CD47 blockade augmentation of trastuzumab antitumor efficacy dependent on antibody-dependent cellular phagocytosis
Li-Chung Tsao, … , Herbert Kim Lyerly, Zachary C. Hartman
Li-Chung Tsao, … , Herbert Kim Lyerly, Zachary C. Hartman
Published December 19, 2019; First published November 5, 2019
Citation Information: JCI Insight. 2019;4(24):e131882. https://doi.org/10.1172/jci.insight.131882.
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Research Article Immunology Oncology

CD47 blockade augmentation of trastuzumab antitumor efficacy dependent on antibody-dependent cellular phagocytosis

Abstract

The HER2-specific monoclonal antibody (mAb), trastuzumab, has been the mainstay of therapy for HER2+ breast cancer (BC) for approximately 20 years. However, its therapeutic mechanism of action (MOA) remains unclear, with antitumor responses to trastuzumab remaining heterogeneous and metastatic HER2+ BC remaining incurable. Consequently, understanding its MOA could enable rational strategies to enhance its efficacy. Using both murine and human versions of trastuzumab, we found its antitumor activity dependent on Fcγ receptor stimulation of tumor-associated macrophages (TAMs) and antibody-dependent cellular phagocytosis (ADCP), but not cellular cytotoxicity (ADCC). Trastuzumab also stimulated TAM activation and expansion, but did not require adaptive immunity, natural killer cells, and/or neutrophils. Moreover, inhibition of the innate immune ADCP checkpoint, CD47, significantly enhanced trastuzumab-mediated ADCP and TAM expansion and activation, resulting in the emergence of a unique hyperphagocytic macrophage population, improved antitumor responses, and prolonged survival. In addition, we found that tumor-associated CD47 expression was inversely associated with survival in HER2+ BC patients and that human HER2+ BC xenografts treated with trastuzumab plus CD47 inhibition underwent complete tumor regression. Collectively, our study identifies trastuzumab-mediated ADCP as an important antitumor MOA that may be clinically enabled by CD47 blockade to augment therapeutic efficacy.

Authors

Li-Chung Tsao, Erika J. Crosby, Timothy N. Trotter, Pankaj Agarwal, Bin-Jin Hwang, Chaitanya Acharya, Casey W. Shuptrine, Tao Wang, Junping Wei, Xiao Yang, Gangjun Lei, Cong-Xiao Liu, Christopher A. Rabiola, Lewis A. Chodosh, William J. Muller, Herbert Kim Lyerly, Zachary C. Hartman

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Figure 1

Generation of murine trastuzumab and its antitumor dependence on antibody-dependent cellular phagocytosis (ADCP) by tumor-associated macrophages (TAMs).

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Generation of murine trastuzumab and its antitumor dependence on antibod...
(A) Cartoon representation of trastuzumab and 4D5 antibodies used in this study. (B) MM3MG cells expressing human HER2Δ16 were implanted into the mammary fat pads (1 × 106 cells) of BALB/c mice. Trastuzumab (human IgG1) or 4D5 (mouse IgG2A) was administered weekly (200 μg per mouse). n = 8–10. (C) Tumors (>1000 mm3 volume) were processed into single-cell suspensions and TAMs (percentage CD11b+F4/80+LY6G–LY6C– of CD45+ cells) were analyzed by FACS. n = 8–10. (D) Experiment as in B was repeated in SCID-beige animals. n = 8. (E) Experiment in SCID-beige was repeated using neutrophil-depleting anti-LY6G antibodies (clone IA8, 300 μg per mouse biweekly). (F and G) To deplete macrophages, SCID-beige mice were pretreated with anti-CSF1R antibody (clone AFS98, 300 μg, 3 times per week) for 2 weeks. (F) Macrophage depletion was verified by FACS. (G) 4D5-IgG2A was injected with anti-CSF1R treatment maintained throughout the experiment. n = 8. (H) Trastuzumab/4D5 induced ADCP of HER2+ breast cancer (BC) cells by bone marrow–derived macrophages (BMDMs). MM3MG-HER2Δ16 cells were labeled with Brilliant Violet 450 Dye and cocultured with BMDMs (3:1 ratio) with control or anti-HER2 antibodies (10 μg/mL). ADCP rates were measured as percentage of BMDM uptake of labeled tumor cells (CD45+ and BV450+), and antibody-dependent cellular cytotoxicity (ADCC) rates were measured as percentage of dying free tumor cells (CD45– and LIVE/DEAD+). ADCP inhibitor (latrunculin A) or ADCC inhibitor (concanamycin A) was added as assay control. n = 3; experiment was repeated 3 separate times. In B, D, E, and G, tumor growth was determined with caliper-based tumor measurement over time. Significance was determined by 2-way ANOVA with Tukey’s multiple-comparisons test (C, F, and H) or 1-way ANOVA test with Tukey’s multiple-comparisons test. All data represent the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.