The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors

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.

Fig. 1.

CD47 is highly expressed on patient solid tumors. (A) Percentage of patient tumor (●) or normal (○) cells that express CD47, as evaluated by flow cytometry, relative to an isotype-matched control. Each symbol represents a different tissue sample. (B) Tumor cells express more CD47 than matched adjacent normal cells, as evaluated by quantitative flow cytometry. Levels of CD47 expression were quantified on live, CD45⁻, CD31⁻, and ESA⁺ cells. Analysis of the kidney samples was performed on live CD45⁻, CD31⁻ cells. See also Fig. S1. GBM, glioblastoma multiforme; HCC, hepatocellular carcinoma.

Fig. 2.

CD47 mRNA expression levels may be a prognostic factor in solid tumors. Increased levels of CD47 mRNA expression were correlated with decreased probability of progression-free survival of ovarian cancer (A and B) and overall survival of glioma (C–E) and glioblastoma (F). See also Table S1.

Fig. 3.

Blockade of CD47–SIRPα interaction enables phagocytosis of patient tumor cells. (A) Representative images of NSG bone marrow-derived macrophages (BMDM) phagocytosing human tumor cells following treatment with the indicated antibody. Arrows point to phagocytosed tumor cells. (B and C) Mouse BMDM (B) and human peripheral blood- (PB) derived macrophages (C) phagocytose patient tumor cells in the presence of blocking anti-CD47 mAbs (B6H12 and Bric126), but not control (IgG1 isotype and anti-HLA) or nonblocking anti-CD47 antibodies (2D3). Each symbol color represents a different ovarian (circle), colon (square), glioblastoma (triangle), or breast (diamond) tumor sample. (D) Anti-CD47 mAbs enable the phagocytosis of colon CSCs (Lineage⁻ESA⁺CD44⁺) by human PB derived (squares) and mouse BMDMs (circles). (E) Blocking anti-CD47 mAbs enable NSG tumor associated macrophages to phagocytose human tumor cells. Each symbol color represents a different tumor. Phagocytic Index indicates the number of tumor cells phagocytosed per 100 macrophages. *P < 0.0001. See also Fig. S2 and Movies S1 and S2. (Magnification, 100×.)

Fig. 4.

Anti-CD47 mAbs inhibit tumor growth in xenotransplantation models. Anti-CD47 mAbs inhibit tumor growth as measured by bioluminescence activity (A) and representative images (B) and improve survival (C) of mice engrafted with ovarian cancer. (D) Representative images of breast cancer engrafted mice treated with anti-CD47 mAbs. (E) Anti-CD47 mAbs inhibited the growth of a second breast cancer sample. (F) Colon tumor growth was inhibited by anti-CD47 mAbs, as evaluated by tumor mass and bioluminescence (Fig. S3A). (G) Glioblastoma growth was inhibited by anti-CD47 mAb treatment. (H) Representative bioluminescence images of treated mice. See also Fig. S3 and Tables S2 and S3.

Fig. 5.

Anti-CD47 mAbs prevent tumor metastasis. (A) The number of mice exhibiting lymph node metastases in each cohort. (B) The number of secondary lymph nodes detected in each mouse. The total number of secondary lymph nodes is indicated. (C) The total number of mice exhibiting lung micrometastases in each cohort. (D) The number of micrometastases in the lungs of each mouse is shown. The total number of lung micrometastases is indicated. (E) Anti-CD47 mAbs prevent head and neck squamous cell carcinoma cells from metastasizing to lymph nodes. The total number of secondary lymph nodes is shown. See also Fig. S4.

Fig. 6.

Anti-CD47 antibodies inhibit tumor growth in immune competent hosts. (A) Representative images of MT1A2 breast tumors at initiation of anti-CD47 mAb therapy. (B) Anti-mouse CD47 mAbs inhibit MT1A2 tumor growth. (C) Representative H&E images of IgG or anti-CD47 mAb treated tumors. (D) Anti-CD47 mAbs (10 μg/mL) do not initiate cell death of MT1A2 cells. See also Fig. S5 and Table S4. (Magnification, 7.8×.)