Abstract CD47, a “don't eat me” signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

Avoiding phagocytosis by tumor-associated macrophages is required for the growth and metastasis of solid tumors (1). Accumulating evidence suggests that cell-surface expression of CD47 is a common mechanism by which cells protect themselves from phagocytosis (1). CD47 expression is required to protect transfused red blood cells, platelets, and lymphocytes from rapid elimination by splenic macrophages (2⇓–4). Mobilized hematopoietic stem cells protect themselves from phagocytosis by increasing CD47 expression as they pass through phagocyte-lined sinusoids and decrease it after relocating to marrow niches (5). Moreover, CD47 expression levels predicted the probability that hematopoietic stem cells would be phagocytosed while circulating (5).

CD47 is a widely expressed transmembrane protein with numerous functions (6). CD47 functions as a ligand for signal regulatory protein-α (SIRPα), a protein expressed on macrophages and dendritic cells (7). Upon binding CD47, SIRPα initiates a signaling cascade that results in the inhibition of phagocytosis (6). This “don't eat me” signal is transmitted by phosphorylation of the immunoreceptor tyrosine-based inhibition motifs present on the cytoplasmic tail of SIRPα (8). Subsequent binding and activation of SHP-1 and SHP-2 [src homology-2 (SH2)-domain containing protein tyrosine phosphatases] blocks phagocytosis, potentially by preventing the accumulation of myosin-IIA at the phagocytic synapse (9⇓⇓–12).

Here we show that CD47 is expressed on all human patient cancer cells tested. To our knowledge, CD47 is a unique nonhousekeeping cell-surface marker expressed by all human cancers. Increased CD47 mRNA expression levels in some solid tumors correlated with a decreased probability of patient survival. Monoclonal antibodies targeted to CD47 enabled the phagocytosis of patient solid tumor cells in vitro, inhibited the growth of orthotopically xenotransplanted human patient tumors, and prevented the metastasis of human patient tumor cells. These results establish CD47 as a critical regulator of innate immune surveillance.

Discussion Here we demonstrate that expression of CD47 is a general mechanism used by human patient solid tumor cells to evade phagocytosis. Blocking mAbs that disrupt the interaction between CD47 and SIRPα enabled the phagocytosis of solid tumor cells in vitro and inhibited tumor growth in several orthotopic xenotransplantation models. Moreover, anti-CD47 antibodies prevented the formation of tumor metastases. These results establish CD47 as a therapeutic target on solid tumor cells. A concern in translating this therapy to human application is the potential for toxicity. CD47 is highly expressed on tumor cells, but also at varying levels on normal (nontumor) cells (6). However, here we demonstrate that blockade of CD47 in immune competent mice produces an effective antitumor response without unacceptable toxicity, albeit with a temporary anemia (Fig. 6, Fig. S6, and Table S4). In a previous report, administering therapeutic doses of blocking anti-mouse CD47 mAbs (clone MIAP301) to normal C57BL/6 mice produced no significant toxic effect except isolated neutropenia (17). In both studies, mice were treated with doses of anti-CD47 mAbs (200–400 μg) that may be in far excess of the minimal effective dose. Furthermore, anti-CD47 mAbs failed to induce phagocytosis of normal (noncancer) cells in vitro (16, 17). It is inferred from this finding that normal healthy tissues lack a secondary prophagocytic “eat me” signal and, in the absence of CD47-SIRPα signaling, are not subject to phagocytosis. Several prophagocytic signals have been identified, including cell surface calreticulin and phosphatidylserine (34, 35). Calreticulin interacts with LDL-receptor-related protein 1 on macrophages and is required for the phagocytosis of tumor cell lines following neutralization of the CD47–SIRPα interaction (36, 37). Cell surface calreticulin is present on a subset of cells in all human leukemias, lymphomas, and solid cancers, but not on nontumor cells (37). The presence of prophagocytic signals on tumor cells may provide a therapeutic window in which to administer anti-CD47 mAbs without causing phagocytosis of healthy cells that lack these “eat me” signals. Thus far, we have evaluated the efficacy of anti-CD47 mAbs to inhibit solid tumor growth as a monotherapy. In our experiments, the efficacy of anti-CD47 therapy was inversely correlated with tumor size at the onset of treatment. Therefore, anti-CD47 antibody therapy may be most effective after the primary mass has been maximally debulked through cytoreductive surgery. Debulking can also be accomplished by chemotherapy or radiotherapy. However, caution must be used in applying anti-CD47 therapy in the context of ongoing or recent cytotoxic or inflammatory therapy, as the translocation of calreticulin to the cell surface upon conditions of cell stress may render normal cells susceptible to phagocytosis by nearby macrophages (38, 39). The therapeutic efficacy of anti-CD47 mAbs may be further enhanced when used concurrently with a second antitumor antibody. In particular, antibodies, such as trastuzumab and cetuximab, which induce antibody-dependent cell-mediated cytotoxicity through the Fc receptor on macrophages, may transmit an “eat-me” signal that enhances the ability of anti-CD47 mAbs to induce phagocytosis (40). Such a strategy has already proven effective in xenotransplantation models of non-Hodgkin's lymphomas, where the combination of anti-CD47 and rituximab antibodies produced a synergistic induction of phagocytosis and therapeutic response (16). Metastasis is the primary reason for failure of local therapies, such as surgery or radiotherapy (41). Anti-CD47 mAb therapy not only inhibited the growth of primary site tumors, but also prevented the formation of tumor metastases in the lymph nodes and lungs, or eliminated them as microtumors. Circulating tumor cells may even be particularly vulnerable to anti-CD47 mAbs if they rely on transmitting a “don't eat me” signal to perivascular macrophages for entry into blood vessels or tether to macrophages for assistance in migration (1, 42). It is also possible that the preventative effect on tumor metastasis is because of disrupting an interaction between CD47 and integrins. CD47 was initially identified as integrin-associated protein and has been shown to bind the α v β 3 , α IIb β 3 , α 2 β 1 integrins (6, 43). The B6H12.2 antibody may affect the ability of CD47 to interact with these integrins, thereby inhibiting their ability to adhere and migrate (44). Future studies will address the ability of anti-CD47 mAbs to eliminate established tumor metastases before and following surgical resection of the primary tumor, mimicking treatment of metastatic disease in the clinical setting. Emerging evidence suggests TAMs support tumor progression and metastasis (1, 28⇓–30, 45). TAMs participate in the development of a microenvironment conducive to tumor growth through remodeling of extracellular matrix and release of factors that promote cell proliferation, angiogenesis, and migration (46). Here, we demonstrated that blockade of CD47 signaling enables TAMs to attack tumor cells that they would otherwise disregard (Fig. 3E). Given that TAMs are present in large numbers within tumors (46), it's possible that anti-CD47 antibody therapy has the potential to restore TAM immunosurveillance and fundamentally alter the role of macrophages in tumor biology. In conclusion, we have found that CD47 is expressed on a wide range of human solid tumors, perhaps as the one constant change in all human aggressive neoplasms. We have also validated one function of CD47 on these cancers, as a “don't eat me” signal. CD47 therefore serves as an attractive target for cancer therapies. We have demonstrated that monoclonal antibodies that block CD47 are effective for treating human solid tumors in vitro and in vivo. We anticipate that these findings will extend to all modalities that interfere with the CD47-SIRPa interaction.

Materials and Methods See SI Materials and Methods for detailed discussion. All animal procedures were approved by the Administrative Panel on Laboratory Animal Care at Stanford University. Human Samples. Tissue specimens were obtained from consented patients as approved by Stanford University Institutional Review Board protocols. Colorectal cancer samples were obtained as previously described (27). Stanford University pathologists defined tumor and matched adjacent normal (nontumor) tissue specimens. Survival Data Analysis. CD47 expression was dichotomized in large (n ≥ 50 samples) solid tumor datasets for which clinical annotations were available. The Maxstat package (version 0.7–13) (47) of the R programming language (version 2.11) was used to define high- and low-CD47 groups. Log-rank P values and hazard ratios with 95% confidence intervals (CI) were derived by Kaplan–Meier analyses. For Affymetrix arrays, data were normalized from raw CEL files using MAS5 with a custom chip definition file-mapping array oligonucleotides to Entrez gene identifiers (48). In Vitro Phagocytosis Assay. For in vitro phagocytosis assay, 5 × 104 macrophages were plated per well in a 24-well tissue-culture plate. Tumor cells were labeled with 2.5 μM carboxyfluorescein succinimidyl ester (CFSE) according to the manufacturer's protocol (Invitrogen). Macrophages were incubated in serum-free medium for 2 h before adding 2 × 105 CFSE-labeled live tumor cells. The indicated antibodies (10 μg/mL) were added and incubated for 2 h at 37°. Macrophages were repeatedly washed and subsequently imaged with an inverted microscope (Leica DMI6000B). The phagocytic index was calculated as the number of phagocytosed CFSE+ cells per 100 macrophages. For ex vivo assays, TAMs (F4/80+; eBioscience) and human tumor cells (GFP+) were isolated by FACS from single-cell suspensions prepared from large (>1 cm3) subcutaneous xenograft tumors established in NSG mice. Purified TAMs and tumor cells and were mixed in the presence of control IgG1 or anti-CD47 (B6H12) antibody (20 μg/mL) and incubated for 2–4 h. Phagocytosis was then determined by flow cytometry detection of GFP+ TAMs. Antibody Preparation, Flow Cytometry Analysis, and Cell Sorting. The anti-hCD47 (B6H12) hybridoma was obtained from the ATCC. Hybridoma cells were cultured under standard conditions and antibodies were purified by Protein G. For quantification of CD47 expression, cells were labeled with a saturating concentration of a 1:1 phycoerythrin- (PE) conjugated anti-CD47 antibody (BD Pharmingen) and analyzed using a BD LSR Fortessa Analyzer. BD QuantiBRITE PE beads (BD Pharmingen) were analyzed at the same settings and conditions as the patient samples. Median absolute CD47 antibody binding for each sample was determined from a calibration curve constructed from the QuantiBRITE bead data using the FlowJo Data Analysis software calibration tool.

Acknowledgments We thank Libuse Jerabek, Rosalind Ravasio, Donna Mahood, Linda Quinn, Rebecca Broome, Amy Erickson, and Cathy Emory for their laboratory assistance and administrative support; Janet Bruno and Jennifer Santos for assisting in tissue acquisition; Kelli Montgomery for tissue staining and scanning; Susan Prohaska for critical discussions and insightful feedback; the Contag laboratory (Stanford University) for bioluminescence imaging reagents; Eric Brown and Hiroshi Morisaki (Genentech) for providing anti-mouse CD47 hybridomas; and Rosey Mushens (International Blood Group Reference Laboratory, United Kingdom) for production and purification of the Bric126 antibody. This work was supported by The Joseph and Laurie Lacob Gynecologic/Ovarian Cancer Fund, The Jim and Carolyn Pride Fund, The Virginia and D. K. Ludwig Fund for Cancer Research, The Weston Havens Foundation, The National Cancer Institute (5P01CA139490), Department of Defense Award W81XWH-07-1-0467, and anonymous donors.

Footnotes Author contributions: S.B.W., J.-P.V., A.J.G., D.S., P.D., S.S.M., J.W., H.C.-T., R. Martin, J.D.C., P.L., M.P.C., K.W., C.T., A.K.V., T.A.S., B.E., S.M.S., P.R., M.A., T.R., D.T., S.J., P.O.E., G.K.S., R. Majeti, M.v.d.R., J.B.S., A.A.A., M.F.C., and I.L.W. designed research; S.B.W., J.-P.V., A.J.G., D.S., P.D., S.S.M., J.W., H.C.-T., R. Martin, J.D.C., P.L., M.P.C., K.W., C.T., A.K.V., T.J.N., T.A.S., B.E., S.M.S., O.M., P.R., R.K.C., D.T., M.v.d.R., and A.A.A. performed research; S.B.W., J.-P.V., A.J.G., D.S., P.D., S.S.M., J.W., H.C.-T., R. Martin, J.D.C., P.L., F.A.S., K.W., A.K.V., T.J.N., T.A.S., A.R.M., B.E., S.M.S., C.K.S., M.-S.C., O.M., P.R., A.C.C., R.K.C., M.A., T.R., D.T., G.K.S., G.L., S.K.S., R. Majeti, G.R.H., M.v.d.R., N.N.H.T., J.B.S., A.A.A., and M.F.C. contributed new reagents/analytic tools; S.B.W., J.-P.V., A.J.G., D.S., S.S.M., J.W., H.C.-T., R. Martin, J.D.C., P.L., M.P.C., K.W., C.T., A.K.V., T.A.S., B.E., S.M.S., D.K., S.J., P.O.E., R. Majeti, M.v.d.R., N.N.H.T., J.B.S., A.A.A., M.F.C., and I.L.W. analyzed data; and S.B.W., J.-P.V., A.A.A., and I.L.W. wrote the paper.

Conflict of interest statement: S.J., M.P.C., R. Majeti, and I.L.W. filed U.S. Patent Application Serial No. 12/321,215 entitled “Methods for Manipulating Phagocytosis Mediated by CD47.” I.L.W. owns Amgen Inc. stock and is a Director of Stem Cells, Inc.

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