Co-phagocytosis of VEGFA with HER2-overexpressing cancer cells induced by HER2-VEGFA–bispecific antibodies improves antitumor responses
Yang Lu, … , Songbo Qiu, Zhen Fan
Yang Lu, … , Songbo Qiu, Zhen Fan
Published September 4, 2025
Citation Information: JCI Insight. 2025;10(20):e194494. https://doi.org/10.1172/jci.insight.194494.
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Research Article Clinical Research Immunology Oncology

Co-phagocytosis of VEGFA with HER2-overexpressing cancer cells induced by HER2-VEGFA–bispecific antibodies improves antitumor responses

Abstract

We conceived of a type of antitumor mechanism of action by which a soluble target in the tumor microenvironment, such as a tumor-driving growth factor, can be phagocytized along with cancer cells via antibody-dependent cellular phagocytosis (ADCP) using an antibody bispecific for the soluble target and a solid target overexpressed on the cancer cell surface. We explored this concept through engineering bispecific antibodies (BsAbs) co-targeting human epidermal growth factor receptor-2 (HER2) and vascular endothelial growth factor A (VEGFA) in an scFv-IgG format (VHS). We showed that the HER2-VEGFA BsAbs but not the parental antibodies alone or in combination induced co-phagocytosis of VEGFA and HER2-overexpressing cancer cells by tumor-associated macrophages via ADCP. In both immunocompromised and immunocompetent mice with aggressive tumors, the BsAbs demonstrated greater anti-metastasis activity and produced a greater survival benefit than the parental antibodies alone or in combination, in a manner dependent on Fcγ receptors on the macrophages. Our results provide proof of the concept that HER2-VEGFA BsAbs achieve enhanced antitumor activity by leveraging HER2 overexpressed on the cancer cell surface to induce co-phagocytosis of VEGFA. Our findings warrant clinical testing of the strategy to treat metastasis and recurrence of HER2-overexpressing solid tumors that respond to anti-VEGFA therapy.

Authors

Yang Lu, Songbo Qiu, Zhen Fan

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Figure 5

Co-phagocytosis of recombinant VEGFA with HER2-overexpressing cancer cells via HER2-VEGFA BsAb–induced ADCP in co-culture with unpolarized and polarized BMDMs.

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Co-phagocytosis of recombinant VEGFA with HER2-overexpressing cancer cel...
(A) BMDMs were treated with IFN-γ (50 ng/mL) and LPS (1:1000 dilution of the stock) for 12 hours or with IL-4, IL-10, and IL-13 (50 ng/mL each) for 24 hours in culture as indicated. The BMDMs were then subjected to FcγR blockade with anti-CD16/CD32 antibody and staining with APC-conjugated anti-F4/80 antibody, PE-conjugated anti-CD86 antibody, and PE/Cy7-conjugated anti-CD206 antibody, or subjected to FcγR blockade with PE-conjugated anti-CD16/CD32 antibody and staining with APC-conjugated anti-F4/80 antibody. The flow cytometry data were gated using FlowJo software (v10) for the levels of CD86, CD206, and FcγRs on F4/80-positive cells. The gating strategy for flow cytometry analysis is shown in Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.194494DS1 (B) BMDMs unpolarized or polarized as shown in A were mixed with D5-HER2 cells (at a 1:2 ratio of cell number) in the presence of mouse IgG control antibody, combination of G6.31.2a and 4D5.2a, or TG-VHS (0.5 μg/mL each). The cell mixture or the BMDMs alone were incubated with a pHrodo green–labeled recombinant VEGFA (0.5 μg/sample) for overnight. The cell samples were then analyzed by flow cytometry after staining with APC-conjugated anti-F4/80 antibody. The flow cytometry data were gated using FlowJo software (v10) for VEGFA green positivity in F4/80-positive cells (BMDMs) and F4/80-negative cells (D5-HER2). The gating strategy for flow cytometry analysis is shown in Supplemental Figure 2. P < 0.0001 by Wilcoxon’s test. (C) Models depicting the processes of phagocytosis and co-phagocytosis of VEGFA opsonized upon the treatments. Created in BioRender (https://BioRender.com/wwe8c21).