Therapeutic anti-CD47 monoclonal antibodies (mAbs) are designed to block the CD47–SIRPα checkpoint and promote immune-mediated recognition and elimination of cancer cells. However, current anti-CD47 mAbs have limitations, including off-tumor toxicity and reduced effectiveness in advanced cancers. Additionally, CD47 serves as a death receptor that mediates programmed cancer cell death (PCCD), a mechanism that has not been fully explored in current therapies. In this study, we introduce CO-001, a chimeric bifunctional IgG4 mAb, and its optimized variant CO-005, a bivalent humanized single-chain fragment variable–fragment crystallizable fusion protein. Both CO-001 and CO-005 promoted phagocytosis and PCCD. CO-005, specifically engineered to overcome the safety limitations associated with anti-CD47 antibodies, demonstrates a superior hematologic safety profile in vitro and ex vivo compared with benchmark anti-CD47 antibodies. Notably, CO-005 exhibited no binding to red blood cells, limited binding to white blood cells, and showed no hemagglutination activity. In preclinical models, CO-005 demonstrated potent antitumor activity in B-cell precursor acute lymphoblastic leukemia and Raji lymphoma xenograft models through the dual action of PCCD induction and enhancement of phagocytosis. The ability of CO-005 to trigger strong PCCD while preserving conventional immune responses provides a novel and promising approach for CD47-targeted cancer therapy. Its favorable safety profile, observed in both in vitro and ex vivo studies, positions CO-005 as a promising candidate with potential therapeutic advantages over existing anti-CD47 treatments.

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©2025 The Authors; Published by the American Association for Cancer Research
2025
American Association for Cancer Research
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First page of Development of a Novel Bifunctional Anti-CD47 Fusion Protein with Improved Efficacy and a Favorable Safety Profile<alt-title alt-title-type="short">Anti-CD47 Fusion Protein with Favorable Safety and Efficacy</alt-title>

Supplementary data

Figure S1

Induction of phagocytosis and PCCD by the humanized versions of CO-001 in Jurkat cells. (A) Phagocytosis activity of humanized versions of CO-001 (1 μg/ml) in Jurkat cell lines using DiO-labelled RAW264.7 macrophages. Phagocytosed target cells were analyzed by flow cytometry as % Celltrace+DiO+ cells (B) Jurkat cells (5×105 cells/ml) were incubated with 1 (left panel) or 10 (right panel) µg/ml CO-002 versions A-L for 3 hours, followed by Annexin V and 7-AAD staining. The cells that stained positive for Annexin V were undergoing PCCD. Data are presented as the mean ± S.D. Significance was determined by ANOVA analysis *p<0.05 relative to isotype control, n = 1 (upper panel), 3 (lower panel).

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Figure S2

Induction of hemagglutination by the humanized versions of CO-001. Incubation of freshly isolated RBCs (2% v/v in PBS) with increasing concentrations of CO-002 candidates A-K, or human IgG4 isotype control. A small punctate circle indicates no hemagglutination, while a diffuse hazy pattern indicates hemagglutination.

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Figure S3

Induction of hemagglutination by the various anti-CD47 mAbs. Incubation of freshly isolated RBCs (2% v/v in PBS) with increasing concentrations of the indicated anti-CD47 mAbs, or human IgG4 and IgG2 isotype control. A small punctate circle indicates no hemagglutination, while a diffuse hazy pattern indicates hemagglutination.

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Figure S4

CO-005 retains the dual functionality of CO-001 in various hematological cancer cell lines. (A) PCCD in MOLT-4, Reh, and CCRF-CEM cell lines after 3 hours of treatment with CO-005 (0.1 and 1 µg/ml. (B) Phagocytosis activity of CO-005 (1 μg/ml) in various hematological cell lines using DiO-labelled RAW264.7 macrophages. (C) CellTrace-labelled Jurkat cells or KG-1a cells (5×105 cells/ml) were co-cultured with DiO-labelled human macrophages differentiated from human PBMCs derived from three healthy human donors and treated with the indicated concentrations of CO-005 or 1 μg/ml of IgG4 for 2 hours. (B and C) Phagocytosed target cells were analyzed by flow cytometry as % Celltrace+DiO+ cells. (A-C) Data represent the mean ± S.D. Significance was determined by ANOVA analysis *p<0.01 relative to isotype control, n = 3-5.

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Figure S5

CO-005 does not bind to murine CD47. The binding of CO-005 to murine CD47 (mCD47) was tested using bone marrow cells harvested from NSG mice. NSG bone marrow cells were incubated with CO-005, Magrolimab, human IgG4 isotype control, miap301 (murine anti-CD47 antibody), or rat IgG2a control at increasing concentrations. Binding was measured using a PE-conjugated Goat anti-human IgG Fc (for HuIgG4, CO-005, and Magrolimab) or Goat anti-Rat (for RatIgG2a and miap301) secondary antibody.

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Table S1

Detailed list of the fluorophore-conjugated antibodies and reagents used in immune cell phenotyping by flow cytometry experiments. All materials were purchased from Thermo Fisher Scientific.

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Table S2

Classification of the positions showing a significant impact on the binding of the CO-001 antibody when mutated. The top line indicates the numbering of the amino acid residues in the CD47 molecule (AF-Q08722-F1, Alphafold). Second line indicates amino acid residues; Grey shading = structural residues. Third line indicates the number of mutations that are forbidden at that position, between 2 and 19. Fourth line: R= red, high impact, between 19 and 14 mutations are forbidden; O= orange, medium impact, between 13 and 7 mutations are forbidden; Y= yellow, low impact, between 2 and 6 mutations are forbidden; W = white, no impact or destructuring. Bottom line: * denotes the residues believed to be part of the CO-001 epitope.

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Table S3

Induction of PCCD and phagocytosis by CO-001 in various hematological cell lines.

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Table S4

The binding affinity of CO-001, CO-004, and CO-005 to recombinant human CD47 using SPR.

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