaaatattc ttattgttat tttcccaatt tttgctatac tcctgttctg gggacagttt 660 ggtattaaaa cacttaaata tagatccggt ggtatggatg agaaaacaat tgctttactt 720 gttgctggac tagtgatcac tgtcattgtc attgttggag ccattctttt cgtcccaggt 780 gaatattcat taaagaatgc tactggcctt ggtttaattg tgacttctac agggatatta 840 atattacttc actactatgt gtttagtaca gcgattggat taacctcctt cgtcattgcc 900 atattggtta ttcaggtgat agcctatatc ctcgctgtgg ttggactgag tctctgtatt 960 gcggcgtgta taccaatgca tggccctctt ctgatttcag gtttgagtat cttagctcta 1020 gcacaattac ttggactagt ttatatgaaa tttgtggctt ccaatcagaa gactatacaa 1080 cctcctagga aagctgtaga ggaacccctt aatgaatac tgagtgag tgatggactc 1140 cgatttggag agtagtaaga cgtgaaagga atacacttgt gtttaagcac catggccttg 1200 atgattcact gttggggaga agaacaga aaagtaactg gttgtcacct atgagaccct 1260 tacgtgattg ttagttaagt ttttattca agcagctgta atttagttaa taaaatatt 1320 atgatctatg ttgtttgccc aattgagatc cagttttg ttgttattt taatcaatta 1380 ggggcaatag tagaatggac aatttccaag aatgatgcct ttcaggtcct agggccctg 1440 gcctctaggt aaccagttta aattggttca gggtgataac tacttagcac tgccctggtg 1500 attack gatatcttg aaaaccagtg gcttccatca aacctttgcc aactcaggtt 1560 height height height height height height height 1620 gggtcaatgg ataataat taagtacagg caggaatttg gttgggagca tcttgtatga 1680 tctccgtag atgtgatatt gatggagata gtggtcctca ttctgggggg ttgccattcc 1740 cacattcccc cttcaacaa cagtgtaca ggtccttccc agatttagg tactttatt 1800 gatggatatg ttttccttt attcacataa ccccttgaaa ccctgtcttg tcctcctgtt 1860 acttgcttct gctgtacaag atgtagcacc ttttctctc tttgaacatg gtctagtgac 1920 acggtagcac cagttgcagg aaggagccag acttgttctc agagcactgt gttcacactt 1980 ttcagcaaaa atagctatgg ttgtaacata tgtattccct tcctctgatt tgaaggcaaa 2040 aatctacagt gtttcttcac ttcttttctg atctggggca tgaaaaaagc aagattgaaa 2100 tttgaactat gagtctcctg catggcaaca aaatgtgtgt caccatcagg ccaacaggcc 2160 agcccttgaa tggggattta ttactgttgt atctatgttg catgataaac attcatcacc 2220 ttcctcctgt agtcctgcct cgtactcccc ttcccctatg attgaaaagt aaacaaaacc 2280 cacatttcct atcctggtta gaaaaatt aatgttctga cagttgtgat cgcctggagt 2340 acttttagac ttttagcatt cgttttttac ctgtttgtgg atgtgtgttt gtatgtgcat 2400 acgtatgaga taggcacatg catcttctgt atggacaaag gtggggtacc tacaggagag 2460 caaaggttaa ttttgtgctt ttagtaaaaa catttaaata caaagttctt tattgggtgg 2520 aattatattt gatgcaaata tttgatcact taaaactttt aaaacttcta ggtaatttgc 2580 cacgcttttt gactgctcac caataccctg taaaaatacg taattcttcc tgtttgtgta 2640 ataagatatt catatttgta gttgcattaa taatagttat ttcttagtcc atcagatgtt 2700 cccgtgtgcc tcttttatgc caaattgatt gtcatatttc atgttgggac caagtagttt 2760 gcccatggca aacctaaatt tatgacctgc tgaggcctct cagaaaactg agcatactag 2820 caagacagct cttcttgaaa aaaaaaaatat gtatacacaa atatatacgt atatctatat 2880 atacgtatgt atatacacac atgtatattc ttccttgatt gtgtagctgt ccaaaataat 2940 aacatatata gagggagctg tattccttta tacaaatctg atggctcctg cagcactttt 3000 tccttctgaa aatatttaca ttttgctaac ctagtttgtt actttaaaaa tcagttttga 3060 tgaaaggagg gaaaagcaga tggacttgaa aaagatccaa gctcctatta gaaaaggtat 3120 gaaaatcttt atagtaaaat tttttataaa ctaaagttgt acctttaat atgtagtaaa 3180 ctctcattta tttggggttc gctcttggat ctcatccatc cattgtgttc tctttaatgc 3240 tgcctgcctt ttgaggcatt cactgcccta gacaatgcca ccagagatag tgggggaaat 3300 gccagatgaa accaactctt gctctcacta gttgtcagct tctctggata agtgaccaca 3360 gaagcaggag tcctcctgct tgggcatcat tgggccagtt ccttctcttt aaatcagatt 3420 tgtaatggct cccaaattcc atcacatcac atttaaattg cagacagtgt tttgcacatc 3480 atgtatctgt tttgtcccat aatatgcttt ttactccctg atcccagttt ctgctgttga 3540 ctcttccatt cagttttatt tattgtgtgt tctcacagtg acaccatttg tccttttctg 3600 caacaacctt tccagctact tttgccaaat tctatttgtc ttctccttca aaacattctc 3660 ctttgcagtt cctcttcatc tgtgtagctg ctcttttgtc tcttaactta ccattcctat 3720 agtactttat gcatctctgc ttagttctat tagttttttg gccttgctct tctccttgat 3780 tttaaaattc cttctatagc tagagctttt ctttctttca ttctctcttc ctgcagtgtt 3840 ttgcatacat cagaagctag gtacataagt taaatgattg agagttggct gtatttagat 3900 ttatcacttt ttaatagggt gagcttgaga gttttctttc tttctgtttt ttttttttgt 3960 tttttttttt tttttttttt tttttttttt tgactaattt cacatgctct aaaaaccttc 4020 aaaggtgatt attttctcc tggaaactcc aggtccattc tgtttaaatc cctaagaatg 4080 tcagaattaa aataacaggg ctatcccgta attgggaaata tttcttttt caggatgcta 4140 tagtcaattt agtaagtgac caccaaattg ttattgcac taacaaagct caaaacacga 4200 taagtttact cctccatctc agtaataaaa attaagctgt aatcaacctt ctaggtttct 4260 cttgtcttaa aatgggtatt caaaaatggg gatctgtggt gtatgtatgg aaacacatac 4320 tccttaattt acctgttgtt ggaactgga gaatgattg tcgggcaacc gtttattttt 4380 tattgtattt tatttggttg aggattttt ttaaaacag ttttacttgt gtcatatttt 4440 aaaattacta actgccatca cctgctgggg tcctttgtta ggtcattttc agtgactaat 4500 agggataatc caggtaactt tgaagagatg agcagtgagt gaccaggcag tttttctgcc 4560 tttagctttg acagttctta attaagatca ttgaagacca gctttctcat aaatttctct 4620 ttttgaaaaa aagaaagcat ttgtactaag ctcctctgta agacaacatc ttaaatctta 4680 aaagtgttgt tatcatgact ggtgagagaa gaaaacattt tgtttttatt aaatggagca 4740 ttatttacaa aaagccattg ttgagaatta gatcccacat cgtataaata tctattaacc 4800 attctaaata aagagaactc cagtgttgct atgtgcaaga tcctctcttg gagctttttt 4860 gcatagcaat taaaggtgtg ctatttgtca gtagccattt ttttgcagtg atttgaagac 4920 caaagttgtt ttacagctgt gttaccgtta aaggtttttt tttttatatg tattaaatca 4980 atttatcact gtttaaagct ttgaatatct gcaatctttg ccaaggtact tttttatta 5040 aaaaaaaaca taactttgta aatattaccc tgtaatatta tatatactta ataaaacatt 5100 ttaagctatt ttgttgggct atttctattg ctgctacagc agaccacaag cacatttctg 5160 aaaaatttaa tttattaatg tatttttaag ttgcttatat tctaggtaac aatgtaaaga 5220 atgatttaaa atattaatta tgaatttttt gagtataata cccaataagc ttttaattag 5280 agcagagttt taattaaaag ttttaaatca gtc 5313 <210> 5 <211> 323 <212> PRT <213> Homo sapiens <400> 5 Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Lys 290 295 300 Ala Val Glu Glu Pro Leu Asn Ala Phe Lys Glu Ser Lys Gly Met Met 305 310 315 320 Asn Asp Glu <210> 6 <211> 305 <212> PRT <213> Homo sapiens <400> 6 Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Asn 290 295 300 Asn 305 <210> 7 <211> 312 <212> PRT <213> Homo sapiens <400> 7 Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Lys 290 295 300 Wing Val Glu Glu Pro Leu Asn Glu 305 310 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 18S RNA forward primer <400> 8 ttgacggaag ggcaccacca g 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 18S RNA reverse primer <400> 9 gcaccaccac ccacggaatc g 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin forward primer <400> 10 ttccttcttg ggtatggaat 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin reverse primer <400> 11 gagcaatgat cttgatcctc 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CD47 forward primer <400> 12 aggccaagtc cagaagcatt c 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CD47 reverse primer <400> 13 aatcattctg ctgctcgttg c 21 <210> 14 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> 3' Neo PCR primer <400> 14 gcatcgcatt gtctgagtag gtgtcattct attc 34 <210> 15 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> 5' IAP PCR primer <400> 15 tcaccttgtt gttcctgtac tacaagca 28 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 3' IAP PCR Primer <400> 16 tgtcacttcg caagtgtagt tcc 23 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> FLT3-ITD PCR Forward Primer 11F <400> 17 gcaatttagg tatgaaagcc agc 23 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> FLT3-ITD PCR Reverse Primer 12R <400> 18 ctttcagcat tttgacggca acc 23

Claims

1. A pharmaceutical for use in the treatment of acute myeloid leukemia (AML) by increasing phagocytosis of AML cells in a human subject, comprising a first antibody that prevents binding of CD47 on AML cells to the SIRPα receptor on phagocytes, thereby targeting the AML cells for phagocytosis, for use in combination with a second antibody that specifically binds to CD33.

2. The pharmaceutical composition of claim 1, wherein the antibody that interferes with the binding of CD47 to SIRPα specifically binds to CD47.

3. The pharmaceutical described in claim 1 or 2, wherein the first antibody is a humanized or chimeric monoclonal antibody.

4. The pharmaceutical described in claim 1 or 2, wherein the second antibody is a humanized or chimeric monoclonal antibody.

5. The pharmaceutical described in claim 1, wherein the first antibody and the second antibody are provided as bispecific antibodies that bind to CD47 and CD33.

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