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 8

Human CD47 gene expression is a prognostic factor in HER2+ breast cancer and limits the therapeutic activity of trastuzumab.

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Human CD47 gene expression is a prognostic factor in HER2+ breast cancer...
(A and B) Kaplan-Meier survival curve for breast cancer (BC) patients’ METABRIC data set. (A) Stratified into low and high groups based on average expression of CD47 in all patients. (B) The same patient stratification based on disease subtype (ER+, HER2+, and triple-negative BC [TNBC]). (C) CD47 knockout in human HER2+ BC line KPL-4 was generated using the CRISPR/Cas9 approach. Control and CD47-KO KPL-4 cells were labeled with Brilliant Violet 450 Dye, and incubated with human monocyte–derived macrophages (hMDMs) at a 3:1 ratio, in the presence of control IgG or trastuzumab (10 μg/mL). Antibody-dependent cellular phagocytosis (ADCP) was measured by percentage of hMDM uptake of labeled KPL-4 cells (CD45+ and BV450+). Data are shown as the mean ± SEM; n = 4 biological replicates. Experiment was repeated using hMDMs generated from 3 healthy PBMC donors. (D) Control or CD47-KO KPL-4 cells were implanted into mammary fat pads of SCID-beige BALB/c mice (5 × 105 cells). Trastuzumab (50 μg) or control human IgG1 was administered weekly and tumor volume was measured. ****P < 0.0001 by 2-way ANOVA with Tukey’s multiple-comparisons test. (E) Tumor-infiltrating macrophage (F4/80+Gr1–CD11b+) populations were analyzed by FACS, except for the CD47-KO plus trastuzumab group, as no tumor growth occurred. Data are shown as the mean ± SEM. n = 7. (F) Tumor-associated macrophages from control-treated and trastuzumab-treated tumors were sorted by FACS (F4/80+Gr1–CD11b+CD45+) and analyzed with RT-qPCR for the expression of pro- and antiinflammatory genes. Data are shown as the mean ± SEM. n = 7. Multiple 2-sided t test. In C and E, significance was determined by 1-way ANOVA with Tukey’s multiple-comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.