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
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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 3

Co-phagocytosis of fibroblast-secreted VEGFA with HER2-overexpressing cancer cells via HER2-VEGFA BsAb–induced ADCP in co-cultures with macrophages.

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Co-phagocytosis of fibroblast-secreted VEGFA with HER2-overexpressing ca...
(A) 4T1-HLC cells and RAW264.7 macrophages (at a 1:5 cell number ratio) were co-cultured in fresh medium supplemented with conditioned medium (at a 1:1 volume ratio) from culture of NIH3T3-G cells or from culture of NIH3T3-VG cells and were treated with one of the antibodies (0.5 μg/mL each) as indicated for 2 hours in a 37°C incubator with 5% CO2. The cells were then harvested and stained with APC-conjugated anti-F4/80 antibody for flow cytometry analysis. Upper panel: The co-cultured cells were gated for mCherry positivity in F4/80-positive cells (indicating ADCP of HER2-overexpressing cells). The cells in Q2 indicate 4T1-HLC cells phagocytized by the macrophages in the co-culture. Phagocytosis (%) = Q2/(Q1 + Q2) × 100. Lower panel: Top row: The co-cultured cells were gated for GFP positivity in F4/80+ cells (indicating VEGFA co-phagocytosis via ADCP of HER2-overexpressing cells). Co-phagocytosis (%) = Q2/(Q1 + Q2) × 100. Middle row: EGFP and F4/80 double-positive cells were further gated for mCherry positivity. Bottom row: The EGFP-positive but F4/80-negative cells were further gated for mCherry positivity. NS, not significant. *P <0.05, **P < 0.01, ****P < 0.0001 by Fisher’s exact test. (B) RAW264.7 cells were stained with CellTrace Far Red fluorescent dye (shown in white) and then seeded on a poly-D-lysine–coated cell culture dish (FluoroDish, World Precision Instruments; https://www.wpiinc.com/var-2827-fluorodish-cell-culture-dish.html) overnight. The next day, 4T1-HLC cells (shown in red) were added to the RAW264.7 cell culture and incubated for 3 hours in the presence of TG-VHS or control antibody as indicated, along with concentrated conditioned medium from NIH3T3-VG cell culture containing VEGFA-EGFP (shown in green). The cells were counterstained with DAPI fluorescent dye (shown in blue) and then fixed with 2% paraformaldehyde prior to confocal cell imaging analysis. Original magnification, ×80. (C) A model depicting the process of VEGFA co-phagocytosis induced by the HER2-VEGFA BsAb. Created in BioRender (https://BioRender.com/6ibpdoc).