Stimulation of natural killer cells with a CD137-specific antibody enhances trastuzumab efficacy in xenotransplant models of breast cancer
Holbrook E. Kohrt, Roch Houot, Kipp Weiskopf, Matthew J. Goldstein, Ferenc Scheeren, Debra Czerwinski, A. Dimitrios Colevas, Wen-Kai Weng, Michael F. Clarke, Robert W. Carlson, Frank E. Stockdale, Joseph A. Mollick, Lieping Chen, Ronald Levy
Holbrook E. Kohrt, Roch Houot, Kipp Weiskopf, Matthew J. Goldstein, Ferenc Scheeren, Debra Czerwinski, A. Dimitrios Colevas, Wen-Kai Weng, Michael F. Clarke, Robert W. Carlson, Frank E. Stockdale, Joseph A. Mollick, Lieping Chen, Ronald Levy
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Research Article Oncology

Stimulation of natural killer cells with a CD137-specific antibody enhances trastuzumab efficacy in xenotransplant models of breast cancer

Abstract

Trastuzumab, a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2; also known as HER-2/neu), is indicated for the treatment of women with either early stage or metastatic HER2+ breast cancer. It kills tumor cells by several mechanisms, including antibody-dependent cellular cytotoxicity (ADCC). Strategies that enhance the activity of ADCC effectors, including NK cells, may improve the efficacy of trastuzumab. Here, we have shown that upon encountering trastuzumab-coated, HER2-overexpressing breast cancer cells, human NK cells become activated and express the costimulatory receptor CD137. CD137 activation, which was dependent on NK cell expression of the FcγRIII receptor, occurred both in vitro and in the peripheral blood of women with HER2-expressing breast cancer after trastuzumab treatment. Stimulation of trastuzumab-activated human NK cells with an agonistic mAb specific for CD137 killed breast cancer cells (including an intrinsically trastuzumab-resistant cell line) more efficiently both in vitro and in vivo in xenotransplant models of human breast cancer, including one using a human primary breast tumor. The enhanced cytotoxicity was restricted to antibody-coated tumor cells. This sequential antibody strategy, combining a tumor-targeting antibody with a second antibody that activates the host innate immune system, may improve the therapeutic effects of antibodies against breast cancer and other HER2-expressing tumors.

Authors

Holbrook E. Kohrt, Roch Houot, Kipp Weiskopf, Matthew J. Goldstein, Ferenc Scheeren, Debra Czerwinski, A. Dimitrios Colevas, Wen-Kai Weng, Michael F. Clarke, Robert W. Carlson, Frank E. Stockdale, Joseph A. Mollick, Lieping Chen, Ronald Levy

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Figure 2

Anti-CD137 agonistic mAbs increase trastuzumab-mediated NK cell cytokine secretion and cytotoxicity on tumor cells as assayed by cell viability.

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Anti-CD137 agonistic mAbs increase trastuzumab-mediated NK cell cytokine...
To evaluate NK cell function, purified NK cells were isolated from 3 independent, healthy donor PBMCs and cultured for 24 hours together with trastuzumab (10 μg/ml) and irradiated (50 Gy) breast cancer cells (SKBR3) at a ratio of 1:1. After 24 hours, NK cells were isolated by negative selection and assessed for purity (>90% purity as defined by CD3–CD56+ flow cytometry) and activation (>50% expression of CD137). Breast cancer cell lines including MCF7 (A and E), BT474M1 (B and F), HER18 (C and G), and SKBR3 (D and H) were cultured for 18 hours with preactivated, purified NK cells in medium alone, or with anti-CD137 mAb (BMS-663513, 10 μg/ml) alone, trastuzumab (10 μg/ml) alone, or trastuzumab plus anti-CD137 mAbs (both at 10 μg/ml), and supernatant was harvested and analyzed by ELISA for IFN-γ (A, MCF7, P = 0.39; B, BT474M1, P = 0.16; C, HER18, *P = 0.017; D, SKBR3, *P = 0.034). Cells were washed and incubated with annexin V and 7-AAD to determine percentage of apoptotic tumor cells by annexin V and 7-AAD staining (E, MCF7, P = 0.43; F, BT474M1, *P = 0.031; G, HER18, *P < 0.001; H, SKBR3, *P < 0.001). Data are shown as mean ± SEM.